Regulatory T Cells and Human Disease.

Naturally occurring CD4+ regulatory T cells (Tregs), which specifically express the transcription factor FoxP3 in the nucleus and CD25 and CTLA-4 on the cell surface, are a functionally distinct T cell subpopulation actively engaged in the maintenance of immunological self-tolerance and homeostasis. Recent studies have facilitated our understanding of the cellular and molecular basis of their generation, function, phenotypic and functional stability, and adaptability. It is under investigation in humans how functional or numerical Treg anomalies, whether genetically determined or environmentally induced, contribute to immunological diseases such as autoimmune diseases. Also being addressed is how Tregs can be targeted to control physiological and pathological immune responses, for example, by depleting them to enhance tumor immunity or by expanding them to treat immunological diseases. This review discusses our current understanding of Treg immunobiology in normal and disease states, with a perspective on the realization of Treg-targeting therapies in the clinic.

CTLA-4-expressing ILC3s restrain interleukin-23-mediated inflammation.

Interleukin (IL-)23 is a major mediator and therapeutic target in chronic inflammatory diseases that also elicits tissue protection in the intestine at homeostasis or following acute infection1-4. However, the mechanisms that shape these beneficial versus pathological outcomes remain poorly understood. To address this gap in knowledge, we performed single-cell RNA sequencing on all IL-23 receptor-expressing cells in the intestine and their acute response to IL-23, revealing a dominance of T cells and group 3 innate lymphoid cells (ILC3s). Unexpectedly, we identified potent upregulation of the immunoregulatory checkpoint molecule cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) on ILC3s. This pathway was activated by gut microbes and IL-23 in a FOXO1- and STAT3-dependent manner. Mice lacking CTLA-4 on ILC3s exhibited reduced regulatory T cells, elevated inflammatory T cells and more-severe intestinal inflammation. IL-23 induction of CTLA-4+ ILC3s was necessary and sufficient to reduce co-stimulatory molecules and increase PD-L1 bioavailability on intestinal myeloid cells. Finally, human ILC3s upregulated CTLA-4 in response to IL-23 or gut inflammation and correlated with immunoregulation in inflammatory bowel disease. These results reveal ILC3-intrinsic CTLA-4 as an essential checkpoint that restrains the pathological outcomes of IL-23, suggesting that disruption of these lymphocytes, which occurs in inflammatory bowel disease5-7, contributes to chronic inflammation.

Human FOXP3+ Regulatory T Cell Heterogeneity and Function in Autoimmunity and Cancer.

Regulatory T (Treg) cells expressing the transcription factor Foxp3 have a critical role in the maintenance of immune homeostasis and prevention of autoimmunity. Recent advances in single cell analyses have revealed a range of Treg cell activation and differentiation states in different human pathologies. Here we review recent progress in the understanding of human Treg cell heterogeneity and function. We discuss these findings within the context of concepts in Treg cell development and function derived from preclinical models and insight from approaches targeting Treg cells in clinical settings. Distinguishing functional Treg cells from other T cells and understanding the context-dependent function(s) of different Treg subsets will be crucial to the development of strategies toward the selective therapeutic manipulation of Treg cells in autoimmunity and cancer.

Control of foreign Ag-specific Ab responses by Treg and Tfr.

Regulatory T cells (Tregs) expressing the transcription factor Foxp3 play a critical role in the control of immune homeostasis including the regulation of humoral immunity. Recently, it has become clear that a specialized subset of Tregs, T-follicular regulatory cells (Tfr), have a particular role in the control of T-follicular helper (Tfh) cell-driven germinal center (GC) responses. Following similar differentiation signals as received by Tfh, Tfr gain expression of characteristic chemokine receptors and transcription factors such as CXCR5 and BCL6 allowing them to travel to the B-cell follicle and deliver in situ suppression. It seems clear that Tfr are critical for the prevention of autoimmune antibody induction. However, their role in the control of foreign antigen-specific antibody responses appears more complex with various reports demonstrating either increased or decreased antigen-specific antibody responses following inhibition of Tfr function. Due to their recent discovery, our understanding of Tfr formation and function still has many gaps. In this review, we discuss our current knowledge of both Tregs and Tfr in the context of humoral immunity and how these cells might be manipulated in order to better control vaccine responses.

Foxp3⁺ T(reg) cells in humoral immunity.

