Differential expression of predisposing HLA-DQ2.5 alleles in DR5/DR7 celiac disease patients affects the pathological immune response to gluten

Abstract

The DR5-DQ7/DR7-DQ2 genotype is very frequent among patients affected by celiac disease (CD), in Europe. This genotype, associated to high risk of CD, carries the HLA-DQA1*05 and HLA-DQB1*02 predisposing alleles, in trans configuration. The alleles encode the DQ2.5 heterodimer responsible of gluten peptide presentation on the surface of antigen-presenting cells (APCs), and consequent pathogenic CD4⁺ T cell activation. We demonstrated that DR5/DR7 APCs induce an anti-gluten CD4⁺ T cell response, of comparable intensity to that observed with APCs carrying DR1/DR3 genotype, which risk alleles are in cis configuration. In addition, we showed that DR5/DR7 APCs from celiac patients stimulated an effector CD4⁺ T cell response higher with respect to that induced by DR5/DR7 APCs from healthy subjects. To explain these findings, we assessed the DQ2.5 RNA and protein quantity. We showed that the expression of DQA1*05 and DQB1*02 risk alleles is much higher than the expression of non-CD-associated alleles, in agreement with the previous results obtained with DR1/DR3 genotype. The differential expression of transcripts influences the quantity of DQα1*05 and DQβ1*02 chains and, as consequence, the cell surface density of DQ2.5 heterodimers. Moreover, both RNA and proteins, are more abundant in APCs from celiac patients than controls. Finally, to unravel the mechanism regulating the expression of predisposing DQA1*05 and DQB1*02 alleles, we quantified the new synthetized RNA and found that the differential expression is explained by their transcription rate. Our results confirmed that the strength of antigen-specific CD4⁺ T cell response is mainly determined by the amount of gluten in the diet and provided a new possible approach for a personalized diagnosis and for risk stratification.

Figure 1

Stimulation of gliadin-reactive T cells by B-LCL with different genotypes. (A) Shows a representative experiment of the dose curve IFN-γ response by a DQ2.5-glia-α1,2-specific TCL obtained from celiac gut mucosa. Intestinal cells were stimulated by B-LCL#5 (from a celiac DR1/DR3), B-LCL#23 (from a celiac DR5/DR7 ), B-LCL#29 (from a control DR5/DR7). The APCs were pulsed with DQ2.5-glia-α1,2 peptide at different concentrations (0.01, 0.1, 1, 10 µM). (B) Shows the average of IFN-γ produced by a DQ2.5-glia-α1,2 -specific TCL at the sub-optimal DQ2.5-glia-α1,2 peptide concentration (1 µM). DR1/DR3 B-LCLs histogram represents the result of N = 5 experiments performed using as APCs celiac B-LCL#5 and B-LCL#6; DR5/DR7 B-LCLs histogram shows the result of N = 9 experiments performed using as APCs celiac B-LCL#23, #25, #26 and #27; DR5/DR7 CTR B-LCLs histogram represents the IFN-γ mean value of N = 9 experiments performed with B-LCL#29, #30, #31, #35 from healthy subjects. Table in panel B displays results of statistical analysis performed using the unpaired Student's t-test to compare B-LCLs with different genotypes, as indicated. *p < 0.05 was considered statistically significant.

Figure 2

Surface expression of DQα1*05 and DQβ1*02 chains. (A,C) Show the MFI values of DQα1*05 anti-DQβ1*02 expression on B-LCL surface membrane for each single CD patient (left) and control (right). (B,D) show the MFI mean of DQα1*05 anti-DQβ1*02 chain staining in B-LCLs from all CD patients and healthy controls. (*p < 0.05, **p < 0.005).

Figure 3

Expression level of DQB1* and DQA1* genes in HLA DR5/DR7 B cell lines. The gene expression is showed as percentages of the total DQA1 and DQB1 transcripts for each B-LCL. (A,C) show the expression of DQA1 alleles in B-LCL from patients (left) and healthy controls (right); (B,D) show the mean percentages of DQA1*05 and DQB1*02 mRNA measured in B-LCLs from all CD patients and healthy controls analyzed (*p < 0.05, ** p < 0.005).

Figure 4

Relative expression of the newly synthetized DQB1* and DQA1* transcripts. The amount of new transcribed DQB1* and DQA1* mRNA in B-LCL#27 is shown as percentages of the total transcript. The relative expression of DQA1*05 versus DQA1*02 and DQB1*02 versus DQB1*03 mRNA is reported in panel A and B, respectively.