1. Immunology and Inflammation
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Lymph node stromal cells constrain immunity via MHC class II self-antigen presentation

  1. Antonio P Baptista
  2. Ramon Roozendaal
  3. Rogier M Reijmers
  4. Jasper J Koning
  5. Wendy W Unger
  6. Mascha Greuter
  7. Eelco D Keuning
  8. Rosalie Molenaar
  9. Gera Goverse
  10. Marlous M S Sneeboer
  11. Joke M M den Haan
  12. Marianne Boes
  13. Reina E Mebius  Is a corresponding author
  1. Vrije Universiteit Medical Center, Netherlands
  2. University of Porto, Portugal
  3. University Medical Center Utrecht, Netherlands
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Cite this article as: eLife 2014;3:e04433 doi: 10.7554/eLife.04433

Abstract

Non-hematopoietic lymph node stromal cells shape immunity by inducing MHC-I-dependent deletion of self-reactive CD8+ T cells and MHC-II-dependent anergy of CD4+ T cells. In this study, we show that MHC-II expression on lymph node stromal cells is additionally required for homeostatic maintenance of regulatory T cells (Tregs) and maintenance of immune quiescence. In the absence of MHC-II expression in lymph node transplants, i.e. on lymph node stromal cells, CD4+ as well as CD8+ T cells became activated, ultimately resulting in transplant rejection. MHC-II self-antigen presentation by lymph node stromal cells allowed the non-proliferative maintenance of antigen-specific Tregs and constrained antigen-specific immunity. Altogether, our results reveal a novel mechanism by which lymph node stromal cells regulate peripheral immunity.

https://doi.org/10.7554/eLife.04433.001

eLife digest

In vertebrates, the immune response that protects against infection and disease is made up of two systems. The body's first line of defense is the innate immune system that attacks invaders rapidly but indiscriminately. If this fails to stop disease progression, the adaptive immune system is activated. Although the adaptive immune response is relatively slow compared with the innate immune response, it is more deliberate and produces cells that specifically target and destroy the pathogen or diseased cells present. The adaptive immune system also produces cells that ‘remember’ the pathogen so that it can be destroyed more quickly if it invades again.

A special type of white blood cell, called a T cell, is key to the adaptive immune response. To activate T cells, fragments of molecules that provoke an immune response—called antigens—must be bound to a ‘major histocompatibility complex’ (MHC) and presented to these cells. This process often occurs in lymph nodes, organs that filter the fluid moving from the body's tissues back into the blood.

Particular cells in the lymph node, called lymph node stromal cells, are essential for the organ's structure; recently, these cells have also been found to play roles in regulating the immune response. For example, lymph node stromal cells can help to destroy self-reactive T cells that attack the host's normal, healthy cells. In addition, some types of lymph node stromal cells produce major histocompatibility complexes, although exactly what these complexes do on these cells was unknown.

Baptista, Roozendaal et al. investigated the role of the major histocompatibility complexes expressed by lymph node stromal cells by transplanting mutant cells that could not produce these complexes into otherwise normal mice. In these mice, T cells became more activated than normal and the transplant was rejected after several weeks.

On further investigation, Baptista, Roozendaal et al. discovered that the major histocompatibility complexes produced by the lymph node stromal cells help to maintain an active population of regulatory T cells. These cells are responsible for shutting down the immune response. This work therefore improves our understanding of how the immune response is regulated and could help to develop new strategies for preventing donor organs being rejected after transplantation.

https://doi.org/10.7554/eLife.04433.002

Introduction

Lymph nodes play a crucial role in the initiation of adaptive immune responses by bringing together antigens, antigen-presenting cells (APCs), and antigen-specific lymphocytes (Junt et al., 2008; Mueller and Germain, 2009; Roozendaal and Mebius, 2011). The interaction between these components within the lymph node parenchyma is fostered by its highly structured architecture, which is dictated by resident non-hematopoietic stromal cells (Katakai et al., 2004). Despite their low frequency among total lymph node cells, lymph node stromal cells have gathered considerable attention recently, as they were found to have important immunoregulatory functions. Indeed, in addition to promoting the communication between dendritic cells (DCs) and lymphocytes by providing anchorage for DCs and guiding T cells via the production of chemokines (Sixt et al., 2005; Bajenoff et al., 2006), lymph node stromal cells were shown to regulate the size of the lymphocyte pool by providing essential survival factors (Link et al., 2007) and to limit T cell activation/proliferation by producing nitric oxide (Lukacs-Kornek et al., 2011; Siegert et al., 2011).

In addition to these broad mechanisms that control the entire lymphocyte pool, lymph node stromal cells were also found to influence lymphocytes in a cognate antigen-dependent manner. Similar as to thymic epithelial cells, several subsets of lymph node stromal cells were shown to express and present peripheral tissue-restricted antigens (PTAs) (Nichols et al., 2007; Cohen et al., 2010; Fletcher et al., 2010). Presentation of PTA-derived peptides in the context of MHC-I molecules leads to the deletion of auto-reactive CD8+ T cells (Lee et al., 2007; Nichols et al., 2007; Magnusson et al., 2008; Yip et al., 2009; Cohen et al., 2010; Fletcher et al., 2010). Furthermore, acquisition of dendritic cell-derived peptide–MHC-II complexes by lymph node stromal cells gives them the ability to negatively regulate CD4+ T cell proliferation and survival (Dubrot et al., 2014).

Importantly, next to the ability to capture MHC-II molecules, lymph node stromal cells were also shown to express their own MHC-II molecules (Malhotra et al., 2012; Dubrot et al., 2014). In this study, we have investigated, for the first time, the function of endogenous MHC-II molecules on lymph node stromal cells. We found MHC-II expression on lymph node stromal cells to be instrumental for the maintenance of FoxP3+ regulatory T cells (Tregs), thereby safeguarding the homeostasis of the immune system by limiting immune reactivity. Altogether, our data add a new layer to the immunoregulatory properties of lymph node stromal cells.

Results

Lymph node stromal cells express surface MHC-II

MHC-II expression was traditionally thought to be restricted to hematopoietic-derived professional antigen-presenting cells, such as dendritic cells, macrophages, and B cells. It is now clear, however, that cells of other origins are also able to express MHC-II molecules and to present antigens to CD4+ T cells (Stagg et al., 2006; Kreisel et al., 2010; Koyama et al., 2012). Amongst these other cells, lymph node stromal cells express MHC-II in the steady-state (Figure 1 and (Malhotra et al., 2012; Dubrot et al., 2014)). By flow cytometry, we found CD45gp38+CD31 fibroblastic reticular cells (FRCs), CD45gp38+CD31+ lymphatic endothelial cells (LECs), and CD45gp38CD31+ blood endothelial cells (BECs) to express MHC-II on their surface (Figure 1A). In agreement, these cells also contained mRNA transcripts for MHC-II (H2-Ab1) itself as well as for the MHC-II-related molecules CD74 (invariant chain—Ii), H2-M (chaperone that catalyzes peptide loading onto MHC-II molecules), and LAMP-1 (marker for endosomes/lysosomes, where antigen processing and MHC-II loading occur) (Figure 1B). Altogether, these data suggest that lymph node stromal cells possess the necessary machinery to process and present antigens in the context of MHC-II molecules.

Lymph node stromal cells express MHC-II in the steady-state.

(A) MHC-II expression on lymph node stromal cells was assessed by flow cytometry. Fibroblastic reticular cells (FRCs) were identified as CD45gp38+CD31 cells; lymphatic endothelial cells (LECs) as CD45gp38+CD31+ cells; and blood endothelial cells (BECs) as CD45gp38CD31+ cells. Filled histograms represent control staining, whereas open histograms represent MHC-II expression. Representative example of five independent experiments performed. (B) mRNA expression of MHC-II (H2-Ab1) and MHC-II-related genes, CD74, H2-M, and LAMP-1, was determined on wild-type FACS-sorted stromal cells by real-time PCR. Total pLN cells (arbitrarily set at one) and FACS-sorted CD45+MHC-II- and CD45+MHC-II+ cells were used as controls. The data represent mean ± SEM; n = 4.

https://doi.org/10.7554/eLife.04433.003

MHC-II expression on lymph node stromal cells regulates T cell activation

To determine the function of MHC-II expression on lymph node stromal cells, we performed lymph node transplantation experiments in which popliteal lymph nodes of wild-type recipient mice were surgically replaced by MCH-II knock-out (KO) lymph nodes. Within 4 weeks, these transplants reconnect to the lymphatic and blood vasculature. While the stromal cell compartment within the transplant remains of donor origin, virtually all immune cells of the donor animal will be replaced by host-derived cells (Wolvers et al., 1999; Hammerschmidt et al., 2008; Molenaar et al., 2009). Therefore, transplantation of MHC-II KO lymph nodes into wild-type recipients resulted in selective absence of stromal cell endogenously expressed MHC-II molecules, whereas MHC-II was normally expressed on the recipient-derived hematopoietic cells that had migrated into the transplanted lymph node (Figure. 2—figure supplement 1). Residual MHC-II expression on stromal cells (Figure. 2—figure supplement 1) was likely due to the acquisition of dendritic cell-derived peptide–MHC-II complexes (Dubrot et al., 2014). Of note, extra-thymic AIRE-expressing cells (ETACs), which were initially characterized as a stromal cell subset (Gardner et al., 2008) but later determined to be hematopoietic-derived (Gardner et al., 2013), expressed CD45, CD11c, EpCAM, as well as MHC-II (Figure 2—figure supplement 2) within MHC-II KO lymph node transplants (Figure 2—figure supplements 1,2). Therefore, ETACs are likely not involved in the phenotypes described below. Ablation of MHC-II on lymph node stromal cells resulted in increased frequencies of CD62LCD44+ activated CD4+ and CD8+ T cells within the transplanted lymph nodes (Figure 2A). T cell activation was restricted to MHC-II KO transplants, as the endogenous lymph nodes of MHC KO lymph node transplant recipients showed similar frequencies of naïve and activated T cells as compared to the endogenous lymph nodes of wild-type transplant recipients (Figure 2—figure supplement 3). Likely as a consequence of local activation, MHC-II KO transplants ended up being rejected, since by week 8 post-transplantation, they could not be recovered. Compared to wild-type transplants, 4 weeks after transplantation, MHC-II KO lymph node transplants showed significant rigidity and tissue damage and the unusual presence of large clusters of CSFR1+CD11b+CD11c+Moma2+F4/80+MHC-II+ macrophages (Figure 2—figure supplement 4). Furthermore, they showed increased expression of several gene transcripts recently associated with a ‘universal’ rejection module (Khatri et al., 2013) and an overall increased common rejection module (CRM) score (Figure 2—figure supplement 5). Altogether, our results suggest that MHC-II expression on lymph node stromal cells regulates T cell activation, exerting a local dampening effect on both CD4+ and CD8+ T cells.

Figure 2 with 7 supplements see all
MHC-II+ lymph node stromal cells regulate T cell activation.

