Leishmania major with Priming of Naive T Cells during Infection ...

The Journal of Immunology

Antigen-Experienced T Cells Limit the Priming of Naive T Cells during Infection with Leishmania major1 Peter M. Gray,* Steven L. Reiner,† Deborah F. Smith,‡ Paul M. Kaye,‡ and Phillip Scott2* One mechanism to control immune responses following infection is to rapidly down-regulate Ag presentation, which has been observed in acute viral and bacterial infections. In this study, we describe experiments designed to address whether Ag presentation is decreased after an initial response to Leishmania major. Naive ␣␤-Leishmania-specific (ABLE) TCR transgenic T cells were adoptively transferred into mice at various times after L. major infection to determine the duration of presentation of parasite-derived Ags. ABLE T cells responded vigorously at the initiation of infection, but the ability to prime these cells quickly diminished, independent of IL-10, regulatory T cells, or Ag load. However, Ag-experienced clonal and polyclonal T cell populations could respond, indicating that the diminution in naive ABLE cell responses was not due to lack of Ag presentation. Because naive T cell priming could be restored by removal of the endogenous T cell population, or adoptive transfer of Ag-pulsed dendritic cells, it appears that T cells that have previously encountered Ag during infection compete with naive Ag-specific T cells. These results suggest that during L. major infection Ag-experienced T cells, rather than naive T cells, may be primarily responsible for sustaining the immune response. The Journal of Immunology, 2006, 177: 925–933.

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ontrol of the intracellular protozoan Leishmania major requires a sustained T cell response, because in contrast to many acute viral and bacterial infections leishmaniasis is associated with a high parasite burden for many weeks. Moreover, after resolution of disease low numbers of parasites persist indefinitely, thus requiring the maintenance of T cell-dependent immunity (1). We found that both central memory and effector CD4⫹ T cells mediate long-term resistance to leishmaniasis, and that L. major-specific central memory T cells could be maintained in the absence of parasites, while the effector T cell population could not (2). The requirement for parasite persistence to maintain effector T cells would suggest that Ag presentation continually occurs during infection. Whether naive T cells contribute to the maintenance of the protective response is unknown, although this would also require continued Ag presentation in persistently infected animals. To date, the issue of how long Ag presentation occurs in leishmaniasis has not been directly investigated. Several recent studies have used adoptive transfer of naive TCR transgenic T cells into previously infected mice as an indicator of ongoing Ag presentation. For example, Ag presentation following infection with Listeria monocytogenes was found to be extremely short-lived (3–5). Thus, within several days, naive Listeria-specific TCR transgenic T cells were unable to proliferate when transferred

*Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104; †Abramson Family Cancer Research Institute and Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104; and ‡ Immunology and Infection Unit, Department of Biology, University of York and Hull York Medical School, York, United Kingdom Received for publication November 29, 2005. Accepted for publication April 20, 2006. 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. 1 This work was supported by National Institutes of Health Grants AI 35914 (to P.S.) and AI 42370 (to S.L.R.), and by grants from the Wellcome Trust (to D.F.S. and P.M.K.) and the British Medical Research Council (to P.M.K.). P.M.G. was supported by National Research Training Award Grant AI 07518. 2 Address correspondence and reprint requests to Dr. Phillip Scott, Department of Pathobiology, School of Veterinary Medicine, 3800 Spruce Street, Philadelphia, PA 19104. E-mail address: [email protected]

Copyright © 2006 by The American Association of Immunologists, Inc.

into Listeria-infected mice, despite the continued presence of bacteria. Similarly, the ability of naive T cells to respond to malaria was lost within 3 days of infection (6). In contrast, another recent study found that following influenza infections, naive TCR transgenic T cells were able to proliferate if transferred into infected animals even after the virus was eliminated, suggesting that Ag presentation was maintained throughout the course of infection and beyond (7). Furthermore, the continued recruitment of naive T cells into the viral response during infection was associated with an increased number of memory T cells, suggesting that recruitment of naive T cells may not only be important in sustaining an ongoing immune response, but important for memory T cell development. Taken together, these divergent results suggest that pathogen-specific characteristics may influence whether Ag presentation is ongoing after infection. Infections induce several immunoregulatory mechanisms that are likely to influence the ability of both previously activated and naive T cells to respond. For example, L. major infection induces the production of IL-10 by macrophages and T cells, in particular regulatory T (Treg)3 cells, which can decrease Ag presentation (8, 9). Another factor that may influence how well naive T cells respond once a primary response has been induced is the degree of competition that occurs between naive and primed T cells. Effector and/or memory T cells differ from naive T cells with respect to faster kinetics in their response to Ag (10, 11), decreased requirement for costimulation (12, 13), their capacity to mediate effector functions (14), and their increased sensitivity to Ag compared with naive cells (12, 15–17). All of these characteristics might make previously activated T cells better able to respond during the course of a L. major infection than naive cells. Indeed, several studies of homeostatic induced proliferation indicate that the responsiveness of naive T cells is significantly influenced by the presence of either additional naive or activated T cells (18 –20). In contrast, because naive T cells migrate through lymph nodes, while effector T cells home to sites of infection, it might be argued that there is little opportunity for competition to occur between 3 Abbreviations used in this paper: Treg, regulatory T; ABLE, ␣␤-Leishmania specific; DC, dendritic cell; dLN, draining lymph node; SLA, soluble leishmanial Ag.

