Infection with the intracellular protozoan parasite Leishmania major induces a state of concomitant immunity wherein secondary immunity is dependent upon the persistence of the original pathogen. Our laboratory has described two populations of Leishmania-induced CD4+ T cells that contribute to immunity: CD62Lhigh central memory T (TCM) cells and CD62Llow effector T cells. To determine whether the prosurvival cytokine IL-7 contributes to maintaining these T cells, we examined expression of the IL7R on CD4+ T cells activated during L. major infection. We found that TCM cells present in chronically infected mice expressed high levels of the IL7R. However, in addition to the expression of the IL7R by TCM cells, CD62Llow cells responding to L. major infection expressed the IL7R. Additional experiments revealed that a large percentage of the IL7RhighCD62Llow cells were Th1 cells, based on transcription at the IFN-γ locus and up-regulation of the Th1-promoting transcription factor T-bet. The up-regulation of T-bet did not prevent IL7R expression by L. major-responding CD4+ T cells, nor did the absence of T-bet result in increased IL7R expression. Finally, blockade of IL7R signaling decreased the number of T-bet+CD4+ T cells, reduced IFN-γ production, and inhibited delayed-type hypersensitivity responses in immune mice challenged with L. major, indicating that IL7R signaling contributes to the maintenance of Th1 effector cells. Thus, both TCM and Th1 effector cells can express the IL7R during chronic L. major infection, which provides a potential means for their long-term survival in addition to the presence of persisting parasites.
Concomitant immunity refers to an immune state in which resistance to a pathogen is dependent upon the persistence of the original pathogen. Such is the case following the infection of C57BL/6 mice with the intracellular protozoan parasite Leishmania major (1). During a primary infection, IL-12 promotes the generation of a protective Th1 response that leads to a reduction in the parasite burden over the course of 10–12 wk and long-term immunity, although parasites persist in these animals for the lifetime of the host (2, 3). Similarly, individuals who have been naturally infected or immunized by a procedure referred to as leishmanization, where small numbers of live parasites are injected at an inconspicuous location, obtain life-long immunity to reinfection, but are believed to continue to harbor low levels of parasites (4). Although our extensive knowledge of the immune response to L. major indicates that Th1 effector cells are critical for immunity (5), how they are maintained once the disease is resolved is not well understood.
Our laboratory has shown that two populations of CD4+ T cells are present in mice that have resolved an infection with L. major (6). One is a population of effector T (TEFF)3 cells that express low levels of the lymph node (LN)-homing molecule CD62L and rapidly produce IFN-γ following Ag restimulation. Once generated in the LNs, these TEFF cells migrate to the site of infection. We also found that central memory T (TCM) cells, which express CD62L and circulate through lymphoid tissues, are present in chronically infected mice. These cells are long lived, as they are able to survive in the absence of parasites but can give rise to CD62Llow TEFF cells following secondary challenge (6). We have also shown that the ability of TCM cells to differentiate into IFN-γ-producing Th1 effector cells following transfer is dependent upon IL-12 production by the recipient (7). Therefore, one model to account for the maintenance of Th1 effector cells in mice that have resolved a primary infection with L. major might be that persistent parasites continually activate some of the long-lived TCM cells to differentiate into short-lived TEFF cells that then mediate concomitant immunity (5).
One way to monitor the potential for the long-term survival of T cells is to assess their expression of the IL7R (CD127). T cells are dependent upon IL-7 for their survival, because signaling through the IL7R promotes cell survival via the up-regulation of antiapoptotic proteins such as Bcl-2 and the glucose transporter Glut1 (8, 9, 10, 11, 12). The IL7R is expressed on naive T cells and is down-regulated following TCR engagement (8, 9, 13, 14, 15, 16, 17, 18, 19, 20, 21). T cells then have the capacity to up-regulate the IL7R for their continued maintenance (22, 23, 24). However, when T cells are continuously stimulated they fail to re-express the IL7R (15). For example, in lymphocytic choriomeningitis virus clone 13 and HIV infections, virus-specific CD8+ T cells that persist during chronic infection exhibit low levels of IL7R expression in addition to impaired function (25, 26, 27, 28). In the case of leishmaniasis, one might similarly predict that the pool of Th1 effector cells maintained by continual stimulation would also express low levels of the IL7R.
