DRB1*0401-restricted human T cell clone specific for the major proinsulin73-90 epitope expresses a down-regulatory T helper 2 phenotype

Abstract

Recently, we have identified proinsulin (P-Ins)73-90 as an immunodominant T cell epitope of HLA-DRB1*0401 (DR4) subjects with ␤-islet cell autoimmunity and of HLA-DR4͞CD4 double-transgenic mice immunized with human P-Ins. We have compared the fine specificities of one human CD4 T cell clone and two mouse T cell hybridoma clones recognizing this epitope, and, although these three clones all recognized the same core region (LALEGSLQK), there were major differences in how they interacted with the peptide (p)͞HLA complex, reflecting the fact that human P-Ins is a foreign antigen in the mouse and an autoantigen in the type 1 diabetes patient. The human T cell clone was forkhead transcription factor 3 (Foxp3)-positive, a marker for regulatory T cell lineages, and secreted predominantly IL-5, IL-10, and low levels of IFN␥ in response to P-Ins73-90. This finding is compatible with the previously detected regulatory cytokine pattern in subjects with ␤-cell autoimmunity. However, added N-or C-terminal amino acids drastically changed HLA and tetramer binding capacity as well as T cell reactivity and the cytokine phenotype of the P-Ins 73-90-specific human CD4 T cell clone, suggesting a potential for this P-Ins epitope as a target for therapeutic intervention in HLA-DR4-positive humans with ␤-islet cell autoimmunity or recent-onset type 1 diabetes.Foxp3 ͉ HLA-DRB1*0401 ͉ type 1 diabetes P roinsulin (P-Ins) is considered an important autoantigen in type 1 diabetes (T1D), because it is the only truly ␤-cellspecific target, and because autoreactivity to P-Ins is very common in T1D patients with the HLA-DRB1*0401 (DR4) DQ8 haplotype (1-6). The differences distinguishing autoreactive from foreign antigen reactive T cell responses to the same immunogenic epitope are still largely unknown (7). To understand the structural requirements for activation of self-reactive P-Ins 73-90 -specific T cells in T1D autoimmunity, we have isolated a Foxp3-positive CD4 ϩ T cell clone from a DR4-homozygous T1D patient and compared the fine specificity of this T cell receptor to those of two murine T cell hybridoma clones derived from HLA-DR4 transgenic mice, in which this epitope is a foreign antigen (8). P-Ins 73-90 is situated at the C terminus of the C peptide and also covers the enzymatic cleavage site of the insulin A chain (8). This site is proteolytically destroyed during the maturation of insulin before secretion and is, therefore, an indication that Proinsulin and not Insulin may be the actual autoantigenic target in T1D.In human studies using purified CD4 ϩ T cells from HLA-DR4-positive subjects with islet autoimmunity, P-Ins 73-90 was also identified as an immunodominant epitope. This epitope was recognized by approximately two thirds of autoantibody-positive subjects, one third of recently diagnosed T1D patients, and a few control subjects (5, 9). The cytokines seen in response to P-Ins 73-90 were predominantly IL-4 and IL-10 in subjects with ␤-cell autoimmunity (9). Moreover, in a consecutive follow up of three high-risk individuals, ...

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

“…To directly test this hypothesis, we analyzed peripheral blood lymphocytes for recognition of HLA class II-proinsulin complexes in subjects with VNTR I/I or VNTR III/x (where x ¼ I or III) genotypes. CD4 þ CD25-peripheral T lymphocytes from 24 Caucasian VNTR I/I and 21 VNTR III/x subjects matched for HLA-DRB1*0401 were cultured with autologous antigen-presenting cells in the presence of PI(76-90), 22 to allow for expansion of antigen-specific cells, and subsequently incubated with soluble DRB1*0401-PI(76-90) complexes conjugated to a phycoerythrin fluorochrome. Out of 45 HLA-DRB1*0401-positive subjects analyzed, 6 were homozygous for DRB1*0401, 12 had in addition DRB1*0301 and the remaining 27 had some other DRB1* allele.…”

“…Tetramers were obtained by incubation with phycoerythrin-labeled streptavidin. PI76-90 immunodominant epitope (SLQPLALEGSLQKRG), 22 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, 4,22 and two irrelevant control peptides, an influenza A hemagglutinin, HA306-318 (KYVKQNTLKLA) 25 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.…”

“…The presence of IL-10 transcripts in the T cells from T1D subjects responding to proinsulin is of interest because this is reminiscent of previous studies in which peripheral blood stimulation with proinsulin peptides was predominantly associated with IL-10 release. 22,26 Figure 4 illustrates mixed T-cell proliferation assays, in which three T-cell clones purified from the sorted '9S' proinsulin tetramer-positive population were added to autologous responder peripheral blood mononuclear cells. In each case, suppression of the bystander T cells, irrespective of additional antigen, was observed.…”

“…PI76-90-specific T-cell clones were isolated from CD4 þ T cells of a diabetic subject, as described previously. 22 They were further characterized in a 3 H thymidine incorporation assay to confirm their PI specificity using both wild-type PI76-90 peptide and PI76-90 S9 substitute peptide. 2,26 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.…”

The therapeutic potential of regulatory T cells for the treatment of autoimmune disease

“…There are several approaches being developed to isolate and induce antigen-specific cells. The most reliable approach is the isolation and expansion of antigen-specific Tregs [93]. The isolation of antigen-specific Treg relies on robust antigen-specific Treg expansion and efficient screening assays.…”