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Ag PRESENTATION DURING Leishmania major INFECTION

naive and effector T cells. Competition might occur in the lymph nodes, however, between naive and central memory or lymph node-homing Ag-experienced T cells (2). In this study, we used Leishmania-specific TCR transgenic mice, termed ␣␤-Leishmania-specific (ABLE) mice, to determine how responses of naive T cells are influenced by an ongoing L. major infection (21). This TCR, which was originally derived from a protective T cell clone (22), recognizes the leishmanial Ag, termed LACK (23). In BALB/c mice, a robust LACK-specific response is initiated shortly after infection with L. major, leading to a 100-fold expansion of LACK-reactive T cells by 4 days (24). By adoptively transferring naive ABLE T cells into mice at various times after L. major, we found that the ability of naive Leishmaniaspecific T cells to proliferate rapidly diminishes following infection, independent of IL-10, Treg, or Ag load. In contrast, previously activated T cells could respond, indicating that the loss of reactivity was not due to lack of Ag presentation. Moreover, after adoptive transfer naive T cells did proliferate in previously infected animals if the endogenous responding T cell population was absent. Taken together, these data indicate that Ag presentation continues after infection, but that T cells that have previously been activated by infection compete with naive Ag-specific T cells. Thus, our results suggest that during L. major infection Ag-experienced T cells, rather than naive T cells, may be primarily responsible for sustaining the immune response.

ferred via the retro-orbital plexus into recipient mice at day 1, 7, 14, or 21 following L. major infection. Ag-experienced ABLE and DO11.10 T cells were generated, as previously described, with minor modifications (31). Briefly, ABLE or DO11.10 lymphocytes were stimulated in vitro at 8 ⫻ 106 cells/ml in 24-well plates with 1 ␮g/ml LACK or OVA peptide, respectively, in the presence of 5 ng/ml rIL-12 and 10 ␮g/ml anti-IL-4 for 4 days. After stimulation, cells were washed three times with PBS and then replated in fresh medium at 1 ⫻ 106 cells/ml in 24-well plates for 5 days. T cells were purified by negative selection using mouse T cell enrichment columns (R&D Systems), and 2 ⫻ 106 purified Ag-experienced ABLE or DO11.10 cells were then CFSE labeled and adoptively transferred into mice.

Materials and Methods Mice BALB/cByJ, C57BL/6, and C57BL/6 Thy-1.1 mice were obtained from The Jackson Laboratory. ABLE TCR transgenic mice bred onto a C␣⫺/⫺ background (21), WT15 TCR transgenic mice (provided by N. Killeen, University of California, San Francisco, CA) (25), BALB/c Thy-1.1 mice, and BALB/c IL-10⫺/⫺ (provided by D. Rennick, DNAX, Palo Alto, CA) were maintained in the animal colony. DO11.10 RAG2⫺/⫺ mice were obtained from Taconic Farms as part of their emerging models program (26). Animals were maintained in a specific pathogen-free environment. All experiments were conducted following the guidelines of the University of Pennsylvania institutional animal care and use committee.

Parasites and Ags L. major parasites (MHOM/IL/80/Friedlin) were grown in Grace’s insect medium (Invitrogen Life Technologies) supplemented with 20% heat-inactivated FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 ␮g/ml streptomycin. Mice were infected in the hind footpad with 5 ⫻ 106 stationary phase promastigotes. Soluble leishmanial Ag (SLA) was prepared, as previously described (22). OVA (OVA323–339) and LACK (LACK156 –173) peptides were obtained from the Protein Chemistry Laboratory at the University of Pennsylvania. CpG DNA (1826) was obtained from Coley Pharmaceutical Group. Immunization of mice was achieved by administering 50 ␮g of SLA along with 50 ␮g of CpG DNA in 50 ␮l of PBS in the hind footpad. Mice were boosted with a second dose of SLA and CpG in the same footpad. Previously described L. major parasites expressing OVA (Leishmania. OVA; Lmj1ˆ8SrRNA::HASPB18OVA) (27) were grown in Schneider’s medium (Sigma-Aldrich) supplemented with 20% heat-inactivated FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 ␮g/ml/streptomycin. Mice were infected in the hind footpad with 5 ⫻ 106 stationary phase promastigotes.

Treg cell depletion Treg cells (CD4⫹CD25⫹) were depleted in vivo with the administration of an anti-CD25 (PC61) mAb, as previously described (28, 29). Briefly, mice were injected i.p. with 1 mg of PC61 7 days before infection. Control mice were treated with 1 mg of an isotype control (rat IgG1) Ab. Depletion of Treg cells was assessed by flow cytometry using a mAb specific for a distinct CD25 epitope (7D4). Depletion was routinely ⬎80% compared with mice treated with the isotype control Ab.

