Nature Immunology

Pain in rheumatoid arthritis (RA) often persists despite effective control of inflammation, suggesting distinct mechanisms driving nociception. In both patients and animal models, pain severity does not strongly correlate with the degree of inflammation1,2. Sensory neurons, with cell bodies located in the dorsal root ganglia (DRG), innervate peripheral tissues, including joints, and transmit pain signals to the central nervous system. Crosstalk between sensory neurons and immune cells occurs at all of these sites. While sensory neurons can be directly activated by immune mediators, it remains unclear whether pain-like behaviour in antibody-induced arthritis models arises independently of immune cell activity, or which immune cell populations and mediators are required to activate pronociceptive mechanisms. Through temporal profiling of the CAIA joint-DRG transcriptomic axis, we identified SEMA4D and OSM signalling as candidate molecular mediators of neutrophil-neuron communication and neuronal sprouting. The joint-DRG atlas also revealed persistent changes in the fibroblast-immune cellular composition of the joint, along with molecular changes in DRG neurons. We showed that mechanical and cold hypersensitivity, as well as sprouting of CGRP+ nociceptive fibers in synovial tissue of mice with collagen antibody-induced arthritis (CAIA), require neutrophils but not macrophages. Analysis of publicly available datasets showed that neutrophils from the synovium of RA patients express high levels of SEMA4D and OSM, and corresponding expression of their receptors, PLEXINB1 and OSMR, in human DRG neurons, underscoring the translational relevance of this axis. Both murine and human-derived DRG neurons sprout in response to OSM. Our findings demonstrate that neutrophils produce molecules that act as cues for nociceptor sensitization and structural remodelling. Targeting these molecules could improve the efficacy of RA treatments by reducing pain while simultaneously preventing disease progression.

Mouse CD8 T cell differentiation has been studied extensively in models of infections and cancer, yet no unified framework spans the full spectrum of immunological contexts. We present the CD8 immgenT framework, integrating >200,000 single-cell transcriptomes and 128-plex surface proteomes from 734 samples spanning multiple perturbations, tissues, and timepoints. Unbiased analysis identifies 21 states encompassing naive, effector, circulating memory, tissue-resident memory, progenitor-exhausted, and terminally-exhausted compartments, among others. These states re-emerge with striking molecular convergence across acute/chronic infections, cancer, autoimmunity, aging, and homeostasis, showing that near-identical transcriptional programs support protective or dysfunctional outcomes depending on developmental history and microenvironment. Classic archetypes map to discrete clusters but exhibit unappreciated heterogeneity and overlap, cautioning against rigid nomenclature. We provide validated combinatorial markers, flow cytometry gating strategies, and immgenT reference-based integration for reproducible annotation of new datasets. This universal coordinate system harmonizes fragmented CD8 T cell literature and clarifies relationships across diverse immune challenges.

Interferons (IFNs) orchestrate diverse immune responses, but distinguishing individual IFN contributions in human transcriptomic data is challenging due to overlapping interferon-stimulated gene (ISG) signatures and limited cell-type-specific datasets. To address this, we generated a single-cell transcriptomic atlas of IFN responses by stimulating primary human T, B, NK, and CD14 monocytes with IFN-I, IFN-II, and IFN-III. This revealed core and cell-type-specific ISG programs across 13 subsets, highlighting distinct functions of IFNs. We developed an algorithm to separate IFN-I and IFN-II activity in transcriptomic data. Applied to multiple myeloma samples, it showed elevated IFN-I and IFN-II responses, with induction therapy reducing only IFN-I. Extending to multiple disease datasets provided a cross-disease overview of IFN-I and IFN-II activities and revealed increased IFN-II activities in T cells during lupus flares. This resource and the accompanying analytical framework enable dissection of IFN-driven transcriptional programs in a cell-type specific manner in human disease.

