Peripheral regulatory T lymphocytes recirculating to the thymus suppress the development of their precursors.

Most T lymphocytes, including regulatory T cells (Treg cells), differentiate in the thymus. The age-dependent involution of this organ leads to decreasing production of T cells. Here we found that the output of new Treg cells from the thymus decreased substantially more than that of conventional T cells. Peripheral mouse and human Treg cells recirculated back to the thymus, where they constituted a large proportion of the pool of Treg cells and displayed an activated and differentiated phenotype. In the thymus, the recirculating cells exerted their regulatory function by inhibiting interleukin 2 (IL-2)-dependent de novo differentiation of Treg cells. Thus, Treg cell development is controlled by a negative feedback loop in which mature progeny cells return to the thymus and restrain development of precursors of Treg cells.

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.

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.

Polarization of circulating Follicular Helper T cells correlates with the severity of Bullous Pemphigoid.

BACKGROUND: T-follicular-helper (Tfh) cells form a distinct population of T-helper cells with different polarizations (type 1, type 2 and type 17) that regulates humoral responses and may participate in the pathophysiology of B-cell mediated autoimmune diseases, such as bullous pemphigoid (BP), a dermatosis mediated by auto-antibodies specific for hemidesmosomal proteins. OBJECTIVES: The aim was to evaluate the impact of superpotent topical corticosteroid (TCS) treatment, which is more effective and safer than high doses of oral corticosteroids and recommended first-line treatment of BP, on circulating Tfh cells. METHODS: We compared by flow cytometry the frequency, polarization and activation of blood Tfh cells from patients with BP at baseline and longitudinally after initiation of TCS treatment to age- and sex-matched healthy subjects. RESULTS: We observed that circulating Tfh cells were more frequent in patients with BP at baseline than in healthy subjects and exhibited an activated phenotype. We further showed a decrease of type 1 and an increase of type 17 Tfh cells in the blood of patients, which resulted in a higher (type 2+type 17) to type 1 Tfh cell ratio. This ratio correlated positively with disease severity as measured by the Bullous Pemphigoid Disease Area Index. Remarkably, along TCS treatment, although the frequency of Tfh cells returned to a level similar to control, the activated phenotype persisted. Interestingly serum IL-21 levels and the Tfh cell subset ratio, similarly to disease activity and serum anti-BP180 and anti-BP230 auto-antibodies, were decreased under the TCS treatment. CONCLUSIONS: Overall, our findings suggest an involvement of the polarization of Tfh cells in the pathophysiology of BP and open the door to a modulation of their activity for therapeutic purposes.

The emergence of circulating activated autoreactive desmoglein 3-specific follicular regulatory T cells is associated with long-term efficacy of rituximab in patients with pemphigus vulgaris.

BACKGROUND: Pemphigus vulgaris (PV) is characterized by autoantibodies targeting keratinocyte adhesion proteins desmoglein (Dsg) 1 and 3, and by the human leukocyte antigen (HLA) predisposition allele HLA-DRB1*0402. Treatment using rituximab (RTX) combined with short-term corticosteroids (CS) allows disease control and long-lasting remission. OBJECTIVES: The principal aim of this study was to evaluate the impact of RTX on the circulating subpopulations of Dsg3-specific T lymphocytes that specifically regulate B-cell responses: follicular helper T (Tfh) and follicular regulatory T (Tfr) lymphocytes. METHODS: Using the HLA-DRB1*0402 tetramer loaded with the Dsg3 immunodominant peptide, we used flow cytometry to analyse the frequency, polarization and activation status of blood Dsg3-specific follicular T-cell populations at baseline, month (M) 6 and long-term follow-up (M60-90) from patients with PV. RESULTS: At baseline, we observed a predominance of Tfh1* and Tfh17 subsets and an underrepresentation of the Tfh2 subset among autoreactive Dsg3-specific Tfh cells compared with nonautoreactive Tfh cells. RTX treatment induced a decrease of autoreactive Tfh cells with no effect on their polarization during follow-up. In parallel, we observed the emergence of a Dsg3-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 nonautoreactive Tfr cells of the same patients with PV. In contrast, very few Dsg3-specific Tfr cells were observed in patients with PV who were treated with CS alone. CONCLUSIONS: Here we show that the emergence of circulating autoreactive Dsg3-specific Tfr cells is associated with the long-term efficacy of RTX in patients with PV.

