Germinal centers (GCs) are transient organized microstructures within secondary lymphoid tissues, which support the development of high-affinity antibodies by B cells. Even though GCs are mainly B cell driven, their formation and maintenance depends on follicular T helper cells (Tfh), a specialized subset of effector CD4 T cells. The differentiation into Tfh initiates in T cell zones of lymphoid organs before GC formation starts. After their interaction with B cells at the T-B border, Tfh locate within GC (GC-Tfh) and regulate the survival of proliferating GC-B cells, which compete for antigen (Ag) and for signals from GC-Tfh to undergo further somatic hypermutation and to mature into high-affinity-antibody-producing plasma cells and memory B cells (Crotty, 2019; Qi, 2016). Thus, GC-Tfh are central players in the regulation of humoral immune responses. Both (i) their differentiation into GC-Tfh and (ii) their survival and clonal expansion within GCs are constantly driven by competition for Ag contacts (Baumjohann et al., 2013; Fazilleau et al., 2009; Hwang et al., 2015; Knowlden and Sant, 2016; Tubo et al., 2013; Merkenschlager et al., 2021).

Here, we asked whether this clonal competition of Tfh for the limited space in GCs involves GC reactions of one or more activated lymphoid organs. This is of special interest for autoimmune conditions when multiple inflamed tissue sites and autoreactive GC reactions exist in distinct lymphoid organs. Tfh can shuttle between GCs of one lymph node or spleen indicating a local competition within one lymphoid organ (Merkenschlager et al., 2021; Shulman et al., 2013). The finding that CXCR5+ Tfh circulate in blood (Brenna et al., 2020; He et al., 2013) opens the possibility for a systemic exchange, in which each Tfh clone could participate in the clonal competition within distinct lymphoid organs in one individual. However, the diversity of individual T cell receptor (TCR) repertoires is extremely high, and it has been shown that individual T cell responses towards Ag are unique and polyclonal (Textor et al., 2018; Thomas et al., 2014; Zarnitsyna et al., 2013). This might be especially valid for auto-Ag because TCR bind with low affinities (Dolton et al., 2018; Rius et al., 2018). In line, the number of autoreactive T cell clones within one individual is particularly low due to the thymic negative selection process, which could support the involvement of multiple cross-reactive T cell clones specific for the same auto-Ag within one individual as it has been suggested previously (Ritvo et al., 2018).

To find out how polyclonal autoreactive Tfh responses are, we studied the distribution of GC-Tfh clones between two activated lymph nodes and challenged the endogenous TCR repertoire by injecting one auto-Ag, directed against a structural protein of murine skin, into the two hind footpads of the same mouse and analyzed the TCRβ sequences of GC-Tfh in both draining popliteal lymph nodes (pln). In contrast to our expectations, we found that the GC-Tfh clonotypes were highly shared between the two pln. This high overlap between both pln was restricted to the dominant GC-Tfh clonotypes and disappeared after establishment of skin pathology even though GC reaction continued. These temporal variations in the distribution of Tfh clones should be considered when utilizing Tfh repertoires to monitor and diagnose autoimmune diseases.

CD4 T cells are pivotal in adaptive immune responses during infection, autoimmune diseases, cancer, and vaccinations. Each CD4 T cell expresses one specific αβ TCR interacting with specific peptide-Ag presented in MHCII complexes (Itano and Jenkins, 2003). Upon interaction of the TCR with peptide:MHCII complexes, naive CD4 T cells undergo proliferation and functional differentiation into distinct T helper subsets such as Th1, Th2, Th17 effector cells, Tfh, and immunosuppressive Treg cells (Zhu et al., 2010). The main factor that regulates CD4 T cell differentiation is the TCR signaling strength, which is controlled by TCR/peptide:MHCII interactions, co-stimulation, and/or optimal dwell times. The differentiation into Tfh depends on strong TCR signals. It is suggested that they are strictly Ag-specific (Hwang et al., 2015; Knowlden and Sant, 2016; Tubo et al., 2013; Merkenschlager et al., 2021). Consistently, it has been shown that the maintenance and expansion of Tfh requires sustained Ag stimulation (Baumjohann et al., 2013; Merkenschlager et al., 2021).

