L cells expressing DQ molecules of the DR3 and DR4 haplotypes: reactivity patterns with mAbs

Abstract

cDNAs coding for the HLA class II DR and DQ alpha and beta chains of the diabetogenic haplotypes DR3 and DR4 were introduced into a mammalian expression vector and transfected into L-cell mouse fibroblasts to produce cells expressing individual human class II molecules. Stable L transfectants were generated expressing each of the DR or DQ isotypes of the cis-encoded alpha and beta chains of the DR3 or DR4 haplotypes, as well as the trans-encoded alpha and beta chains of the DQ molecules of the two haplotypes. However, isotype mismatched combinations (DR alpha/DQ beta or DQ alpha/DR beta) did not result in any stable transfectants. The stable DQ L-cell transfectants obtained, along with homozygous B-cell lines expressing the DQ2 and DQ8 specificities, were tested against a large panel of twentyone anti-HLA class II monoclonal antibodies (mAbs). Their unusual reactivity patterns are described including the failure of most "pan-DQ" mAbs to react with all DQ expressing L-cell transfectants. Interestingly, some mAbs react with certain alpha beta heterodimers expressed on B-LCL but fail to recognize the same heterodimers expressed on the transfectants. This is suggestive of minor structural modifications that class II molecules undergo depending on the cells they are expressed on.

HLA class I peptide polymorphisms contribute to class II DQβ0603:DQα0103 antibody specificity

“…However, transfection of HLA DMA and DMB genes into 721.174(DR3) cells restored the reactivity of the monoclonal antibody with the DR3 protein 15 . Similar observations have been reported with certain anti-DQ monoclonal antibodies failed to recognize the DQ proteins expressed on either mouse L cell or human HeLa cell transfectants most likely due to the lack of appropriate peptides bound to the binding groove of the DQ protein 16 . Patient sera that react to the DQβ0603:DQα0103 heterodimer without cross-reacting with other HLA DQ antigens are frequently observed.…”

Comparative organization and function of the major histocompatibility complex of domesticated cattle

“…Similar results were reported for human DQ transfectants. They are difficult to produce and maintain, and some DQ gene pairs showed altered reactivity with monoclonal antibodies, possibly due to minor structural modifications that were cell line dependent (62).…”

Questionable expression of unstable DQ heterodimer containing HLA‐DQA1*01:07

“…The DQA1*01:07‐ stable cells were transduced with a graded concentration of retrovirus particles that contained pMXs‐IG/ DQB1 . After 48 h of transduction with DQB1 , cytosolic expression of GFP and cell‐surface expression of DQ (in the GFP‐positive cells) were measured by flow cytometry (EPICS‐XL; Beckman Coulter Inc., Fullerton, CA, USA), using monomorphic anti‐HLA class II β mAb (WR18) (MorphoSys AG, Martinsried, Germany) or isotype control (mouse IgG2a [6H3]; Medical & Biological Laboratories, Co. Ltd., Nagoya, Japan) with phycoerythrin‐conjugated anti‐mouse IgG (Rockland Immunochemicals Inc., Pottstown, PA, USA or Southern Biotechnology Associates Inc., Birmingham, AL, USA). The mean fluorescent intensity (MFI) of HLA and GFP was plotted to calculate the increase in the MFI of HLA relative to the MFI of GFP (ΔMHC).…”