Wild-type mice were transplanted with either wild-type (wt Tx) or MHC-II KO (MHC-II KO Tx) lymph nodes. After 4 weeks, host-derived CD4+ and CD8+ T cells present within the transplants were characterized by flow cytometry (A). In (B), transplanted animals were depleted of CD4+ T cells by administration of the antibody GK1.5. For easier comparison, the data of Figure 2A regarding CD62LCD44+CD8+ T cells are duplicated here. Representative contour plots are shown; the numbers in the plots indicate the frequency of cells within the drawn gates. The data represent mean ± SEM; n = 4; *p ≤ 0.05, **p ≤ 0.01.

https://doi.org/10.7554/eLife.04433.004

CD4+ T cells restrict CD8+ T cell activation in MHC-II KO lymph node transplants

As MHC-II molecules are not thought to directly mediate cellular interactions with CD8+ T cells, we reasoned that CD8+ T cell activation in the absence of lymph node stromal cell MHC-II expression could be an indirect effect of local CD4+ T cell activation. We tested this hypothesis by depleting CD4+ cells with bi-weekly intraperitoneal injections of the anti-CD4 antibody GK1.5, starting 1 week before transplantation until the time of analysis (4 weeks after transplantation) (Figure 2—figure supplement 6). In contrast to our expectation, CD4+ T cell depletion led to a further increase in the frequency of activated CD62LCD44+ CD8+ T cells in MHC-II KO transplants (Figure 2B). These results therefore suggested that CD8+ T cell activation in the absence of MHC-II expressing lymph node stromal cells was not a direct consequence of deregulated CD4+ T cell activation, but rather appeared to be constrained by CD4+ T cells. Further supporting this notion, also within the endogenous lymph nodes of CD4-depleted mice receiving MHC-II KO lymph node transplants, a significant increase of CD62LCD44+ CD8+ T cells was observed when compared to wild-type lymph node transplant recipients (Figure 2—figure supplement 7). Thus, it appears that in contrast to our initial hypothesis, CD4+ T cells not only restrain local CD8+ T cell activation in transplanted lymph nodes in a manner that is dependent on lymph node stromal cell endogenous MHC-II expression but are also required to prevent its systemic spreading.

MHC-II+ stromal cells support FoxP3+ Treg proliferation

T cell activation is largely kept in check by Treg cells, thereby safeguarding the homeostasis of the immune system. Since Treg frequency was reduced in MHC-II KO lymph node transplants (Figure 2—figure supplement 6) and Treg development and maintenance involves agonistic selection on MHC-II presented peptides (Josefowicz et al., 2012), we decided to assess directly whether lymph node stromal cell endogenous MHC-II deficiency would affect Tregs. By transferring CFSE-labeled T cells into Rag2−/− mice transplanted with either MHC-II KO or wild-type lymph nodes, we found the homeostatic proliferation of CD4+FoxP3+ cells to critically depend on stromal cell endogenous MHC-II expression. As compared to wild-type transplants, CD4+FoxP3+ cells proliferated roughly 3 times less efficiently in MHC-II KO lymph node transplants (Figure 3A,B and Figure 3—figure supplement 1), resulting in a threefold reduction in Treg frequency (Figure 3C). A reduction in the proliferation of CD4+FoxP3 cells was also observed in MHC-II KO lymph node transplants, although this did not reach statistical significance (Figure 3B and Figure 3—figure supplement 1). Overall, these results revealed a pivotal role for lymph node stromal cell MHC-II expression on the homeostatic proliferation of CD4+ T cells, which seemed particularly relevant for the homeostatic maintenance of CD4+Foxp3+ Tregs.

Figure 3 with 1 supplement see all
MHC-II+ lymph node stromal cells support Treg homeostatic proliferation.

Rag2-deficient mice transplanted with either wild-type (wt Tx) or MHC-II KO (MHC-II KO Tx) lymph nodes were injected with 107 CFSE-labeled wild-type lymphocytes. 48 hr later, mice were sacrificed and the transferred cells within the lymph node transplants analyzed by flow cytometry. The CFSE profile of transferred Foxp3+CD4+ T cells is shown in (A). In (B), the ratio between the division indexes of wild-type (B6) and MHC-II KO lymph node transplant recovered CD4+Foxp3+ Tregs and CD4+Foxp3 conventional T cells is shown. The frequency of CD4+Foxp3+ Tregs recovered from wild-type and MHC-II KO lymph node transplant is shown in (C). The data represent mean ± SEM; n = 2 independent experiments with 2–3 animals per group; *p ≤ 0.05, **p ≤ 0.01.

https://doi.org/10.7554/eLife.04433.012

Presentation of endogenous self-antigens by lymph node stromal cells supports Treg maintenance in vitro

The observation that MHC-II expression on lymph node stromal cells impacted on the peripheral maintenance of Tregs implied that cognate ligands for the T cell receptor (TCR) of Tregs are expressed and presented by the lymph node stromal cell compartment. In support of such hypothesis, previous research has reported expression of several peripheral tissue-restricted antigens (PTAs) in lymph node stromal cells (Nichols et al., 2007; Cohen et al., 2010; Fletcher et al., 2010). To assess whether lymph node stromal cells were able to present endogenous antigens in the context of MHC-II molecules, we used K14-mOVA transgenic mice, in which ovalbumin (OVA) expression is driven by the human keratin 14 promoter (Bianchi et al., 2009). In these mice, OVA is expressed in the skin and thymus (Bianchi et al., 2009) as well as in the 3 major lymph node stromal cell subsets (Figure 4A). As all primary lymph node stromal cell cultures established contained large amounts of contaminating hematopoietic cells (data not shown), which precluded our in vitro antigen presentation assays, we generated distinct lymph node stromal cell lines by long-term in vitro culture of lymph node single cell suspensions of K14-mOVA mice on collagen matrixes. Using this approach, we generated one cell line (K14-mOVAneg), resembling FRCs, which did not express detectable OVA mRNA transcripts and was thus used as a control cell line, and another cell line (K14-mOVApos), resembling LECs, which expressed OVA transcripts abundantly (Figure 4B and Figure 4—figure supplement 1). Co-culture of these cell lines with OVA-specific CD8+ OT-I and CD4+ OT-II T cells revealed that OVA-derived peptides could be presented by lymph node stromal cells in MHC-I as well as MHC-II molecules, as both OT-I and OT-II T cells showed increased CD25 expression when cultured with K14-mOVApos but not with K14-mOVAneg cells (Figure 4—figure supplement 2). Neither lymph node stromal cell line induced OT-I or OT-II T cell proliferation, however (Figure 4—figure supplement 2). To directly address whether self-antigen presentation by lymph node stromal cells in the context of MHC-II molecules influenced Tregs, we repeated our co-culture experiments and stained CD4+ T cells for Foxp3 and Helios. As compared to culture in medium alone, co-culture of CD4+ T cells with either cell line increased the survival of Foxp3+ cells irrespective of their TCR specificity (Figure 4C), which is suggestive of the production of T cell survival factors by both cell lines (Link et al., 2007). More importantly, K14-mOVApos cells significantly increased the recovery of Foxp3+ OT-II T cells as compared to the K14-mOVAneg cell line (Figure 4C). This effect was not apparent in co-cultures with wild-type CD4+ T cells and could be blocked by the addition of the MHC-II blocking antibody M5/114 (Figure 4C), indicating that the presentation of OVA-derived peptides in the context of MHC-II molecules by the K14-mOVApos cell line was the driver of increased Foxp3+ OT-II T cell survival in our assays. Of significance, CD4+Foxp3 conventional OT-II T cells, in the exact same conditions, did not show similar behavior (Figure 4—figure supplement 3). Overall, our data suggest that MHC-II-mediated self-antigen presentation by lymph node stromal cells drives CD4+Foxp3+ Treg maintenance.

Figure 4 with 3 supplements see all
Endogenous OVA presentation by lymph node stromal cells promotes Treg maintenance in vitro.

OVA mRNA expression in primary FACS-sorted stromal cells (A; n = 2) and in vitro-generated stromal cell lines derived from K14-mOVA mice (B; n = 5) was determined by real-time PCR. Peripheral lymph nodes (pLN) from K14-mOVA mice were used as controls. (C) MACS-sorted CD4+ wild-type or OT-II transgenic cells were cultured together with in vitro-generated stromal cell lines of K14-mOVA origin in the absence or presence of the MHC-II blocking antibody M5/114. After 72 hr of co-culture, OT-II cells were characterized by flow cytometry. Representative counterplots are shown; the numbers in the plots represent the frequency of CD4+Fox3+ T cells. Graphs depict the number of CD4+Fox3+ T cells in the beginning and at the end of culture. Data represent mean ± SEM; n = 3 for wild-type CD4+ T cells; n = 8 for OT-II cells; and n = 3 for OT-II cells + M5/114. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

https://doi.org/10.7554/eLife.04433.014

Presentation of endogenous self-antigens by lymph node stromal cells supports peripheral maintenance of Tregs in vivo

To address the effect of self-antigen presentation by lymph node stromal cells on antigen-specific Tregs in vivo, we transplanted lymph nodes of K14-mOVA mice into wild-type hosts and analyzed the fate of transferred OVA-specific OT-II CD4+ T cells. In this transplantation setting, OVA expression became confined to the transplanted lymph node stromal cells. Transplantation of K14-mOVA lymph nodes did not affect the endogenous pools of CD8+ T cells, CD4+ T cells, or CD4+FoxP3+ Tregs, as in the transplants as well as in the endogenous lymph nodes of the different ‘chimeric’ mice, the frequencies of the various T cell subsets were comparable (Figure 5—figure supplement 1). Similarly, transferred OT-II T cells showed a comparable distribution of naïve CD62L+CD44 and activated CD62LCD44+ phenotypes in wild-type and K14-mOVA lymph node transplants as well as in wild-type endogenous lymph nodes (Figure 5A,B). Confirming our in vitro data, OT-II T cells did not proliferate (data not shown). Presentation of OVA by the lymph node stroma, however, led to a significant increase in the number of CD4+FoxP3+ OT-II T cells, particularly of the naïve-like CD62L+ subtype, within K14-mOVA transplants as compared to wild-type lymph node transplants (Figure 5C). Higher frequencies of total and CD62L+ naïve-like CD4+FoxP3+ OT-II Tregs were also evident in the endogenous lymph nodes of recipients of K14-mOVA lymph node transplants as compared to wild-type lymph node transplant recipient mice, although this did not translate in increased cell numbers (Figure 5D). In conclusion, our data suggest that the presentation of endogenous antigens by the lymph node stroma contributes to the selective maintenance of antigen-specific Tregs.

Figure 5 with 2 supplements see all
Endogenous OVA presentation by lymph node stromal cells promotes Treg maintenance in vivo.

Wild-type mice transplanted with either wild-type (wt Tx) or K14-mOVA transgenic (K14-mOVA Tx) lymph nodes were injected with 107 CD45.1+ OT-II cells. 3 days after the transfer, mice were sacrificed and the transferred OT-II cells in both the transplanted lymph nodes (A, C) and the endogenous lymph nodes (B, D) analyzed by flow cytometry. Naïve and activated OT-II T cells were defined as CD62L+CD44- and CD62LCD44+ cells, respectively (A, B); naïve-like OT-II Tregs as CD4+CD62L+Foxp3+ cells (C, D). Representative contour plots of the analysis performed are shown on the left; the numbers in the plots indicate the frequency of cells within the drawn gates. The graphs shown on the right represent the mean ± SEM of 2 independent experiments; n = 6 and n = 8 for wild-type and K14-mOVA lymph node transplanted animals, respectively; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

https://doi.org/10.7554/eLife.04433.018

Presentation of endogenous self-antigens by lymph node stromal cells constrains immune reactivity

The increased frequencies and numbers of CD4+FoxP3+ OT-II T cells in K14-mOVA lymph node transplant recipients suggested that the presentation of OVA as a self-antigen by lymph node stromal cells may promote the development of tolerance towards OVA. To test this hypothesis, we measured OVA-specific delayed-type hypersensitivity (DTH) responses in mice transplanted with either K14-mOVA or wild-type lymph nodes. As determined by ear swelling, DTH responses towards OVA were significantly reduced in mice transplanted with K14-mOVA lymph nodes as compared to mice transplanted with wild-type lymph nodes (Figure 6A), suggesting that OVA presentation by lymph node stromal cells restrained immune reactivity. In this setting, control of the immune response did not seem to emerge from reduced T cell priming or antigen-specific T cell deletion as comparable frequencies of IFNγ-producing effector CD8+ and CD4+ T cells were found in K14-mOVA lymph node and wild-type lymph node recipients, upon in vitro re-stimulation of splenic cells with OVA peptides (% of IFNγ+ CD8+ and CD4+ T cells in wild-type lymph node transplant recipients vs K14-mOVA lymph node transplant recipients: 0.23 ± 0.08 vs 0.37 ± 0.17, p = 0.28; 0.28 ± 0.09 vs 0.55 ± 0.22, p = 0.47).

