Immune responses to citrullinated and homocitrullinated peptides in healthy donors are not restricted to the HLA SE shared allele and can be selected into the memory pool.

Citrullination and homocitrullination are stress induced post-translational modifications (siPTMs) which can be recognized by T cells. Peripheral blood mononuclear cells isolated from healthy donors and rheumatoid arthritis (RA) patients were stimulated with nine siPTM-peptides. CD45RA/CD45RO depletion was employed to determine if peptide-specific responses are naïve or memory. Human leucocyte antigen (HLA)-DP4 and HLA-DR4 transgenic mice were immunized with siPTM-peptides and immune responses were determined with ex vivo ELISpot assays. The majority (24 out of 25) of healthy donors showed CD4 T cell-specific proliferation to at least 1 siPTM-peptide, 19 to 2 siPTM-peptides, 14 to 3 siPTM-peptides, 9 to 4 siPTM-peptides, 6 to 5 siPTM-peptides and 4 to 6 siPTM-peptides. More donors responded to Vim28-49cit (68%) and Bip189-208cit (75%) compared with Vim415-433cit (33%). In RA patients, the presentation of citrullinated epitopes is associated with HLA-SE alleles; however, we witnessed responses in healthy donors who did not express the SE allele. The majority of responding T cells were effector memory cells with a Th1/cytotoxic phenotype. Responses to Vim28-49cit and Eno241-260cit originated in the memory pool, while the response to Vim415-433cit was naïve. In the HLA-DP4 and HLA-DR4 transgenic models, Vim28cit generated a memory response. Peptide-specific T cells were capable of Epstein-Barr virus transformed lymphoblastoid cell line recognition suggesting a link with stress due to infection. These results suggest siPTM-peptides are presented under conditions of cellular stress and inflammation and drive cytotoxic CD4 T cell responses that aid in the removal of stressed cells. The presentation of such siPTM-peptides is not restricted to HLA-SE in both humans and animal models.

Autoantibodies against ATP4A are a feature of the abundant autoimmunity that develops in first-degree relatives of patients with type 1 diabetes.

OBJECTIVE: Type 1 diabetes is associated with autoantibodies to different organs that include the gut. The objective of the study was to determine the risk of developing gastric parietal cell autoimmunity in relation to other autoimmunity in individuals with a family history of type 1 diabetes. METHODS: Autoantibodies to the parietal cell autoantigen, H+ /K+ ATPase subunit A (ATP4A) was measured in 2218 first-degree relatives of patients with type 1 diabetes, who were prospectively followed from birth for a median of 14.5 years. All were also tested regularly for the development of islet autoantibodies, transglutaminase autoantibodies, and thyroid peroxidase autoantibodies. RESULTS: The cumulative risk to develop ATP4A autoantibodies was 8.1% (95% CI, 6.6-9.6) by age 20 years with a maximum incidence observed at age 2 years. Risk was increased in females (HR, 1.9; 95% CI, 1.3-2.8; p = 0.0004), relatives with the HLA DR4-DQ8/DR4-DQ8 genotype (HR, 3.4; 95% CI, 1.9-5.9; p < 0.0001) and in participants who also had thyroid peroxidase autoantibodies (HR, 3.7; 95% CI, 2.5-5.5; p < 0.0001). Risk for at least one of ATP4A-, islet-, transglutaminase-, or thyroid peroxidase-autoantibodies was 24.7% (95% CI, 22.6-26.7) by age 20 years and was 47.3% (95% CI, 41.3-53.3) in relatives who had an HLA DR3/DR4-DQ8, DR4-DQ8/DR4-DQ8, or DR3/DR3 genotype (p < 0.0001 vs. other genotypes). CONCLUSIONS: Relatives of patients with type 1 diabetes who have risk genotypes are at very high risk for the development of autoimmunity against gastric and other organs.

Peptide-MHC Binding Reveals Conserved Allosteric Sites in MHC Class I- and Class II-Restricted T Cell Receptors (TCRs).

