Koen Venken

To broadly measure the spectrum of cellular self-antigens for natural killer T cells (NKT), we developed a sensitive lipidomics system to analyze lipids trapped between CD1d and NKT T cell receptors (TCRs). We captured diverse antigen complexes formed in cells from natural endogenous lipids, with or without inducing endoplasmic reticulum (ER) stress. After separating protein complexes with no, low, or high CD1d–TCR interaction, we eluted lipids to establish the spectrum of self-lipids that facilitate this interaction. Although this unbiased approach identified fifteen molecules, they clustered into only two related groups: previously known phospholipid antigens and unexpected neutral lipid antigens. Mass spectrometry studies identified the neutral lipids as ceramides, deoxyceramides, and diacylglycerols, which can be considered headless lipids because they lack polar headgroups that usually form the TCR epitope. The crystal structure of the TCR–ceramide–CD1d complex showed how the missing headgroup allowed the TCR to predominantly contact CD1d, supporting a model of CD1d autoreactivity. Ceramide and related headless antigens mediated physiological TCR binding affinity, weak NKT cell responses, and tetramer binding to polyclonal human and mouse NKT cells. Ceramide and sphingomyelin are oppositely regulated components of the “sphingomyelin cycle” that are altered during apoptosis, transformation, and ER stress. Thus, the unique molecular link of ceramide to NKT cell response, along with the recent identification of sphingomyelin blockers of NKT cell activation, provide two mutually reinforcing links for NKT cell response to sterile cellular stress conditions.

Immunomodulatory T cells play a pivotal role in protection against (auto)immune‐mediated diseases that open perspectives for therapeutic modulation. However, how immune regulatory networks operate in vivo is less understood. To this end, we focused on FOXP3+CD4+CD25+ regulatory T cells (Tregs) and invariant natural killer T (iNKT) cells, two lymphocyte populations that independently regulate adaptive and innate immune responses. In vitro, a functional interplay between Tregs and iNKT cells has been described, but whether Tregs modulate the function and phenotype of iNKT cell subsets in vivo and whether this controls iNKT‐mediated autoimmunity is unclear. Taking advantage of the conditional depletion of Tregs, we examined the in vivo interplay between iNKT and Treg cells in steady state and in preclinical models of liver and gut autoimmunity. Under non‐inflamed conditions, Treg depletion enhanced glycolipid‐mediated iNKT cell responses, with a general impact on Type 1, 2 and 17 iNKT subsets. Moreover, in vivo iNKT activation in the absence of Tregs suppressed the induction of iNKT anergy, consistent with a reduction in programmed cell death receptor 1 (PD‐1) expression. Importantly, we unveiled a clear role for an in vivo Treg‐iNKT crosstalk both in concanavalin A‐induced acute hepatitis and oxazolone‐induced colitis. Here, the absence of Tregs led to a markedly enhanced liver and gut pathology, which was not observed in iNKT‐deficient mice. Taken together, these results provide evidence for a functional interplay between regulatory T cell subsets critical in controlling the onset of autoimmune disease.

Objective: Patients with spondyloarthritis (SpA) often present with microscopic signs of gut inflammation, a risk factor for progressive disease. We investigated whether mucosal innate‐like T cells are involved in dysregulated interleukin‐23 (IL‐23)/IL‐17 responses in the gut‐joint axis in SpA.Methods: Ileal and colonic intraepithelial lymphocytes (IELs), lamina propria lymphocytes (LPLs), and paired peripheral blood mononuclear cells (PBMCs) were isolated from treatment‐naive patients with nonradiographic axial SpA with (n = 11) and without (n = 14) microscopic gut inflammation and healthy controls (n = 15) undergoing ileocolonoscopy. The presence of gut inflammation was assessed histopathologically. Immunophenotyping of innate‐like T cells and conventional T cells was performed using intracellular flow cytometry. Unsupervised clustering analysis was done by FlowSOM technology. Serum IL‐17A levels were measured via Luminex.Results: Microscopic gut inflammation in nonradiographic axial SpA was characterized by increased ileal intraepithelial γδ‐hi T cells, a γδ‐T cell subset with elevated γδ‐T cell receptor expression. γδ‐hi T cells were also increased in PBMCs of patients with nonradiographic axial SpA versus healthy controls and were strongly associated with Ankylosing Spondylitis Disease Activity Score. The abundance of mucosal‐associated invariant T cells and invariant natural killer T cells was unaltered. Innate‐like T cells in the inflamed gut showed increased RORγt, IL‐17A, and IL‐22 levels with loss of T‐bet, a signature that was less pronounced in conventional T cells. Presence of gut inflammation was associated with higher serum IL‐17A levels. In patients treated with tumor necrosis factor blockade, the proportion of γδ‐hi cells and RORγt expression in blood was completely restored.Conclusion: Intestinal innate‐like T cells display marked type 17 skewing in the inflamed gut mucosa of patients with nonradiographic axial SpA. γδ‐hi T cells are linked to intestinal inflammation and disease activity in SpA.