To determine the potential for long-term survival of the CD4+ T cells activated by L. major infection, we have characterized their expression of the IL7R. We found that TCM cells express high levels of the IL7R during chronic infection, which is consistent with their ability to survive long-term. Moreover, a population of CD4+ IL7Rhigh T cells emerged within the first 2 wk despite the continued presence of parasites. Additional studies revealed that both CD62Lhigh and CD62Llow CD4+ T cells expressed the IL7R. The presence of Leishmania-responsive CD62Llow T cells expressing high levels of the IL7R was unexpected and led us to further characterize these cells. By using IFN-γ reporter mice, we found that almost half of the Th1 cells expressed the IL7R. We also showed that the ability of CD62Llow CD4+ T cells to express the IL7R was not inhibited by the up-regulation of the Th1-promoting transcription factor T-bet, nor did the absence of T-bet promote elevated IL7R expression. Lastly, we observed a significant decrease in the number of T-bet+CD4+ T cells in immune mice treated with Abs that block IL7R signaling, as well as a reduction in Leishmania-specific IFN-γ production and delayed-type hypersensitivity (DTH) responses. Taken together, these results indicate that concomitant immunity to L. major may be maintained not only by a population of TCM cells that can differentiate into short-lived effector cells, but also by a pool of resting Th1 effector cells with the ability to access IL-7 to promote their survival.
Materials and Methods
C57BL/6J, B6.SJL-Ptprca Pepcb/BoyJ (CD45.1), and B6.129S6-Tbx21tm1Glm/J (T-bet knockout (KO)) mice were purchased from The Jackson Laboratory or the National Cancer Institute (Fredricksburg, MD). IFN-γ reporter (Yeti) mice were provided by M. Mohrs (Trudeau Institute, Saranac Lake, NY) (29). Animals were maintained in a pathogen-free environment, and experiments were performed in accordance with the guidelines of the University of Pennsylvania Institutional Animal Care and Use Committee.
Parasites and infections
L. major (MHOM/IL/80/Friedlin) parasites were grown in Schneider’s insect medium supplemented with 20% heat-inactivated FBS and 2 mM glutamine. Infectious stage metacyclic parasites were enriched using density gradient centrifugation (30). Mice were infected in the hind footpad or the ear dermis with 1–2 × 106 parasites with similar results. For IL-12 treatments, IL-12 (0.5 μg) was administered with the parasites and additional IL-12 treatments were given on days 3 and 7 i.p. For secondary challenge, mice were infected in the contralateral footpad. DTH was determined by measuring footpad swelling 48 h postchallenge with a digital caliper (Mitutoyo) and subtracting the average size of the footpad before infection. To quantify parasite burden in the lesion, single cell suspensions of parasites were prepared from the tissue and plated in serial 10-fold dilutions. Each sample was plated in quadruplicate and the mean of the negative log parasite titer was determined after 7 days of culture at 26°C.
CD4+ T cells were enriched from the lymphoid tissue of donor mice before transfer either by using MACS (Miltenyi Biotec) or a FACSAria flow cytometer (BD Biosciences). All cells (except those isolated from Yeti mice) were CFSE labeled (1.25 μM) (Molecular Probes), and 5 × 106 cells were transferred to congenic recipients unless noted otherwise as in the Fig. 1 and Fig. 3 legends.
In vivo Ab treatment
Mice were treated with 200 μg of A7R34 (anti-IL7R; Bio X Cell) i.p. every 2–3 days over a 2-wk period. This treatment resulted in a loss of IL-7-dependent B cell precursors in the bone marrow and an inability to stain for surface expression of the IL7R (data not shown).
The following Abs used to detect cell surface markers were purchased from eBioscience: CD4, CD45.2, CD45.1, CD44, CD127 (IL7Rα) (PE or allophycocyanin only), and CD62L (PE, allophycocyanin, or PerCP-Cy5.5 only). Before intracellular cytokine staining, cells were stimulated with PMA, ionomycin, and brefeldin A for 4 h in vitro and fixed with 2% paraformaldehyde in PBS. For intracellular detection of cytokines and T-bet, cells were permeabilized with 0.2% saponin and stained with IL-2-allophycocyanin and IFN-γ-PE-Cy7 or T-bet-Alexa Fluor 647 (eBioscience). Data were acquired on an LSR II or a FACSCanto flow cytometer (BD Biosciences). Analysis was performed using FlowJo software (Tree Star). For all samples, gating was established using a combination of isotype and fluorescence-minus-one controls (31).
