Abatacept increases T cell exhaustion in early RA individuals who carry HLA risk alleles.

Exhausted CD8 T cells (TEX) are associated with worse outcome in cancer yet better outcome in autoimmunity. Building on our past findings of increased TIGIT+KLRG1+ TEX with teplizumab therapy in type 1 diabetes (T1D), in the absence of treatment we found that the frequency of TIGIT+KLRG1+ TEX is stable within an individual but differs across individuals in both T1D and healthy control (HC) cohorts. This TIGIT+KLRG1+ CD8 TEX population shares an exhaustion-associated EOMES gene signature in HC, T1D, rheumatoid arthritis (RA), and cancer subjects, expresses multiple inhibitory receptors, and is hyporesponsive in vitro, together suggesting co-expression of TIGIT and KLRG1 may broadly define human peripheral exhausted cells. In HC and RA subjects, lower levels of EOMES transcriptional modules and frequency of TIGIT+KLRG1+ TEX were associated with RA HLA risk alleles (DR0401, 0404, 0405, 0408, 1001) even when considering disease status and cytomegalovirus (CMV) seropositivity. Moreover, the frequency of TIGIT+KLRG1+ TEX was significantly increased in RA HLA risk but not non-risk subjects treated with abatacept (CTLA4Ig). The DR4 association and selective modulation with abatacept suggests that therapeutic modulation of TEX may be more effective in DR4 subjects and TEX may be indirectly influenced by cellular interactions that are blocked by abatacept.

GNTI-122: an autologous antigen-specific engineered Treg cell therapy for type 1 diabetes.

Tregs have the potential to establish long-term immune tolerance in patients recently diagnosed with type 1 diabetes (T1D) by preserving ß cell function. Adoptive transfer of autologous thymic Tregs, although safe, exhibited limited efficacy in previous T1D clinical trials, likely reflecting a lack of tissue specificity, limited IL-2 signaling support, and in vivo plasticity of Tregs. Here, we report a cell engineering strategy using bulk CD4+ T cells to generate a Treg cell therapy (GNTI-122) that stably expresses FOXP3, targets the pancreas and draining lymph nodes, and incorporates a chemically inducible signaling complex (CISC). GNTI-122 cells maintained an expression profile consistent with Treg phenotype and function. Activation of CISC using rapamycin mediated concentration-dependent STAT5 phosphorylation and, in concert with T cell receptor engagement, promoted cell proliferation. In response to the cognate antigen, GNTI-122 exhibited direct and bystander suppression of polyclonal, islet-specific effector T cells from patients with T1D. In an adoptive transfer mouse model of T1D, a mouse engineered-Treg analog of GNTI-122 trafficked to the pancreas, decreased the severity of insulitis, and prevented progression to diabetes. Taken together, these findings demonstrate in vitro and in vivo activity and support further development of GNTI-122 as a potential treatment for T1D.

The IFIH1-A946T risk variant promotes diabetes in a sex-dependent manner.

Type 1 diabetes (T1D) is an autoimmune disease in which pancreatic islet ß-cells are attacked by the immune system, resulting in insulin deficiency and hyperglycemia. One of the top non-synonymous single-nucleotide polymorphisms (SNP) associated with T1D is in the interferon-induced helicase C domain-containing protein 1 (IFIH1), which encodes an anti-viral cytosolic RNA sensor. This SNP results in an alanine to threonine substitution at amino acid 946 (IFIH1A946T) and confers an increased risk for several autoimmune diseases, including T1D. We hypothesized that the IFIH1A946T risk variant, (IFIH1R) would promote T1D pathogenesis by stimulating type I interferon (IFN I) signaling leading to immune cell alterations. To test this, we developed Ifih1R knock-in mice on the non-obese diabetic (NOD) mouse background, a spontaneous T1D model. Our results revealed a modest increase in diabetes incidence and insulitis in Ifih1R compared to non-risk Ifih1 (Ifih1NR) mice and a significant acceleration of diabetes onset in Ifih1R females. Ifih1R mice exhibited a significantly enhanced interferon stimulated gene (ISG) signature compared to Ifih1NR, indicative of increased IFN I signaling. Ifih1R mice exhibited an increased frequency of plasma cells as well as tissue-dependent changes in the frequency and activation of CD8+ T cells. Our results indicate that IFIH1R may contribute to T1D pathogenesis by altering the frequency and activation of immune cells. These findings advance our knowledge on the connection between the rs1990760 variant and T1D. Further, these data are the first to demonstrate effects of Ifih1R in NOD mice, which will be important to consider for the development of therapeutics for T1D.

