Characterization of Blood Mucosal-Associated Invariant T Cells in Patients With Axial Spondyloarthritis and of Resident Mucosal-Associated Invariant T Cells From the Axial Entheses of Non-Axial Spondyloarthritis Control Patients

Abstract

Objective: The importance of interleukin-17A (IL-17A) in the pathogenesis of axial spondyloarthritis (SpA) has been demonstrated by the success of IL-17A blockade. However, the nature of the cell populations that produce this important proinflammatory cytokine remains poorly defined. We undertook this study to characterize the major IL-17A-producing blood cell populations in the peripheral blood of patients with axial SpA, with a focus on mucosal-associated invariant T (MAIT) cells, a population known to be capable of producing IL-17.

Methods: We evaluated IL-17A production from 5 sorted peripheral blood cell populations, namely, MAIT cells, γδ T cells, CD4+ T cells, CD8+ T cells, and neutrophils, before and after stimulation with phorbol myristate acetate, the calcium ionophore A23187, and β-1,3-glucan. Expression of IL-17A transcripts and protein were determined using nCounter and ultra-sensitive Simoa technology, respectively. MAIT cells from the axial entheses of non-axial SpA control patients (n = 5) were further characterized using flow cytometric immunophenotyping and quantitative polymerase chain reaction, and the production of IL-17 was assessed following stimulation.

Results: On a per-cell basis, MAIT cells from peripheral blood produced the most IL-17A compared to CD4+ T cells (P < 0.01), CD8+ T cells (P < 0.0001), and γδ T cells (P < 0.0001). IL-17A was not produced by neutrophils. Gene expression analysis also revealed significantly higher expression of IL17A and IL23R in MAIT cells. Stimulation of peripheral blood MAIT cells with anti-CD3/CD28 and IL-7 and/or IL-18 induced strong expression of IL17F. MAIT cells were present in the normal, unaffected entheses of control patients who did not have axial SpA and showed elevated AHR, JAK1, STAT4, and TGFB1 transcript expression with inducible IL-17A protein. IL-18 protein expression was evident in spinal enthesis digests.

Conclusion: Both peripheral blood MAIT cells and resident MAIT cells in normal axial entheses contribute to the production of IL-17 and may play important roles in the pathogenesis of axial SpA.

Figure 1

Heatmaps showing the expression patterns of genes associated with ankylosing spondylitis (AS) and of genes of the interleukin‐23 (IL‐23)/IL‐17 pathway in T cell subpopulations isolated from the peripheral blood of patients with axial spondyloarthritis (SpA). A, Expression levels of 36 genes associated with AS susceptibility in T cells from axial SpA patients. B, Messenger RNA expression levels of 29 genes associated with the IL‐23/IL‐17 pathway, selected from the Molecular Signatures Database. T cells were stimulated for 2 hours with phorbol myristate acetate (50 ng/ml), calcium ionophore A23187 (5 μM), and β‐1,3‐glucan (50 μg/ml). Heatmaps show hierarchical clustering of genes among T cell populations from individual patient samples (n = 9). Gene expression data are log₂ transformed, centered to a mean value of 0, and scaled to unit variance. The color key on the left denotes the scale of gene expression, ranging from lower levels (blue) to higher levels (yellow).

Figure 2

IL‐17A protein production after 18 hours of stimulation (A) and IL17A transcript levels (normalized expression) after 2 hours of stimulation (B) were assessed in sorted CD4+ T cells, mucosal‐associated invariant T (MAIT) cells, CD8+ T cells, γδ T cells, and neutrophils from the peripheral blood of axial SpA patients. Cells were stimulated with phorbol myristate acetate (50 ng/ml), calcium ionophore A23187 (5 μM), and β‐1,3‐glucan (50 μg/ml). Symbols represent individual samples. Bars show the mean ± SEM. * = P < 0.05; ** = P < 0.01, by Mann‐Whitney test. *** = P < 0.001, by Wilcoxon‐Mann‐Whitney test. ns = not significant (see Figure 1 for other definitions).

Figure 3

Sorted CD4+ T cells, MAIT cells, CD8+ T cells, γδ T cells, and neutrophils from the peripheral blood of axial SpA patients were analyzed for gene expression of IL17F (A), IL23R (B), and IFNG (C). Results are presented as normalized transcript levels. Cells were stimulated with phorbol myristate acetate (50 ng/ml), calcium ionophore A23187 (5 μM), and β‐1,3‐glucan (50 μg/ml). Symbols represent individual samples. Bars show the mean ± SEM. * = P < 0.05; ** = P < 0.01; *** = P < 0.001, by Wilxocon‐Mann‐Whitney test. See Figure 2 for definitions.

Figure 4

Gene expression of IL17A (A), IL17F (B), and IFNG (C) in sorted mucosal‐associated invariant T (MAIT) cells and CD4+CCR6+ T cells from non‐axial SpA patients (n = 3) after 36 hours of stimulation in 6 different conditions, including unstimulated, stimulation with anti‐CD3/anti‐CD28, stimulation with anti‐CD3/anti‐CD28 and interleukin‐7 (IL‐7) (20 ng/ml), stimulation with anti‐CD3/anti‐CD28 and IL‐18 (50 ng/ml), and stimulation with anti‐CD3/anti‐CD28 and both IL‐7 (20 ng/ml) and IL‐18 (50 ng/ml). Results are presented as the mean ± SEM normalized transcript levels. Symbols represent individual samples.

Figure 5

Transcriptional profiling of entheseal mucosal‐associated invariant T (MAIT) cells and proinflammatory cytokine induction. A, Stratification of MAIT cell subsets based on their expression of T cell receptor Vα7.2 and CD161 in entheseal soft tissue (EST), perientheseal bone (PEB), and peripheral blood (PB) from non‐axial spondyloarthritis patients. MAIT cells expressing tissue resident/memory markers were identified by CD69 expression, and naive/circulating MAIT cells were identified by CD45RA expression. Results are shown as the mean percentage (n = 5). B, Basal expression of cytokines, chemokines, growth factors, signaling molecules, and tissue residency markers. Expression values are the log₁₀ change in threshold cycle relative to the values for hypoxanthine guanine phosphoribosyltransferase (n = 7). The color key denotes differential gene expression, in which values <–1 indicate lower relative expression and values >1 indicate higher relative expression. Gray indicates absence of expression. P = 0.038 by 2‐tailed t‐test for independent samples for the difference in expression of CCR6 by MAIT cells from peripheral blood and MAIT cells from perientheseal bone. C, Intracellular tumor necrosis factor (TNF) and interleukin‐17 (IL‐17) cytokine expression in conditions with or without stimulation with phorbol myristate acetate (50 ng/ml) and ionomycin (1 μg/ml) for 3 hours in the presence of BD GolgiPlug protein transport inhibitor in perientheseal bone–derived MAIT cells (n = 2).