The emergence of circulating activated autoreactive Dsg3-specific Tfr cells is associated with long-term efficacy of RTX in PV patients.

BACKGROUND: Pemphigus vulgaris (PV) is characterized by autoantibodies targeting keratinocytes adhesion proteins desmoglein (Dsg) 1 and 3, and by the HLA-DRB1-0402 predisposition allele. Treatment using rituximab (RTX) combined with short-term corticosteroids (CS) allows disease control and long-lasting remission. OBJECTIVE: The principal aim of this study is to evaluate the impact of RTX on the circulating subpopulations of Dsg-3-specific T lymphocytes that specifically regulate B cell responses: follicular helper (Tfh) and follicular regulatory T (Tfr) lymphocytes. METHODS: Using the HLA-DRB1-0402 tetramer loaded with the Dsg-3 immunodominant peptide, we analysed by flow cytometry the frequency, the polarisation and the activation status of blood Dsg-3-specific follicular T cell populations at baseline, Month 6 and long-term follow-up (Month 60-90) from PV patients. RESULTS: At baseline, we observed a predominance of Tfh1* and Tfh17 subsets and an underrepresentation of the Tfh2 subset among autoreactive Dsg-3-specific Tfh cells as compared with non-autoreactive Tfh cells. RTX treatment induced a decrease of autoreactive Tfh cells with no effect on their polarisation during patients' follow-up. In parallel, we observed the emergence of a Dsg-3-specific Tfr subpopulation with a significant overexpression of the surface activation markers PD1, ICOS, and CD25 that was not observed at the surface of autoreactive Tfh and non-autoreactive Tfr cells of the same PV patients. In contrast, a very few Dsg-3 specific Tfr cells were observed in PV patients treated with CS alone. CONCLUSION: Here we show that the emergence of circulating autoreactive Dsg-3-specific Tfr cells is associated with the long-term efficacy of RTX in PV patients.

THEMIS promotes T cell development and maintenance by rising the signaling threshold of the inhibitory receptor BTLA.

The current paradigm about the function of T cell immune checkpoints is that these receptors switch on inhibitory signals upon cognate ligand interaction. We here revisit this simple switch model and provide evidence that the T cell lineage protein THEMIS enhances the signaling threshold at which the immune checkpoint BTLA (B- and T-lymphocyte attenuator) represses T cell responses. THEMIS is recruited to the cytoplasmic domain of BTLA and blocks its signaling capacity by promoting/stabilizing the oxidation of the catalytic cysteine of the tyrosine phosphatase SHP-1. In contrast, THEMIS has no detectable effect on signaling pathways regulated by PD-1 (Programmed cell death protein 1), which depend mainly on the tyrosine phosphatase SHP-2. BTLA inhibitory signaling is tuned according to the THEMIS expression level, making CD8+ T cells more resistant to BTLA-mediated inhibition than CD4+ T cells. In the absence of THEMIS, the signaling capacity of BTLA is exacerbated, which results in the attenuation of signals driven by the T cell antigen receptor and by receptors for IL-2 and IL-15, consequently hampering thymocyte positive selection and peripheral CD8+ T cell maintenance. By characterizing the pivotal role of THEMIS in restricting the transmission of BTLA signals, our study suggests that immune checkpoint operability is conditioned by intracellular signal attenuators.

Proenkephalin deletion in hematopoietic cells induces intestinal barrier failure resulting in clinical feature similarities with irritable bowel syndrome in mice.

Opioid-dependent immune-mediated analgesic effects have been broadly reported upon inflammation. In preclinical mouse models of intestinal inflammatory diseases, the local release of enkephalins (endogenous opioids) by colitogenic T lymphocytes alleviate inflammation-induced pain by down-modulating gut-innervating nociceptor activation in periphery. In this study, we wondered whether this immune cell-derived enkephalin-mediated regulation of the nociceptor activity also operates under steady state conditions. Here, we show that chimeric mice engrafted with enkephalin-deficient bone marrow cells exhibit not only visceral hypersensitivity but also an increase in both epithelial paracellular and transcellular permeability, an alteration of the microbial topography resulting in increased bacteria-epithelium interactions and a higher frequency of IgA-producing plasma cells in Peyer's patches. All these alterations of the intestinal homeostasis are associated with an anxiety-like behavior despite the absence of an overt inflammation as observed in patients with irritable bowel syndrome. Thus, our results show that immune cell-derived enkephalins play a pivotal role in maintaining gut homeostasis and normal behavior in mice. Because a defect in the mucosal opioid system remarkably mimics some major clinical symptoms of the irritable bowel syndrome, its identification might help to stratify subgroups of patients.

