Long-Lived Skin-Resident Memory T Cells Contribute to Concomitant Immunity in Cutaneous Leishmaniasis

Abstract

Memory T cells, which protect against reinfection in many diseases, have predominantly been characterized in models of acute viral or bacterial infection. In contrast, memory T cells are less well understood in diseases where pathogens persist following disease resolution, such as leishmaniasis, in spite of the fact that these infections often lead to immunity to reinfection, termed concomitant immunity. Defining the T cells that mediate concomitant immunity is an important step in developing vaccines for these diseases. One set of protective T cells are short-lived effector T cells requiring constant stimulation, which would be difficult to maintain by vaccination. However, parasite-independent memory T cells, including central memory T cells (Tcm) and skin-resident T cells (Trm) have recently been described in leishmaniasis. Given their location, Trm cells are particularly suited for protection, and were found to globally seed the skin following Leishmania infection or immunization. Upon challenge, Trm cells rapidly respond to reduce the parasite burden, suggesting that developing strategies to generate parasite-independent Trm cells will be an important step in the quest for a successful leishmaniasis vaccine.

The maintenance of a residual pool of pathogen-specific memory T cells following infection is critical for resistance to reinfection, and characterization of these memory T cells and elucidation of the mechanisms involved in their generation and maintenance is of particular importance in the development of vaccines. A substantial research effort has revealed that memory T cells are not simply cells that escape activation-induced cell death following infection but rather are a heterogenous population that can develop at different times during the immune response, and have distinct life spans, migration patterns, and functions (Kaech and Wherry 2007; Sallusto et al. 2010; Pepper and Jenkins 2011; Kaech and Cui 2012; Clark 2015; Laidlaw et al. 2016; Mueller and Mackay 2016; Macallan et al. 2017; Ho and Kupper 2019). However, despite these advances, significant gaps remain in our understanding of how memory T cells can be generated following vaccination, which is in large part the reason it has been so difficult to develop vaccines for diseases not controlled by antibody.

The most popular experimental models for characterizing memory T cells have been acute viral (e.g., lymphocytic choriomeningitis virus [LCMV]) or bacterial (listeria) infections. These have been particularly useful models for understanding memory T cells as sterile immunity is induced following disease resolution, and thus the T cells that survive are by definition memory T cells (Kaech and Wherry 2007; Condotta et al. 2012). In contrast to these acute infections, multiple infections follow a pattern in which clinical disease resolves, but a low level of pathogens persist. For example, tuberculosis patients can maintain low levels of bacteria for many years (Flynn and Chan 2001). Similarly, resolution of disease from protozoal infections caused by Trypanosoma cruzi or Toxoplasma gondii is followed by long-term persistence of the parasites, and recently there has been renewed interest in viral infections where persistent virus can be observed long after clinical cure (McCarthy and Morrison 2017; Randall and Griffin 2017). One of the best-studied examples of this type of infection is experimental cutaneous leishmaniasis caused by Leishmania major, the model that was first used to define the in vivo factors that promote Th1 and Th2 cell development (Scott et al. 1988; Heinzel et al. 1989). Following resolution of a primary infection, C57BL/6 mice are immune to a challenge infection but maintain a low level of persistent parasites. This immunity, referred to as concomitant immunity, protects against subsequent infections and is considered the “gold standard” for protection, although it fails to result in sterile immunity following a challenge infection (Mandell and Beverley 2016). In all of these persistent infections, the presence of persistent antigen complicates the functional identification of memory T cells, leaving open the question of how to define memory T cells when pathogens persist, and whether memory T cells even develop under such conditions.

Immunity to Cutaneous Leishmaniasis

Leishmania are parasitic protozoa that are transmitted by sand flies and cause a wide spectrum of diseases, ranging from self-healing lesions to chronic metastatic disease that is difficult to treat with current chemotherapeutics (Scott and Novais 2016). The parasites primarily replicate in macrophages and are controlled when macrophages are activated by interferon (IFN)-γ from CD4⁺ Th1 cells. There is no role for antibodies in protection, which is an advantage in studying T-cell memory, as protection against a secondary challenge is solely dependent on T cells. CD8 T cells may play a dual role in cutaneous leishmaniasis, as production of IFN-γ by CD8 T cells can be protective (Belkaid et al. 2002; Uzonna et al. 2004), while cytolytic CD8 T cells in lesions fail to kill the parasites but rather promote NLRP3 inflammasome activation and release of proinflammatory interleukin (IL)-1β, thereby exacerbating disease (Novais et al. 2013, 2017). Host genetics strongly influences the outcome of infection. For example, C57BL/6 mice infected with L. major develop a strong Th1 response and resolve their lesions in 8 to 12 weeks, while BALB/c mice develop a Th2 response and an uncontrolled infection leading to metastatic lesions and eventual death (Scott and Novais 2016). Although this stark dichotomy may be less evident in patients, control of the parasites (although not always control of the disease) in human leishmaniasis is completely dependent upon a Th1-type response (Scott and Novais 2016).