Pemphigus vulgaris (PV) is an autoimmune disease mediated by pathogenic antibodies directed against the body's own tissues (called autoantibodies). In this condition, autoantibodies bind to and inhibit a protein called desmoglein 3 (Dsg3), which is responsible for the adhesion of keratinocytes (cells that make up the superficial layers of the skin and mucous membranes). As a result, PV causes painful blistering. People with PV are commonly treated with low doses of oral corticosteroids (prednisone) and rituximab (administered via infusions), which have significantly improved patient outcomes. Rituximab temporarily (for 6­9 months) depletes B lymphocytes, which are immune cells that differentiate into cells producing anti-Dsg3 antibodies. While autoimmune diseases can be lifelong, this study looked at why some people achieve long-lasting remissions without ongoing treatment, even after the return of B lymphocytes. We analysed anti-Dsg3 follicular T cells, which regulate B-cell differentiation. Among these, we observed the emergence of regulatory (or inhibiting) T cells over time following treatment, accompanied by a decrease in conventional (or activating) follicular T cells. We also found that these autoimmune follicular regulatory T cells were more activated compared with conventional follicular T cells or other T cells not directed against the Dsg3 protein. Overall, our findings suggest that the emergence of these follicular regulatory T cells may be responsible for the sustained remission observed in some people with PV and could be promoted by the use of rituximab.

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.

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.

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.

Plasma cells negatively regulate the follicular helper T cell program.

B lymphocytes differentiate into antibody-secreting cells under the antigen-specific control of follicular helper T cells (T(FH) cells). Here we demonstrate that isotype-switched plasma cells expressed major histocompatibility complex (MHC) class II, the costimulatory molecules CD80 and CD86, and the intracellular machinery required for antigen presentation. Antigen-specific plasma cells accessed, processed and presented sufficient antigen in vivo to induce multiple helper T cell functions. Notably, antigen-primed plasma cells failed to induce interleukin 21 (IL-21) or the transcriptional repressor Bcl-6 in naive helper T cells and actively decreased these key molecules in antigen-activated T(FH) cells. Mice lacking plasma cells showed altered T(FH) cell activity, which provided evidence of this negative feedback loop. Hence, antigen presentation by plasma cells defines a previously unknown layer of cognate regulation that limits the antigen-specific T(FH) cell program that controls ongoing B cell immunity.

The function of follicular helper T cells is regulated by the strength of T cell antigen receptor binding.

How follicular helper T cells (T(FH) cells) differentiate to regulate B cell immunity is critical for effective protein vaccination. Here we define three transcription factor T-bet-expressing antigen-specific effector helper T cell subsets with distinguishable function, migratory properties and developmental programming in vivo. Expression of the transcriptional repressor Blimp-1 distinguished T zone 'lymphoid' effector helper T cells (CD62L(hi)CCR7(hi)) from CXCR5(lo) 'emigrant' effector helper T cells and CXCR5(hi) 'resident' T(FH) cells expressing the transcriptional repressor Bcl-6 (CD62L(lo)CCR7(lo)). We then show by adoptive transfer and intact polyclonal responses that helper T cells with the highest specific binding of peptide-major histocompatibility complex class II and the most restricted T cell antigen receptor junctional diversity 'preferentially' developed into the antigen-specific effector T(FH) compartment. Our studies demonstrate a central function for differences in the binding strength of the T cell antigen receptor in the antigen-specific mechanisms that 'program' specialized effector T(FH) function in vivo.

The strength of BCR signaling shapes terminal development of follicular helper T cells in mice.

