The immune system helps to protect us from cancer, infection by microbes and other diseases. There are several different types of immune cells that each have particular roles. For example, cytotoxic T cells can kill other cells in the body that are damaged or infected. These cells are found in various locations around the body—including a region of the spleen known as the white pulp—where they wait in an inactive state until they detect signals from a damaged or infected cell. These T cells divide and mature to produce populations of active T cells known as effector cytotoxic lymphoid cells (or CTLs for short), a process which is thought to occur within the white pulp.

A small protein called cytokine IL-12 is involved in the production of CTLs. The cytokine is released from other immune cells and causes the activated T cells to divide and mature. It has long been believed that IL-12 produced in the white pulp early on in the process is sufficient to drive this process, but more recent work suggests that sustained production of IL-12 in other areas of the spleen that are accessible to the bloodstream may be needed.

Here, Shah et al. studied the generation of cytotoxic T cells in mice that had been exposed to a vaccine against a disease called Toxoplasmosis. Their experiments show that IL-12 drives both the early and late stages of CTL production. In the early stages, the T cells respond to IL-12 that is secreted by a group of ‘lymphoid dendritic’ cells in the white pulp. However, in the later stages, the T cells move away from the white pulp to other parts of the spleen known as the marginal zone and red pulp, where a distinct group of ‘myeloid dendritic’ cells also produce IL-12 and direct the final maturation of the CTLs.

Shah et al.'s findings also show that the process in which cytotoxic T cells divide and later mature to produce CTLs involves a series of tightly controlled events that mostly occur outside of the white pulp. These observations provide a new perspective on how to develop vaccines and other treatments that more efficiently generate the CTLs needed to protect against infections and cancer.

The activation of naïve CD8⁺ T cells occurs in the T cell areas of secondary lymphoid organs (Mempel et al., 2004). Once activated, CD8⁺ T cells are thought to undergo rapid clonal expansion and differentiate into IFN-γ-producing effector cytotoxic lymphoid cells (CTLs) through an ‘autopilot’ mechanism (Mercado et al., 2000; Bevan and Fink, 2001; Kaech and Ahmed, 2001; van Stipdonk et al., 2001). Consistent with this paradigm, CD8α⁺ dendritic cells (DCs), which are resident in the splenic white pulp, are critical for both antigen cross-presentation (den Haan et al., 2000) and production of a key pro-inflammatory cytokine, IL-12 (Reis e Sousa et al., 1997; Mashayekhi et al., 2011). IL-12 is an important signal 3 cytokine that drives clonal proliferation of activated CD8⁺ T cells in vitro (Curtsinger et al., 1999) as well as their effector cytokine production in vivo (Schmidt and Mescher, 1999, 2002; Valenzuela et al., 2002; Liu et al., 2006; Wilson et al., 2008). However, following their activation and migration into the inner PALS (Reis e Sousa et al., 1997), CD8α⁺ DCs become hyporesponsive and cease IL-12 production (Reis e Sousa et al., 1999), raising the question of whether this initial early burst of IL-12 is sufficient to drive end-stage effector CD8⁺ T cell differentiation to completion.

Recently, there has been mounting evidence that additional events occurring outside the T cell area are critical for primary T_H1 and CTL effector differentiation. CXCR3 deficiency results in an inefficient generation of cytokine-producing effector cells and instead favors a response skewed towards memory cells (Kohlmeier et al., 2011; Kurachi et al., 2011; Groom et al., 2012). CXCR3-mediated outmigration of T cells into the splenic marginal zone (MZ) (Kurachi et al., 2011) or the peripheral medullary areas of lymph nodes (Groom et al., 2012) enables interactions with IL-12 producing myeloid cells. The role of CXCR3 in lymphocyte peripheralization also extends to the memory response and is essential for secondary effector T cell generation from central memory T cells (Sung et al., 2012; Kastenmuller et al., 2013). These recent findings argue that an initial exposure to IL-12 and other signal 3 cytokines during the early stages of T cell activation may not be sufficient for effector T cell differentiation. Instead, a subsequent exposure to IL-12 at extrafollicular sites appears to drive effector CD8⁺ T cell differentiation to completion. It is also possible that this requirement may stem from the initial lack of a robust CD8α⁺ DC- derived IL-12 during viral and bacterial immune responses (Dalod et al., 2003; Edelson et al., 2011).