Human papilloma virus-16-specific CD8+ T-cell expansions characterize different clinical forms of lichen planus and not lichen sclerosus et atrophicus.

Lichen planus (LP) is a cutaneomucosal chronic inflammatory disease characterized by a CD8+ cytotoxic T-lymphocytes (CTL) infiltrate. In erosive oral LP, we found HPV16-specific activated CTL in lesions, supporting a pathogenic contribution of HPV16. Here, we investigated whether a similar scenario occurs in other clinical forms of LP and in lichen sclerosus et atrophicus (LSA), another chronic disease also affecting the mucosa and/or the skin. Blood CTL from LP and LSA patients expressed significant higher levels of granzyme B, perforin and CD107a proteins than healthy donors. Expansions of TCRVß3+ CTL, with presence of TCR clonotypes identical to those previously detected in erosive oral LP, were found both in blood and mucosal/skin lesions of LP, and not of LSA patients. These expansions were enriched with HPV16-specific CD8+ T-cells as shown by their recognition of the E711-20 immunodominant epitope. In LSA patients, the peripheral repertoire of CTL was oligoclonal for TCRVß6+ CTL. Finally, although patients with LP and LSA have developed antibodies against HPV16 capsid L1, antibodies against HPV16 E6 were only observed in patients with LP. Overall, our data collectively suggest an involvement of HPV16-specific CTL in different clinical forms of LP, not only in erosive oral LP, while a different scenario operates in LSA.

A selective LIS1 requirement for mitotic spindle assembly discriminates distinct T-cell division mechanisms within the T-cell lineage.

The ability to proliferate is a common feature of most T-cell populations. However, proliferation follows different cell-cycle dynamics and is coupled to different functional outcomes according to T-cell subsets. Whether the mitotic machineries supporting these qualitatively distinct proliferative responses are identical remains unknown. Here, we show that disruption of the microtubule-associated protein LIS1 in mouse models leads to proliferative defects associated with a blockade of T-cell development after ß-selection and of peripheral CD4+ T-cell expansion after antigen priming. In contrast, cell divisions in CD8+ T cells occurred independently of LIS1 following T-cell antigen receptor stimulation, although LIS1 was required for proliferation elicited by pharmacological activation. In thymocytes and CD4+ T cells, LIS1 deficiency did not affect signaling events leading to activation but led to an interruption of proliferation after the initial round of division and to p53-induced cell death. Proliferative defects resulted from a mitotic failure, characterized by the presence of extra-centrosomes and the formation of multipolar spindles, causing abnormal chromosomes congression during metaphase and separation during telophase. LIS1 was required to stabilize dynein/dynactin complexes, which promote chromosome attachment to mitotic spindles and ensure centrosome integrity. Together, these results suggest that proliferative responses are supported by distinct mitotic machineries across T-cell subsets.

THEMIS enhances the magnitude of normal and neuroinflammatory type 1 immune responses by promoting TCR-independent signals.

Signals that determine the differentiation of naïve CD4+ T helper (TH) cells into specific effector cell subsets are primarily stimulated by cytokines, but additional signals are required to adjust the magnitude of TH cell responses and set the balance between effective immunity and immunological tolerance. By inducing the post-thymic deletion of the T cell lineage signaling protein THEMIS, we showed that THEMIS promoted the development of optimal type 1 immune responses to foreign antigens but stimulated signals that favored encephalitogenic responses to self-neuroantigens. THEMIS was required to stimulate the expression of the gene encoding the transcriptional regulator T-BET and the production of the cytokine interferon-γ (IFN-γ), and it enhanced the ability of encephalitogenic CD4+ T cells to migrate into the central nervous system. Consistently, analysis of THEMIS expression in polarized CD4+ T cells showed that THEMIS was selectively increased in abundance in TH1 cells. The stimulation of predifferentiated effector CD4+ T cells with antigen-presenting cells revealed a stimulatory function for THEMIS on type 1 cytokine responses, similar to those observed ex vivo after immunization. In contrast, THEMIS exerted opposing effects on naïve CD4+ T cells in vitro by inhibiting the T cell receptor (TCR)-mediated signals that lead to TH1 cell responses. These data suggest that THEMIS exerts TCR-independent functions in effector T cells, which increase the magnitude of normal and pathogenic TH1 cell-mediated responses.

