Human in vitro-induced IL-17A+ CD8+ T-cells exert pro-inflammatory effects on synovial fibroblasts.

IL-17A+ CD8+ T-cells, termed Tc17 cells, have been identified at sites of inflammation in several immune-mediated inflammatory diseases. However, the biological function of human IL-17A+ CD8+ T-cells is not well characterized, likely due in part to the relative scarcity of these cells. Here, we expanded IL-17A+ CD8+ T-cells from healthy donor PBMC or bulk CD8+ T-cell populations using an in vitro polarization protocol. We show that T-cell activation in the presence of IL-1ß and IL-23 significantly increased the frequencies of IL-17A+ CD8+ T-cells, which was not further enhanced by IL-6, IL-2, or anti-IFNγ mAb addition. In vitro-generated IL-17A+ CD8+ T-cells displayed a distinct type-17 profile compared with IL-17A- CD8+ T-cells, as defined by transcriptional signature (IL17A, IL17F, RORC, RORA, MAF, IL23R, CCR6), high surface expression of CCR6 and CD161, and polyfunctional production of IL-17A, IL-17F, IL-22, IFNγ, TNFα, and GM-CSF. A significant proportion of in vitro-induced IL-17A+ CD8+ T-cells expressed TCRVα7.2 and bound MR1 tetramers indicative of MAIT cells, indicating that our protocol expanded both conventional and unconventional IL-17A+ CD8+ T-cells. Using an IL-17A secretion assay, we sorted the in vitro-generated IL-17A+ CD8+ T-cells for functional analysis. Both conventional and unconventional IL-17A+ CD8+ T-cells were able to induce pro-inflammatory IL-6 and IL-8 production by synovial fibroblasts from patients with psoriatic arthritis, which was reduced upon addition of anti-TNFα and anti-IL-17A neutralizing antibodies. Collectively, these data demonstrate that human in vitro-generated IL-17A+ CD8+ T-cells are biologically functional and that their pro-inflammatory function can be targeted, at least in vitro, using existing immunotherapy.

Drivers of Inflammation in Psoriatic Arthritis: the Old and the New.

PURPOSE OF REVIEW: The recognition that IL-17 is produced by many lymphoid-like cells other than CD4+ T helper (Th17) cells raises the potential for new pathogenic pathways in IBD/psoriasis/SpA. We review recent knowledge concerning the role of unconventional and conventional lymphocytes expressing IL-17 in human PsA and axSpA. RECENT FINDINGS: Innate-like lymphoid cells, namely gamma delta (γδ) T-cells, invariant natural killer T (iNKT) cells and mucosal-associated invariant T (MAIT) cells, together with innate lymphoid cells (ILCs) are found at sites of disease in PsA/SpA. These cells are often skewed to Type-17 profiles and may significantly contribute to IL-17 production. Non-IL-23 dependent IL-17 production pathways, utilising cytokines such as IL-7 and IL-9, also characterise these cells. Both conventional CD4 and CD8 lymphocytes show pathogenic phenotypes at sites of disease. A variety of innate-like lymphoid cells and conventional lymphocytes contribute towards IL-17-mediated pathology in PsA/SpA. The responses of these cells to non-conventional immune and non-immune stimuli may explain characteristic clinical features of these diseases and potential therapeutic mechanisms of therapies such as Jak inhibitors.

Single cell RNA and T-cell receptor sequencing reveals substantive similarities between synovial fluid and synovial tissue T-cells in inflammatory arthritis.

OBJECTIVE: Synovial fluid (SF) derived T-cells are frequently studied as a proxy for investigating the synovial tissue (ST) T-cell infiltrate in inflammatory arthritis. However, since ST is the primary site of inflammatory activity, there is debate as to whether SF provides a true reflection of the ST T-cell population. METHODS: In this study, we used single cell RNA sequencing paired with single cell T-cell receptor (TCR) sequencing to directly compare memory T-cells from paired samples of SF and ST from 6 patients with inflammatory arthritis to investigate their similarity in terms of TCR repertoire and T-cell subset composition. RESULTS: The TCR repertoires of SF and ST T-cells were strikingly similar, particularly for CD8+ T-cells. A median of 49% of the total CD8+ TCR repertoire in SF was shared with ST, compared to 20% shared with blood. Similarly, 47% of the ST CD8+ TCR repertoire was shared with SF compared to 25% with blood. Furthermore, once the effect of collagenase digestion on gene expression by ST T-cells had been accounted for, the frequencies of specific CD8+ and CD4+ T-cell subsets were, in general, similar in SF and ST and were distinct from blood. CONCLUSION: Our results suggest that T-cells migrate and equilibrate between SF and ST and maintain similar phenotypes in both sites. We conclude that SF is an appropriate proxy for investigating the T-cell infiltrate in inflamed synovium, particularly in terms of investigating the TCR repertoire.

Psoriatic and rheumatoid arthritis joints differ in the composition of CD8+ tissue-resident memory T cell subsets.

