Immunology of Diabetes Society T-Cell Workshop: HLA class I tetramer-directed epitope validation initiative T-Cell Workshop Report-HLA Class I Tetramer Validation Initiative.

BACKGROUND: Identification of T-cell reactivity to ß-cell antigen epitopes is an important goal for studying pathogenesis and for designing and monitoring of immunotherapeutic interventions in type 1 diabetes (T1D). METHODS: We performed a multicentre validation of known human leukocyte antigen (HLA) class I CD8+ T-cell epitopes. To this end, peripheral blood T-cell responses were measured in 35 recently (<2 years) diagnosed HLA-A*02:01+ T1D patients using blind-coded HLA-A2 tetramers (TMrs) and pentamers (PMrs), encompassing two epitopes of preproinsulin (PPI; PPIA12-20 and PPIB10-18) and two epitopes of glutamic acid decarboxylase (GAD; GAD114-122 and GAD536-545). We also compared the readout of TMrs and PMrs with a CD8+ T-cell interferon-γ enzyme-linked immunospot assay. RESULTS: Despite the minute frequencies of autoreactive cells detected by TMrs/PMrs, most (73-77%) T1D patients had responses to one or more of the epitopes used. All four epitopes were recognized by T1D patients, with a prevalence ranging from 5 to 25%. TMrs and PMrs detected more positive responses to the ß-cell epitopes than CD8+ T-cell interferon-γ enzyme-linked immunospot. However, concordance between positive responses to TMrs and PMrs was limited. CONCLUSIONS: Using a multicentre blind-coded setup and three different T-cell assays, we have validated PPI and GAD epitopes as commonly recognized CD8+ T-cell targets in recently diagnosed T1D patients. Both TMrs and PMrs showed higher detection sensitivity than the CD8+ T-cell interferon-γ enzyme-linked immunospot assay. However, there are some important methodological issues that need to be addressed in using these sensitive techniques for detecting low frequency responses.

Comparison of cryopreservation methods on T-cell responses to islet and control antigens from type 1 diabetic patients and controls.

BACKGROUND: Type 1 diabetes (T1D) is a cell-mediated autoimmune disease characterized by destruction of the pancreatic islet cells. The use of cryopreserved cells is preferable to the use of freshly isolated cells to monitor clinical trials to decrease assay and laboratory variability. METHODS: The T-Cell Workshop Committee of the Immunology of Diabetes Society compared two widely accepted T-cell freezing protocols (warm and cold) to freshly isolated peripheral blood mononuclear cells from patients with T1D and controls in terms of recovery, viability, cell subset composition, and performance in functional assays currently in use in T1D-related research. Nine laboratories participated in the study with four different functional assays included. RESULTS: The cold freezing method yielded higher recovery and viability compared with the warm freezing method. Irrespective of freezing protocol, B cells and CD8+ T cells were enriched, monocyte fraction decreased, and islet antigen-reactive responses were lower in frozen versus fresh cells. However, these results need to take in to account that the overall response to islet autoantigens was low in some assays. CONCLUSIONS: In the current study, none of the tested T-cell functional assays performed well using frozen samples. More research is required to identify a freezing method and a T-cell functional assay that will produce responses in patients with T1D comparable to responses using fresh peripheral blood mononuclear cells.

Isolation and preservation of peripheral blood mononuclear cells for analysis of islet antigen-reactive T cell responses: position statement of the T-Cell Workshop Committee of the Immunology of Diabetes Society.

Autoimmune T cell responses directed against insulin-producing ß cells are central to the pathogenesis of type 1 diabetes (T1D). Detection of such responses is therefore critical to provide novel biomarkers for T1D 'immune staging' and to understand the mechanisms underlying the disease. While different T cell assays are being developed for these purposes, it is important to optimize and standardize methods for processing human blood samples for these assays. To this end, we review data relevant to critical parameters in peripheral blood mononuclear cell (PBMC) isolation, (cryo)preservation, distribution and usage for detecting antigen-specific T cell responses. Based on these data, we propose recommendations on processing blood samples for T cell assays and identify gaps in knowledge that need to be addressed. These recommendations may be relevant not only for the analysis of T cell responses in autoimmune disease, but also in cancer and infectious disease, particularly in the context of clinical trials.

