CXCR5 and TIM-3 expressions define distinct exhausted T cell subsets in experimental cutaneous infection with Leishmania mexicana.

T-cell exhaustion is a key stage in chronic infections since it limits immunopathology, but also hinders the elimination of pathogens. Exhausted T (Tex) cells encompass dynamic subsets, including progenitor cells that sustain long-term immunity through their memory/stem like properties, and terminally-differentiated cells, resembling the so-called Tex cells. The presence of Tex cells in chronic leishmaniasis has been reported in humans and murine models, yet their heterogeneity remains unexplored. Using flow cytometry, we identified Tex cells subtypes based on PD-1, CXCR5 and TIM-3 expressions in draining lymph nodes (dLNs) and lesion sites of C57BL/6 mice infected with L. mexicana at 30-, 60- and 90-days post-infection. We showed that infected mice developed a chronic infection characterized by non-healing lesions with a high parasite load and impaired Th1/Th2 cytokine production. Throughout the infection, PD-1+ cells were observed in dLNs, in addition to an enhanced expression of PD-1 in both CD4+ and CD8+ T lymphocytes. We demonstrated that CD4+ and CD8+ T cells were subdivided into PD-1+CXCR5+TIM-3- (CXCR5+), PD-1+CXCR5+TIM-3+ (CXCR5+TIM-3+), and PD-1+CXCR5-TIM-3+ (TIM-3+) subsets. CXCR5+ Tex cells were detected in dLNs during the whole course of the infection, whereas TIM-3+ cells were predominantly localized in the infection sites at day 90. CXCR5+TIM-3+ cells only increased at 30 and 60 days of infection in dLNs, whereas no increase was observed in the lesions. Phenotypic analysis revealed that CXCR5+ cells expressed significantly higher levels of CCR7 and lower levels of CX3CR1, PD-1, TIM-3, and CD39 compared to the TIM-3+ subset. CXCR5+TIM-3+ cells expressed the highest levels of all exhaustion-associated markers and of CX3CR1. In agreement with a less exhausted phenotype, the frequency of proliferating Ki-67 and IFN-γ expressing cells was significantly higher in the CXCR5+ subset within both CD4+ and CD8+ T cells compared to their respective TIM-3+ subsets, whereas CD8+CXCR5+TIM-3+ and CD8+TIM-3+ subsets showed an enhanced frequency of degranulating CD107a+ cells. In summary, we identified a novel, less-differentiated CXCR5+ Tex subset in experimental cutaneous leishmaniasis caused by L. mexicana. Targeting these cells through immune checkpoint inhibitors such as anti-PD-1 or anti PD-L1 might improve the current treatment for patients with the chronic forms of leishmaniasis.

Alpha-galactosylceramide as adjuvant induces protective cell-mediated immunity against Leishmania mexicana infection in vaccinated BALB/c mice.

Adjuvants represent a promising strategy to improve vaccine effectiveness against infectious diseases such as leishmaniasis. Vaccination with the invariant natural killer T cell ligand α-galactosylceramide (αGalCer) has been used successfully as adjuvant, generating a Th1-biased immunomodulation. This glycolipid enhances experimental vaccination platforms against intracellular parasites including Plasmodium yoelii and Mycobacterium tuberculosis. In the present study, we assessed the protective immunity induced by a single-dose intraperitoneal injection of αGalCer (2 µg) co-administrated with a lysate antigen of amastigotes (100 µg) against Leishmania mexicana infection in BALB/c mice. The prophylactic vaccination led to 5.0-fold reduction of parasite load at the infection site, compared to non-vaccinated mice. A predominant pro-inflammatory response was observed in challenged vaccinated mice, represented by a 1.9 and 2.8-fold-increase of IL-1ß and IFN-γ producing cells, respectively, in the lesions, and by 23.7-fold-increase of IFN-γ production in supernatants of restimulated splenocytes, all compared to control groups. The co-administration of αGalCer also stimulated the maturation of splenic dendritic cells and modulated a Th1-skewed immune response, with high amounts of IFN-γ production in serum. Furthermore, peritoneal cells of αGalCer-immunized mice exhibited an elevated expression of Ly6G and MHCII. These findings indicate that αGalCer improves protection against cutaneous leishmaniasis, supporting evidence for its potential use as adjuvant in Leishmania-vaccines.

