Tolerance of skin allografts incompatible at the entire H-2 complex induced in adult mice by the post-transplant treatment.

Permanent tolerance of the entire H-2 complex incompatible B10 skin allografts was induced in adult B10.A mice by post-transplant treatment. Recipient mice were treated with heterologous antithymocyte serum (ATS) and specific tissue extracts or monoclonal antibodies anti-Thy-1.2. Combination of treatments with the specific tissue extracts and monoclonal antibodies leads to a high degree of tolerance in the majority of ATS-treated animals. Results thus show that it is possible to induce long-term tolerance of the entire H-2 complex incompatible skin allografts in mice using specific and non-specific immunosuppression given in the post-transplant period.

Prolongation of skin allograft survival in ATS-treated mice by post-transplant injections of species antigenic extracts.

B10.A male mice were grafted with H-2-incompatible murine B10.A(2R) skin allografts and treated with antithymocyte serum on days 2, 4, and 7 after transplantation. Repeated injections of cell-free tissue extracts from livers or spleens of B10.A(2R) mice were given in the standard doses, starting on the day of transplantation or on day 14 or day 28 after transplantation. The standard doses were the equivalents of material extracted from 40 mg or 80 mg of wet weight of liver or spleen tissue. Almost all of the regimens used in which antigen injections were begun on day 14 or day 28 after transplantation were successful and led to a marked prolongation in skin allograft survival. In some experimental groups most of the grafts survived 100 days after grafting and 8--33% grafts showed long-term survival in individual groups. The mechanism of this tolerance is mediated by suppressor cells which were characterized by means of anti-Thy 1.2 antibodies as T lymphocytes. the in vitro experiments have shown that cytotoxic cell precursors may be present in long-term tolerant mice and that they may be reactive to the tolerated antigens after sensitization.

Mechanisms of tolerance of the Ia antigen disparate skin allografts.

Transplantation tolerance was induced in A.TL mice to donors having disparity in the Ia antigens and identity at the H-2K, H-2D and non-H-2 antigens. After neonatal injection of 12 X 10(6) semiallogeneic lymphoid cells, permanent survival of A.TH skin allografts was observed in more than 90% of the recipients. Adoptive transfer of 50 X 10(6) lymphoid cells from normal A.TL donors to tolerant mice resulted in rejection of the tolerated grafts only in half of the animals. When cells from tolerant mice were tested in MLC and GVH assays, they reacted positively as did cells from normal mice. After sensitization in vitro, cells from tolerant mice were cytotoxic to A.TH antigens. Serum from tolerant mice did not inhibit cell proliferation in MLC assay nor blocked cytotoxic reaction in vitro and also did not enhance survival of A.TH skin grafts in normal A.TL mice. When the enhancing effect of this serum was tested in the recipients treated with ALS, prolonged survival of allografts was observed. Attempts to prolong survival of A.TH skin allografts by transfer of spleen cells from tolerant donors failed in normal A.TL recipients, while they were successful in the recipients treated with ALS. Long-term tolerated A.TH allografts, when transferred to normal A.TL recipients, were rejected. The findings show that loss of antigenicity of the tolerated skin allografts is not the mechanism of tolerance in this model and that cells capable of recognizing antigens of the tolerated allografts and reacting against them are still present in tolerant animals. Tolerance of skin allografts disparate only in Ia antigens, as has been shown at least for the strain combination tested, is probably mediated by the positive serum and cell suppressor mechanisms that block in vivo the effector phase of allotransplantation reaction.

Adoptive transfers of transplantation tolerance in genetically different strain combinations of mice.

Neonatal transplantation tolerance was induced in strain combinations of mice involving differences in the H-2D or H-2K regions, in the K or D ends of H-2, or in the central I region of the H-2 complex. Attempts were made to transfer the tolerance adoptively by suppressor cells to syngeneic non-immunosuppressed recipients. Adoptive transfer of tolerance was successful only in the combination with H-2D region disparity, and significant but short-lasting prolongation of skin allograft survival time was also obtained in the combination disparate at the D end of H-2. Transfers of tolerance were not successful in the combinations involving differences in the K or I regions of the H-2 complex irrespective of whether cells were transferred one day before or four days after skin grafting. The results are discussed with respect to hitherto known antigenic and tolerogenic properties of individual H-2 regions.

The regulatory role of I-J subregion in neonatal tolerance induction to H-2D alloantigens.

The mechanism of neonatally induced transplantation tolerance was studied in two mouse strain combinations involving differences at the D region of the H-2 complex only or at the same D region plus I-J subregion (including I-E, I-C, S and G regions). In the strain combination with the H-2D difference only, cells from tolerant mice proliferated markedly in the MLR assay when incubated with antigens tolerated in vivo, whereas the MLR reactions were negative in the combination with D plus I-J region disparities. In the latter combination cells from tolerant mice also did not respond to third-party antigens and their incubation with the tolerated antigens led to the suppression of cell proliferation. This non-specific suppression was absent in cells from tolerant mice in the strain combination, which differed in I-C, S, G and D alloantigens. Specific suppressor cells, which inhibited the development of cytotoxic cells, were demonstrated in tolerant mice of both strain combinations. The results show that, in additon to the specific suppressor cells induced by H-2K or H-2D alloantigens, nonspecific suppressor cells induced by the I-J region disparity that may regulate the resultant activity against H-2D (and probably also H-2K) alloantigens are involved in transplantation tolerance.

Comparison of neonatally and adoptively induced transplantation tolerance in mice.

