Transformation of LMTK- cells with purified HLA class I genes. II. Serologic characterization of HLA-A3 and CW3 molecules.

The expression of two different HLA class I genes was observed after transformation of LMTK- cells. The corresponding class I molecules reacted differentially with monomorphic monoclonal antibodies (m.Ab). Absorption and elution studies of the human alloantibodies reacting with the transformed cells and cellular radioimmunoassay of these cells with polymorphic m.Ab resulted in the identification of HLA-A3 and CW3 molecules. These transformed cells were used to immunize C3H mice and induce the production of xenogeneic antisera, which, following absorption, showed polymorphic reactivity with human cells, suggesting that some of these sera could be used as typing reagents.

Affinity and inhibitory capacity of T-cell proliferation of monoclonal anti-Ia antibodies.

The rates of dissociation from the surface of Ia-positive spleen cells of three different anti-Ia monoclonal antibodies were evaluated and compared with their inhibitory effects on T-lymphocyte proliferative responses involving the same spleen cell population, either as a source of accessory cells (responses to soluble antigens) or as allogeneic stimulating cells. In general, a direct relationship was observed between the affinity with which these monoclonal antibodies interact with Ia-positive cells and the magnitude of their inhibitory effects, whether soluble antigen- or alloantigen-induced responses were tested. These results indicate that the affinity of monoclonal antibodies is a factor that has to be considered to the same extent as their fine specificity when interpreting the results of inhibitory studies of T-cell responses by anti-Ia monoclonal antibodies.

Analysis of unexpected inhibitions of T lymphocyte proliferation to soluble antigen, alloantigen and mitogen by unfragmented anti-I-Ak or anti-I-E/Ck monoclonal antibodies.

We investigated the capacity of anti-I-Ak and anti-I-E/Ck monoclonal antibodies (m.Ab.) to inhibit T lymphocyte proliferative responses to soluble antigen (Keyhole limpet hemocyanin), alloantigens (H-2 or non-H-2 related) and a mitogen (Concanavalin A). Surprisingly, specific inhibition was observed in all circumstances, and with both anti-I-Ak and anti-I-E/Ck m.Ab., whether the responses tested were I restricted in cell mixing experiments or not. The significance of the inhibition by anti-Ia m.Ab. of non-Ia-restricted responses is still not completely understood. These results, however, strongly suggest that in vitro inhibition by anti-Ia antibodies of T cell proliferative responses does not necessarily indicate I restriction of the presentation to T lymphocytes of the corresponding antigen.

Identification on I-Ak molecules of a functional site recognized by proliferating T-lymphocytes.

The inhibitory capacity of 17 monoclonal antibodies (m.Ab.) specific for the products of the I-Ak subregion was evaluated in proliferative responses of B10.BR T-lymphocytes to GAT, Keyhole limpet hemocyanin, and ovalbumin. Considered in isolation, each m.Ab. mediated inhibitory effects of comparable magnitude on these three different proliferative responses. On the other hand, clear differences were observed when the magnitude of the inhibitory effects was compared from one m.Ab. to another. The m.Ab. were consequently classified as strong or moderate-to-weak inhibitors of T-cell proliferative responses. Evidence was simultaneously gained indicating the following: (a) the determinants recognized by different m.Ab. were expressed on the same molecules; (b) the differences in affinity of the m.Ab. for I-Ak positive cells did not explain their differences in inhibitory capacities; (c) conversely, the inhibitory capacity of each m.Ab. followed its ability to inhibit the cell surface fixation of Ia.17-specific 10-2.16 m.Ab; (d) the strong inhibitory capacity of some m.Ab. was not related to a special ability to modulate cell surface Ia molecules. These results suggest that antigen recognition by T lymphocytes is preferentially restricted by a functional site of the I-Ak molecules related to the Ia.17 and Ia.1 specificities.

KLH-specific, I-E/C-restricted clones of proliferating T lymphocytes.

The recognition of keyhole limpet hemocyanin by a substantial proportion of proliferating clones of murine T lymphocytes was found to be restricted by the I-E/Ck molecule, which is a combinatorial product of genes located in the I-A (Ae) and I-E/C (E alpha) subregions of the murine major histocompatibility complex. The respective roles of the Ae (polymorphic) and E alpha (oligomorphic) gene products in the expression of the structures which are used as restriction elements by these T-cell clones was analyzed by mating parental strains unable to present the antigen and bearing selected Ae and E alpha alleles. Efficient complementation for antigen presentation was found to require the expression by accessory cells of the Aek-gene product, whereas all E alpha allelic molecules were functionally equivalent. These results (a) indicate that the immunoregulatory role of I-region gene products, initially described for molecules selected for their limited number of antigenic epitopes, also applies to complex multiepitopic antigens; (b) illustrate the advantage which results from the diversity of the Ia molecules expressed by accessory cells for the development of potent immune responses; and (c) suggest that a correlation might exist between the degree of polymorphism of a given family of H-2 allelic molecules and their ability to be used as restriction elements for antigen recognition by T lymphocytes.

Cell surface fixation of alloantigen bearing plasma vesicles in the presence of polyethylene glycol.

The fixation of plasma vesicles at the surface of intact mouse spleen or tumor cells was studied in order to introduce the foreign alloantigens of the vesicles into the plasma membrane of these cells. A 3-6-fold increase of fixation of radioiodinated vesicles was obtained when cells and vesicles were incubated in the presence of polyethylene glycol 1500 (PEG 1500). The fixation of vesicles on the surface of cells was demonstrated by scanning electron microscopy. Cells treated with vesicles in the presence of PEG acquired the corresponding membrane alloantigens, as demonstrated by cellular binding radioimmunoassay. However, sensitivity to antibody-dependent lysis was obtained only when vesicle fixation was achieved in the presence of both wheat germ agglutinin and polyethylene glycol. The introduction of foreign alloantigens in the plasma membrane of the treated cells might help to define the functional properties of these molecules.