Primary Leishmania major infection typically produces cutaneous lesions that not only heal but also harbor persistent parasites. While the opposing roles of CD4+ T-cell-derived IFN-γ and IL-10 in promoting parasite killing and persistence have been well established, how these responses develop from naïve precursors has not been directly monitored throughout the course of infection. We used peptide:Major Histocompatibility Complex class II (pMHCII) tetramers to investigate the endogenous, parasite-specific primary CD4+ T-cell response to L. major in mice resistant to infection. Maximal frequencies of IFN-γ+ CD4+ T cells were observed in the spleen and infected ears within a month after infection and were maintained into the chronic phase. In contrast, peak frequencies of IL-10+ CD4+ T cells emerged within 2 weeks of infection, persisted into the chronic phase, and accumulated in the infected ears but not the spleen, via a process that depended on local antigen presentation. T helper type-1 (Th1) cells, not Foxp3+ regulatory T cells, were the chief producers of IL-10 and were not exhausted. Therefore, tracking antigen-specific CD4+ T cells revealed that IL-10 production by Th1 cells is not due to persistent T-cell antigen receptor stimulation, but rather driven by early antigen encounter at the site of infection.
The adaptive immune response to intracellular parasites or bacteria is in large part dependent upon the generation of primed CD4+ T cells in secondary lymphoid organs. The clonal proliferation that accompanies priming provides for a greater number of antigen-specific cells that can differentiate into effector cells capable of homing to sites of infection. In the case of Leishmania major (Lm), the differentiation of CD4+ T cells into Th1 cells that can migrate to the site of parasite inoculation by infected sand fly bite in the skin is required to activate infected macrophages and promote healing of the cutaneous lesion . The number and function of Th1 cells can be limited by the activation of regulatory T (Treg) cells and other populations of IL-10 secreting cells, including Th1 cells, which may restrain immunopathology while preventing the complete elimination of the parasite . In chronically infected mice, the number of persistent organisms and the severity of the chronic cutaneous lesions are determined by the balance of immune activating and deactivating cells and cytokines in the site .
The expansion of parasite-specific CD4+ T cells, their homing to and function within peripheral sites of infection, and their maintenance, contraction, or exhaustion during the chronic stage, remain poorly characterized in any Leishmania infection model. The adoptive transfer of naïve TCR transgenic CD4+ T cells responding to OVA expressing Lm (Lm-OVA), revealed their early proliferation in the draining lymph node and generation of both central and effector memory cells during the first 14 days of infection . This study did not follow the cells in the inoculation site, nor their fate during the chronic stage of infection. Furthermore, the transfer of large, non-physiologic numbers of T cells with the same specificity can distort their proliferation and differentiation programs [5, 6], a phenomenon borne out in the response of OT-II transgenic T cells to Lm OVA . Peptide-MHC class II (pMHCII) tetramer-based approaches that permit detection of rare endogenous precursors in naïve mice have been applied in Lm infection models to study the activation and expansion of CD4+ T cells specific for the Lm antigen LACK . The studies were again confined to lymph node cells during the acute stage of infection, and did not consider the polyfunctionality of these cells.
Here we used a sensitive pMHCII tetramer-based approach that allowed detection of polyclonal pMHCII-specific CD4+ T cells in mice after intradermal infection with Lm. We applied this approach to enumerate the expansion, contraction, and tissue distribution of parasite-specific CD4+ T cells throughout the course of the infection. Most informatively, we were able to define the dynamics of IFN-γ and IL-10-secreting effector and Treg cells that contribute to the chronicity of the infection, and to the balance of immunity and pathology in the inflammatory site.
