M.C.J.A. van Eggermond

An HLA-DQ alpha allele identified at DNA and protein level is strongly associated with celiac disease

An HLA-DQ alpha cDNA probe showed upon hybridization a highly significant discrepancy between the RFLP of celiac disease patients and healthy controls. The 4.0-kb Bgl II restriction fragment was present in 97% of celiac disease patients (n = 30), compared to 56% in a healthy control population (n = 72) (RR = 14.9; p less than 0.0005). At the product level all celiac disease patients tested to date have one DQ alpha chain in common, designated HLA-DQ alpha 2.3, which is associated with the 4.0-kb Bgl II fragment. This HLA-DQ alpha allele identified at the DNA level and product level seems to be a better marker for genetic susceptibility to develop celiac disease than those available to date.

TCR V alpha- and V beta-gene segment use in T-cell subcultures derived from a type-III bare lymphocyte syndrome patient deficient in MHC class-II expression.

Previously, we and others have shown that MHC class-II deficient humans have greatly reduced numbers of CD4+CD8– peripheral T cells. These type-III Bare Lymphocyte Syndrome patients lack MHC class-II and have an impaired MHC class-I antigen expression. In this study, we analyzed the impact of the MHC class-II deficient environment on the TCR V-gene segment usage in this reduced CD4+CD8– T-cell subset. For these studies, we employed TcR V-region-specific monoclonal antibodies (mAbs) and a semiquantitative PCR technique with V α and V ß amplimers, specific for each of the most known V α- and V ß;-gene region families. The results of our studies demonstrate that some of the V α-gene segments are used less frequent in the CD4+CD8– T-cell subset of the patient, whereas the majority of the TCR V α- and V ß-gene segments investigated were used with similar frequencies in both subsets in the type-III Bare Lymphocyte Syndrome patient compared to healthy control family members. Interestingly, the frequency of TcR V α12 transcripts was greatly diminished in the patient, both in the CD4+CD8– as well as in the CD4–CD8+ compartment, whereas this gene segment could easily be detected in the healthy family controls. On the basis of the results obtained in this study, it is concluded that within the reduced CD4+CD8– T-cell subset of this patient, most of the TCR V-gene segments tested for are employed. However, a skewing in the usage frequency of some of the V α-gene segments toward the CD4–CD8+ T-cell subset was noticeable in the MHC class-II deficient patient that differed from those observed in the healthy family controls.

Unambiguous typing for HLA-DQ TA10 and 2B3 specificities using specific oligonucleotide probes.

Oligonucleotide probes specific for the serologically defined TA10 and 2B3 specificities were selected based on a comparison of the available HLA-DQ beta sequences. Panel and family segregation studies confirm a complete correlation between the reactivities of the selected probes and the TA10/IIB3 antibodies. The Glu residue at position 45 of the HLA-DQ beta chain is specific for the TA10 determinants, and a DQ beta Gly-Val-Tyr sequence is found at position 45-47 for all 2B3-positive DQ beta chains.

Analysis of the peripheral T-cell compartment in the MHC class II deficiency syndrome.

To analyse the impact of lack of MHC class II expression on the composition of the peripheral T-cell compartment in man, the expression characteristics of several membrane antigens were examined on peripheral blood lymphocytes (PBL) and cultured T cells derived from an MHC-class-II-deficient patient. No MHC class II expression could be detected on either PBL or activated T cells. Moreover, the expression of MHC class I was reduced both on PBL and in vitro activated T cells compared to the healthy control. However, the reduced expression of CD26 observed on the PBL of the patient was restored after in vitro expansion. Despite the presumably class-II-deficient thymic environment, a distinct but reduced single CD4+ T-cell population was observed in the PBL of the patient. After in vitro expansion, the percentage of CD4+ cells dropped even further, most likely due to a proliferative disadvantage, compared to the single CD8+ T-cell population. However, proliferation analysis showed that T-cell activation via the TcR/CD3 pathway is not affected by the MHC class II deficiency.

HLA class II DNA analysis by RFLP reveals novel class II polymorphism

Polymorphism of the HLA-D/DR region has been defined by serologic and cellular methods. Additionally, protein and DNA analyses not only confirmed and refined the definition of the established polymorphisms but also revealed further polymorphisms for which no serologic or cellular correlate are known (yet). To study these in more detail, we analyzed the banding patterns obtained from Southern blot hybridizations with DR beta and DQ alpha cDNA probes. Specific fragments reflecting already defined polymorphisms could be identified. A refined HLA-D/DR definition based upon the presence of DNA subtypes could be introduced. Fragments have also been identified that are associated with the DR/Dw specificities. Moreover, individuals with different DR types may also share fragments in hybridization assays. These shared hybridizing fragments (SHFs) are, for instance, found in individuals typed as DR4, DR5, DR7, and DRw8. In total, 20 SHFs were found using two restriction enzymes and the DR beta and DQ alpha cDNA probes. Some of these SHFs correlate with antigenic determinants defined by broad reacting alloantisera, such as DRw52, but for 14 of these SHFs, no serologic equivalent has been found so far. Thus, SHFs reflect a conservation at the DNA level of the HLA class II region, which suggests that the polymorphic class II genes may be more conserved than previously thought. The possible biologic implications of conserved sequences in the HLA class II genes will be discussed.

10 AN HLA-DQ ALPHA ALLELE IDENTIFIED AT DNA AND PROTEIN LEVEL IS STRONGLY ASSOCIATED WITH COELIAC DISEASE

Susceptibility genes for coeliac disease are associated with certain HLA-DR genotypes. More recent studies also show strong association with some HLA-DQ alleles. We have examined the presence of disease associated HLA-DQ alleles by restriction fragment length polymorphism (RFPL) with the help of a HLA-DQ alpha cDNA probe. This probe showed upon hybridization a highly significant discrepancy between the RFLP of coeliac disease patients and healthy controls. The 4.0 kb. Bgl II restriction fragment was present in 97% of coeliac disease patients (n=30), compared to 56% in a healthy control population (n=72) (RR= 14.9; p<0.0005). This fragment is associated with HLA-DR3, -5, some DRw8, and DRw14Dw16. This was confirmed by the analysis of the panel of 72 homozygous typing cells obtained from the Xth International Workshop on Histocompatibility Testing.At the product level all coeliac disease patients tested sofar have one DQ alpha chain in common, designated HLA-DQ alpha 2.3, which is associated with the 4.0 kb Bgl II fragment. This HLA-DQ alpha allele identified at the DNA level and product level seems to be a better marker for genetic susceptibility to develop coeliac disease than those available sofar.