Figure 6 with 1 supplement see all
Endogenous OVA presentation by lymph node stromal cells constrains immune reactivity.

(A) Wild-type mice transplanted with either wild-type (wt Tx) or K14-mOVA transgenic (K14-mOVA Tx) lymph nodes were immunized with OVA in incomplete Freund's adjuvant (IFA) in the tail base and re-challenged with OVA alone in both ears. In vivo delayed-type hypersensitivity (DTH) responses were determined by ear swelling. The data represent mean ± SEM; n = 5 mice per group; *p ≤ 0.05. (B) Wild-type mice either left untreated or transplanted with wild-type or K14-mOVA transgenic lymph nodes, at week -4, were transplanted with either wild-type or K14-mOVA skin on day 0. 4 weeks after skin transplantation, skin grafts were isolated and mRNA transcripts belonging to the common rejection module (CRM) analyzed by real-time PCR. The aggregate CRM score is shown. n = 6 mice per group; *p < 0.05, ***p ≤ 0.001.

https://doi.org/10.7554/eLife.04433.021

To address whether self-antigen presentation by lymph node stromal cells could have clinical relevance, we used a dual transplantation system in which we assessed whether transplantation of K14-mOVA lymph nodes would improve K14-mOVA skin graft acceptance. In these experiments, wild-type age-matched recipients were left untreated or were transplanted with either wild-type or K14-mOVA lymph nodes at week -4, followed by a skin transplant at day 0, after which they were monitored for 4 additional weeks. As compared to wild-type skin grafts, K14-mOVA skin grafts showed increased thickness and rigidity (not depicted) and an overall increased CRM score (Figure 6B), suggesting an ongoing process of rejection. Expression of CRM genes was significantly reduced, by more than 50%, by prior transplantation of K14-mOVA, but not wild-type lymph nodes (Figure 6B and figure 6—figure supplement 1), thus confirming the regulatory properties of self-antigen-expressing lymph node stromal cells. Remarkably, in contrast to K14-mOVA skin transplants, K14-mOVA lymph node transplants showed unaltered CRM scores when compared to wild-type transplants or to endogenous non-transplanted wild-type lymph nodes (Figure 2—figure supplement 5), which suggest that lymph node stromal cells are specifically endowed with regulatory potential. As assessed by the CRM, this capacity seemed to be, at least partially, dependent on MHC-II expression (Figure 2—figure supplement 5). Taken together, our data suggest that antigen-presenting lymph node stromal cells constrain immune responses in vivo independently of antigen-specific T cell deletion.

Discussion

The last years have been instrumental in uncovering the crucial role of non-hematopoietic lymph node stromal cells in the maintenance of immune tolerance. Lymph node stromal cells have been shown to regulate the proliferation of activated T cells by inhibiting T cell homotypic interactions and restraining entry into cell cycle (Lukacs-Kornek et al., 2011; Siegert et al., 2011). In addition, they were shown to induce the deletion of auto-reactive CD8+ T cells via their capacity to produce, process, and present PTAs on MHC-I molecules (Lee et al., 2007; Nichols et al., 2007; Magnusson et al., 2008; Cohen et al., 2010; Fletcher et al., 2010) and to induce CD4+ T cell dysfunction via acquired peptide–MHC-II complexes (Dubrot et al., 2014). In the present study, we have investigated the role of these cells as antigen-presenting cells via endogenous, non-acquired MHC-II. We have shown that MHC-II-mediated antigen presentation by lymph node stromal cells promotes the maintenance of Tregs contributing to the preservation of immune quiescence.

MHC-II molecules were expressed on the surface of FRCs, LECs, and BECs and endowed these cells with the ability to control immune reactivity. As evidenced by transplantation of MHC-II KO lymph nodes, which resulted in selective MHC-II deficiency on the transplanted lymph node stromal cell compartment, MHC-II expression on lymph node stromal cells controlled the activation of CD4+ as well as CD8+ T cells. Both T cell subsets showed an increased frequency of CD62LCD44+ activated cells in MHC-II KO lymph node transplants as compared to wild-type lymph node transplants. These results are particularly relevant, as in the context of the ability of lymph node stromal cells to capture dendritic cell-derived peptide–MHC-II complexes (Dubrot et al., 2014), they reveal a critical role for lymph node stromal cell endogenously expressed MHC-II in immune regulation. The effect of stromal cell MHC-II deficiency on CD8+ T cell activation was rather unexpected as MHC-II molecules are not thought to mediate cellular interactions with CD8+ T cells. Additional experiments, however, showed that this effect resulted from the lack of regulation by CD4+ T cells. In line with this, we found that in the absence of endogenous MHC-II expression on lymph node stromal cells, the maintenance as well as the proliferation of Tregs was impaired. Together, these data showed that for the homeostatic regulation of Tregs, similar as for other lymphocyte populations (Cyster et al., 1994; Dummer et al., 2001), access to the lymph node parenchyma is required. Importantly, our data also suggest that the maintenance of Tregs and their control of T cell activation are spatially linked and must occur within the same location. In fact, even though Treg numbers were adequately maintained in secondary lymphoid organs other than the transplant, the inability to maintain them within MHC-II KO lymph node transplants resulted in the local activation of T cells, which in the long-term resulted in the rejection of the MHC-II KO transplants. This is significant as it suggests that, in order to minimize host vs graft disease in transplanted individuals, local maintenance of Tregs should be enforced.

Of significance, as ETACs persisted and still expressed MHC-II in MHC-II KO lymph node transplants, we can conclude that, despite their ability to inactivate CD4+ and CD8+ T cells (Gardner et al., 2008, 2013), these cells cannot fully compensate for the lack of lymph node stromal cell-imposed regulation.

One could argue that rejection of MHC-II-deficient transplants could result from the introduction of MHC-I defects or unappreciated antigens during genetic targeting to generate MHC-II KO mice. In our experiments, we used MHC-II KO animals that were established by the insertion of a hygromycin-resistance cassette at the deletion site, spanning from the second exon of H2-Ab1 to the third exon of the H2-Ea gene, into 129S2/SvPas-derived H1 embryonic stem cells (Madsen et al., 1999). As the H2 locus of C56BL/6J and 129Sv strains is identical (b haplotype) and our MHC-II KO animals show normal MHC-I expression (Madsen et al., 1999), rejection of MHC-II KO lymph nodes could not have been caused by impaired recognition of MHC-I molecules. Indeed, if faulty recognition of MHC-I molecules would be responsible for MHC-II KO transplant rejection, one would not expect CD8+ T cell activation to be present and augmented by CD4+ T cell deletion. Unappreciated antigens derived from the hygromycin-resistance cassette could play a role in MHC-II KO lymph node rejection, as recipient mice had never been exposed to such antigens. However, previous experience with lymph nodes harboring GFP constructs (Molenaar et al., 2009) and presently also with the K14-mOVA transgene suggest that possibility to be remote. Taken these considerations as a whole, our data strongly support the notion that endogenous MHC-II expression on lymph node stromal cells is critical for maintaining low CRM scores and thus safeguarding tolerance.

Treg development in the thymus occurs through agonist selection on MHC-II presented peptides (Josefowicz et al., 2012). Similarly, our data with the K14-mOVA transgenic lymph node transplantation and OT-II T cell transfer system confirmed that the peripheral maintenance of the Treg pool required MHC-II-mediated presentation of endogenous antigens as well. Presentation of OVA-derived peptides by the transplanted lymph node stromal cell compartment led to enhanced numbers of OT-II Tregs within the lymph node transplant, which was particularly evident for CD62L-expressing CD4+Foxp3+ Tregs. Determination of the origin of the expanded cells warrants further research. Given that a large fraction of the expanded Treg population expressed Helios (Figure 5—figure supplement 2), a transcription factor originally associated with Treg development in the thymus (Thornton et al., 2010), it may seem that transferred thymus-derived OT-II Tregs were specifically maintained via cognate interactions with the K14-mOVA lymph node stroma. Alternatively, as Helios expression was more recently shown to be induced upon T cell activation (Akimova et al., 2011) preceding Foxp3 induction on peripherally induced Tregs (Gottschalk et al., 2012), it may be that our expanded Treg population reflects peripheral differentiation of naïve OT-II T cells into OT-II Tregs. In either case, the increase in OT-II Tregs did not involve cellular proliferation. This contrasts with the effect of self-antigen recognition in peripheral tissues, such as the skin, which induces vigorous Treg proliferation (Rosenblum et al., 2011). Overall, our in vitro and in vivo data suggest that the antigen-mediated interaction between lymph node stromal cells and Tregs provides specific survival signals to the latter cells that may allow antigen-stimulated Tregs to outcompete Tregs that have not seen their cognate antigen. If present, such a mechanism would most likely select the Treg repertoire to match the peripheral need for immune regulation. Supporting this hypothesis, it was previously shown that the peripheral Treg repertoire differs significantly between different anatomical locations (Lathrop et al., 2008), a situation that may reflect differences in regional lymph nodes (Wolvers et al., 1999; Hammerschmidt et al., 2008).

Transplantation of K14-mOVA transgenic lymph nodes was associated with in vivo development of OVA unresponsiveness. Importantly, in contrast with previous reports showing that PTA expression by lymph node stromal cells drives the deletion of self-reactive CD8+ T cells (Lee et al., 2007; Nichols et al., 2007; Gardner et al., 2008; Magnusson et al., 2008; Cohen et al., 2010; Fletcher et al., 2010), in our system OVA unresponsiveness did not seem to be related to this mechanism. We observed comparable frequencies of OVA-specific IFNγ-producing CD8+ T cells between mice transplanted with K14-mOVA transgenic lymph nodes and mice transplanted with wild-type lymph nodes. Unresponsiveness did not seem to arise from the deletion of OVA-specific IFNγ-producing CD4+ T cells either. This is in agreement with the recent observation by Magnusson et al., showing that self-antigen presentation by lymph node stromal cells does not promote the deletion of self-reactive CD4+ T cells (Magnusson et al., 2008). This is however, in contrast with the findings that LECs induce antigen-specific CD4+ T cell apoptosis (Dubrot et al., 2014). The reasons for such disparate results are currently unknown, but may relate to the use of different experimental systems and/or to putative differences in self-antigen processing by lymph node stromal cells and dendritic cells. Regardless of the origin of such discrepancies, together these results highlight the existence of multiple regulatory mechanisms that in concert safeguard the homeostasis of the immune system.