In this study, we asked how this particular CD4 T cell subset is distributed within one individual at the clonal level. We used an autoantibody-mediated disease model of the skin, epidermolysis bullosa acquisita, and induced GC-Tfh in two separate draining pln by injecting auto-Ag in adjuvant (Ag1 or Ag2 group) or adjuvant only (PBS group).

Our study shows two major findings. First, we found a high overlap of dominant GC-Tfh clonotypes in both pln of one mouse (Figure 2a, b). This data demonstrates that even though the response to the auto-Ag was polyclonal in each mouse, a group of almost identical GC-Tfh clonotypes became dominant in each draining pln. Thus, exposures to the auto-Ag (Ag1 or Ag2) decreased the diversity of the endogenous Tfh repertoire compared to the PBS group. To establish this finding, we used multiplex iRepertiore PCRs and subsequent MiTCR analysis for preparing libraries and annotating TCRβ sequences (Bolotin et al., 2013), which ensures to yield the highest possible diversity of TCRβ clonotypes (Afzal et al., 2019). To confirm this decrease in diversity of the endogenous Tfh repertoire, we reanalyzed our data for Ag1 with a more precise analysis tool MiXCR that is advantageous for error corrections and adjusts for a more accurate clonal composition (Bolotin et al., 2013; Bolotin et al., 2015; Team I, 2019). The high overlap of the dominant Tfh clonotypes in the Ag1 group was clearly confirmed (Figure 2—figure supplement 1). To further compare both analysis tools, we calculated the MHI from samples of the Ag1 and PBS groups analyzed either with MiTCR or MiXCR. Data show a high similarity in both the Ag1 group (0.70 ± 0.04) and the PBS group (0.69 ± 0.039). Obviously, our finding that Tfh clonotypes highly overlap in contralateral pln is very robust independent of the analysis tools. On the other side, the suitability of the multiplex PCRs can be seen by the correct detections of the deletions in the TRBV genes of SJL mice (Figure 3c; Behlke et al., 1986). In summary, this data that the endogenous Tfh repertoire decreased after Ag exposure indicates that the number of T cell clones specific for the injected auto-Ag is restricted per mouse and supports previous reports demonstrating that the precursor frequency specific for auto-Ag within the T cell population of an individual is rather low (Jenkins and Moon, 2012). However, the precursor frequency of naive T cells specific for different peptide:MHC complexes varies in size. Therefore, it will be interesting whether this high clonal overlap would be found after injection of xenogeneic or pathogenic Ag. Moreover, because both Ag used in this study are polypeptides of a relatively large size (200–400 aa) and clearly differ from the size of peptides (13–25 aa) that are usually presented in MHCII (Natarajan et al., 2018), it is likely that they contain more than one antigenic epitope. It is reasonable to assume that injecting smaller peptides will further narrow the diversity of the individual GC-Tfh repertoire. In an initial experiment as an example for a recombinant xenogeneic protein antigen, we injected the Schistosoma mansoni-derived glutathione-s-transferase (GST)-tag of Ag1 (Rao et al., 2003), emulsified in TM into both footpads, and analyzed the distribution of Tfh clonotypes in both draining pln 4 weeks p.i. (Figure 2—figure supplement 2, Supplementary file 6). We found that the distribution of the GST-specific Tfh clonotypes between both pln was less controlled compared to the auto-Ag1. Dot plot diagrams showed a higher variability between the mice. In line, the MHI was not significantly different from the PBS group (Figure 2—figure supplement 2) and the number of overlapping Tfh clonotypes is lower in the GST group compared to the Ag1 group (Figure 3—figure supplement 1). Analysis of the V/J usage revealed significant differences in expression of TRBV3. The J2-4-segment was significantly less abundant after GST/TM immunization compared to the PBS/-group and the J2-5 segment was jointly higher abundant after GST/TM and Ag1 immunization compared to the control group (Figure 3—figure supplement 1). These data confirm the strict Ag specificity of Tfh cells in GCs, especially during the first 4 weeks p.i. The higher inconsistency in the number of overlapping Tfh clonotypes in the GST group could be explained by an earlier consumption of the Ag or the presence of more high-affinity T cell clones. These findings might be strongly influenced by the time point and the Ag concentration, which should be further investigated in future studies.