Adoptive transfers Lymphocytes from the spleens and lymph nodes of mice were stained with CFSE, as previously described (30), and various doses of cells were trans-

Dendritic cell (DC) generation and transfer DCs were generated, as previously described (32). Briefly, bone marrow was collected from the femurs of BALB/cByJ mice, and clusters within the bone marrow suspensions were dispersed by passing through a 20-gauge needle using a syringe. Cells were seeded into 6-well plates at ⬃2.5 ⫻ 105/ml in 2.5 ml of medium (RPMI 1640; Invitrogen Life Technologies) supplemented with 10% heat-inactivated FBS (HyClone), 2 mM L-glutamine (Mediatech), 100 U/ml penicillin plus 100 ␮g/ml streptomycin (Mediatech), and 50 ␮M 2-ME (Invitrogen Life Technologies). In addition, 20 ng/ml GM-CSF (PeproTech) was added. At days 3, 6, and 8, a further 2.5 ml of medium containing 20 ng/ml GM-CSF was added. At day 10, 90 –95% of cells were comprised of DCs (MHCII⫹, CD11c⫹). For maturation of DCs, cells were harvested at day 10 and incubated with 100 ng/ml LPS (Sigma-Aldrich) for 18 –24 h in the presence of 5 ng/ml GM-CSF in round-bottom polypropylene tubes. Maturation of cells was confirmed by surface staining of cells for CD40 (HM40-3), MHCII (M5/114,15.2), CD80 (16-10A1), and CD86 (GL1), and analyzed by flow cytometry. Cell surface marker expression was compared with immature DCs that were cultured at day 10 in the absence of LPS. Adoptive transfer of DCs was performed, as previously described (33, 34). Briefly, 5 ⫻ 106 CFSE-labeled ABLE Thy-1.1 cells were transferred into day 7 L. major-infected mice. One day later, mice were given 5 ⫻ 105 LPS-matured DCs that had been pulsed with 5 ␮g/ml LACK156 –173 in the same footpad that had been infected. After 3 days, ABLE cells in nondraining and draining lymph nodes (dLN) were analyzed by flow cytometry. Negative control mice were given DCs that were unpulsed.

Flow cytometry, intracellular cytokine staining, and analysis Cells isolated from dLN and non-dLN were analyzed by flow cytometry directly ex vivo, as previously described (35). Briefly, cells were washed in staining buffer (PBS containing 0.1% BSA and 0.1% sodium azide) and incubated with Fc block (50 ␮g/ml 2.4G2 and 500 ␮g/ml rat Ig) before incubation with specific fluorochrome-conjugated mAbs against CD4 (RM4-5), CD44 (IM7), Thy-1.2 (53-2.1), Thy-1.1 (OX-7), or isotype control Abs (all from BD Pharmingen). Cells stained with anti-mouse DO11.10 TCR (KJ1-26) (Caltag Laboratories) were preincubated with normal mouse serum before incubation with specific mAbs. For intracellular cytokine staining, cells were stimulated with 50 ng/ml PMA, 500 ng/ml ionomycin, and 10 ␮g/ml brefeldin A (all from Sigma-Aldrich) for 4 h before surface staining. Fixed and surface-stained cells were permeabilized with 0.2% saponin (Sigma-Aldrich) in staining buffer before staining with specific fluorochrome-conjugated mAbs against IFN-␥ (XMG1.2) (BD Pharmingen). Samples were acquired on a FACSCalibur and analyzed using CellQuest Pro (BD Biosciences). T cells were labeled with CFSE, and the subsequent dilution of this fluorescent dye was detected by flow cytometry and used to calculate the responder frequency (number of donor T cells that divided due to stimulus) and the proliferative capacity (average number of daughter cells generated per responder), as described in detail in previous work (36).

Results Activation of naive T cells diminishes following L. major infection Our initial goal was to use Leishmania-specific TCR transgenic (ABLE) T cells as a monitor of ongoing Ag presentation following L. major infection, as has been done with several other pathogens (3, 6, 7). BALB/c Thy-1.1 mice were infected, and at different times following infection, CFSE-labeled ABLE T cells were transferred into the animals. After 3 days, the donor cells in the lymph nodes were analyzed by flow cytometry for proliferation and IFN-␥ production. No proliferation of ABLE T cells was observed

The Journal of Immunology in mice that were not infected. When ABLE T cells were adoptively transferred into mice infected with L. major for 1 day, ⬎50% of the cells diluted CFSE in the dLN, and 14% of the cells acquired the ability to produce IFN-␥ (Fig. 1A). However, this robust response was significantly diminished, although still detectable, when the naive ABLE T cells were transferred into mice that were infected for 1 wk or more with L. major. The significance, if any, of the detected response in mice that received cells 1 wk after infection is currently unknown. Similar results were obtained with

FIGURE 1. The ability to activate naive L. major-specific T cells rapidly diminishes following infection. BALB/c recipient mice (Thy-1.1⫹) were infected with 5 ⫻ 106 stationary phase L. major promastigotes, and CFSE-labeled ABLE transgenic T cells (Thy-1.2⫹) were transferred at the indicated times following infection. Three days following transfer, cells were harvested and analyzed by flow cytometry. A, Histogram and dot plots were gated on donor cells (Thy-1.2⫹). The number in the corner represents the percentage of donor cells that were CFSEdim in the non-dLN or dLN. The numbers in the corners of the dot plots indicated the percentage of donor cells that acquired the ability to produce IFN-␥ in the dLN of the recipient animals. B, BALB/c recipient mice (Thy-1.2⫹) were infected with transgenic Leishmania promastigotes expressing OVA (LeishmaniaOVA) and CFSE-labeled DO11.10 transgenic T cells were transferred at the indicated times following infection. Three days following transfer, cells were harvested and analyzed by flow cytometry. Histogram plots were gated on donor cells (KJ1-26⫹CD4⫹). The number in the corner represents the percentage of donor cells that were CFSEdim in the dLN. C, The percentage of ABLE cells that responded by proliferating (responder frequency) in the dLN at each time point was calculated (see Materials and Methods). D, Whole dLN from mice that received ABLE cells 7 days after infection with L. major and harvested 3 days later were isolated and restimulated in vitro with 1 ␮g of LACK peptide for 3 days. Donor cells were gated on by Thy-1.2 expression. The data are representative of three or more experiments.