Ag-specific cytokine production
Splenocytes from naive and L. major-infected mice were plated at 4 × 106 cells/well of a 24-well plate and cultured with medium or freeze-thawed parasite Ag (FTAg) for 72 h. FTAg was prepared from stationary phase promastigotes subjected to four cycles of freezing at −150°C and thawing at 37°C. Detection of IFN-γ in the supernatant was determined by sandwich ELISA.
Statistical analysis was performed using the Student’s t test with Prism (GraphPad Software), and a value of p < 0.05 was considered statistically significant.
TCM cells generated during L. major infection express the IL7R
We previously characterized the T cells responding to L. major infection by phenotyping proliferating T cells following the adoptive transfer of CFSE-labeled polyclonal CD4+ T cells to naive congenic mice that were subsequently infected with L. major (6). We found that a population of TCM cells contributed to immunity to reinfection and could be maintained in the absence of persistent parasites. To determine whether those TCM cells could use IL-7 to survive, we characterized the IL7R expression of TCM cells in chronically infected mice. CD4+ T cells purified from C57BL/6 mice that had resolved a primary infection (referred to as immune mice) were CFSE labeled and transferred to naive congenic recipients. Recipient mice were infected the following day and sacrificed 2 wk following challenge. Although some cells from immune mice that proliferated in response to infection expressed low levels of CD62L, a T cell population expressing CD62L was maintained that we have previously identified as TCM cells (Fig. 1 A) (6). Many of these CD62Lhigh cells expressed the IL7R at a level equivalent to that of resting CD4+ T cells from uninfected mice, suggesting that TCM cells have the ability to maintain or re-express the IL7R following secondary challenge.
To demonstrate that these IL7R-expressing TCM cells were able to give rise to cytokine-producing TEFF cells, CD4+CD62LhighIL7Rhigh cells from immune or naive mice were transferred to naive recipients and subsequently infected as described above. Polyclonal CD62LhighIL7Rhigh cells from both naive and immune mice contained a population of Leishmania-specific cells that proliferated in response to infection and, as expected, the percentage of cells that were CFSEdim was significantly greater when the cells were derived from immune donor cells (Fig. 1,B) (6). This difference most likely reflects an increase in the total number of Leishmania-specific cells within the donor pool of immune mice. Although cells from both categories of donor mice gave rise to IL-2-producing cells, CD62LhighIL7Rhigh cells from immune mice gave rise to a significantly larger population of IFN-γ-producing cells than their naive counterparts (Fig. 1 C). These results are consistent with our previous findings demonstrating that TCM cells can proliferate, lose expression of CD62L, and become IFN-γ producers following reactivation (6). Thus, one way that concomitant immunity can be maintained is by sustaining a pool of TCM cells that can become Th1 cells as needed.
Both CD62Llow and CD62Lhigh Leishmania-activated CD4+ T cells can express the IL7R during an ongoing infection
A stereotypical response of T cells to TCR stimulation is the rapid down-regulation of the IL7R and, in the case of acute infections where the pathogen is cleared, this down-regulation is transient at the population level (8, 9, 13, 14, 16, 17, 18, 19, 20, 24). Although our data indicate that TCM cells express the IL7R in L. major immune mice, TCM cells represent a minority of the T cells responding to infection, and we predicted that a large percentage of the responding T cells would fail to re-express the IL7R due to continuous stimulation by persistent parasites. To characterize IL7R expression on CD4+ T cells following in vivo infection with L. major, we first looked at the Ag-experienced CD4+ T cells present in the LN draining the site of infection (dLN) by staining cells for the activation-induced adhesion molecule CD44. As an internal control, we compared the level of IL7R expression on the CD44high cells to that of the naive CD44low cells, which are known to be IL7Rhigh. We infected C57BL/6 mice with L. major and isolated the dLN at the indicated time points. At 1 wk postinfection (pi) we observed a global down-regulation of the IL7R on the pool of CD44high cells (Fig. 2 A). However, as early as 2 wk pi, when the parasite burden is still increasing, a population of IL7Rhigh cells was evident and gradually came to dominate the pool of activated cells over the next several weeks. These results illustrate that activated CD4+ T cells in the dLN down-regulate the IL7R early following infection, similar to the findings in other viral and bacterial infection models. However, they begin to express increased levels of the IL7R soon after infection despite the ongoing inflammatory response and parasite burden.