During CD8⁺ T cell priming, the inflammatory milieu governs effector CD8⁺ T cell generation. In particular, the cytokine IL-12 potently promotes CD8⁺ T cell effector differentiation and function, through T-bet-dependent induction of KLRG1 expression and IFN-γ production (Joshi et al., 2007; Wilson et al., 2008, 2010). In addition to these effects, IL-12 appears to also control the migratory potential of primary and effector T cells, by negatively regulating CXCR3 expression (Slutter et al., 2013). Given the possibility of IL-12 being produced both within the T cell area and at extrafollicular sites, it remains unclear how IL-12 temporarily and spatially controls these key facets of the primary effector CTL response. Furthermore, although it is assumed that these IL-12 effects are CD8⁺ T cell-autonomous, the possibility exists that regulation may be indirectly mediated through the actions of this cytokine on other IL-12 responsive immune cells.

In order to address these questions, we used a Toxoplasma gondii vaccination model to conduct a detailed analysis of the role of IL-12 in the generation, function and migratory potential of parasite-specific effector CD8⁺ T cells. We had previously identified this tgd057-reactive H-2K^b-restricted CD8⁺ T cell response in mice vaccinated with an attenuated, uracil auxotrophic strain of T. gondii (CPS), known to elicit CD8⁺ T cell-dependent protective immunity (Fox and Bzik, 2002) and have demonstrated a strict in vivo requirement for IL-12 to generate KLRG1⁺ effector CTLs (Wilson et al., 2008, 2010). Our results reveal that the sequence of differentiative events that culminate in the production of primary end-stage effector CD8⁺ T cells occurs over a protracted period and that IL-12 exerts regulatory functions at both early and late phases of effector cell generation. The effects of IL-12 in upregulating KLRG1 expression and priming for IFN-γ production require CD8⁺ T cell intrinsic cytokine signaling. In contrast, we found that the belated downregulation of CXCR3 on effector CD8⁺ T cells is indirectly regulated by IL-12 and is instead controlled by a pathway in which IFN-γ and IFN-γ-inducible chemokines mediate this downmodulation. Using an in vivo intravascular staining method (Olson et al., 2013; Anderson et al., 2014), we were able to reveal that these later stages of effector CD8⁺ T cell differentiation occur extrafollicularly, involving DCs as cellular sources of both non-CD8α⁺ DC-derived IL-12 and CXCR3-ligands. Surprisingly, we also found extensive proliferation of both KLRG1⁻ and KLRG1⁺ CD8⁺ T cells in the MZ and red pulp (RP). Taken together with earlier studies (Lauvau et al., 2001; Cockburn et al., 2010), our findings argue against the notion that effector CTL generation occurs through an ‘autopilot’ sequence and, instead, involves a multi-leveled progression of effector T cell precursors through distinct splenic microenvironments, where their differentiation is controlled by a complex interplay with locally positioned activated immune cells.