Current approaches to measuring human islet-antigen specific T cell function in type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease caused by the T cell-mediated destruction of the pancreatic insulin-producing beta cells. Currently there are no widely accepted and standardized assays available to analyse the function of autoreactive T cells involved in T1D. The development of such an assay would greatly aid efforts to understand the pathogenesis of T1D and is also urgently required to guide the development of antigen-based therapies intended to prevent, or cure, T1D. Here we describe some of the assays used currently to detect autoreactive T cells in human blood and review critically their strengths and weaknesses. The challenges and future prospects for the T cell assays are discussed.

Insulin gene VNTR genotype associates with frequency and phenotype of the autoimmune response to proinsulin.

Immune responses to autoantigens are in part controlled by deletion of autoreactive cells through genetically regulated selection mechanisms. We have directly analyzed peripheral CD4+ proinsulin (PI) 76-90 (SLQPLALEGSLQKRG)-specific T cells using soluble fluorescent major histocompatibility complex class II tetramers. Subjects with type I diabetes and healthy controls with high levels of peripheral proinsulin-specific T cells were characterized by the presence of a disease-susceptible polymorphism in the insulin variable number of tandem repeats (INS-VNTR) gene. Conversely, subjects with a 'protective' polymorphism in the INS-VNTR gene had nearly undetectable levels of proinsulin tetramer-positive T cells. These results strongly imply a direct relationship between genetic control of autoantigen expression and peripheral autoreactivity, in which proinsulin genotype restricts the quantity and quality of the potential T-cell response. Using a modified tetramer to isolate low-avidity proinsulin-specific T cells from subjects with the susceptible genotype, transcript arrays identified several induced pro-apoptotic genes in the control, but not diabetic subjects, likely representing a second peripheral mechanism for maintenance of tolerance to self antigens.

Pro- and anti-inflammatory cytokine production by autoimmune T cells against preproinsulin in HLA-DRB1*04, DQ8 Type 1 diabetes.

AIMS/HYPOTHESIS: Preproinsulin is a target T cell autoantigen in human Type 1 diabetes. This study analyses the phenotype and epitope recognition of preproinsulin reactive T cells in subjects with a high genetic risk of diabetes [HLA-DRB1*04, DQ8 with Ab+ (autoantibody-positive) or without islet autoantibodies (control subjects)], and in HLA-matched diabetic patients. METHODS: A preproinsulin peptide library approach was used to screen for cytokine profiles and epitope specificities in human peripheral blood lymphocytes, and CD4(+)CD45RA(-) and CD4(+)CD45RA(+) T cell subfractions, representing memory and naive and recently primed T cells respectively. RESULTS: In CD4(+) T cell subsets we identified immunodominant epitopes and cytokine production patterns that differed profoundly between patients, Ab+ subjects and non-diabetic HLA-matched control subjects. In Ab+ subjects, a C-peptide epitope C13-29 and insulin B-chain epitope B11-27 were preferentially recognised, whereas insulin-treated Type 1 diabetic patients reacted to native insulin and B-chain epitope B1-16. In peripheral blood lymphocytes of Ab+ subjects, an increase in T helper (Th) 1 (IFNgamma, IL-2) and Th2 (IL-4) cytokines was detectable, wheras in CD45RA(+) and CD45RA(-) subsets, IL-4 and IL-10 phenotypes dominated, compatible with the contribution of non-CD4 cells to IFNgamma content. In insulin-treated Type 1 diabetic patients, naive and recently primed CD4(+) cells were characterised by increasd IFNgamma, TNFalpha, and IL-5. CONCLUSIONS/INTERPRETATION: Our data show that T cell reactivity to preproinsulin in CD45RA subsets is Th2-dominant in Ab+ subjects, challenging the Th1 paradigm in Type 1 diabetes. Characteristic immunodominant epitopes and cytokine patterns distinguish diabetic patients and Ab+ subjects from HLA-matched healthy individuals. This could prove useful in monitoring of T-cell immunity in clinical diabetes intervention trials.

