Ah-Kau Ng

Heterogeneous distribution of the determinants defined by monoclonal antibodies on HLA-A and B antigens bearing molecules.

The monoclonal antibody (MoAb) 6/31, an IgG2a, and the MoAb Cr-1, an IgGl, identify determinants expressed on the heavy chain of subpopulations of HLA-A and B antigens while the MoAb NAMB-1, an IgGl, reacts with human 2-microglobulin (2-m). The MoAb 6/31 and CR-1 reacted with lymphocytes from all of the 20 HLA-typed donors and the 6 homozygous lymphoid cell lines tested, indicating that the corresponding determinants are different from those defining the conventional serological polymorphism. The distribution of the determinants recognized by the MoAb 6/31 and CR-1 on molecules carrying HLA-A and B allospecificites was investigated by incubating NP-40 extracts of cultured lymphoid cells with insolubilized monoclonal antibodies and then measuring the level of HLA-A and B antigens in the supernatant: these studies showed that the determinant defined by the MoAb CR-1 is expressed on all HLA-A and B allospecificities tested, while that defined by MoAb 6/31 is detectable only on some of the HLA-A and B allospecificities. Inhibition-binding assays with radiolabeled monoclonal antibodies and Fab2-blocking assays indicate that the antigenic determinant identified by the MoAb CR-1 is spatially close to those recognized by the MoAb 6/31 and by the MoAb NAMB-1; the latter two antibodies react with spatially distant antigenic determinants. Furthermore, Pab2-blocking assays indicate that the determinants recognized by each monoclonal antibody is spatially close to the conventional serologically defined allotypic determinants only on some of the HLA-A and B allospecificities tested, and that sera with the same specificity may recognize different determinants on a given HLA-A and B alloantigen.

A late-differentiation antigen associated with the helper inducer function of human T cells

T lymphocytes possessing helper function produce soluble factors that greatly augment B-cell proliferation and differentiation into antibody-secreting cells1. In humans the subset of T lymphocytes bearing the T4 surface antigen comprises most of the cells that display helper activity and recognize class II antigens of the major histocompatibility complex (MHC), while the subset bearing the T8 antigen comprises T cells recognizing class I MHC antigens and exhibiting cytotoxic or suppressor function1–3. Monoclonal antibodies to T4 or T8 greatly inhibit the cognitive and effector function of cells with the corresponding phenotype1–4. This function/phenotype correlation is not absolute, however, for there are many examples of T8-positive clones that recognize MHC class II antigens and have helper activity, as well as of T4-positive clones with suppressor or cytotoxic function5–9. Recently a family of cell-surface neoantigens, which might be relevant to T-cell function and which are present on activated but not on resting T lymphocytes, has been identified in mouse and humans using monoclonal antibodies10. Some of these antibodies block the cytolytic activity of alloreactive T-cell clones, suggesting the possible involvement of such molecules in the activation of cytotoxic T-cell clones or in the lytic process itself10–13. We now describe a similar late-differentiation antigen (LDA1) that is expressed by human T lymphocytes only following activation and is recognized by a monoclonal antibody that inhibits the antibody-inducing helper function of T lymphocytes.

ADCC of Cultured Human Melanoma Cells: Analysis with Monoclonal Antibodies to Human Melanoma-associated Antigens

Four monoclonal antibodies to human melanoma‐associated antigens (MAA) were found to be able to mediate specific cell‐dependent lysis (ADCC) of cultured human melanoma cells. The extent of specific lysis of melanoma cells was influenced by the effector to target cell ratio, the amount of antibody added to the reaction mixture, and the incubation time. Cultured melanoma cells treated for 16 h with puromycin or cycloheximide (final concentration, 1.0 μg/ml) displayed increased Susceptibility to ADCC even though the cell surface expression of MAA was not changed. On the other hand, treatment of melanoma cells with tunicamycin (final concentration, 1.0 μg/ml) reduced the expression of MAA but did not affect susceptibility to ADCC. These findings suggest that other properties of the target cell membrane besides antigen density play a role in the outcome of ADCC reaction.

Serological, functional, and immunochemical characterization of a monoclonal antibody (MoAb Q2/70) to human Ia-like antigens

Serological and immunochemical studies showed that monoclonal antibody Q2/70 (MoAb Q2/70), produced by the hybridoma technique, is specific for human Ia-like antigens. This antibody recognizes an antigenic determinant which is different from those defining the serologic polymorphism of Ia-like antigens, and is expressed on subsets of human Ia-like molecules and on lymphoid cells from other species. MoAb Q2/70 inhibits unidirectional MLRs* between allogenic human lymphocytes, but not between murine and human lymphocytes. In ADCC* assays. MoAb Q2/70 mediates lysis of cultured human B lymphoid cells RPMI 4098, effected by murine splenocytes. The antibody is suitable to isolate immunologically functional B lymphocytes from human peripheral blood.

Ontogeny of human Ia antigens

Abstract Indirect immunofluorescence (IIP) staining of tissues from human fetuses (ages ranging from 8 to 32 weeks of intrauterine life) with monoclonal antibodies (MoAb) to monomorphic determinants of Ia antigens and HLA-A,B,C antigens has shown that both types of antigens are already detectable in tissues of 8-week-old fetuses. Ia antigens and HLA-A,B,C antigens reach their almost-complete tissue distribution after 32 and 24 weeks of intrauterine life, respectively. The structure of Ia antigens synthesized by fetal thymus cells is similar to that of B-lymphoid cell-derived Ia antigens. Ia antigen-bearing thymic fetal cells can stimulate allogeneic lymphocytes in mixed lymphocyte reactions (MLRs). These reactions are blocked by monoclonal antibodies to monomorphic determinants of human Ia antigens and of HLA-A,B, antigens.