Because we found that TCM cells express the IL7R, we initially hypothesized that the IL7Rhigh cells dominant in the CD44high population in the dLN as the infection progresses were developing TCM cells. To ascertain whether this was the case, we examined CD62L expression on the IL7R-expressing cells seen at 2, 4, and 6 wk pi (Fig. 2 B). The CD44lowIL7Rhigh naive cells were largely CD62Lhigh, and within the CD44highIL7Rhigh cells there was a population of CD62Lhigh cells that most likely represents the Leishmania-specific TCM population. However, there was also a large percentage of CD44highIL7Rhigh cells that were not expressing CD62L both at 2 wk and at later time points. Thus, our results not only indicate that the IL7R is down-regulated during the first few weeks of infection and relatively soon thereafter re-expressed, but that both TCM cells (CD62Lhigh) and CD62Llow cells express the IL7R.
Th1-polarized cells have an IL7Rhigh phenotype
Our data indicate that in addition to a population of TCM cells, a population of CD62Llow CD4+ T cells activated by L. major infection express the IL7R as early as 2 wk after infection. To determine whether these CD62Llow IL7Rhigh T cells were effector cells, we asked whether they were able to make IFN-γ. We isolated the dLN from infected animals as in Fig. 2 to assess the capacity of the activated cells to produce IFN-γ ex vivo. In comparison to the LN of an uninfected mouse, there was an increase in both the percent CD44high and also the percent IFN-γ+CD4+ T cells following L. major infection (Fig. 3,A). We then compared IL7R expression on the activated (CD44high) cells vs the naive (CD44low) cells of infected mice. We once again observed both IL7Rhigh and IL7Rlow cells 2 wk pi, but we were surprised to find a percentage of IL7Rhigh cells that were capable of producing IFN-γ (Fig. 3,B). As a second approach to address this question without having to restimulate the cells in vitro, which can influence IL7R expression, we used the Yeti IFN-γ reporter mouse (29). By using this approach, we could also ensure that all eYFP/IFN-γ expression was the result of L. major infection. We enriched for naive T cells by sorting for CD4+CD44loweYFP− cells from Yeti mice and transferred the cells to congenic recipients before infection with L. major. Two weeks following infection, we assessed CD44 up-regulation and eYFP/IFN-γ expression within the donor cell population in the dLN. Similar to the dilution of CFSE in the above experiments, little CD44 up-regulation was observed in the donor T cell population of uninfected mice over the 2-wk period. However, there was a large increase in the percentage and total number of CD44high cells in L. major-infected mice (Fig. 3,C). Analysis of the CD44high cells from infected mice showed at least four distinct populations of cells, including one group of cells that expressed eYFP and also expressed high levels of the IL7R (Fig. 3 D). This indicates that a population of Th1-polarized cells express the IL7R relatively soon after infection.
L. major-specific CD4+ T cells coexpress T-bet and the IL7R
As an alternative approach to test for the polarization of IL7R-expressing cells following L. major infection, we asked whether the Th1-promoting transcription factor T-bet, which is required for IFN-γ production by CD4+ T cells, was up-regulated in IL7RhighCD4+ T cells (32). We analyzed the response of both naive and immune CD4+ T cells to L. major by transferring CFSE-labeled cells into naive congenic recipients that were subsequently challenged with L. major as in Fig. 1. After 2 wk, we sacrificed the mice and analyzed the donor T cells within the dLN. We gated on the CFSEdim cells and found that CD62Llow cells were both IL7Rhigh and IL7Rlow (Fig. 4,A). This was consistent with our finding that the IL7R was expressed on CD44high cells directly ex vivo following infection (Figs. 2,B and 3,B). Next, by analyzing both the CD62Lhigh and CD62Llow cells for expression of T-bet and the IL7R, we observed a small but definable population of cells that coexpressed T-bet and the IL7R 2 wk following L. major infection (Fig. 4,A). These results were somewhat surprising, as recent studies have suggested that the up-regulation of T-bet induces a down-regulation of the IL7R (33, 34). One possible explanation for this discrepancy was that the up-regulation of T-bet that we observed following a primary L. major infection was not high enough to induce IL7R down-regulation (34). However, cells from immune mice that have resolved a primary infection also coexpressed T-bet and the IL7R following a secondary L. major challenge (Fig. 4,B). To further test whether enhanced T-bet expression might down-regulate the IL7R, we treated mice with IL-12 at the time of infection to induce a strong polarization of the primary response (Fig. 4,C). Under these conditions, there was a significant increase in both the percentage and total number of IL7Rhigh T-bet-expressing cells (Fig. 4, C and D). Therefore, we found that T-bet up-regulation, even under IL-12-induced inflammatory conditions, was not associated with the down-regulation of the IL7R. Together, these findings suggest that Th1 cells generated during L. major infection have the potential to use IL-7-IL7R signaling to promote their long-term survival rather than face the eminent cell death typically associated with the acquisition of effector function.
