Stem-like T cells are associated with the pathogenesis of ulcerative colitis in humans.

To understand the role of T cells in the pathogenesis of ulcerative colitis (UC), we analyzed colonic T cells isolated from patients with UC and controls. Here we identified colonic CD4+ and CD8+ T lymphocyte subsets with gene expression profiles resembling stem-like progenitors, previously reported in several mouse models of autoimmune disease. Stem-like T cells were increased in inflamed areas compared to non-inflamed regions from the same patients. Furthermore, TCR sequence analysis indicated stem-like T cells were clonally related to proinflammatory T cells, suggesting their involvement in sustaining effectors that drive inflammation. Using an adoptive transfer colitis model in mice, we demonstrated that CD4+ T cells deficient in either BCL-6 or TCF1, transcription factors that promote T cell stemness, had decreased colon T cells and diminished pathogenicity. Our results establish a strong association between stem-like T cell populations and UC pathogenesis, highlighting the potential of targeting this population to improve clinical outcomes.

Impact of Genetic Polymorphisms on Human Immune Cell Gene Expression.

While many genetic variants have been associated with risk for human diseases, how these variants affect gene expression in various cell types remains largely unknown. To address this gap, the DICE (database of immune cell expression, expression quantitative trait loci [eQTLs], and epigenomics) project was established. Considering all human immune cell types and conditions studied, we identified cis-eQTLs for a total of 12,254 unique genes, which represent 61% of all protein-coding genes expressed in these cell types. Strikingly, a large fraction (41%) of these genes showed a strong cis-association with genotype only in a single cell type. We also found that biological sex is associated with major differences in immune cell gene expression in a highly cell-specific manner. These datasets will help reveal the effects of disease risk-associated genetic polymorphisms on specific immune cell types, providing mechanistic insights into how they might influence pathogenesis (https://dice-database.org).

A regulatory circuit controlled by extranuclear and nuclear retinoic acid receptor α determines T cell activation and function.

Ligation of retinoic acid receptor alpha (RARα) by RA promotes varied transcriptional programs associated with immune activation and tolerance, but genetic deletion approaches suggest the impact of RARα on TCR signaling. Here, we examined whether RARα would exert roles beyond transcriptional regulation. Specific deletion of the nuclear isoform of RARα revealed an RARα isoform in the cytoplasm of T cells. Extranuclear RARα was rapidly phosphorylated upon TCR stimulation and recruited to the TCR signalosome. RA interfered with extranuclear RARα signaling, causing suboptimal TCR activation while enhancing FOXP3+ regulatory T cell conversion. TCR activation induced the expression of CRABP2, which translocates RA to the nucleus. Deletion of Crabp2 led to increased RA in the cytoplasm and interfered with signalosome-RARα, resulting in impaired anti-pathogen immunity and suppressed autoimmune disease. Our findings underscore the significance of subcellular RA/RARα signaling in T cells and identify extranuclear RARα as a component of the TCR signalosome and a determinant of immune responses.

Publisher Correction: Innate-like functions of natural killer T cell subsets result from highly divergent gene programs.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Innate-like functions of natural killer T cell subsets result from highly divergent gene programs.

Natural killer T cells (NKT cells) have stimulatory or inhibitory effects on the immune response that can be attributed in part to the existence of functional subsets of NKT cells. These subsets have been characterized only on the basis of the differential expression of a few transcription factors and cell-surface molecules. Here we have analyzed purified populations of thymic NKT cell subsets at both the transcriptomic level and epigenomic level and by single-cell RNA sequencing. Our data indicated that despite their similar antigen specificity, the functional NKT cell subsets were highly divergent populations with many gene-expression and epigenetic differences. Therefore, the thymus 'imprints' distinct gene programs on subsets of innate-like NKT cells that probably impart differences in proliferative capacity, homing, and effector functions.

The HVEM-BTLA Axis Restrains T Cell Help to Germinal Center B Cells and Functions as a Cell-Extrinsic Suppressor in Lymphomagenesis.