Importantly, as lymph node stromal cells enwrap the tubular system that connects the incoming afferent lymphatic vessels with the inner core of lymph nodes (Sixt et al., 2005), it will be important to assess in the future whether exogenous antigens can be taken up from the lymph node conduits by lymph node stromal cells and presented to T cells. This may be particularly relevant in the context of immune responses as presentation of exogenous antigen by lymph node stromal cells may contribute to reactive CD8+ T cell deletion, to CD4+ T cell dysfunction, and/or to Treg expansion and therefore prevent excessive inflammation or contribute to the contraction of the immune response. Alternatively, as lymph node stromal cells express toll-like receptors (TLRs) (Fletcher et al., 2010), it may be that during immune responses antigen processing gets redirected (Blander and Medzhitov, 2006) and that lymph node stromal cells, like dendritic cells (Reis e Sousa, 2006), mature from a tolerogenic phenotype towards an immunogenic one.

In conclusion, we showed a hitherto unrecognized role for lymph node stromal cells in the maintenance of peripheral Tregs and immune quiescence. MHC-II-mediated antigen presentation by lymph node stromal cells was essential for the steady-state maintenance of Tregs as well as for their homeostatic recovery in lymphocyte-depleted environments. Tregs, in turn, prevented immune activation, which was required for graft acceptance. Importantly, in contrast with previous reports (Lee et al., 2007; Nichols et al., 2007; Magnusson et al., 2008; Cohen et al., 2010; Fletcher et al., 2010), in vivo immune regulation by self-antigen-expressing lymph node stromal cells in our transplantation setting was not related to self-reactive T cell deletion, suggesting that lymph node stromal cells may use multiple mechanisms to control self-reactivity.

Materials and methods

Mice

C57BL/6J (wild-type), C57BL/6.129S2-H2delAb1−Ea/J (MHC-II KO), C57BL/6-Rag2tm1Cgn/J (Rag2−/−), and human keratin 14 membrane-bound ovalbumin (K14-mOVA), C57BL/6-Tg(TcraTcrb)1100MjB/J (OT-I), and C57BL/6-Tg(TcraTcrb)425Cbn/J(OT-II) transgenic mice were kept at the Vrije University Medical Center animal facility under SPF conditions. All animal experiments were reviewed and approved by the Vrije University Scientific and Ethics Committees.

Lymph node transplantation

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Lymph node transplants were performed as previously described (Mebius et al., 1993). Briefly, wild-type or Rag2−/− recipient mice were anaesthetized with xylazine and ketamine and their popliteal lymph nodes removed and replaced by peripheral (axillary, brachial, or inguinal) lymph nodes of donor origin (wild-type, MHC-II KO, or K14-mOVA). Each recipient mouse received two identical lymph nodes. Transplants were allowed to reconnect to the blood and lymphatic vasculatures for at least 4 weeks, upon which their function was tested.

Skin transplantation

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Wild-type recipient mice were anaesthetized with xylazine and ketamine and shaved on the flank, after which a skin flap of 1 cm2 was removed. Back skin from shaved, sex-matched wild-type or K14-mOVA donor mice was removed and cut into 1 cm2 pieces, which were sutured to the recipients' skin.

In vivo T cell depletion

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In lymph node transplant recipients of wild-type origin, depletion of CD4+ T cells was achieved by intraperitoneal treatment with the anti-CD4 antibody GK1.5. Treatment was renewed twice per week starting 1 week before the performance of lymph node transplants until the end of the experiments. The first two injections contained each 200 μg of antibody, whereas all the remaining only 100 μg. In Rag2−/− recipient mice, homeostatic expansion of T cells co-transplanted with the donor lymph nodes was prevented by treatment with the anti-CD4 and anti-CD8 antibodies GK1.5 and 2.43. Each mouse received 3 intraperitoneal injections of 200 μg of each antibody evenly distributed during the first week after transplantation.

Cell isolation, labeling, and transfer

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To obtain single cell suspensions for in vivo transfer, lymph nodes and spleens of wild-type and OT-II transgenic mice were squeezed through cell strainers. Erythrocytes were lysed with ammonium-chloride–potassium (ACK) lysis buffer. OT-II T cells were isolated with the CD4+ cell negative isolation kit from Miltenyi (Leiden, The Netherlands) following the manufacturer's instructions—purity was on average >85%. CFSE (Invitrogen, Breda, The Netherlands) labeling was performed as previously described (Wolvers et al., 1999). Briefly, cells were resuspended at 40 × 106 cells/ml and incubated with 5 μM CFSE (Molecular Probes, Breda, the Netherlands) for 10 min at 37°C. 107 CFSE-labeled cells were transferred into the tail vein of transplanted mice.

Delayed-type hypersensitivity (DTH) response

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4 weeks after lymph node transplantation, transplanted mice were immunized with 100 μg OVA (Sigma-Aldrich, St Louis, MO) in 25 μl incomplete Freund's adjuvant (Sigma–Aldrich) plus 25 μl PBS (B. Braun, Euterpehof, The Netherlands) subcutaneously in the tail base. 5 days later, the immunized mice were injected intradermally in both ears with 10 μl of PBS containing 10 μg of OVA. Ear thickness was measured with a micrometer (Mitutoyo, Tokyo, Japan) before and 24 hr after secondary challenge in a blinded fashion. Ear swelling was calculated as the difference in ear thickness at 0 and 24 hr. Each mouse provided two independent measurements (two ears) that were averaged to determine the average ear swelling. Ex vivo evaluation of effector T cells was performed 1 day after the last ear measurement. Briefly, lymphocytes were isolated from spleens and re-stimulated with the OVA peptides SIINFEKL (OVA257-264—0.2 μg/ml) and EKLTEWTSSNVMEER (OVA265-279—200 μg/ml) for 24 hr. The last 4 hr of culture were performed in the presence of Golgiplug (BD Biosciences, Breda, The Netherlands).

Primary lymph node stromal cell sorting and flow cytometry

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Peripheral lymph node single cell suspensions for cell sorting and flow cytometry were obtained by lymph node enzymatic digestion with 0.2 mg/ml collagenase P (Roche, Penzberg, Germany), 0.8 mg/ml dispase II (Roche), and 0.1 mg/ml DNAse I (Roche) as described by Fletcher et al. (2011). Prior to cell sorting, the single cell preparation was enriched for non-hematopoietic stromal cells by negative selection of hematopoietic cells using the α-mouse CD45 (clone MP33) PE.Cy7 antibody from eBioscience (Halle-Zoersel, Belgium) and the mouse PE selection kit from StemCell Technologies (Grenoble, France), accordingly to the manufacturer's instructions. Surface stainings were performed on ice for 30 min in PBS/2%FCS/5 mM EDTA. Intracellular stainings were performed in permeabilization buffer, upon fixation in fixation/permeabilization buffer (both from eBioscience) for 30 min. The antibodies used were: α-CD31 (clone ERMP12) and α-MHC-II (M5/114) labeled with AlexaFluor 555 and 488 (Invitrogen), respectively; unlabeled hamster α-mouse gp38 (8.1.1) developed with goat anti-hamster AlexaFluor 647 (Invitrogen); biotin-conjugated α-mouse CD45.1 (A20; eBioscience) and α-mouse TER-119 (eBioscience) developed with streptavidin AlexaFluor 488 (Invitrogen) and PE.Cy7 (eBioscience), respectively; α-CD3 (17A2) eFluor 660, α-CD4 (GK1.5) AlexaFluor 488, PerCP.Cy5 and PE.Cy7, α-CD8 (53-6.7) PE.Cy7 and APC.eFluor 780, α-CD11c (N418) APC, α-CD25 (PC61.5) PE, α-CD44 (IM7) PE, α-CD45 (MP33) PE.Cy7 and APC.Cy7, α-CD62L (MEL-14) PE.Cy7, α-EpCAM (G8.8) PE, α-FoxP3 (FJK-16s) AlexaFluor 647, and α-IFNγ (XMG1.2) APC purchased from eBiosciences; and α-Helios (22F6) PacificBlue obtained from Biolegend (Fell, Germany). Live and dead cells were discriminated either with 7AAD, SytoxBlue, or live/dead Near-IR fixable dead cell stain (all from Invitrogen). Cells were sorted on a MoFlo sorter (DakoCytomation, Heverlee, Belgium) or analyzed on either a Cyan ADP (DakoCytomation) or a CantoII (BD Biosciences) flow cytometer with FlowJo software (TreeStar, Ashland, OR). Fluorescence minus one (FMO) and isotype control stain sets were used to assess detection thresholds.

Immunofluorescence

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Lymph node transplants were embedded in OCT compound (Sakura Finetek, Leiden, the Netherlands) and snap frozen in liquid nitrogen. Frozen blocks were cut into 7-μm sections. Sections were stained by incubation with the relevant antibodies for periods of 45 min at room temperature; and when needed, further incubated with appropriate secondary antibodies/reagents for 30 min. The antibodies used were: α-B220 (6B2), α-CD31 (ERMP12), α-MHC-II (M5/114), and Moma2 labeled in house with either Alexa Fluor 647 or AlexaFluor 555 (Invitrogen); unlabeled rat anti-mouse ERTR7 developed with anti-rat AlexaFluor 488 (Invitrogen); unlabeled rabbit anti-mouse collagen type I (polyclonal; AbCAM, Cambridge, UK) and anti-mouse CSFR1 (polyclonal, Sigma–Aldrich, St Louis, MO) developed with anti-rabbit AlexaFluor 647 and anti-rabbit AlexaFluor 555 (Invitrogen), respectively; biotin-labeled α-mouse CD11c (N418; BioLegend, Fell, Germany) developed with streptavidin conjugated to AlexaFluor488 (Invitrogen); and directly labeled α-CD3 (17A2) eFluor 660, α-F4/80 (BM8) eFluor 660, and α-Lyve-1 (ALY7) AlexaFluor 488 (all from eBioscience). Pictures were taken on a DM6000 Leica immunofluorescence microscope (Leica Microsystems, Rijwijk, the Netherlands). Analysis of the area occupied by CD11c+CSFR1+ clusters was performed with ImageJ.

In vitro generation of lymph node stromal cell lines

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Peripheral lymph node single cell suspensions for culture purposes were obtained by enzymatic digestion as described above. To obtain enriched primary stromal cell cultures, the resulting cell suspensions were plated on collagen-coated flasks and cultured in RPMI (Invitrogen) containing 10% heat-inactivated FCS (Gibco, Breda, The Netherlands), 2% glutamine (Lonza, Basel, Switzerland), 2% penicillin–streptomycin (Lonza), and 50 μM 2-mercapethanol (Merck, Haarlen, The Netherlands). Stromal cells were allowed to adhere to the collagen matrix overnight, after which non-adherent immune cells were washed away. Long-term culture and regular fractioning of these cultures permitted the establishment of immortalized lymph node stromal cell lines that were subsequently sorted and repeatedly characterized by flow cytometry to ensure the maintenance of stable phenotypes and the absence of CD45+ hematopoietic cells.

In vitro T cell and lymph node stromal cell co-cultures

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To isolate T cells for in vitro culture, lymph nodes and spleens from wild-type, OT-I, and OT-II mice were dissected and squeezed through cell strainers. Erythrocytes were lysed with ammonium-chloride–potassium (ACK) lysis buffer. CD4+ wild-type and OT-II T cells were magnetically isolated with the CD4+ cell negative isolation kit and CD8+ OT-I T cells with the CD8+ cell negative isolation kit (both from Miltenyi), following the manufacture's protocols. To determine T cell activation/proliferation, T cells were cultured together with lymph node stromal cell lines derived from K14-mOVA mice in the presence of 200 U/ml mouse recombinant IL2 (BioLegend). As a positive control for T cell activation/proliferation, T cells were cultured with CD3/CD28 T cell expander Dynabeads (Invitrogen) at a 1/1 ratio. To assess the role of lymph node stromal cells in antigen-specific Treg maintenance, T cells were cultured together with lymph node stromal cell lines derived from K14-mOVA mice in the absence of recombinant IL2. In some experiments, the MHC-II blocking antibody M5/114 (10 μg/ml) was added to the co-cultures.

