Michael B. Widmer

Genetic and Immunological Complexity of Major Histocompatibility Regions

There are genetic differences within the major histocompatibility complex of the mouse which lead to skin graft rejection but which cannot be detected serologically. When confronted with these differences on allogeneic cells, lymphocytes proliferate in vitro. In other cases, in vitro lymphocyte proliferation but no skin graft rejection is associated with loci that are linked to but genetically separable from the loci controlling the serologically defined antigens.

Mixed lymphocyte culture reactivity and H-2 histocompatibility loci differences

The relationship between the major histocompatibility systems as defined serologically and by the mixed leukocyte culture (MLC) test is one which has received attention in both man (1) and mouse (2). In man, studies within families suggest that cells of siblings who inherit the same HL-A chromosomes from their parents and thus are serologically identical at HL-A do not stimulate in the MLC test. Cells of siblings who inherit different HL-A chromosomes do stimulate. This, combined with the finding that cells of all of several hundred unrelated pairs stimulate in the MLC test, suggests that a single genetic system controls reactivity in MLC, that this system is highly polymorphic and either the same as that coding for the HL-A antigens or very closely linked to the HL-A (1). Similarly, differences at the H-2 system in the mouse result in MLC activation (2). The H-2 system in the mouse can be divided into two separate regions, H-2K and H-2D (3). Recently Rychlikova et al. (4) studied a series of mouse recombinant strains that differed only for the H-2D region or only for the H-2K region and other inbred strains that differed at both the H-2D and the H-2K regions. Their findings suggested that differences for H-2K alone or for both H-2K and H-2D resulted in MLC activation. Differences for the H-2D region alone. however, did not result in MLC activation in their study. We have repeated these studies using a number of recombinant strains tested in

Analysis of cloned T cell function. II: Differential blockade of various cloned T cell functions by cyclosporine

Cyclosporine has profound suppressive effects on selected in vitro functions of cloned T lymphocytes. Cyclosporine inhibits the antigen-induced proliferation of the helper T cell clone 12-11. The effective dose required to reduce this response by 50% (ED50) is 28 ng/ml. In contrast, the proliferation of clone 12-11 induced by exogenous growth factors in secondary mixed lymphocyte culture supernatant (2 degrees MLC SN), is relatively insensitive to cyclosporine (ED50 = 4600 ng/ml). Furthermore, cyclosporine abrogates both antigen-induced and mitogen-induced secretion of lymphokines by clone 12-11, indicating that cloned helper T cell function is sensitive to cyclosporine even when interactions between specific alloantigens and their cell surface receptors are bypassed with mitogen. The suppressive effect of cyclosporine is not limited to helper T cell clones. The cytolytic T lymphocyte (CTL) clone 5MD2-2 is also sensitive to cyclosporine. Again, cyclosporine (100 ng/ml) blocks the antigen-driven, but not the exogenous lymphokine-driven, component of clone 5MD2-2 proliferation. This suppression does not result from the occlusion of antigen receptors or from antigen deformation by cyclosporine, because clone 5MD2-2 remains capable of antigen-specific cytolysis in the presence of cyclosporine concentrations that can suppress its proliferation. Finally, the ability of clone 5MD2-2 to remove IL-2 activity from culture media, a function that is significantly enhanced by contact with specific alloantigen, is not influenced by suppressive cyclosporine concentrations.

Lymphocyte reactivity in vitro. VII. The effect of polymorphonuclear leukocytes on lymphocyte response.

SUMMARY One-way mixed leukocyte culture (MLC) tests were performed using, in all cases, mitomycin C-treated stimulating cells from healthy university students. Responding cells from four groups were tested: hospitalized patients with malignancy, age- and sex-matched hospitalized patients without known malignancy, age- and sex-matched nonhospitalized controls, and students who were younger than the persons in the other three groups. Whereas approximately 30% of the variation in response was unaccounted for by known variables, the great majority of the remaining variation could be accounted for by the number of polymorphonuclear leukocytes present in the culture. These results suggest that quantitation of MLC tests may be better after removal of polymorphonuclear leukocytes from the culture—a step we have now instituted in our laboratory.