T(reg) cells are essential for the maintenance of immune homeostasis and prevention of autoimmunity. In humoral immune responses, loss of T(reg) cell function causes increased levels of serum autoantibodies, hyper-IgE, spontaneous generation of germinal centres, and enhanced numbers of specialised T follicular helper cells (T(fh) cells) controlled by the lineage-defining transcription factor BCL-6 (B-cell lymphoma 6). Recent studies have demonstrated that a subset of T(reg) cells [T follicular regulatory (T(freg)) cells] are able to co-opt the follicular T-cell program by gaining expression of BCL-6 and travelling to the follicle where they have an important role in the control of expansion of T(fh) cells and the germinal centre reaction. However, the mechanisms by which they exert this control are still under investigation. In this review, we discuss the effects of T(reg) cells on humoral immunity and the mechanisms by which they exert their regulatory function.

Two modes of immune suppression by Foxp3(+) regulatory T cells under inflammatory or non-inflammatory conditions.

Foxp3-expressing regulatory T cells (Tregs) play a crucial role in maintaining immune tolerance and homeostasis. One of the key issues for understanding Treg immunobiology is to determine how they suppress excessive or aberrant immune responses. Although a number of molecules have been reported to contribute to Treg suppressive function, the importance and precise role of each molecule is not clear. In this review, we propose and discuss that two modes of suppression can be distinguished. In the physiological and steady state, activation of naïve T cells can be suppressed by natural Tregs via deprivation of activation signals including CD28 signal and IL-2 from antigen-reactive T cells, keeping the latter in a naïve state in lymphoid tissues. These deprivation mechanisms are transiently abrogated in inflammatory conditions, allowing T cells to respond to antigen. In contrast, in highly inflammatory environments, for example, in microbial infection, activated Tregs acquire the capacity to kill or inactivate effector T cells and antigen-presenting cells, for example, via granzyme/perforin formation and IL-10 secretion, thereby actively damping excessive immune responses. Understanding these processes will help effectively controlling physiological and pathological immune responses via Tregs.

Contact dependent suppression of CD4 T cell activation and proliferation by B cells activated through IgD cross-linking.

Although the co-stimulatory interaction between B and T cells is well defined, recent evidence suggests that B cells also have a regulatory role. Here, we show that B cells activated using anti-IgD conjugated to dextran (α-δ-dex) directly inhibit TCR-induced CD4 T cell activation, proliferation and cytokine production. This effect was observed in CD4 T cells activated both with and without CD28 co-stimulation. T cell viability was unaffected, and the T cell suppressive effect was mediated by contact with IgD activated purified B cells and not by IL-10 or other soluble factors. This is the first evidence of IgD activated B cells mediating inhibition of activation and proliferation of CD4 T cells in humans. This article is protected by copyright. All rights reserved.

A genetic method specifically delineates Th1-type Treg cells and their roles in tumor immunity.

Regulatory T (Treg) cells expressing the transcription factor (TF) Foxp3 also express other TFs shared by T helper (Th) subsets under certain conditions. Here, to determine the roles of T-bet-expressing Treg cells, we generate a mouse strain, called VeDTR, in which T-bet/Foxp3 double-positive cells are engineered to be specifically labeled and depleted by a combination of Cre- and Flp-recombinase-dependent gene expression control. Characterization of T-bet+Foxp3+ cells using VeDTR mice reveals high resistance under oxidative stress, which is involved in accumulation of T-bet+Foxp3+ cells in tumor tissues. Moreover, short-term depletion of T-bet+Foxp3+ cells leads to anti-tumor immunity but not autoimmunity, whereas that of whole Treg cells does both. Although ablation of T-bet+Foxp3+ cells during Toxoplasma infection slightly enhances Th1 immune responses, it does not affect the course of the infection. Collectively, the intersectional genetic method reveals the specific roles of T-bet+Foxp3+ cells in suppressing tumor immunity.

Celastrol suppresses humoral immune responses and autoimmunity by targeting the COMMD3/8 complex.