IL7R down-regulation is independent of T-bet expression
The experiments described above demonstrate that in the presence of physiological levels of T-bet, the IL7R can be expressed by CD4+ T cells. However, this did not exclude the possibility that higher levels of the IL7R might be expressed in the absence of T-bet. To test this possibility, we performed adoptive transfer experiments as described above. CD4+ T cells from either wild-type (WT) or T-bet KO donors were CFSE labeled, transferred to naive congenic recipients, and infected with L. major following transfer. At 2 wk pi proliferation was not significantly different between WT- and KO-derived CD4+ T cells, but in contrast to the WT donor cells, none of the proliferating T-bet KO cells made IFN-γ (Fig. 5,A). IL7R expression was identical in both the WT- and KO-derived cells, suggesting not only that the IL7R can be expressed concurrently with T-bet in L. major infection, but also that no increase in IL7R expression is evident when T-bet is absent (Fig. 5, B and C). Taken together, these data suggest that T-bet has little influence on the IL7R expression of CD4+ T cells responding to an L. major infection.
IL-7 maintains a population of CD4+ effector T cells in immune mice
Our data suggest that a population of Th1 effector cells may be dependent upon IL-7 for their survival. To test this hypothesis, we treated immune mice for 2 wk with Abs that block signaling through the IL7R (anti-IL7R; A7R34). These mice exhibited a decrease in the total T cell pool as well as a decrease in both the naive CD44low and activated CD44high CD4+ T cell populations (Fig. 6,A). Within the population of activated T cells, there was a significant decrease in the CD62Lhigh TCM cells and the T-bet+ Th1 effector cells (Fig. 6, B and C). To determine whether the absence of IL7R signaling had a negative effect on the function of Th1 effector cells, we first assayed for the production of parasite-specific IFN-γ following in vitro restimulation with FTAg. When we normalized for the overall reduction in spleen size, we found a significant decrease in the amount of Leishmania-specific IFN-γ produced per spleen of those mice treated with the anti-IL7R mAb (Fig. 6,D). To determine whether there was a decrease in Th1 effector cell function in vivo, we measured the DTH response in immune mice where IL-7R signaling was blocked and found a significant reduction in the DTH response of treated mice (Fig. 6,E). As expected, because not all of the CD62Llow or IFN-γ+ CD4+ T cells expressed the IL-7R, the response was not totally ablated. In addition, there was no reduction in the ability of treated mice to mediate resistance to a secondary challenge (Fig. 6 F). This was consistent not only with the presence of IL7R− Th1 effector cells but also with previous data suggesting that only a low number of TEFF cells may be required to mediate protection (35). Nevertheless, these data indicate that, in agreement with its expression of the IL7R, a population of Th1 effector cells uses IL-7 to survive during L. major infection.
Concomitant immunity is responsible for the substantial resistance to reinfection seen following the resolution of a primary infection with Leishmania major, but how this immunity is maintained is not well understood. Our previous data indicate that there is a pool of Ag-reactive CD4+CD62Lhigh TCM cells in immune mice that contribute to concomitant immunity (6). In this study we show that these CD4+ TCM cells express high levels of the IL7R and are able to give rise to IFN-γ-producing Th1 cells after secondary challenge. Because IL-7 and IL7R signaling are required for T cell survival (8, 9, 10), these results are consistent with the ability of TCM cells to persist long term in the absence of parasites (6). In contrast, because Th1 effector cells generated following L. major infection are believed to be short lived (6, 36, 37), we anticipated that Th1 cells generated during infection would not express the IL7R. This result would be consistent with studies in other chronic infections, where the presence of persisting pathogens is associated with Ag-specific T cells that do not express the IL7R (15, 25, 26, 27, 28). However, we identified a population of infection-induced CD4+ cells in L. major-infected mice that were CD62Llow and expressed high levels of the IL7R. We characterized these T cells and found that a percentage of them were Th1 cells, based on their ability to make IFN-γ and express T-bet. Finally, by treating infected mice with blocking Abs against the IL7R, we found that both TCM and Th1 effector cells are reduced in number in the absence of IL7R signaling, and this reduction correlated with a reduction in the magnitude of the immune response as measured by IFN-γ production and a DTH response. Thus, these results describe a population of CD4+IL7RhighCD62LlowT-bet+ T cells that we believe can not only contribute to concomitant immunity, but have the potential to be maintained by IL7R signaling.
