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W2987267675 abstract "Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract The proinflammatory cytokine IL-12 drives the generation of terminally differentiated KLRG1+ effector CD8+ T cells. Using a Toxoplasma vaccination model, we delineate the sequence of events that naïve CD8+ T cells undergo to become terminal effectors and the differentiation steps controlled by IL-12. We demonstrate that direct IL-12 signaling on CD8+ T cells is essential for the induction of KLRG1 and IFN-γ, but the subsequent downregulation of CXCR3 is controlled by IL-12 indirectly through the actions of IFN-γ and IFN-γ-inducible chemokines. Differentiation of nascent effectors occurs in an extrafollicular splenic compartment and is driven by late IL-12 production by DCs distinct from the classical CD8α+ DC. Unexpectedly, we also found extensive proliferation of both KLRG1− and KLRG1+ CD8+ T cells in the marginal zone and red pulp, which ceases prior to the final KLRG1Hi CXCR3Lo stage. Our findings highlight the notion of an extrafollicular pathway for effector T cell generation. https://doi.org/10.7554/eLife.09017.001 eLife digest 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. https://doi.org/10.7554/eLife.09017.002 Introduction 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 TH1 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-2Kb-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. Results CD8+ T cell proliferative response is IL-12 independent while effector cell differentiation is IL-12 dependent To determine the early effects of IL-12 on CD8+ T cell proliferation and differentiation during T. gondii infection, we used a tetramer-based enrichment method (Klenerman et al., 2002; Moon et al., 2007) to enumerate H-2Kb-restricted CD8+ T cells specific for the T. gondii antigen tgd057 (Wilson et al., 2010) in wild-type (WT) and IL-12p35 deficient hosts following CPS vaccination. The tetramer-based enrichment method allows for a ∼2-log increase in detection of tgd057-specific CD8+ T cells (Figure1—figure supplement 1). To accurately enumerate the exact numbers of tgd057-specific CD8+ T cells when their frequencies are very low we mixed a known number of naïve Thy1.1+-marked tgd057-specific CD8+ T cells from a somatic cell nuclear transfer (SCNT) mouse (Kirak et al., 2010a) with spleen cells from individual naïve mice (Figure 1—figure supplement 2). We are able to detect 2093 ± 273 and 1200 ± 138 endogenous tgd057-specific CD8+ T cells in spleens of naïve WT and IL-12p35 deficient mice, respectively (Figure 1—figure supplement 2). Figure 1A shows that there is little change in absolute cell numbers of tgd057-specific CD8+ T cells between day 0 and day 4 post-vaccination in the spleen. However, we can detect a 10-fold clonal expansion occurring between days 4 and 5-post infection in spleens of both WT and IL-12 deficient mice. The absolute numbers of tgd057-specific CD8+ T cells continue to increase at similar rates through day 7 in WT and IL-12 deficient mice. These results indicate that the lack of IL-12 signals does not affect the timing or the magnitude of the proliferative response of tgd057-specific CD8+ T cells during T. gondii vaccination. Figure 1 with 2 supplements see all Download asset Open asset CD8+ T cell proliferative response is IL-12 independent while effector cell differentiation is IL-12 dependent. (A) Absolute numbers of tgd057-specific CD8+ T cells in spleen after CPS vaccination in wild-type (WT) and IL-12p35−/− mice. (B) tgd057-specific CD8+ T cells were analyzed for CD44, CD62L and KLRG1 cell surface expression. Pie charts represent frequency averages of F1 (CD62L+ KLRG1−), F2 (CD62L−, KLRG1−), F3 (CD62L−, KLRG1+), and F4 (CD62L+, KLRG1+) subsets of tgd057-specific CD8+ T cells from WT and IL-12p35−/− spleens D0–D7 post CPS vaccination. Data shown include only CD44hi cells on D4–D7 and include all tgd057-specific CD8+ T cells on D0–D3. (C) Absolute numbers of tgd057-specific CD8+ T cells in peritoneal exudate cells (PECs) of WT and IL-12p35−/− mice after CPS vaccination. Cells were characterized based on tgd057-specific CD8+ T cells. (D) Average of frequencies of F1 (CD62L+ KLRG1−), F2 (CD62L−, KLRG1−), F3 (CD62L−, KLRG1+), and F4 (CD62L+, KLRG1+) subsets of tgd057-specific CD8+ T cells in PECs D0–D7. Data represent 5 independent experiments with 3–5 mice