The Use of Peptide-MHCII Tetramers to Identify Antigen-Specific T Follicular Helper and T Follicular Regulatory Cells.

Characterization and counting of the different immune cell subpopulations are largely used in order to predict the quality of vaccination or the progression of diseases. As such, flow cytometry is a valuable technology to perform an exact cartography of the immune cell subsets. In the context of B-cell responses, specialized structures emerge in B-follicles of second lymphoid organs where B-cells "undergo maturation processes under the guidance of specific T-cells, follicular helper T-cells, and follicular regulatory T-cells. Thus, tracking these cell types is of high interest, especially in the context of protein vaccination. In this purpose, we describe here, how we can track antigen-specific follicular helper T-cells and follicular regulatory T-cells by flow cytometry after protein vaccination in nonmodified wild-type mice, which ultimately provides a comprehensive way to better understand the function of these particular cells in vivo.

CD4+ T cells that help B cells - a proposal for uniform nomenclature.

T follicular helper (Tfh) cells cognately guide differentiation of antigen-primed B cells in secondary lymphoid tissues. 'Tfh-like' populations not expressing the canonical Tfh cell transcription factor BCL6 have also been described, which can aid particular aspects of B cell differentiation. Tfh and Tfh-like cells are essential for protective and pathological humoral immunity. These CD4+ T cells that help B cells are polarized to produce diverse combinations of cytokines and chemokine receptors and can be grouped into distinct subsets that promote antibodies of different isotype, affinity, and duration, according to the nature of immune challenge. However, unified nomenclature to describe the distinct functional Tfh and Tfh-like cells does not exist. While explicitly acknowledging cellular plasticity, we propose categorizing these cell states into three groups based on phenotype and function, paired with their anatomical site of action.

Bromodomain and extraterminal (BET) protein inhibition of IgG/IgE production in murine B cells is counter-balanced by a strong Th2 bias.

OBJECTIVES: Inhibitors of bromodomain and extra terminal domain (BET) proteins are a new and growing class of anti-cancer drugs, which decrease oncogene expression by targeting superenhancers. Antibody production is another physiological process relying on superenhancers, and it remains to be clarified whether potential immunomodulatory properties of BET inhibitors might impact humoral immunity and allergy. METHODS: We thus evaluated humoral immune responses and their Th2 context in vitro and in vivo in mice following treatment with the classical BET-inhibitor JQ1. We quantified immunoglobulin (Ig) and antibody production by B cells either stimulated in vitro or obtained from immunised mice. JQ1 effects on class switching and activation-induced deaminase loading were determined, together with modifications of B, T follicular helper (Tfh) and T helper 2 (Th2) populations. JQ1 was finally tested in B-cell-dependent models of immune disorders. RESULTS: Bromodomain and extra terminal domain inhibition reduced class switching, Ig expression on B cells and antibody secretion and was correlated with decreased numbers of Tfh cells. However, JQ1 strongly increased the proportion of GATA3+ Th2 cells and the secretion of corresponding cytokines. In a mouse allergic model of lung inflammation, JQ1 did not affect eosinophil infiltration or mucus production but enhanced Th2 cytokine production and aggravated clinical manifestations. CONCLUSION: Altogether, BET inhibition thus interweaves intrinsic negative effects on B cells with a parallel complex reshaping of T-cell polarisation which can increase type 2 cytokines and eventually promote B-cell-dependent immunopathology. These opposite and potentially hazardous immunomodulatory effects raise concerns for clinical use of BET inhibitors in patients with immune disorders.