The IFIH1-A946T risk variant promotes diabetes in a sex-dependent manner.

Type 1 diabetes (T1D) is an autoimmune disease in which pancreatic islet ß-cells are attacked by the immune system, resulting in insulin deficiency and hyperglycemia. One of the top non-synonymous single-nucleotide polymorphisms (SNP) associated with T1D is in the interferon-induced helicase C domain-containing protein 1 ( IFIH1 ), which encodes an anti-viral cytosolic RNA sensor. This SNP results in an alanine to threonine substitution at amino acid 946 (IFIH1 A946T ) and confers an increased risk for several autoimmune diseases, including T1D. We hypothesized that the IFIH1 A946T risk variant, ( IFIH1 R ) would promote T1D pathogenesis by stimulating type I interferon (IFN I) signaling leading to immune cell alterations. To test this, we developed Ifih1 R knock-in mice on the non-obese diabetic (NOD) mouse background, a spontaneous T1D model. Our results revealed a modest increase in diabetes incidence and insulitis in Ifih1 R compared to non-risk Ifih1 ( Ifih1 NR ) mice and a significant acceleration of diabetes onset in Ifih1 R females. Ifih1 R mice exhibited a significantly enhanced interferon stimulated gene (ISG) signature compared to Ifih1 NR , indicative of increased IFN I signaling. Ifih1 R mice exhibited an increased frequency of plasma cells as well as tissue-dependent changes in the frequency and activation of CD8 + T cells. Our results indicate that IFIH1 R may contribute to T1D pathogenesis by altering the frequency and activation of immune cells. These findings advance our knowledge on the connection between the rs1990760 variant and T1D. Further, these data are the first to demonstrate effects of Ifih1 R in NOD mice, which will be important to consider for the development of therapeutics for T1D.

Expansion of HLA-DR Positive Peripheral Helper T and Naive B cells in Anticitrullinated Protein Antibody-Positive Individuals At Risk for Rheumatoid Arthritis.

OBJECTIVE: To investigate immune dysregulation in the peripheral blood that contributes to the pre-rheumatoid arthritis (RA) stage of RA development in anticitrullinated protein antibody (ACPA)+ individuals. METHODS: Using 37 markers by mass cytometry, we investigated peripheral blood mononuclear cells (PBMCs) from ACPA+ at-risk individuals, ACPA+ early untreated patients with RA, and ACPA- controls in the Tokyo Women's Medical University cohort (n = 17 in each group). Computational algorithms, FlowSOM and Optimized t-Distributed Stochastic Neighbor Embedding, were employed to explore specific immunologic differences between study groups. These findings were further evaluated, and longitudinal changes were explored, using flow cytometry and PBMCs from the US-based Targeting Immune Responses for Prevention of RA cohort that included 11 ACPA+ individuals who later developed RA (pre-RA), of which 9 had post-RA diagnosis PBMCs (post-RA), and 11 ACPA- controls. RESULTS: HLA-DR+ peripheral helper T (Tph) cells, activated regulatory T cells, PD-1hi CD8+ T cells, and CXCR5-CD11c-CD38+ naive B cells were significantly expanded in PBMCs from at-risk individuals and patients with early RA from the Tokyo Women's Medical University cohort. Expansion of HLA-DR+ Tph cells and CXCR5-CD11c-CD38+ naive B cells was likewise found in both pre-RA and post-RA time points in the Targeting Immune Responses for Prevention of RA cohort. CONCLUSION: The expansion of HLA-DR+ Tph cells and CXCR5-CD11c-CD38+ naive B cells in ACPA+ individuals, including those who developed inflammatory arthritis and classified RA, supports a key role of these cells in transition from pre-RA to classified RA. These findings may identify a new mechanistic target for treatment and prevention in RA.

Lupus IgA1 autoantibodies synergize with IgG to enhance pDC responses to RNA-containing immune complexes.