Cell signalling networks govern fundamental cellular processes yet remain incompletely defined. Moreover, what is known is biased toward a limited subset of well-characterised components. Phosphoprotein-based interrogation methods, including mass spectrometry and targeted phosphosite panels, have limited utility in physiological settings dependent on cell-cell interactions because the signalling fluxes can be difficult to detect despite producing robust functional responses. Here we developed a perturbation-based experimental framework that infers signalling pathway architecture using quantitative functional outputs rather than direct measurements of effector state, e.g., phosphorylation levels. Using antigen-specific, NF-{kappa}B-GFP reporter-expressing transformed T-cells co-cultured with cellular targets, we performed an arrayed CRISPR-Cas9 screen targeting a curated signalome of kinases, phosphatases, adaptor and scaffolding proteins, totalling 706 genes. Quantitative effect-size profiling recovered canonical T-cell receptor regulators and revealed unequal, family-specific patterns of control over NF-{kappa}B activation. Comparing T-cell stimulation with low- and high-affinity antigen uncovered signal-strength-dependent buffering of proximal signalling nodes, exemplified by reduced sensitivity to perturbation of LCK under high-intensity stimulation. Targeted perturbation in primary human CD8 T-cells validated our findings and identified TRRAP and CTDSPL2 as negative regulators of T-cell effector output, whose disruption enhanced cytotoxicity, degranulation, and cytokine production in both polyclonal and TCR-engineered T cells. Together, these results establish a scalable strategy for mapping signalling pathway architecture in the setting of physiological T-cell activation.

Tumor-infiltrating lymphocytes (TIL) often fail to restrain tumor growth due to progressive differentiation to an exhausted state. In healthy tissues, tissue-resident memory T cells (TRM) maintain protection for years, and patient tumors that contain TIL with TRM features are associated with better prognosis. Proteomic and transcriptomic profiling of T cell populations identified proteostasis as a significant factor distinguishing TRM and progenitor-exhausted TIL from terminally-exhausted TIL, including loss of E3 ubiquitin ligases NEURL3, RNF149, and WSB1, with accumulation of unfolded proteins in spite of functional proteasome activity. Enforced expression of these ligases by TIL preserved stem-like TCF1+ populations and improved anti-tumor function, whereas their knockout impaired TIL and altered T cell differentiation in acute infection. Sustained ligase expression rescued accumulation of unfolded proteins in TIL and improved immunotherapy outcome in preclinical models, highlighting the critical role of proteostasis in TIL function and identifying new avenues for advancing cancer immunotherapy.

Cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction is traditionally framed within a dichotomy of health and disease, yet its systemic immune consequences across the spectrum of CFTR activity remain incompletely defined. Using multimodal immune profiling, we constructed a single-cell atlas of circulating immune cells in people with cystic fibrosis (pwCF), healthy F508del carriers and non-carriers. In pwCF, circulating immunity was markedly altered following treatment with elexacaftor-tezacaftor-ivacaftor, with broad reductions in pro-inflammatory cytokines and immune changes linked to improved clinical outcomes. Strikingly, healthy F508del carriers exhibited a CF-like immune signature characterised by low-grade systemic inflammation, including elevated IL-6, reduced mucosal-associated invariant T cells, and inflammatory monocyte features overlapping with pwCF. Together, these findings show that CFTR dysfunction spans a spectrum of systemic immune dysregulation, challenging a strict dichotomy between health and disease.

Fibroblasts play a dual role in shaping tissue homeostasis and immune responses during inflammatory perturbations. Manipulating fibroblast behavior has therefore emerged as a promising strategy for autoimmune diseases. Here, through integrated multimodal single-cell transcriptomic and proteomic profiling of synovial tissue combined with prospective clinical data from 54 patients with rheumatoid arthritis, we identify C-X-C motif chemokine 12 (CXCL12)hi Apolipoprotein C1 (APOC1)+ fibroblasts as a pathogenic cell population driving refractory synovitis. CXCL12hi APOC1+ fibroblasts construct local niche in spatial coordinates with plasmablasts via the CXCL12-CXCR4 axis. APOC1 orchestrates senescent inflammatory cancer-associated fibroblast(iCAF)-like properties of this cluster through activation of the STAT3-C/EBP pathway. Therapeutic elimination of senescent cells, either alone or in combination with TNF inhibition, significantly ameliorates experimental arthritis. Together, these findings uncover a mechanistic basis for treatment resistance in rheumatoid arthritis and highlight senescent iCAF-like fibroblasts as a promising therapeutic target.

