Multiple autoimmune-associated variants confer decreased IL-2R signaling in CD4+ CD25(hi) T cells of type 1 diabetic and multiple sclerosis patients

Abstract

IL-2 receptor (IL-2R) signaling is essential for optimal stability and function of CD4(+)CD25(hi)FOXP3(+) regulatory T cells (Treg); a cell type that plays an integral role in maintaining tolerance. Thus, we hypothesized that decreased response to IL-2 may be a common phenotype of subjects who have autoimmune diseases associated with variants in the IL2RA locus, including T1D and MS, particularly in cells expressing the high affinity IL-2R alpha chain (IL-2RA or CD25). To examine this question we used phosphorylation of STAT5 (pSTAT5) as a downstream measure of IL-2R signaling, and found a decreased response to IL-2 in CD4(+)CD25(hi) T cells of T1D and MS, but not SLE patients. Since the IL2RArs2104286 haplotype is associated with T1D and MS, we measured pSTAT5 in controls carrying the rs2104286 risk haplotype to test whether this variant contributed to reduced IL-2 responsiveness. Consistent with this, we found decreased pSTAT5 in subjects carrying the rs2104286 risk haplotype. Reduced IL-2R signaling did not result from lower CD25 expression on CD25(hi) cells; instead we detected increased CD25 expression on naive Treg from controls carrying the rs2104286 risk haplotype, and subjects with T1D and MS. However the rs2104286 risk haplotype correlated with increased soluble IL-2RA levels, suggesting that shedding of the IL-2R may account in part for the reduced IL-2R signaling associated with the rs2104286 risk haplotype. In addition to risk variants in IL2RA, we found that the T1D-associated risk variant of PTPN2rs1893217 independently contributed to diminished IL-2R signaling. However, even when holding genotype constant at IL2RA and PTPN2, we still observed a significant signaling defect in T1D and MS patients. Together, these data suggest that multiple mechanisms converge in disease leading to decreased response to IL-2, a phenotype that may eventually lead to loss of tolerance and autoimmunity.

Figure 1. Decreased response to IL-2 in CD4⁺CD25⁺ T cells is a phenotype common to T1D and MS, but not SLE subjects.

PBMC were thawed, rested and stimulated with media alone or IL-2 (100 IU/ml) prior to fixation, permeabilization and staining with CD4, CD25, CD45RO and pSTAT5. (A) The frequency of pSTAT5⁺ cells of CD25^hi cells was determined by comparing media to 25 IU IL-2 stimulation for 20min. (B) In an independent cohort, the frequency of pSTAT5⁺ cells of CD25^lo cells was determined by comparing media to 100 IU IL-2 stimulation for 10min. In all experiments, patients and controls were prospectively matched with controls for age and ethnicity. Groups in (A) and (B) differed by multiple parameter testing using a Kruskal-Wallis test with p-values of 0.0008 and 0.0198, respectively. Statistical significance for individual pairings using a Mann-Whitney test are shown. Differences shown in (A) were verified with permutation testing where control vs T1D and control vs MS remained significant (<0.001). For (B), after permutation testing the control vs T1D (0.005), T1D vs MS (0.03) and T1D vs SLE (0.01) pairings remained significant. There was one outlier in the T1D cohort in (B). By ANOVA, age, gender, race, disease status or immune treatment did not significantly contribute to differences in % pSTAT5 for any cohort (therapy and disease status analyses are shown in Figure S3). The mean pSTAT5 response is indicated for each group. IL2RArs2104286 genotype is noted by solid black (A/A), solid grey (A/G) and open (G/G) symbols.

Figure 2. Decreased response to IL-2 in CD4⁺CD25^hi T cells correlates with an IL2RA risk haplotype associated with T1D and MS.

PBMC were thawed and stained as in Figure 1. (A) The frequency and MFI fold increase of pSTAT5⁺ cells of CD25⁺ gated cells was determined by comparing media to IL-2 stimulated genotyped control samples. (B) The frequency of pSTAT5⁺ cells of CD25^lo was determined following stimulation of genotyped controls with IL-2 or IL-15 (200pg/ml) for 10min. The mean pSTAT5 response is indicated for each group and subjects with IL2RArs2104286 genotypes G/G (▴), A/G (○) and A/A (▪) are differentiated by symbol shapes. Statistical significance was determined using a Mann Whitney test. All controls tested were Caucasian with no significant difference in age or gender between genotypes. *denotes the risk haplotype determined by looking at variation at the rs21042856 allele while holding the rs12722495 allele constant at T/T and the rs11594656 allele constant at T/T.