RNA isolation, complementary DNA (cDNA) synthesis, and real-time PCR

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To analyze gene expression in transplanted lymph nodes and skin, transplanted tissues were dissected and immediately stored in Trizol (Gibco) at −80°C. Tissue homogenization was performed with an Ultra-Turrax T10 disperser (IKA, Staufen, Germany). mRNA was isolated and cDNA synthesized, as previously described (Baptista et al., 2013).To analyze the transcript expression in sorted cells, mRNA was isolated with the mRNA capture kit from Roche and cDNA synthesized with the Reverse Transcription System from Promega (Leiden, The Netherlands), according to the manufacturers' instructions. In both cases, real-time PCR was performed on StepOne real-time PCR systems from Applied Biosystems (Bleiswijk, The Netherlands). The expression of each transcript was analyzed and normalized for the expression of selected housekeeping genes with geNORM v3.4 software (Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium).

Data analysis and statistics

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T cell proliferation was analyzed with FlowJo software (Tree Star). Division indexes represent the average number of cell divisions that a cell in the original population has undergone according to the formula: division index = total number of cell divisions/total number of precursor cells present at the beginning of the assay. The common rejection module (CRM) score represents the geometric mean expression of the genes comprised in the CRM (Khatri et al., 2013). Statistical analysis was performed with GraphPad Prim v.4 (GraphPad Software, San Diego, CA). One-way non-parametric analysis of variance (Kruskal–Wallis test) coupled to Dunn's or Tukey's multiple comparison tests, non-parametric repeated measures analysis of variance (Friedman's test) coupled to Dunn's multiple comparison tests, and non-parametric unpaired and paired t-tests were used as required. Data in Figure 6B were subjected to a Box–Cox transformation, to approximate it to a normal distribution, after which one-way Anova statistical analysis was performed on the transformed data, as described above. All data is expressed as mean ± SEM. p-values <0.05 were considered significant.

References

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    Intranasally induced immunological tolerance is determined by characteristics of the draining lymph nodes: studies with OVA and human cartilage gp-39
    1. DA Wolvers
    2. CJ Coenen-de Roo
    3. RE Mebius
    4. MJ van der Cammen
    5. F Tirion
    6. AM Miltenburg
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    Journal of Immunology 162:1994–1998.
  41. 41

Decision letter

  1. Emil R Unanue
    Reviewing Editor; Washington University School of Medicine, United States

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

Thank you for sending your work entitled “Lymph node stromal cells constrain immunity via MHC class II self-antigen presentation” for consideration at eLife. Your article has been favorably evaluated by Tadatsugu Taniguchi (Senior editor), a guest Reviewing editor, and 3 reviewers, one of whom, Victor Engelhard (reviewer #2), has agreed to reveal his identity.

The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.

Your paper is of definite interest to the editors and the reviewers. There were a number of important issues that were raised. We would like to proceed with your paper provided that you are willing to consider and answer many of their comments. The following are issues that you should consider of importance and that will require further analysis.

Although you need to consider the various issues raised by the three reviewers there are some major ones to pay attention to.

For one, the examination of MHC-II expression on the lymph nodes was brought up by the three reviewers and it is clearly one that you need to examine at depth. Note comment 1 of Reviewer 1 and Reviewer 2, and the comment regarding Figure 1 from Reviewer 3.

It is also important that you give us your interpretation of Reviewer 2 #5 concerning the possible transfer of pMHC-II complexes from one cell to another. This is also brought up by Reviewer 3 in the comment on Figure 1. Please address this important point including new experiments so as to clarify this issue.

We trust that you will be able to evaluate the reviewers’ comments. And that we can see a revised version of your interesting paper.

Extract from Reviewer 1:

Figure 5 shows an increase in the % and number of Tregs in subjects that received K14-OVA lymph node transplants compared with WT lymph node transplants. Figure 6 shows that K14-OVA expression in transplanted lymph nodes reduces the DTH response (as measured by ear swelling and CRM score) relative to OVA-free control transplanted lymph nodes. However, no analysis of Tregs is provided in this part of the study. The data in Figures 5 and 6 suggest that some form of immunoregulation is conferred by OVA expression in the transplanted lymph node however both donor and host tissues express MHC class II in these experiments. OVA expressed in the donor lymph node could have been presented by either donor lymph node cells (stroma), through the direct antigen presentation pathway, or by host antigen presenting cells that enter the graft (dendritic cells, monocytes) and present via an indirect pathway (endocytosis or phagocytosis of OVA expressing cells or via membrane transfer). Ideally, these transplant experiments would have been done in hosts with MHC class II deficiency in bone marrow derived antigen presenting cells such that the only MHC class II expressing cells would come from the donor tissue. The requirement for MHC class II in CD4 T cell and Treg development however makes it impossible for the authors to do these experiments using the MHC class II global knockout mice at hand. However, a definitive analysis of the cells presenting MHC class II-OVA peptides and Treg maintenance in their experimental system would be necessary to substantiate the authors' claims and conclusions. Putting these issues aside, the critical comparison in Figure 6b shows an interesting trend in the CRM score when transferred lymph node contains cells expressing OVA but importantly this difference does not reach statistical significance and therefore does not support the claims made by the authors.

Additional points:

1) The MHC class II analysis in Figure 1 should be performed transplanted lymph nodes and extended to include various subsets of bone marrow derived antigen presenting cell types in a manner where the donor and host cells can be parsed. This is critical as it allows the reader to assess precisely which cell subsets lack MHC class II within the transplanted lymph node.

2) The images in Figure 2 are provided to show structural damage to the transplanted lymph node when the donor tissue lacks MHC class II. However, this data cannot support the authors' claim without providing images for the control tissues (transplanted lymph node when the donor tissue is MHC class II+). In addition, quantification of the structural damage depicted in the images should be provided.

3) In two separate places (Figure 1–figure supplement 1; Figure 2–figure supplement 4) data for ETAC-like cells were included in the Figures but not described in the manuscript until the Discussion.

4) Characterization including phenotype and purity of the stromal cell lines (both control and K14-derived) must be provided. Control and antigen-expressing stromal cell lines should belong to the same lineage.

5) A major point of this paper is that lymph node stromal cells support Treg maintenance however the data in Figure 4 indicate that the number of Tregs that persist in a culture with lymph node stromal cells is extremely low. How does this efficiency compare with dendritic cells? Representative flow cytometry data should accompany the graph in Figure 4c.

6) Flow cytometry data for the efficiency of CD4 or CD8 T cell depletion in the donor tissues must be shown.

7) Statistical analysis of data in Figure 2–figure supplement 2 must be shown.

8) Figure 3a, the y-axis is missing the values for cell counts.

Reviewer 2 (pay particular attention to #1):

1) There is no characterization of cell population distributions and MHC-II expression patterns in transplanted WT LN or MHC-IIko LN. These are essential to understand the differences in T cell behavior in these two LN, and to support the assertions in the Results section and the significance of Figure 2–figure supplement 1.

2) What is the basis for rejection of MHC-IIneg LN? The analyses of T cells in an MHC-IIneg LN are in an environment of inflammation and tissue damage, raising the question of whether their characteristics are causing the pathology or are affected by it, apart from any influence of Treg.

3) The authors conclude that lack of MHC-II molecules on LNSC leads to diminished antigen-specific Treg maintenance/function and enables more robust activation of conventional CD4 and CD8 T cells. An alternative is based on the observation that MHC-II molecules are antigen-nonspecific ligands for the LAG-3 inhibitory receptor. This could affect the activation state of CD8 T cells and non-regulatory CD4 T cells independently of any influence on Treg representation. The data in right panels of Figure 2b and Figure 2b–figure supplement 3 comparing WT and MHC-IIko LN in GK1.5 treated mice are strongly suggestive of this.

4) Long-term LNSC lines are problematic, as the lineages have different survival capabilities and often lose differentiated characteristics. The authors acknowledge that their ova negative line is FRC like, while the ova pos line is LEC like. Thus, comparing their effects on T cells to establish a role for antigen (Figure 4) is very problematic. In Figure 4, even OVAneg LNSC lines are associated with a substantial representation of FoxP3+ cells in both bulk CD4 and OT-II T cells. Based on Figure 4–figure supplement 1, this is not due to proliferation, and is associated with upregulation of CD25 in CD8 T cells, but not OT-II cells. The authors state that this is an increase in survival of FoxP3+ cells, but FoxP3neg cells should also be examined, and there should be a zero time control to establish whether antigen is associated with selective survival or differentiation. Regardless, the distinct phenotypes of the cell lines will make it difficult to definitively conclude that antigen expression is responsible.

5) The authors interpret the results in Figure 5 as though only LNSC present antigen, but do not account for the possibility that ova positive LNSC transfer antigen to MHC-II positive hematopoetic cells in the same LN, or transfer preformed MHC-II antigen complexes. Thus, the results do not unambiguously establish that LNSC are responsible for any increase in representation of Treg or Treg subsets via direct presentation of antigen.

6) Figure 5 and Figure 5–figure supplement 1 show several instances in which changes in absolute numbers of Treg or Treg subsets change, but frequency or representation does not - or the converse. This must mean that other cell populations are also changing in response to characteristics of the transplanted LN. This needs to be clarified, as the manuscript text indicates that LN stroma contribute to the selective maintenance of antigen-specific.

Reviewer 3 (comment on Figure 1 needs to be prioritized):

1) Figure 1: the authors claim a “selective absence of MHCII on LNSC from Tx MHCII-/- mice (transplanted in WT)”. Given the recent publication showing that MHCII are not only endogenously expressed by LNSC, but also acquired from DC, this result is quite surprising. It would be mandatory to show this result and compare with the situation in which DC do not express MHCII. For instance, show an absence of MHCII expression by LNSC from MHCII-/- LN Tx into chimeric mice (MHCII-/- BM into irradiated WT). Importantly, can the authors demonstrate any difference in Treg proliferation in the context of MHCII-/- LN Tx compared to endogenous distal LN?

2) Figure 2 shows that T cell activation is increased in MHCII-/- LN Tx. As a demonstration of an effect happening locally in Tx LN, it is important to provide not only WT LN Tx, but distal endogenous LN fron MHCII-/- LN Tx mice as well.

3) Figure 3 shows T cell (Treg and non Treg) proliferation 48h after OTII transfer. Is the observed effect still happening at later time points? Here as well, a distal endogenous LN should be provided. In addition, the division index ratio is quite confusing, since we do not have any indication of the proliferation rate of the cells. Please provide % of T cell (Treg and non Treg) proliferation, as well as % of Tregs. Moreover, dendritic cells were shown to be responsible for homeostatic Treg proliferation (Nussensweig's lab). How do the authors explain their results in that context? If, as shown, there are less Tregs in MHCII-/- LN Tx, we would have expected increased non Treg T cell proliferation, whereas opposite results are observed. Can the authors comment on that?

4) Figure 4: The cell lines obtained and used by the authors (“FRC like OVAneg” and “LEC like OVApos”) represent a quite artificial system and are poorly relevant in the context of the paper (most experiments were performed in vivo). It is true that primary LEC / FRC cultures are contaminated by hematopoietic cells. However, in case the authors think these in vitro experiments are relevant for the paper, they need to sort LECs and FRC from those primary cultures to repeat the experiments. Furthermore, it is quite surprising that no CD8+ T cell proliferation was observed in these in vitro settings, since many studies have described early and transient CD8 T cell proliferation (followed by T cell deletion) after culture with by MHCI-Ag presenting LNSC.

