CD8+ T cells as a source of IFN-γ production in human cutaneous leishmaniasis

Abstract

Background: In human leishmaniasis Th1/Th2 dichotomy similar to murine model is not clearly defined and surrogate marker(s) of protection is not yet known. In this study, Th1/Th2 cytokines (IL-5, IL-10, IL-13 and IFN-γ) profile induced by purified CD4(+)/CD8(+) T cells in response to Leishmania antigens were assessed at transcript and protein levels in 14 volunteers with a history of self-healing cutaneous leishmaniasis (HCL) and compared with 18 healthy control volunteers.

Methodology/principal findings: CD4(+)/CD8(+)/CD14(+) cells were purified from peripheral blood using magnetic beads; CD4(+)/CD8(+) T cells were co-cultured with autologous CD14(+) monocytes in the presence of soluble Leishmania antigens (SLA). Stimulation of either CD4(+) T cells or CD8(+) T cells of HCL volunteers with SLA induced a significantly (P<0.05) higher IFN-γ production compared with the cells of controls. Upregulation of IFN-γ gene expression in CD4(+) cells (P<0.001) and CD8(+) cells (P = 0.006) of HCL volunteers was significantly more than that of controls. Significantly (P<0.05) higher fold-expression of IFN-γ gene was seen in CD4(+) cells than in CD8(+) cells. In HCL volunteers a significantly (P = 0.014) higher number of CD4(+) cells were positive for intracellular IFN-γ production than CD8(+) cells.

Conclusions/significance: Collectively, the volunteers have shown maintenance of specific long-term immune responses characterized by a strong reaction to leishmanin skin test and IFN-γ production. The dominant IFN-γ response was the result of expansion of both CD4(+) and CD8(+) T cells. The results suggested that immune response in protected individuals with a history of zoonotic cutaneous leishmaniasis (ZCL) due to L. major is mediated not only through the expansion of antigen-specific IFN-γ producing CD4(+) Th1 cells, but also through IFN-γ producing CD8(+) T cells.

Figure 1. The purity of peripheral blood enriched CD4⁺ and CD8⁺ T cell populations after magnetic beads isolation.

CD4⁺ (A) and CD8⁺ (B) lymphocytes were isolated from a cell suspension of PBMC by positive selection using CD4/CD8 cocktail Abs and anti-CD4 or anti-CD8 coated magnetic nanoparticles. The purity of yielded T cell populations was analysed by flow cytometry using conjugated mAbs.

Figure 2. Cytokine profile of CD4⁺ and CD8⁺ T cells after SLA stimulation.

Purified CD4⁺ and CD8⁺ T cells were adjusted to 1–2×10⁵ cells/well in a U-bottomed 96-well plates and co-cultured with 1∶10 of mitomycin treated autologous monocytes in cRPMI 1640 supplemented with 10% human AB Rh+ serum. IFN-γ, IL-5, IL-10, and IL-13 were titrated on supernatant of SLA stimulated of CD4⁺ (A) and CD8⁺ (B) T cells at 72 hrs of culture using sandwich ELISA method. The amount of IL-5 was not detectable in the culture supernatants. Filled symbols represent HCL volunteers; Open symbols represent healthy controls.

Figure 3. Relative expression of cytokine genes in SLA stimulated CD4⁺ and CD8⁺ T cells to unstimulated cells (blank wells) of culture.

Total RNA was extracted from stimulated CD4⁺ and CD8⁺ T cells of culture and reverse transcription of mRNA to cDNA was performed using M-MuLV enzyme. Two steps Real-time PCR was set on cDNA samples using SYBR Green I system and specific primer pairs. Threshold cycles (Cts) of each amplicon was used for further analysis. The relative quantities of the target genes were normalized against the relative quantities of the internal standard (GAPDH). Fold-expression changes were calculated using the equation 2^−ΔΔCT. A) relative expression of cytokine genes in CD4⁺ T cells B) relative expression of cytokine genes in CD8⁺ T cells.

Figure 4. Frequency of purified CD4⁺/CD8⁺ T cells producing intracellular IFN-γ.

A portion of the cells at 72 hrs of SLA stimulation was used for ICS assay. Cells were adjusted at about 5×10⁵/ml and stimulated with PMA + Ionomycin for 5–6 hrs. Monensin was added during the last 4–5 hrs of culture. Cells were permeabilized and stained for intracellular IFN-γ with conjugated mAbs. A) One representative flow cytometry plot showing intracellular IFN-γ positive fractions of gated populations of CD4⁺ and CD8⁺ T cells in HCL volunteers. B) One representative flow cytometry plot showing intracellular IFN-γ positive fractions of gated populations of CD4⁺ and CD8⁺ T cells in healthy controls. C) Flow cytometry data of all volunteers were pooled and are shown as Median (horizontal line) with interquartile ranges (box) and range (whiskers) of intracellular IFN-γ positive cells.