Leishmania mexicana: Novel Insights of Immune Modulation through Amastigote Exosomes.

Exosomes are extracellular microvesicles of endosomal origin (multivesicular bodies, MVBs) constitutively released by eukaryotic cells by fusion of MVBs to the plasma membrane. The exosomes from Leishmania parasites contain an array of parasite molecules such as virulence factors and survival messengers, capable of modulating the host immune response and thereby favoring the infection of the host. We here show that exosomes of L. mexicana amastigotes (aExo) contain the virulence proteins gp63 and PP2C. The incubation of aExo with bone marrow-derived macrophages (BMMs) infected with L. mexicana led to their internalization and were found to colocalize with the cellular tetraspanin CD63. Furthermore, aExo inhibited nitric oxide production of infected BMMs, permitting enhanced intracellular parasite survival. Expressions of antigen-presenting (major histocompatibility complex class I, MHC-I, and CD1d) and costimulatory (CD86 and PD-L1) molecules were modulated in a dose-dependent fashion. Whereas MHC-I, CD86 and PD-L1 expressions were diminished by exosomes, CD1d was enhanced. We conclude that aExo of L. mexicana are capable of decreasing microbicidal mechanisms of infected macrophages by inhibiting nitric oxide production, thereby enabling parasite survival. They also hamper the cellular immune response by diminishing MHC-I and CD86 on an important antigen-presenting cell, which potentially interferes with CD8 T cell activation. The enhanced CD1d expression in combination with reduction of PD-L1 on BMMs point to a potential shift of the activation route towards lipid presentations, yet the effectivity of this immune activation is not evident, since in the absence of costimulatory molecules, cellular anergy and tolerance would be expected.

TNF contributes to T-cell exhaustion in chronic L. mexicana infections of mice through PD-L1 up-regulation.

Leishmania mexicana can produce chronic infections leading to exhausted T cell phenotypes, mediated by PD-1/PD-L1. Little is known on mechanisms that induce these inhibitory molecules in chronic leishmaniasis. We analyzed factors that contribute to exhausted phenotypes in chronic L. mexicana infections of mice. Our results show that draining lymph node cells express enhanced levels of PD-1/PD-L1. T lymphocytes producing low cytokine levels were also found. L. mexicana infection of dendritic cells (DCs) produced elevated amounts of TNF and showed up-regulation of PD-L1 expression. We provide evidence that T cells of chronic L. mexicana infections in mice are functionally exhausted due to chronic TNF production, which leads to PD-L1 up-regulation in DCs. We conclude that TNF has a fundamental role in promoting T cell exhaustion during chronic L. mexicana infections, which contributes to the inability of T cells to proliferate and produce pro-inflammatory cytokines, thus favoring disease progression.

The mKate fluorescent protein expressed by Leishmania mexicana modifies the parasite immunopathogenicity in BALB/c mice.

Parasites have been engineered to express fluorescent reporter proteins, yet the impact of red fluorescent proteins on Leishmania infections remains largely unknown. We analysed the infection outcome of Leishmania mexicana parasites engineered for the constitutive expression of mKate protein and evaluated their immunogenicity in BALB/c mice. Infection of BALB/c mice with mKate transfected L. mexicana (LmexmKate ) parasites caused enlarged lesion sizes, leading to ulceration, and containing more parasites, as compared to LmexWT . The mKate protein showed immunogenic properties inducing antibody production against the mKate protein, as well as enhancing antibody production against the parasite. The augmented lesion sizes and ulcers, together with the more elevated antibody production, were related to an enhanced number of TNF-α and IL-1ß producing cells in the infected tissues. We conclude that mKate red fluorescent protein is an immunogenic protein, capable of modifying disease evolution of L. mexicana.