In this report, we identify the sequence of events (schematically depicted in Figure 9) that naïve CD8⁺ T cells undergo as they differentiate into effector CD8⁺ T cells in response to infection with the intracellular protozoan pathogen, T. gondii. We further delineate the steps where the pro-inflammatory cytokine, IL-12, is required for effector CD8⁺ T cell differentiation. Upon activation, CD44, a surface receptor necessary for T cell adhesion to extracellular matrix proteoglycans, is rapidly upregulated and then L-selectin (CD62L) is downregulated in an IL-12-independent manner (Figure 1). CXCR3 is expressed early in the CD44^Hi, CD62L⁺ (F1) CD8⁺ T cell stage, and remains highly expressed through the CD44^Hi, CD62L⁻ (F2) CD8⁺ T cell stage (Figure 2). This early expression of CXCR3 is T-bet dependent but IL-12 independent (Figure 2). While still in the white pulp and even prior to CD8⁺ T cell expansion (Figures 1, 3), a few CD44^Hi, CD62L⁻ CD8⁺ T cells begin to acquire KLRG1 surface expression in an IL-12 dependent manner. As CD8⁺ T cell numbers increase and outmigrate to the RP, KLRG1 expression escalates (Figure 3). As shown in Figure 8 we are able to reveal, for the first time, that CD8⁺ T cell proliferation does not only occur exclusively in the white pulp (Figure 8A–D). Unexpectedly, we found that most of the proliferating CD8⁺ T cells are CD62L⁻ (F2 and F3 stage CD8⁺ T cells) and many of these cycling effector precursors are observed to be outmigrating into the MZ (Figure 8) via bridging channels (Figure 8—figure supplement 1) (Khanna et al., 2007). Finally, these extrafollicular KLRG1⁺ precursor effector CD8⁺ T cells downregulate CXCR3 expression in an IL-12 dependent fashion. CXCR3 downregulation by IL-12 is indirectly mediated by IFN-γ and IFN-γ-dependent chemokines, which are produced by other T lymphocytes and mDCs, respectively (Figure 7), in the MZ where the extrafollicular CD8⁺ T cells have been shown to cluster and interact with inflammatory myeloid cells producing IL-12 in a CXCR3 dependent manner (Kurachi et al., 2011). Our report outlines a detailed order of events during the differentiation of naïve CD8⁺ T cells to KLRG1⁺ CXCR3⁻ effector CD8⁺ T cells and, together with several recent reports (Khanna et al., 2007; Kohlmeier et al., 2011; Kurachi et al., 2011; Groom et al., 2012; Sung et al., 2012), highlights the importance of the MZ and RP as extrafollicular sites for their proliferation and differentiation.

Figure 9

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Our in-depth analysis of IL-12 regulated checkpoints during the early effector CD8⁺ T cell programming in response to T. gondii vaccination has provided several new insights. While IL-12 has been thought to be the major signal 3 cytokine needed for in vitro proliferation (Curtsinger et al., 1999; Valenzuela et al., 2002) and augmentation of the immune synapse (Markiewicz et al., 2009), we found that IL-12 does not provide an obligatory signal for T. gondii-specific CD8⁺ T cell clonal expansion in vivo, but rather is critical for the early KLRG1 expression on precursor effector CD8⁺ T cells. Although KLRG1, IFN-γ and CXCR3 are under transcriptional control of T-bet, the expression pattern of CXCR3 is opposite to the expression patterns of KLRG1 and IFN-γ, such that CXCR3 expression is increased early during CD62L⁺ F1 and CD62L⁻ F2 stages of CD8⁺ T cell differentiation, while KLRG1 is expressed during the later stages of effector differentiation (Figure 2). While CXCR3 is expressed early and its upregulation is IL-12 independent, we show that that once precursor effector CD8⁺ T cells upregulate KLRG1, CXCR3 expression is downregulated in an IL-12 dependent manner (Figures 2, 4). Even though IL-12 is required for both the upregulation of KLRG1 and the downregulation of CXCR3, these two effects can be temporally and spatially dissociated (Figures 3, 4). Furthermore, while the IL-12 effects on KLRG1 and IFN-γ are acting on the CD8⁺ T cells themselves, we found, surprisingly, that downregulation of CXCR3 is regulated by IL-12 in a non-CD8⁺ T cell autonomous fashion (Figure 5). This latter finding seems to be at odds with a previous report that IL-12 directly regulates the suppression of CXCR3 expression of effector CD8⁺ T cells (Slutter et al., 2013). In this previous study, Slutter et al. (2013) also used a chimeric approach with IL-12rβ1 deficient and WT OT-1 cells to demonstrate that IL-12 receptor signaling has negative regulatory effects on CXCR3 expression during influenza priming and that this control is CD8⁺ T cell-intrinsic. Our results indicate a similar conclusion when we analyzed CD8⁺ T cells without regard for KLRG1 expression (Figure 5). However, when we analyze CXCR3 expression on KLRG1⁻ vs KLRG1⁺ CD8⁺ T cells expression, we arrive at a different conclusion (Figure 5). In the absence of IL-12 receptor signaling, we show that KLRG1⁺ CD8⁺ T cells maintain low CXCR3 expression (Figure 5). The appearance of CD8⁺ T cell autonomous control can be explained by population skewing at the expense of KLRG1⁺ CD8⁺ T cells, which is the only stage where CXCR3 is decreased. Thus, even in the influenza system, IL-12 probably indirectly downregulates CXCR3.