Inflammasomes are multiprotein complexes that form and activate after exposure to pathogenic microbes and host danger signals that trigger an inflammatory response. Although NLRP1 and NLRP3 inflammasomes share structural similarities and can be activated by similar stimuli, no evidence of heterotypic inflammasome assemblies has been reported. Here, we identify a unique interaction between NLRP1 and NLRP3 in human cells, forming a hybrid inflammasome, to drive inflammation. NLRP1 is essential for this hydrid inflammasome activation and NLRP3-mediated Caspase-1 activation and release of IL-1{beta} and IL-18. The presence of the heterocomplex inflammasome was confirmed in blood samples from patients after kidney transplantation and is associated with inflammatory responses driven by NLRP3 and MEFV mutations that cause inflammasomopathies. Our findings reveal an unexpected level of intricacy in inflammasome composition, pinpointing hybrid targets that may pave the way for innovative pharmacological treatments for inflammatory disorders. Significance StatementPrevious findings showed interactions between NLRP3 and NLRC4 or NLRP3 and NLRP11 showing that would be possible the interaction of the inflammasomes as supercomplexes and not working alone. Now, we show a new inflammasome heterocomplex inflammasome between NLRP1 and NLRP3 which is associated to the inflammatory profile in autoimmune diseases patietns and transplanted patients. These findings could open a new research topic for the design of dual inflammasomes inhibitors.

The identification of specific markers to distinguish resident microglia from infiltrating monocytes has been a long-standing challenge in neuroscience. Recently, proteins such as P2RY12, TMEM119, and FCRLS have been proposed as microglia-specific and are now widely used to define microglial populations in health and disease. The specificity of these markers was predicated on the assumption that circulating monocytes retain their distinct signatures after entering the central nervous system (CNS). Here, we challenge this paradigm. Using a combination of bone marrow chimeras, single-cell RNA sequencing, ATAC-seq, flow cytometry, and immunohistochemistry, we demonstrate that monocytes engrafting into the CNS acquire de novo expression of these established microglia markers. This phenotypic conversion is driven by profound epigenetic reprogramming, characterized by dynamic changes in chromatin accessibility at key gene loci, including P2ry12, Tmem119, and Aif1 (Iba1), and a shift in transcription factor binding motifs toward a microglial profile. We show this process occurs in the retina following injury and, remarkably, under physiological conditions in the brain and spinal cord, where blood-derived monocytes progressively contribute to the resident myeloid pool. Furthermore, engrafted monocytes downregulate canonical monocyte markers (Ly6C, CD45), eventually becoming indistinguishable from embryonic microglia based on conventional phenotyping. Our findings reveal that infiltrating monocytes undergo extensive epigenetic and transcriptional remodeling to adopt a microglia-like fate, challenging the specificity of current markers and necessitating a re-evaluation of the distinct roles of these two cell populations in CNS pathology. Significance StatementDistinguishing resident CNS microglia from infiltrating monocytes is fundamental to understanding neuro-inflammation. This study reveals that widely used "microglia-specific" markers are not exclusive, as monocytes entering the central nervous system are epigenetically reprogrammed to express them. This mimicry invalidates long-held assumptions about microglial identity and demonstrates that many cells previously identified as microglia may have a peripheral origin. Our work underscores the critical need for more reliable methods to differentiate these populations to accurately define their respective contributions to CNS health and disease.

Heat is a cardinal feature of inflammation. Despite temperature variability and dependence of enzymes and complexes, how heat and fever affect immune cells remains uncertain. We found that heat broadly increased inflammatory activity of CD4+ T cell subsets and decreased Treg suppressive function. Th1 cells, however, also selectively developed mitochondrial dysfunction with high levels of ROS production and DNA damage. This led Th1 cells to undergo Tp53-dependent death, which was required to minimize the accumulation of mutations in heat and inflammation. Th1 cells with similar DNA damage signatures were also detected in Crohns disease and rheumatoid arthritis. Fever and inflammation-associated heat thus selectively induce mitochondrial stress and DNA damage in activated Th1 cells that requires p53 to maintain genomic integrity of the T cell repertoire. One Sentence SummaryFever temperatures augment CD4+ T cell-mediated inflammation but induce differential metabolic stress and DNA damage in T cell subsets, with Th1 cells selectively sensitive and dependent on p53 to induce apoptosis and maintain genomic integrity.