Detection of CD4+ T cells specific for an Lm-derived model antigen
We generated Lm parasites (Lm 2W) that express a secreted chimeric protein consisting of the 2W peptide and the L. donovanii 3′ nucleotidase/nuclease, an antigen expressed in the promastigote and amastigote stages  to directly visualize an endogenous polyclonal, antigen-specific CD4+ T-cell response to Lm. The 2W peptide is a variant of peptide 52–68 from the I-E alpha chain (EAWGALANWAVDSA) . The relatively large naive precursor size of the 2W:I-Ab-specific T-cell repertoire provided a technical advantage over tracking CD4+ T cells specific for a chicken ovalbumin-derived peptide (323–339) bound to I-Ab [10, 11] with a previously generated recombinant L. major strain . C57BL/6 mice were primed in the footpad with either Lm 2W or Lm SP-OVA and boosted in the ears 8 weeks later with the homologous recombinant L. major strain used in the primary infection to determine if endogenous CD4+ T-cell responses to L. major-derived antigens could be detected with I-Ab tetramers. One week after boosting, we stained single-cell suspensions with fluorochrome-conjugated 2W:I-Ab and pooled OVA323–339:I-Ab and OVA265–280:I-Ab tetramers , and used anti-fluorochrome magnetic beads to enrich specific T cells from the ears and ear-draining lymph nodes (dLNs) of uninfected and Lm-infected mice. T cells in the enriched fraction were identified as CD3+ cells that did not bind a cocktail of lineage-specific antibodies (Fig. 1A). 2W:I-Ab-specific T cells were detected in the CD4+ T-cell compartment but not among the MHC class I-restricted CD8+ T cells (Fig. 1B and C), suggesting that the tetramer bound to T cells via the TCR. 2W:I-Ab-specific CD4+ T cells were found in the dLNs of uninfected and SP-OVA-infected mice, but were absent in the ears of those mice (Fig. 1C). Furthermore, the tetramer-binding cells in these mice were CD44low, as expected for naive cells. In contrast, large numbers of CD44high 2W:I-Ab-specific CD4+ T cells were detected in the dLN and ears of Lm-2W-infected mice (Fig. 1C). Importantly, the expanded 2W:I-Ab+ T-cell population did not bind the OVAp:I-Ab tetramers (Fig. 1D), again indicating the tetramer staining was TCR-specific. These results indicate that endogenous pMHCII-specific T-cell responses to L. major-derived model antigens can be monitored with I-Ab tetramers.
Kinetics of primary 2W:I-Ab-specific T-cell response to Lm-2W infection
The course of lesion development as well as parasite growth and clearance obtained following intra-dermal ear injection of 105 metacyclic promastigotes of the Lm-2W parasites were comparable with that of the wild type Lm FV1 strain, with pathology peaking at 6–8 weeks, and mean parasite numbers peaking at 4–5 weeks post-infection in the ear and in the local dLN (data not shown). At 5 weeks post-infection, none of the four Lm FV1 infected mice and only one of four of the Lm 2W infected mice presented any detectable parasites in the spleen. The maintenance of a low number of Lm 2W parasites in the skin following healing of the lesion, comparable to the number reported for the wild type strain , was confirmed by detection of between 1.28 × 102 and 2.48 × 103 parasites in a total of six ears from mice examined at 11 and 17 weeks post-infection.
We enumerated 2W:I-Ab-specific CD4+ T cells in the ear-draining lymph nodes, spleen, and ears in response to intra-dermal infection with Lm-2W to gain insight into the development, maintenance, and tissue distribution of the parasite-specific CD4+ T-cell response. 2W:I-Ab-specific T cells were detected in the spleen and dLN and absent from the ears of uninfected mice (Fig. 2A and B), and as expected for naive cells, these were virtually all CD44low (Fig. 2A). CD44high 2W:I-Ab-specific T cells were detected in the dLN on day 3 post-infection (Fig. 2B), indicating that priming was underway. This is consistent with a previous report showing expansion of LACKp:I-Ad-specific T cells in BALB/c mice . On day 3, however, no 2W:I-Ab-specific CD4+ T cells had reached the ears (Fig. 2B). By day 7, the number of 2W:I-Ab-specific T cells had increased about 10-fold in the ear-draining lymph nodes and spleen over those in naive mice, and about 70-fold 2W:I-Ab-specific T cells had accumulated in the ears (Fig. 2A and B). 2W:I-Ab-specific T cells expanded about 100-fold in the dLNs within the first 2 weeks of the infection and then declined by day 32 to about 9% of peak numbers. By day 56, the number of 2W:I-Ab-specific T cells was about 3.5% of peak numbers, a value that was maintained for at least 2 months into the chronic phase of the infection. In contrast, peak numbers of 2W:I-Ab-specific T cells in the spleen and ears were achieved 3 weeks post-infection and were at least three times greater than in the lymph nodes. 2W:I-Ab-specific T cells in the spleen and ears gradually declined during the following 35 days to about 11% of peak numbers, values that were maintained for at least 2 months. Most of the 2W:I-Ab-specific T cells enumerated at any given time point were found in the spleen, indicating that this tissue is a large reservoir of parasite-specific T cells. These results demonstrate that parasite-specific CD4+ T cells undergo expansion, contraction, and numerically stable phases in response to a persistent infection, and that the tempos of these processes differ for the dLN, spleen, and ears.