Evidence for tmTNF reverse signaling in vivo: Implications for an arginase-1-mediated therapeutic effect of TNF inhibitors during inflammation.

In order to ascertain the significance of transmembrane tumor necrosis factor (tmTNF) reverse signaling in vivo, we generated a triple transgenic mouse model (3TG, TNFR1-/-, TNFR2-/-, and tmTNFKI/KI) in which all canonical tumor necrosis factor (TNF) signaling was abolished. In bone-marrow-derived macrophages harvested from these mice, various anti-TNF biologics induced the expression of genes characteristic of alternative macrophages and also inhibited the expression of pro-inflammatory cytokines mainly through the upregulation of arginase-1. Injections of TNF inhibitors during arthritis increased pro-resolutive markers in bone marrow precursors and joint cells leading to a decrease in arthritis score. These results demonstrate that the binding of anti-TNF biologics to tmTNF results in decreased arthritis severity. Collectively, our data provide evidence for the significance of tmTNF reverse signaling in the modulation of arthritis. They suggest a complementary interpretation of anti-TNF biologics effects in the treatment of inflammatory diseases and pave the way to studies focused on new arginase-1-dependent therapeutic targets.

Ageing of T-dependent B cell responses.

The human immune system is in continuous interaction with environmental factors (pathogens, exercise, stress, pollutants, diet, vaccines, and therapeutics) that condition its efficiency by promoting or moderating multiple immune mechanisms. While the deleterious impact of external factors can be avoided or limited, the immune system itself grows weaker with age. Immune cells persist in the elderly, and the observed decline of cellular immunity is related to cellular senescence. Immunosenescence, which affects both T and B cells, erodes lymphocyte-dependent responses to vaccines and pathogens. Germinal centers (GCs), the organized lymphoid structures where B cells engage in affinity maturation, are regulated by follicular helper (Tfh) and follicular regulatory (Tfr) T cells, the major T cell components of GCs. This review discusses how age-related changes affect Tfh and Tfr cells as key components of B cell immunity, and how they ultimately shape the response of the ageing immune system to vaccines and infectious challenges.

Antigen-dependent multistep differentiation of T follicular helper cells and its role in SARS-CoV-2 infection and vaccination.

T follicular helper (Tfh) cells play an essential role in regulating the GC reaction and, consequently, the generation of high-affinity antibodies and memory B cells. Therefore, Tfh cells are critical for potent humoral immune responses against various pathogens and their dysregulation has been linked to autoimmunity and cancer. Tfh cell differentiation is a multistep process, in which cognate interactions with different APC types, costimulatory and coinhibitory pathways, as well as cytokines are involved. However, it is still not fully understood how a subset of activated CD4+ T cells begins to express the Tfh cell-defining chemokine receptor CXCR5 during the early stage of the immune response, how some CXCR5+ pre-Tfh cells enter the B-cell follicles and mature further into GC Tfh cells, and how Tfh cells are maintained in the memory compartment. In this review, we discuss recent advances on how antigen and cognate interactions are important for Tfh cell differentiation and long-term persistence of Tfh cell memory, and how this is relevant to the current understanding of COVID-19 pathogenesis and the development of potent SARS-CoV-2 vaccines.

Rituximab and Corticosteroid Effect on Desmoglein-Specific B Cells and Desmoglein-Specific T Follicular Helper Cells in Pemphigus.

Pemphigus is an autoimmune blistering disease mediated by autoantibodies directed against desmogleins (DSGs). We recently showed that first-line treatment with rituximab (RTX) enables more patients to achieve long-lasting remission off therapy than corticosteroids alone. To understand the immunological mechanisms that mediate long-lasting clinical remission after RTX treatment, we analyzed the phenotype of DSG-specific memory B cells and DSG-specific T follicular helper cells by flow cytometry and measured antibody-secreting cells by enzyme-linked immune absorbent spot in patients treated with corticosteroids alone or RTX. This post hoc analysis of the RITUX3 trial showed that RTX induced a significant decrease of IgG-switched DSG-specific memory B cells. Accordingly, anti-DSG antibody-secreting cells were no longer detected in patients in complete remission after RTX. In contrast, corticosteroids did not modify the frequency or the phenotype of DSG-specific memory B cells, and anti-DSG antibody-secreting cells were still detected after treatment, even in patients in remission. Using peptide-HLADRB1∗0402 tetramer staining, we identified DSG-3-specific T follicular helper cells, which dramatically decreased after RTX, while remaining stable after corticosteroid treatment. Our findings suggest that long-lasting response to RTX in pemphigus relies on the decrease of DSG-specific circulating T follicular helper cells, which correlates with a sustained depletion of IgG-switched memory autoreactive B cells, leading to the disappearance of anti-DSG antibody-secreting cells.