927 cells from another LACK-specific TCR transgenic mouse (data not shown) (25). It remained possible that the inability to optimally activate naive LACK-specific T cells was peculiar to the LACK Ag. Therefore, experiments were also performed using transgenic L. major parasites that expressed the model Ag, OVA (Leishmania-OVA). In these experiments, instead of transferring ABLE T cells, OVA-specific, DO11.10 T cells were adoptively transferred 1 or 7 days following infection with Leishmania-OVA. Similar to recognition by ABLE T cells of LACK Ag in wild-type parasites, the Leishmania-OVA experiments showed a similar phenotype in that there was a reduction in proliferation in DO11.10 cells when transferred at day 7 (6%) compared with day 1 (40%) following infection (Fig. 1B). The diminished activation of naive ABLE T cells observed after L. major infection was reflected in the percentage of donor cells in the dLN that responded by proliferating (responder frequency) (Fig. 1C), although an analysis of the proliferative capacity (the number of daughter cells produced by each dividing cell) (36) indicated that once cells initiated their proliferative cycle they responded similarly (data not shown). These results suggest that the activation signal received by cells 7 days following infection is similar to the signal received by cells 1 day after infection, but fewer of the cells after 7 days of infection are receiving this activation signal. The diminished ability of naive T cells to proliferate during L. major infection was not due to any intrinsic deficit in the T cells adoptively transferred into L. majorinfected mice, because LACK peptide induced substantial in vitro proliferation by ABLE T that had been parked in L. major-infected mice from day 7 to day 10 (Fig. 1D). Because transfer of lower doses of TCR transgenic T cells has been shown to increase proliferation (37, 38), we also decreased the number of ABLE T cells transferred into infected animals. At the lowest dose of donor T cells that could still be detected in the lymph nodes, proliferation of naive TCR transgenic cells was also diminished at day 7 of infection compared with day 1 (data not shown). One explanation of these results could be that Ag presentation has ceased because of decreased levels of Ag. To test this, mice were given a second administration of live parasites 6 days after the initial infection, and CFSE-labeled ABLE cells were adoptively transferred 1 day later. As with a single infection, the ABLE T cell response was significantly diminished at day 7 when compared with cells that were transferred into mice on day 1 (Fig. 2A). Taken together, these data indicated that during infection with L. major the ability to optimally activate naive T cells is a transient event. Finally, to determine whether the diminished response by naive T cells during infection was limited to the dLN of the primary infection, we assessed the ability of naive T cells to respond in another site following a secondary infection. Mice were infected with a second dose of parasites in the contralateral footpad before the adoptive transfer of ABLE cells 7 days following primary infection. In lymph nodes draining the primary and secondary sites of infection, a lower frequency of donor cells responded by proliferating compared with mice that received ABLE cells 1 day after primary infection (Fig. 2B). This was also associated with a decrease in IFN-␥⫹ donor cells following secondary exposure compared with the activation of ABLE cells observed 1 day following infection. Although there is only a modest decrease in the frequency of IFN-␥⫹ cells at day 7 compared with day 1 (Fig. 2B) (17% compared with 21%, respectively), a comparison of all the mice tested at each time point showed a significant difference (13.1 ⫾ 1.7 at day 7 compared with 21.6 ⫾ 1.2 at day 1; p ⬍ 0.0001). These data suggest that the diminished response of naive T cells that occurs during L. major infection is a systemic event.

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FIGURE 2. Subsequent exposure to live parasites does not rescue the inability to activate naive cells on day 7. BALB/c recipient mice (Thy1.1⫹) were infected, as described in Fig. 1. At day 6 postprimary infection, one group of mice was reinfected in the same (A) or contralateral (B) footpad with L. major, and control mice were given PBS in place of parasites at day 6. CFSE-labeled ABLE transgenic T cells (Thy-1.2⫹) were adoptively transferred following infection, as indicated. Cells were harvested and analyzed as in Fig. 1, with the exception that the primary (left) and secondary (right) dLN were analyzed from mice challenged in the contralateral footpad. Donor cells were gated on by Thy-1.2 expression. The data are representative of three experiments.

The diminished response of naive T cells in L. major-infected animals is not due to immunosuppression Both specific and nonspecific mechanisms of immunosuppression operate during infection with L. major, and may contribute to the reduced response of naive T cells once a L. major infection is established (8, 9, 39, 40). Therefore, we tested whether naive T cells with specificity to an Ag unrelated to Leishmania exhibited decreased responses in mice with an established L. major infection. BALB/c mice infected with L. major for 6 days, or naive animals, were immunized with OVA protein. The following day, CFSE-labeled DO11.10 and ABLE Thy-1.1 transgenic T cells were adoptively transferred into the immunized mice, and proliferation of the donor cells in the dLN was analyzed by flow cytometry. The proliferation of DO11.10 cells in L. major-infected mice that were immunized with OVA 6 days after infection (day 6 OVA/day 7 L. major) was similar to control mice that were immunized with OVA alone. However, the ABLE cells were unable to respond in both cases, suggesting that a global decrease in Ag presentation was not responsible for the diminished response of naive T cells in L. major-infected mice (Fig. 3). Importantly, donor cells responded to their cognate Ag in control mice that were im-