Preferential expansion of T-bet+Foxp3− CD4+ T cells in response to Lm-2W infection
We then sought to determine which T-cell subsets were present within the tetramer-binding cells in the chronic phase of the infection. We focused our analysis on Th1 cells and Foxp3+ Treg cells because Lm infection in B6 mice induces a potent Th1 response  and thymus-derived parasite-specific Treg cells have been implicated in parasite persistence [14, 15]. The 2W:I-Ab-specific CD4+ T-cell repertoire is amenable for these studies because it can generate Th1 cells  and about 8% of the tetramer-binding cells in naïve B6 mice are Helios+Foxp3+ Treg cells , suggesting a thymic origin . Naive CD44low 2W:I-Ab-specific T cells in the secondary lymphoid tissues do not express T-bet . Seventy-seven to ninety-four percent of the CD44high 2W:I-Ab-specific T cells in the ears, dLN, and spleen respectively, expressed T-bet and not Foxp3 8 weeks post-infection (Fig. 3B and C), indicating that these were Th1 cells  and not Treg cells [20, 21]. In contrast, 40–65% of the CD44hightetramer− CD4+ T cells in the ears, dLN, and spleen were Th1 cells, demonstrating that the tetramer-binding T cells are enriched for these cells. Less than 7% of the tetramer-binding cells in these tissues expressed Foxp3, and a small fraction co-expressed T-bet (Fig. 3B and C), as has been described for Mycobacterium tuberculosis infection . Contrary to 2W:I-Ab+ cells, 5–27% of the tetramer− compartment was comprised of Treg cells. 2W:I-Ab+ Th1 cells were 20–100 times more abundant than 2W:I-Ab+ Treg cells in the sampled tissues (Fig. 3D). Similar results were observed by tracking OVAp:I-Ab-specific T cells in response to SP-OVA infection (Fig. 3E), demonstrating that they were not unique to the 2W:I-Ab-specific CD4+ T-cell repertoire.
To explore the possibility that expansion of parasite-specific Treg cells may have occurred earlier in infection, Foxp3 and T-bet expression were analyzed on 2W:I-Ab+ and 2W:I-Ab− cells recovered at 3 weeks post-infection, and in comparison to the frequency of these cells in naïve mice. Again, less than 5% of the tetramer binding cells in the dLN, spleen or ear expressed Foxp3 (Fig. 3F), significantly less than the frequency of the Foxp3+ cells in the tetramer− population (9–26%), and less than the frequency of these cells in the tetramer binding population of cells present in the dLN and spleen of uninfected mice (note that no tetramer binding cells were detected in the naïve ear). In addition, no increase in the total number of tetramer-binding Foxp3+ cells was observed in the dLN or spleen compared with that in naïve mice (Fig. 3G). The low but detectable numbers of 2W:I-Ab-specific Foxp3+ cells in the infected skin likely reflects inflammation-induced recruitment of these cells. By contrast, there was a massive expansion of the 2W:I-Ab-specific Foxp3−T-bet+ cells in dLN and spleen over the numbers present in naïve mice, and they were 29 times more abundant than the 2W:I-Ab+ Treg cells in the ear.
These findings indicate that Th1 cells comprise by far the largest T-cell subset both early and during the chronic stage of the infection, and suggest that parasite-specific thymic Treg cells are not preferentially expanded in response to this infection.