The data from both our chimeric transfer and late in vivo IFN-γ neutralization experiments indicate that IL-12 does not directly regulate CXCR3 downmodulation during precursor effector CD8⁺ T cell development (Figures 6, 7), but instead acts through an indirect pathway by which IFN-γ suppresses CXCR3 expression. In support of this model, we find that there is production of IFN-γ by bystander T_H1 T cells and that IFN-γ maintains CXCR3 suppression in vitro. Thus, we propose a three-cell model (Figure 7) for CXCR3 downregulation, in which the developing effector CD8⁺ T cell is the direct target of IFN-γ derived from bystander T_H1 T cells and IFN-γ-induced chemokines produced by mDCs. In this model, IL-12 acts by inducing IFN-γ production from bystander T_H1 T cells rather than the developing effector CD8⁺ T cells themselves. The mechanism of CXCR3 suppression by IFN-γ is currently not clear, but it will be interesting to find out whether it uses the same ID2-mediated pathway described for IL-12 (Yang et al., 2011; Knell et al., 2013). It is also unclear what signals drive the production of IL-12 by mDCs at later time points after the initial IL-12 production by CD8α⁺ DCs has waned. It is possible that residual non-replicating T. gondii parasites persist (Dupont et al., 2014) and, in addition, IFN-γ itself acting together with accessory signals from bystander lymphocytes provides inductive signals for mDC production of IL-12 (Ariotti et al., 2014; Slutter and Harty, 2014). Regardless of these uncertainties, our identification of IFN-γ as the critical mediator for CXCR3 downregulation provides a common mechanism for how multiple proinflammatory cytokines (Yang et al., 2011) can redundantly downmodulate CXCR3 on developing effector CD8⁺ T cells.