Resolution of leishmanial lesions is followed by the development of lifelong immunity to reinfection. Experimentally, this immunity is evident following resolution of L. major infection of C57BL/6 mice, which upon challenge exhibit a delayed-type hypersensitivity (DTH) response at the challenge site, but fail to develop lesions. The noted resistance to disease developed following resolution of a cutaneous lesion in humans provided the basis for a procedure termed leishmanization, during which individuals were infected with live parasites in an innocuous site to prevent subsequent infections leading to disfiguring facial lesions (Senekji and Beattie 1941; Khamesipour et al. 2005). Although discontinued because of complications where lesions failed to resolve, as well as the potential for the development of severe disease if the individual became immunosuppressed, these real-world results provided clear evidence that immunity is induced following resolution of leishmanial lesions not only in experimental animals but also in humans. However, whether that immunity depended upon persistent parasites was impossible to assess in these “immunized” individuals.

THE ROLE OF PERSISTENT PARASITES IN MAINTAINING IMMUNITY

The persistence of leishmania—or any other pathogen—and the development of concomitant immunity following resolution of clinical disease raises several questions. First, what T cells provide protection? Second, how do these persistent parasites survive in the face a strong concomitant immunity that protects against challenge? Third, why do these persistent parasites fail to induce T-cell exhaustion, thus limiting resistance to reinfection? Finally, is concomitant immunity totally dependent upon persistent parasites or can protective memory T cells develop in a parasite-independent manner? Results from many laboratories found that following leishmanial lesion resolution circulating effector T cells (Teff) that can provide protection to challenge are present in mice (Alexander and Phillips 1980; Powrie et al. 1994; Zaph et al. 2004; Mou et al. 2014; Peters et al. 2014). These were mostly CD4⁺ T cells, although double-negative class II–restricted T cells were also found to provide protection in adoptive transfer experiments (Mou et al. 2014). These circulating CD4⁺ Teff cells were characterized as short-lived T-bet⁺, Ly6C⁺ T cells that migrate to the site of a challenge infection within days, thus providing protection to challenge (Peters et al. 2014; Hohman and Peters 2019) but were not able to be maintained in the absence of persistent parasites.

One attractive idea for how parasites may persist in the face of a strong Teff cell response is that they may be able to hide in fibroblasts, neutrophils, and/or M2 macrophages, all of which have been identified as potential safe havens (Bogdan et al. 2000; Peters et al. 2008; Lee et al. 2018). However, while some parasites may hide from the immune response, many appear to continually replicate in macrophages, where an equilibrium between parasite destruction and survival leads to a relatively constant low number of parasites (Mandell and Beverley 2017). It remains unclear why not all of the parasites are killed, but there is strong support for the notion that regulatory mechanisms limit the Teff-cell response. Such regulation may also potentially explain why Teff cells do not become functionally exhausted. The most convincing evidence that regulatory mechanisms prevent complete T cell–mediated elimination of parasites comes from the observation that some mice infected with low doses of L. major are able to clear all the parasites in the absence of IL-10 (Belkaid et al. 2001).

Because Teff cells provide protection to challenge infection, and presumably would require constant stimulation, a logical conclusion is that persistent parasites are required to maintain immunity. Consistent with this possibility are studies suggesting that when parasites are completely eliminated, as can occur following challenge with a very low number of parasites or in the absence of IL-10, protection is lost (Uzonna et al. 2001; Belkaid et al. 2002). These results suggest that maintenance of immunity requires persistent parasites, and it would be expected that the absence of persistent parasites would deplete the pool of circulating Teff cells, which would lessen resistance. However, whether a small population of memory T cells survive that are insufficient to provide protection is unknown. Alternatively, it is possible that memory T cells develop during a normal infection but that the absence of IL-10 compromises their development. This would be consistent with the finding that IL-10 and STAT3 deficiency limits memory T–cell development (Cui et al. 2011; Laidlaw et al. 2015). Thus, to conclusively address this issue will require other approaches to eliminate persistent parasites in experimental animals following an infection in which the presence of memory T cells can be assessed.