Kinetics of IFN-γ and IL-10 production during primary Lm-2W infection
Helper T-cell-derived IFN-γ and IL-10 exert opposite effects on parasite burden . We evaluated IFN-γ and IL-10 production by 2W:I-Ab-specific T cells throughout the infection to determine when these responses develop and assessed the tissue distribution of the cells capable of making these cytokines upon restimulation in vivo. Both Lm-specific Th1 cells and Lm-specific Treg cells can produce IL-10 [14, 15, 23]. Since most of the tetramer+ cells in the chronic phase expressed T-bet and not Foxp3, Th1 cells were the most likely source of IL-10. Within the first 2 months of infection, about 15% of the 2W:I-Ab-specific T cells in the ears made IFN-γ in the absence of in vivo peptide re-stimulation above the levels of those cells found in the spleen (Fig. 4A–C). This is in agreement with a report of IFN-γ production by CD4+ T cells in the skin of mice immunized intra-dermally with protein emulsified in Incomplete Freund's adjuvant . In contrast, no IL-10 could be detected without the intravenous injection of 2W peptide (Fig. 4A, B, and D). Two to four hours after intravenous injection of 2W peptide, IFN-γ and IL-10 could be detected in tetramer-binding cells. The frequency of IFN-γ+ 2W:I-Ab+ T cells in the spleen rose from 25% on day 14 to 65% on day 21, and then remained at around 50% for at least 70 more days (Fig. 4A and C). The frequency of IFN-γ+ 2W:I-Ab+ T cells in the ears also increased sharply between days 14 and 28, from 45% to 65%, and then gradually increased to about 80% by day 91 after infection. About 5% of the tetramer-binding cells in the spleen 14 or 21 days post infection made IL-10 after peptide stimulation (Fig. 4A and D). By day 28 the frequency dropped to background levels. In contrast, ∼20% of the tetramer-binding cells in the ears at all of the time points analyzed made IL-10 after peptide stimulation (Fig. 5B and D). On day 14, about half of the IL-10+ cells in the spleen and ears were IFN-γ+ (Fig. 4A–C). However, by day 21, IFN-γ+ cells were the major source of IL-10 within tetramer-binding cells. This trend became more pronounced by day 28 in the ears.
IL-10 production by CD8+ T cells responding to a chronic LCMV infection has been associated with greater levels of T-cell exhaustion, including a reduced capacity to make IFN-γ on a per cell level . To determine if this property applied to CD4+ T cells responding to Lm, we measured IFN-γ mean fluorescence intensities in IL-10+IFN-γ+ and IL-10−IFN-γ+ 2W:I-Ab-specific T cells in the ears at different times after Lm 2W infection (Fig. 4E). IL-10+ cells were unimpaired in their capacity to produce IFN-γ after peptide stimulation in vivo. Collectively, these results show that the elaboration of an optimal Th1 response to Lm requires at least 3–4 weeks, while that of the IL-10 response occurs 1–2 weeks earlier. Furthermore, parasite-specific CD4+ T cells with enhanced cytokine production potential, in particular for IL-10, were enriched in the ears compared to the spleen.
IL-10 producing CD4+ T cells are enriched in the site of antigen-presentation
The observation that cytokine-producing 2W:I-Ab-specific T cells were enriched in the infected ears compared with those in the spleen prompted us to test whether cognate antigen presentation regulated the tissue distribution of these cells. Mice were simultaneously infected with Lm SP-OVA in one ear and Lm 2W in the other, and 2W:I-Ab-specific T cells in each ear and the spleen were evaluated for IFN-γ and IL-10 production 4 or 12 weeks post infection. In the absence of exogenous peptide, about 10% of the tetramer-binding T cells in the ear infected with Lm 2W produced IFN-γ above the levels of those achieved in the ear infected with Lm SP-OVA (Fig. 5), suggesting that this basal level of IFN-γ is due to in situ pMHCII presentation . This experiment also demonstrated that only a fraction of the ear-infiltrating cells are making detectable amounts of IFN-γ at any given time. Virtually no IL-10+ cells could be detected in the absence of exogenous 2W peptide (Fig. 5). Two to four hours after intravenous injection of 2W peptide, ∼40% of the 2W:I-Ab-specific T cells in the spleen made IFN-γ, whereas ∼60% of those in the Lm SP-OVA and Lm 2W ears made IFN-γ. However, the differences between the spleen and each ear were not statistically significant. Importantly, the observation that similar frequencies of 2W:I-Ab-specific T cells in the Lm SP-OVA and Lm 2W ears produced IFN-γ after peptide re-stimulation suggests that IFN-γ production potential is not enhanced by local antigen presentation. In contrast, IL-10+ Th1 cells were largely restricted to the Lm 2W ear, suggesting that IL-10 production potential is indeed regulated by local antigen presentation.