Autoantibodies to nuclear antigens are hallmarks of the autoimmune disease systemic lupus erythematosus (SLE) where they contribute to pathogenesis. However, there remains a gap in our knowledge regarding how different isotypes of autoantibodies contribute to disease, including the production of the critical type I interferon (IFN) cytokines by plasmacytoid dendritic cells (pDCs) in response to immune complexes (ICs). We focused on IgA, which is the second most prevalent isotype in serum, and along with IgG is deposited in glomeruli in lupus nephritis. Here, we show that individuals with SLE have IgA autoantibodies against most nuclear antigens, correlating with IgG against the same antigen. We investigated whether IgA autoantibodies against a major SLE autoantigen, Smith ribonucleoproteins (Sm/RNPs), play a role in IC activation of pDCs. We found that pDCs express the IgA-specific Fc receptor, FcαR, and there was a striking ability of IgA1 autoantibodies to synergize with IgG in RNA-containing ICs to generate robust pDC IFNα responses. pDC responses to these ICs required both FcαR and FcγRIIa, showing a potent synergy between these Fc receptors. Sm/RNP IC binding to and internalization by pDCs were greater when ICs contained both IgA1 and IgG. pDCs from individuals with SLE had higher binding of IgA1-containing ICs and higher expression of FcαR than pDCs from healthy control individuals. Whereas pDC FcαR expression correlated with blood ISG signature in SLE, TLR7 agonists, but not IFNα, upregulated pDC FcαR expression in vitro. Together, we show a new mechanism by which IgA1 autoantibodies contribute to SLE pathogenesis.

Multifaceted immune dysregulation characterizes individuals at-risk for rheumatoid arthritis.

Molecular markers of autoimmunity, such as antibodies to citrullinated protein antigens (ACPA), are detectable prior to inflammatory arthritis (IA) in rheumatoid arthritis (RA) and may define a state that is 'at-risk' for future RA. Here we present a cross-sectional comparative analysis among three groups that include ACPA positive individuals without IA (At-Risk), ACPA negative individuals and individuals with early, ACPA positive clinical RA (Early RA). Differential methylation analysis among the groups identifies non-specific dysregulation in peripheral B, memory and naïve T cells in At-Risk participants, with more specific immunological pathway abnormalities in Early RA. Tetramer studies show increased abundance of T cells recognizing citrullinated (cit) epitopes in At-Risk participants, including expansion of T cells reactive to citrullinated cartilage intermediate layer protein I (cit-CILP); these T cells have Th1, Th17, and T stem cell memory-like phenotypes. Antibody-antigen array analyses show that antibodies targeting cit-clusterin, cit-fibrinogen and cit-histone H4 are elevated in At-Risk and Early RA participants, with the highest levels of antibodies detected in those with Early RA. These findings indicate that an ACPA positive at-risk state is associated with multifaceted immune dysregulation that may represent a potential opportunity for targeted intervention.

Trimodal single-cell profiling reveals a novel pediatric CD8αα+ T cell subset and broad age-related molecular reprogramming across the T cell compartment.

Age-associated changes in the T cell compartment are well described. However, limitations of current single-modal or bimodal single-cell assays, including flow cytometry, RNA-seq (RNA sequencing) and CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing), have restricted our ability to deconvolve more complex cellular and molecular changes. Here, we profile >300,000 single T cells from healthy children (aged 11-13 years) and older adults (aged 55-65 years) by using the trimodal assay TEA-seq (single-cell analysis of mRNA transcripts, surface protein epitopes and chromatin accessibility), which revealed that molecular programming of T cell subsets shifts toward a more activated basal state with age. Naive CD4+ T cells, considered relatively resistant to aging, exhibited pronounced transcriptional and epigenetic reprogramming. Moreover, we discovered a novel CD8αα+ T cell subset lost with age that is epigenetically poised for rapid effector responses and has distinct inhibitory, costimulatory and tissue-homing properties. Together, these data reveal new insights into age-associated changes in the T cell compartment that may contribute to differential immune responses.

Shifts in isoform usage underlie transcriptional differences in regulatory T cells in type 1 diabetes.

