Compartmentalized cytotoxic immune response leads to distinct pathogenic roles of natural killer and senescent CD8⁺ T cells in human cutaneous leishmaniasis

Cytotoxic activity mediated by CD8+ T cells is the main signature of the immunopathogenesis of cutaneous leishmaniasis (CL). Here, we performed a broad evaluation of natural killer (NK) cell phenotypic and functional features during cutaneous leishmaniasis. We demonstrate for the first time that CL patients present the accumulation of circulating NK cells with multiple features of replicative senescence including low proliferative capacity and shorter telomeres, elevated expression of CD57, KLRG1 but diminished CD27 stimulatory receptor expression. Moreover, they exhibited higher cytotoxic and inflammatory potential than age‐matched controls. The accumulation of circulating senescent NK cells (CD56dim CD57bright) correlated positively with skin lesion size in the same patients, suggesting that they, like circulating senescent CD8+ T cells, may contribute to the immunopathology of CL. However, this senescent population had lower cutaneous lymphocyte antigen expression and so had diminished skin‐homing potential compared with total or senescent CD8+ T cells. This was confirmed in CL skin lesions where we found a predominance of CD8+ T cells (both senescent and non‐senescent) that correlated with the severity of the disease. Although there was also a correlation between the proportions of senescent NK cells (CD56+ CD57+) in the skin and lesion size, this was less evident. Collectively our results demonstrate first‐hand that senescent cytotoxic cells may mediate skin pathology during human cutaneous leishmaniasis. However, as senescent cytotoxic CD8+ T cells predominate in the skin lesions, they may have a greater role than NK cells in mediating the non‐specific skin damage in CL

PD-1 Blockade Modulates Functional Activities of Exhausted-Like T Cell in Patients With Cutaneous Leishmaniasis

Patients infected by Leishmania braziliensis develop debilitating skin lesions. The role of inhibitory checkpoint receptors (ICRs) that induce T cell exhaustion during this disease is not known. Transcriptional profiling identified increased expression of ICRs including PD-1, PDL-1, PDL-2, TIM-3, and CTLA-4 in skin lesions of patients that was confirmed by immunohistology where there was increased expression of PD-1, TIM-3, and CTLA-4 in both CD4^{+} and CD8^{+} T cell subsets. Moreover, PDL-1/PDL-2 ligands were increased on skin macrophages compared to healthy controls. The proportions PD1^{+}, but not TIM-3 or CTLA-4 expressing T cells in the circulation were positively correlated with those in the lesions of the same patients, suggesting that PD-1 may regulate T cell function equally in both compartments. Blocking PD-1 signaling in circulating T cells enhanced their proliferative capacity and IFN-γ production, but not TNF-α secretion in response to L. braziliensis recall antigen challenge in vitro. While we previously showed a significant correlation between the accumulation of senescent CD8^{+}CD45RA^{+}CD27^{-} T cells in the circulation and skin lesion size in the patients, there was no such correlation between the extent of PD-1 expression by circulating on T cells and the magnitude of skin lesions suggesting that exhausted-like T cells may not contribute to the cutaneous immunopathology. Nevertheless, we identified exhausted-like T cells in both skin lesions and in the blood. Targeting this population by PD-1 blockade may improve T cell function and thus accelerate parasite clearance that would reduce the cutaneous pathology in cutaneous leishmaniasis

Genetic diversity of Leishmania amazonensis strains isolated in northeastern Brazil as revealed by DNA sequencing, PCR-based analyses and molecular karyotyping

Abstract\ud \ud \ud \ud Background\ud \ud Leishmania (Leishmania) amazonensis infection in man results in a clinical spectrum of disease manifestations ranging from cutaneous to mucosal or visceral involvement. In the present study, we have investigated the genetic variability of 18 L. amazonensis strains isolated in northeastern Brazil from patients with different clinical manifestations of leishmaniasis. Parasite DNA was analyzed by sequencing of the ITS flanking the 5.8 S subunit of the ribosomal RNA genes, by RAPD and SSR-PCR and by PFGE followed by hybridization with gene-specific probes.\ud \ud \ud \ud Results\ud \ud ITS sequencing and PCR-based methods revealed genetic heterogeneity among the L. amazonensis isolates examined and molecular karyotyping also showed variation in the chromosome size of different isolates. Unrooted genetic trees separated strains into different groups.\ud \ud \ud \ud Conclusion\ud \ud These results indicate that L. amazonensis strains isolated from leishmaniasis patients from northeastern Brazil are genetically diverse, however, no correlation between genetic polymorphism and phenotype were found.We thank Lucile FloeterWinter for critical reading of the manuscript and Artur T.L. de Queiroz for initial help with phylogenetic analysis. This work is supported by grants from CNPq, FAPESB and PAPES/FIOCRUZ. J.P.C. de Oliveira was supported by a CNPq fellowship; C.I.O. and F.M.C.F were supported by a FAPESB fellowship. AAC, AB, and CIO are senior investigators from CNPq. AB is a senior investigator for Instituto de Investigação em Imunologia (iii).We thank Lucile Floeter-Winter for critical reading of the manuscript and Artur T.L. de Queiroz for initial help with phylogenetic analysis. This work is supported by grants from CNPq, FAPESB and PAPES/FIOCRUZ. J.P.C. de Oliveira was supported by a CNPq fellowship; C.I.O. and F.M.C.F were supported by a FAPESB fellowship. AAC, AB, and CIO are senior investigators from CNPq. AB is a senior investigator for Instituto de Investigação em Imunologia (iii)

Genome of the Avirulent Human-Infective Trypanosome—Trypanosoma rangeli

Background: Trypanosoma rangeli is a hemoflagellate protozoan parasite infecting humans and other wild and domestic mammals across Central and South America. It does not cause human disease, but it can be mistaken for the etiologic agent of Chagas disease, Trypanosoma cruzi. We have sequenced the T. rangeli genome to provide new tools for elucidating the distinct and intriguing biology of this species and the key pathways related to interaction with its arthropod and mammalian hosts.  Methodology/Principal Findings: The T. rangeli haploid genome is ,24 Mb in length, and is the smallest and least repetitive trypanosomatid genome sequenced thus far. This parasite genome has shorter subtelomeric sequences compared to those of T. cruzi and T. brucei; displays intraspecific karyotype variability and lacks minichromosomes. Of the predicted 7,613 protein coding sequences, functional annotations could be determined for 2,415, while 5,043 are hypothetical proteins, some with evidence of protein expression. 7,101 genes (93%) are shared with other trypanosomatids that infect humans. An ortholog of the dcl2 gene involved in the T. brucei RNAi pathway was found in T. rangeli, but the RNAi machinery is non-functional since the other genes in this pathway are pseudogenized. T. rangeli is highly susceptible to oxidative stress, a phenotype that may be explained by a smaller number of anti-oxidant defense enzymes and heatshock proteins.  Conclusions/Significance: Phylogenetic comparison of nuclear and mitochondrial genes indicates that T. rangeli and T. cruzi are equidistant from T. brucei. In addition to revealing new aspects of trypanosome co-evolution within the vertebrate and invertebrate hosts, comparative genomic analysis with pathogenic trypanosomatids provides valuable new information that can be further explored with the aim of developing better diagnostic tools and/or therapeutic targets