To address the possibility that the inability to detect 2W:I-Ab-specific T-cell IL-10 production in the skin in the absence of exogenous peptide administration was due to the relative insensitivity of the intracellular IL-10 staining, we infected bicistronic IL-10 GFP reporter mice, designated Vert-X . Reporter expression was observed in 5–8.6% of the CD4+CD44high 2W:I-Ab- cells recovered from the uninfected ears of Vert-X mice (Fig. 6A and B). No tetramer binding cells were observed in the ears of the uninfected reporter mice, similar to the wild type mice (data not shown). Following 5 weeks infection with Lm 2W in one ear and Lm SP-OVA in the contra-lateral ear, 7–12% of the 2W:I-Ab-specific T cells were IL-10 GFP+ in the 2W ear compared with 0–5.2% in the OVA ear (Fig. 6A and B). A greater absolute number of the CD4+CD44high T cells in the 2W:I-Ab- compartment in both ears acquired a IL-10 GFP+ phenotype, although their frequencies did not increase over those observed in the uninfected Vert-X mice. These data reinforce the conclusion that IL-10 production is regulated by local antigen presentation.
The present study describes how Lm-specific CD4+ T-cell immunity develops. Parasite-specific CD4+ T cells expand in the infection-draining LN over the course of a few weeks, where they gradually polarize into IFN-γ-producing Th1 cells. After priming, these cells migrate to the infected tissue and spleen. A fraction of the parasite-specific CD4+ T cells rapidly acquire the capacity to make IL-10, which is largely restricted to the site of infection. IL-10+ IFN-γ− T cells appear early in the infection site but do not persist into the chronic phase. In contrast, IL-10+ Th1 cells preserve the ability to produce IFN-γ and IL-10 well into the chronic phase of the infection. Thus, the parasite-specific IL-10 and IFN-γ CD4+ T-cell responses differ in their development and maintenance kinetics.
Enumerating tetramer-binding cells throughout the course of Lm infection revealed that the antigen-specific CD4+ T-cell response to this pathogen was protracted compared with acute infections that induce Th1 responses. Tetramer-binding cells reached the peak of expansion about 1–2 weeks later than endogenous pMHCII-specific responses against LCMV Armstrong and L. monocytogenes and declined more slowly than T cells primed during these other infections [16, 27, 28]. The delayed kinetics of clonal expansion might be a consequence of slow pathogen growth compared with LCMV and L. monocytogenes, thereby limiting the amount of antigen available early in the infection, as has been demonstrated for M. tuberculosis infection . It is likely that using a lower, more physiological inoculum of Lm would produce an even more delayed CD4+ T-cell response . A stable population of pathogen-specific CD4+ T cells has also been documented in response to L. donovani  and M. tuberculosis infection , suggesting that this might be a common feature of locally persistent infections. Sustained antigen presentation in the chronic phase might contribute to the numeric stability of the parasite-specific T-cell pool by inducing iterative rounds of proliferation and/or recruiting new thymic emigrants [4, 32], although naïve Leishmania-specific T cells have been shown to respond poorly in the presence of previously primed cells . Since sustained antigen presentation supports continued CD4+ T-cell expansion [34-36], it is reasonable to surmise that this mechanism could contribute to the stability of pathogen-specific CD4+ T-cell numbers in a chronic infection.