Ageing promotes early T follicular helper cell differentiation by modulating expression of RBPJ.

Ageing profoundly changes our immune system and is thought to be a driving factor in the morbidity and mortality associated with infectious disease in older people. We have previously shown that the impaired immunity to vaccination that occurs in aged individuals is partly attributed to the effect of age on T follicular helper (Tfh) cell formation. In this study, we examined how age intrinsically affects Tfh cell formation in both mice and humans. We show increased formation of Tfh precursors (pre-Tfh) but no associated increase in germinal centre (GC)-Tfh cells in aged mice, suggesting age-driven promotion of only early Tfh cell differentiation. Mechanistically, we show that ageing alters TCR signalling which drives expression of the Notch-associated transcription factor, RBPJ. Genetic or chemical modulation of RBPJ or Notch rescues this age-associated early Tfh cell differentiation, and increased intrinsic Notch activity recapitulates this phenomenon in younger mice. Our data offer mechanistic insight into the age-induced changes in T-cell activation that affects the differentiation and ultimately the function of effector T cells.

CD5 signalosome coordinates antagonist TCR signals to control the generation of Treg cells induced by foreign antigens.

CD5 is characterized as an inhibitory coreceptor with an important regulatory role during T cell development. The molecular mechanism by which CD5 operates has been puzzling and its function in mature T cells suggests promoting rather than repressing effects on immune responses. Here, we combined quantitative mass spectrometry and genetic studies to analyze the components and the activity of the CD5 signaling machinery in primary T cells. We found that T cell receptor (TCR) engagement induces the selective phosphorylation of CD5 tyrosine 429, which serves as a docking site for proteins with adaptor functions (c-Cbl, CIN85, CRKL), connecting CD5 to positive (PI3K) and negative (UBASH3A, SHIP1) regulators of TCR signaling. c-CBL acts as a coordinator in this complex enabling CD5 to synchronize positive and negative feedbacks on TCR signaling through the other components. Disruption of CD5 signalosome in mutant mice reveals that it modulates TCR signal outputs to selectively repress the transactivation of Foxp3 and limit the inopportune induction of peripherally induced regulatory T cells during immune responses against foreign antigen. Our findings bring insights into the paradigm of coreceptor signaling, suggesting that, in addition to providing dualistic enhancing or dampening inputs, coreceptors can engage concomitant stimulatory and inhibitory signaling events, which act together to promote specific functional outcomes.

A 3-week nonalcoholic steatohepatitis mouse model shows elafibranor benefits on hepatic inflammation and cell death.

The long duration of animal models represents a clear limitation to quickly evaluate the efficacy of drugs targeting nonalcoholic steatohepatitis (NASH). We, therefore, developed a rapid mouse model of liver inflammation (i.e., the mouse fed a high-fat/high-cholesterol diet, where cyclodextrin is co-administered to favor hepatic cholesterol loading, liver inflammation, and NASH within 3 weeks), and evaluated the effects of the dual peroxisome proliferator-activated receptor alpha/delta agonist elafibranor (ELA). C57BL6/J mice were fed a 60% high-fat, 1.25% cholesterol, and 0.5% cholic acid diet with 2% cyclodextrin in drinking water (HFCC/CDX diet) for 3 weeks. After 1 week of the diet, mice were treated orally with vehicle or ELA 20 mg/kg q.d. for 2 weeks. Compared with vehicle, ELA markedly reduced liver lipids and nonalcoholic fatty liver disease activity scoring, through steatosis, inflammation, and fibrosis (all P < 0.01 vs. vehicle). Flow cytometry analysis showed that ELA significantly improved the HFCC/CDX diet-induced liver inflammation by preventing the increase in total number of immune cells (CD45+), Kupffer cells, dendritic cells, and monocytes population, as well as the reduction in natural killer and natural killer T cells, and by blocking conversion of T cells in regulatory T cells. ELA did not alter pyroptosis (Gasdermin D), but significantly reduced necroptosis (cleaved RIP3) and apoptosis (cleaved caspase 3) in the liver. In conclusion, ELA showed strong benefits on NASH, including improvement in hepatic inflammation, necroptosis, and apoptosis in the 3-week NASH mouse. This preclinical model will be useful to rapidly detect the effects of novel drugs targeting NASH.