per group per experiment. Data were analyzed using two-way ANOVA and (A) Holms-Sidak or (C) Bonferroni post-hoc tests; *p < 0.05. See Figure 1—figure supplements 1, 2 for the methodology employed to enumerate absolute numbers of tgd057-specific CD8+ T cells. https://doi.org/10.7554/eLife.09017.003 Despite the apparent lack of a role for IL-12 in the proliferative recruitment of tgd057- specific CD8+ T cells, IL-12 could play a crucial role in the early diversification of CD8+ T cells into end-stage effector CD8+ T cells vs memory precursor type cells. Indeed, previous studies in our laboratory and others have shown that IL-12 is crucial for the differentiation of KLRG1+ effector CTLs through T-bet dependent induction of effector genes (Joshi et al., 2007; Wilson et al., 2008, 2010). To determine how IL-12 signals affect the early differentiation of tgd057-specific CD8+ T cells, we monitored the expression of CD62L and KLRG1 following T. gondii vaccination. We have previously used these cell surface markers to define four specific CD8+ T cell stages: F1 (TCM: CD62L+, KLRG1−), F2 (TEM: CD62L−, KLRG1−), F3 (TEFF: CD62L−, KLRG1+) and F4 (CD62L+, KLRG1+) (Wilson et al., 2008, 2010). While F1 and F2 are determined to be central and effector memory CD8+ T cells, respectively; F3 are the late stage highly IFN-γ-producing effector CD8+ T cells, little is still known about the phenotype and function of the F4 stage CD8+ T cells. We do not observe consistent changes in CD8+ T cell stage distribution until day 3 in either WT or IL-12 deficient hosts (Figure 1B). However, by day 3, ∼7–8% of the tgd057-specific CD8+ T cells start to commit to putative TEM and TEFF cells by downregulating CD62L and increasing KLRG1 expression in the WT hosts (Figure 1B). In IL-12p35 deficient hosts, L-selectin downregulation is also observed starting on day 3, however KLRG1 upregulation is absent (Figure 1B). On day 4, there is a dramatic increase in the frequency of tgd057-specific CD8+ T cells that have downregulated CD62L, a third of which also acquire KLRG1 in WT mice. However, in the absence of IL-12, there is an attenuation of KLRG1 expression resulting in an even larger frequency of F2 tgd057-specific CD8+ T cells (Figure 1B). The frequency of KLRG1+ tgd057-specific CD8+ T cells continues to increase through day 7 post-vaccination in WT hosts; yet, in IL-12 deficient mice, KLRG1 upregulation remains attenuated and the frequencies of F3 stage tgd057-specific CD8+ T cells do not reach levels seen WT hosts (Figure 1B). These results indicate that IL-12 signaling has an important role on the differentiation of KLRG1+ effector tgd057-specific CD8+ T cells, and that this occurs earlier than the proliferative burst seen after day 4. In contrast, downregulation of CD62L and subsequent clonal expansion are IL-12 independent. During effector CD8+ T cell differentiation, expression of CXCR3 is downregulated in an IL-12 dependent manner In spite of the fact that IL-12 signals did not play a crucial role in proliferation of tgd057-specific CD8+ T cells in the spleen, we observed that case to be different at the site of infection. We enumerated tgd057-specific CD8+ T cells in the peritoneal exudate cells (PECs) using the SVLAFRRL:H-2Kb tetramer (Figure 1C). Surprisingly, we found that the absolute numbers of tgd057-specific CD8+ T cells in the PECs in an IL-12 deficient environment by day 6 were attenuated by one log in absolute numbers as the height of the adaptive immune response was reached (Figure 1C). The observation that tgd057-specific CD8+ T cells traffic to the peritoneum is attenuated when IL-12 signals are lacking prompted us to investigate the role of IL-12 for effector tgd057-specific CD8+ T cells migration. The T-box protein, T-bet, is a master regulator of the differentiation and functional activity of effector CD8+ T cells. It has been shown to associate with the promoters of genes for KLRG1 (Joshi et al., 2007), IFN-γ (Joshi et al., 2007), as well as the chemokine receptor, CXCR3 (Beima et al., 2006; Harms Pritchard et al., 2015). We hypothesized that since KLRG1 expression and the production of IFN-γ by F3 late stage effector CD8+ T cells is IL-12 dependent (Wilson et al., 2008, 2010), CXCR3 expression may also be IL-12 dependent, possibly explaining the attenuation of effector cells to the site of infection in our model. Analysis of CXCR3 expression