IMMUNITY WITHOUT PERSISTENT PARASITES

Memory T cells are functionally defined as those T cells that remain long term once an infection is cleared. Thus, while it is relatively easy to identify Tms following sterile resolution of an acute infection, it is more challenging to do so in the case of concomitant immunity. In the latter case, the most straightforward approach to identify memory T cells would be to eliminate the persistent pathogens and determine whether pathogen-specific T cells remain. As discussed above, blocking the regulatory influence of IL-10 in L. major infections can lead to parasite clearance and loss of immunity, but has the caveat that IL-10 depletion may influence memory T–cell development (Cui et al. 2011; Laidlaw et al. 2015). A better approach might be to eliminate the persistent parasites by anti-leishmania drug treatment. Unfortunately, even sustained drug treatment in L. major–infected mice fails to eliminate all the parasites (P Scott, unpubl.). However, this approach has been used successfully in T. cruzi infections (Bustamante et al. 2008; Rosenberg et al. 2016), where they found that long-term antiparasite drug treatment, which completely eliminated the parasite, rendered mice resistant to reinfection, indicating that memory T cells contributed to concomitant immunity following T. cruzi infection independent of persistent parasites.

We took an alternative approach and infected mice with an attenuated L. major parasite, which was generated by deletion of the dhfr-ts (dihydrofolate reductase–thymidylate synthase) gene (Titus et al. 1995). These dhfr-ts-deficient L. major parasites failed to survive long term in vivo, and following their clearance a population of Leishmania-specific CD4 T cells, with the phenotype of central memory T cells (Tcm) (CD62L^hi, CCR7^hi), were present (Zaph et al. 2004). Adoptive transfer of these persisting T cells to naive mice provided protection against L. major challenge, although the protection was delayed as compared to that observed in mice with persistent parasites. These results demonstrate that Tcm cells can indeed survive in the absence of persistent leishmania, although the protection they afford was, as might be expected, less than that mediated by Teff cells.

Tcm cells in leishmaniasis appear to be generated early after infection, and to cease active proliferation after a few rounds of division (Colpitts and Scott 2010). In addition, they maintain their ability to develop into either Th1- or Th2-type cells, suggesting they are not derived from committed CD4⁺ Th1 cells (Pakpour et al. 2008). This raises challenges in targeting them in a vaccine, as adjuvants that are known to promote Th1-cell development, such as IL-12 (Afonso et al. 1994; Pearce and Shen 2007), may promote expansion of short-lived Th1 cells while compromising development of memory T cells (Pearce and Shen 2007).

Taken together, these studies demonstrate that there are at least two circulating populations of Leishmania-specific CD4⁺ T cells present in L. major–infected mice. One subset, Teff cells, fails to survive in the absence of persistent parasites, while a population of Tcm cells develop early after infection and are long-lived in the absence of parasites (Fig. 1). These Tcm cells migrate through other lymph nodes and are poised to respond upon a secondary challenge leading to an expanded pool of Teff cells. The protective capacity of the Tcm cells was demonstrated in adoptive transfer experiments, where Tcm cells isolated from previously infected mice provided resistance to reinfection when transferred to naive mice (Zaph et al. 2004). However, as might be expected, this resistance is delayed compared with the rapid protection afforded mice that have a preexisting population of Teff cells. Whether Tcm cells are the only circulating Leishmania-specific T cells that might be genuine memory T cells is unknown, although in addition to Teff cells and Tcm cells, a population with an effector memory phenotype (CD4⁺, CD62L^low, IL-7R^hi) and a population of class II–restricted, double-negative (CD4^–D8^–) T cells are present in immune mice and humans, but their longevity in the absence of parasites has not been tested (Antonelli et al. 2006; Colpitts et al. 2009; Mou et al. 2014).

Figure 1.