Delayed rejection of soluble tumor necrosis factor receptor-secreting tumor allografts.

BACKGROUND Exogenous soluble tumor necrosis factor receptor (TNFR) has been shown to be an effective immunosuppressant. It has yet to be tested whether tissues secreting soluble TNFR, when transplanted into a foreign host, could locally generate immunosuppression and therefore manifest prolonged survival. METHODS A murine tumor line was transfected with the gene encoding a chimeric protein consisting of the extracellular domain of the human 75-kDa TNFR fused to the Fc region of the human IgG1 heavy chain. This tumor line was then injected into allogeneic recipients. RESULTS Transfected tumor cells were shown to secrete soluble TNFR. When transplanted into minor histocompatibility antigen-disparate allogeneic recipients, these tumor cells grew as a solid tumor and resisted rejection, whereas untransfected tumors and interleukin-4 receptor transfectant controls were rejected within 4 weeks. The resistance to rejection could be reversed by coadministration of an anti-TNFR monoclonal antibody. CONCLUSIONS Prolongation of graft survival can be achieved by genetically altering transplanted tissue to secrete soluble cytokine receptors.

Multi-Locus control of MLC reactions in rhesus monkeys

The genetic basis for MLC reactivity in rhesus monkeys was further investigated. Cells from anintra-RhLA recombinant monkey were MLC reactive with those of an SD-identical sib and with each of two sets of homozygous typing cells carrying either paternal haplotype. Also, cells from a pair ofRhLA- identical sibs reacted in MLC with each other, as well as with three genotypically different siblings. This excludes control of MLC reactivity by the conventionalD locus only. Thus the results selected for presentation provide formal evidence for the existence of at least one additional MLC locus, namedD′. The possibility thatD′ exerts its influence on MLC reactivity only after alloimmunization is discussed.

Antigen specificity and helper characteristics of antigen-driven helper-cell-independent cytolytic T lymphocytes

Antigen-driven, helper-cell-independent cytolytic T lymphocytes, which proliferate in response to antigenic stimulation independently of exogenous lymphokines, were characterized with regard to antigen specificity and helper factor production. Mapping data indicate that the determinants recognized by such cells for cytotoxicity and proliferation induction are identical. In addition, the cells were shown to function like helper cells in that they secrete soluble IL-2-like factor upon stimulation with the sensitizing antigens. The potential importance of this cell type in the immune response is discussed.

Mediation of cellular recruitment in vivo by functionally distinct T cell clones.

The ability of functionally distinct alloreactive T cell clones to mediate celliular recruitment in vivo was examined in a modified sponge matrix allograft model. Changes in cellular recruitment to paired healed s.c urethane sponge grafts injected with cytolytic T lymphocyte (CTL), helper, or helper-independent CTL clones, or bulk resting mlixed leukocyte culture (MLC) cells, and allogeneic or syngeneic blasts were studied. Injection of indium-111-labeled unsensitized cells i.v. was used to assess cellular recruitment to the graft site. All three aloreactive T cell clones and bulk MLC cells mediated preferential recruitment of circulating labeled cells when injected with allogeneic cells. The helper clone mediated significantly greater recruitment than the CTL clone. These results confirm at the clonal level our previous observations that populations of allosensitized cells enriched for either cytolytic or noncytolytic T lymphocytes can mediate cellular recruitment in vivo and extends them to include helper-cell-independent cytolytic T lymphocytes.

Lymphocyte-Defined Histocompatibility Differences as a Model for Tumor Antigens

Reactivity of lymphocytes in mixed leukocyte culture (MLC) [2, 8] for the most part reflects disparity for the major histocompatibility complex (MHC). The MHC contains loci which control the serologically defined antigens (H-2 of mouse or HL-A of man). However, evidence in the mouse points to the existence of other MHC differences which cannot be detected serologically, but which can lead to MLC activation, and in some cases to graft rejection. This evidence has been extensively reviewed in previous publications [4, 5] and will only be summarized here; the possible relationship of these findings to the detection and definition of tumor antigens will be discussed.