Figure 3. Increased expression of CD25 on naïve Treg is common to controls carrying the IL2RArs2104286 risk haplotype and MS and T1D subjects.

PBMC were thawed and stained for CD4, CD25, FOXP3, helios and CD45RA as described in materials and methods. (A) Representative gating of CD4⁺ T cells on FOXP3⁻ Teff and FOXP3⁺ Treg. CD25 MFI was measured on CD45RA⁺ naïve and CD45RA⁻ memory Teff (B) and Treg (C) populations of controls and (D) autoimmune subjects. The mean pSTAT5 response is indicated for each group. For (B) and (C), all controls tested were Caucasian with no significant difference in age or gender between IL2RArs2104286 genotypes. G/G (▴), A/G (○) and A/A (▪) subjects are differentiated by symbol shapes. In (D), patients and controls were prospectively matched with controls for age and ethnicity. In (B-C), statistical significance between pairings was determined using a student’s t- test after assessing normality of the data as determined using the Shapiro-Wilk test. P-values were then adjusted using the Benjamini Hochberg false discovery rate procedure. Adjusted p values where significant are shown. Groups in (D) differed by multiple parameter testing using a Kruskal-Wallis test (0.0085). Statistical significance for individual pairings using a Mann-Whitney test is shown. Differences were verified with permutation testing where control vs T1D (0.015) and control vs MS (0.01) remained significant. There were two outliers in the MS cohort. *denotes the risk haplotype determined by looking at variation at the IL2RArs21042856 allele while holding the rs12722495 allele constant at T/T and the rs11594656 allele constant at T/T. By ANOVA, age, gender, race, disease status or immune treatment did not significantly contribute to differences in CD25 MFI for any cohort. IL2RArs2104286 genotype is noted by solid black (A/A), solid grey (A/G) and open (G/G) symbols.

Figure 4. Increased serum sIL-2RA correlates with lower response to IL-2.

PBMC and serum samples from the same blood draw were assayed for response to IL-2 as in Figure 1 and serum sIL-2RA using Luminex. All controls tested were Caucasian with no significant difference in age or gender between genotypes. Multivariable analysis was performed using linear regression and is shown in the Figure. To confirm these results we performed several additional tests. Random sampling of 80% of the data 1000 times resulted in a Pearson correlation of −0.516 and a p-value of 0.0015.Significance was also tested using a non-parametric correlation (Spearman test = −0.581, p-value of 0.0003) verifying that outliers are not the reason for statistical significance.

Figure 5. IL2RA and PTPN2 genotype independently contribute to decreased IL-2R signaling in controls.

PBMC were stained as in Figure 1. The frequency of pSTAT5 of CD25^hi T cells in control subjects were stratified by PTPN2rs1893217 and IL2RArs2104286 haplotype. The mean pSTAT5 response is indicated for each group. Statistical significance between rs2104286 haplotypes within groups defined by PTPN2 genotype was determined using a Mann Whitney test. When analyzed as covariates in PLINK, age and gender did not significantly contribute to this phenotype (p = 0.4459 and p = 0.4954, respectively) and there was no evidence for interaction between IL2RA and PTPN2 genotype (p = 0.7688). *denotes the risk haplotype determined by looking at variation at the rs21042856 allele while holding the rs12722495 allele constant at T/T and the rs11594656 allele constant at T/T.

Figure 6. Factors beyond IL2RA and PTPN2 variants contribute to reduced IL2R signaling in T1D and MS subjects.

(A) T1D and (B) MS subjects carrying the protective IL2RArs2104286 haplotype and the protective PTPN2rs1893217 SNP were compared to genotype, age and ethnicity matched controls. T1D subjects and matching controls were stimulated with 100 IU/ml IL-2 for 10min while MS samples and matching controls were stimulated with 25 IU/ml IL-2 for 20min. The mean pSTAT5 response is indicated for each group. Statistical significance was determined using a Mann Whitney test. *denotes the risk haplotype determined by looking at variation at the rs21042856 allele while holding the rs12722495 allele constant at T/T and the rs11594656 allele constant at T/T.