Avidity-dependent programming of autoreactive T cells in T1D

Abstract

Fate determination for autoreactive T cells relies on a series of avidity-dependent interactions during T cell selection, represented by two general types of signals, one based on antigen expression and density during T cell development, and one based on genes that interpret the avidity of TCR interaction to guide developmental outcome. We used proinsulin-specific HLA class II tetramers to purify and determine transcriptional signatures for autoreactive T cells under differential selection in type 1 diabetes (T1D), in which insulin (INS) genotypes consist of protective and susceptible alleles that regulate the level of proinsulin expression in the thymus. Upregulation of steroid nuclear receptor family 4A (NR4A) and early growth response family genes in proinsulin-specific T cells was observed in individuals with susceptible INS-VNTR genotypes, suggesting a mechanism for avidity-dependent fate determination of the T cell repertoire in T1D. The NR4A genes act as translators of TCR signal strength that guide central and peripheral T cell fate decisions through transcriptional modification. We propose that maintenance of an NR4A-guided program in low avidity autoreactive T cells in T1D reflects their prior developmental experience influenced by proinsulin expression, identifying a pathway permissive for autoimmunity.

Figure 1. Influence of INS genotype on the frequency of proinsulin-specific CD4+ T cells.

These results are shown for all subjects (left panel), healthy control subjects (middle panel), and T1D subjects (right panel), matched for HLA-DRB1*04:01 and tested using the proinsulin 88S tetramer. Differences in the distribution of tetramer positivity among the groups were calculated using Mann-Whitney U test; an earlier study including some of these subjects has been reported (7). Each symbol represents one tested subject. Black horizontal lines represent medians and inter-quartile ranges. Dashed horizontal lines indicate threshold binding of the negative-control tetramer. Susceptible INS gene I  =  INS VNTR I/I; Protective INS gene III  =  INS VNTR I/III or III/III.

Figure 2. Relative expression panels for all transcripts of CD4 T cells binding the proinsulin 88S tetramer.

Expression panels show significant differences between VNTR I and VNTR III subjects for all subjects (A), healthy individuals (B), and T1D patients (C). Symbols represent individual RNA samples tested in duplicate. The differences in the level of transcript expression between INS genotypes were calculated using a Mann-Whitney U test. Longer horizontal lines indicate the medians and small horizontal lines indicate the inter-quartile ranges. Only plots reaching statistically significant difference among the groups are shown. Susceptible INS-gene I  =  INS-VNTR I/I; protective INS-gene III  =  INS-VNTR I/III or III/III.

Figure 3. Relative expression plots for significant genes that distinguish between healthy individuals and T1D patients.

Patients with both INS genotypes (A), those with the VNTR I-susceptible INS genotype (B), and VNTR III-protective INS genotype (C) are shown. Horizontal lines indicate medians and inter-quartile ranges. The differences in the level of transcript-expression between healthy individuals and T1D patients were calculated using a Mann-Whitney U test. Only plots reaching significant difference between the groups are shown.

Figure 4. Flow cytometry profile of CD4+ T cells.

A T1D patient with the susceptible INS VNTR I/I genotype is shown (A). Staining with the proinsulin 76–90 tetramer (left) and the 88S tetramer (right) is shown. Influence of INS genotype on the frequency of proinsulin-specific CD4+ T cells (B), shown for all subjects (left panel), healthy control subjects (middle panel), and T1D subjects (right panel), matched for HLA-DRB1*04:01 and tested using the proinsulin 79–90 tetramer. Differences in the distribution of tetramer positivity among the groups were calculated using a Mann-Whitney U test; an earlier study including some of these subjects has been reported (7). Solid horizontal lines represent medians and inter-quartile ranges. Dashed horizontal lines indicate threshold binding of the negative control tetramer. Susceptible INS gene I  =  INS VNTR I/I; protective INS gene III  =  INS VNTR I/III or III/III.

Figure 5. Influence of INS genotype on expression profiles of proinsulin-specific CD4+ T cells.

Relative expression profiles are shown for all subjects matched for HLA-DRB1*04:01 and selected for high antigen avidity using the proinsulin 76–90 tetramer (A). Several genes showed significant transcript differences between control and T1D subjects matched for the INS VNTR III diabetes-protective genotype, all p<0.01. (B).