Genome-wide association studies have identified numerous loci with allelic associations to Type 1 Diabetes (T1D) risk. Most disease-associated variants are enriched in regulatory sequences active in lymphoid cell types, suggesting that lymphocyte gene expression is altered in T1D. Here we assay gene expression between T1D cases and healthy controls in two autoimmunity-relevant lymphocyte cell types, memory CD4+/CD25+ regulatory T cells (Treg) and memory CD4+/CD25- T cells, using a splicing event-based approach to characterize tissue-specific transcriptomes. Limited differences in isoform usage between T1D cases and controls are observed in memory CD4+/CD25- T-cells. In Tregs, 402 genes demonstrate differences in isoform usage between cases and controls, particularly RNA recognition and splicing factor genes. Many of these genes are regulated by the variable inclusion of exons that can trigger nonsense mediated decay. Our results suggest that dysregulation of gene expression, through shifts in alternative splicing in Tregs, contributes to T1D pathophysiology.

Hydroxychloroquine in Stage 1 Type 1 Diabetes.

OBJECTIVE: Innate immune responses may be involved in the earliest phases of type 1 diabetes (T1D). RESEARCH DESIGN AND METHODS: To test whether blocking innate immaune cells modulated progression of the disease, we randomly assigned 273 individuals with stage 1 T1D to treatment with hydroxychloroquine (n = 183; 5 mg/kg per day to a maximum of 400 mg) or placebo (n = 90) and assessed whether hydroxychloroquine treatment delayed or prevented progression to stage 2 T1D (i.e., two or more islet autoantibodies with abnormal glucose tolerance). RESULTS: After a median follow-up of 23.3 months, the trial was stopped prematurely by the data safety monitoring board because of futility. There were no safety concerns in the hydroxychloroquine arm, including in annual ophthalmologic examinations. Preplanned secondary analyses showed a transient decrease in the glucose average area under the curve to oral glucose in the hydroxychloroquine-treated arm at month 6 and reduced titers of anti-GAD and anti-insulin autoantibodies and acquisition of positive autoantibodies in the hydroxychloroquine arm (P = 0.032). CONCLUSIONS: We conclude that hydroxychloroquine does not delay progression to stage 2 T1D in individuals with stage 1 disease. Drug treatment reduces the acquisition of additional autoantibodies and the titers of autoantibodies to GAD and insulin.

Dysregulated IFN-γ signals promote autoimmunity in STAT1 gain-of-function syndrome.

Heterozygous signal transducer and activator of transcription 1 (STAT1) gain-of-function (GOF) mutations promote a clinical syndrome of immune dysregulation characterized by recurrent infections and predisposition to humoral autoimmunity. To gain insights into immune characteristics of STAT1-driven inflammation, we performed deep immunophenotyping of pediatric patients with STAT1 GOF syndrome and age-matched controls. Affected individuals exhibited dysregulated CD4+ T cell and B cell activation, including expansion of TH1-skewed CXCR3+ populations that correlated with serum autoantibody titers. To dissect underlying immune mechanisms, we generated Stat1 GOF transgenic mice (Stat1GOF mice) and confirmed the development of spontaneous humoral autoimmunity that recapitulated the human phenotype. Despite clinical resemblance to human regulatory T cell (Treg) deficiency, Stat1GOF mice and humans with STAT1 GOF syndrome exhibited normal Treg development and function. In contrast, STAT1 GOF autoimmunity was characterized by adaptive immune activation driven by dysregulated STAT1-dependent signals downstream of the type 1 and type 2 interferon (IFN) receptors. However, in contrast to the prevailing type 1 IFN-centric model for STAT1 GOF autoimmunity, Stat1GOF mice lacking the type 1 IFN receptor were only partially protected from STAT1-driven systemic inflammation, whereas loss of type 2 IFN (IFN-γ) signals abrogated autoimmunity. Last, germline STAT1 GOF alleles are thought to enhance transcriptional activity by increasing total STAT1 protein, but the underlying biochemical mechanisms have not been defined. We showed that IFN-γ receptor deletion normalized total STAT1 expression across immune lineages, highlighting IFN-γ as the critical driver of feedforward STAT1 elevation in STAT1 GOF syndrome.

Immune-related adverse events after immune check point inhibitors: Understanding the intersection with autoimmunity.

