HLA-DQA1 and HLA-DQB1 Alleles, Conferring Susceptibility to Celiac Disease and Type 1 Diabetes, are More Expressed Than Non-Predisposing Alleles and are Coordinately Regulated

Abstract

HLA DQA1*05 and DQB1*02 alleles encoding the DQ2.5 molecule and HLA DQA1*03 and DQB1*03 alleles encoding DQ8 molecules are strongly associated with celiac disease (CD) and type 1 diabetes (T1D), two common autoimmune diseases (AD). We previously demonstrated that DQ2.5 genes showed a higher expression with respect to non-CD associated alleles in heterozygous DQ2.5 positive (HLA DR1/DR3) antigen presenting cells (APC) of CD patients. This differential expression affected the level of the encoded DQ2.5 molecules on the APC surface and established the strength of gluten-specific CD4⁺ T cells response. Here, we expanded the expression analysis of risk alleles in patients affected by T1D or by T1D and CD comorbidity. In agreement with previous findings, we found that DQ2.5 and DQ8 risk alleles are more expressed than non-associated alleles also in T1D patients and favor the self-antigen presentation. To investigate the mechanism causing the high expression of risk alleles, we focused on HLA DQA1*05 and DQB1*02 alleles and, by ectopic expression of a single mRNA, we modified the quantitative equilibrium among the two transcripts. After transfection of DR7/DR14 B-LCL with HLA-DQA1*05 cDNA, we observed an overexpression of the endogenous DQB1*02 allele. The DQ2.5 heterodimer synthesized was functional and able to present gluten antigens to cognate CD4⁺ T cells. Our results indicated that the high expression of alpha and beta transcripts, encoding for the DQ2.5 heterodimeric molecules, was strictly coordinated by a mechanism acting at a transcriptional level. These findings suggested that, in addition to the predisposing HLA-DQ genotype, also the expression of risk alleles contributed to the establishment of autoimmunity.

Keywords: autoimmunity; expression; regulation; risk genes.

Figure 1

Expression of DQB1 and DQA1 genes. The gene expression is showed as percentages of the total DQA1 and DQB1 transcripts for each APC. Panel A and C show the expression of DQA1 alleles in B-LCL and PBMC, respectively; panel B and D show the expression of DQB1 alleles in B-LCL and PBMC, respectively. The APC are grouped by their genotypes; the significance, among the expression values of two alleles, was calculated for each patient and for the group with at least two samples (* p < 0.05, ** p < 0.005).

Figure 1

Expression of DQB1 and DQA1 genes. The gene expression is showed as percentages of the total DQA1 and DQB1 transcripts for each APC. Panel A and C show the expression of DQA1 alleles in B-LCL and PBMC, respectively; panel B and D show the expression of DQB1 alleles in B-LCL and PBMC, respectively. The APC are grouped by their genotypes; the significance, among the expression values of two alleles, was calculated for each patient and for the group with at least two samples (* p < 0.05, ** p < 0.005).

Figure 2

Coregulated expression of DQA1*05 and DQB1*02 CD risk alleles in DR1/DR3 B-LCL#5. This cell line was transfected with pcDNA, pDQA105, pDQA105Δ, and pDQB102 constructs. The surface expression of DQα1*05 (panel A) and DQ1β*02 (panel B) was measured by flow cytometry and reported as mean fluorescence intensity (MFI). The amount of DQA1*05 and DQB1*02 mRNA related to diseases, as well as the DQA1*01 and DQB1*05 mRNA, non-associated to pathologies, was reported as copy number in panel C and as fold variation in panel D. The p-value was calculated respect to cells transfected with empty vectors (* p < 0.05).

Figure 3

Coregulated expression of DQA1*05 and DQB1*02 CD risk alleles in DR7/DR14 B-LCL and nascent RNA. This cell line was transfected with pcDNA and pDQA105 constructs. In panel A we show the MFI assessed by flow cytometry. Panel B reports the fold variation of endogenous DQA1*01, DQA1*02, DQB1*02 and DQB1*05 mRNA. P value was calculated respect to cells transfected with empty vector (* p < 0.05). Panel C shows the quantity of nascent endogenous mRNA following pDQA105 transfection. The left histogram shows the ectopic DQA1*05 mRNA in addition to the endogenous DQA1*01 and DQA1*02 mRNA, as percentage of total DQA1 transcript. The right histogram reported the amount of nascent endogenous DQB1*02 and DQB1*05 mRNA as percentage of total DQB1 transcript.

Figure 4

The antigen presenting properties of B-LCLs transfected with pDQA105. The antigen presenting capability of B-LCL pulsed with immunodominant DQ2.5-γ1 gliadin peptide were measured by assessing the activation of intestinal CD4⁺ T cell line. Panel A shows the IFN-γ production by CD4 stimulated by DR3/DR3 B-LCL#1 and DR1/DR3 B-LCL#5 transfected with pcDNA or pDQA105 constructs. Panel B shows the IFN-γ production by CD4 stimulated with DR3/DR3 B-LCL#1, DR1/DR3 B-LCL#5, and DR7/DR14 B-LCL transfected with pcDNA or pDQA105 constructs.