T cells are vital for adaptive immune responses that protect against pathogens and cancers. The T cell receptor (TCR)-CD3 complex comprises a diverse αß TCR heterodimer in noncovalent association with three invariant CD3 dimers. The TCR is responsible for recognizing antigenic peptides bound to MHC molecules (pMHC), while the CD3 dimers relay activation signals to the T cell. However, the mechanisms by which TCR engagement by pMHC is transmitted to CD3 remain mysterious, although there is growing evidence that mechanosensing and allostery both play a role. Here, we carried out NMR analysis of a human autoimmune TCR (MS2-3C8) that recognizes a self-peptide from myelin basic protein presented by the MHC class II molecule HLA-DR4. We observed pMHC-induced NMR signal perturbations in MS2-3C8 that indicate long-range effects on TCR ß chain conformation and dynamics. Our results demonstrate that, in addition to expected changes in the NMR resonances of pMHC-contacting residues, perturbations extend to the Vß/Vα, Vß/Cß, and Cß/Cα interfacial regions. Moreover, the pattern of long-range perturbations is similar to that detected previously in the ß chains of two MHC class I-restricted TCRs, thereby revealing a common allosteric pathway among three unrelated TCRs. Molecular dynamics (MD) simulations predict similar pMHC-induced effects. Taken together, our results demonstrate that pMHC binding induces long-range allosteric changes in the TCR ß chain at conserved sites in both representative MHC class I- and class II-restricted TCRs, and that these sites may play a role in the transmission of signaling information.

Variants in IL23R-C1orf141 and ADO-ZNF365-EGR2 are associated with susceptibility to Vogt-Koyanagi-Harada disease in Japanese population.

Vogt-Koyanagi-Harada (VKH) disease is a systemic inflammatory disorder that affects pigment cell-containing organs such as the eye (e.g., chronic and/or recurrent granulomatous panuveitis). While the exact etiology and pathogenic mechanism of VKH disease are unclear, HLA-DR4 alleles have been documented to be strongly associated with VKH disease in various ethnic groups. Recently, a genome-wide association study (GWAS) found two new genetic risk factors (IL23R-C1orf141 and ADO-ZNF365-EGR2) in a non-HLA region from a Han Chinese population. In this study, we replicated these GWAS findings in a Japanese population. A total of 1,643 Japanese samples (380 cases with VKH disease and 1,263 healthy controls) were recruited. We assessed four single nucleotide polymorphisms (SNPs) shown in previous GWAS: rs78377598 and rs117633859 in IL23R-C1orf141, and rs442309 and rs224058 in ADO-ZNF365-EGR2. A significant allelic association with VKH disease was observed for all of the four SNPs (rs78377598: pc = 0.0057; rs117633859: pc = 0.0017; rs442309: pc = 0.021; rs224058: pc = 0.035). In genotypic association analysis, the minor alleles of IL23R-C1orf141 rs78377598 and rs117633859 had the strongest association with disease susceptibility under the additive model (pc = 0.0075 and pc = 0.0026, respectively). The minor alleles of ADO-ZNF365-EGR2 rs442309 and rs224058 were most strongly associated with disease susceptibility under the dominant model (pc = 0.00099 and pc = 0.0023, respectively). The meta-analysis of the current and previous studies found that all of the four SNPs exhibited a significantly strong association with VKH disease (meta-p < 0.00001: rs78377598, meta-odds ratio (OR) = 1.69; rs1176338, meta-OR = 1.82; rs442309, meta-OR = 1.34; rs224058, meta-OR = 1.33). In summary, our study replicated significant associations with VKH disease susceptibility reported in a previous GWAS. Thus, the IL23R-C1orf141 and ADO-ZNF365-EGR2 loci may play important roles in the development of VKH disease through genetic polymorphisms.

DNA methylation near the INS gene is associated with INS genetic variation (rs689) and type 1 diabetes in the Diabetes Autoimmunity Study in the Young.