The gastrointestinal tract is continuously exposed to foreign antigens in food and commensal microbes with potential to induce adaptive immune responses. Peripherally induced T regulatory (pTreg) cells are essential for mitigating inflammatory responses to these agents1-4. While ROR{gamma}t+ antigen-presenting cells (ROR{gamma}t-APCs) were shown to program gut microbiota-specific pTreg5-7, their definition remains incomplete, and the APC responsible for food tolerance has remained elusive. Here, we identify a distinct subset of ROR{gamma}t-APCs, designated tolerogenic dendritic cells (tDC), required for differentiation of both food- and microbiota-specific pTreg cells and for establishment of oral tolerance. tDC development and function require expression of the transcription factors Prdm16 and ROR{gamma}t, as well as a unique Rorc(t) cis-regulatory element. Gene expression, chromatin accessibility, and surface marker analysis establish tDC as myeloid in origin, distinct from ILC3, and sharing epigenetic profiles with classical DC. Upon genetic perturbation of tDC, we observe a substantial increase in food antigen-specific T helper 2 (Th2) cells in lieu of pTreg, leading to compromised tolerance in mouse models of asthma and food allergy. Single-cell analyses of freshly resected mesenteric lymph nodes from a human organ donor, as well as multiple specimens of human intestine and tonsil, reveal candidate tDC with co-expression of PRDM16 and RORC and an extensive transcriptome shared with mice, highlighting an evolutionarily conserved role across species. Our findings suggest that a better understanding of how tDC develop and how they regulate T cell responses to food and microbial antigens could offer new insights into developing therapeutic strategies for autoimmune and allergic diseases as well as organ transplant tolerance.

Meningeal immune cells have recently emerged as critical modulators of neural function in both physiological and pathological states1,2. In particular, immune cells within the meninges surrounding the dorsal root ganglia (DRG) have been implicated in the pathogenesis of neuropathic pain3-5. Yet, the cellular complexity of the meningeal immune landscape and the neuroimmune interactions linking this niche to neuropathic pain remain poorly understood. Here, we show that peripheral nerve injury induces both the recruitment of leukocytes to the DRG meninges and their transcriptional reprogramming across innate (primarily myeloid cells) and adaptive immune compartments. The accumulation of myeloid cells, predominantly neutrophils and classical monocytes, within the DRG meninges originates from local vertebral bone marrow (BM) emergency myelopoiesis, followed by their migration through ossified vertebral channels. Further analysis identified meningeal granulocyte-macrophage colony- stimulating factor (GM-CSF), produced mainly by group 2 innate lymphoid cells (ILC2s), as a key mediator that instructs vertebral BM emergency myelopoiesis after peripheral nerve injury, thereby promoting neuropathic pain. These findings uncover a fundamental process linking meningeal immunity to vertebral BM emergency myelopoiesis in the pathophysiological cascade of neuropathic pain and highlight meningeal GM-CSF as the instructive signal orchestrating this neuroimmune axis.

Adaptive immune responses are induced by vaccination and infection, yet little is known about how CD4+ T cell memory differs when primed in these two contexts. Notably, viral infection is generally associated with higher levels of systemic inflammation than is vaccination. To assess whether the inflammatory milieu at the time of CD4+ T cell priming has long-term effects on memory, we compared Spike-specific memory CD4+ T cells in 22 individuals around the time of the participants third SARS-CoV-2 mRNA vaccination, with stratification by whether the participants first exposure to Spike was via virus or mRNA vaccine. Multimodal single-cell profiling of Spike-specific CD4+ T cells revealed 755 differentially expressed genes that distinguished infection- and vaccine-primed memory CD4+ T cells. Spike-specific CD4+ T cells from infection-primed individuals had strong enrichment for cytotoxicity and interferon signaling genes, whereas Spike-specific CD4+ T cells from vaccine-primed individuals were enriched for proliferative pathways by gene set enrichment analysis. Moreover, Spike-specific memory CD4+ T cells established by infection had distinct epigenetic landscapes driven by enrichment of IRF-family transcription factors, relative to T cells established by mRNA vaccination. This transcriptional imprint was minimally altered following subsequent mRNA vaccination or breakthrough infection, reflecting the strong bias induced by the inflammatory environment during initial memory differentiation. Together, these data suggest that the inflammatory context during CD4+ T cell priming is durably imprinted in the memory state at transcriptional and epigenetic levels, which has implications for personalization of vaccination based on prior infection history. One Sentence SummarySARS-CoV-2 infection versus SARS-CoV-2 mRNA vaccination prime durable transcriptionally and epigenetically distinct Spike-specific CD4+ T cell memory landscapes.