Isolation of Ia-like antigen-bearing cells from human peripheral lymphocytes through the use of a monoclonal antibody to framework determinants of Ia-like antigens.

Abstract A non-complement fixing monoclonal antibody (Q2/70) to framework determinants of human Ia-like antigens was used to develop a method for isolating Ia-like antigen bearing, i.e., Ia-like (+) cells and cells lacking these antigens, i.e. Ia-like (−), from human peripheral blood lymphocytes (PBL). The method was based on sensitization of PBL with the antibody Q2/70, followed by rosetting with sheep (ShE) coated with purified rabbit anti-mouse Ig antibodies, differential centrifugation on a Ficoll-Hypaque gradient, and finally recovery of Ia-like (+) and Ia-like (−) cells from the bottom and the interface of the gradient respectively. Marker analysis of the two cell subpopulations showed that the majority of the bottom cell fraction were Ia-like (+) and carried B cell markers such as membrane bound immunoglobulins (MbIg) and C3 receptors. On the other hand, the majority of the interface cell fraction were Ia-like (−) and carried T cell markers such as receptors for 2-aminoethylisothiouronium treated sheep erythrocytes (AETShE) and goat erythrocytes (GoE). Serological and functional studies showed that the Ia-like (+) cells (1) were useful targets in complement mediated cytotoxicity assays for HLA-DR typing; (2) served as stimulator but not as responder in unidirectional mixed lymphocyte reactions (MLRs); (3) did not display lytic activity in natural killer (NK) cell cytotoxicity and in antibody dependent cellular cytotoxicity (ADCC); and (4) proliferated in response to pokeweed mitogen (PWM) stimulation in the presence of helper T cells. On the other hand, the Ia-like (−) cells (1) responded to but failed to stimulate allogeneic lymphocytes in the MLRs; (2) were highly active in NK and ADCC assays; and (3) provided helper activity in PWM stimulation of purified B cells.

Effect of Tunicamycin on the Assembly and Antigenicity of HLA Antigens: Analysis with Monoclonal Antibodies

The effect of glycosylation on the assembly and antigenicity of HLA antigens was investigated by examining HLA antigens synthesized in the presence of the antibiotic tunicamycin, an inhibitor of asparagine‐linked oligosaccharide addition, with monoclonal antibodies specific for a variety of antigenic determinants. The monoclonal antibody Q5/13 reactive with a determinant expressed on the β chain of human Ia‐like antigens immunoprecipitated α and β subunits with reduced apparent molecular weights from tunicamycin‐treated cells, indicating that glycosylation is not required for association of the Ia‐like antigen α and β subunits. Immunoprecipitation of HLA‐A,B,C antigens from tunicamycin‐treated cells with four monoclonal antibodies specific for the heavy chain and one specific for β2‐microglobulin showed that the heavy‐chain determinant detected by the antibody Q6/64 is absent from the non‐glycosylated molecule. This is the first demonstration that carbohydrate addition during biosynthesis affects the protein conformation of the HLA‐A,B,C heavy chain.

An antiglobulin microcytotoxicity assay to analyze non-complement fixing monoclonal antibodies to human histocompatibility antigens.

An antiglobulin microcytotoxicity assay has been used to analyze non-complement fixing monoclonal antibodies to human histocompatibility antigens. The assay utilizes methodology similar to that of the widely used microcytotoxicity assay for HLA typing, requires low numbers of target cells and is suitable to test large numbers of samples. The sensitivity of the assay is influenced by the anti-mouse Ig antiserum used, by the sequence of addition of the various reagents and by the incubation time. The assay is suitable to screen supernatants of clones derived from hybridization experiments and to characterize the serological specificity of anti-HLA monoclonal antibodies.

Immunochemical and functional analysis of Ia-like antigens on human monocyte-macrophages with monoclonal antibodies.

Abstract The distribution, structural profile and functional properties of Ia-like antigens synthesized by human monocyte-macrophages have been analyzed using monoclonal antibodies to common determinants of these antigens. Up to 45 and 70%- of monocyte-macrophages isolated from the fluid of blisters induced with cantharidin and from peripheral blood, respectively, react with monoclonal antibodies to human Ia-like antigens. The level of Ia-like antigens on monocytes-macrophages appears to be similar to that on cultured B lymphoid cells. Monoclonal antibodies to common determinants of Ia-like antigens specifically block antigen presentation by monocyte-macrophages to T lymphocytes as well as proliferative response of T lymphocytes to autologous and allogeneic monocytes-macrophages. These results indicate that common determinants of Ia-like antigens play a role in the interaction of monocytes-macrophages with T lymphocytes.

Immunochemical analysis of anti-HLA-A2, HLA-A3, and HLA-B27 xenoantisera elicited with hybrids between human and murine cells

Rabbits were immunized with hybrids constructed with human and murine cells. Serological and immunochemical studies showed that xenoantiserum 1595 is operationally specific for HLA-A2, xenoantisera 0806 and 0746 for HLA-A3 and xenoantiserum 0745 for HLA-1327. The Fab2 blocking assay suggests a spatial relationship between allotypic determinants recognized by the xenoantisera and those reacting with the HLA-A, B-specific monoclonal antibodies (MoAb) Q1/28,6/3 1, Q6/64, and CR-1 and the anti-β2-microglobulin (β2-μ) MoAb NAMB-1.