Humoral and cellular immune parameters before and during immunosuppressive therapy of a patient with stiff-man syndrome and insulin dependent diabetes mellitus.

OBJECTIVES: Humoral and cellular immune reactivity are reported for two neuroendocrine autoantigens-glutamic acid decarboxylase (GAD) and the protein tyrosine phosphatase IA-2-in a patient with the autoimmune type of stiff-man syndrome and insulin dependent diabetes (IDDM). METHODS: Antibodies and T cell proliferation against GAD and IA-2 and cytokine release of antigen stimulated T cells (IFN-gamma) were determined before and several times during immunosuppressive therapy with prednisolone. RESULTS: Raised GAD antibodies against full length GAD65 or chimeric constructs were detected before therapy and they remained at a high concentration despite a marked clinical improvement during cortisone treatment. Antibodies to IA-2 were undetectable, but weak T cell responses to both GAD and IA-2 were seen before therapy and once on reduction of high cortisone dosages when the patient showed signs of clinical deterioration. Cytokine profiles showed increased IFN-gamma production after stimulation with GAD or IA-2 suggesting increased activation of TH1 cells. CONCLUSION: Immunosuppressive therapy --even with extremely high doses of 500 mg a day--does not lead to the reduction of antibody concentrations in the periphery nor to a switch in epitope recognition of such antibodies despite clinical improvement. The amount of T cell reactivity to various antigens, however, may be a useful marker to monitor the effectiveness of immunotherapy.

Autoimmune diabetes: the role of T cells, MHC molecules and autoantigens.

Type 1 diabetes (IDDM) is a T cell mediated autoimmune disease which in part is determined genetically by its association with major histocompatibility complex (MHC) class II alleles. The major role of MHC molecules is the regulation of immune responses through the presentation of peptide epitopes of processed protein antigens to the immune system. Recently it has been demonstrated that MHC molecules associated with autoimmune diseases preferentially present peptides of other endogenous MHC proteins, that often mimic autoantigen-derived peptides. Hence, these MHC-derived peptides might represent potential targets for autoreactive T cells. It has consistently been shown that humoral autoimmunity to insulin predominantly occurs in early childhood. The cellular immune response to insulin is relatively low in the peripheral blood of patients with IDDM. Studies in NOD mice however have shown, that lymphocytes isolated from pancreatic islet infiltrates display a high reactivity to insulin and in particular to an insulin peptide B 9-23. Furthermore we have evidence that cellular autoimmunity to insulin is higher in young pre-diabetic individuals, whereas cellular reactivity to other autoantigens is equally distributed in younger and older subjects. This implicates that insulin, in human childhood IDDM and animal autoimmune diabetes, acts as an important early antigen which may target the autoimmune response to pancreatic beta cells. Moreover, we observed that in the vast majority of newly diagnosed diabetic patients or individuals at risk for IDDM, T cell reactivity to various autoantigens occurs simultaneously. In contrast, cellular reactivity to a single autoantigen is found with equal frequency in (pre)-type 1 diabetic individuals as well as in control subjects. Therefore the autoimmune response in the inductive phase of IDDM may be targeted to pancreatic islets by the cellular and humoral reactivity to one beta-cell specific autoantigen, but spreading to a set of different antigens may be a prerequisite for progression to destructive insulitis and clinical disease. Due to mimic epitopes shared by autoantigen(s), autologous MHC molecules and environmental antigens autoimmunity may spread, intramolecularly and intermolecularly and amplify upon repeated reexposure to mimic epitopes of environmental triggers.

Relation between cellular and humoral immunity to islet cell antigens in type 1 diabetes.