FIGURE 3. Ag presentation to an unrelated Ag is intact at 7 days following L. major infection. BALB/c recipient mice (Thy-1.2⫹) were infected with L. major. Six days following infection, one group of mice was injected with 25 ␮g of OVA protein in the same footpad (d6-OVA/d7-L. major). As controls, mice were given protein alone (d1-OVA), infected with L. major for 1 or 7 days (d1-L. major and d7-L. major, respectively), or infected and immunized at the same time (d1-L. major/OVA). CFSElabeled OVA-specific (DO11.10) transgenic T and ABLE Thy-1.1⫹ cells were adoptively transferred into the recipient mice at the indicated time points. The dLN were harvested and analyzed by flow cytometry 3 days following transfer. Histogram plots were gated on KJ1-26⫹CD4⫹ (DO11.10) or Thy-1.1⫹ (ABLE) donor cells in the dLN of recipient mice. The data are representative of three experiments.

munized with OVA protein, infected with L. major, or immunized and infected at the same time 1 day before receiving donor cells. L. major infection is associated with a Treg response and increased production of IL-10, both of which may significantly impair T cell responses (8, 9). Therefore, we tested whether IL-10 or Treg cells might modulate the responses of naive T cells during L. major infection. IL-10⫺/⫺ animals were infected and received CFSE-labeled ABLE T cells at 1 and 7 days following infection, as described in Fig. 1. Three days following transfer, the mice were sacrificed, and dLN were harvested and analyzed for proliferation and IFN-␥ production by flow cytometry. In the absence of IL-10 ABLE cells exhibited slightly enhanced proliferation, but naive T cells transferred at 7 days of infection still displayed decreased proliferation compared with cells transferred at the initiation of the infection (Fig. 4A). Depletion of IL-4 also had no effect on the ability of naive T cells to respond at day 7 (data not shown). In a similar set of experiments, we depleted mice of Treg cells before infection. Depletion of Treg cells failed to enhance the ability of naive ABLE T cells to respond when transferred into mice infected for 1 wk with L. major (Fig. 4B), although the depletion of CD25⫹ cells also led to a more robust proliferative and IFN-␥ response 1 day after infection compared with control mice. Taken together, these data demonstrate that the diminished response of naive T cells in L. major-infected mice is unrelated to IL-10, IL-4, or Treg cells.

The Journal of Immunology

FIGURE 4. The suboptimal ability to activate naive T cells at 7 days following L. major infection is not dependent upon immunosuppression. A, BALB/c IL-10⫺/⫺ recipient mice (Thy-1.2⫹) were infected with L. major and CFSE-labeled ABLE transgenic T cells (Thy-1.1⫹) were transferred at 1 or 7 days following infection. As controls, experiments were also performed with wild-type recipient mice, as described in Fig. 1. Dot plots are gated on donor cells in the dLN of recipient mice (Thy-1.1⫹). The numbers in the corners of the dot plots represent the percentage of events in either of the CFSEdim quadrants, and the numbers in parentheses represent the percentage of cells that produce IFN-␥ that are CFSEdim. B, BALB/c recipient mice (Thy-1.2) were depleted of Treg cells by administration of 1 mg of anti-CD25 (PC61) Ab i.p. 7 days before infection, and control mice were treated with 1 mg of rat IgG. Following infection, CFSE-labeled ABLE transgenic T cells (Thy-1.1⫹) were transferred, harvested, and analyzed, as described in Fig. 1. Dot plots were gated on donor cells in the dLN of recipient mice (Thy-1.1⫹).

Ag-experienced T cells are more efficiently activated during L. major infection compared with naive cells It is widely accepted that Ag-experienced T cells have a lower threshold for activation compared with naive T cells (12, 15–17). Therefore, we considered the possibility that our results were not due to loss of Ag presentation, but competition between naive and primed T cells. To test this, we first asked whether Ag-experienced T cells responded better during an ongoing infection. Thus, ABLE transgenic T cells that had been stimulated in vitro with LACK peptide were adoptively transferred into infected mice. There were robust responses elicited in mice that received either naive or previously activated cells 1 day following infection (Fig. 5A). In mice that received Ag-experienced cells 7 days after infection, 40% of the cells had diluted CFSE, while only 10% of the naive cell counterparts had done so. Thus, while the proliferative response by primed T cells was less at day 7 compared with day 1, the response was significantly enhanced compared with naive T cells. To determine whether polyclonal, Ag-experienced T cells would proliferate in L. major-infected mice, T cells from C57BL/6 Thy1.1 mice that had resolved infection (⬎12 wk) were harvested, CFSE labeled, and then adoptively transferred into C57BL/6 mice that had been infected for 1 or 7 days. When harvested 1 wk later, ⬃30% of the polyclonal immune cells transferred into mice 1 day following infection had diluted CFSE in the dLN and ⬎10% of those cells had acquired the ability to produce IFN-␥ (Fig. 5B). In mice that received immune cells after being infected for 7 days, ⬎40% of the donor cells in the dLN had diluted CFSE. In addition, the percentage of IFN-␥-producing cells was also enhanced in mice infected for 7 days compared with 1 day (Fig. 5B). However,

