CD4 T cell activation by B cells in human Leishmania (Viannia) infection

Abstract

Background: An effective adaptive immune response requires activation of specific CD4 T cells. The capacity of B cells to activate CD4 T cells in human cutaneous leishmaniasis caused by Leishmania (Viannia) has not been evaluated.

Methods: CD4 T cell activation by B cells of cutaneous leishmaniasis patients was evaluated by culture of PBMCs or purified B cells and CD4 T cells with Leishmania panamensis antigens. CD4 T cell and B cell activation markers were evaluated by flow cytometry and 13 cytokines were measured in supernatants with a bead-based capture assay. The effect of Leishmania antigens on BCR-mediated endocytosis of ovalbumin was evaluated in the Ramos human B cell line by targeting the antigen with anti-IgM-biotin and anti-biotin-ovalbumin-FITC.

Results: Culture of PBMCs from cutaneous leishmaniasis patients with Leishmania antigens resulted in upregulation of the activation markers CD25 and CD69 as well as increased frequency of CD25hiCD127- cells among CD4 T cells. Concomitantly, B cells upregulated the costimulatory molecule CD86. These changes were not observed in PBMCs from healthy subjects, indicating participation of Leishmania-specific lymphocytes expanded in vivo. Purified B cells from these patients, when interacting with purified CD4 T cells and Leishmania antigens, were capable of inducing significant increases in CD25 and CD69 expression and CD25hiCD127- frequency in CD4 T cells. These changes were associated with upregulation of CD86 in B cells. Comparison of changes in CD4 T cell activation parameters between PBMC and B cell/CD4 T cell cultures showed no statistically significant differences; further, significant secretion of IFN-γ, TNF-α, IL-6 and IL-13 was induced in both types of cultures. Additionally, culture with Leishmania antigens enhanced BCR-mediated endocytosis of ovalbumin in Ramos human B cells.

Conclusions: The capacity of B cells specific for Leishmania antigens in peripheral blood of cutaneous leishmaniasis patients to activate CD4 T cells and induce cytokine secretion is similar to that of all cell populations present in PBMCs. This capacity implicates B cells as a plausible target for modulation of the immune response to Leishmania infection as a therapeutic strategy.

Figure 1

Stimulation with pLAg induces upregulation of activation/regulatory markers in CD4 T cells from CL patients. PBMCs were isolated from peripheral blood of CL patients (n = 10) and healthy subjects (n = 4), incubated with promastigote L. panamensis antigen (pLAg) for 5 days and stained for CD4, CD25, CD127, and CD69. A. Contour plots of cells within the CD4⁺ lymphocyte gate from one representative CL patient showing the effects of pLAg on the expression of CD25, CD127, and CD69. B. Expression of CD25 and CD69, and percentage of CD25^hiCD127^- cells within the CD4⁺ gate for healthy subjects and CL patients. **p < 0.01.

Figure 2

Stimulation with pLAg induces upregulation of costimulatory molecules in APCs from CL patients. PBMCs were isolated from peripheral blood of CL patients (CLP, n = 10) and healthy subjects (HS, n = 4), incubated with promastigote L. panamensis antigen (pLAg) for 5 days and stained for CD20, CD11c, CD86, CD80 and HLA-DR. A. Histograms of cells within the CD20⁺ lymphocyte gate (B cells, top panels) and CD11c^hiFS^hi gate (DCs, bottom panels) from representative HS and CLP showing the effects of pLAg on expression of CD86, CD80 and HLA-DR. B. Percentages of variation in mean fluorescence intensity (MFI) of each marker in cells incubated with pLAg in relation to controls for HS and CLP. **p < 0.01, ***p < 0.001.

Figure 3

Stimulation with pLAg induces cytokine secretion in PBMCs from CL patients. PBMCs were isolated from peripheral blood of CL patients (n = 10), incubated with promastigote L. panamensis antigen (pLAg) for 5 days and the indicated cytokines were measured in supernatants with a bead-based assay. *p < 0.05, **p < 0.01.

Figure 4

Purified B cells induce CD4 T cell activation in CL patients. B cells and CD4 T cells were purified from PBMCs of CL patients (n = 10), incubated for 5 days separately or together, with or without promastigote L. panamensis antigen (pLAg), and stained for CD4, CD25, CD127, and CD69. A. Representative plots for each culture condition showing the expression of CD25, CD127 and CD69 in the CD4⁺ gate. B. Expression of CD25 within the CD4⁺ gate. C. Expression of CD69 within the CD4⁺ gate. D. Percentage of CD25^hiCD127^- cells within the CD4⁺ gate. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

B cells upregulate CD86 in the presence of CD4 T cells. B cells and CD4 T cells were purified from PBMCs of CL patients (n = 10), incubated for 5 days separately or together, with or without promastigote L. panamensis antigen (pLAg), and stained for CD20, CD86, CD80, and HLA-DR. A. Expression of CD86 within the CD20⁺ gate. B. Expression of CD80 within the CD20⁺ gate. C. Expression of HLA-DR within the CD20⁺ gate. *p < 0.05, **p < 0.01.

Figure 6

Stimulation with pLAg induces cytokine secretion in B cell/CD4 T cell co-cultures from CL patients. B cells and CD4 T cells were purified from PBMCs of CL patients (n = 10), incubated for 5 days separately or together, with or without promastigote L. panamensis antigen (pLAg) and the indicated cytokines were measured in supernatants with a bead-based assay. *p < 0.05, **p < 0.01.

Figure 7

L. panamensis upregulates costimulatory and MHC molecules and enhances BCR-mediated endocytosis in Ramos cells. Ramos cells were cultured without stimulus or treated with CpG or promastigote L. panamensis antigen (pLAg) for 48 hours and then incubated with anti-IgM-biotin at 4°C and Ova-FITC-anti-biotin at 37°C for up to 3 hours. A. Expression of CD86, CD80 and HLA-DR after 48 hours of treatment. B. Means ± SD of four experiments are shown. C. FACS analysis of the frequency of Ova-FITC positive cells after 1 hour of endocytosis. Representative plots for one experiment are shown. D. Mean fluorescence intensity (MFI) for Ova-FITC in Ramos cells after 5, 10, 15, 30, 60, 120 and 180 minutes of endocytosis. One representative experiment of two is shown. E. Frequency of cells positive for Ova-FITC after 1 hour of endocytosis. Means ± SD of four experiments are shown. *p < 0.05; **p < 0.01; ***p < 0.001.