5) Figure 5 and 6 are elegant and nice. The major issue I have is to understand whether the authors want to claim a local or a systemic effect of LNSC in dampening self-reactive T cell responses. Indeed, Figure 1 shows a local effect, whereas Figure 5 and 6 show a more general effect. Figure 5d shows a significant Treg increase in endogenous distal LN as well (although discrepancies exist between FACS dot plots (1.17% vs 1.7%) and histograms (about 3% vs >4% Treg). The authors need to clarify this point and, in case they think the effect is systemic, explain how it might work.

https://doi.org/10.7554/eLife.04433.023

Author response

Extract from Reviewer 1:

Figure 5 shows an increase in the % and number of Tregs in subjects that received K14-OVA lymph node transplants compared with WT lymph node transplants. Figure 6 shows that K14-OVA expression in transplanted lymph nodes reduces the DTH response (as measured by ear swelling and CRM score) relative to OVA-free control transplanted lymph nodes. However, no analysis of Tregs is provided in this part of the study. The data in Figures 5 and 6 suggest that some form of immunoregulation is conferred by OVA expression in the transplanted lymph node however both donor and host tissues express MHC class II in these experiments. OVA expressed in the donor lymph node could have been presented by either donor lymph node cells (stroma), through the direct antigen presentation pathway, or by host antigen presenting cells that enter the graft (dendritic cells, monocytes) and present via an indirect pathway (endocytosis or phagocytosis of OVA expressing cells or via membrane transfer). Ideally, these transplant experiments would have been done in hosts with MHC class II deficiency in bone marrow derived antigen presenting cells such that the only MHC class II expressing cells would come from the donor tissue. The requirement for MHC class II in CD4 T cell and Treg development however makes it impossible for the authors to do these experiments using the MHC class II global knockout mice at hand. However, a definitive analysis of the cells presenting MHC class II-OVA peptides and Treg maintenance in their experimental system would be necessary to substantiate the authors' claims and conclusions. Putting these issues aside, the critical comparison in Figure 6b shows an interesting trend in the CRM score when transferred lymph node contains cells expressing OVA but importantly this difference does not reach statistical significance and therefore does not support the claims made by the authors.

We agree with this reviewer that in the experiments shown in Figures 5 and 6 we were not able to distinguish whether direct antigen presentation by stromal cells or indirect antigen presentation, via host antigen presenting cells, is responsible for the increase in % and number of Treg cells and the reduction in DTH response and CRM score. However, together with the data presented in Figure 4, in which we show that antigen presentation by lymph node stromal cells in vitro leads to the selective survival of antigen-specific CD4+FoxP3+ Treg cells in a MHC-II-dependent manner, we have established that lymph node stromal cells can directly present antigens to T cells. We fully realize that the transfer of MHC-II-antigen peptide complexes from dendritic cells to lymph node stromal cells, as published by Dubrot et al (JEM, 2014) is a mechanism by which lymph node stromal cells can also regulate their antigen presenting function. This mechanism would however not play a significant role in these settings, as dendritic cells are circulating and host-derived cells (as also shown in Figure 2–figure supplement 1 and further explained under point 1) and thus are not expressing the K14-OVA transgene. Reversely, the possibility that MHCII-peptide complexes are transferred from donor-derived stromal cells to circulating dendritic cells is indeed a possibility, and may occur in addition to the direct presentation of antigen by stromal cells, as we have shown in Figure 4.

As this reviewer pointed that the data shown in Figure 6 was interesting but not statistically significant, and therefore not relevant, we consulted a statistician to more closely have a look at our statistical analysis. Importantly, after careful re-analysis, the statistician pointed out that our data was not distributed normally, and therefore should undergo a Box-Cox transformation, to convert it to a normal distribution. Subsequently, applying one-way Anova on the transformed data revealed that the differences between the animals transplanted with wild-type lymph nodes and K14mOVA skin and animals transplanted with K14mOVA lymph nodes and skin reached a p-value of 0.036, and thus statistically significantly different from each other. Moreover, an even less stringent correction is allowed according to the statistician, approaching the data in a one-tailed manner, resulting in p-value of 0.018 between these two groups. We feel that this data is very relevant for the manuscript, as this shows the consequences of antigen presentation by lymph node stromal cells. In the revised manuscript, we updated Figure 6B and the statistical analysis entry on the Materials and methods section. We have also acknowledged the statisticians’ help.

Additional points:

1) The MHC class II analysis in Figure 1 should be performed transplanted lymph nodes and extended to include various subsets of bone marrow derived antigen presenting cell types in a manner where the donor and host cells can be parsed. This is critical as it allows the reader to assess precisely which cell subsets lack MHC class II within the transplanted lymph node.

We agree with this reviewer that analysis of MHC-II expression in transplanted lymph nodes is of critical importance. Therefore, we now include such analysis in Figure 2–figure supplement 1. Whereas dendritic cells and extrathymic AIRE-expressing cells (ETACs) remain MHC-II positive (similar % of MHC-II+ cells within CD11c+ and EpCAM+ cells in wild-type and MHC-II-/- transplants), lymph node stromal cells exhibit a significant reduction in MHC-II expression in MHC-II-/- transplants as compared to wild-type transplants. MHC-II+ stromal cells in MHC-II-/- transplants are likely to have acquired MHC-II expression from dendritic cells as shown by Dubrot et al, JEM 2014. It is noteworthy to point out that gp38-CD31- (double negative) cells do not express MHC-II even in wild-type intact animals and therefore contribute to the negative peak present in both wild-type and MHC-II-/- transplants.

2) The images in Figure 2 are provided to show structural damage to the transplanted lymph node when the donor tissue lacks MHC class II. However, this data cannot support the authors' claim without providing images for the control tissues (transplanted lymph node when the donor tissue is MHC class II+). In addition, quantification of the structural damage depicted in the images should be provided.

We provide the analysis requested by showing images from wild-type transplants in Figure 2–figure supplement 3. In addition we have quantified the amount of structural damage by assessing the area occupied by CD11c+CSFR1+ macrophage clusters in multiple sections. We express these data as percentage of total lymph node area.

3) In two separate places (Figure 1–figure supplement 1; Figure 2–figure supplement 4) data for ETAC-like cells were included in the Figures but not described in the manuscript until the Discussion.

In the course of our experiments, we analyzed EpCAM+ ETAC-like cells given the original publication stating that these cells were of stromal origin (Gardner et al. Science 2008), but found that they were most likely a subset of dendritic cells as they expressed high levels of CD45, CD11c and MHC-II. More importantly, these cells could be observed within MHC-II-/- lymph node transplants and expressed normal MHC-II levels (see Figure 2–figure supplement 1). Together, these data suggested that ETACs were not contributing to the effects on T cell activation and lymph node rejection that we observed. We stated these data in the discussion and showed it in supplementary figures. In the revised version of our manuscript, we highlight these data in the Results section, as to clarify from early on that ETACs are likely not involved in the phenotypes described.

4) Characterization including phenotype and purity of the stromal cell lines (both control and K14-derived) must be provided. Control and antigen-expressing stromal cell lines should belong to the same lineage.

We agree with this reviewer that the stromal cell lines used in our experiments should have belonged to the same lineage. However, despite several attempts to generate large panels of stromal cell lines representing each lineage and of different genetic backgrounds, we have not managed to do so. Indeed, in our hands FACS-sorting of stromal cells immediately after their enzymatic isolation resulted in highly damaged cells that quickly died in culture. Thus, we have generated our cell lines by culture of unfractionated lymph node cells, which were allowed to adhere to the culture plate O/N and were subsequently repeatedly washed to remove non-adherent immune cells. Only after a long period of time (usually more than a month) we had enough cells that could be FACS sorted and would survive such procedure. These cultures seemed to favor the development of FRC-like cells. LEC development was extremely rare and BEC or DN stromal cell development was never observed. To fully disclose the origin of our stromal cell lines, we provide their phenotypic characterization in Figure 4–figure supplement 1.

5) A major point of this paper is that lymph node stromal cells support Treg maintenance however the data in Figure 4 indicate that the number of Tregs that persist in a culture with lymph node stromal cells is extremely low. How does this efficiency compare with dendritic cells? Representative flow cytometry data should accompany the graph in Figure 4c.

The number of Tregs that persist in our cultures is low, indeed. Treg cell death in these cultures results from the absence of exogenously added survival factors, such as recombinant IL2. Recombinant IL2 was purposely left out as these experiments were designed to specifically address the role of self-antigen-presentation by stromal cells in the survival/maintenance of Tregs, which could have been masked by the addition of exogenous survival factors. Representative FACS plots of Treg recovery were added to Figure 4c. We have not compared Treg persistence upon co-culture with stromal cells to co-culture with dendritic cells pulsed with OVA peptide. Given the critical role of TCR engagement in T cell survival, the higher levels of MHC-II surface expression on DCs and the need to add processed OVA peptides, such an experiment will likely result in higher T cell survival than when T cells are co-cultured with stromal cells expressing endogenously-derived OVA peptides. Indeed, even if OVA peptides were to be exogenously added to stromal cells and DCs, DCs would most likely outperform stromal cells, again given their higher MHC-II expression. Similar results are to be expected if full length OVA was to be added and antigen presentation would rely on antigen acquisition and processing, given the role of DCs as professional antigen-presenting cells.

6) Flow cytometry data for the efficiency of CD4 or CD8 T cell depletion in the donor tissues must be shown.

In addition to the graphs depicting CD4+ T cell depletion in the recipient’s own tissues, we now also provide data concerning CD4+ T cell depletion in the transplants in Figure 2–figure supplement 6. Determination of CD4+Foxp3+ T cell depletion in the transplants was not possible due to the very low number of CD4+ T cells retrieved from those lymph nodes. This precluded accurate analysis.

7) Statistical analysis of data in Figure 2–figure supplement 2 must be shown.

The statistical analysis of this data is now provided. In the revised manuscript this figure is Figure 2–figure supplement 5.

8) Figure 3a, the y-axis is missing the values for cell counts.

In Figure 3a an offset histogram overlay is shown to more easily visualize the difference between Treg homeostatic proliferation in wild-type and MHC-/- lymph node transplants. Such representation makes the axes of both curves distinct, thereby precluding the possibility to add a single scale on the Y-axis indicating cell counts. We have adjusted the label from ‘counts’ to ‘cell counts’, to be clearer. An indication of relative cell numbers can be deferred from Figure 3c, showing that the frequency of Treg in MHC-II-/- lymph nodes is significantly reduced as compared to their frequency in wild-type transplants.

Reviewer 2 (pay particular attention to #1):

1) There is no characterization of cell population distributions and MHC-II expression patterns in transplanted WT LN or MHC-IIko LN. These are essential to understand the differences in T cell behavior in these two LN, and to support the assertions in the Results section and the significance of Figure 2–figure supplement 1.

We agree with this reviewer that analysis of MHC-II expression in transplanted lymph nodes is critical. We have included that analysis in Figure 2–figure supplement 1. Whereas dendritic cells and ETAC-like cells remain MHC-II positive, lymph node stromal cells exhibit a significant decrease in MHC-II expression in MHC-II-/- transplants as compared to wild-type transplants. The MHC-II+ stromal cells that we detected in MHC-II-/- transplants are likely to have acquired MHC-II expression from dendritic cells as shown by Dubrot et al, JEM 2014.

2) What is the basis for rejection of MHC-IIneg LN? The analyses of T cells in an MHC-IIneg LN are in an environment of inflammation and tissue damage, raising the question of whether their characteristics are causing the pathology or are affected by it, apart from any influence of Treg.

