T helper17 signatures in SF of SLE patients
Focusing on T-cell phenotypes, we visualized the distribution by tSNE analysis of the CD3+ T cells. As depicted in Fig. 2C, PBMC and SFMC T-cell cluster in different directions. Within the CD4+ compartment, we observed higher frequencies of CCR4+, CXCR5+, CCR6+ and similar levels of CXCR3+ in SF compared to PB. The most prominent T-helper subset in SF was CCR6+ cells, a marker for Th17 cells. 35-55% of CD4+ T cells expressed CCR6 (Fig. 2C, Supplementary Fig. 3D), with ~40% co-expressing CCR4. Moreover, Tregs (CD25+ FoxP3+), Eomes+, Granzyme A+ (GZMA), were also enriched in SF as compared to PB (Fig. 2C). PD1+HLA-DR+ cells were also abundant, and we identified a distinct population of T-peripheral helper (TPH; PD1highHLA-DRhigh) cells in SF, of which ~30% co-expressed CCR6 (Fig. 3). In SF, ~70-80% of CD8+ T cells expressed Eomes and GZMA and a high proportion of CD8+ PD-1+ HLA-DR+/- cells were detected in all SF samples (Fig. 2C, Supplementary Fig. S3D). CX3CR1 was not expressed on CD4+ nor CD8+ T cells in SF. SF T-cell phenotypes in SLE, RA and SpA, presented similar patterns.
From one SLE patient, SFMC from two timepoints were available (Supplementary Fig.S3E). For most CD8+ and CD4+ subsets, similar frequencies were detected with the exception of GZMA+ Eomes- T cells which were more prominent at the first timepoint. In line with the other SLE patient, CCR6+ CD4+ T cells was one of the most prominent subsets in SF.
Since we observed both IL-17A and increased CCR6+ CD4+ T cells in SF of SLE patients, we investigated the capacity of SF-derived T cells to produce IL-17A. Following in vitro stimulation of SFMCs from one SLE and one SpA sample, intracellular IL-17A and IFNγ production was analyzed. In both samples, IL-17A production was induced and confined to the CCR6+ CD4+ cells (Fig.4, Supplementary Fig.S4). We also observed abundant IFNγ production in IL-17-negative CD4+ T cells. Of note CD8+ T cells also produced IFNγ, but not IL-17A.