as CD8+ T cells differentiate from F1 (TCM) to F2 (TEM) cells in the spleen show that CXCR3 expression is highly expressed when CD8+ T cells are CD62L+ or CD62L− (Figure 2A,B). Interestingly, our data demonstrate that the early expression of CXCR3 is IL-12 independent, but T-bet dependent (Figure 2A,B) and as these cells differentiate into end-stage effector IFN-γ producing KLRG1+ CD8+ T cells, they downregulate CXCR3 (Figure 2A,B), indicating that IL-12 has late downregulatory effects on CXCR3 expression. Figure 2 Download asset Open asset During effector CD8+ T cell differentiation, expression of CXCR3 is downregulated in an IL-12 dependent manner. CD62L and KLRG1 surface expression (A) and CXCR3 surface expression (B) on total CD8+ T cells were assessed by flow cytometry 7 days post-vaccination. Data represent 3–4 independent experiments with 4–5 mice per group per experiment. Mean ± SEM, data were analyzed using unpaired t test, and Holms-Sidak post-hoc test, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. https://doi.org/10.7554/eLife.09017.006 Downregulation of CXCR3 expression on CD8+ T cells occurs following KLRG1 induction in the splenic RP We next wished to determine where in the spleen KLRG1 upregulation and CXCR3 downregulation occurs as tgd057-specific CD8+ T cells differentiate into late-stage effectors. In order to study the white pulp and RP distribution of the CD8+ T cells within the spleen, we intravenously injected mice with a fluorescently conjugated anti-CD8α antibody on days 4 and 7 post CPS vaccination (Figure 3A). Tissue was isolated after a brief delay to quickly label only cells exposed to the blood and RP within the spleen, but excluded cells in white pulp (Olson et al., 2013; Anderson et al., 2014). Interestingly, our results suggest that on day 4, tgd057-specific CD8+ T cells upregulate KLRG1 expression after trafficking to the RP (Figure 3B). By day 7 post-vaccination, the tgd057-specific CD8+ T cells are found to be equally localized to the white pulp and the RP prior to increasing their KLRG1 expression, where, once KLRG1 expression is upregulated, the majority (∼80%) of the cells is located in the RP. Thus, the early upregulation of KLRG1 expression on tgd057-specific CD8+ T cells occurs mainly in the RP, where they may be encountering a secondary signal of the pro-inflammatory cytokine, IL-12, for later differentiation into end-stage effector CD8+ T cells. Because the preferential localization of CD62L− KLRG1− and CD62L− KLRG1+ tgd057-specific CD8+ T cells is in the RP on day 4, we next determined whether CXCR3 is concomitantly downregulated as KLRG1 expression is increased. As Figure 3C indicates, at day 4 CXCR3 expression in both KLRG1− and KLRG1+ CD8+ T cells is not substantially decreased, but by day 7, the KLRG1+ CD8+ T cells which are predominantly RP-localized have completely downregulated CXCR3. Taken together, these results demonstrate that prior to the upregulation of KLRG1, effector precursor CD8+ T cells expressing high levels of CXCR3 can migrate to the RP, where they upregulate KLRG1 and then downregulate CXCR3 as they differentiate into late-stage effector CD8+ T cells. Figure 3 Download asset Open asset Downregulation of CXCR3 expression on CD8+ T cells occurs following KLRG1 induction in the splenic red pulp (RP). (A) Representative FACS profile identifying splenic RP and white pulp by differential staining with i.v injected APC conjugated anti-CD8α antibody. (B) Compiled data of RP distribution of F1, F2 and F3 tgd057-specific CD8+ T cells in spleen on D4 and D7 post CPS vaccination. (C) CXCR3 expression on F2-KLRG1− and F3-KLRG1+ tgd057-specific CD8+ T cells on D4 and D7 post CPS vaccination. Data are representative of 3 independent experiments with 6–8 mice per experiment. Mean ± SEM, data were analyzed using unpaired t test, and Holms-Sidak post-hoc test, ***p ≤ 0.001. https://doi.org/10.7554/eLife.09017.007 Late effects of IL-12 on the CD8+ T cell differentiation, function and chemokine receptor expression Our data thus far suggest that IL-12 exerts early effects on the differentiation of effector tgd057-specific CD8+ T cells during T. gondii infection by upregulating KLRG1 even prior to clonal expansion (Figure 1), but also plays a later role in the downregulation of CXCR3 expression (Figure 2). IL-12 may be produced only early during vaccine priming and programs the