Immune checkpoint inhibitor therapies act through blockade of inhibitory molecules involved in the regulation of T cells, thus releasing tumor specific T cells to destroy their tumor targets. However, immune checkpoint inhibitors (ICI) can also lead to a breach in self-tolerance resulting in immune-related adverse events (irAEs) that include tissue-specific autoimmunity. This review addresses the question of whether the mechanisms that drive ICI-induced irAEs are shared or distinct with those driving spontaneous autoimmunity, focusing on ICI-induced diabetes, ICI-induced arthritis, and ICI-induced thyroiditis due to the wealth of knowledge about the development of autoimmunity in type 1 diabetes, rheumatoid arthritis, and Hashimoto's thyroiditis. It reviews current knowledge about role of genetics and autoantibodies in the development of ICI-induced irAEs and presents new studies utilizing single-cell omics approaches to identify T-cell signatures associated with ICI-induced irAEs. Collectively, these studies indicate that there are similarities and differences between ICI-induced irAEs and autoimmune disease and that studying them in parallel will provide important insight into the mechanisms critical for maintaining immune tolerance.

Diminished responses to mRNA-based SARS-CoV-2 vaccines in individuals with rheumatoid arthritis on immune-modifying therapies.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disorder that causes debilitating swelling and destruction of the joints. People with RA are treated with drugs that actively suppress one or more parts of their immune system, and these may alter the response to vaccination against SARS-CoV-2. In this study, we analyzed blood samples from a cohort of patients with RA after receiving a 2-dose mRNA COVID-19 vaccine regimen. Our data show that individuals on the cytotoxic T lymphocyte antigen 4-Ig therapy abatacept had reduced levels of SARS-CoV-2-neutralizing antibodies after vaccination. At the cellular level, these patients showed reduced activation and class switching of SARS-CoV-2-specific B cells, as well as reduced numbers and impaired helper cytokine production by SARS-CoV-2-specific CD4+ T cells. Individuals on methotrexate showed similar but less severe defects in vaccine response, whereas individuals on the B cell-depleting therapy rituximab had a near-total loss of antibody production after vaccination. These data define a specific cellular phenotype associated with impaired response to SARS-CoV-2 vaccination in patients with RA on different immune-modifying therapies and help inform efforts to improve vaccination strategies in this vulnerable population.

Translational immunology: Applying fundamental discoveries to human health and autoimmune diseases.

Studying the human immune system is challenging. These challenges stem from the complexity of the immune system itself, the heterogeneity of the immune system between individuals, and the many factors that lead to this heterogeneity including the influence of genetics, environment, and immune experience. Studies of the human immune system in the context of disease are increased in complexity as multiple combinations and variations in immune pathways can lead to a single disease. Thus, although individuals with a disease may share clinical features, the underlying disease mechanisms and resulting pathophysiology can be diverse among individuals with the same disease diagnosis. This has consequences for the treatment of diseases, as no single therapy will work for everyone, therapeutic efficacy varies among patients, and targeting a single immune pathway is rarely 100% effective. This review discusses how to address these challenges by identifying and managing the sources of variation, improving access to high-quality, well-curated biological samples by building cohorts, applying new technologies such as single-cell omics and imaging technologies to interrogate samples, and bringing to bear computational expertise in conjunction with immunologists and clinicians to interpret those results. The review has a focus on autoimmune diseases, including rheumatoid arthritis, MS, systemic lupus erythematosus, and type 1 diabetes, but its recommendations are also applicable to studies of other immune-mediated diseases.

SARS-CoV-2 mRNA vaccines decouple anti-viral immunity from humoral autoimmunity.

mRNA-based vaccines dramatically reduce the occurrence and severity of COVID-19, but are associated with rare vaccine-related adverse effects. These toxicities, coupled with observations that SARS-CoV-2 infection is associated with autoantibody development, raise questions whether COVID-19 vaccines may also promote the development of autoantibodies, particularly in autoimmune patients. Here we used Rapid Extracellular Antigen Profiling to characterize self- and viral-directed humoral responses after SARS-CoV-2 mRNA vaccination in 145 healthy individuals, 38 patients with autoimmune diseases, and 8 patients with mRNA vaccine-associated myocarditis. We confirm that most individuals generated robust virus-specific antibody responses post vaccination, but that the quality of this response is impaired in autoimmune patients on certain modes of immunosuppression. Autoantibody dynamics are remarkably stable in all vaccinated patients compared to COVID-19 patients that exhibit an increased prevalence of new autoantibody reactivities. Patients with vaccine-associated myocarditis do not have increased autoantibody reactivities relative to controls. In summary, our findings indicate that mRNA vaccines decouple SARS-CoV-2 immunity from autoantibody responses observed during acute COVID-19.