Antibody production is key for effective immune response and relies on follicular helper T (Tfh) cells. B cell-Tfh cell interactions result either in an extra-follicular low affinity B-cell response or in germinal center reactions producing high-affinity memory B cells and long-lived plasma cells. As Tfh cells influence B-cell commitment, it also became clear that B cells influence these interactions in ways that still remain unresolved. We observed that strong BCR signals decreased Tfh-cell differentiation in vitro, which correlated with decreased expression of ICOS-L at the surface of stimulated B cells. Further, we comprehensively demonstrated that ICOS-L expression correlated with the level of Tfh differentiation irrespective of antigen presentation at the surface of activated B cells. Our in vivo experiments could show that immunization with a high-affinity antigen for B cells resulted in much less Tfh development than immunization with low-affinity antigen. Furthermore, blocking ICOS-L in vivo inhibited Tfh development when using low-affinity antigen. Altogether, these results indicate that BCR affinity shapes Tfh-cell development in part through ICOS/ICOS-L interactions. Ultimately, we reveal new depths in the B cell-Tfh cell crosstalk that could eventually result in better vaccine protocols.

Circulating follicular helper T cells exhibit reduced ICOS expression and impaired function in narcolepsy type 1 patients.

Despite genetic and epidemiological evidence strongly supporting an autoimmune basis for narcolepsy type 1, the mechanisms involved have remained largely unknown. Here, we aimed to investigate whether the frequency and function of circulating follicular helper and follicular regulatory T cells are altered in narcolepsy type 1. Peripheral blood mononuclear cells were collected from 32 patients with narcolepsy type 1, including 11 who developed disease after Pandemrix® vaccination, and 32 age-, sex-, and HLA-DQB1*06:02-matched healthy individuals. The frequency and phenotype of the different circulating B cell and follicular T cell subsets were examined by flow cytometry. The function of follicular helper T cells was evaluated by assessing the differentiation of naïve and memory B cells in a co-culture assay. We revealed a notable increase in the frequency of circulating B cells and CD4+CXCR5+ follicular T cells in narcolepsy patients compared to age-, sex- and HLA-matched healthy controls. However, the inducible T-cell costimulator molecule, ICOS, was selectively down-regulated on follicular T cells from patients. Reduced frequency of activated ICOS+ and PD1high blood follicular T cells was also observed in the narcolepsy group. Importantly, follicular T cells isolated from patients with narcolepsy type 1 had a reduced capacity to drive the proliferation/survival and differentiation of memory B cells. Our results provide novel insights into the potential involvement of T cell-dependent B cell responses in the pathogenesis of narcolepsy type 1 in which down-regulation of ICOS expression on follicular helper T cells correlates with their reduced function. We hypothesize that these changes contribute to regulation of the deleterious autoimmune process after disease onset.

Follicular helper T cells: lineage and location.

Follicular helper T (Tfh) cells are the class of effector T helper cells that regulates the step-wise development of antigen-specific B cell immunity in vivo. Deployment of CXCR5+ Tfh cells to B cell zones of lymphoid tissues and stable cognate interactions with B cells are central to the delivery of antigen-specific Tfh cell function. Here, we review recent advances that have helped to unravel distinctive elements of developmental programming for Tfh cells and unique effector Tfh cell functions focused on antigen-primed B cells. Understanding the regulatory functions of Tfh cells in the germinal center and the subsequent regulation of memory B cell responses to antigen recall represent the frontiers of this research area with the potential to alter fundamentally the design of future vaccines.

Myelin oligodendrocyte glycoprotein induces incomplete tolerance of CD4(+) T cells specific for both a myelin and a neuronal self-antigen in mice.

T-cell polyspecificity, predicting that individual T cells recognize a continuum of related ligands, implies that multiple antigens can tolerize T cells specific for a given self-antigen. We previously showed in C57BL/6 mice that part of the CD4(+) T-cell repertoire specific for myelin oligodendrocyte glycoprotein (MOG) 35-55 also recognizes the neuronal antigen neurofilament medium (NF-M) 15-35. Such bi-specific CD4(+) T cells are frequent and produce inflammatory cytokines after stimulation. Since T cells recognizing two self-antigens would be expected to be tolerized more efficiently, this finding prompted us to study how polyspecificity impacts tolerance. We found that similar to MOG, NF-M is expressed in the thymus by medullary thymic epithelial cells, a tolerogenic population. Nevertheless, the frequency, phenotype, and capacity to transfer experimental autoimmune encephalomyelitis (EAE) of MOG35-55 -reactive CD4(+) T cells were increased in MOG-deficient but not in NF-M-deficient mice. We found that presentation of NF-M15-35 by I-A(b) on dendritic cells is of short duration, suggesting unstable MHC class II binding. Consistently, introducing an MHC-anchoring residue into NF-M15-35 (NF-M15-35 T20Y) increased its immunogenicity, activating a repertoire able to induce EAE. Our results show that in C57BL/6 mice bi-specific encephalitogenic T cells manage to escape tolerization due to inefficient exposure to two self-antigens.