Celastrol, a bioactive molecule extracted from the Tripterygium wilfordii plant, has been shown to exhibit anti-inflammatory properties. However, its mechanism of action has not been fully elucidated. Here, we show that celastrol suppresses humoral immune responses and autoimmunity by disabling a protein complex consisting of copper metabolism MURR1 domain-containing (COMMD) 3 and COMMD8 (COMMD3/8 complex), a signaling adaptor for chemoattractant receptors. Having demonstrated the involvement of the COMMD3/8 complex in a mouse model of rheumatoid arthritis, we identified celastrol as a compound that covalently bound to and dissociated the COMMD3/8 complex. Celastrol inhibited B cell migration, reduced antibody responses, and blocked arthritis progression, recapitulating deficiency of the COMMD3/8 complex. These effects of celastrol were abolished in mice expressing a celastrol-resistant mutant of the COMMD3/8 complex. These findings establish that celastrol exerts immunosuppressive activity by targeting the COMMD3/8 complex. Our study suggests that the COMMD3/8 complex is a potentially druggable target in autoimmune diseases and points to celastrol as a lead pharmacologic candidate in this capacity.

Single-cell analyses and host genetics highlight the role of innate immune cells in COVID-19 severity.

Mechanisms underpinning the dysfunctional immune response in severe acute respiratory syndrome coronavirus 2 infection are elusive. We analyzed single-cell transcriptomes and T and B cell receptors (BCR) of >895,000 peripheral blood mononuclear cells from 73 coronavirus disease 2019 (COVID-19) patients and 75 healthy controls of Japanese ancestry with host genetic data. COVID-19 patients showed a low fraction of nonclassical monocytes (ncMono). We report downregulated cell transitions from classical monocytes to ncMono in COVID-19 with reduced CXCL10 expression in ncMono in severe disease. Cell-cell communication analysis inferred decreased cellular interactions involving ncMono in severe COVID-19. Clonal expansions of BCR were evident in the plasmablasts of patients. Putative disease genes identified by COVID-19 genome-wide association study showed cell type-specific expressions in monocytes and dendritic cells. A COVID-19-associated risk variant at the IFNAR2 locus (rs13050728) had context-specific and monocyte-specific expression quantitative trait loci effects. Our study highlights biological and host genetic involvement of innate immune cells in COVID-19 severity.

TCR diversity and Treg cells, sometimes more is more.

Treg cells are critical for the maintenance of immune homeostasis and suppression of naturally occurring self-reactive T cells; however, in order to induce suppression Treg cells must first be activated via their T-cell receptor by recognition of specific antigen-MHC complexes. In this issue of the European Journal of Immunology, Föhse et al. [Eur. J. Immunol. 2011. 41: 3101-3113.] shed light on the important question of the role of TCR diversity on Treg-cell function by demonstrating that high TCR diversity is crucial for optimal Treg-cell expansion, peripheral reshaping of the Treg-cell TCR repertoire and in vivo suppressive capacity. In this Commentary, we discuss these findings and also propose a simple mathematical model to aid in the understanding of the relationship between Treg-cell TCR diversity and the level of suppression delivered by Treg cells in vivo.

Using Mass Cytometry to Address Tfh and Tfr Heterogeneity.

Recent advances in single-cell technologies have made it possible to gather increased amounts of information about even rare cell subtypes. In particular, mass cytometry is able to assess the expression of 30-50 proteins on millions of cells. Within CD4 T-cells, T-follicular helper cells (Tfh) and their regulatory counterpart, T-follicular regulatory cells (Tfr), localize to the B-cell follicle and have specialized roles in the maintenance and regulation of B-cell antibody production. The frequency of Tfh and Tfr in circulation has also been associated with ongoing antibody responses.In this chapter, we detail methods to analyze the frequency and phenotype of the populations of Tfh and Tfr found in humans by mass cytometry.

Symbiotic bacteria-dependent expansion of MR1-reactive T cells causes autoimmunity in the absence of Bcl11b.

MHC class I-related protein 1 (MR1) is a metabolite-presenting molecule that restricts MR1-reactive T cells including mucosal-associated invariant T (MAIT) cells. In contrast to MAIT cells, the function of other MR1-restricted T cell subsets is largely unknown. Here, we report that mice in which a T cell-specific transcription factor, B-cell lymphoma/leukemia 11B (Bcl11b), was ablated in immature thymocytes (Bcl11b∆iThy mice) develop chronic inflammation. Bcl11b∆iThy mice lack conventional T cells and MAIT cells, whereas CD4+IL-18R+ αß T cells expressing skewed Traj33 (Jα33)+ T cell receptors (TCR) accumulate in the periphery, which are necessary and sufficient for the pathogenesis. The disorders observed in Bcl11b∆iThy mice are ameliorated by MR1-deficiency, transfer of conventional T cells, or germ-free conditions. We further show the crystal structure of the TCR expressed by Traj33+ T cells expanded in Bcl11b∆iThy mice. Overall, we establish that MR1-reactive T cells have pathogenic potential.