OBJECTIVE: Mechanisms underlying the role of non-human leukocyte antigen (HLA) genetic risk variants in type 1 diabetes (T1D) are poorly understood. We aimed to test the association between methylation and non-HLA genetic risk. METHODS: We conducted a methylation quantitative trait loci (mQTL) analysis in a nested case-control study from the Dietary Autoimmunity Study in the Young. Controls (n = 83) were frequency-matched to T1D cases (n = 83) based on age, race/ethnicity, and sample availability. We evaluated 13 non-HLA genetic markers known be associated with T1D. Genome-wide methylation profiling was performed on peripheral blood samples collected prior to T1D using the Illumina 450 K (discovery set) and infinium methylation EPIC beadchip (EPIC validation) platforms. Linear regression models, adjusting for age and sex, were used to test to each single nucleotide polymorphism (SNP) -probe combination. Logistic regression models were used to test the association between T1D and methylation levels among probes with a significant mQTL. A meta-analysis was used to combine odds ratios from the two platforms. RESULTS: We identified 10 SNP-methylation probe pairs (false discovery rate (FDR) adjusted P < .05 and validation P < .05). Probes were associated with the GSDMB, C1QTNF6, IL27, and INS genes. The cg03366382 (OR: 1.9, meta-P = .0495), cg21574853 (OR: 2.5, meta-P = .0232), and cg25336198 (odds ratio: 6.6, meta-P = .0081) probes were significantly associated with T1D. The three probes were located upstream from the INS transcription start site. CONCLUSIONS: We confirmed an association between DNA methylation and rs689 that has been identified in related studies. Measurements in our study preceded the onset of T1D suggesting methylation may have a role in the relationship between INS variation and T1D development.

Glucan-Chitin Particles Enhance Th17 Response and Improve Protective Efficacy of a Multivalent Antigen (rCpa1) against Pulmonary Coccidioides posadasii Infection.

Developing an effective and safe recombinant vaccine requires microbe-specific antigens combined with an adjuvant/delivery system to strengthen protective immunity. In this study, we designed and expressed a multivalent recombinant Coccidioides polypeptide antigen (rCpa1) that consists of three previously identified antigens (i.e., Ag2/Pra, Cs-Ag, and Pmp1) and five pathogen-derived peptides with high affinity for human major histocompatibility complex class II (MHC-II) molecules. The purified rCpa1 was encapsulated into four types of yeast cell wall particles containing ß-glucan, mannan, and chitin in various proportions or was mixed with an oligonucleotide (ODN) containing two methylated dinucleotide CpG motifs. This multivalent antigen encapsulated into glucan-chitin particles (GCP-rCpa1) showed significantly greater reduction of fungal burden for human HLA-DR4 transgenic mice than the other adjuvant-rCpa1 formulations tested. Among the adjuvants tested, both GCPs and ß-glucan particles (GPs) were capable of stimulating a mixed Th1 and Th17 response. Mice vaccinated with GCP-rCpa1 showed higher levels of interleukin 17 (IL-17) production in T-cell recall assays and earlier lung infiltration by activated Th1 and Th17 cells than GP-rCpa1-vaccinated mice. Both C57BL/6 and HLA-DR4 transgenic mice that were vaccinated with the GCP-rCpa1 vaccine showed higher survival rates than mice that received GCPs alone. Concurrently, the GCP-rCpa1 vaccine stimulated greater infiltration of the injection sites by macrophages, which engulf and process the vaccine for antigen presentation, than the GP-rCpa1 vaccine. This is the first attempt to systematically characterize the presentation of a multivalent coccidioidomycosis vaccine encapsulated with selected adjuvants that enhance the protective cellular immune response to infection.

Collateral Damage: Insulin-Dependent Diabetes Induced With Checkpoint Inhibitors.