The autoimmune disease insulin-dependent diabetes is thought to result from T-cell mediated destruction of pancreatic beta cells. We analysed the relation between humoral and cellular immunity to multiple islet cell antigens, including human insulin, glutamate decarboxylase GAD65, tyrosine phosphatase (ICA512/IA2), human pancreas and RIN cells in 28 patients with newly diagnosed type 1 diabetes and 9 antibody-positive (Ab+) relatives at high risk for type 1 diabetes. Of newly diagnosed patients, all showed reactivity to at least one recombinant islet cell antigen, by elevated cellular or humoral (or both) immune responses. Fifty-seven percent of patients and relatives showed T-cell reactivity to more than one islet cell antigen and 68% revealed humoral immunity to more than one islet cell antigen. Increased T-cell response to one single islet cell antigen was observed in 32% and positive antibody response in 25% of diabetic patients and relatives. Further-more, we found that T-cell reactivity to GAD was associated with T-cell reactivity to RIN cells, whereas reactivity to ICA512 and insulin was not associated with any other T-cell response. Likewise, antibody response to ICA512/IA2 correlated with antibodies to human pancreas (ICA), whereas antibody response to GAD or insulin was not related to any other antibody response. No positive or inverse correlation, however, was detected between T cell and humoral immunity, except for a positive association of antibodies and T-cell reactivity to insulin. Our data suggest that both humoral and cellular immune reactivity to multiple islet cell antigens are present in patients with newly diagnosed type 1 diabetes and in high risk relatives, but the two immune responses are individually activated.

Cellular immune response to diverse islet cell antigens in IDDM.

In IDDM, T-cells are postulated to mediate the destruction of pancreatic beta-cells. We analyzed peripheral blood mononuclear cell (PBMC) responses to human insulin, glutamate decarboxylase GAD65, tyrosine phosphatase ICA512, glucagon, membrane preparations of RIN cells and human pancreas, and three control antigens (La = nuclear cell antigen, tetanus toxoid, and phytohemagglutinin). A total of 28 patients with newly diagnosed IDDM, 9 antibody-positive (Ab+) first-degree relatives, and 16 healthy control subjects were included. Increased proliferative responses to pancreatic islet cell antigens were observed in diabetic patients and in Ab+ relatives compared with control subjects, whereas T-cell reactivity to nonpancreatic control antigens was similar between the study groups. The highest differences in the magnitude of proliferative responses were seen for ICA512, followed by membrane preparations of RIN cells, GAD65, and human pancreas. Few subjects reacted with insulin or glucagon. Interestingly, Ab+ relatives showed higher T-cell reactivity with respect to stimulation indexes and prevalences than newly diagnosed diabetic patients, and as many as 89% of Ab+ relatives showed proliferation to more than one islet cell antigen preparation in comparison to 43% of newly diagnosed diabetic patients and none of the control subjects. Statistical analysis revealed significant positive correlation of insulin autoantibody levels with the levels of insulin-specific T-cells in Ab+ relatives, but no relation of PBMC responses to age, sex, or HLA-DR haplotypes. Our results demonstrate the simultaneous existence of various autoreactive T-cells specific for islet cell antigens in the prediabetic period. These T-cells may play a significant role in the pathogenesis of the disease.

The T-cell receptor beta chain CDR3 region of BV8S1/BJ1S5 transcripts in type 1 diabetes.