The tumor necrosis factor receptor superfamily member HVEM is one of the most frequently mutated surface proteins in germinal center (GC)-derived B cell lymphomas. We found that HVEM deficiency increased B cell competitiveness during pre-GC and GC responses. The immunoglobulin (Ig) superfamily protein BTLA regulated HVEM-expressing B cell responses independently of B-cell-intrinsic signaling via HVEM or BTLA. BTLA signaling into T cells through the phosphatase SHP1 reduced T cell receptor (TCR) signaling and preformed CD40 ligand mobilization to the immunological synapse, thus diminishing the help delivered to B cells. Moreover, T cell deficiency in BTLA cooperated with B cell Bcl-2 overexpression, leading to GC B cell outgrowth. These results establish that HVEM restrains the T helper signals delivered to B cells to influence GC selection outcomes, and they suggest that BTLA functions as a cell-extrinsic suppressor of GC B cell lymphomagenesis.

Intermittent PI3Kδ inhibition sustains anti-tumour immunity and curbs irAEs.

Phosphoinositide 3-kinase δ (PI3Kδ) has a key role in lymphocytes, and inhibitors that target this PI3K have been approved for treatment of B cell malignancies1-3. Although studies in mouse models of solid tumours have demonstrated that PI3Kδ inhibitors (PI3Kδi) can induce anti-tumour immunity4,5, its effect on solid tumours in humans remains unclear. Here we assessed the effects of the PI3Kδi AMG319 in human patients with head and neck cancer in a neoadjuvant, double-blind, placebo-controlled randomized phase II trial (EudraCT no. 2014-004388-20). PI3Kδ inhibition decreased the number of tumour-infiltrating regulatory T (Treg) cells and enhanced the cytotoxic potential of tumour-infiltrating T cells. At the tested doses of AMG319, immune-related adverse events (irAEs) required treatment to be discontinued in 12 out of 21 of patients treated with AMG319, suggestive of systemic effects on Treg cells. Accordingly, in mouse models, PI3Kδi decreased the number of Treg cells systemically and caused colitis. Single-cell RNA-sequencing analysis revealed a PI3Kδi-driven loss of tissue-resident colonic ST2 Treg cells, accompanied by expansion of pathogenic T helper 17 (TH17) and type 17 CD8+ T (TC17) cells, which probably contributed to toxicity; this points towards a specific mode of action for the emergence of irAEs. A modified treatment regimen with intermittent dosing of PI3Kδi in mouse models led to a significant decrease in tumour growth without inducing pathogenic T cells in colonic tissue, indicating that alternative dosing regimens might limit toxicity.

IL-27 regulates the differentiation of follicular helper NKT cells via metabolic adaptation of mitochondria.

Invariant natural killer T (iNKT) cells are innate-like T lymphocytes that express an invariant T cell receptor α chain and contribute to bridging innate and acquired immunity with rapid production of large amounts of cytokines after stimulation. Among effecter subsets of iNKT cells, follicular helper NKT (NKTFH) cells are specialized to help B cells. However, the mechanisms of NKTFH cell differentiation remain to be elucidated. In this report, we studied the mechanism of NKTFH cell differentiation induced by pneumococcal surface protein A and α-galactosylceramide (P/A) vaccination. We found that Gr-1+ cells helped iNKT cell proliferation and NKTFH cell differentiation in the spleen by producing interleukin-27 (IL-27) in the early phase after vaccination. The neutralization of IL-27 impaired NKTFH cell differentiation, which resulted in compromised antibody production and diminished protection against Streptococcus pneumoniae infection by the P/A vaccine. Our data indicated that Gr-1+ cell-derived IL-27 stimulated mitochondrial metabolism, meeting the energic demand required for iNKT cells to differentiate into NKTFH cells. Interestingly, Gr-1+ cell-derived IL-27 was induced by iNKT cells via interferon-γ production. Collectively, our findings suggest that optimizing the metabolism of iNKT cells was essential for acquiring specific effector functions, and they provide beneficial knowledge on iNKT cell-mediated vaccination-mediated therapeutic strategies.

Protein kinase C-η controls CTLA-4-mediated regulatory T cell function.

Regulatory T (Treg) cells, which maintain immune homeostasis and self-tolerance, form an immunological synapse (IS) with antigen-presenting cells (APCs). However, signaling events at the Treg cell IS remain unknown. Here we show that the kinase PKC-η associated with CTLA-4 and was recruited to the Treg cell IS. PKC-η-deficient Treg cells displayed defective suppressive activity, including suppression of tumor immunity but not of autoimmune colitis. Phosphoproteomic and biochemical analysis revealed an association between CTLA-4-PKC-η and the GIT2-αPIX-PAK complex, an IS-localized focal adhesion complex. Defective activation of this complex in PKC-η-deficient Treg cells was associated with reduced depletion of CD86 from APCs by Treg cells. These results reveal a CTLA-4-PKC-η signaling axis required for contact-dependent suppression and implicate this pathway as a potential cancer immunotherapy target.

