Marrie J. Kardol

Human monoclonal HLA antibodies reveal interspecies crossreactive swine MHC class I epitopes relevant for xenotransplantation.

Crossreactivity of anti-HLA antibodies with SLA alleles may limit the use of pig xenografts in some highly sensitized patients. An understanding of the molecular basis for this crossreactivity may allow better selection of xenograft donors. We have tested 68 human monoclonal HLA class I antibodies (mAbs) for reactivity with pig lymphocytes from SLA defined pigs and found nine to be crossreactive. Eight of nine were broadly HLA reactive IgM-mAbs. The putative HLA epitopes for seven mAbs. were conserved in the aminoacid sequence of the SLA alleles studied. The lack of reactivity of a large number of mAbs largely correlated with the absence of the putative epitopes in the SLA alleles studied. We conclude that most patients with anti-HLA class I antibodies should be able to find pig donors lacking SLA antigens that cross react with their antibodies and that many of the crossreacting epitopes can be defined by analysis of shared epitopes in the aminoacid sequence of human and pig MHC antigens.

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

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

MHC class I molecules expressed on cell surfaces are composed of H chain, β2-microglobulin and any of a vast array of peptides. The role of peptide in the recognition of HLA class I by serum HLA Abs is unknown. In this study, the solid-phase assay of a series (n = 11) of HLA-A2-reactive, pregnancy-induced, human mAbs on a panel (n = 12) of recombinant monomeric HLA-A2 molecules, each containing a single peptide, revealed peptide selectivity of the mAbs. The flow cytometry membrane staining intensities on the HLA-A2-transduced cell line K562, caused by these mAbs, correlated with the number of monomer species detected by the mAbs. Flow cytometry staining on HLA-A2-bearing cell lines of a variety of lineages was indicative of tissue selectivity of these HLA-A2 mAbs. This tissue selectivity suggests that the deleterious effect on allografts is confined to alloantibodies recognizing only HLA class I loaded with peptides that are derived from tissue-specific and household proteins. Since Abs that are only reactive with HLA loaded with irrelevant peptides are expected to be harmless toward allografts, the practice of HLA Ab determination on lymphocyte-derived HLA deserves reconsideration.

The Influence of the Workshop Monoclonal Antibodies on CML, AgTR, PLT, ADCC, and NK Cell Activity. Functional Studies with Workshop Antibodies

The introduction of the hybridoma technique (1) has made it possible to produce monoclonal antibodies against functionally different (T-) lymphocyte subsets. The antigens recognized by monoclonal antibodies from the CD4, CD8, and LFA-1 class appear to play a role in the recognition—adhesion of the target/stimulator cell by the effector cell (for review see Ref. 2). The T3 antigen, associated with the T cell receptor (3), is not involved in the recognition—adhesion step but might transmit signals for the lethal hit or mitogenesis. Recently, it has been found that sheep red blood cell receptors, defined by CD2 mAbs, can also play a role in the activation of T cells (4) while earlier reports indicated that the cytolytic activity of cytotoxic T lymphocytes could be partially blocked by CD2 mAbs (5,6).