Insulin-dependent diabetes may occur in patients with cancers who are treated with checkpoint inhibitors (CPIs). We reviewed cases occurring over a 6-year period at two academic institutions and identified 27 patients in whom this developed, or an incidence of 0.9%. The patients had a variety of solid-organ cancers, but all had received either anti-PD-1 or anti-PD-L1 antibodies. Diabetes presented with ketoacidosis in 59%, and 42% had evidence of pancreatitis in the peridiagnosis period. Forty percent had at least one positive autoantibody and 21% had two or more. There was a predominance of HLA-DR4, which was present in 76% of patients. Other immune adverse events were seen in 70%, and endocrine adverse events in 44%. We conclude that autoimmune, insulin-dependent diabetes occurs in close to 1% of patients treated with anti-PD-1 or -PD-L1 CPIs. This syndrome has similarities and differences compared with classic type 1 diabetes. The dominance of HLA-DR4 suggests an opportunity to identify those at highest risk of these complications and to discover insights into the mechanisms of this adverse event.

Isolation of human lymphocytes with high yield and viability from the gastrointestinal and female reproductive tract of a humanized DRAG mouse.

The mucosal tissues of the gut and female reproductive tract (FRT) are susceptible to pathogen infections including bacteria, viruses, and parasites, and are also the targets for immune disorders such as Crohn's disease, inflammatory bowel disease (IBD), and many types of cancers. However, the role of the mucosal immune cells to control these diseases is largely unknown. The limited availability of human mucosal biopsy tissue and the low number of cells that can be isolated from these tissues hampers the characterization of the phenotype and function of human mucosal immune cell subsets. Therefore, human-immune-system humanized mice are surrogate models to investigate the human mucosal immune cell responses during the course of the disease. The current protocols used to harvest the immune cells from the mucosal tissues, however, result in low recovery of cells with poor viability. We have established a novel protocol, which results in a high yield of human lymphocytes with high viability to overcome this issue. The immune cells obtained from a single DRAG mouse by our protocol were sufficient for conducting functional assays and for flow cytometry analyses including phenotypic, exhaustion, and functional panels.

Accumulation of HLA-DR4 in Colonic Epithelial Cells Causes Severe Colitis in Homozygous HLA-DR4 Transgenic Mice.

BACKGROUND: Homozygous HLA-DR4/I-E transgenic mice (tgm) spontaneously developed colitis similar to human ulcerative colitis. We explored whether endoplasmic reticulum stress in colonic epithelial cells due to overexpression of HLA-DR4/I-E was involved in the pathogenesis of colitis. METHODS: Major histocompatibility complex class II transactivator-knockout (CIITAKO) background tgm were established to test the involvement of HLA-DR4/I-E expression in the pathogenesis of colitis. Histological and cellular analyses were performed and the effect of oral administration of the molecular chaperone tauroursodeoxycholic acid (TUDCA) and antibiotics were investigated. IgA content of feces and serum and presence of IgA-coated fecal bacteria were also investigated. RESULTS: Aberrantly accumulated HLA-DR4/I-E molecules in colonic epithelial cells were observed only in the colitic homozygous tgm, which was accompanied by upregulation of the endoplasmic reticulum stress marker Binding immunoglobulin protein (BiP) and reduced mucus. Homozygous tgm with CIITAKO, and thus absent of HLA-DR4/I-E expression, did not develop colitis. Oral administration of TUDCA to homozygotes reduced HLA-DR4/I-E and BiP expression in colonic epithelial cells and restored the barrier function of the intestinal tract. The IgA content of feces and serum, and numbers of IgA-coated fecal bacteria were higher in the colitic tgm, and antibiotic administration suppressed the expression of HLA-DR4/I-E and colitis. CONCLUSIONS: The pathogenesis of the colitis observed in the homozygous tgm was likely due to endoplasmic reticulum stress, resulting in goblet cell damage and compromised mucus production in the colonic epithelial cells in which HLA-DR4/I-E molecules were heavily accumulated. Commensal bacteria seemed to be involved in the accumulation of HLA-DR4/I-E, leading to development of the colitis.

Major Histocompatibility Complex Class II Alleles Influence Induction of Pathogenic Antiphospholipid Antibodies in a Mouse Model of Thrombosis.