CD69+CD103+ tissue-resident memory T (TRM) cells are important drivers of inflammation. To decipher their role in inflammatory arthritis, we apply single-cell, high-dimensional profiling to T cells from the joints of patients with psoriatic arthritis (PsA) or rheumatoid arthritis (RA). We identify three groups of synovial CD8+CD69+CD103+ TRM cells: cytotoxic and regulatory T (Treg)-like TRM cells are present in both PsA and RA, while CD161+CCR6+ type 17-like TRM cells with a pro-inflammatory cytokine profile (IL-17A+TNFα+IFNγ+) are specifically enriched in PsA. In contrast, only one population of CD4+CD69+CD103+ TRM cells is detected and at similarly low frequencies in both diseases. Type 17-like CD8+ TRM cells have a distinct transcriptomic signature and a polyclonal, but distinct, TCR repertoire. Type 17-like cells are also enriched in CD8+CD103- T cells in PsA compared with RA. These findings illustrate differences in the immunopathology of PsA and RA, with a particular enrichment for type 17 CD8+ T cells in the PsA joint.

Polyfunctional, Proinflammatory, Tissue-Resident Memory Phenotype and Function of Synovial Interleukin-17A+CD8+ T Cells in Psoriatic Arthritis.

OBJECTIVE: Genetic associations imply a role for CD8+ T cells and the interleukin-23 (IL-23)/IL-17 axis in psoriatic arthritis (PsA) and other spondyloarthritides (SpA). IL-17A+CD8+ (Tc17) T cells are enriched in the synovial fluid (SF) of patients with PsA, and IL-17A blockade is clinically efficacious in PsA/SpA. This study was undertaken to determine the immunophenotype, molecular profile, and function of synovial Tc17 cells in order to elucidate their role in PsA/SpA pathogenesis. METHODS: Peripheral blood (PB) and SF mononuclear cells were isolated from patients with PsA or other types of SpA. Cells were phenotypically, transcriptionally, and functionally analyzed by flow cytometry (n = 6-18), T cell receptor ß (TCRß) sequencing (n = 3), RNA-Seq (n = 3), quantitative reverse transcriptase-polymerase chain reaction (n = 4), and Luminex or enzyme-linked immunosorbent assay (n = 4-16). RESULTS: IL-17A+CD8+ T cells were predominantly TCRαß+ and their frequencies were increased in the SF versus the PB of patients with established PsA (P < 0.0001) or other SpA (P = 0.0009). TCRß sequencing showed that these cells were polyclonal in PsA (median clonality 0.08), while RNA-Seq and deep immunophenotyping revealed that PsA synovial Tc17 cells had hallmarks of Th17 cells (RORC/IL23R/CCR6/CD161) and Tc1 cells (granzyme A/B). Synovial Tc17 cells showed a strong tissue-resident memory T (Trm) cell signature and secreted a range of proinflammatory cytokines. We identified CXCR6 as a marker for synovial Tc17 cells, and increased levels of CXCR6 ligand CXCL16 in PsA SF (P = 0.0005), which may contribute to their retention in the joint. CONCLUSION: Our results identify synovial Tc17 cells as a polyclonal subset of Trm cells characterized by polyfunctional, proinflammatory mediator production and CXCR6 expression. The molecular signature and functional profiling of these cells may help explain how Tc17 cells can contribute to synovial inflammation and disease persistence in PsA and possibly other types of SpA.

TNF Blockade Maintains an IL-10+ Phenotype in Human Effector CD4+ and CD8+ T Cells.

CD4+ and CD8+ effector T cell subpopulations can display regulatory potential characterized by expression of the prototypically anti-inflammatory cytokine IL-10. However, the underlying cellular mechanisms that regulate expression of IL-10 in different T cell subpopulations are not yet fully elucidated. We recently showed that TNF inhibitors (TNFi) promote IL-10 expression in human CD4+ T cells, including IL-17+ CD4+ T cells. Here, we further characterized the regulation of IL-10 expression via blockade of TNF signaling or other cytokine/co-stimulatory pathways, in human T cell subpopulations. Addition of the TNFi drug adalimumab to anti-CD3-stimulated human CD4+ T cell/monocyte cocultures led to increased percentages of IL-10+ cells in pro-inflammatory IL-17+, IFNγ+, TNFα+, GM-CSF+, and IL-4+ CD4+ T cell subpopulations. Conversely, exogenous TNFα strongly decreased IL-10+ cell frequencies. TNF blockade also regulated IL-10 expression in CD4+ T cells upon antigenic stimulation. Using time course experiments in whole peripheral blood mononuclear cell (PBMC) cultures, we show that TNF blockade maintained, rather than increased, IL-10+ cell frequencies in both CD4+ and CD8+ T cells following in vitro stimulation in a dose- and time-dependent manner. Blockade of IL-17, IFNγ, IL-6R, or CD80/CD86-mediated co-stimulation did not significantly regulate IL-10 expression within CD4+ or CD8+ T cell subpopulations. We show that TNF blockade acts directly on effector CD4+ T cells, in the absence of monocytes or CD4+ CD25highCD127low regulatory T cells and independently of IL-27, resulting in higher IL-10+ frequencies after 3 days in culture. IL-10/IL-10R blockade reduced the frequency of IL-10-expressing cells both in the presence and absence of TNF blockade. Addition of recombinant IL-10 alone was insufficient to drive an increase in IL-10+ CD4+ T cell frequencies in 3-day CD4+ T cell/monocyte cocultures, but resulted in increased IL-10 expression at later time points in whole PBMC cultures. Together, these data provide additional insights into the regulation of IL-10 expression in human T cells by TNF blockade. The maintenance of an IL-10+ phenotype across a broad range of effector T cell subsets may represent an underappreciated mechanism of action underlying this widely used therapeutic strategy.