We recently described the T-cell receptor (TCR) beta chain CDR3 motif S-SDRLG-NQPQH (BV8S1-BJ1S5) in an islet-specific T-cell clone (K2.12) from a type 1 diabetic patient (AS). A similar motif (RLGNQ) was also reported in a T-cell clone of non-obese diabetic (NOD) mice by others. In order to determine the frequency of our motif in selected and unselected T-cell populations, we cloned and sequenced the CDR3 region of BV8S1-BJ1S5 transcripts. These transcripts were derived from unstimulated peripheral blood T lymphocytes from two type 1 diabetic patients (AS and FS) and their non-diabetic sibling (WS), as well as from an islet-specific T-cell line of one of the patients. In addition, we compared the structure and composition of the CDR3 region in BV8S1-BJ1S5 transcripts from peripheral blood T cells between the patients and their non-diabetic sibling (>50 sequences each). We found that 30% of the islet-specific T-cell line cDNA clones expressed the entire sequence-motif, whereas it was absent in the clones of unstimulated peripheral blood T cells from both patients and their non-diabetic sibling. The average length of the CDR3 region was shorter in the patients (mean AS 9.9, FS 9.9, versus WS 10.7, p = 0.0037) and the number of inserted nucleotides in N nucleotide addition at the DJ-junction lower (mean AS 3.5, FS 3. 2, versus WS 5.2, P = <10(-4)) as compared with their non-diabetic sibling. Moreover, the pattern of amino acid usage in the CDR3 region was dissimilar at positions 5 and 6, where polar amino acids predominated in both diabetic siblings. In contrast, basic amino acids are preferentially used at position 5 in the clones of the non-diabetic sibling. These data provide information on the general structure of the TCR(BV8S1-BJ1S5) CDR3 region in type 1 diabetes and may indicate differences in the amino and nucleic acid composition of the TCR beta chain CDR3 region between two type 1 diabetic patients and their non-diabetic sibling.

HLA-DQ-restricted, islet-specific T-cell clones of a type I diabetic patient. T-cell receptor sequence similarities to insulitis-inducing T-cells of nonobese diabetic mice.

We established a T-cell line and 20 CD4+ T-cell clones from the peripheral blood lymphocytes of a type I diabetic patient using a membrane preparation of a rat insulinoma cell line (beta membrane antigen [BMA]) as a source of antigen. The T-cell line and three selected clones proliferated specifically to stimulation with BMA and to membranes prepared from human islets, rat pancreas, and NOD pancreas, but not to control antigens. Proliferation-inhibition studies using human leukocyte antigen (HLA)-specific monoclonal antibodies revealed HLA-DQw1-restricted recognition of BMA. An analysis of the T-cell receptor (TCR) repertoire of the T-cell line after 8 and 40 days of culture showed a prominent usage of the V alpha 1 and V alpha 12 gene families. Although sequencing of the TCR V alpha and V beta chains of the three clones demonstrated that each used different V alpha and V beta gene segments, structural similarities were found in complementary-determining region 3 (CDR3), the region that is postulated to interact with the peptide component of the TCR ligand. When we compared these TCR sequences with published sequences of disease-inducing T-cells of NOD mice, highly related TCR V beta families were detected. Furthermore, stretches of identical amino acids within the CDR3 region were found between two pairs of human and mouse T-cells. If one considers the species differences in TCR genes and sequence differences in the restriction elements for these cells (HLA-DQ vs. H-2 I-A nod), these sequence similarities are striking and may be useful for pinpointing T-cells of primary importance in the development of disease.

[Immunologic and clinical definition of the rejection reaction in kidney transplantation]. / Imunolosko i klinicko definiranje reakcija odbacivanja transplantiranog bubrega.

In the evaluation of the immunological state of a kidney transplant there is no reliable diagnostic test. Therefore, several various tests should always be applied. The aim of this study was to define the type of immunological reaction (cellular or humoral) that causes the rejection of transplanted kidney. A group of 45 patients with a kidney transplant were tested 175 times, in various phases of the kidney transplant, starting on the day of transplantation up to several months posttransplant. Specific antibodies (SA) and specific cytotoxic lymphocytes (SCL) directed against donor cells in lymphocyte-mediated cytolysis tests in the presence of antibodies (ADLMC) or without them (LMC) were determined. LMC was significantly correlated with acute rejection (85%). In the phase of clinical quiscence of the kidney transplant LMC was positive in 4% of the cases. During the period of chronic rejection it was positive in 75% of the cases. ADLMC test is significantly correlated with chronic rejection (88%). In the period of acute rejection it was positive in 27% while in clinical quiscence in 11% of the cases. Twenty-two patients with non-immunological disorders of the kidney transplant were tested 62 times. LMC test was always negative, while ADLMC test was positive in 6.4% of the cases. These tests prove to be good parameters for defining immunological reaction. Thus, they can be of great importance in determining adequate immunosuppressive therapy. Negative results of tests in non-immunological disorders of graft function are highly significant.