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.

Monitoring of antitumor immunity in patients with larynx cancer by tube leukocyte adherence inhibition assay.

The presence of specific antitumor immunity was examined by the tube leukocyte adherence inhibition (LAI) assay in patients with carcinomas of the larynx. Peripheral blood leukocytes (PBL) from patients with larynx cancer gave positive reactions in the LAI assay with the antigen prepared from the carcinoma of the larynx but not with antigen from normal larynx tissue. Adherence of PBL from normal donors did not differ significantly irrespective of whether antigens from tumor or normal larynx tissue were used. With a panel of tumor extracts tested, PBL of some patients reacted to only one antigen, wheras PBL of other patients reacted to several antigens. LAI reactivity disappeared after surgery, reappeared at 4 weeks after surgery, and then declined when patients were tumor free. These results show that the tube LAI assay may be of value in diagnosing larynx cancer and in following up its treatment.

Passive immunological enhancement of muscle allografts in rats.

The effect of the passive administration of antiserum obtained from recipients immunized with antigens from H-1 + non-H-1 incompatible donors on muscle allograft survival in rats was studied. In transplantation across the non-H-1 antigenic barrier (H-1 compatible but non-H-1 incompatible) a satisfactory degree of immunological enhancement was achieved. Recovery of contractile properties of muscle allografts after different immunological treatments of the recipients was compared.

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.

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.

Attempts to compare the effectiveness of blocking factors and enhancing antibodies in vivo and in vitro.

Sera from rats carrying tolerated skin allografts were tested for the presence of blocking activity in vitro. Sera with blocking activity had no effect on transplantation tolerance induction in newborn animals. Immunological enhancement of tumor growth was procured by passive transfer of serum from tolerant animals bearing skin allografts. It made no difference whether or not the serum contained blocking activity in vitro. These results suggest that there is no relationship between blocking factors and enhancing activity in vivo.

A contribution to the question of genetic transmission of immunological tolerance.

In view of the discrepancy in the results concerning the question of inheritance of immunological tolerance, we tried to enhance the genetic effect of tolerization by tolerizing male mice over three succeeding generations and by testing the ability of their progeny to reject an allogeneic tumour graft. This highly sensitive in vivo test, even of partial tolerance, did not reveal any difference in allogeneic tumour growth between progeny of control and tolerized males. Thus, the results again did not confirm genetic transmission of induced tolerance. The possible causes of the discrepancy between the results reported here and the results obtained by others are considered.

Different states of transplantation tolerance, and the problem of inheritance of tolerance.

In agreement with previous results, we failed to observe hereditary transmission of transplantation tolerance in the progeny of tolerant male mice when tested by skin grafting. At the same time we showed that the detection of the tolerance state depended on the test of immunity used. The possibility has not excluded that positive results with hereditary transmission of tolerance via fathers [1-3] may be due to the appropriate detection systems.

Maintenance of allogeneic cell recognition in allograft tolerant mice.

The organ distribution of 51Cr-labelled lymph node cells following transfer to syngeneic, allogeneic and specifically tolerant allogeneic recipients were compared. Neonatal induction of transplantation tolerance in several donor-recipient combinations involving different H-2 haplotypes had no effect on the reduction of homing into lymph nodes and the values of the homing did not differ from those of the untreated allogeneic recipients. In both cases, they amounted to 45-55% of the syngeneic controls. Even those neonatally treated animals, which had not been rendered tolerant at birth and rejected test skin grafts, were not capable of removing the transferred allogeneic cells by a typical immune elimination. The results indicate that animals recognize allogeneic cells, irrespective of whether or not they reject test skin grafts.

Effect of heterologous antimacrophage serum on growth of Rous virus-induced sarcoma in the allogeneic and syngeneic system.

The effect of heterologous antimacrophage serum (AMS) and normal rabbit serum (NRS) on the immune reaction against Rous virus-induced sarcoma (RSL) was studied in rats. The growth of RSL sarcoma transplanted against the H-1 barrier in AMS-treated rats was more progressive than in the untreated or NRS-treated control group. On the other hand, the growth of RSL sarcoma was significantly suppressed in syngeneic AMS- or NRS-treated recipients compared to the untreated control rats.

Application of tube LAI assay in larynx cancer patients.

The tube LAI assay was used for long-term follow-up of specific antitumor immunity in larynx cancer patients. A high percentage of patients examined before surgery exhibited positive LAI reactivity, and the success of treatment was reflected in the disappearance of positive LAI reactions. The tube LAI assay may be useful in diagnosing the precancerous states. Finally, positive LAI reactions were observed in hospital staff members exposed to contact with cancer patients over several years.

Comparison of the hemocytometer and tube modifications of the leukocyte adherence inhibition assay -- II. Application of both modifications for detection of anti-tumor immunity in man.

The hemocytometer and the tube modifications of the leukocyte adherence inhibition (LAI) assay were compared under identical experimental conditions in patients with carcinoma of the larynx. In the direct test, both modifications showed adherence inhibition if patients peripheral blood leukocytes (PBL) were incubated with a larynx cancer antigen preparation. Cell-free supernatants obtained by cocultivation of patients PBL with the antigenic extract yielded positive results only in the hemocytometer modification. Those supernatants inhibited the adherence of both normal human PBL and guinea pig peritoneal cells. Human encephalitogenic protein used as antigenic preparation in both direct and indirect hemocytometer modifications caused changes in adherence too. The presented results confirm the opinion that the hemocytometer and tube LAI assays base on different mechanisms.