Very few Treg cells were found within the parasite-specific T-cell populations tested, either at 3 or 8 weeks post-infection. The few Foxp3+ cells within the 2W:I-Ab+ and OVAp:I-Ab+ compartments of uninfected mice expanded much less than the Foxp3− cells in response to infection, and the numbers of 2W:I-Ab+ Treg cells appeared not to increase relative to the numbers found in the dLN and spleen of mice. These findings are surprising in light of the thymic-derived Lm-specific Treg cells that have been previously described in B6 mice infected with the wild type Lm strain of the transgenic Lm used in this study . The present findings suggest that the majority of the Treg cells found in the infected skin are not parasite-specific. We cannot exclude the possibility that Treg cells might be more abundant within other Lm-specific populations besides those tested. Formally addressing this point will depend on the generation of pMHCII tetramers to directly track a variety of CD4+ T-cell responses to endogenous Lm antigens. However, considering that 2W:I-Ab-specific Foxp3+ Treg cells have been observed after intradermal immunization with peptide emulsified in Incomplete Freund's adjuvant , the absence of 2W:I-Ab-specific Foxp3+ Treg cells in the infected dermis points to likely differences in the cytokine milieu of Lm infection and IFA immunization that result in activation of functionally distinct CD4+ T-cell subsets within the 2W:I-Ab-specific repertoire.
Most of the parasite-specific T cells detected with tetramer differentiated into Th1 cells, as illustrated by their expression of T-bet and ability to produce IFN-γ after peptide re-stimulation in vivo, and a fraction of these also made IL-10. Our results validate the well-established observation that Lm infection induces a robust Th1 response in mice genetically resistant to infection . IL-10+ Th1 cells that can be recovered from the inflammatory site have also been described in the context of both healing and non-healing Lm infections [13, 23], and have been described in other chronic infections including visceral leishmaniasis , toxoplasmosis  and malaria [2, 39]. The frequency of IFN-γ+ tetramer+ cells increased with time, supporting a model of progressive Th1-cell differentiation . In contrast, the frequency of IL-10+ cells peaked early and was maintained thereafter. This result suggests that IL-10 synthesis is not necessarily a result of long-term stimulation. It is interesting that within the first 2 weeks of the infection, there were IL-10+ cells in the skin that did not make IFN-γ. It was also surprising to see that IL-10+ Th1 cells were not exhausted. This might be explained by the fact that in contrast to the CD8+ and CD4+ T-cell exhaustion that occurs following chronic, systemic viral infections , Lm infection in B6 mice is restricted to the site of infection and to local LNs, such that circulating T cells might be only transiently stimulated. Similarly, parasite-specific CD8+ T cells in mice infected with Trypanosoma cruzi, which chiefly localize to muscle during the chronic phase, did not show impaired effector function . Thus, Lm infection induces a robust Th1 response that develops slowly and is maintained in the chronic phase of the infection.
Our study extends our understanding of how IL-10 production by Th1 cells is regulated in vivo. Following peptide re-stimulation in vivo, IL-10+ Th1 cells were largely restricted to the Lm 2W infected ear. The 2W:I-Ab-specific T cells present in the Lm OVA infected ear presumably home there as a consequence of the inflammation, similar to that described for human leishmaniasis skin lesions found to contain non-antigen specific T cells . But upon their arrival, they do not appear to be locally activated by cognate antigen recognition to produce IL-10 in this site. Similar observations were made using the IL-10 GFP reporter mice in which even in the absence of exogenous peptide 2W:I-Ab-specific T cells that acquired an IL-10 GFP phenotype could be detected that were significantly more frequent in the Lm 2W infected ear compared with the Lm SP-OVA infected ear. Thus IL-10 production potential is chiefly restricted to the site of infection and implicates local antigen presentation in this process. Previous reports indicate that high and/or repeated TCR ligation can induce Th1 cells to make IL-10 [38, 44]. Of particular relevance may be the finding that rested T. gondii-specific IL-10+ Th1 clones produced IL-10 with delayed kinetics compared with recently stimulated ones , suggesting that IL-10 production might be transiently induced at local sites of antigen presentation and lost after cessation of cognate pMHCII-TCR interaction. IL-27, the IL-21R, ICOS, the aryl hydrocarbon receptor, and the transcription factor c-maf have also been shown to drive IL-10 expression [44-48]. Of note, the IL-27R was required for IL-10 production by Th1 cells in a non-healing model of Lm infection . Collectively, our data and the published literature on IL-10 regulation suggest that signals emanating from the TCR, co-stimulatory receptors, and cytokine receptors during CD4+ T cell priming induce IL-10, while continued TCR signaling at the site of infection sustain IL-10 production potential.
