Identification, Isolation, and Culture of HLA-A2-Specific B Lymphocytes Using MHC Class I Tetramers

Abstract

Characterizing the individual B cells that participate in the production of anti-HLA Abs requires isolation and culture of these cells and a suitable assay for detection of Abs produced in these B cell cultures. We previously showed that B cell precursors, programmed for anti-HLA Ab secretion, are present at measurable frequencies in peripheral blood of women immunized by pregnancy. In this study, we show that tetrameric HLA-A2, although designed for characterization of CTLs, provides a suitable affinity ligand for isolation of allospecific B cells, which subsequently can be induced to produce HLA-A2 Ab in a CD40-driven culture system. The validity of this concept was established by assaying human hybridomas, producing anti-HLA Abs, for specific tetrameric HLA-A2 binding. The availability of anti-HLA Ab-producing B cell cultures that are established without immortalization will be of value when T-B cell interaction is studied at an alloantigen-specific level.

Impact of Peptides on the Recognition of HLA Class I Molecules by Human HLA Antibodies

“…The findings of these two groups may have been fortuitous, as our present observation of five differentially HLA-A2-reactive human mAbs, which were derived from three different immune donors, indicates that peptide selectivity of HLA Abs is no isolated incident. This is further supported by preliminary data demonstrating that more than one-half of the HLA Abs present in supernatants of monoclonally seeded B cells of donor ROU, cultured after tetrameric HLA-A2-guided isolation (25), were peptide selective. (C. Eijsink and A. Mulder, unpublished observations).…”

Two Novel Assays of Alloantibody-Secreting Cells Demonstrating Resistance to Desensitization With IVIG and rATG

“…allo-specific B cells from peripheral blood (30,31). While these studies have yielded important information, we contend that it is likely that they have primarily involved memory B cells and/or other B cell types in peripheral blood that express cell surface immunoglobulin rather than the ASCs responsible for persistent alloantibody production.…”

In situ identification of allospecific B cells using pentamers

“…The middle panels in Figure 1 There are recent reports of MHC or HLA class I tetramers being used to detect allospecific B cells by flow cytometry in mice 2 and in humans. [3][4][5] To our knowledge, this is the first report of pentamers being used for this purpose, and the first time that allospecific B cells have been detected in situ. We view this as a significant advance because it now paves the way for one to ask site-specific questions to obtain spatial and geographical information concerning the sites of alloantibody production and the cellular interactions required for this sensitization in this rodent model.…”

“…Our previous findings suggested that Asp36Tyr is a dominant factor overriding the dose-reducing effects of other known CYP2C9 and VKORC1 markers and contributing as much as 18% to warfarin dose-response variability. 3 We believe that inclusion of the VKORC1 Asp36Tyr marker in the future pharmacogenetic approach to personalized warfarin therapy is important, particularly for prevention of risks of rethrombosis. …”