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

Number and subtypes of natural killer cells in patients with allergic rhinitis in comparison to healthy subjects

"n Normal 0 false false false EN-US X-NONE AR-SA MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Arial; mso-bidi-theme-font:minor-bidi;} Background: Allergic rhinitis is a common disorder with great morbidity. Its prevalence has increased during recent years, therefore attracting attentions to its mechanisms. Type 2 cytokines play a major role in allergies. It has been proposed that Natural killer (NK) cells may be able to produce type 2 cytokines. This study was done to evaluate NK cells number and subtypes in patients with allergic rhinitis, comparing healthy subjects."n"nMethods: In a case control study, patients with allergic rhinitis were compared to healthy non-atopic subjects. Allergic rhinitis was diagnosed according to ARIA guidelines. NK cells quantity was studied by staining of peripheral blood mono nuclear cells with anti-CD16-FITC and anti-CD56-PE and evaluated by two color flowcytometry. Intracellular cytokines were evaluated by tri-color flowcytometry. NK cells were separated by magnetic beads, and cultured for 72 hours. Secretion of IL-4, IL-5, IL-10, IL-13, and IFN-γ was measured by ELISA, in stimulated and unstimulated conditions."n"nResults: Patients had more CD16+ CD56+ NK cells than control group. IL-4+ NK cells were significantly higher in patients (p<0.001), but the number of IFN-γ+ NK cells was not different. Cytokine secretion of NK cells was similar in case and control groups. Although IL-13 level after stimulation seemed higher in patients, the difference was not significant."n"nConclusion: NK cells number is increased in patients with allergic rhinitis and a considerable number of them produce IL-4

B-cell Lineage Study in Patients with Juvenile Idiopathic Arthritis

Objective: Juvenile idiopathic arthritis (JIA) is the most common rheumatic disease in children. The exact causes of disease are still poorly understood. It seems that B cells have several functions in JIA, including production of autoantibodies, antigen presentation, production of cytokines, and activation of T cells. Here, we aimed to evaluate B-cell lineage and its precursors in the bone marrow of patients with JIA. Methods: Twenty consecutive patients with JIA were enrolled in this study. JIA is subdivided into three groups of Pauciarticular, Polyarticular, and Systemic JIA. Bone marrow mononuclear cells were separated. Then we analyzed the immunophenotype of the JIA patients by flow cytometry. After separation, the mononuclear cells were stained specific for B cell lineage [CD10, CD19 and CD20], T cell lineage [CD3] and non specific lineage [CD34, HLA-DR and TdT]. Findings: Flow cytometric study of bone marrow showed that JIA patients had low level of CD10, CD19, and CD20. Polyarticular patients had lower level of D10, CD19, and CD20 than pauciarticular JIA patients and systemic onset JIA patients had lower levels than both of them. Conclusion: Decreasing of B cell precursor in bone marrow is one of mechanisms for pathogenesis of JIA and the more decreased B cell precursors in bone marrow are, the worst severity of the disease is. Significant differences in CD10 content of bone marrow were detected between the polyarticular and pauciarticular groups. So, it seems that polyarticular JIA patients had lower percentage of pre B cell stage

Sequence of primer pairs for different amplicons.

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

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

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