B cell-intrinsic TBK1 is essential for germinal center formation during infection and vaccination in mice.

The germinal center (GC) is a site where somatic hypermutation and clonal selection are coupled for antibody affinity maturation against infections. However, how GCs are formed and regulated is incompletely understood. Here, we identified an unexpected role of Tank-binding kinase-1 (TBK1) as a crucial B cell-intrinsic factor for GC formation. Using immunization and malaria infection models, we show that TBK1-deficient B cells failed to form GC despite normal Tfh cell differentiation, although some malaria-infected B cell-specific TBK1-deficient mice could survive by GC-independent mechanisms. Mechanistically, TBK1 phosphorylation elevates in B cells during GC differentiation and regulates the balance of IRF4/BCL6 expression by limiting CD40 and BCR activation through noncanonical NF-κB and AKTT308 signaling. In the absence of TBK1, CD40 and BCR signaling synergistically enhanced IRF4 expression in Pre-GC, leading to BCL6 suppression, and therefore failed to form GCs. As a result, memory B cells generated from TBK1-deficient B cells fail to confer sterile immunity upon reinfection, suggesting that TBK1 determines B cell fate to promote long-lasting humoral immunity.

Consecutive BNT162b2 mRNA vaccination induces short-term epigenetic memory in innate immune cells.

Consecutive mRNA vaccinations against SARS-CoV-2 reinforced both innate and adaptive immune responses. However, it remains unclear whether the enhanced innate immune responses are mediated by epigenetic regulation and, if so, whether these effects persist. Using mass cytometry, RNA-Seq, and ATAC-Seq, we show that BNT162b2 mRNA vaccination upregulated antiviral and IFN-stimulated gene expression in monocytes with greater effects after the second vaccination than those after the first vaccination. Transcription factor-binding motif analysis also revealed enriched IFN regulatory factors and PU.1 motifs in accessible chromatin regions. Importantly, although consecutive BNT162b2 mRNA vaccinations boosted innate immune responses and caused epigenetic changes in isolated monocytes, we show that these effects occurred only transiently and disappeared 4 weeks after the second vaccination. Furthermore, single-cell RNA-Seq analysis revealed that a similar gene signature was impaired in the monocytes of unvaccinated patients with COVID-19 with acute respiratory distress syndrome. These results reinforce the importance of the innate immune response in the determination of COVID-19 severity but indicate that, unlike adaptive immunity, innate immunity is not unexpectedly sustained even after consecutive vaccination. This study, which focuses on innate immune memory, may provide novel insights into the vaccine development against infectious diseases.

Adult survivors of invasive pneumococcal disease exhibit defective B cell function.

Adults who have recovered from an episode of invasive pneumococcal disease demonstrate defective B cell activation in response to αδ-dex, a polyclonal polysaccharide mimic, compared with matched control subjects. The defect is not overcome by CD4(+) T cell assistance and may explain the relatively poor response to pneumococcal vaccination in survivors of invasive pneumococcal disease.

Kinetics of immune responses to nasal challenge with meningococcal polysaccharide one year after serogroup-C glycoconjugate vaccination.

BACKGROUND: Recipients of serogroup-C glycoconjugate meningococcal vaccine (MCC) exhibit waning of serum bactericidal antibody (SBA) titers, but the rate of decline and the speed of their immunological memory in response to new meningococcal nasopharyngeal colonization are unknown. METHODS: In a prospective challenge study, we measured persistence of SBA and anti-Neisseria meningitidis serogroup-C (MenC) immunoglobulin (Ig) G and IgA in adults aged 18-39, 28 days and 12 months after receiving MCC. Volunteers were then challenged intranasally with 50 µg MenC polysaccharide to mimic meningococcal colonization, and systemic and mucosal antibody responses were measured. RESULTS: All subjects had protective SBA titers (≥8) 28 days after MCC vaccination, but 12.3% and 20.2% had unprotective (<8) or low (<128) levels, respectively, after 12 months. Following rechallenge (12 months postvaccination) and measurement of antibody responses after 4, 7, and 10 days, rises in SBA titers were only observed in subjects with low (<128) or nonprotective (<8) prerechallenge SBA titers. In subjects with pre rechallenge SBA titers <8, the majority did not reach a protective SBA titer until 7 days post-rechallenge. MenC-specific IgG levels rose in both serum and saliva in correlation with SBA titers. No detectable rise in salivary IgA was observed. CONCLUSIONS: In those individuals who fail to retain protective SBA 12 months after MCC, immunological memory fails to generate protective systemic and mucosal antibodies until 7 days post intranasal challenge with cognate meningococcal polysaccharide. This is likely too slow to protect from natural meningococcal infection. MCC vaccinees rely on persistence of antibody levels rather than immunological memory for sustained protection.