Central memory T cells (Tcm) provide a pool of Leishmania-reactive T cells poised to respond and differentiate into effector T cells (Teff) upon rechallenge. Following infection, activated CD4 T cells in the draining lymph nodes develop into either Teff cells that migrate to the site of infection or Tcm cells that circulate though lymph nodes (1). Upon a secondary challenge, Tcm cells in lymph nodes draining the site of the challenge proliferate and differentiate into Teff cells (2). These Teff cells then migrate to the secondary site of infection to assist in control of the parasites (3). (DC) dendritic cell, (IFN) interferon.

SKIN-RESIDENT MEMORY T CELLS PROVIDE PROTECTION

A hallmark of T cells is their ability to constantly circulate in the blood and lymphatics, which provides them with the capacity to respond wherever an infection might occur. This is a critical characteristic of naive T cells, as the frequency of any one T cell specificity is low, and it is only by circulating that the T cells are able to find their cognate antigen. Once activated, many T cells continue to circulate, but a subset of these activated T cells migrate into tissues and become resident (Masopust et al. 2010; Schenkel and Masopust 2014; Clark 2015; Park and Kupper 2015; Mueller and Mackay 2016; Liu et al. 2018; Ho and Kupper 2019). The most well-characterized resident memory T cells (Trm) are CD8⁺ T cells, which have been identified in almost every tissue in the body, and mediate protection against infections or in some cases promote autoimmunity (Willemsen et al. 2019). In contrast, while our understanding of CD4⁺ Trm cells is limited compared with CD8⁺ Trm cells, CD4⁺ Trm cells have been described in several tissues, including the lung, gut, and skin (Teijaro et al. 2011; Jiang et al. 2012; Mackay et al. 2012; Clark 2015; Beura et al. 2019). A large number of Trm cells reside in the skin (Clark 2015), a location ideal for responding rapidly to infection. In leishmaniasis, a resident population of CD4⁺ T cells that produce IFN-γ upon restimulation are present in the skin, and, similar to other studies, these Trm cells are not restricted to the site of infection but are present globally throughout the skin (Jiang et al. 2012; Glennie et al. 2015; Davies et al. 2017).

Functional analysis of Trm cells in the skin is uniquely enabled by the ability to transfer the cells by skin graft to naive recipients to assess their protective role in the absence of an existing population of circulating Teff cells. Using this technique, we grafted skin isolated at a distance from the initial site of L. major infection (containing no parasites) or skin from naive mice onto naive mice. Significant protection to a challenge infection was observed when the skin graft from immune mice was challenged with L. major when compared with skin grafts from naive animals (Glennie et al. 2015). Importantly, Trm cells were maintained in the skin graft for more than a month, indicating both that the CD4 Trm cells were indeed resident, and also that they did not require the presence of persistent parasites to be maintained. In fact, the striking aspect of these results is that substantial protection was obtained without any preexisting Leishmania-specific Teff cells. Even more striking, RAG mice receiving a skin graft from immune mice and subsequently challenged with Leishmania were significantly protected, clearly establishing a central role for these Leishmania-specific Trm cells in protection. On further confirmation, parabiosis experiments in which naive and immune mice were surgically joined to allow circulating cells to equilibrate between the mice, but in which Trm cells stayed in the skin of the immune mice, revealed that upon challenge with L. major, the immune parabiont was significantly protected within 72 hours, while the naive parabiont containing only circulating Teff cells exhibited no protection at this early time point (Glennie et al. 2017).

A critical aspect of Leishmania-specific Trm cells is their ability to enter and reside in noninflamed skin distant from the lesions, thereby allowing them to protect against challenge infections at distal sites. Entry of T cells into noninflamed skin is mediated at least in part by the expression of P and E selectin ligands, which bind to endothelial expressed selectins (Jiang et al. 2012). Once in the skin, other factors promote T-cell residency. T-cell exit from lymphoid organs and tissues is highly regulated by several factors, and the presence or absence of some of these molecules can predict residency of T cells in specific tissues. One of these, the sphingosine-1-phosphate-1 receptor (S1PR1) promotes egress of T cells, and is counterbalanced by the expression of the transmembrane C-type lectin CD69 (Shiow et al. 2006). Thus, CD69 expression on T cells in the tissues can be indicative of T-cell residency, while expression of S1PR1, as well as the transcription factor Kruppel-like factor 2 (KFL-2), which promotes S1P1R expression, stimulates T-cell migration and are more likely to be expressed on circulating cells (Carlson et al. 2006). When CD4⁺ T cells were sorted into CD69⁺ and CD69^– cells and assessed for gene expression, CD4⁺CD69⁺ T cells expressed low levels of both S1pr1 and Klf2, while CD69^– T cells in tissues or T cells in the draining lymph node expressed higher levels of these molecules (Fig. 2). The transcription factor Blimp-1, encoded by Prdm1, and the related transcription factor, Hobit, encoded by Zfp683, are associated with residency of T cells (Mackay et al. 2016), and both were found to be preferentially expressed by T cells from leishmanial lesions compared with the draining lymph node, with Blimp-1 most highly expressed in CD69⁺ T cells. These results suggest that leishmanial lesions contain a population of CD4⁺ T cells that are most likely migrating through the tissues (CD69^–), and another population of resident T cells that are CD69⁺.