Regulation of B cell responses by distinct populations of CD4 T cells.

Maturation of B cells in Germinal Centers (GC) is a hallmark in adaptive immunity and the basis of successful vaccines that protect us against lethal infections. Nonetheless, vaccination efficacy is very much reduced in aged population and against highly mutagenic viruses. Therefore, it is key to understand how B cell selection takes place in GC in order to develop new and fully protective vaccines. The cellular mechanisms that control selection of GC B cells are performed by different T cell populations. On one side, cognate entanglement of B cells with T follicular helper (Tfh) cells through cytokines and co-stimulatory signals promotes survival, proliferation, mutagenesis and terminal differentiation of GC B cells. On the other hand, regulatory T cells have also been reported within GC and interfere with T cell help for antibody production. These cells have been classified as a distinct T cell sub-population called T Follicular regulatory cells (Tfr). In this review, we investigate the phenotype, function and differentiation of these two cell populations. In addition, based on the different functions of these cell subsets, we highlight the open questions surrounding their heterogeneity.

A Natural Variant of the Signaling Molecule Vav1 Enhances Susceptibility to Myasthenia Gravis and Influences the T Cell Receptor Repertoire.

The guanine nucleotide exchange factor Vav1 is essential for transducing T cell receptor (TCR) signals and plays an important role in T cell development and activation. Previous genetic studies identified a natural variant of Vav1 characterized by the substitution of an arginine (R) residue by a tryptophane (W) at position 63 (Vav1R63W). This variant impacts Vav1 adaptor functions and controls susceptibility to T cell-mediated neuroinflammation. To assess the implication of this Vav1 variant on the susceptibility to antibody-mediated diseases, we used the animal model of myasthenia gravis, experimental autoimmune myasthenia gravis (EAMG). To this end, we generated a knock-in (KI) mouse model bearing a R to W substitution in the Vav1 gene (Vav1R63W) and immunized it with either torpedo acetylcholine receptor (tAChR) or the α146-162 immunodominant peptide. We observed that the Vav1R63W conferred increased susceptibility to EAMG, revealed by a higher AChR loss together with an increased production of effector cytokines (IFN-γ, IL-17A, GM-CSF) by antigen-specific CD4+ T cells, as well as an increased frequency of antigen-specific CD4+ T cells. This correlated with the emergence of a dominant antigen-specific T cell clone in KI mice that was not present in wild-type mice, suggesting an impact on thymic selection and/or a different clonal selection threshold following antigen encounter. Our results highlight the key role of Vav1 in the pathophysiology of EAMG and this was associated with an impact on the TCR repertoire of AChR reactive T lymphocytes.

Several Follicular Regulatory T Cell Subsets With Distinct Phenotype and Function Emerge During Germinal Center Reactions.

An efficient B cell immunity requires a dynamic equilibrium between positive and negative signals. In germinal centers (GCs), T follicular helper cells are supposed to be the positive regulator while T follicular regulatory (Tfr) cells were assigned to be the negative regulators. Indeed, Tfr cells are considered as a homogenous cell population dedicated to dampen the GC extent. Moreover, Tfr cells prevent autoimmunity since their dysregulation leads to production of self-reactive antibodies (Ab). However, a growing corpus of evidence has revealed additional and unexpected functions for Tfr cells in the regulation of B cell responses. This review provides an overview of the Tfr cell contribution and presents Tfr cell proprieties in the context of vaccination.