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A comparison has been made between the alloantibody response evoked by graded doses of cells being allogeneic and semiallogeneic with the host. The responses were measured by hemagglutination and complement-dependent lymphocytotoxicity. In the strain combination of rats used (DA leads to HO), a marked difference in the optimal dose was found after i.v. injection of lymphocytes. Although semiallogeneic cells gave the better antigenic stimulus at lower cell doses (0.3 and 3 X 10(6) cells), allogeneic cells were better at higher cell doses (30 and 150 X 10(6) cells). Immunization experiments with allogeneic and semiallogeneic erythrocytes or mitomycin- or heat-treated lymphocytes indicated that the antigenicity of the cells was not directly related to the cell surface concentration of antigen and was independent on the proliferative capacity of the lymphocytes; it was, however, curtailed by heat treatment of these cells. The 3 X 10(6), but not the 0.3 or 30 X 10(6) dose of semiallogeneic cells primed efficiently for a secondary hemagglutinin and cytotoxic response. I.v. injected 51Cr-labelled allogeneic and semiallogeneic lymphocytes showed different localized patterns. Between 4 and 24 hr after injection allogeneic lymphocytes were apparently more rapidly lost from the recipient lymph nodes. Collectively, these data indicate that the different immunizing properties of i.v. injected allogeneic and semiallogeneic lymphocytes are not simple consequences of the antigen dose transferred, but suggest that different localization patterns might influence the ability of these cells to induce cytotoxic and hemagglutinating alloantibody formation.
By exploiting congenic rat strains (HO.B2 and PVG/c) cell-mediated immune responses against Ag-B antigens alone were measured and compared with responses against (1) non-Ag-B antigens and (2) Ag-B and non-Ag-B antigens in combination. It was confirmed that multiple non-Ag-B antigens provoke prompt first-set skin graft rejections, but are much weaker than Ag-B antigens in stimulating both graft-versus-host (GVH) and cytotoxic activity. No evidence of synergistic interaction was found between anti-Ag-B and anti-non-Ag-B responses either by GVH assay or in the generation of cytotoxic cells. Specific partitioning of cytotoxic cells on antigenic monolayers suggested that cytotoxic cells on antigenic monolayers suggested that cytotoxicity is predominantly directed against Ag-B antigens. The measurements of GVH activity consolidate previous work, which suggested that 4.5 to 12% of nonimmune T cells can respond to each Ag-B determined antigenic complex and eliminate the possibility that most of these cells were responding to non-Ag-B antigens. Two principles for measuring GVH activity were compared: (1) 3H-thymidine incorporation into donor lymphocytes at 24 hr after transfer to irradiated F1 hybrid recipients and (2) the popliteal lymph node assay, which depends on a secondary phase of host cell proliferation.
When an "isolated" single lymph node is challenged with irradiated allogeneic lymphocytes, there is a change in the reactivity of the lymphocytes flowing out of the node when they are cultured in vitro in unidirectional mixed lymphocyte cultures (MLC) against the immunizing lymphocytes. These changes in the reactivity of the recipient lymphocytes are shortlived, follow a set time sequence in relation to the cell traffic changes accompanying the immune response, are the property of small lymphocytes and not blast cells, are exhibited by surface Ig-negative cells, and they are specific for the donor lymphocytes. It is suggested that antigen causes the selective retention of antigen-specific lymphocytes within the stimulated node followed by the proliferation and differentiation of large numbers of antigen-specific cells, which then leave the lymph node as small lymphocytes.
The ability of skin epidermal cells to induce allogeneic lymphocytes into proliferation was examined in mixed skin cell-lymphocyte culture reaction (MSLR). The stimulating capacity of skin cells was reduced significantly by trypsin digestion, although the damage was repaired by incubation at 37 C for 3 hr. The optimal concentration of mitomycin C for treatment of stimulating cells in the MSLR differed from that in mixed lymphocyte culture reaction (MLR). Irradiation rendered them three to four times more stimulatory than did mitomycin C. Removal of adherent cells from responding cells by passage through a nylon-wool column gave a substantial elevation of the MSLR. The lymphocytes cocultured with skin cells in the primary MSLR incorporated 3H-thymidine, with the peak at the 6th day of culture. If the lymphocytes primed in the MSLR were restimulated with skin cells from the same stimulating strain, the primed lymphocytes responded promptly and in great magnitude.
The response of normal single-born calves to the s.c. injection of allogeneic lymphocytes and that of chimeric calves to allogeneic and cotwin cells were studied. Whereas allogeneic cells produced local dermal swelling along with an efflux of blast cells and specific cytolytic antibody in the regional lymph, the injection of cotwin lymphocytes into chimeric calves failed to produce responses in excess of those of controls. There was little indication of specific reduction in responsiveness of the lymphocytes of normal calves following challenge with allogeneic cells.
Intravenous injection of 2,000 rad of irradiated allogeneic cells can suppress the development of antigraft delayed-type hypersensitivity (DTH) to major and minor histocompatibility (H) antigens which normally arises after s.c. immunization. Secondary type DTH responses to minor H antigens were also largely suppressed by an i.v. injection of irradiated allogeneic cells 1 week preceding the s.c. priming injection. The extent of suppression of primary DTH to allogeneic H-2-incompatible cells depended on the dose of i.v. injected irradiated cells. After a dose of 1 x 10(7) irradiated spleen cells i.v., the suppression persisted for at least 40 days. Intravenous injection of cells incompatible for minor H antigens could not suppress the DTH to H-2 alloantigens and vice versa. Suppression of DTH to H-2 alloantigens was haplotype specific. Proliferation studies indicated that the immunosuppressed mice do not respond upon s.c. immunization with an increased proliferative activity in the draining lymph nodes, in contrast to nonsuppressed mice. The data suggest that i.v. preimmunization with allogeneic cells induces specific suppression of antigraft immunity acting at the induction stage of the immune response.
The ability of the popliteal lymph node assay (PLNA) To detect host-versus-graft activity in response to H-Y antigens was investigated in the rat by using presensitized recipients. It was shown that significant popliteal lymph node (PLN) enlargement in response to H-Y-incompatible lymphocytes from syngeneic donors could be obtained in female recipients that had been preimmunized by the weekly i.p. injection of 1 x 10(7) lymphocytes from males of the same strain. In one rat strain, it was also shown that when male recipients had been similarly preimmunized with lymphocytes from females of the same strain, significant PLN enlargement could be obtained in response to lymphocytes from syngeneic females. The results that we obtained using the PLNA are discussed in comparison with those that have been reported when H-Y-incompatible skin grafts have been used to investigate H-Y antigen in the rat. These results indicate that, in the rat, the PLNA provides a useful alternative method for the investigation of H-Y antigen and may also provide a useful means to investigate the response of male rats to lymphocytes from syngeneic females.
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