Transplantation tolerance induced by the semiallogeneic cells in newborn mice or by the adoptive transfer of syngeneic spleen cells from neonatally tolerant donors in adult mice was studied in the strain combination with the H-2D region disparity (B10.A recipients--B10.A(2R) donors). Tolerance could be transfered adoptively already from 3-week-old mice that had been rendered tolerant at birth and the ability for the transfer of tolerance persisted for long periods even when neonatally tolerant animals were not skin grafted. Both neonatally and adoptively induced tolerance could not be abolished by the adoptive transfer of 100 x 10(6) immunocompetent cells from normal syngeneic donors. It was observed in the in vitro experiments that cells from tolerant mice in the two types of tolerance reacted to the tolerated antigens in the mixed lymphocyte culture, did not react to the tolerated antigens in the microcytotoxicity test (only some mice with adoptively induced tolerance showed a certain degree of reactivity), but cells from both types of tolerant mice inhibited the in vitro sensitization of cells from normal syngeneic animals. This suppression was stronger with cells from neonatally tolerant mice.

Cross-reactivity between Bacillus Calmette-Guérin and Rous Virus-induced sarcoma detected in rats by tube leukocyte adherence inhibition assay.

Specific immunity to Rous virus-induced sarcoma (RSL) was investigated by the tube leukocyte adherence inhibition (LAI) assay in rats immunized with Bacillus Calmette-Guérin (BCG). Peritoneal cells from Lewis rats immunized s.c. with BCG gave positive reactions in the tube LAI assay with the antigen prepared from RSL in Lewis rats. Lymph node cells from Lewis rats immunized with BCG had no cytotoxic effect on RSL cells in vitro, whereas peritoneal cells from the same rats were strongly cytotoxic for RSL cells. Growth of RSL tumors in vivo was not inhibited in BCG-treated rats as compared to that in untreated rats. The results show that LAI reactivity correlates with cytotoxicity of peritoneal cells and does not correlate with the cytotoxicity of lymph node cells and that positive LAI reactions with tumor antigen need not be reflected in the suppression of growth of the tumor in vivo.

Resistance of transplantation tolerance to X-irradiation.

With regard to the previous finding that suppressor cells participating in the state of transplantation tolerance were radiosensitive, the possibility was investigated whether tolerance can be abolished by irradiation. In the rat model used (AVN recipients, Lewis donors), both neonatally induced tolerance and tolerance induced in adult life by the transfer of suppressor cells were found to be radioresistant.

Transplantation unresponsiveness induced by allogeneic serum in combination with allogeneic cells and hydrocortisone in adult rats.

Attempts were made to optimize the treatment by using serum as antigen source for the induction of transplantation unresponsiveness in adult rats. With the scheme described it was possible to induce by serum injections the transplantation unresponsiveness not only in the week non-H-1, but also in the strong H-1 antigenic difference; this shows that there is no essential difference in the presence of H-1 and non-H-1 antigens in the serum. Allogeneic serum in conjunction with allogeneic cells was most effective in the non-H-1-different strain combination, whereas the combined treatment consisting of allogeneic serum, allogeneic cells, and hydrocortisone produced the longest skin graft survival in the H-1 antigenic difference. Some paradoxical results are discussed.

Suppression of allosensitization in vitro by cells from tolerant rats.

Lymph node cells from AVN rats tolerant to Lew antigens after neonatal induction of tolerance, which did not react in the mixed lymphocyte culture and cytotoxicity assays, were tested for the presence of suppressor cells. Sensitization of lymphocytes from normal rats was inhibited by the addition of lymph node cells from these tolerant rats. The mixed lymphocyte culture reaction of lymph node cells from normal AVN rats with the irradiated Lew cells was inhibited when lymphocytes from tolerant rats were added. In contrast, the cytotoxicity of immune lymphocytes was not inhibited by the addition of lymph node cells from tolerant rats. Neither did a 24-h or 48-h co-cultivation of immune lymph node cells with lymphocytes from tolerant AVN rats suppress the cytotoxicity.

Individual variability and correlation of the in vitro tests in tolerant rats.

Cytotoxicity of lymphocytes, mixed lymphocyte cultures, stimulation with PHA, suppressive activity of cells in vitro and efficacy of the adoptive transfer of cells in vivo were simultaneously investigated in rats bearing tolerated skin allografts. Cells from animals which did not react in MLC were simultaneously hyporeactive when stimulated with PHA. On the other hand, cytotoxicity of lymphocytes did not correlate with their ability to respond to specific antigens in MLC. None of the in vitro tests correlated with the efficacy of the adoptively transferred syngeneic lymphoid cells.

Immunological enhancement of muscle allografts induced by serum from tolerant rats.

Serum from tolerant animals induced immunological enhancement of muscle allografts. Serum from both animals bearing tolerated skin allografts and animals in which tolerance to skin grafts disappeared was effective. No significant differences between these two types of allografts were found with respect to contractile and histochemical behaviour. Serum from animals in which skin allografts survived transiently following non-specific treatment induced weak immunological enhancement. The changes in contractile and histochemical properties of muscle allografts serve as adequate parameters for assessment of the success of immunological enhancement.

Radiosensitivity of suppressor cells in neonatally tolerant rats.

Specific suppressor cells were demonstrated in rats that had carried tolerated skin allografts for long periods of time after being rendered tolerant at birth. These suppressor cells were able to transfer tolerance to sublethally irradiated syngeneic recipients and to inhibit cytotoxic antibody production in normal syngeneic recipients. Suppressive activity of these cells was shown to be radiosensitive. The presence of suppressor cells in tolerant animals was attributable to neonatal tolerance induction and not to skin grafting of neonatally treated animals. In some cases spleen cells from tolerant animals transferred adoptively or induced permenent tolerance to skin grafts, which suggests a long-lasting active mechanism of tolerance.