Another arising question is when this overlap of T cell clonotypes between both pln of one mouse starts. For example, T cells could travel between pln and the clonal selection could be a continuous organism-wide systemic process. The presence of circulating Tfh cells has been described in humans and mice (Brenna et al., 2020; He et al., 2013); however, it is not clear whether these circulating Tfh cells can enter ongoing GC reactions. Alternatively, it could take place during the priming phase. In this case, either the naive TCR repertoire is sufficiently broad that the Ag-driven selection for the GC reactions in both pln would robustly yield groups of similar clones independently or recently activated CD4 T cell clones would exchange between both pln after priming before GC reaction starts. To address this, we performed an initial experiment and identified TCRβ sequences of entire left and right pln of the same mouse (naive mice and mice that had been exposed to Ag1 for 1 or 3 days; please find detailed information in Supplementary file 5) and compared the relative percentage of overlapping T cell clonotypes between left and right pln by using the MHI. We found that the MHI did not differ between naive mice and Ag-exposed mice at 1 day p.i. (0.2035 + 0.01962 in naive mice, 0.2113 + 0.009 at 1 day p.i.), but a significant increased MHI at 3 days p.i. (0.3716 + 0.031; p<0.01, Kruskal–Wallis test, mean + SD for n = 3 mice, Figure 4—figure supplement 1). In parallel, the number of proliferating T cells and the mRNA expression for the proliferation marker Ki67 increased significantly (Figure 4—figure supplement 1). One may speculate that a high number of progenies emerges from the high-affinity T cell clones 3 days p.i. (Cho et al., 2017), which then migrate into other activated lymphoid tissues before differentiating into GC-Tfh. In this case, the initial progeny of activated T cell clones could originate from identical precursors, which would enter the circulation and migrate into the other pln, in which they outcompete low-affinity competitors and develop into GC-Tfh. However, on the other side it is assumed that Tfh stay resident in lymph nodes to provide B cell help (Crotty, 2019). One possibility to observe potential exchanges of Tfh cells between lymph nodes would be treatment with the T cell trafficking blocker FTY720. However, previous studies revealed no effect in this autoimmune mouse model (Niebuhr et al., 2017; Thieme et al., 2019). The second major finding of our study is that the PBS group, which received only adjuvant, develops GCs to a similar extent as the Ag group. Here, in contrast to the Ag group, the GC-Tfh repertoire remains diverse. No overlap of dominant GC-Tfh clonotypes was found between both pln. TM is a water-in-oil adjuvant that contains squalene among other components but no peptides that could be presented in MHCII (TiterMax Inc). It is proposed that adjuvants stress or kill local cells, which leads to the release of endogenous peptide-epitopes that are subsequently presented to T cells (Riteau et al., 2016) and induce the formation of GC reactions as observed in this study (Figure 1c). In this scenario, numerous endogenous peptide-epitopes at low concentrations might be released by the local inflammation, which leads to the diverse GC-Tfh repertoire in both pln (Figure 2a, b). However, to break the tolerance in our model, both auto-Ag had to be injected at very high concentrations (60 µg/Ag1 or 120 µg/Ag2, respectively) (Sitaru et al., 2006). This data leads to the hypothesis that the concentration of the Ag is critically involved in the emergence of overlapping GC-Tfh repertoires in separate pln (summarized in Figure 6). It will be interesting to find out how low Ag concentrations at distinct tissue sites affect the GC-Tfh repertoires. Moreover, it will be important to study the adjuvant-stimulated peptide-epitopes in future studies, especially because squalene compounds are frequently used in human vaccines and might evoke unwanted autoimmune responses (Pellegrini et al., 2009).

