CD8+ T Cells as a Source of IFN-γ Production in Human Cutaneous Leishmaniasis

Cutaneous leishmaniasis (CL) is usually a self-healing skin lesion caused by different species of Leishmania parasite. Resistance and susceptibility of mice to Leishmania major infection is associated with two types of CD4+ T lymphocytes development: Th1 type response with production of cytokine IFN-γ is associated with resistance, whereas Th2 type response with production of cytokines IL-4 and IL-5 is associated with susceptibility. A clear Th1/Th2 dichotomy similar to murine model is not defined in human leishmaniasis and we need as much information as possible to define marker(s) of protection. We purified CD4+/CD8+ T cells, stimulated them with Leishmania antigens and analysed gene and protein expression of Th1/Th2 cytokines in volunteers with a history of self-healing CL who are presumed to be protected against further Leishmania infection. We have seen significant upregulation of IFN-γ gene expression and high IFN-γ production in the Leishmania stimulated CD4+ T cells and CD8+ T cells. We concluded that both antigen-specific IFN-γ producing CD4+ Th1 cells and IFN-γ producing CD8+ T cells contribute to the long term protection in individuals with a history of CL. This proves the importance of CD8+ T cells as a source of IFN-γ in Th1-like immune responses

Emergence of African Swine Fever Virus, Northwestern Iran

In 2008, African swine fever was introduced into Georgia, after which it spread to neighboring Armenia, Azerbaijan, and the Russian Federation. That same year, PCR and sequence analysis identified African swine fever virus in samples from 3 dead female wild boars in northwestern Iran. Wild boars may serve as a reservoir

Sequence of primer pairs for different amplicons.

<p>Sequence of primer pairs for different amplicons.</p

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

<p>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.</p

Basic information of the volunteers.

<p>Age and LST: Mean±S.D.</p><p>Others: Median (Range).</p><p>NA = not applicable.</p

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

<p>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.</p

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

<p>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.</p

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

<p>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.</p