Autoantibody Response to Islet Transplantation in Type 1 Diabetes

Islet allotransplantation into patients with autoimmune type 1 diabetes represents a reexposure to autoantigen. Here, measurement of antibodies to GAD and IA-2 autoantigens before and after islet transplantation in 36 patients (33 receiving islet plus kidney grafts with cyclosporin and steroid-based immunosuppression, and 3 receiving solitary islet transplants with mycophenolate but cyclosporin-free immunosuppression) demonstrated marked rises in GAD antibodies within 7 days posttransplantation in 5 patients (3 receiving islet after kidney transplants, and 2 receiving solitary islet transplants) and within 30 days in the third patient receiving solitary islet transplantation. GAD antibodies were of the IgG1 subclass, against major autoantigenic epitopes, and in cases of islet after kidney transplants, the responses were short-lived and not accompanied by HLA antibodies. Two of these patients had subsequent marked rises of IA-2 antibodies, and an additional patient had a marked rise in IgM-GAD antibodies 3 years after transplantation. Insulin independence was not achieved in patients with autoantibody elevations and was significantly less frequent in these patients. These data are consistent with a reactivation of autoimmunity that may be dependent on immunosuppression therapy and is associated with impaired graft function

Rapamycin does not adversely affect intrahepatic islet engraftment in mice and improves early islet engraftment in humans.

Objective: In this study we examined the effect of rapamycin (RAPA), a key component of the immunosuppressive regimen in clinical islet transplantation, on islet engraftment and function in vivo. Methods and results: Diabetic C57BL/6 or BALB/C recipient mice were transplanted with 350 syngeneic islets through the portal vein (PV-Tx; C57BL/6 n = 60; BALB/C n = 22) and treated with once-daily oral RAPA (1 mg/kg) or vehicle. No differences in post-transplant blood glucose concentrations and glucose tolerance were observed between RAPA-and vehicle-treated mice. The impact of RAPA on human islet engraftment was assessed in 10 patients with type 1 diabetes treated with 0.1 mg/kg/day rapamycin before islet transplantation. Compared to non pre-treated islet transplant recipents (n = 12), RAPA pre-treated patients had increased blood RAPA concentrations (p = 0.006) and fasting C-peptide concentrations (p = 0.005) in the two weeks post-transplant. RAPA pre-treatment was associated with a reduction in chemokines CCL2 and CCL3 concentrations pre-transplant (p < 0.01), and a dampened chemokine response (p = 0.005) post-transplant. Concordantly, in vitro RAPA inhibited the secretion of CCL2 and CCL3 by monocytes. Conclusion: Rapamycin does not adversely affect intrahepatic islet engraftment in the mouse, and potentially improves islet engraftment in humans by an anti-inflammatory mechanism

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

Distinguishing activated T regulatory cell and T conventional cells by single-cell technologies

Resting conventional T cells (Tconv) can be distinguished from T regulatory cells (Treg) by the canonical markers FOXP3, CD25 and CD127. However, the expression of these proteins alters after T-cell activation leading to overlap between Tconv and Treg. The objective of this study was to distinguish resting and antigen-responsive T effector (Tconv) and Treg using single-cell technologies. CD4+ Treg and Tconv cells were stimulated with antigen and responsive and non-responsive populations processed for targeted and non-targeted single-cell RNAseq. Machine learning was used to generate a limited set of genes that could distinguish responding and non-responding Treg and Tconv cells and which was used for single-cell multiplex qPCR and to design a flow cytometry panel. Targeted scRNAseq clearly distinguished the four-cell populations. A minimal set of 27 genes was identified by machine learning algorithms to provide discrimination of the four populations at >95% accuracy. In all, 15 of the genes were validated to be differentially expressed by single-cell multiplex qPCR. Discrimination of responding Treg from responding Tconv could be achieved by a flow cytometry strategy that included staining for CD25, CD127, FOXP3, IKZF2, ITGA4, and the novel marker TRIM which was strongly expressed in Tconv and weakly expressed in both responding and non-responding Treg. A minimal set of genes was identified that discriminates responding and non-responding CD4+ Treg and Tconv cells and, which have identified TRIM as a marker to distinguish Treg by flow cytometry

Inducible IL-7 Hyperexpression Influences Lymphocyte Homeostasis and Function and Increases Allograft Rejection