We have concluded that rejection of MHC-II-/- lymph node transplants was due to inefficient control of T cell activation caused by impaired maintenance of regulatory T cells. We acknowledge the fact that transplantation is accompanied by inflammation due to surgical trauma and tissue damage due to inefficient blood supply of the transplanted lymph node in the first days after transplantation. Such early inflammation and tissue damage can promote T cell activation. However, in MHC-II sufficient transplants such early T cell activation subsides as the healing process progresses. In MHC-II-/- transplants, the same T cell activation event seems to progress uncontrolled (due to Treg underrepresentation) and likely results in transplant rejection. In sum, we cannot exclude that T cell activation may contribute to and may get fuelled by tissue inflammation and damage. Indeed, the two events are interconnected and not easily separated.

3) The authors conclude that lack of MHC-II molecules on LNSC leads to diminished antigen-specific Treg maintenance/function and enables more robust activation of conventional CD4 and CD8 T cells. An alternative is based on the observation that MHC-II molecules are antigen-nonspecific ligands for the LAG-3 inhibitory receptor. This could affect the activation state of CD8 T cells and non-regulatory CD4 T cells independently of any influence on Treg representation. The data in right panels of Figure 2b and Figure 2b-figure supplement 3 comparing WT and MHC-IIko LN in GK1.5 treated mice are strongly suggestive of this.

We agree with this reviewer that the MHC-II/LAG-3 axis can play an important role in our experimental system and thus contribute to the T cell activation seen in MHC-II-/- transplants independently of any Treg influence. Although we cannot rule out this possibility completely, our results regarding decreased Treg frequency (Figure 2–figure supplement 6) and inefficient Tregs expansion in MHC-II-/- lymph nodes (Figure 3) and improved survival of antigen-specific Tregs upon antigen recognition on stromal cells in vitro (Figure 4) and in vivo (Figure 5) strongly support a dominant role for Tregs in our assays. Indeed, in the in vitro assays we document an increase in antigen-specific Tregs induced by antigen-presentation by LECs that could be blocked by an anti-MHC-II antibody, and that non-antigen presenting FRCs were unable to induce.

Furthermore, when lack of MHC-II molecules would directly act on LAG-3 inhibitory receptors expressed on CD8 T cells and non-regulatory CD4 T cells, the enhanced activity state of CD8 cells, observed upon CD4 T cell depletion could not be easily explained. In our opinion, the data presented in Figures 2b and Figure 2–figure –supplement 3 (now Figure 2–figure supplement 7) is more conclusive with CD4+ T cell depletion releasing a break, most likely imposed by Tregs, on CD8+ T cell activation.

4) Long-term LNSC lines are problematic, as the lineages have different survival capabilities and often lose differentiated characteristics. The authors acknowledge that their ova negative line is FRC like, while the ova pos line is LEC like. Thus, comparing their effects on T cells to establish a role for antigen (Figure 4) is very problematic. In Figure 4, even OVAneg LNSC lines are associated with a substantial representation of FoxP3+ cells in both bulk CD4 and OT-II T cells. Based on Figure 4–figure supplement 1, this is not due to proliferation, and is associated with upregulation of CD25 in CD8 T cells, but not OT-II cells. The authors state that this is an increase in survival of FoxP3+ cells, but FoxP3neg cells should also be examined, and there should be a zero time control to establish whether antigen is associated with selective survival or differentiation. Regardless, the distinct phenotypes of the cell lines will make it difficult to definitively conclude that antigen expression is responsible.

We agree with this reviewer that long-term maintenance of stromal cells is problematic because these cells lose many of the characteristics that they exhibit in vivo, namely chemokine production. However, the use of these cellular models as surrogate stromal cells is presently the best solution. In our hands, FACS-sorting stromal cells directly from lymph nodes always damaged them to the point that we could not culture them for the duration of our experiments.

As suggested by this reviewer, we added data characterizing the stromal cell lines used in our experiments in Figure 4–figure supplement 1 and data regarding the number of Foxp3- and Foxp3+ CD4+ T cells in the input in Figures 4c and Figure 4–figure supplement 3. In Figure 4–figure supplement 3, we also added data regarding the behavior of CD4+Foxp3- conventional T cells, showing that these cells are not affected by co-culture with stromal cells in the same manner as CD4+Foxp3+ cells are. Importantly, the data regarding conventional T cells and Tregs were obtained in the exact same conditions, as we performed our stromal cell-T cell co-cultures with unfractionated CD4+ T cells containing both Foxp3- and Foxp3+ cells.

Finally, in order to prove that antigen-presentation is the driving force that leads to increased numbers of Foxp3+ T cells in our assays, we have blocked antigen-presentation with the MHC-II specific antibody M5/114. As seen in Figure 4c, blockade of MHC-II-mediated presentation prevented the K14mOVApos cell line to promote increased CD4+Foxp3+ OT-II T cell recovery to a point where “K14mOVApos cell line-induced” CD4+Foxp3+ OT-II T cell numbers were no longer distinct from “K14mOVApos cell line-induced” CD4+Foxp3+ wild-type T cell numbers. Thus, altogether, we believe that despite the intrinsic differences between our cell lines, i.e. between FRC- and LEC-like cell lines, our data accurately reflects the effect of stromal cell-mediated self-antigen presentation in Treg maintenance.

5) The authors interpret the results in Figure 5 as though only LNSC present antigen, but do not account for the possibility that ova positive LNSC transfer antigen to MHC-II positive hematopoetic cells in the same LN, or transfer preformed MHC-II antigen complexes. Thus, the results do not unambiguously establish that LNSC are responsible for any increase in representation of Treg or Treg subsets via direct presentation of antigen.

We agree with this reviewer that our in vivo experiments with K14mOVA lymph node transplants do not unambiguously establish that lymph node stromal cell-mediated antigen presentation is responsible for the increase in Treg representation and “immune tolerance”. However, they do establish that stromal cell-derived antigens can promote Treg expansion and thus provide protection. This can be caused by both direct and indirect (via other cells) antigen-presentation. Our in vitro experiments, however, clearly establish that lymph node stromal cells can present antigen in the context of MHC-I (as shown by others as well) and MHC-II molecules, with stromal cell MHC-II-mediated presentation contributing to the homeostasis of antigen-specific Tregs.

6) Figure 5 and Figure 5-figure supplement 1 show several instances in which changes in absolute numbers of Treg or Treg subsets change, but frequency or representation does not - or the converse. This must mean that other cell populations are also changing in response to characteristics of the transplanted LN. This needs to be clarified, as the manuscript text indicates that LN stroma contribute to the selective maintenance of antigen-specific.

There were instances in which the proportion of Foxp3+ OT-II T cells increased in K14mOVA lymph node transplant recipients while the absolute number of these cells did not. We attribute these discrepancies to the selective retention of OT-II T cells in K14mOVA transplants (see Author response image 1 below – left panel). Antigen recognition in K14mOVA transplants possibly induces TCR-mediated stop signals (TCR-mediated CD69 upregulation would drive intracellular retention of the egress receptor S1P1 and thus trap OT-II T cells in the transplant (Shiow et al, Nature 2006)). Such an event induces a generalized increased in OT-II T cell numbers in K14mOVA transplants, including Foxp3+ OT-II Tregs (Figure 5c), without necessarily changing the balance between Foxp3- and Foxp3+ OT-II T cells (Figure 5c). In K14mOVA transplants, Foxp3+ OT-II T cells will receive enhanced survival factors, which will enhance their overall maintenance. As in the context of antigen-driven activation, CD4+Foxp3+ Tregs downregulate S1P1 at a slower pace as compared to conventional CD4+Foxp3- T cells (Liu et al, NatImmunol 2009), they will egress from the K14mOVA transplants faster than their conventional counterparts and thus better populate distal lymph nodes. This leads to an increase in the percentage of CD4+Foxp3+ OT-II T cells in the endogenous lymph nodes of K14mOVA lymph node recipients. However, as the bulk of OT-II T cells are being actively sequestered in the K14mOVA transplants, and thus underrepresented in the endogenous lymph nodes of K14mOVA transplant recipients (see Author response image 1 below – right panel), the increased frequency of CD4+Foxp3+ OT-II T cells in the endogenous lymph nodes of K14mOVA transplant recipients is not accompanied by an increase in the total number of these cells.

Furthermore, as far as our analysis of host-derived immune cells went, we have not observed significant differences between wild-type and K14mOVA transplant recipients.

Author response image 1

Reviewer 3 (comment on Figure 1 needs to be prioritized):

1) Figure 1: the authors claim a “selective absence of MHCII on LNSC from Tx MHCII-/- mice (transplanted in WT)”. Given the recent publication showing that MHCII are not only endogenously expressed by LNSC, but also acquired from DC, this result is quite surprising. It would be mandatory to show this result and compare with the situation in which DC do not express MHCII. For instance, show an absence of MHCII expression by LNSC from MHCII-/- LN Tx into chimeric mice (MHCII-/- BM into irradiated WT). Importantly, can the authors demonstrate any difference in Treg proliferation in the context of MHCII-/- LN Tx compared to endogenous distal LN?

We agree with this reviewer that analysis of MHC-II expression in transplanted lymph nodes is critical. We have now included such analysis in Figure 2–figure supplement 1. We show that whereas dendritic cells and ETAC-like cells remain MHC-II positive, lymph node stromal cells exhibit a significant decrease in MHC-II expression in MHC-II-/- transplants as compared to wild-type transplants. As the reviewer points out, the MHC-II expression detected in MHC-II-/- transplanted stromal cells is likely to have been acquired from dendritic cells (Dubrot et al. JEM 2014). The analysis suggested by the reviewer to address this point, unfortunately, could not be performed, as transfer of MHC-II-/- bone marrow into wild-type recipients leads to severe disease and death of the chimeric animals (Marguerat et al, JImmunol 1999). Finally, as shown in the revised Figure 3–figure supplement 1, no difference in Treg proliferation in the endogenous lymph nodes of wild-type and MHC-II-/- transplant recipients was observed. Thus, direct comparison of Treg proliferation between MHC-II-/- transplants and the endogenous lymph nodes of MHC-II-/-transplant recipients, showed a significant reduction in Treg proliferation in the former (see Author response image 2). Together, these data suggest that Treg proliferation was specifically impaired in MHC-II-/- transplants.

Author response image 2

2) Figure 2 shows that T cell activation is increased in MHCII-/- LN Tx. As a demonstration of an effect happening locally in Tx LN, it is important to provide not only WT LN Tx, but distal endogenous LN fron MHCII-/- LN Tx mice as well.

We agree with this reviewer that additional clarification of the local effect of MHC-II deficiency is needed. We provided such data in Figure 2–figure supplement 4, which shows that no alterations in T cell activation were observed in the endogenous lymph nodes of MHC-II-/- lymph node transplant recipients as compared to wild-type lymph node transplant recipients.

3) Figure 3 shows T cell (Treg and non Treg) proliferation 48h after OTII transfer. Is the observed effect still happening at later time points? Here as well, a distal endogenous LN should be provided. In addition, the division index ratio is quite confusing, since we do not have any indication of the proliferation rate of the cells. Please provide % of T cell (Treg and non Treg) proliferation, as well as % of Tregs. Moreover, dendritic cells were shown to be responsible for homeostatic Treg proliferation (Nussensweig's lab). How do the authors explain their results in that context? If, as shown, there are less Tregs in MHCII-/- LN Tx, we would have expected increased non Treg T cell proliferation, whereas opposite results are observed. Can the authors comment on that?