Vaccine adjuvants alter TCR-based selection thresholds.

How T cell receptor (TCR) specificity evolves in vivo after protein vaccination is central to the development of helper T (Th) cell function. Most models of clonal selection in the Th cell compartment favor TCR affinity-based thresholds. Here, we demonstrated that depot-forming vaccine adjuvants did not require Toll-like receptor (TLR) agonists to induce clonal dominance in antigen-specific Th cell responses. However, readily dispersible adjuvants using TLR-9 and TLR-4 agonists skewed TCR repertoire usage by increasing TCR selection thresholds and enhancing antigen-specific clonal expansion. In this manner, vaccine adjuvants control the local accumulation of Th cells expressing TCR with the highest peptide MHC class II binding. Clonal composition was altered by mechanisms that blocked the local propagation of clonotypes independently of antigen dose and not as a consequence of interclonal competition. This capacity of adjuvants to modify antigen-specific Th cell clonal composition has fundamental implications for the design of future protein subunit vaccines.

Central tolerance spares the private high-avidity CD4(+) T-cell repertoire specific for an islet antigen in NOD mice.

Although central tolerance induces the deletion of most autoreactive T cells, some autoreactive T cells escape thymic censorship. Whether potentially harmful autoreactive T cells present distinct TCRαß features remains unclear. Here, we analyzed the TCRαß repertoire of CD4(+) T cells specific for the S100ß protein, an islet antigen associated with type 1 diabetes. We found that diabetes-resistant NOD mice deficient for thymus specific serine protease (TSSP), a protease that impairs class II antigen presentation by thymic stromal cells, were hyporesponsive to the immunodominant S100ß1-15 epitope, as compared to wild-type NOD mice, due to intrathymic negative selection. In both TSSP-deficient and wild-type NOD mice, the TCRαß repertoire of S100ß-specific CD4(+) T cells though diverse showed a specific bias for dominant TCRα rearrangements with limited CDR3α diversity. These dominant TCRα chains were public since they were found in all mice. They were of intermediate- to low-avidity. In contrast, high-avidity T cells expressed unique TCRs specific to each individual (private TCRs) and were only found in wild-type NOD mice. Hence, in NOD mice, the autoreactive CD4(+) T-cell compartment has two major components, a dominant and public low-avidity TCRα repertoire and a private high-avidity CD4(+) T-cell repertoire; the latter is deleted by re-enforced negative selection.

Autoimmune regulator (AIRE)-deficient CD8+CD28low regulatory T lymphocytes fail to control experimental colitis.

Mutations in the gene encoding the transcription factor autoimmune regulator (AIRE) are responsible for autoimmune polyendocrinopathy candidiasis ectodermal dystrophy syndrome. AIRE directs expression of tissue-restricted antigens in the thymic medulla and in lymph node stromal cells and thereby substantially contributes to induction of immunological tolerance to self-antigens. Data from experimental mouse models showed that AIRE deficiency leads to impaired deletion of autospecific T-cell precursors. However, a potential role for AIRE in the function of regulatory T-cell populations, which are known to play a central role in prevention of immunopathology, has remained elusive. Regulatory T cells of CD8(+)CD28(low) phenotype efficiently control immune responses in experimental autoimmune and colitis models in mice. Here we show that CD8(+)CD28(low) regulatory T lymphocytes from AIRE-deficient mice are transcriptionally and phenotypically normal and exert efficient suppression of in vitro immune responses, but completely fail to prevent experimental colitis in vivo. Our data therefore demonstrate that AIRE plays an important role in the in vivo function of a naturally occurring regulatory T-cell population.

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.