One of the principal roles of IL-7 is to promote the survival of both naive and memory T cells, and expression of the IL7R following infection is thought to contribute to the dynamic changes in the quantitative T cell response during the expansion and contraction phases of an immune response (8, 38). For example, at the peak of the expansion phase to an acute viral or bacterial infection, the majority of the Ag-specific cells are IL7Rlow and are destined to be a short-lived population that can execute effector function and need not be maintained after the pathogen is cleared (8, 17, 18, 19, 21). In contrast, after contraction occurs the remaining Ag-specific T cells express high levels of the IL7R, which promotes their continued maintenance. Although these results suggest that IL7R expression plays a role in T cell contraction, recent studies have shown that while expression of the IL7R may be necessary for T cell survival, it does not guarantee the long-term survival of T cells (39, 40). This may in part be due to the fact that the amount of IL-7 available to cells is limited. However, our findings that both TCM and Th1 cells express the IL7R suggest that both cell types have the potential to be maintained by IL-7. In support of this hypothesis, we found a global decrease in the CD4+ T cell populations of immune mice subjected to IL7R-signaling blockade, which correlated with a decrease in IFN-γ production and the DTH response to a secondary challenge. A practical consequence of IL7R expression on these infection-induced T cell populations is that they could be expanded by exogenous treatment with IL-7, which has been shown to enhance the maintenance of memory T cells in other systems (16, 41, 42, 43).
One of the unexpected findings of this study was that despite the presence of persistent L. major parasites, immune mice contain L. major-responsive CD4+ T cells that express the IL7R. This is in contrast to certain chronic viral infections such as lymphocytic choriomeningitis virus clone 13 and HIV, where T cells become functionally exhausted, are impaired in their ability to survive, proliferate, or secrete effector cytokines, and fail to express the IL7R (25, 26, 27, 28, 44, 45, 46). However, the maintenance of IL7R-expressing T cells under conditions of concomitant immunity is not unique to L. major. For example, following infection with the related protozoan parasite Trypanosoma cruzi, where parasites persist at low levels in multiple tissues, a small percentage of T cells express the IL7R (23, 47, 48). Similarly, latent γ-herpesvirus persists for the life of the host under T cell-mediated control, and a percentage of the virus-specific CD8+ T cells express high levels of the IL7R and provide functional secondary immunity (49, 50). The factors that determine whether chronic infections lead to the generation of functional IL7R-expressing memory T cells vs an exhausted T cell population that fails to express the IL7R are unknown. However, a likely difference may be the degree to which the Ag-responsive T cells are continuously stimulated (51). The fact that the IL7R is down-regulated upon TCR engagement suggests that many of the responding T cells present in L. major-infected mice were not recently stimulated through their TCR. Indeed, although L. major-infected mice harbor parasites following resolution of the disease, the number of parasites is extremely low, and many Ag-specific T cells may not be coming in contact with parasites. Moreover, the ability of T cells to be stimulated by parasites may be even further reduced due to the fact that L. major-infected macrophages have been shown to function poorly as APCs compared with noninfected cells (52). Thus, our data may suggest that many of the parasites are sequestered in a way that mimics pathogen clearance, allowing for the emergence of a population of IL-7R-expressing Th1 cells.