OBJECTIVE: Both environmental and genetic factors are important in the development of antiphospholipid antibodies (aPL) in patients with antiphospholipid syndrome (APS). Currently, the only available data on predisposing genetic factors have been obtained from epidemiologic studies, without mechanistic evidence. Therefore, we studied the influence of major histocompatibility complex (MHC) class II alleles on the production of aPL in a mouse model of APS. METHODS: Three groups of mice, MHC class II-deficient (MHCII-/- ) mice, MHCII-/- mice transgenic for human HLA-DQ6 (DQ6), DQ8, or DR4 alleles, and the corresponding wild-type (WT) mouse strains were immunized; half were immunized with human ß2 -glycoprotein I (ß2 GPI), and the other half were immunized with control ovalbumin (OVA) protein. Thrombus formation in vivo, tissue factor activity in carotid and peritoneal macrophages, and serum levels of tumor necrosis factor (TNF), IgG anticardiolipin (aCL), antibodies, and anti-OVA antibodies were determined. RESULTS: Immunization with ß2 GPI induced significant production of aCL and anti-ß2 GPI in WT mice compared with control mice immunized with OVA (P < 0.001) but diminished aCL (P < 0.001) and anti-ß2 GPI (P = 0.016) production in MHCII-/- mice. Anti-ß2 GPI production was fully restored in DQ6 and DQ8 mice, while levels of anti-ß2 GPI in DR4 mice and aCL in all transgenic lines were only partially restored (P < 0.001 to P < 0.046). Thrombus size in WT mice was twice that in MHCII-/- mice (P < 0.001) but similar to that in all transgenic lines. Carotid and peritoneal macrophage tissue factor levels decreased by >50% in MHCII-/- mice compared with wild-type B6 mice and were restored in DQ8 mice but not DR4 mice or DQ6 mice. TNF levels decreased 4-fold in MHCII-/- mice (P < 0.001) and were not restored in transgenic mice. CONCLUSION: Our mechanistic study is the first to show that MHC class II alleles influence not only quantitative aPL production but also the pathogenic capacity of induced aPL.

A case of fulminant Type 1 diabetes following anti-PD1 immunotherapy in a genetically susceptible patient.

Programmed cell death-1 protein (PD-1) is an immune checkpoint that has gained popularity in the treatment of several advanced cancers. Inhibiting this checkpoint is known to enhance immune response, but is also known to diminish immune tolerance and to increase autoimmune toxicity. We discuss a case of rapid onset fulminant Type 1 diabetes induced by treatment with anti-programmed cell death-1 monoclonal antibody, nivolumab, in a patient with late-stage non-small-cell lung adenocarcinoma. The patient had no history of previous diabetes but did reveal a high-risk genotype for Type 1 diabetes development (DR3-DQ2; DR4-DQ8). This finding supports that acute Type 1 diabetes can be an important adverse effect of immunotherapies targeting T-cell activation regulation. Because of the severity of this adverse effect, physicians should be aware of it, and studies directed to the detection of new biomarkers for early risk stratification (e.g., HLA) should be sought.

A humanized HLA-DR4 mouse model for autoimmune myocarditis.

Myocarditis, the principal cause of dilated cardiomyopathy and heart failure in young adults, is associated with autoimmunity to human cardiac α-myosin (hCAM) and the DR4 allele of human major histocompatibility II (MHCII). We developed an hCAM-induced myocarditis model in human HLA-DR4 transgenic mice that lack all mouse MHCII genes, demonstrating that immunization for 3weeks significantly increased splenic T-cell proliferative responses and titres of IgG1 and IgG2c antibodies, abolished weight gain, provoked cardiac inflammation and significantly impaired cardiac output and fractional shortening, by echocardiography, compared to adjuvant-injected mice. Neither cardiac dilatation nor fibrosis occurred at this time point but prolonging the experiment was associated with mortality. Treatment with mixtures of hCAM derived peptides predicted to have high affinity for DR4 significantly preserved ejection fraction and fractional shortening. Our new humanized mouse model of autoimmune cardiomyopathy should be useful to refine hCAM-derived peptide treatment.