As suggested by this reviewer, in Figure 3–figure supplement 1 of the revised manuscript we provide data regarding the proliferation of conventional and Foxp3+ T cells separately, where one can see that Treg proliferation is significantly impaired in the absence of stromal cell-derived MHC-II, whereas proliferation of conventional T cells is not. Furthermore, we have also included data regarding T cell proliferation in the endogenous lymph nodes of transplant recipients, where one can observe that neither transplantation of wild-type nor MHC-II-/- lymph nodes interferes with the homeostatic proliferation of T cells in distant lymph nodes. In Figure 3c, we also included the analysis of Treg frequency, which indicates that impaired proliferation of Tregs leads to a significant reduction of these cells in MHC-II-/- transplants.

We have not analyzed DCs in the same experiments in which we determine Treg proliferation. However, we observed an increase in the frequency of DC in MHC-II-/- transplants as compared to wild-type transplants (see Author response image 3 below). This is in agreement with data by the Nussensweig's lab that showed that Treg depletion results in increased DC numbers by a Flt3 ligand-dependent mechanism (Lui et al, Science 2009) and likely reflects a compensatory mechanism in response to the diminished Treg numbers, which was however insufficient in the context of stromal MHC-II deficiency. In addition, one should mention that MHC-II expression in DCs is required to sustain Treg proliferation (Darasse-Jeze et al, JEM 2009) and in MHC-II-/- lymph node transplants, infiltrating dendritic cells expressed normal levels of MHC-II molecules (Figure 2–figure supplement 1). Altogether, these data suggest that lymph node stromal cell MHC-II expression plays a major role in the homeostasis of Tregs, independently of dendritic cell MHC-II expression.

Author response image 3

Despite reduced Treg numbers, we did not observe an increase in CD4+ conventional T cell proliferation in the absence of stromal cell MHC-II expression (Figure 3–figure supplement 1). We agree that this may seem contradictory at first, given that Tregs will negatively regulate T cell expansion. However, it should be acknowledged that self-recognition is required for the maintenance of peripheral T cells as a whole (Takada and Jameson, NatRevImmunol 2009). Thus, lack of proper self-antigen presentation in the context of stromal cell MHC-II deficiency will interfere with antigen recognition by the overall CD4+ T compartment, thereby influencing the homeostatic reconstitution of the CD4+ conventional T cell pool as well. Notwithstanding, our data clearly shows that the homeostatic proliferation of CD4+ conventional T cell is less affected by the absence of stromal-derived MHC-II molecules than the homeostatic proliferation of CD4+Foxp3+ T cells.

4) Figure 4: The cell lines obtained and used by the authors (“FRC like OVAneg” and “LEC like OVApos”) represent a quite artificial system and are poorly relevant in the context of the paper (most experiments were performed in vivo). It is true that primary LEC / FRC cultures are contaminated by hematopoietic cells. However, in case the authors think these in vitro experiments are relevant for the paper, they need to sort LECs and FRC from those primary cultures to repeat the experiments. Furthermore, it is quite surprising that no CD8+ T cell proliferation was observed in these in vitro settings, since many studies have described early and transient CD8 T cell proliferation (followed by T cell deletion) after culture with by MHCI-Ag presenting LNSC.

We agree with this reviewer that stromal cell lines are a quite artificial system because long-term culture of lymph node stromal cells results in loss of many of their distinctive characteristics. Stromal cells sorted directly ex vivo from lymph nodes would be preferable, however, in our hands, stromal cell sorting results in highly damaged cells that invariably die in culture. The in vitro experiments are however of significance, as we show that in the absence of dendritic cells, antigen presentation by stromal cells clearly affects T cells in an antigen- and MHC-II-dependent manner. Regarding the lack of CD8+ T cell proliferation in our experiments, we can only speculate that model antigen expression driven by the human keratin14 promoter may be lower than when driven by the promoters used in other studies (iFABP-tOVA; GFAP-HA), thus resulting in less efficient peptide-MHC-I presentation.

5) Figure 5 and 6 are elegant and nice. The major issue I have is to understand whether the authors want to claim a local or a systemic effect of LNSC in dampening self-reactive T cell responses. Indeed, Figure 1 shows a local effect, whereas Figure 5 and 6 show a more general effect. Figure 5d shows a significant Treg increase in endogenous distal LN as well (although discrepancies exist between FACS dot plots (1.17% vs 1.7%) and histograms (about 3% vs >4% Treg). The authors need to clarify this point and, in case they think the effect is systemic, explain how it might work.

The effect of stromal cell MHC-II deficiency is local. It seems that the homeostatic maintenance of Tregs numbers (and possibly their suppressive function) requires continuous and appropriate recognition of peptide-MHC-II complexes in stromal cells. As Tregs egress from MHC-II-/- transplants and recirculate through other secondary lymphoid organs, they will enter MHC-II sufficient environments that are able to rescue any imbalances to which Tregs may have been subjected in the transplant. Consequently, the Treg imbalance and T cell activation remains restricted to the transplant. Such local effect may, however, have systemic consequences when a given antigen is expressed only in one particular lymph node in which stromal cell-mediated presentation may become defective, resulting in the failure to generate antigen-specific Treg maintenance.

In contrast, the effect of K14mOVA lymph node transplantation is systemic. We attribute this to the recirculation of Tregs. In the K14mOVA transplant, lymph node stromal cell-derived OVA provides a survival advantage to OVA-specific Tregs allowing them to greatly expand. Such expansion coupled with Treg recirculation leads to a systemic overrepresentation of OVA-specific Tregs and thus to an increased ability to constrain immune responses.

The apparent discrepancy between the FACS plots and the summarizing graphs in Figure 5d arises from the selection of the two FACS plots with lower frequency of Foxp3+CD62L+ Tregs. The range of Foxp3+CD62L+ Tregs frequencies in wild-type transplants and K14mOVA transplants is 1.17-3.74% and 1.70-7.31%, respectively. To more faithfully represent our data, in the revised Figure, we have replaced those plots by plots showing percentages of Foxp3+CD62L+ Tregs closer to the averages observed.

https://doi.org/10.7554/eLife.04433.024

Article and author information

Author details

  1. Antonio P Baptista

    1. Department of Molecular Cell Biology and Immunology, Vrije Universiteit Medical Center, Amsterdam, Netherlands
    2. Graduate Program in Areas of Basic and Applied Biology, University of Porto, Porto, Portugal
    Contribution
    APB, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Contributed equally with
    Ramon Roozendaal
    Competing interests
    The authors declare that no competing interests exist.
  2. Ramon Roozendaal

    Department of Molecular Cell Biology and Immunology, Vrije Universiteit Medical Center, Amsterdam, Netherlands
    Present address
    Infectious Diseases and Vaccines Therapeutic Area, Janssen Pharmaceutical Companies of Johnson and Johnson, Leiden, Netherlands
    Contribution
    RR, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Contributed equally with
    Antonio P Baptista
    Competing interests
    The authors declare that no competing interests exist.
  3. Rogier M Reijmers

    Department of Molecular Cell Biology and Immunology, Vrije Universiteit Medical Center, Amsterdam, Netherlands
    Contribution
    RMR, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents
    Competing interests
    The authors declare that no competing interests exist.
  4. Jasper J Koning

    Department of Molecular Cell Biology and Immunology, Vrije Universiteit Medical Center, Amsterdam, Netherlands
    Contribution
    JJK, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents
    Competing interests
    The authors declare that no competing interests exist.
  5. Wendy W Unger

    Department of Molecular Cell Biology and Immunology, Vrije Universiteit Medical Center, Amsterdam, Netherlands
    Contribution
    WWU, Acquisition of data, Analysis and interpretation of data
    Competing interests
    The authors declare that no competing interests exist.
  6. Mascha Greuter

    Department of Molecular Cell Biology and Immunology, Vrije Universiteit Medical Center, Amsterdam, Netherlands
    Contribution
    MG, Acquisition of data, Analysis and interpretation of data
    Competing interests
    The authors declare that no competing interests exist.
  7. Eelco D Keuning

    Department of Molecular Cell Biology and Immunology, Vrije Universiteit Medical Center, Amsterdam, Netherlands
    Contribution
    EDK, Acquisition of data, Analysis and interpretation of data
    Competing interests
    The authors declare that no competing interests exist.
  8. Rosalie Molenaar

    Department of Molecular Cell Biology and Immunology, Vrije Universiteit Medical Center, Amsterdam, Netherlands
    Contribution
    RM, Acquisition of data, Analysis and interpretation of data
    Competing interests
    The authors declare that no competing interests exist.
  9. Gera Goverse

    Department of Molecular Cell Biology and Immunology, Vrije Universiteit Medical Center, Amsterdam, Netherlands
    Contribution
    GG, Acquisition of data, Analysis and interpretation of data
    Competing interests
    The authors declare that no competing interests exist.
  10. Marlous M S Sneeboer

    Department of Molecular Cell Biology and Immunology, Vrije Universiteit Medical Center, Amsterdam, Netherlands
    Contribution
    MMSS, Acquisition of data, Analysis and interpretation of data
    Competing interests
    The authors declare that no competing interests exist.
  11. Joke M M den Haan

    Department of Molecular Cell Biology and Immunology, Vrije Universiteit Medical Center, Amsterdam, Netherlands
    Contribution
    JMMH, Provided MHC-II KO mice, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  12. Marianne Boes

    Department of Pediatric Immunology, Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht, Netherlands
    Contribution
    MB, Provided K14-mOVA mice, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  13. Reina E Mebius

    Department of Molecular Cell Biology and Immunology, Vrije Universiteit Medical Center, Amsterdam, Netherlands
    Contribution
    REM, Conception and design, Analysis and interpretation of data, Drafting or revising the article
    For correspondence
    r.mebius@vumc.nl
    Competing interests
    The authors declare that no competing interests exist.

Funding

Nederlandse Organisatie voor Wetenschappelijk Onderzoek (VICI grant 918.56.612)

  • Reina E Mebius

Nederlandse Organisatie voor Wetenschappelijk Onderzoek (ALW program grant 820.02.004)

  • Ramon Roozendaal

Nederlandse Organisatie voor Wetenschappelijk Onderzoek (ALW program grant 823.02.011)

  • Jasper J Koning

Nederlandse Organisatie voor Wetenschappelijk Onderzoek (VENI grant 916.13.011)

  • Rogier M Reijmers

Fundação para a Ciência e a Tecnologia (PhD Scholarship SFRH/BD/33247/2007)

  • Antonio P Baptista

Nederlandse Organisatie voor Wetenschappelijk Onderzoek (ALW program grant 854.10.055)

  • Gera Goverse

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Wessel N van Wieringen (Dept. Epidemiology and Biostatistics, VUMC, Amsterdam, the Netherlands) for his help with statistical analysis.

This work was supported by grants from Fundação para a Ciência e Tecnologia—Portugal (SFRH/BD/33247/2007 to APB)—and the Netherlands Organization for Scientific Research (VICI grant 918.56.612 to REM, ALW program grants 820.02.004 to RR, 823.02.011 to JJK, and 854.10.005 to GG, and VENI grant 916.13.011 to RMR).

Ethics

Animal experimentation: All animal experiments were reviewed and approved by the Vrije University Scientific and Ethics Committees (protocols MCB09-35, MCB10-01 and MCB13-06). All surgery was performed under xylazine and ketamine anesthesia.

Reviewing Editor

  1. Emil R Unanue, Washington University School of Medicine, United States

Publication history

  1. Received: August 21, 2014
  2. Accepted: November 19, 2014
  3. Accepted Manuscript published: November 19, 2014 (version 1)
  4. Version of Record published: December 10, 2014 (version 2)

Copyright

© 2014, Baptista et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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