Richard T. Maziarz

The human HLA specific monoclonal antibody W6/32 recognizes a discontinuous epitope within the α2 domain of murine H-2Db

W6/32 is a monoclonal antibody (mAb) that recognizes a monomorphic antigenic determinant expressed on HLAA, -B, and -C major histocompatibility antigens (Brodsky et al. 1979). Recently, this mAb has been shown to bind to mouse lymphocytes that express the H-2D b antigen (Ivanyi and Van de Meugheuvel 1984). In this study, hybrid H-2 molecules have been used to localize the domain that expresses the epitope recognized by W6/32 on H-2D b. A number of L-cell lines [derived from the C3H mouse (H-2k)] that have been transfected with either human or mouse class I MHC genes were used in this study (Allen et al. 1984, Maziarz et al. 1985). Transformed L cells that expressed either the HLA-A2 antigens or the H-2D b antigens bound the W6/32 mAb while another transformant that expressed H-2K b did not (Fig. 1). This is consistent with previous observations (Ivanyi and Van de Meugheuvel 1984). In addition, transformants that expressed H-2L d or H-2D d antigens also did not bind the W6/32 mAb (data not shown). Previously, the antigenic determinant defined by W6/32 had been shown to be located within the HLA domains al and a2 rather than in or3 by the use ofinterspecies hybrid MHC molecules (Maziarz et al. 1985, Engelhard et al. 1985). Similarly, hybrid class I genes have been constructed between the H-2D b and H-2K b genomic clones and have been expressed in L cells (Allen et al. 1984). As shown in Figure 2, W6/32 binding was equivalent to the binding of the H-2Db-specific mAb 28-8-6S on line 6-2 (which expresses the al and ~2 domains of H-2D b and the cd domain of H-2Kb; Fig. 2D) but did not bind to the reciprocal protein expressed on line 5-6 (which expresses the od and o~2 domains of H-2Kb; Fig. 2B). Further localization of the antigenic determinant recognized by W6/32 to the c~2 domain was demonstrated by the binding of the mAb

Regulation of cytotoxic T lymphocyte-mediated graft rejection following bone marrow transplantation.

Cytotoxic T lymphocytes have been implicated as the effector cell mediating graft rejection following human allogeneic bone marrow transplantation. We have studied a BMT patient who rejected haploidentical T cell-depleted marrow. In vitro studies demonstrated that the circulating lymphocytes were CD3+ and CD8+, of recipient origin, and exhibited selective cytotoxicity against donor-specific class I major histocompatibility complex antigens. Cytotoxicity was inhibited by monoclonal antibodies directed against CD3, CD8, CD2, and lymphocyte function-associated antigen-1 on the T cell, and against MHC class I proteins on the target cell. Furthermore, these circulating cells inhibited the in vitro growth and differentiation of enriched donor bone marrow progenitor cells, an inhibition that was partially reversed by anti-CD3 MAb. Donor-specific recipient-derived CTL may mediate resistance to engraftment, and CTL activity may be inhibited by a number of MAb. The implications of these findings for host preparation and treatment are discussed.

Distinct effects of interferon-γ and MHC class I surface antigen levels on resistance of the K562 tumor cell line to natural killer-mediated lysis

Various investigators have examined the relationship between tumor cell susceptibility to natural killer (NK) cell lysis and the expression of HLA class I antigens on the tumor cell. There is controversy as to whether or not an inverse relationship exists, and if so, the basis of the relationship between these two phenomena remains undefined. To address these questions, the genomic clones for two HLA antigens were transfected into the erythroleukemia cell line K562, a cell line that is used as the standard to assess human NK and major histocompatibility complex (MHC) nonrestricted cytolysis. Susceptibility to NK lysis was not affected by the de novo expression of HLA antigens on the K562 after DNA mediated gene transfer. Interferon-gamma (IFN-gamma) treatment of K562 induced levels of MHC class I antigen surface expression comparable to those found on the transfected cells; however, the IFN-gamma-treated cells were resistant to NK lysis. When very high levels of surface HLA antigens were induced on the transfectants, a potential effect of class I MHC expression on K562 lysis could be discerned that was distinct from the resistance to NK lysis induced by IFN-gamma-treatment.

Reversal of Infection with Mycobacterium avium intracellulare by Treatment with Alpha-Interferon in a Patient with Hairy Cell Leukemia

A patient with debilitating hairy cell leukemia and documented Mycobacterium avium intracellulare infection unresponsive to standard antituberculous therapy who was treated with alpha-interferon is described. A rapid clinical response with correction of underlying pancytopenia and eradication of the atypical mycobacteria infection was found. No deleterious effects from alpha-interferon therapy were found. The associated resolution of anergy and the sterilization of bone marrow suggest that the reversal of host cellular immune defects led to the eventual control of this patients infection.

The regulation of exogenous and endogenous class I MHC genes in a human tumor cell line, K562

Previous studies have implied the existence of a trans-dominant intracellular repressor able to down-regulate the expression of the entire family of class I MHC genes in the genome of the K562 erythroleukemia cell line. This study demonstrates, however, that the transfection of human or murine class I genes into K562 cells leads to the cell surface expression of the transfected MHC gene product in all situations, even when several kilobases of 5 flanking sequence were included in the transfected genes. The endogenous cellular class I MHC genes remained repressed in the transfected cells. These findings suggest that repression of class I MHC gene expression in K562 may not be mediated predominantly by a trans-dominant repressor of MHC gene expression; rather, other more complex regulatory influences might exist.

β2-microglobulin from serum associates with several class I antigens expressed on the surface of mouse L-cells

Abstract Bovine β 2 -microglobulin (β2-m) present in fetal calf serum (FCS) is able to replace endogenous β2-m associated with several class I antigens from human and mouse cells maintained in culture [Bernabeu et al . (1984) Nature, Lond . 308 , 642–645]. Here we show that human HLA-A2 and HLA-B7, as well as mouse H-2L d and H-2D d heavy chains expressed after gene transfer in mouse L-cells, associate to a large extent with bovine β2-m. We also demonstrate that bovine β2-m associated with the endogenous H-2K k /D k heavy chains generates an antibody response when L-cells are injected into syngeneic C3H mice.

Viral restricted cytolytic T lymphocyte recognition of hybrid human-murine class I histocompatibility antigens

Hybrid human-murine major histocompatibility antigens have been constructed and expressed on the surface of both human RD and murine L cell lines after DNA mediated gene transfer. These antigens linked the polymorphic domains (alpha 1 and alpha 2) of H-2Kb and the carboxy-terminal domains (alpha 3, transmembrane, and intracellular) of HLA-A2. Previously we demonstrated that these antigens were serologically intact and were recognized by allospecific cytolytic T lymphocytes. However, the cell lines expressing the hybrid antigen were less well lysed than the native H-2Kb expressing cell lines. In this study, we extend these observations and demonstrate that virally restricted cytolytic T lymphocytes specific for vesicular stomatitis virus and for Sendai virus can recognize cell lines expressing the hybrid antigen, whether expressed on murine (L cell) or human (RD cell) lines. Furthermore, the data show a profound influence by the carboxy-terminal domains upon the polymorphic T-cell restricting epitopes.