Mem Inst T-cell responses associated with resistance to Leishmania infection in individuals from endemic areas for Leishmania (Viannia) braziliensis

Abstract

Subclinical or asymptomatic infection is documented in individuals living in endemic areas for leishmaniasis suggesting that the development of an appropriate immune response can control parasite replication and maintain tissue integrity. A low morbidity indicates that intrinsic factors could favor resistance to Leishmania infection. Herein, leishmanial T-cell responses induced in subjects with low susceptibility to leishmaniasis as asymptomatic subjects were compared to those observed in cured cutaneous leishmaniasis (CCL) patients, who controlled the disease after antimonial therapy. All of them have shown maintenance of specific long-term immune responses characterized by expansion of higher proportions of CD4 + as compared to CD8 + Leishmania reactive T-lymphocytes. Asymptomatic subjects had lower indexes of in vitro Leishmania induced lymphoproliferative responses and interferon-gamma (IFN-γ) production in comparison to CCL patients. On the other hand, interleukin (IL-10) production was much higher in asymptomatics than in CCL, while no differences in IL-5 levels were found. In conclusion, long lived T-cell responses achieved by asymptomatic individuals differed from those who had developed symptomatic leishmaniasis in terms of intensity of lymphocyte activation (proliferation or IFN-γ) and regulatory mechanisms (IL-10). The absence of the disease in asymptomatics could be explained by their intrinsic ability to create a balance between immunoregulatory (IL-10) and effector cytokines (IFN-γ), leading to parasite destruction without producing skin tissue damage. The establishment of profiles of cell-mediated immune responses associated with resistance against Leishmania infection is likely to make new inroads into understanding the long-lived immune protection against the disease. Key words: asymptomatic infection -Leishmania (Viannia) braziliensis -cured leishmaniasis -cytokines -T-cell subsetslong term immunity (Grimaldi Jr & MacMahon-Pratt 1991). The spectrum of the clinical presentation ranges from self-healing or benign cutaneous lesions to more severe forms, such as disseminated lesions or mucosal involvement Studies conducted in mice and humans have unequivocally shown that a major T-cell driven component underlies the establishment of acquired immunity and protection against re-infection ( The majority of ATL patients develop cutaneous leishmaniasis (CL) infection or asymptomatic individuals in endemic areas suggests that infected individuals can control Leishmania replication preventing the development of the disease. In this connection, the maintenance of Leishmania specific long-term immunity in asymptomatic subjects reinforces the idea that frequent parasite stimuli can confer protection against Leishmania re-infection or reactivation in individuals from endemic areas In asymptomatic individuals low levels of IFN-γ and TNF-α are induced contrasting with the strong production of these cytokines observed during active leishmaniasis In this study, leishmanial T-cell responses induced in asymptomatic subjects were compared to those observed in clinically cured patients. Our aim was to establish profiles of T-cell phenotypes and cytokines associated with resistance to leishmaniasis. We believe that such profiles can provide a better insight into the immunological mechanisms associated with protection against the disease. SUBJECTS, MATERIALS, AND METHODS Studied population -Twenty-eight individuals from endemic areas for L. (V.) braziliensis (Lb) infection in the state of Rio de Janeiro were studied. The subjects were divided into two groups: 11 asymptomatic subjects (4 males and 7 females, mean age ± SD = 40.2 ± 21 years, median = 37 years), 17 cured CL patients (CCL) evaluated 1-17 years after the end of therapy (8 males and 9 females, mean age ± SD = 42.5 ± 14.2 years, median = 40 years). Asymptomatic individuals had no clinical past history of skin ulcer suggestive of leishmaniasis. Subclinical infection was determined by in vitro evidence of induction of cellular responses to leishmanial antigens (lymphocyte activation-proliferation and/or IFN-γ production). CCL patients were diagnosed with leishmaniasis confirmed by clinical, parasitological, and/ or immunological tests and achieved clinical cure after successful antimonial therapy