The present finding that the protective IFN-γ+ T-cell response develops slowly while the immunosuppresive IL-10+ response emerges relatively early and is sustained locally has important implications for achieving a sterile cure. It suggests that early mobilization of large numbers of Th1 cells, preferably those lacking IL-10 potential, might be necessary to rapidly curtail Lm expansion.
Materials and methods
Female C57BL/6 mice were purchased from Taconic Laboratories. Vert-X (C57BL/6 IL-10/eGFP) mice were generated as described  and were bred in the NIAID animal breeding facility. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Animal Care and Use Committee of the NIAID, NIH (protocol number LPD 68E). All mice were maintained at the NIAID animal care facility under specific pathogen-free conditions.
Parasites and infections
Lm SP-OVA transgenic parasites were generated by transfection of Lm FV1 (MHOM/IL/80/Friedlin) promastigotes as previously described . For the generation of transgenic Lm expressing the 2W peptide, the pKS NEO plasmid was used to express a chimeric protein between the L. donovani 3′-nucleotidase/nuclease (Ld3′NT/NU)  and the C terminus of the 2W peptide (EAWGALANWAVDSA) . To that end, the Ld3′NT/NU gene was amplified by PCR using the following primers. The forward primer, 5′-TGGACTAGTATG GCTCGAGCTCGTTTCCT TCAG-3′ contains a SpeI site followed by the first 24 nucleotides of the Ld3NT/NU gene. The reverse primer, 5′-CCAACTAGTC TACGCCGAGTCCACCGCCCAGTTCGCCAGCGCGCCCCACGCCTCA GCGCTGATGCCTTTCTGATCGTAG-3′ contains nucleotide 981 to 1005 of the Ld3′NT/NU gene followed by 42 nucleotides encoding the 2W peptide, a stop codon, and a SpeI cloning site. The codons used for the 2W peptide sequence in the reverse primer were chosen to reflect the GC-rich Leishmania codon bias. The PCR amplified product was cloned into the SpeI site of the pKS NEO plasmid and the sequence of the NT::2W open reading frame in the pKS NEO NT::2W plasmid was verified by nucleotide sequencing. Lm FV1 (MHOM/IL/80/Friedlin) promastigotes were transfected with pKS NEO NT::2W plasmid by electroporation and selected for growth at 26°C in medium 199 (Gibco BRL) supplemented with 20% heat-inactivated FBS (Gemini), 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, 40 mM HEPES 0.1 mM adenine (in 50 mM HEPES), 5 mg/ml hemin (in 50%) triethanolamine, and 1 mg/ml 6-biotin (Sigma) in presence of geneticin (G418) (Sigma, St. Louis, MO) as previously described . Expression of the chimeric protein was confirmed by SDS-PAGE and Western blotting of total parasite cell lysates and cultures supernatants using the anti-Ld3′NT/NU antibody as previously described .
For mouse infections, infective-stage metacyclic promastigotes were isolated from 4–5 day-old stationary cultures by density centrifugation on a Ficoll gradient . Metacyclic promastigotes (1 × 105) were inoculated in the left hind footpad in a volume of 40 μl, or intra-dermally into the ear using a 27.5-gauge needle in a volume of ∼5 μl. Parasite loads in infected tissues were quantified by serial dilution in biphasic promastigote growth medium as previously described .
pMHCII tetramer production
Soluble 2W:I-Ab, OVA323–339:I-Ab, OVA265–280:I-Ab, and peptide-register trapped OVA-2:I-Ab and OVA-3:I-Ab monomers corresponding to registers 325–335 and 327–338 of chicken ovalbumin, respectively, were produced and biotinylated in Drosophila melanogaster S2 cells and then combined with streptavidin-allophycocyanin or streptavidin-phycoerythrin (Prozyme) to make tetramers, as previously described [11, 17].