Calcium signals regulate the functional differentiation of thymic iNKT cells.

How natural or innate-like lymphocytes generate the capacity to produce IL-4 and other cytokines characteristic of type 2 immunity remains unknown. Invariant natural killer T (iNKT) cells differentiate in the thymus into NKT1, NKT2, and NKT17 subsets, similar to mature, peripheral CD4+ T helper cells. The mechanism for this differentiation was not fully understood. Here, we show that NKT2 cells required higher and prolonged calcium (Ca2+ ) signals and continuing activity of the calcium release-activated calcium (CRAC) channel, than their NKT1 counterparts. The sustained Ca2+ entry via CRAC pathway in NKT2 cells was apparently mediated by ORAI and controlled in part by the large mitochondrial Ca2+ uptake. Unique properties of mitochondria in NKT2 cells, including high activity of oxidative phosphorylation, may regulate mitochondrial Ca2+ buffering in NKT2 cells. In addition, the low Ca2+ extrusion rate may also contribute to the higher Ca2+ level in NKT2 cells. Altogether, we identified ORAI-dependent Ca2+ signaling connected with mitochondria and cellular metabolism, as a central regulatory pathway for the differentiation of NKT2 cells.

Transcriptional reprogramming of mature CD4⁺ helper T cells generates distinct MHC class II-restricted cytotoxic T lymphocytes.

TCRαß thymocytes differentiate into either CD8αß(+) cytotoxic T lymphocytes or CD4(+) helper T cells. This functional dichotomy is controlled by key transcription factors, including the helper T cell master regulator ThPOK, which suppresses the cytolytic program in major histocompatibility complex (MHC) class II-restricted CD4(+) thymocytes. ThPOK continues to repress genes of the CD8 lineage in mature CD4(+) T cells, even as they differentiate into effector helper T cell subsets. Here we found that the helper T cell fate was not fixed and that mature, antigen-stimulated CD4(+) T cells terminated expression of the gene encoding ThPOK and reactivated genes of the CD8 lineage. This unexpected plasticity resulted in the post-thymic termination of the helper T cell program and the functional differentiation of distinct MHC class II-restricted CD4(+) cytotoxic T lymphocytes.

Mucosal memory CD8⁺ T cells are selected in the periphery by an MHC class I molecule.

The presence of immune memory at pathogen-entry sites is a prerequisite for protection. Nevertheless, the mechanisms that warrant immunity at peripheral interfaces are not understood. Here we show that the nonclassical major histocompatibility complex (MHC) class I molecule thymus leukemia antigen (TL), induced on dendritic cells interacting with CD8αα on activated CD8αß(+) T cells, mediated affinity-based selection of memory precursor cells. Furthermore, constitutive expression of TL on epithelial cells led to continued selection of mature CD8αß(+) memory T cells. The memory process driven by TL and CD8αα was essential for the generation of CD8αß(+) memory T cells in the intestine and the accumulation of highly antigen-sensitive CD8αß(+) memory T cells that form the first line of defense at the largest entry port for pathogens.

Invariant natural killer T cells recognize glycolipids from pathogenic Gram-positive bacteria.

Natural killer T cells (NKT cells) recognize glycolipid antigens presented by CD1d. These cells express an evolutionarily conserved, invariant T cell antigen receptor (TCR), but the forces that drive TCR conservation have remained uncertain. Here we show that NKT cells recognized diacylglycerol-containing glycolipids from Streptococcus pneumoniae, the leading cause of community-acquired pneumonia, and group B Streptococcus, which causes neonatal sepsis and meningitis. Furthermore, CD1d-dependent responses by NKT cells were required for activation and host protection. The glycolipid response was dependent on vaccenic acid, which is present in low concentrations in mammalian cells. Our results show how microbial lipids position the sugar for recognition by the invariant TCR and, most notably, extend the range of microbes recognized by this conserved TCR to several clinically important bacteria.