Acute graft-versus-host disease (aGVHD) is a significant complication of allogeneic hematopoietic stem cell transplantation (aHSCT), driven by alloreactive donor T cells in the gut. However, the roles of additional donor and host cells in this process are not fully understood. We conducted multiplexed imaging on 59 biopsies from patients with gastrointestinal GVHD and 10 healthy controls, revealing key pathological changes, including fibrosis, crypt alterations, loss of Paneth cells, accumulation of endocrine cells, and disrupted immune organization, particularly a reduction in IgA-secreting plasma cells. Interestingly, CD8T cells were enriched only in a subset of patients, while others exhibited non-canonical enrichments of macrophages and neutrophils. Post-transplantation time significantly influenced immune composition, with host cells dominating plasma and T cell compartments long after transplantation. This spatial atlas of healthy duodenum and GVHD uncovers non-canonical immune dynamics, offering insights into disease pathophysiology and potential clinical applications in GVHD and other inflammatory bowel diseases.

Synovial tissue inflammation is the hallmark of rheumatoid arthritis (RA). Recent work has identified prominent pathogenic cell states in inflamed RA synovial tissue, such as T peripheral helper cells; however, the epigenetic regulation of these states has yet to be defined. We measured genome-wide open chromatin at single cell resolution from 30 synovial tissue samples, including 12 samples with transcriptional data in multimodal experiments. We identified 24 chromatin classes and predicted their associated transcription factors, including a CD8+ GZMK+ class associated with EOMES and a lining fibroblast class associated with AP-1. By integrating an RA tissue transcriptional atlas, we found that the chromatin classes represented superstates corresponding to multiple transcriptional cell states. Finally, we demonstrated the utility of this RA tissue chromatin atlas through the associations between disease phenotypes and chromatin class abundance as well as the nomination of classes mediating the effects of putatively causal RA genetic variants.

Initiation of B-cell receptor (BCR)1 signaling, and subsequent antigen-encounter in germinal centers2,3 represent milestones of B-lymphocyte development that are both marked by sharp increases of CD25 surface-expression. Oncogenic signaling in B-cell leukemia (B-ALL)4 and lymphoma5 also induced CD25-surface expression. While CD25 is known as an IL2-receptor chain on T- and NK-cells6-9, the significance of its expression on B-cells was unclear. Our experiments based on genetic mouse models and engineered patient-derived xenografts revealed that, rather than functioning as an IL2-receptor chain, CD25 expressed on B-cells assembled an inhibitory complex including PKC{delta} and SHIP1 and SHP1 phosphatases for feedback control of BCR-signaling or its oncogenic mimics. Recapitulating phenotypes of genetic ablation of PKC{delta}10-12, SHIP113,14 and SHP114, 15,16, conditional CD25-deletion decimated early B-cell subsets but expanded mature B-cell populations and induced autoimmunity. In B-cell malignancies arising from early (B-ALL) and late (lymphoma) stages of B-cell development, CD25-loss induced cell death in the former and accelerated proliferation in the latter. Clinical outcome annotations mirrored opposite effects of CD25-deletion: high CD25 expression levels predicted poor clinical outcomes for patients with B-ALL, in contrast to favorable outcomes for lymphoma-patients. Biochemical and interactome studies revealed a critical role of CD25 in BCR-feedback regulation: BCR-signaling induced PKC{delta}-mediated phosphorylation of CD25 on its cytoplasmic tail (S268). Genetic rescue experiments identified CD25-S268 tail-phosphorylation as central structural requirement to recruit SHIP1 and SHP1 phosphatases to curb BCR-signaling. A single point mutation CD25S268A abolished recruitment and activation of SHIP1 and SHP1 to limit duration and strength of BCR-signaling. Loss of phosphatase-function, autonomous BCR-signaling and Ca2+-oscillations induced anergy and negative selection during early B-cell development, as opposed to excessive proliferation and autoantibody production in mature B-cells. These findings highlight the previously unrecognized role of CD25 in assembling inhibitory phosphatases to control oncogenic signaling in B-cell malignancies and negative selection to prevent autoimmune disease.