Isolation and Characterization of Both Human and Mouse Tfh/Tfr Cells.

Regulatory T cells (Tregs) expressing the transcription factor Foxp3 have a critical role for the control of immune homeostasis. The Treg subgroup T follicular regulatory cells (Tfr) have a specialized function to travel to the B cell follicle and control antibody responses. While Tfr may be identified by their protein or gene expression profiles, the use of in vitro functional assays to determine their suppressive capacity is important to further characterize these cells. Here we present methods for the identification and purification of Tfr from both mice and humans followed by co-culture with B cells and T follicular helper cells (Tfh). The suppressive activity of the Tfr is then assessed by the ability to prevent Tfh-dependent B cell class switching and plasma blast formation measured by flow cytometry and immunoglobulin production in culture supernatants measured by enzyme-linked immunosorbent assay. These assays will also provide in-depth characterization of the functional suppressive capacity of any isolated Tfr or Treg population. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Isolation of murine T follicular regulatory cells Basic Protocol 2: Measurement of murine T follicular regulatory cell suppressive function Basic Protocol 3: Isolation of human T follicular regulatory cells Basic Protocol 4: Measurement of human T follicular regulatory cell suppressive function.

Arid5a Promotes Immune Evasion by Augmenting Tryptophan Metabolism and Chemokine Expression.

The acquisition of mesenchymal traits leads to immune evasion in various cancers, but the underlying molecular mechanisms remain unclear. In this study, we found that the expression levels of AT-rich interaction domain-containing protein 5a (Arid5a), an RNA-binding protein, were substantially increased in mesenchymal tumor subtypes. The deletion of Arid5a in tumor cell lines enhanced antitumor immunity in immunocompetent mice, but not in immunodeficient mice, suggesting a role for Arid5a in immune evasion. Furthermore, an Arid5a-deficient tumor microenvironment was shown to have robust antitumor immunity, as manifested by suppressed infiltration of granulocytic myeloid-derived suppressor cells and regulatory T cells. In addition, infiltrated T cells were more cytotoxic and less exhausted. Mechanistically, Arid5a stabilized Ido1 and Ccl2 mRNAs and augmented their expression, resulting in enhanced tryptophan catabolism and an immunosuppressive tumor microenvironment. Thus, our findings demonstrate the role of Arid5a beyond inflammatory diseases and suggest Arid5a as a promising target for the treatment of immunotolerant malignant tumors.See related Spotlight by Van den Eynde, p. 854.

CTLA-4 expression by B-1a B cells is essential for immune tolerance.

CTLA-4 is an important regulator of T-cell function. Here, we report that expression of this immune-regulator in mouse B-1a cells has a critical function in maintaining self-tolerance by regulating these early-developing B cells that express a repertoire enriched for auto-reactivity. Selective deletion of CTLA-4 from B cells results in mice that spontaneously develop autoantibodies, T follicular helper (Tfh) cells and germinal centers (GCs) in the spleen, and autoimmune pathology later in life. This impaired immune homeostasis results from B-1a cell dysfunction upon loss of CTLA-4. Therefore, CTLA-4-deficient B-1a cells up-regulate epigenetic and transcriptional activation programs and show increased self-replenishment. These activated cells further internalize surface IgM, differentiate into antigen-presenting cells and, when reconstituted in normal IgH-allotype congenic recipient mice, induce GCs and Tfh cells expressing a highly selected repertoire. These findings show that CTLA-4 regulation of B-1a cells is a crucial immune-regulatory mechanism.