Vogt-Koyanagi-Harada-like Syndrome Complicating Pembrolizumab Treatment for Metastatic Melanoma.

Vogt-Koyanagi-Harada (VKH) syndrome is a rare condition implicating systemic immune reaction against melanocytes. The pathophysiology is unclear. A genetic predisposition has been suggested as HLA-DR4/DRB1*04 is more common among VKH patients. Drug induced VKH syndrome has been reported in advanced melanoma patients receiving immunotherapy, including ipilimumab and adoptive cell transfer of Tumor-Infiltrating Lymphocyte associated with IL-2. To date, no case of anti PD-1 -induced VKH syndrome has been described. We report here the case of a HLA-DR4/DRB1*04 patient successfully treated with anti PD-1 for advanced melanoma who developed a systemic immune reaction against melanocytes for whom we discuss a VKH-like syndrome diagnosis in a potentially genetically predisposed patient.

SCIB1, a huIgG1 antibody DNA vaccination, combined with PD-1 blockade induced efficient therapy of poorly immunogenic tumors.

PURPOSE: We have previously shown that supraoptimal signaling of high avidity T cells leads to high expression of PD-1 and inhibition of proliferation. This study was designed to see if this effect could be mitigated by combining a vaccine that stimulates high avidity T cells with PD-1 blockade. EXPERIMENTAL DESIGN: We investigated the anti-tumor effect of a huIgG1 antibody DNA vaccine (SCIB1) and PD-1 blockade. RESULTS: Vaccination of HLA-DR4 transgenic mice with SCIB1 induced high frequency and avidity T cell responses that resulted in survival (40%) of mice with established B16F1-DR4 tumors. SCIB1 vaccination was associated with increased infiltration of CD4 and CD8 T cells within the tumor but was also associated with upregulation of PD-L1 within the tumor environment. PD-1 blockade also resulted in increased CD8 T cell infiltration and an anti-tumor response with 50% of mice showing long term survival. In line with our hypothesis that PD-1/PD-L1 signaling results in inhibition of proliferation of high avidity T cells at the tumor site, the combination of PD-1 blockade with vaccination, enhanced the number and proliferation of the CD8 tumor infiltrate. This resulted in a potent anti-tumor response with 80% survival of the mice. CONCLUSIONS: There is a benefit in combining PD-1 blockade with vaccines that induce high avidity T cell responses and in particular with SCIB1.

KIR and HLA Genotypes Implicated in Reduced Killer Lymphocytes Immunity Are Associated with Vogt-Koyanagi-Harada Disease.

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells are killer lymphocytes that provide defense against viral infections and tumor transformation. Analogous to that of CTL, interactions of killer-cell immunoglobulin-like receptors (KIR) with specific human leukocyte antigen (HLA) class I ligands calibrate NK cell education and response. Gene families encoding KIRs and HLA ligands are located on different chromosomes, and feature variation in the number and type of genes. The independent segregation of KIR and HLA genes results in variable KIR-HLA interactions in individuals, which may impact disease susceptibility. We tested whether KIR-HLA combinations are associated with Vogt-Koyanagi-Harada (VKH) disease, a bilateral granulomatous panuveitis that has strong association with HLA-DR4. We present a case control study of 196 VKH patients and 209 controls from a highly homogeneous native population of Japan. KIR and HLA class I genes were typed using oligonucleotide hybridization method and analyzed using two-tailed Fisher's exact probabilities. The incidence of Bx-KIR genotypes was decreased in VKH patients (odds ratio [OR] 0.58, P = 0.007), due primarily to a decrease in centromeric B-KIR motif and its associated KIRs 2DS2, 2DL2, 2DS3, and 2DL5B. HLA-B22, implicated in poor immune response, was increased in VKH (OR = 4.25, P = 0.0001). HLA-Bw4, the ligand for KIR3DL1, was decreased in VKH (OR = 0.59, P = 0.01). The KIR-HLA combinations 2DL2+C1/C2 and 3DL1+Bw4, which function in NK education, were also decreased in VKH (OR = 0.49, P = 0.012; OR = 0.59, P = 0.013). Genotypes missing these two inhibitory KIR-HLA combinations in addition to missing activating KIRs 2DS2 and 2DS3 were more common in VKH (OR = 1.90, P = 0.002). These results suggest that synergistic hyporesponsiveness of NK cells (due to poor NK education along with missing of activating KIRs) and CTL (due to HLA-B22 restriction) fail to mount an effective immune response against viral-infection that may trigger VKH pathogenesis in genetically susceptible individuals, such as HLA-DR4 carriers.