Sera from asymptomatic subjects or cured patients were non-reactive for the presence of Leishmania specific IgM and IgG immunoglobulins by indirect immunofluorescence. This study was approved by the Ethic Committee of the Fundação Oswaldo Cruz. Informed consent was obtained from all individuals. Lymphocyte proliferative response (LPR) assaysPeripheral blood mononuclear cells (PBMC) were used in LPR assays as previously described Phenotypic analysis -For obtaining leishmanial antigen-reactive T-cells, PBMC (3 × 10 6 per well) were in vitro cultured in 24-well flat-bottomed plates (Nunc) in the presence of the equivalent of 5 × 10 6 disrupted Lb promastigotes under conditions previously described. After five days in culture, cells were harvested and washed, and then blast cells were separated by centrifugation over discontinuous Percoll gradient (Sigma). For phenotypic analysis, Lb-reactive blast T-cells were incubated in the presence of 5 µl of monoclonal antibodies (Coulter Corporation, Hialeah, FL, US) for CD3 + (CD3-RD1), CD4 + (T4-FITC), and CD8 + (T8-RD1). After incubation, the cells were washed three times and resuspended in a fixing solution containing 1% paraformaldehyde in PBS prior to flow cytometric analysis. Blast cells were defined by forward and side-scatter gating. Each sample was run and data was analyzed with EXPO32 software in an EPICS ALTRA flow cytometer (Beckman-Coulter, Miami, US). Each culture's supernatant was collected on day 3 to test IL-5 and IL-10 concentrations and on day 5 to test IFN-γ concentration. Supernatants were stored at -20°C until use. Cytokine measurement -Cytokine assays were performed by enzyme-linked immunosorbent assay (ELISA). Monoclonal antibodies and recombinant cytokines were purchased from BD Biosciences Pharmingen, San Diego, CA, US. All samples were tested in duplicate and compared to standard curves to determine the cytokine concentration. The procedures were performed according to the manufacture's instructions, and the concentration was analyzed using SOFTmax PRO 4.0 program (Life Sciences Edition, Molecular Devices Corporation, US). Results were expressed in picograms per milliliter. The minimum cytokine levels detected were 62.5 pg/ml for IFNγ, 31.2 pg/ml for IL-10, and 15.6 pg/ml for IL-5. Statistical analysis -The Mann-Whitney test was used to compare the results for three groups. The analysis was performed by GraphPad InstatV2.04 (GraphPad Software, San Diego, CA, US) and SPSS (8.0 for Windows) softwares. The results were expressed as mean ± standard deviation and/or median. Mem Inst Oswaldo Cruz, Rio de Janeiro RESULTS LPR of PBMC stimulated in vitro with Lb-Ag -The LPR induced by Lb-Ag was positive (stimulation index ≥ 2.5) in all CCL patients (SI = 15.7 ± 14.7, median = 10.4, n = 17) and asymptomatic subjects (SI = 9.3 ± 9.8, median = 6.2, n = 11) Phenotypic characterization of Lb-reactive T-cells -T lymphocytes preferentially proliferated in response to leishmanial antigens: asymptomatic individuals (T CD3 + = 78 ± 14.7%, median = 78%, n = 02) and CCL patients (T CD3 + = 50 ± 21.7%, median = 49.4%, n = 14). A clear preferential induction of CD4 + over CD8 + T cells was observed in all analyzed groups: asymptomatic individuals -T CD4 + = 43.8 ± 19.8% (median = 47.6%, n = 10) and T CD8 + = 37.6 ± 13% (median = 35.3%, n = 10); CCL patients -T CD4 + = 29.5 ± 20% (median = 23.2%, n = 14), and T CD8 + = 13.8 ± 6.7% (median = 11.5%, n = 14). Higher percentages of CD4 + (p = 0.02) and CD8 + T-cells (p < 0.0001) were observed in asymptomatic individuals as compared to CCL Cytokine production by PBMC stimulated in vitro with Lb-Ag -The mean levels of IFN-γ in the cell culture supernatants from asymptomatic individuals (1282 ± 972 pg/ml, median = 1064 pg/ml, n = 11) were lower than those observed CCL patients (1975 ± 2054 pg/ml, median = 1670 pg/ml, n = 17) (p > 0.05). We found that among CCL there were high and low IFN-γ producers, and this cytokine was not detectable in three patients In contrast, IL-10 levels were significantly higher for asymptomatic individuals (733 ± 233 pg/ml, median = 698 pg/ml, n = 7) in comparison to CCL patients (416 ± 188 pg/ml, median = 403 pg/ml, n = 10) (p = 0.009). IL-5 production was observed in CCL patients (91.3 ± 71.3 pg/ml, median = 42 pg/ml, n = 11), but this cytokine was only detected in two out of nine asymptomatic subjects (45.2 ± 17.8 pg/ml). Immunity in asymptomatic human leishmaniasis • Rita C Bittar et al