Sample preparation, pMHCII tetramer staining, and magnetic enrichment
Individual spleens and retromaxillary (ear-draining) lymph nodes were removed and mechanically dissociated using a syringe plunger. Individual ear tissue was prepared by digestion with DMEM containing 200 μg Liberase CI enzyme blend (Roche Diagnostics Corp.) as previously described . Single cell suspensions of tissue homogenates were filtered using a 70 mm-pore size Falcon cell strainer (BD Biosciences). 2W:I-Ab and OVAp:I-Ab tetramer staining and magnetic enrichment were performed as previously described . Briefly, single cell suspensions of spleen, lymph nodes, or ears were stained with 10 nM allophycocyanin- or phycoerythrin-labeled 2W:I-Ab-streptavidin or OVAp:I-Ab-streptavidin tetramers for 1 h at room temperature. Samples were then incubated with magnetic anti-fluorochrome microbeads and run through a magnetized LS column (Miltenyi Biotec).
Peptide re-stimulation for measurement of cytokine production in vivo
Mice were injected intravenously with 100 μg of 2W peptide (EAWGALANWAVDSA) (GenScript). Spleen and ears were harvested 2–4 h later and homogenized on ice in media supplemented with Brefeldin A (10 ug/ml) (SIGMA) .
Antibodies and flow cytometry
All antibodies were from eBioscience unless indicated. Samples were stained for 30 min at 4°C with Pacific Blue-, eFluor 450-, or PerCP-Cy5.5-conjugated anti-B220 (RA3–6B2), anti-CD11b (MI-70), anti-CD11c (N418), and anti-F4/80 (BM8, Invitrogen), Pacific Orange-conjugated anti-CD8α (5H10, Invitrogen), fluorescein isothiocyanate-conjugated anti-CD3ε (145–2C11), peridinin chlorophyll protein-cyanine 5.5-conjugated anti-CD3ε (145–2C11), or anti-CD4 (RM4–5), Alexa Fluor-conjugated anti-CD44 (IM7), allophycocyanin-Alexa Fluor 750 or allophycocyanin-eFluor 780-conjugated anti-CD4 (RM4–5). In some experiments, samples were treated with the Foxp3 Fixation/Permeabilization Buffer (eBioscience) per manufacturer's instructions, and then stained with eF450-conjugated anti-Foxp3 (FJK-16s) and PE- or AlexaFluor-647-conjugated anti-Tbet (eBio4B10). In other experiments, samples were fixed and permeabilized with BD Cytofix/Cytoperm (Becton-Dickinson) according to the manufacturer's instructions, and subsequently stained for 1 h at 4°C with PE-conjugated anti-IL-10 (JES5–16E3) and PE-Cy7-conjugated anti-IFNγ (XMG1.2). Samples were run on LSRII or Fortessa flow cytometers (Becton-Dickinson) and analyzed with FlowJo (Tree Star).
Statistical tests were performed in Microsoft Excel or GraphPad Prism. Comparisons of absolute cell numbers were done on the log10 of each value to minimize differences in statistical variance of the raw values due to exponential growth. p values <0.05 were considered statistically significant. The two-tailed, unpaired Student's t test was used when comparing two groups, and a one-way analysis of variance (ANOVA) with Bonferroni's post-test was performed when comparing three groups.
We thank Kim Beacht, J. Walter, and R. Speier for expert technical assistance. This work was supported in part by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health, and by grants from the US National Institutes of Health, R37 AI027998, R01 AI039614, and R01 AI066016 (M.K.J.), T32 AI07313 (A.J.P.), and T32 CA9138 (M.P.), and a Frieda M. Kunze Fellowship from the Minnesota Medical Foundation (A.J.P.).
Conflict of interest
The authors declare no financial or commercial conflict of interest.