The IL-7/IL-7R pathway is essential for lymphocyte development and disturbances in the pathway can lead to immune deficiency or T cell mediated destruction. Here, the effect of transient hyperexpression of IL-7 was investigated on immune regulation and allograft rejection under immunosuppression. An experimental in vivo immunosuppressive mouse model of IL-7 hyperexpression was developed using transgenic mice (C57BL/6 background) carrying a tetracycline inducible IL-7 expression cassette, which allowed the temporally controlled induction of IL-7 hyperexpression by Dexamethasone and Doxycycline treatment. Upon induction of IL-7, the B220+ c-kit+ Pro/Pre-B I compartment in the bone marrow increased as compared to control mice in a serum IL-7 concentration-correlated manner. IL-7 hyperexpression also preferentially increased the population size of memory CD8+ T cells in secondary lymphoid organs, and reduced the proportion of CD4+Foxp3+ T regulatory cells. Of relevance to disease, conventional CD4+ T cells from an IL-7-rich milieu escaped T regulatory cell-mediated suppression in vitro and in a model of autoimmune diabetes in vivo. These findings were validated using an IL-7/anti-IL7 complex treatment mouse model to create an IL-7 rich environment. To study the effect of IL-7 on islet graft survival in a mismatched allograft model, BALB/c mice were rendered diabetic by streptozotocin und transplanted with IL-7-inducible or control islets from C57BL/6 mice. As expected, Dexamethasone and Doxycycline treatment prolonged graft median survival as compared to the untreated control group in this transplantation mouse model. However, upon induction of local IL-7 hyperexpression in the transplanted islets, graft survival time was decreased and this was accompanied by an increased CD4+ and CD8+ T cell infiltration in the islets. Altogether, the findings show that transient elevations of IL-7 can impair immune regulation and lead to graft loss also under immune suppression

BMI at Age 8 Years Is Influenced by the Type 2 Diabetes Susceptibility Genes HHEX-IDE and CDKAL1

OBJECTIVE: To determine whether HHEX-IDE and CDKAL1 genes, which are associated with birth weight and susceptibility to type 2 diabetes, continue to influence growth during childhood. RESEARCH DESIGN AND METHODS: BMI, weight, and height at age 8 years expressed as age- and sex-corrected standard deviation scores (SDS) against national reference data and single-nucleotide polymorphism genotyping of HHEX-IDE and CDKAL1 loci were analyzed in 646 prospectively followed children in the German BABYDIAB cohort. All children were singleton full-term births; 386 had mothers with type 1 diabetes, and 260 had fathers with type 1 diabetes and a nondiabetic mother. RESULTS: Type 2 diabetes risk alleles at the HHEX-IDE locus were associated with reduced BMI-SDS at age 8 years (0.17 SDS per allele; P = 0.004). After stratification for birth weight, both HHEX-IDE and CDKAL1 risk alleles were associated with reduced BMI-SDS (0.45 SDS, P = 0.0002; 0.52 SDS, P = 0.0001) and weight-SDS (0.22 SDS, P = 0.04; 0.56 SDS, P = 0.0002) in children born large for gestational age (&gt;90th percentile) but not children born small or appropriate for gestational age. Within children born large for gestational age, BMI and weight decreased with each additional type 2 diabetes risk allele ( approximately -2 kg per allele; &gt;8 kg overall). Findings were consistent in children of mothers with type 1 diabetes (P &lt; 0.0001) and children of nondiabetic mothers (P = 0.008). CONCLUSIONS: The type 2 diabetes susceptibility alleles at HHEX-IDE and CDKAL1 loci are associated with low BMI at age 8 years in children who were born large for gestational age

Two Distinctly HLA-Associated Contiguous Linear Epitopes Uniquely Expressed Within the Islet Antigen 2 Molecule Are Major Autoantibody Epitopes of the Diabetes-Specific Tyrosine Phosphatase-Like Protein Autoantigens

AbstractThe related tyrosine phosphatase-like proteins islet Ag (IA)-2 and IA-2β are autoantigens of type 1 diabetes in humans. Autoantibodies are predominantly against IA-2, and IA-2-specific epitopes are major autoantibody targets. We used the close homology of IA-2 and IA-2β to design chimeras and mutants to identify humoral IA-2-specific epitopes. Two major IA-2 epitopes that are absent from the related autoantigens IA-2β and IA-2Δ 13 splice variant ICA512.bdc were found contiguous to each other within IA-2 juxtamembrane amino acids 611–620 (epitope JM1) and 621–630 (epitope JM2). JM1 and JM2 are recognized by sera from 67% of patients with IA-2 Abs, and relatives of patients with type 1 diabetes having Abs to either JM epitope had a &gt;50% risk for developing type 1 diabetes within 6 years, even in the absence of diabetes-associated HLA genotypes. Remarkably, the presence of Abs to one of these two epitopes was mutually exclusive of the other; JM2 Abs and not JM1 Abs were found in relatives with HLA DR3/4, DR4/13, or DR1/4 genotypes; and the binding of autoantibodies to the JM2 epitope, but not the JM1 epitope, markedly affected proteolysis of IA-2. This is a unique demonstration of HLA-associated B cell responses to epitopes within a single autoantigen in humans and is consistent with modification of Ag processing by specific Ab-influencing peptide presentation by HLA molecules