T-cell receptor Vβ repertoire of CD8+ T-lymphocyte subpopulations in cutaneous leishmaniasis patients from the state of Rio de Janeiro, Brazil

In human cutaneous leishmaniasis (CL), the immune response is mainly mediated by T-cells. The role of CD8⁺ T-lymphocytes, which are related to healing or deleterious functions, in affecting clinical outcome is controversial. The aim of this study was to evaluate T-cell receptor diversity in late-differentiated effector (LDE) and memory CD8⁺ T-cell subsets in order to create a profile of specific clones engaged in deleterious or protective CL immune responses. Healthy subjects, patients with active disease (PAD) and clinically cured patients were enrolled in the study. Total CD8⁺ T-lymphocytes showed a disturbance in the expression of the Vβ2, Vβ9, Vβ13.2, Vβ18 and Vβ23 families. The analyses of CD8⁺T-lymphocyte subsets showed high frequencies of LDE CD8⁺T-lymphocytes expressing Vβ12 and Vβ22 in PAD, as well as effector-memory CD8⁺ T-cells expressing Vβ22. We also observed low frequencies of effector and central-memory CD8⁺ T-cells expressing Vβ2 in PAD, which correlated with a greater lesion size. Particular Vβ expansions point to CD8⁺ T-cell clones that are selected during CL immune responses, suggesting that CD8⁺ T-lymphocytes expressing Vβ12 or Vβ22 are involved in a LDE response and that Vβ2 contractions in memory CD8⁺T-cells are associated with larger lesions.

The sample processing time interval as an influential factor in flow cytometry analysis of lymphocyte subsets

The objective of this paper is to propose a protocol to analyze blood samples in yellow fever 17DD vaccinated which developed serious adverse events. We investigated whether or not the time between sample collection and sample processing could interfere in lymphocyte subset percentage, for it is often impossible to analyze blood samples immediately after collection due to transport delay from collection places to the flow cytometry facility. CD4+CD38+ T, CD8+CD38+ T, CD3+ T, CD19+ B lymphocyte subsets were analyzed by flow cytometry in nine healthy volunteers immediately after blood collection and after intervals of 24 and 48 h. The whole blood lysis method and gradient sedimentation by Histopaque were applied to isolate peripheral blood mononuclear cells for flow cytometry analyses. With the lysis method, there was no significant change in lymphocyte subset percentage between the two time intervals (24 and 48 h). In contrast, when blood samples were processed by Histopaque gradient sedimentation, time intervals for sample processing influenced the percentage in T lymphocyte subsets but not in B cells. From the results obtained, we could conclude that the whole blood lysis method is more appropriate than gradient sedimentation by Histopaque for immunophenotyping of blood samples collected after serious adverse events, due to less variation in the lymphocyte subset levels with respect to the time factor.

Lymphocyte subset analyses in healthy adults vaccinated with yellow fever 17DD virus

In this study the kinetics of humoral and cellular immune responses in first-time vaccinees and re-vaccinees with the yellow fever 17DD vaccine virus was analyzed. Flow cytometric analyses were used to determine percentual values of T and B cells in parallel to the yellow fever neutralizing antibody production. All lymphocyte subsets analyzed were augmented around the 30th post vaccination day, both for first-time vaccinees and re-vaccinees. CD3+ T cells increased from 30.8 percent (SE ± 4 percent) to 61.15 percent (SE ± 4.2 percent), CD4+ T cells from 22.4 percent (SE ± 3.6 percent) to 39.17 percent (SE ± 2 percent) with 43 percent of these cells corresponding to CD4+CD45RO+ T cells, CD8+ T cells from 15.2 percent (SE ± 2.9 percent) to 27 percent (SE ± 3 percent) with 70 percent corresponding to CD8+CD45RO+ T cells in first-time vaccinees. In re-vaccinees, the CD3+ T cells increased from 50.7 percent (SE ± 3 percent) to 80 percent (SE ± 2.3 percent), CD4+ T cells from 24.9 percent (SE ± 1.4 percent) to 40 percent (SE ± 3 percent) presenting a percentage of 95 percent CD4+CD45RO+ T cells, CD8+ T cells from 19.7 percent (SE ± 1.8 percent) to 25 percent (SE ± 2 percent). Among CD8+CD38+ T cells there could be observed an increase from 15 to 41.6 percent in first-time vaccinees and 20.7 to 62.6 percent in re-vaccinees. Regarding neutralizing antibodies, the re-vaccinees presented high titers even before re-vaccination. The levels of neutralizing antibodies of first-time vaccinees were similar to those presented by re-vaccinees at day 30 after vaccination, indicating the success of primary vaccination. Our data provide a basis for further studies on immunological behavior of the YF 17DD vaccine.