Complement protein C3 is crucial for immune responses in mucosal sites such as the lung, where it aids in microbe elimination and enhances inflammation. While trained immunity - enhanced secondary responses of innate immune cells after prior exposure - is well-studied, the role of the complement system in trained immune responses remains unclear. We investigated the role of C3 in trained immunity and found that in vivo, trained wild-type mice showed significantly elevated pro-inflammatory cytokines and increased C3a levels upon a second stimulus, whereas C3-deficient mice exhibited a blunted cytokine response and heightened evidence of lung injury. Ex vivo, C3-deficient alveolar macrophages (AMs) displayed reduced chemokine and cytokine output after training, which was restored by exogenous C3 but not by C3a. Inhibiting C3aR, both pharmacologically and with a genetic C3aR knockout, prevented this restoration, indicating the necessity of C3aR engagement. Mechanistically, trained WT AMs demonstrated enhanced glycolytic activity compared to C3-deficient AMs - a defect corrected by exogenous C3 in a C3aR-dependent manner. These findings reveal that C3 modulates trained immunity in AMs through C3aR signaling, affecting cytokine production and metabolic reprogramming, and highlight a novel role for C3 in trained immunity.

Type I interferon (IFN-I) and interferon-{gamma} (IFN{gamma}) are central regulators of antiviral immunity, yet how they cooperatively govern CD8 T cell fate during chronic infection remains unresolved. Here, we uncover a previously unrecognized, spatially encoded interferon circuit that actively constrains progenitor exhausted CD8 T cells (Tpex) during chronic LCMV infection. Persistent IFN-I signaling indirectly restricts Tpex expansion by enforcing their sequestration within PDL1-rich B cell niches of lymphoid tissue and by suppressing T cell-derived IFN{gamma}. Blockade of IFN-I signaling enables Tpex migration into T cell zones of splenic follicles driving IFN{gamma} production, which in turn sustains PDL1 expression on myeloid cells to re-impose local inhibitory pressure. Combined IFN-I and IFN{gamma} blockade disrupts this feedback, promoting coordinated niche redistribution of Tpex and checkpoint remodeling that drives robust Tpex expansion. Single-cell transcriptomics reveal that this layered IFN-I-IFN{gamma} interplay establishes a regulatory balance that constrains Tpex proliferation while preserving effector-like transcriptional programs in their progeny effector CD8 T cells, ultimately preventing premature terminal differentiation. Thus, interferons orchestrate the coordinated T cell-myeloid regulatory circuit that integrates tissue organization, cytokine feedback, and checkpoint control to regulate CD8 T cell exhaustion during chronic infection.

Knowledge of the mechanisms underpinning the development of protective immunity conferred by mRNA vaccines is fragmentary. Here we investigated responses to COVID-19 mRNA vaccination via ultra-low-volume sampling and high-temporal-resolution transcriptome profiling (23 subjects across 22 timepoints, and with 117 COVID-19 patients used as comparators). There were marked differences in the timing and amplitude of the responses to the priming and booster doses. Notably, we identified two distinct interferon signatures. The first signature (A28/S1) was robustly induced both post-prime and post-boost and in both cases correlated with the subsequent development of antibody responses. In contrast, the second interferon signature (A28/S2) was robustly induced only post-boost, where it coincided with a transient inflammation peak. In COVID19 patients, a distinct phenotype dominated by A28/S2 was associated with longer duration of intensive care. In summary, high-temporal-resolution transcriptomic permitted the identification of post- vaccination phenotypes that are determinants of the course of COVID-19 disease.

Three recent contemporaneously published papers1-3 showed that antigen-presenting cells (APC) expressing the nuclear receptor ROR{gamma}t instruct peripheral regulatory T (pTreg) cell differentiation and establish tolerance to the gut microbiota. These studies identified a spectrum of ROR{gamma}t+ APCs, distinct from dendritic cells, that included type 3 innate lymphoid cells (ILC3s) as well as novel APC types. Given the discordant conclusions as to the nature of the pTreg-inducing APC and the divergent nomenclature used in these three studies, there is a clear need to reconcile these analyses and the identity of the cell types described. Our reanalysis of the single cell RNA-seq data from these studies revealed the presence of four distinct subsets of non-ILC3 ROR{gamma}t+ APCs, present in all three datasets reported, and confirmed expression of Itgb8, the critical factor for ROR{gamma}t+ APC mediated pTreg induction, in cells synonymous with the non-Aire expressing Thetis cell subset, TC IV.