Figure 6

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Another conclusion of our study is that the total number of GC-Tfh within one individual is regulated by the number of lymphoid tissues that drain Ag-exposed tissue sites. This data suggests that Ag injection at two or more tissue sites could improve the development of high-affinity antibodies. We believe that this information will be highly useful for the development of new and more efficient vaccination strategies.

Finally, we wish to highlight that this in vivo approach could help to identify new peptide-specific TCR sequences and confirm already established datasets (Teraguchi et al., 2020). Even though TCR datasets are growing, the number of TCRs with known Ag specificity and function is extremely low (Shugay et al., 2018; Zvyagin et al., 2020). Especially the detection of self-reactive CD4 TCR is problematic due to the low binding affinities between TCR and autoantigenic-peptide:MHCII complexes (Dolton et al., 2018; Rius et al., 2018). Thus, even though our approach is restricted to mouse models, the use of HLA-transgenic mice might enable to link TCR sequences also to human antigenic epitopes.

T cell receptor-RNA sequencing data generated in this study are deposited in the sequence read archive hosted at https://www.ncbi.nlm.nih.gov/sra under the primary accession code PRJNA731654. Some of the data (skin effector T cells) have been deposited under the accession code PRJNA586880.

1. 1. Kalies K
   2. Niebuhr M

   (2021) NCBI BioProject

   ID PRJNA731654. TRBV sequences of murine follicular T helper cells in experimental epidermolysis bullosa acquisita.

   https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA731654
2. 1. Kalies K
   2. Niebuhr M

   (2021) NCBI BioProject

   ID PRJNA586880. TCRb-seq data from dermal T cells in SJL mice upon immunization.

   https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA586880

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Author details

1. Markus Niebuhr

   Institute for Anatomy, University of Lübeck, Lübeck, Germany

   Contribution

   Data curation, Software, Formal analysis, Validation, Investigation, Methodology

   Competing interests

   No competing interests declared

2. Julia Belde

   Institute for Anatomy, University of Lübeck, Lübeck, Germany

   Contribution

   Data curation, Investigation

   Competing interests

   No competing interests declared

3. Anke Fähnrich

   1. Institute for Anatomy, University of Lübeck, Lübeck, Germany
   2. Lübeck Institute of Experimental Dermatology, University of Lübeck, Lübeck, Germany

   Contribution

   Data curation, Formal analysis, Validation, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1904-9544

4. Arnauld Serge

   Laboratoire Adhésion et Inflammation, Inserm U1067 CNRS, Aix-Marseille Université, Marseille, France

   Contribution

   Data curation, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1280-6277

5. Magali Irla

   Centre d'Immunologie de Marseille Luminy (CIML), INSERM U1104, Aix-Marseille Université UM2, Marseille, France

   Contribution

   Data curation, Investigation, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8803-9708

6. Christoph T Ellebrecht

   1. Institute for Anatomy, University of Lübeck, Lübeck, Germany
   2. Department of Dermatology, University of Pennsylvania, Philadelphia, United States

   Contribution

   Data curation, Methodology

   Competing interests

   No competing interests declared

7. Christoph M Hammers

   1. Institute for Anatomy, University of Lübeck, Lübeck, Germany
   2. Department of Dermatology, University of Lübeck, Lübeck, Germany

   Contribution

   Data curation, Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5631-2415

8. Katja Bieber

   Lübeck Institute of Experimental Dermatology, University of Lübeck, Lübeck, Germany

   Contribution

   Data curation, Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3855-6683

9. Jürgen Westermann

   Institute for Anatomy, University of Lübeck, Lübeck, Germany

   Contribution

   Resources, Funding acquisition

   Competing interests

   No competing interests declared

10. Kathrin Kalies

    Institute for Anatomy, University of Lübeck, Lübeck, Germany

    Contribution

    Conceptualization, Supervision, Funding acquisition, Investigation, Methodology, Writing - original draft, Project administration, Writing - review and editing

    For correspondence

    kathrin.kalies@uni-luebeck.de

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-8929-4249

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