Investigation Of Human IL-17-producing γδ T Cell Subsets And Induced Differentiation Conditions

Background:Tuberculosis (TB) that caused by the infection of Mycobacterium tuberculosis (Mtb)has been a global health issue that received much more concerns all the time, but thepathogenesis of TB, and the mechanism of host immunity to Mtb infection has not beenfully elucidated to date. In recent years, the role of innate immune cell-like effects of γδT cells in tuberculosis infection has been confirmed by experimental studies and clinicalobservations of many researchers. IL-17is an important pro-inflammatory cytokine,recent studies have found that γδ T cells were the main source of IL-17-secreting cells,in a mouse model of Mtb and BCG infection. Our previous study found that human γδ Tcells producing IL-17in normal human peripheral blood, and the proportion of thesecells increased in active TB patients, suggesting that IL-17-secreting γδ T cells may beinvolved in the TB pathogenesis. The studies indicate that human γδ T cells werestimulated with the phosphate antigen in the presence of inducible cytokines to inducethe secretion of IL-17, but the role and influence of Mtb peptide antigen, the Mtbheat-resistant antigen (Mtb-HAg) and polyclonal stimulus, the anti-TCRγδ antibodystimulate human γδ T cells in the presence of inducible cytokines to produce IL-17arenot clear. In addition, the culture conditions of differentiation of human γδ T cellsproducing IL-17, which subset of γδ T cells to secrete IL-17and the connection withother subsets are subject to further investigation.Objective:To investigate the effects of Mtb-HAg and anti-TCRγδ antibody on the inductionand differentiation of human γδ T cells to produce IL-17, and to explore the differencein IL-17secretion of the subsets of γδ T cells in the culture conditions, so as tounderstand the roles of different γδ T cell subsets to produce IL-17.Methods:1. Peripheral blood mononuclear cells (PBMC) were isolated from fresh healthy human blood, and γδ T cells were purified from PBMC by magnetic beads (MACS),and then stimulated with Mtb-HAg or anti-TCRγδ antibody, and with or withoutanti-CD28antibody, and cultured in the presence or absence of the cytokine cocktail(IL-1β, IL-6, TGF-β and IL-23) for10to12days. The number of IL-17producingcells among γδ T cells was detected by ELISPOT assay induced by stimulation withPMA and Ionomycin.2. PBMC isolated from buffy coat cells of healthy volunteers were frozen andpreserved. γδ T cells were purified from cryopreserved PBMC by MACS, culturedin RPMI-1640medium or IMDM medium supplemented with10%NBS or humanAB serum, and stimulated with anti-TCRγδ antibody and anti-CD28antibody in thepresence of the cytokine cocktail (IL-1β, IL-6, TGF-β and IL-23) for10to12days.The number of IL-17producing cells among γδ T cells was detected by ELISPOTassay induced by stimulation with PMA and Ionomycin.3. From cryopreserved PBMC, Vδ2+γδ T cells and Vδ2ˉγδ T cells were purified byusing the magnetic activated cell sorting method among γδ T cells, the two subsetswere cultured in IMDM medium supplemented with10%human AB serum, withstimulation of anti-TCRγδ antibody and anti-CD28antibody in the presence of thecytokine cocktail (IL-1β, IL-6, TGF-β and IL-23) for10to12days. The number ofIL-17producing cells among γδ T cells was detected by ELISPOT assay induced bystimulation with PMA and Ionomycin.Results:1. After different stimulation and polarization conditions, the number of IL-17producing cells (per105γδ T cells) in anti-TCRγδ+CKs group (67±33) wassignificantly higher than that in the anti-TCRγδ group (6±3)(P＜0.01). Meanwhile,the IL-17producing γδ T cells number in anti-TCRγδ+anti-CD28+CKs group(185±54) was significantly higher than that in anti-TCRγδ+CKs group (67±33)(P＜0.01) and that in Mtb-HAg+anti-CD28+CKs group (74±41) was significantlylower than that in anti-TCRγδ+anti-CD28+CKs group (185±54)(P＜0.01), buthigher than that in Mtb-HAg+anti-CD28group (19±12)(P＜0.05).2. Purified γδ T cells were activated and polarized as above described for10to12days,the IL-17producing γδ T cells number (per105γδ T cells) in the IMDM group(333±46) was significantly higher than that in RPMI-1640group (56±38)(P＜0.01).3. Purified γδ T cells from fresh or cryopreserved PBMC were activated and polarized as above described for10to12days, the IL-17producing γδ T cells number in thefresh PBMC group was significantly higher than that in cryopreserved PBMC group(P＜0.01).4. Purified γδ T cells from cryopreserved PBMC were activated and polarized as abovedescribed for10to12days, using IMDM that containing10%NBS or10%humanAB＋serum as culture medium, the IL-17producing γδ T cells number in the10%human AB＋serum group was significantly higher than that in10%NBS group (P＜0.01).5. When the proportion of Vδ3-8subsets in the cultured Vδ2－γδ T cells>50%, theIL-17producing γδ T cells number (per105γδ T cells) in the Vδ2－γδ T cells group(1537±1193) was significant higher than that in the Vδ2+γδ T cells group (579±420)(P＜0.05). When the proportion of Vδ3-8subsets in the cultured Vδ2－γδ T cells<50%, the IL-17producing γδ T cells number in the Vδ2－γδ T cells group(491±745) was significantly lower compared with that in the Vδ2+γδ T cellsgroup (1729±1315)(P＜0.01).Conclusion：1. The cytokines (IL-1β, IL-6, TGF-β and IL-23) required for the differentiation ofTh17can also induce the differentiation of IL-17+γδ T cells after activated withMtb-HAg or anti-TCRγδ antibody. In addition, CD28co-stimulation can enhancedthe differentiation of IL-17from activated γδ T cells.2. The anti-TCRγδ antibody has a stronger activity than Mtb-HAg to active thedifferentiation of IL-17+γδ T cells.3. The IMDM medium promote the differentiation of IL-17-producing γδ T cells,suggested that it is related with the aryl hydrocarbon receptor.4. The main subsets category of IL-17-producing γδ T cells is Vδ3-8subsets, followedby the Vδ2subsets, at least Vδ1subsets.