Figure 2.

CD4 T cells in lesions exhibit gene expression patterns consistent with resident memory T cells. CD4⁺CD69⁺ and CD4⁺CD69^– T cells were sorted from Leishmania major lesions (ear) and CD4⁺CD44⁺ T cells sorted from the draining lymph nodes 5 weeks postinfection. Quantitative polymerase chain reaction (qPCR) was performed to identify genes associated with T-cell residency (ND Glennie, FO Novais, and P Scott, unpubl.).

Following a primary infection with L. major, Leishmania-specific Th1 Teff cells proliferate in the draining lymph nodes and subsequently migrate to the site of infection (Fig. 3). Teff cells also migrate into noninflamed skin distant from the primary lesion site, where some transit through the skin and return to the circulation via the lymph nodes, while others become Trm cells. Upon secondary challenge, Trm cells act as a first line of defense by producing IFN-γ that can activate macrophages to kill the parasites. The activated Trm cells also induce the production of CCL2 and CCL7, leading to the recruitment of inflammatory monocytes (Goncalves et al. 2011; Glennie et al. 2017). These monocytes are primed to kill the parasites by production of both nitric oxide and reactive oxygen species (Goncalves et al. 2011; Glennie et al. 2017; Romano et al. 2017), thus providing an early level of parasite control. The ensuing inflammatory response promotes Teff recruitment to the lesions, which provides further protection via the production of IFN-γ.

Figure 3.

Resident memory T cells (Trm) in the skin rapidly respond to challenge, leading to recruitment of inflammatory monocytes and effector T cells (Teff) from the blood. Following infection, activated CD4T cells in the draining lymph nodes develop into Teff cells that migrate to the site of infection (1). Some migrate into noninflamed skin distant from the lesion and become Trm cells, while others recirculate back to the circulation via the lymphatics (2). Upon challenge, Trm cells respond, produce interferon (IFN)-γ, and stimulate the production of chemokines that recruit inflammatory monocytes as well as Teff cells (3). In the lymph node draining the site of secondary challenge, Tcm cells differentiate into Teff cells that contribute to the circulating Teff pool (not shown). (DC) dendritic cell, (NO) nitric oxide, (ROS) reactive oxygen species.

VACCINES

Cutaneous leishmaniasis poses a conundrum for vaccine development. On the one hand, control of a primary infection is highly protective, a prospect that bodes well for any potential vaccine. On the other hand, the parasite is never fully cleared, and it could be argued that maintaining the most robust immunity would require the presence of persistent parasites. While a live vaccine that provides persistent stimulation of T cells provides excellent protection (leishmanization), it would be problematic to deliver a vaccine that results in the continued presence of parasites that in the future might cause disease. And while strategies to deliver antigen on some recurring basis might be developed, it is not clear how that would be practically achieved. Fortunately, such a protocol may not be necessary, as there are examples of more traditional approaches promoting long-term resistance (Coler et al. 2015; Mou et al. 2015; Duthie et al. 2017; Osman et al. 2017; Louis et al. 2019).

Because our current successful vaccines depend upon the induction of a strong antibody response, and as antibodies play no role in protection to leishmaniasis, it is not surprising that we have yet to develop a Leishmania vaccine. Indeed, this failure is not unique to leishmaniasis, but is true for many infections that depend upon maintaining a memory T–cell response. Nevertheless, there are examples of experimental leishmaniasis vaccines that are very promising, and have already been shown to exhibit immunogenicity in humans (Coler et al. 2015; Mou et al. 2015; Duthie et al. 2017; Osman et al. 2017). Additionally, the success of these vaccines in experimental models demonstrates the feasibility of generating long-term immunity via immunization.