PTPN22 R620W gene editing in T cells enhances low-avidity TCR responses.

A genetic variant in the gene PTPN22 (R620W, rs2476601) is strongly associated with increased risk for multiple autoimmune diseases and linked to altered TCR regulation and T cell activation. Here, we utilize Crispr/Cas9 gene editing with donor DNA repair templates in human cord blood-derived, naive T cells to generate PTPN22 risk edited (620W), non-risk edited (620R), or knockout T cells from the same donor. PTPN22 risk edited cells exhibited increased activation marker expression following non-specific TCR engagement, findings that mimicked PTPN22 KO cells. Next, using lentiviral delivery of T1D patient-derived TCRs against the pancreatic autoantigen, islet-specific glucose-6 phosphatase catalytic subunit-related protein (IGRP), we demonstrate that loss of PTPN22 function led to enhanced signaling in T cells expressing a lower avidity self-reactive TCR, but not a high-avidity TCR. In this setting, loss of PTPN22 mediated enhanced proliferation and Th1 skewing. Importantly, expression of the risk variant in association with a lower avidity TCR also increased proliferation relative to PTPN22 non-risk T cells. Together, these findings suggest that, in primary human T cells, PTPN22 rs2476601 contributes to autoimmunity risk by permitting increased TCR signaling and activation in mildly self-reactive T cells, thereby potentially expanding the self-reactive T cell pool and skewing this population toward an inflammatory phenotype.

Cytotoxic CD8+ T cells target citrullinated antigens in rheumatoid arthritis.

The immune mechanisms that mediate synovitis and joint destruction in rheumatoid arthritis (RA) remain poorly defined. Although increased levels of CD8+ T cells have been described in RA, their function in pathogenesis remains unclear. Here we perform single cell transcriptome and T cell receptor (TCR) sequencing of CD8+ T cells derived from anti-citrullinated protein antibodies (ACPA)+ RA blood. We identify GZMB+CD8+ subpopulations containing large clonal lineage expansions that express cytotoxic and tissue homing transcriptional programs, while a GZMK+CD8+ memory subpopulation comprises smaller clonal expansions that express effector T cell transcriptional programs. We demonstrate RA citrullinated autoantigens presented by MHC class I activate RA blood-derived GZMB+CD8+ T cells to expand, express cytotoxic mediators, and mediate killing of target cells. We also demonstrate that these clonally expanded GZMB+CD8+ cells are present in RA synovium. These findings suggest that cytotoxic CD8+ T cells targeting citrullinated antigens contribute to synovitis and joint tissue destruction in ACPA+ RA.

Regulatory T cells suppress CD4+ effector T cell activation by controlling protein synthesis.

Regulatory T cells (Tregs) suppress the activation and subsequent effector functions of CD4 effector T cells (Teffs). However, molecular mechanisms that enforce Treg-mediated suppression in CD4 Teff are unclear. We found that Tregs suppressed activation-induced global protein synthesis in CD4 Teffs prior to cell division. We analyzed genome-wide changes in the transcriptome and translatome of activated CD4 Teffs. We show that mRNAs encoding for the protein synthesis machinery are regulated at the level of translation in activated CD4 Teffs by Tregs. Tregs suppressed global protein synthesis of CD4 Teffs by specifically inhibiting mRNAs of the translation machinery at the level of mTORC1-mediated translation control through concerted action of immunosuppressive cytokines IL-10 and TGFß. Lastly, we found that the therapeutic targeting of protein synthesis with the RNA helicase eIF4A inhibitor rocaglamide A can alleviate inflammatory CD4 Teff activation caused by acute Treg depletion in vivo. These data show that peripheral tolerance is enforced by Tregs through mRNA translational control in CD4 Teffs.