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FIGURE 5. Previously activated ABLE transgenic T cells and immune polyclonal cells are less susceptible to the suboptimal activation observed 7 days following infection. A, BALB/c recipient mice (Thy-1.1⫹) were infected, as described in Fig. 1. CFSE-labeled ABLE transgenic naive or previously activated (primed) (Thy-1.2⫹) (see Materials and Methods) were adoptively transferred into recipient mice at day 1 or 7 following infection. The dLN were harvested and analyzed by flow cytometry 3 days following transfer. Histogram plots were gated on donor cells (Thy-1.2⫹). The number in the corner represents the percentage of donor cells that were CFSEdim in the dLN. The numbers in the corners of the dot plots indicated the percentage of donor cells that acquired the ability to produce IFN-␥ in the dLN of the recipient animals. B, Naive C57BL/6 mice (Thy-1.2⫹) were infected, as described in Fig. 1. Immune cells from C57BL/6 mice (Thy1.1⫹) that had healed infection (⬎12 wk following infection) were transferred at the indicated times following infection. Seven days following transfer, cells were harvested and analyzed by flow cytometry. Donor cells were gated on by Thy-1.1 expression. C, BALB/c recipient mice were infected with Leishmania-OVA. CFSE-labeled DO11.10 transgenic naive or previously activated (primed) (see Materials and Methods) were adoptively transferred into recipient mice at day 1 or 7 following infection. The dLN were harvested and analyzed by flow cytometry 3 days following transfer. Histogram plots were gated on donor cells (KJ1-26⫹CD4⫹). The data are representative of three experiments.

when similar experiments were attempted with the transfer of naive polyclonal cells into mice infected for 1 or 7 days, there was no detectable proliferation in either group at 3 or even 7 days following transfer (data not shown). It is likely that this is due to the low precursor frequency of Ag-specific cells present in a naive polyclonal population. Lastly, it remained possible that the decrease in activation of ABLE and the decreased sensitivity of primed cells to this effect were specific to LACK-reactive T cells. Therefore, to discount this, naive and previously activated DO11.10 cells were

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adoptively transferred into mice that were infected with Leishmania-OVA for 1 or 7 days. Similar to ABLE cells, in mice that received Ag-experienced DO11.10 cells 7 days after infection, 25% of the cells had diluted CFSE, while only 5% of the naive cell counterparts had done so (Fig. 5C). Taken together, these data indicate that both TCR transgenic T cells and polyclonal Ag-experienced T cells are efficiently recruited into the response even when transferred following infection, suggesting that Ag-experienced cells have an advantage compared with naive T cells in maintaining the immune response to L. major.

not shown). Together, these data provide evidence that naive T cells are refractory to stimulation in the presence of Ag-experienced cells and indicate that during an ongoing immune response to a given Ag, previously activated cells may preferentially respond. They also suggest that Ag presentation is unimpaired during the first week of L. major infection.

Endogenous responding cells inhibit the activation/expansion of naive Ag-specific T cells Although our results with primed T cells indicate that Ag presentation does not cease after infection, it leaves open the question of why naive T cells are less able to proliferate when transferred to infected mice. It is well known that following L. major infection in BALB/c mice, a LACK-specific T cell response is initiated (24, 39). Having determined that Ag-experienced T cells are more readily activated than naive T cells after infection, we wanted to test whether competition by the endogenously responding cells was responsible for the suboptimal activation of the naive T cells. Therefore, naive ABLE T cells were adoptively transferred into DO11.10 RAG2⫺/⫺ mice infected with L. major. Because DO11.10 RAG2⫺/⫺ mice only have T cells specific for an OVA epitope, the Leishmania-specific endogenous response has been effectively removed. When naive ABLE cells were transferred into DO11.10-infected mice that had been infected for 1 day, ⬎70% of the ABLE cells detected in the dLN of DO11.10 mice had diluted CFSE and almost 60% of the donor cells had acquired effector function (Fig. 6). Interestingly, in DO11.10 mice that had received donor cells at 7 days following infection, nearly 70% of the ABLE cells were CFSEdim in the dLN and 76% of the cells had gained the ability to produce IFN-␥. The expansion observed in infected animals was not due to homeostatic proliferation because none was detected upon adoptive transfer of ABLE cells into uninfected control mice. In addition, in vitro experiments were performed investigating the activation of naive ABLE T cells in the presence of previously activated ABLE T cells, and found that in the presence of primed cells, naive T cells exhibit a diminished response (data

FIGURE 6. The previously activated Ag-specific cells prevent the optimal activation of naive Ag-reactive cells. DO11.10 RAG2⫺/⫺ recipient mice (Thy-1.2⫹) were infected, as described in Fig. 1. CFSE-labeled ABLE transgenic cells (Thy-1.1⫹) were adoptively transferred into recipient mice at day 1 or 7 following infection. The dLN of recipient mice were harvested and analyzed by flow cytometry 3 days following transfer. Dot plots were gated on donor cells in the dLN of recipient mice (Thy-1.1⫹). The numbers in the corners represent the percentage of events in either of the CFSEdim quadrants, and the numbers in parentheses represent the percentage of cells that produce IFN-␥ that are CFSEdim. The data are representative of three experiments.