entire effector differentiation pathway over time. Alternatively, IL-12 may be produced at both early and late time points, potentially by distinct APCs. To address the latter scenario, we neutralized IL-12 late (D3) and compared its effects on CD8+ T cell differentiation to early (D0) and continuous neutralization (D0 and D3) following CPS vaccination. Consistent with our earlier results, the blockade of IL-12 at early, late or both time points did not affect absolute tgd057-specific CTL numbers (Figure 4A). When IL-12 is neutralized only on day 3, KLRG1 expression and IFN-γ-production were still attenuated, albeit less markedly compared to the effects of early and continuous blockade of the cytokine (Figure 4B,C). Importantly, we find that late neutralization of IL-12 interrupts the downregulation of CXCR3 expression on late-stage effector tgd057-specific CD8+ T cells (Figure 4D). These results confirm that IL-12 is required for early programming of effector differentiation, and indicate that IL-12 production occurs days after initial CPS vaccine exposure and continues to induce further differentiation of effector-fated CD8+ T cells. Figure 4 Download asset Open asset IL-12 exerts both early and late effects on CD8+ T cell differentiation, function and chemokine receptor expression. Anti-IL-12p40 mAb was administered i.p. on D0, D3 or both days post CPS vaccination. Spleens were harvested on D5 post CPS vaccination. (A) Absolute numbers of tgd057-specific CD8+ T cells, (B) average frequency of F1–F4 stages defined by cell surface expression of CD62L and KLGR1, (C) IFN-γ production and (D) CXCR3 downregulation on tgd057-specific CD8+ T cells. Data are from 3 independent experiments with 3–4 mice per group per experiment. Mean ± SEM, data were analyzed using multiple unpaired t test, and Holm-Sidak post-hoc test, *p ≤ 0.05, **p ≤ 0.01. https://doi.org/10.7554/eLife.09017.008 IL-12 mediates surface CXCR3 downregulation in a CD8+ T cell-extrinsic manner The observation that IL-12-mediated downregulation of CXCR3 can be temporally dissociated from its effects on KLRG1 upregulation (Figure 2B,C), which was previously shown to be a CD8+ T cell-autonomous effect of the cytokine (Slutter et al., 2013), prompted us to ask if the IL-12 effects are CD8+ T cell-intrinsic or extrinsic. In addition, CXCR3 downregulation on KLRG1+ CD8+ T cells occurs in the RP (Figure 3B,C), where IL-12 producing cell types other than CD8α+ DCs may be involved. Specifically, the late-acting IL-12 may be secreted by inflammatory-monocyte derived DCs, and has been shown to require NK (Goldszmid et al., 2012) or TH1-CXCR3+ CD4+ T cell -derived IFN-γ (Cohen et al., 2013) for DC activation. This scenario raises the possibility that other cells besides the developing effector CD8+ T cells may be the targets of IL-12 signaling. In order to determine if IL-12 mediates CXCR3 downregulation directly, we adoptively transferred a 1:1 mixture of naïve WT and IL-12rβ2 deficient monoclonal tgd057-specific CD8+ T cells (derived via SCNT cloning) into a CD45.1+ WT B6 host (Figure 5A). 7 days after CPS-vaccination, we observe that the lack of IL-12 signaling does not alter the frequency of IL-12rβ2 deficient donor CD8+ T cells (Figure 5B), but CXCR3 downregulation is inhibited (Figure 5C). These results confirm earlier conclusions that IL-12 is not essential for T cell expansion in vivo and are consistent with the notion that IL-12 directly signals the downregulation of CXCR3 expression. However, when we further compared CXCR3 expression between KLRG1− vs KLRG1+ stages, we found that the F3 stage KLRG1+ CD8+ T cells had downregulated CXCR3 regardless of their ability to signal IL-12 (Figure 5D). We conclude that CXCR3 is downregulated in a non-CD8+ T cell autonomous manner, which is not consistent with previous results (Slutter et al., 2013). The apparent lack of CXCR3 downregulation is due to the predominant deficiency in KLRG1 expression on the IL-12Rβ2 deficient CD8+ T cells (Figure 5E). Unlike CXCR3, KLRG1 expression and IFN-γ production, that are known to be directly regulated by IL-12 signaling, behave in a cell autonomous manner (Figure 5E,F). Figure 5 Download asset Open asset IL-12 mediates CXCR3 downregulation in a CD8+ T cell-extrinsic manner. (A) A schematic representation of adoptive co-transfer of naive 500 WT somatic cell nuclear transfer (SCNT, CD45.2+, Thy1.1+) and