The vaccines already tested for immunogenicity in humans contain recombinant antigens identified by antibodies from leishmaniasis patients or identified as they were expressed on the parasite surface, and are designed to target either CD4 or CD8 T cells. An alternative approach to identifying potentially protective Leishmania antigens was to identify peptides being presented to T cells by Leishmania-infected, antigen-presenting cells (Mou et al. 2015). This approach identified the first immunodominant Leishmania antigen. This protein, phosphoenolpyruvate carboxykinase (PEPCK), is recognized by ∼20% of L. major–specific CD4T cells at the peak of infection and has broad cross-reactivity with different Leishmania species. Importantly, T cells from leishmaniasis patients responded to this protein, and immunization in mice with PEPCK using a variety of different strategies led to long-term protection against a challenge with leishmaniasis. These results further indicate that the presence of persistent parasites is not required to generate long-term memory responses.

Because CD4 Trm cells are maintained long term in the absence of persistent parasites, they would appear to be excellent targets for a vaccine. The question then is how best to induce these cells. Studies with vaccinia demonstrated that scarification of the skin and exposure to the virus was particularly effective at generating skin Trm cells (Jiang et al. 2012; Park and Kupper 2015; Ho and Kupper 2019). To determine whether exposure via the skin was critical for generating Leishmania-specific immunity, mice were immunized intradermally or intramuscularly with PEPCK DNA followed by electroporation (Louis et al. 2019). While both routes induced a strong systemic T-cell response, only the intradermal route led to the generation of Trm cells in the skin. These were present both at the site of immunization but also at distant skin sites. When intradermally immunized mice were challenged with L. major long after immunization they exhibited significant protection, while mice immunized by the intramuscular route did not (Louis et al. 2019). More studies need to be done to characterize the T-cell populations stimulated by this immunization, but one can hypothesize that similar to what occurs in L. major–infected mice, the immunization induces CD4⁺ T-cell proliferation in the draining lymph node, followed by circulation of the T cells in the blood and entry into noninflamed skin sites where they differentiate into Trm cells. One can predict that upon challenge, the Trm cells recruit inflammatory monocytes to the infection site that provide early protection (Glennie et al. 2017), followed by priming of T cells to become Teff cells in the draining lymph nodes that circulate and further act to limit parasite proliferation. Whether Tcm cells are generated by this immunization and contribute to the protection observed in the intradermally immunized mice has yet to be tested.

CONCLUDING REMARKS

When an infection is completely cleared it is easy to identify memory T cells, as by definition these are the cells that are maintained long term following resolution of disease. In contrast, in some infections, low levels of residual pathogens are maintained, and concomitant immunity protects against reinfection. This raises the question of whether bona fide memory T cells develop in these situations. This is not simply an academic question, as the development of vaccines for these diseases will depend upon generating protective memory T cells. Whereas it would be expected that circulating Teff cells might depend upon continued stimulation, it is now clear that protective memory T cells do indeed develop during these infections, and that these T cells can be maintained in the absence of persistent pathogens. This was directly demonstrated in T. cruzi–infected mice, which maintained immunity when all the parasites were cleared by drug treatment (Bustamante et al. 2008; Rosenberg et al. 2016). Similarly, the data are clear in leishmaniasis, where persistent parasites are not required to maintain a population of CD4⁺ Tcm or Trm cells (Zaph et al. 2004; Glennie et al. 2015, 2017; Louis et al. 2019), and experimentally immunization can promote the generation of Trm cells and long-term resistance to reinfection (Louis et al. 2019).

The development of a leishmanial vaccine will have a profound impact on the disease burden for individuals living in Leishmania-endemic regions, as currently the drugs for controlling the parasites are often ineffective and toxic. Whereas a human vaccine has yet to be developed, as described above several experimental vaccines show promise. Notably, the induction of protective long-lived skin Trm cells that are not dependent upon persistent parasites following infection or immunization is encouraging and suggests that these cells poised to respond at the very site where a challenge infection will occur may be the best T-cell targets for a vaccine. While concomitant immunity has a component that is pathogen dependent, it does not preclude the generation of pathogen-independent memory T cells by infection or immunization. This is good news for the prospect of developing a leishmaniasis vaccine.