Naive and Ag-experienced T cells compete for access to APCs Because naive ABLE T cells were able to respond both at the initiation and during L. major infection in DO11.10 mice that contained no other Leishmania-specific cells, and because primed T cells have a lower threshold for activation than naive T cells, it would appear that competition for APCs is a likely explanation for the diminished naive T cell response observed during infection. Therefore, to determine whether APCs were limiting after infection, we asked whether DCs could overcome the diminished naive T cell response seen during infection. BALB/c mice were infected with L. major, and at day 7 CFSE-labeled ABLE Thy-1.1 T cells were adoptively transferred into the infected mice. One day later, infected mice that had received ABLE cells were injected with LACK-pulsed DC. Three days after the adoptive transfer of DCs, the draining and non-dLN were harvested and analyzed by flow cytometry. Infected animals that received unpulsed DCs had limited activation of naive ABLE T cells in the dLN compared with positive control mice that were uninfected, but received Ag-pulsed DCs (Fig. 7). In contrast, injection of LACK-pulsed DCs restored the ability to effectively induce proliferation of naive T cells. This was also reflected in the acquisition of effector function (Fig. 7). These data indicate that naive and Ag-experienced cells compete for access to APCs during infection with L. major.

FIGURE 7. Naive and Ag-experienced T cells compete for access to APCs. Naive BALB/c mice (Thy-1.2⫹) were either infected with 5e6 stationary phase promastigotes or left uninfected. Naive CFSE-labeled ABLE transgenic T cells (Thy-1.1⫹) were transferred into naive or day 7 infected mice. One day after transfer, LPS-matured bone marrow-derived DC pulsed with 5 ␮g of LACK peptide or left unpulsed were adoptively transferred directly into the recipient via the footpad. Three days following transfer of bone marrow-derived DC, cells were harvested and analyzed by flow cytometry. Histogram plots were gated on donor cells (Thy-1.1⫹). The number in the corner represents the percentage of donor cells that were CFSEdim in the dLN. The numbers in the corners of the dot plots indicated the percentage of donor cells that acquired the ability to produce IFN-␥ in the dLN of the recipient animals. The data are representative of two experiments.

The Journal of Immunology Secondary immunizations do not recruit naive T cells into the immune response A logical extension of our findings is that the competition between naive and primed T cells that we observed during infection might also influence the ability to recruit naive T cells into an ongoing immune response induced by immunization. To assess this issue, we immunized mice with an SLA fraction and the adjuvant CpG, boosted them by administration of a second injection, and assessed the ability of transferred ABLE TCR transgenic T cells to proliferate. As expected, ABLE cells transferred into mice that had received SLA and CpG 1 day earlier exhibited a significant proliferative response (Fig. 8). However, when naive T cells were transferred into mice that were given a second administration of Ag, there was an 80% reduction in the ability of the donor T cells to dilute CFSE.

Discussion Analysis of the proliferation of TCR transgenic T cells following adoptive transfer into infected mice can be used as an indirect measure to assess how long Ag presentation continues after infection. A recent study using this approach found that Ag presentation persists for at least as long as the pathogen survives (7), while other studies indicate that Ag presentation is rapidly down-regulated after infection (3, 4, 6, 41). Using a similar approach, we found that adoptively transferred naive TCR transgenic cells that recognize the leishmanial Ag LACK do not proliferate during an ongoing infection with L. major as well as at the initiation of the response. However, our data show that this is not due to a loss of Ag presentation, but instead due to competition between naive and Ag-experienced T cells. Infection of BALB/c mice with L. major is associated with increased IL-10 production (42, 43) and Treg cell activity (8), but neither of these was associated with the decreased naive T cell response seen in L. major-infected mice. However, elimination of endogenous T cells was able to restore the naive T cell response to levels observed in mice infected for only 1 day, suggesting that the activated endogenous Leishmania-specific CD4⫹ T cells are competing with the naive donor T cell population. In other systems, T cell competition has been shown to play a role during homeostatic proliferation (18, 20, 44), as well as priming of T cells of the same

FIGURE 8. Secondary immunizations recruit few naive T cells into the immune response. BALB/c recipient mice (Thy-1.2⫹) were immunized in the hind footpad with 50 ␮g of SLA and 50 ␮g of CpG DNA. In addition, a third group of mice was boosted with a second administration of SLA and CpG DNA in the same footpad at day 6 following primary immunization (boost). CFSE-labeled ABLE transgenic T cells (Thy-1.1⫹) were transferred at either day 1 or 7 following the primary immunization. As a negative control, ABLE transgenic T cells were transferred into naive BALB/c mice. The data are representative of two independent experiments.