naïve 500 il12rβ2−/− SCNT (CD45.2+, Thy1.2+) CD8+ T cells into naïve CD45.1+ WT recipients 1 hr prior to CPS vaccination. (B) Representative FACS profile shows frequency of antigen specific polyclonal endogenous and monoclonal donor CD8+ T cells D7 post CPS vaccination. (C) CXCR3 expression on total tgd057-specific donor CD8+ T cells on D7 post vaccination (left) and expressed as MFI (right). (D) tgd057-specific donor CD8+ T cells were analyzed for CXCR3 expression by first gating for CD62L and KLRG1 cell surface expression, then studying expression of CXCR3 in F2 and F3 stages. (E) Frequency of F1–F4 stages of WT CD8+ T cells and IL-12rβ2−/− SCNT CD8+ T cells D7 post-CPS vaccination. (F) IFN-γ production by WT SCNT and IL-12rβ2−/− SCNT CD8+ T cells was analyzed after CPS restimulation. Data are from 3 independent experiments consisting of 4–8 mice per group per experiment. Mean ± SEM. Data were analyzed using unpaired t test, and (D) Bonferroni post-hoc test. (E) Mann–Whitney post-doc test; *p < 0.01, ***p < 0.001. https://doi.org/10.7554/eLife.09017.009 An alternative pathway involving IFN-γ and IFN-γ-inducible chemokines mediates suppression of CXCR3 on late-stage effector CD8+ T cell precursors To explore the mechanism of CD8+ T cell-extrinsic downregulation of CXCR3 expression, we considered the role of IFN-γ because it promotes the expression of CXCR3 chemokine ligands and because IL-12 may be acting indirectly through IFN-γ production by non-CD8+ T cells. To first test if blocking IFN-γ alone can prevent the downregulation of CXCR3 on CD8+ T cells in vivo, we neutralized IFN-γ late (D3) during CPS vaccination. We found that in vivo blockade of IFN-γ as late as D3 disrupts the downregulation of CXCR3 expression in KLRG1-expressing tgd057-specific CD8+ T cells (Figure 6A) without attenuating KLRG1 upregulation or IFN-γ production (Figure 6B,C). Figure 6 Download asset Open asset Neutralization of IFN-γ is sufficient to prevent CXCR3 downregulation on effector CD8+ T cell precursors. Anti-IFN-γ mAb was administered i.p. on D3 post-CPS vaccination. Mice were sacrificed on D5 post-CPS vaccination and tgd057-specific CD8+ T cells were analyzed for (A) CXCR3 expression, (B) CD62L and KLRG1 surface expression on tgd057-specific CD8+ T cells and (C) IFN-γ production post in vitro CPS restimulation in tgd057-specific CD8+ T cells. Data are from 2 experiments consisting of 3–5 mice per group per experiment. Mean ± SEM, paired t test and Holm-Sidak post-hoc test, ***p < 0.001. https://doi.org/10.7554/eLife.09017.010 The fact that blockade of IFN-γ can also prevent the downregulation of CXCR3 on KLRG1+ CD8+ T cells to the same extent as neutralization of IL-12 does, prompted us to ask if IFN-γ can suppress CXCR3 on CD8+ T cells similarly to IL-12. We exploited the fact that IL-12 deficient F3 stage KLRG1+ CD8+ T cells fail to decrease CXCR3 and used these cells as an in vitro model system to directly interrogate what factors can cause CXCR3 downregulation. Upon exposure and binding to its chemokine ligands, surface expressed CXCR3 is rapidly downregulated, endocytosed and degraded, but is quickly replenished by newly synthesized protein (Meiser et al., 2008). We therefore used CXCL10 exposure to ‘strip’ pre-existing CXCR3 from D7 CPS vaccinated IL-12p35 deficient splenocytes and determined whether IFN-γ or IL-12 can maintain the expression of CXCR3 on CD8+ T cells at a low level. As expected, the addition of IP-10 rapidly decreases the expression of CXCR3 on CD8+ T cells lacking IL-12 signals (Figure 7A, left), but importantly, the addition of IFN-γ and not the addition of CXCL10 alone, maintains the low levels of CXCR3 on these CD8+ T cells, to a similar extent as the addition of IL-12 (Figure 7A, right). This result suggests that IFN-γ may be acting directly on the developing CD8+ T cell to suppress CXCR3 expression downstream of IL-12. Consistent with this notion, addition of IFN-γ alone to highly purified CD8α+ TCRβ+ T cells from D7 CPS vaccinated IL-12p35 deficient spleens still maintains the low levels of CXCR3 expression (Figure 7B) without the need for" @default.
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W2987267675 title "Author response: An extrafollicular pathway for the generation of effector CD8+ T cells driven by the proinflammatory cytokine, IL-12" @default.
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