Our report highlights that the expression pattern of CXCR3 is highly complex as CD8⁺ T cells progress through distinct stages of differentiation. Although CXCR3 expression has been classically associated with IL-12 driven type-1 effector adaptive immune responses, we show that CXCR3 is expressed very early in the CD62L⁺ KLRG1⁻ F1 stage and thus allowing CD62L⁻ KLRG1⁻ F2 stage effector precursor CD8⁺ T cells to outmigrate to the MZ and the RP, where they are further exposed to proinflammatory signals and differentiate into effector CD8⁺ T cells. The same CXCR3 mediated outmigration has been reported for primary CD4⁺, CD8⁺ T cells and central memory T cells (Kurachi et al., 2011; Groom et al., 2012; Sung et al., 2012; Kastenmuller et al., 2013), arguing for the existence of a common extrafollicular pathway for type-1 effector cell generation. Following proinflammatory cytokine and chemokine exposure KLRG1⁺ CXCR3^Hi precursor effector CD8⁺ T cells gain the ability to produce high levels of IFN-γ while downregulating CXCR3 expression. The observation that outmigrating differentiated effector CD8⁺ T cells found in the splenic RP are CXCR3^Lo while published studies have clearly shown that tissue associated effector CD8⁺ T cells are mostly CXCR3^Hi (Kohlmeier et al., 2011; Harris et al., 2012; Slutter et al., 2013; Ochiai et al., 2015) raises the question of whether they reacquire CXCR3 once they reach infected tissue sites. A recent study of Mtb-infected lung tissues has demonstrated the existence of two distinct subpopulations of CD4⁺ T_H1 cells that differ in their accessibility to an intravenous fluorescent antibody tracer (Sakai et al., 2014). The tracer-accessible subpopulation resembled our F3 stage effector CD8⁺ T cells being KLRG1⁺ CXCR3^Lo CX3CR1^Hi and was IFN-γ^Hi. In contrast, the other subpopulation more deeply localized in the tissue parenchyma were CXCR3^Hi and exhibited a highly activated phenotype with expression of CD69 and PD-1, but lower in KLRG1 expression and IFN-γ production. Therefore, one interesting scenario is by virtue of their CX3CR1 expression, the F3 stage effector cell circulates in the blood and serves a patrolling function similar to CX3CR1^Hi monocytes (Auffray et al., 2007). They could then regain CXCR3 expression and function as effector CD8⁺ T cells in parasite-infected tissues. Future studies should investigate the reciprocal patterns of expression of CXCR3 and CX3CR1 and their counter regulation.

Using immunohistochemistry methods Khanna et al. (2007) demonstrated the outmigration of antigen-specific CD8⁺ T cells through ‘bridging channels’ into the MZ and splenic RP. Kurachi et al. (2011) further reported that CXCR3 enabled CD8⁺ T cells to cluster with IL-12 producing CD11c⁺ DCs in the MZ and RP. Now, we show that precursor effector CD8⁺ T cells in the bridging channels and MZ are, in fact, actively proliferating and that they receive IL-12 signals to complete their differentiation in the RP. Collectively, these findings highlight a previously unrecognized extrafollicular pathway for the generation and maturation of primary effector CD8⁺ T cells. Interestingly, in the B cell response, there is an analogous and well-recognized extrafollicular differentiation pathway of IgG class switched plasma cells producing the primary antibody response to T cell-dependent antigens (MacLennan et al., 2003; Kurosaki et al., 2015). By analogy to the way that the non-germinal center plasma cell response is favored by strong B cell receptor signaling and antigen presentation by MZ localized DCs (Paus et al., 2006; Phan et al., 2006; Chappell et al., 2012), the extrafollicular generation of effector CD8⁺ T cells is also likely regulated by differential T cell receptor signaling (Bernhard et al., 2015) and local cytokine and chemokine milieu created by specialized pathogen-engaged APCs interacting with bystander lymphocytes in the MZ and RP. The exposure of effector precursors to this extrafollicular tissue niche could provide major extrinsic factors that determine the observed interclonal and, perhaps, intraclonal variability in effector lymphocyte fates that is independent of their intrinsic TCR-peptide:MHC affinity and dwell time (Plumlee et al., 2013; Tubo et al., 2013).

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Author details

1. Suhagi Shah

   Center for Immunity and Inflammation, New Jersey Medical School, Rutgers University, Newark, United States

   Contribution

   SS, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Gijsbert M Grotenbreg

   Immunology Programme, Departments of Microbiology and Biological Sciences, National University of Singapore, Singapore, Singapore

   Present address

   121 Bio LLC, Cambridge, United States

   Contribution

   GMG, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

3. Amariliz Rivera

   Center for Immunity and Inflammation, New Jersey Medical School, Rutgers University, Newark, United States

   Contribution

   AR, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

4. George S Yap

   Center for Immunity and Inflammation, New Jersey Medical School, Rutgers University, Newark, United States

   Contribution

   GSY, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   yapgs@njms.rutgers.edu

   Competing interests

   The authors declare that no competing interests exist.

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