931 (45, 46) and different (47, 48) specificities during responses to Ag. In leishmaniasis, there is a substantial expansion of LACK-reactive T cells within 4 days (24, 49), which are likely to be responsible for inhibiting the activation of naive LACK-specific TCR transgenic cells. This inhibition was specific, because there was no decrease in the response of naive T cells to OVA during L. major infection. Therefore, it appears that competition for particular class II molecules presenting LACK epitopes is responsible for the decreased naive T cell response. Consistent with this hypothesis was our ability to enhance the naive T cell response with LACK-pulsed DCs. Thus, our findings are similar to those showing that the activation of naive T cells specific for a particular epitope can be inhibited in the presence of Ag-experienced cells of the same specificity through competition for access to APCs (20, 47, 48, 50). The inability to rescue the phenotype by the administration of more parasites or parasite-derived Ag indicates that the paucity of Ag is not responsible for the transient capacity to activate naive T cells. Rather, because adoptive transfer of Ag-pulsed DCs fully rescues the ability to prime naive Leishmania-specific T cells, it would appear that LACK-presenting APCs are limiting, as the number of Ag-specific T cells expands during infection. Given the fact that there is a limited amount of surface area on DCs with which to interact with T cells, and it has been suggested that T cells with a lower threshold for activation preferentially occupy this space, preventing the activation of T cells with a higher threshold for activation (48), it is likely that this plays a significant role in T cell activation. In addition, the ability of primed T cells to respond better than naive T cells to APCs other than DCs, such as macrophages and B cells, may also contribute to the capacity of primed T cells to respond. This hypothesis is consistent with our data especially because the ability of previously activated T cells to respond to lower doses of Ag (12, 15–17), as well as their less stringent requirements for costimulation (12, 13), may make them better able to respond when DCs are limiting compared with naive T cells. Why Ag presentation is maintained in some infections, but not others, is unknown. In the case of Listeria, it was found that Ag presentation to CD8 T cells is short-lived due to the ability of CTLs to eliminate APCs (3, 4). This is not the mechanism operating in leishmaniasis, because primed L. major T cells proliferated after transfer to L. major-infected mice. Moreover, depletion of CD8⫹ T cells had no effect on the ability of naive cells to proliferate (data not shown). The most likely explanation for the difference between the Listeria and L. major infections is that the magnitude of the CD8 T cell response is less following L. major infection, although we have not directly investigated this issue. Similar to Listeria, the CD8 response to a sporozoite Ag of malaria was also found to be short-lived (6). However, in this case, whether APCs were killed due to the cytolytic activity of CD8 cells was not tested, and it is possible that competition between primed and naive T cells may have contributed to this response. In contrast, results with influenza virus suggest that competition does not always occur (7). Thus, when naive influenza-specific CD4⫹ T cells were transferred into mice at different times after infection, they were able to proliferate for several weeks. It is unclear why the competition that we observed with L. major was not evident in the influenza system, although it might be explained by either a lower frequency of hemagglutinin-specific T cells within the activated endogenous T cell population or differences in the affinity of the TCR-peptide interactions. Taken together, these studies indicate that the ability to recruit naive T cells into an ongoing immune response will vary, presumably depending upon specific characteristics of the infection. Similarly, adoptive transfer of naive OT-I cells into mice infected for 3 wk with OVA-transgenic Leishmania

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donovani was host protective, albeit with delayed kinetics compared with transfer of previously activated OT-I cells (51). One implication of our results is that continuous boosting with vaccines may not expand the pool of naive Ag-specific cells beyond those that were primed initially. Rather, primed cells will have a significant advantage over those cells that have not encountered Ag. Our studies with both parasites and SLA suggest that once an endogenous pool of primed T cells is established, additional injections of Ag will recruit few naive T cells into the immune response. This occurred in the lymph node given a primary infection, as well as in other lymph nodes draining secondary sites of stimulation. These data suggest that Ag-experienced T cells from the initial priming event in the recipient mouse home to a secondary lymph node upon reinfection, and compete with naive T cells that were transferred into the mice (Fig. 2). These cells may be the precursors of central memory T cells, which we have shown to be critical in maintaining immunity in the absence of persistent parasites (2). Whether this population is depleted with continued Ag administration has not yet been tested, but because few additional naive T cells are recruited into the response, it is possible that additional injections of Ag will increase the pool of short-lived effector T cells at the expense of central memory T cells. In contrast, it is possible that those few naive T cells that do respond in the face of competition by primed cells are better able to survive. In studies with L. monocytogenes, it was shown that competition decreased the attrition of CD8 T cells, and increased the percentage of cells that survived and became central memory T cells (5). Thus, those cells that received reduced stimulation were at a distinct advantage, consistent with the idea that signal strength dictates cell survival (52). Although there was no evidence of competition in studies of CD4 T cells stimulated by influenza, naive T cells that responded later in the infection, thus receiving less stimulation due to lower viral loads, were better memory T cells (7). Consequently, blocking the recruitment of those naive T cells into the response diminished immunity. Although our studies do not address this issue, they help highlight the importance of determining whether continued boosting leads to expansion of more memory T cells or depletion of a central memory T cell pool. The finding that competition inhibited the ability of naive T cells to respond following infection was unexpected, and indicates that the use of adoptively transferred naive TCR transgenic T cells to assess Ag presentation may not always be appropriate. Based on our adoptive transfers, we might have incorrectly concluded that Ag presentation ceased after the first week of L. major infection. At the same time, however, these studies allowed us to discover that significant competition exists between naive T cells and previously primed T cells following L. major infection. Future studies will be required to determine whether this is unique to LACK, or an attribute of other leishmanial Ags. However, the data with Leishmania-OVA would suggest it is not peculiar to LACK. In either event, the current study has important implications to consider for the optimal recruitment of T cells into a response following both a natural infection and a prime and boost regimen of immunization.

References

Acknowledgments We thank Karen Joyce for the maintenance of the mouse colonies. We thank Nigel Killeen for providing the WT15 TCR transgenic mice. We thank members of the Scott laboratory for helpful discussions.

Disclosures The authors have no financial conflict of interest.

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