Differential effect of HLA class-I versus class-II transgenes on human T and B cell reconstitution and function in NRG mice.

Humanized mice expressing Human Leukocyte Antigen (HLA) class I or II transgenes have been generated, but the role of class I vs class II on human T and B cell reconstitution and function has not been investigated in detail. Herein we show that NRG (NOD.RagKO.IL2RγcKO) mice expressing HLA-DR4 molecules (DRAG mice) and those co-expressing HLA-DR4 and HLA-A2 molecules (DRAGA mice) did not differ in their ability to develop human T and B cells, to reconstitute cytokine-secreting CD4 T and CD8 T cells, or to undergo immunoglobulin class switching. In contrast, NRG mice expressing only HLA-A2 molecules (A2 mice) reconstituted lower numbers of CD4 T cells but similar numbers of CD8 T cells. The T cells from A2 mice were deficient at secreting cytokines, and their B cells could not undergo immunoglobulin class switching. The inability of A2 mice to undergo immunoglobulin class switching is due to deficient CD4 helper T cell function. Upon immunization, the frequency and cytotoxicity of antigen-specific CD8 T cells in DRAGA mice was significantly higher than in A2 mice. The results indicated a multifactorial effect of the HLA-DR4 transgene on development and function of human CD4 T cells, antigen-specific human CD8 T cells, and immunoglobulin class switching.

Of the multiple mechanisms leading to type 1 diabetes, T cell receptor revision may play a prominent role (is type 1 diabetes more than a single disease?).

A single determinant factor for autoimmunity does not exist; disease development probably involves contributions from genetics, the environment and immune dysfunction. Type 1 diabetes is no exception. Genomewide-associated studies (GWAS) analysis in T1D has proved disappointing in revealing contributors to disease prediction; the only reliable marker has been human leucocyte antigen (HLA). Specific HLAs include DR3/DR4/DQ2/DQ8, for example. Because HLA molecules present antigen to T cells, it is reasonable that certain HLA molecules have a higher affinity to present self-antigen. Recent studies have shown that additional polymorphisms in HLA that are restricted to autoimmune conditions are further contributory. A caveat is that not all individuals with the appropriate 'pro-autoimmune' HLA develop an autoimmune disease. Another crucial component is autoaggressive T cells. Finding a biomarker to discriminate autoaggressive T cells has been elusive. However, a subset of CD4 helper cells that express the CD40 receptor have been described as becoming pathogenic. An interesting function of CD40 on T cells is to induce the recombination-activating gene (RAG)1/RAG2 T cell receptor recombination machinery. This observation is contrary to immunology paradigms that changes in TCR molecules cannot take place outside the thymic microenvironment. Alteration in TCR, called TCR revision, not only occurs, but may help to account for the development of autoaggressive T cells. Another interesting facet is that type 1 diabetes (T1D) may be more than a single disease; that is, multiple cellular components contribute uniquely, but result ultimately in the same clinical outcome, T1D. This review considers the process of T cell maturation and how that could favor auto-aggressive T cell development in T1D. The potential contribution of TCR revision to autoimmunity is also considered.

Genetically modified human CD4(+) T cells can be evaluated in vivo without lethal graft-versus-host disease.

