PMC

PI-specific T cells exert bystander suppression. Shown are three representative clones that proliferated specifically to native and 9S PI peptides (10 μg ml⁻¹). Addition to responder T cells suppressed proliferation, irrespective of antigen exposure. The s.d. bars represent average of three replicate wells. PI76–90-specific T-cell clones were isolated from CD4+ T cells of a diabetic subject, as described previously. They were further characterized in a ³H thymidine incorporation assay to confirm their PI specificity using both wild-type PI76–90 peptide and PI76–90 S9 substitute peptide.^, For suppression assays, 20 000 clone T cells per well were activated with irradiated DRB1^*0401 PBMCs (60 000 per well) as antigen-presenting cells (APCs) pulsed with either of the PI peptides or alone. Activated cells were then added to a responder cell culture consisting of CD25+-depleted CD4+ T cells of a DRB1^*0401 donor activated by 5 μg ml⁻¹ soluble anti-CD3 (UCHT1; BD Biosciences Pharmingen, San Diego, CA, USA), 5 μg ml⁻¹ soluble anti-CD28 (CD28.2; Pharmingen) and irradiated autologous APCs (non-CD4 cells). The ability of each T-cell clone to suppress proliferation of the responder cells was determined by ³H thymidine incorporation during the final 16 h of the 5-day assay.

Flow cytometry profiles of CD4 T cells of T1D and control subjects used for RNA isolation and subsequent transcript arrays, showing tetramer binding with the PI 76–90 9S modified tetramer. Parallel HA306–319 tetramer-positive binding (foreign control antigen) and an irrelevant control tetramer, outer surface protein A (OspA) 163–175, are also shown. The numbers in the upper right quadrant represent percentages of the tetramer-positive cells in the CD4+CD25− T-cell subset after 14 days of culture. Major histocompatibility complex (MHC) class II tetramers were produced by constructing the expression vectors and generating soluble biotinylated HLA-DRB1^*0401 molecules, as described elsewhere. HLA molecules were loaded with peptides by detergent-facilitated exchange and dialyzed to remove the unbound fractions. Tetramers were obtained by incubation with phycoerythrin-labeled streptavidin. PI76–90 immunodominant epitope (SLQPLALEGSLQKRG), a PI76–90 9S substitute peptide (SLQPLALEGSLQSRG), in which substitution of K88 to S in peptide position 9 (9P) enhanced its binding affinity and agonistic activity,^, and two irrelevant control peptides, an influenza A hemagglutinin, HA306–318 (KYVKQNTLKLA) as a positive control and Borrelia burgdorferi OspA163–175 (KSYVLEGTLTAEK) as a negative control were each loaded into HLA-DR4 molecules assembled into tetramers. After 12–14 days of culture, T cells were stained with tetramers for flow cytometry analysis as described.

Partial expression profile of PI-specific, tetramer-sorted cells. Upregulated transcripts for FASLG, tumor necrosis factor (TNF), TNFSF11 and IL-2, which are commonly associated with peripheral tolerance, distinguish T1D subjects (a) from control subjects (b). Similar associations were not observed for control HA306–318-specific cells (c, d). Relative transcript expression was calculated as fold up- or downregulation in peptide-stimulated cultures relative to autologous non-stimulated cultures. Gene expression profiling was performed on tetramer-positive cells isolated by flow cytometry using a BD FACSVantage cell sorter (BD Biosciences, San Jose, CA, USA). From the sorted tetramer-positive cells, total RNA was extracted using a MagMAX Total RNA Isolation Kit (Applied Biosystems, Inc., Foster City, CA, USA). First-strand complementary DNA was synthesized with the RT² first-strand kit (SABiosciences, Corp., Frederick, MD, USA) and samples plated on RT² profiler PCR arrays for common human cytokines (SABiosciences), followed by the amplification by real-time PCR on a StepOne Plus or an ABI7000 sequence detection system (Applied Biosystems, Inc.). Amplification was carried out in a total volume of 25 μl for 40 cycles of 15 s at 95 °C and 1 min at 60 °C each. Expression of each gene was determined by normalizing the expression to five housekeeping genes. Relative gene expression for each subject was calculated by comparing peptide-stimulated samples to an unstimulated autologous sample to calculate a fold-change value.

Relationship between INS-VNTR genotype and detection of CD4 T cells, which bind PI76–90 tetramers. Subjects with VNTR class I alleles have high levels of peripheral blood T cells recognizing soluble DRB1^*0401-PI76–90 tetramer compared with the subjects having the protective class III alleles (a). The same T-cell samples were analyzed using the PI76–90 9S modified tetramer; no difference between the two groups of subjects was detected, implying presence of low-avidity PI-reactive T cells in both (b). In addition, no difference in tetramer positivity between the two groups of subjects was found when tetramers specific for another T1D autoantigen, GAD555–567, or foreign antigen, HA306–318, were used, indicating proinsulin specificity of the VNTR association (c, d). ***P=0.0004, differences in the level of tetramer-reactive T cells were calculated using Mann–Whitney U test. Solid horizontal lines represent means. Dashed horizontal lines indicate 0.5% binding of negative control tetramer. ^$INS-VNTR III/x where x=I or III. Typing for HLA-DRB1^*04 subtypes was performed using sequence-specific primers and probes in conjunction with real-time PCR. A total of 45 HLA-DRB1^*0401-positive subjects were analyzed out of which 6 were homozygous for DRB1^*0401, 12 had in addition DRB1^*0301 and the remaining 27 had in addition some other DRB1^* allele. T1D patients of INS-VNTR I/I as well as INS-VNTR I/III genotype were of comparable age (average 25.8±8.9 years vs 26.2±11.6 years, respectively) and had similar duration of the disease (3.4±1.9 years vs 5.5±1.5 years, respectively) as were autoantibody-positive subjects (36.3±10.4 vs 42±12.8) and healthy controls (40.4±14.5 vs 50.5±14.6). The VNTR genotype was assigned based on the genotype at the −23 A/T single-nucleotide polymorphism (SNP) at the INS promoter.^, Peripheral blood mononuclear cells were isolated from heparinized blood and CD4+CD25− T cells enriched in two steps. In the first step CD4+ T cells were separated from all non-CD4 cells using CD4+ T cell isolation kit (Miltenyi Biotec, Auburn, CA, USA). In the second step CD25+ T cells were depleted using anti-CD25-coated micro beads. CD4+CD25− T cells were stimulated in vitro for 12–14 days in 48-well tissue culture plates with autologous adherent non-CD4+ cells prepulsed with either PI peptides, hemagglutinin (HA) peptide (10 μg ml⁻¹) or without peptides. At days 6, 8 and 10, human recombinant IL-2 (10 IU ml⁻¹, Chiron Corporation, Emeryville, CA, USA) was added to the cultures.