Objectives Gut and joint inflammation commonly co-occur in spondyloarthritis (SpA) which strongly restricts therapeutic modalities. The immunobiology underlying differences between gut and joint immune regulation, however, is poorly understood. We therefore assessed the immunoregulatory role of CD4⁺FOXP3⁺ regulatory T (Treg) cells in a model of Crohn’s-like ileitis and concomitant arthritis. Methods RNA-sequencing and flow cytometry was performed on inflamed gut and joint samples and tissue-derived Tregs from tumour necrosis factor (TNF)^∆ARE mice. In situ hybridisation of TNF and its receptors (TNFR) was applied to human SpA gut biopsies. Soluble TNFR (sTNFR) levels were measured in serum of mice and patients with SpA and controls. Treg function was explored by in vitro cocultures and in vivo by conditional Treg depletion. Results Chronic TNF exposure induced several TNF superfamily (TNFSF) members (4-1BBL, TWEAK and TRAIL) in synovium and ileum in a site-specific manner. Elevated TNFR2 messenger RNA levels were noted in TNF^∆ARE/+ mice leading to increased sTNFR2 release. Likewise, sTNFR2 levels were higher in patients with SpA with gut inflammation and distinct from inflammatory and healthy controls. Tregs accumulated at both gut and joints of TNF^∆ARE mice, yet their TNFR2 expression and suppressive function was significantly lower in synovium versus ileum. In line herewith, synovial and intestinal Tregs displayed a distinct transcriptional profile with tissue-restricted TNFSF receptor and p38MAPK gene expression. Conclusions These data point to profound differences in immune-regulation between Crohn’s ileitis and peripheral arthritis. Whereas Tregs control ileitis they fail to dampen joint inflammation. Synovial resident Tregs are particularly maladapted to chronic TNF exposure.

Divergent therapeutic outcomes on different disease domains have been noted with interleukin (IL)-23 and IL-17A-blockade in psoriatic arthritis (PsA). Therefore, elucidating the role of RORγt, the master regulator of type 17 immune responses, is of potential therapeutic interest. To this end, RORγt inhibition was assessed in combined skin, joint and gut inflammation in vivo, using a PsA model. We tested the efficacy of a RORγt antagonist in B10.RIII mice challenged with systemic overexpression of IL-23 by hydrodynamic injection of IL-23 enhanced episomal vector (IL-23 EEV). Clinical outcomes were evaluated by histopathology. Bone density and surface erosions were examined using micro-computed tomography. Cytokine production was measured in serum and by intracellular flow cytometry. Gene expression in PsA-related tissues was analysed by qPCR. RORγt-blockade significantly ameliorated psoriasis, peripheral arthritis and colitis development in IL-23 EEV mice (improvement of clinical scores and weight loss respectively by 91.8%, 58.2% and 7.0%, p< 0.001), in line with profound suppression of an enhanced type IL-17 immune signature in PsA-affected tissues. Moreover, inflammation-induced bone loss and bone erosions were reduced (p< 0.05 in calcaneus, p< 0.01 in tibia). Sustained IL-23 overexpression resulted in only mild signs of sacroiliitis. Gamma-delta (γδ)-T cells, the dominant source of T cell-derived IL-17A and IL-22, were expanded during IL-23 overexpression, and together with Th17 cells, clearly countered by RORγt inhibition (p< 0.001). RORγt-blockade shows therapeutic efficacy in a preclinical PsA model with protection towards extra-musculoskeletal manifestations, reflected by a clear attenuation of type 17 cytokine responses by γδ-T cells and Th17 cells.

Recent progress in immunobiology has led to the observation that, among cells classically categorized as the typical representatives of the adaptive immune system, i.e., T cells, some possess the phenotype of innate cells. Invariant T cells are characterized by T cell receptors recognizing a limited range of non-peptide antigens, presented only in the context of particular molecules. Mucosal-associated invariant T cells (MAIT cells) are an example of such unconventional cells. In humans, they constitute between 1% and 8% of the peripheral blood T lymphocytes and are further enriched in mucosal tissues, mesenteric lymph nodes, and liver, where they can account for even 40% of all the T cells. MAIT cells recognize antigens in the context of major histocompatibility complex class I-related protein (MR1). Upon activation, they instantly release pro-inflammatory cytokines and mediate cytolytic function towards bacterially infected cells. As such, they have been a rapidly evolving research topic not only in the field of infectious diseases but also in the context of many chronic inflammatory diseases and, more recently, in immuno-oncology. Novel findings suggest that MAIT cells function could also be modulated by endogenous ligands and drugs, making them an attractive target for therapeutic approaches. In this review, we summarize the current understanding of MAIT cell biology, their role in health and disease and discuss their future potential in cancer immunotherapy. This is discussed through the prism of knowledge and experiences with invariant natural killer T cells (iNKT)—another prominent unconventional T cell subset that shares many features with MAIT cells.