Adoptive cell immunotherapy for human diseases, including the use of T cells modified to express an anti-tumour T-cell receptor (TCR) or chimeric antigen receptor, is showing promise as an effective treatment modality. Further advances would be accelerated by the availability of a mouse model that would permit human T-cell engineering protocols and proposed genetic modifications to be evaluated in vivo. NOD-scid IL2rγ(null) (NSG) mice accept the engraftment of mature human T cells; however, long-term evaluation of transferred cells has been hampered by the xenogeneic graft-versus-host disease (GVHD) that occurs soon after cell transfer. We modified human primary CD4(+) T cells by lentiviral transduction to express a human TCR that recognizes a pancreatic beta cell-derived peptide in the context of HLA-DR4. The TCR-transduced cells were transferred to NSG mice engineered to express HLA-DR4 and to be deficient for murine class II MHC molecules. CD4(+) T-cell-depleted peripheral blood mononuclear cells were also transferred to facilitate engraftment. The transduced cells exhibited long-term survival (up to 3 months post-transfer) and lethal GVHD was not observed. This favourable outcome was dependent upon the pre-transfer T-cell transduction and culture conditions, which influenced both the kinetics of engraftment and the development of GVHD. This approach should now permit human T-cell transduction protocols and genetic modifications to be evaluated in vivo, and it should also facilitate the development of human disease models that incorporate human T cells.

CD4+ T-cell epitope prediction using antigen processing constraints.

T-cell CD4+ epitopes are important targets of immunity against infectious diseases and cancer. State-of-the-art methods for MHC class II epitope prediction rely on supervised learning methods in which an implicit or explicit model of sequence specificity is constructed using a training set of peptides with experimentally tested MHC class II binding affinity. In this paper we present a novel method for CD4+ T-cell eptitope prediction based on modeling antigen-processing constraints. Previous work indicates that dominant CD4+ T-cell epitopes tend to occur adjacent to sites of initial proteolytic cleavage. Given an antigen with known three-dimensional structure, our algorithm first aggregates four types of conformational stability data in order to construct a profile of stability that allows us to identify regions of the protein that are most accessible to proteolysis. Using this profile, we then construct a profile of epitope likelihood based on the pattern of transitions from unstable to stable regions. We validate our method using 35 datasets of experimentally measured CD4+ T cell responses of mice bearing I-Ab or HLA-DR4 alleles as well as of human subjects. Overall, our results show that antigen processing constraints provide a significant source of predictive power. For epitope prediction in single-allele systems, our approach can be combined with sequence-based methods, or used in instances where little or no training data is available. In multiple-allele systems, sequence-based methods can only be used if the allele distribution of a population is known. In contrast, our approach does not make use of MHC binding prediction, and is thus agnostic to MHC class II genotypes.

Citrullinated Vimentin Presented on MHC-II in Tumor Cells Is a Target for CD4+ T-Cell-Mediated Antitumor Immunity.

Stressful conditions in the harsh tumor microenvironment induce autophagy in cancer cells as a mechanism to promote their survival. However, autophagy also causes post-translational modification of proteins that are recognized by the immune system. In particular, modified self-antigens can trigger CD4(+) T-cell responses that might be exploited to boost antitumor immune defenses. In this study, we investigated the ability of CD4 cells to target tumor-specific self-antigens modified by citrullination, which converts arginine residues in proteins to citrulline. Focusing on the intermediate filament protein vimentin, which is frequently citrullinated in cells during epithelial-to-mesenchymal transition of metastasizing epithelial tumors, we generated citrullinated vimentin peptides for immunization experiments in mice. Immunization with these peptides induced IFNγ- and granzyme B-secreting CD4 T cells in response to autophagic tumor targets. Remarkably, a single immunization with modified peptide, up to 14 days after tumor implant, resulted in long-term survival in 60% to 90% of animals with no associated toxicity. This antitumor response was dependent on CD4 cells and not CD8(+) T cells. These results show how CD4 cells can mediate potent antitumor responses against modified self-epitopes presented on tumor cells, and they illustrate for the first time how the citrullinated peptides may offer especially attractive vaccine targets for cancer therapy.