The optimal Th1 immune response would include the generation of sufficient effector T cells to control the infection while also generating memory T cells. One way that this might occur is by asymmetrical cell division at the initiation of the infection, whereby one daughter cell becomes a TEFF cell while the other adopts memory characteristics (53). This model could explain how TCM cells generated in leishmanial infection develop. However, the factors promoting the strongest Th1 response might be predicted to preclude the development of Th1 effector memory cells. In addition to the fact that IFN-γ and IL-12 have detrimental effects on T cell survival and/or memory T cell development (54, 55, 56, 57), it has recently been demonstrated that the transcription factor required for commitment to the Th1 lineage, T-bet, inhibits IL7R expression (33, 34). Thus, in CD8+ T cells, high levels of T-bet were associated with low levels of IL7R expression and the generation of short-lived effector cells; similarly, overexpression of T-bet inhibited IL7R expression in CD4+ T cells (33, 34). Nevertheless, we found that CD4+ T cells that have up-regulated T-bet can also express high levels of the IL7R during both a primary and secondary challenge with L. major. Moreover, even when we administered IL-12 during infection to further boost T-bet expression, IL7R expression was maintained. Why we failed to observe an inverse correlation between T-bet and IL7R is unknown, although one possibility is that IL7R expression is regulated differently in CD4+ and CD8+ T cells (58, 59). However, regardless of the reasons, our data clearly indicate that T-bet expression does not prohibit the development of IL7R-expressing Th1 cells. Thus, while these and previous studies indicate that CD4+ TCM cells in L. major infections may develop before the effector stage (7), our current results are consistent with studies indicating that TEFF cells can also develop the capacity to survive long term (22, 23, 60, 61).
There is no successful vaccine for human leishmaniasis even though it is well known that individuals who have resolved a primary infection are resistant to reinfection. Indeed, for centuries people in regions endemic for cutaneous leishmaniasis have protected themselves from disfiguring lesions by intentionally infecting themselves with live parasites in an inconspicuous site, a process known as leishmanization (4). The goal for a leishmanial vaccine is to mimic the quality of protective immunity elicited by infection with WT parasites without having to resort to infections with virulent parasites; defining the T cells involved in this protection is one step in that direction. In our previous studies we showed that mice immunized with a thymidine auxotrophic L. major lacking dihydrofolate reductase-thymidylate synthase (dhfr-ts−) generated a long-lived CD4+ TCM cell population and were protected against L. major challenge (6, 62). However, the resistance was not as effective as that associated with the resolution of a primary infection with virulent L. major, which was attributed to the absence of CD4+CD62Llow Th1 effector cells in the dhfr-ts−-immunized mice (6). We hypothesized that CD62Llow cells required persisting parasites to be maintained, although given our new results it is tempting to speculate that the CD4+CD62LlowIL7RhighT-bet+ T cell population we have identified in immune mice fails to be generated following immunization with dhfr-ts−, an issue we are currently investigating. Nevertheless, a key to developing a better vaccine may require increasing the frequency of IL7RhighT-bet+ T cells over a yet undefined threshold (63). Additionally, while we have focused on IL7R expression in this study, other characteristics of the CD4+CD62LlowT-bet+ T cell population may be important, such as the ability of these T cells to produce multiple cytokines such as IL-2, TNF-α, and IFN-γ (64, 65). The challenge of future studies is to identify various adjuvants and boosting regimes, including the administration of IL-7, that enhance the generation of both TCM cells and CD4+CD62LlowIL7RhighT-bet+ cells and thus promote more robust immunity from a vaccine.
In summary, we have demonstrated for the first time that the IL7R is expressed on several populations of CD4+ T cells over the course of infection with L. major. Expression of the IL7R on the population of TCM cells that is maintained in chronically infected mice is consistent with previous studies showing that TCM cells can use IL-7 to promote their long-term survival. However, in the current study we show that the presence of low levels of L. major parasites does not hinder the ability of these cells to express the IL7R. Moreover, the emergence of an IL7R-expressing population of Th1-polarized effector cells was observed early following infection, during the period of high parasite burden concurrent with the onset of the inflammatory response. Our results show that Th1 cells with the potential to signal through the IL7R contribute to concomitant immunity and suggest that the elicitation of this population may improve the efficacy of future vaccines to protect against cutaneous leishmaniasis.
We thank Markus Mohrs and Ed Pearce for providing the Yeti mice and the Penn Flow Cytometry & Cell Sorting Facility for technical assistance.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by National Institutes of Health Grant AI35914 (to P.S.).
Abbreviations used in this paper: TEFF, effector T; TCM, central memory T; DTH, delayed-type hypersensitivity; eYFP, enhanced yellow fluorescence protein; FTAg, freeze-thawed Ag; LN, lymph node; dLN, draining lymph node; pi, postinfection; WT, wild type; KO, knockout; dhfr-ts, dihydrofolate reductase-thymidylate synthase.