Next, we sought to investigate the outcome of αβ- and γδ-specific TCR stimulation on isolated iIEL in ex vivo stimulation assays. Since systemic γδ T cells in lymph nodes, spleen and circulation 19, 21, 34 as well as intraepithelial γδ T cells in the skin 35 have been described to be biased to produce IL-17A, we tested whether this pro-inflammatory cytokine was produced by intestinal γδ iIEL. We found that, irrespective of CD8α expression, γδ iIEL did not produce IL-17A upon stimulation with anti-TCR mAb or PMA/ionomycin (Fig. 2). This is in accordance with a recent report showing that intestinal γδ IEL are not ‘pre-wired’ toward a specific lineage 36. Therefore, we focused in this study on the well-established γδ IEL effector molecules CC chemokine ligand 4 (CCL4) and IFN-γ. Chemokine and cytokine production of αβ, γδ and total iIEL from WT mice was monitored by stimulation with plate-bound anti-γδ TCR (GL3 and GL4), anti-αβ TCR (H57-597, called H57) and anti-CD3 (2C11), respectively, followed by cytokine measurement in the supernatants. Here, αβ or γδ TCR triggering induced similar concentrations of CCL4 (Fig. 3A, upper panel), whereas higher amounts of IFN-γ were produced through anti-αβ TCR stimulation (Fig. 3A, lower panel). In addition, matching results were obtained in different iIEL populations from WT mice by stimulation with plate-bound anti-CD3 (2C11), anti-αβ TCR (H57) and anti-γδ TCR (GL3) followed by intracellular staining. TCR engagement induced CCL4 production in both αβ and γδ iIEL populations (Fig. 3B, left panel), whereas more αβ iIEL than γδ iIEL produced IFN-γ (Fig. 3B, right panel). These results clearly showed that iIEL were not anergic in these assays and that the TCR in αβ and γδ iIEL was functional. These findings were also in line with previous reports 37, 38 that showed cytokine production by iIEL during TCR complex activation. Moreover, downstream of TCR engagement, activation of the cells with the Ca2+ ionophore ionomycin showed that γδ iIEL populations had a better capacity to produce CCL4 (Fig. 3C, left panel) and αβ iIEL populations a better ability to produce IFN-γ in response to ionomycin-induced Ca2+-flux (Fig. 3C, right panel). Interestingly, direct comparison revealed that mAb-mediated TCR stimulation was significantly more efficient than PMA/ionomycin incubation in inducing CCL4 and IFN-γ production in γδCD8αα+ iIEL (Fig. 3D). In contrast to γδ iIEL, αβ iIEL populations showed similar activation behavior either with PMA/ionomycin or TCR stimulation (Fig. 3E); however, αβ+CD4+ iIEL produced IFN-γ more efficiently after PMA/ionomycin stimulation than via TCR complex triggering. These findings show the diverse responsiveness of each iIEL population upon the TCR complex activation and underline the role of the intracellular Ca2+ increase in the activation process. On the other hand, the importance of the γδ TCR, especially in γδCD8αα+ iIEL population, highlights a central role of this receptor for the function of γδ iIEL.
Figure 3. CCL4 and IFN-γ production by iIEL populations depends on TCR complex stimulation and correlates with ionomycin-induced intracellular [Ca2+] increase. Freshly isolated iIEL suspensions were stimulated in vitro. Subsequently, various T-cell populations were gated according to expression of TCR αβ or TCR γδ as well as co-receptors CD8α, CD8β, and CD4 and analyzed by intracellular cytokine staining as detailed in Supporting Information Fig. 2 and 3. (A) Representative quantification of CCL4 (upper panel) and IFN-γ (lower panel) in supernatants of iIEL stimulated by plate-bound anti-γδ TCR (clones GL3 and GL4), anti-αβ TCR (clone H57-597), anti-CD3 (clone 145-2C11) measured by cytokine bead array. N/S: no stimulation. (B) Intracellular FACS analysis of CCL4 (left panel) and IFN-γ (right panel) production in iIEL populations incubated on plates coated with anti-γδ TCR (clone GL3), anti-αβ TCR (clone H57), anti-CD3 (clone 2C11). Columns show mean±SEM, n=3 independent experiments. (C) Intracellular FACS analysis of CCL4 (left panel) and IFN-γ (right panel) production in the presence (black bars) or absence (white bars) of ionomycin (2 μg/mL) in the same iIEL populations. Columns show mean±SEM, n=3 independent experiments. (D) Direct comparison of CCL4 (left panel) and IFN-γ (right panel) production after stimulation with PMA/ionomycein ionomycin (black), anti-CD3 (red) or anti-γδ TCR (green) of γδ+CD8αα+ and γδ+DN iIEL populations by intracellular FACS analysis. N/S: no stimulation. Columns show mean±SEM, n=3 independent experiments. (E) Analysis as in (D) for αβ+CD4+, αβ+CD4+CD8α+, αβ+CD8αβ+ and αβ+CD8αα+ iIEL populations. All experiments were carried out with cells derived from WT C57BL/6 mice.
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