αßT cell receptors expressed by CD4(-)CD8αß(-) intraepithelial T cells drive their fate into a unique lineage with unusual MHC reactivities.

Coreceptor CD4 and CD8αß double-negative (DN) TCRαß(+) intraepithelial T cells, although numerous, have been greatly overlooked and their contribution to the immune response is not known. Here we used T cell receptor (TCR) sequencing of single cells combined with retrogenic expression of TCRs to study the fate and the major histocompatibility complex (MHC) restriction of DN TCRαß(+) intraepithelial T cells. The data show that commitment of thymic precursors to the DN TCRαß(+) lineage is imprinted by their TCR specificity. Moreover, the TCRs they express display a diverse and unusual pattern of MHC restriction that is nonoverlapping with that of CD4(+) or CD8αß(+) T cells, indicating that they sense antigens that are not recognized by the conventional T cell subsets. The new insights indicate that DN TCRαß(+) T cells form a third lineage of TCRαß T lymphocytes expressing a variable TCR repertoire, which serve nonredundant immune functions.

The identification of the endogenous ligands of natural killer T cells reveals the presence of mammalian α-linked glycosylceramides.

Glycosylceramides in mammalian species are thought to be present in the form of ß-anomers. This conclusion was reinforced by the identification of only one glucosylceramide and one galactosylceramide synthase, both ß-transferases, in mammalian genomes. Thus, the possibility that small amounts of α-anomers could be produced by an alternative enzymatic pathway, by an unfaithful enzyme, or spontaneously in unusual cellular compartments has not been examined in detail. We approached the question by taking advantage of the exquisite specificity of T and B lymphocytes and combined it with the specificity of catabolic enzymes of the sphingolipid pathway. Here, we demonstrate that mammalian immune cells produce constitutively very small quantities of α-glycosylceramides, which are the major endogenous ligands of natural killer T cells. Catabolic enzymes of the ceramide and glycolipid pathway tightly control the amount of these α-glycosylceramides. The exploitation of this pathway to manipulate the immune response will create new therapeutic opportunities.

Thymus-Derived CD4+CD8+ Cells Reside in Mediastinal Adipose Tissue and the Aortic Arch.

Double-positive CD4+CD8αß+ (DP) cells are thought to reside as T cell progenitors exclusively within the thymus. We recently discovered an unexpected CD4+ and CD8αß+ immune cell population in healthy and atherosclerotic mice by single-cell RNA sequencing. Transcriptomically, these cells resembled thymic DPs. Flow cytometry and three-dimensional whole-mount imaging confirmed DPs in thymus, mediastinal adipose tissue, and aortic adventitia, but nowhere else. Deep transcriptional profiling revealed differences between DP cells isolated from the three locations. All DPs were dependent on RAG2 expression and the presence of the thymus. Mediastinal adipose tissue DPs resided in close vicinity to invariant NKT cells, which they could activate in vitro. Thymus transplantation failed to reconstitute extrathymic DPs, and frequencies of extrathymic DPs were unaltered by pharmacologic inhibition of S1P1, suggesting that their migration may be locally confined. Our results define two new, transcriptionally distinct subsets of extrathymic DPs that may play a role in aortic vascular homeostasis.

NKT cells turn ten.

It has been 10 years since the first workshop on natural killer T cells helped to launch a growth phase for this field of research.

Interleukin 10 acts on regulatory T cells to maintain expression of the transcription factor Foxp3 and suppressive function in mice with colitis.

Regulatory T cells (T(reg) cells) that express the transcription factor Foxp3 suppress the activity of other cells. Here we show that interleukin 10 (IL-10) produced by CD11b(+) myeloid cells in recombination-activating gene 1-deficient (Rag1(-/-)) recipient mice was needed to prevent the colitis induced by transferred CD4(+)CD45RB(hi) T cells. In Il10(-/-)Rag1(-/-) mice, T(reg) cells failed to maintain Foxp3 expression and regulatory activity. The loss of Foxp3 expression occurred only in recipients with colitis, which indicates that the requirement for IL-10 is manifested in the presence of inflammation. IL-10 receptor-deficient (Il10rb(-/-)) T(reg) cells also failed to maintain Foxp3 expression, which suggested that host IL-10 acted directly on the T(reg) cells. Our data indicate that IL-10 released from myeloid cells acts in a paracrine manner on T(reg) cells to maintain Foxp3 expression.