Immunological response of patients with tumors of the urogenital tract: a follow-up of cell-mediated antitumor immunity by the LAI test.

Antigen-induced inhibition of leukocyte adherence is a suitable method providing information on immunological response of the tumor bearers. Leukocytes from patients' blood were tested by an in vitro method to detect cell-mediated antitumor immunity. Positive reactions of leukocytes to the specific antigen were found in carcinoma of the uterine cervix, vulva and ovary in 13 patients. The modified LAI test evaluated on the basis of free cells in Eagle's medium using the specific antigen is a simplified reversed method which, however, keeps the specificity.

Prolonged or permanent survival of skin allografts induced by monoclonal antibodies against lymphocyte subpopulations.

The effect of cytotoxic monoclonal antibodies against T lymphocyte subpopulations on skin allograft survival in mice was compared. Antibodies against cytotoxic lymphocytes (anti-Lyt-2.2) suppressed the allotransplantation reaction more effectively than antibodies against helper/inducer T lymphocytes (monoab anti-Lyt-1.2 and GK1.5). The suppressive effect of anti-Lyt-2.2 antibodies was comparable with that of the antibodies against the whole T lymphocyte population (monoab anti-Thy-1.2). Antibodies anti-Lyt-2.2 led up to a permanent survival (in 40% of animals) of H-2 incompatible skin allografts, when administered for long periods since the day of birth. The effect of the different antibodies, however, depends on the timing of application in relation to skin grafting. The results indicate that prolongation of survival and almost permanent tolerance of skin allografts can be obtained by the specific elimination of T lymphocyte subpopulations by the use of monoab and that Lyt-2 positive T lymphocytes play an important role in rejection allotransplantation reaction.

Prolonged survival of skin allografts in rats treated with antigen and hydrocortisone and the trend in graft-versus-host reactions.

The mechanism of prolongation of skin allograft survival in rats treated with specific antigen and hydrocortisone was studied in the strain combination with the strong (H-1 + non-H-1) and weak (non-H-1) antigenic difference. The grafts differing from the recipients at the weak non-H-1 loci only showed the longest survival although unresponsiveness to the strong (H-1) antigens of the donor type had already disappeared. In both strain combinations, immunological reactivity of lymphoid cells from treated animals was demonstrated in local GVH reactions at the end of the treatment (i.e., 12 days after transplantation). Lymphoid cells isolated from long surviving (100 days) AVN recipients of non-H-1 different grafts did not react in the GVH test. Attempts to obtain prolongation of skin allograft survival by passive transfer of serum removed after termination of immunization with strong (H-1 + non-H-1) or only weak (non-H-1) antigens were not successful.

Suppressor factor from a permanent T cell line inhibits allotransplantation reactions in vivo.

A permanent T cell line producing an antigen-non-specific suppressor factor was established. This factor inhibits the proliferation of lymphoid cells in vitro and, when injected in vivo, it suppresses the allotransplantation reaction. The line established may represent one of the cell populations playing an active role in the induction and maintenance of immunological tolerance.

Recognition of syngeneic and allogeneic histocompatibility antigens by peripheral blood leucocytes from non-immunized rats.

Peripheral blood leucocytes from normal, non-immunized adult rats recognize syngeneic and allogeneic tissue antigens, and this recognition can be detected by the tube leucocyte adherence inhibition assay. We show that PBL from newborn rats have the same and even a stronger ability for recognition and that the recognition is preserved in rats bearing tolerated skin allografts after neonatal induction of transplantation tolerance. Testing of tissue extracts prepared from the congenic strain of rats suggested that PBL from normal animals recognize histocompatibility antigens.

Quantitative aspects of abolition of transplantation tolerance by the adoptive transfer of lymphoid cells.

It was found that with neonatally induced transplantation tolerance about 50% of the rats are insensitive, or little sensitive, to the adoptive transfer of immunity. The proportion of rejected and unrejected skin grafts in individual experiments did not show a direct dependence on the dose of transferred cells, and immune lymphocytes were as effective as non-immune cells. No dependence on the time post-transplantation was found. Only the results of the adoptive transfers performed after more than 120 days after transplantation indicated an increased resistance of the tolerant state. Also repeated transfers in which increasing doses of cells were used showed a relatively stable state of tolerance in some animals. It is discussed that the difference in the sensitivity to the adoptive transfer might reflect at least quantitative individual differences in the mechanism of the state of unresponsiveness in neonatally induced tolerance.

Tumour cell destruction by macrophages from conventional animals.

Peritoneal exudate cells (PEC) from conventional donors displayed in vitro the cytotocix effects on the rat sarcoma cell lines LWI3K2, LW13K3, LW13K4 and RSL but they were not cytotoxic to fibroblasts derived from normal rats of the same strains. PEC were also cytotoxic to human urinary bladder carcinoma cells (T-24) grown in vitro. Cytotoxicity was detectable already at the effector to target cell ratio of 1 : 1, and the effector cells from syngeneic, allogeneic and xenogeneic donors were equally effective. The cytotoxic effect was apparently exerted by macrophages because the addition of lymphocytes from PEC donors did not change the target cells. The addition of serum from PEC donors to a mixture of target and effector cells protected the tumour cells from the destruction by macrophages. Cytotoxicity was blocked by serum from all PEC donors tested (syngeneic, allogeneic and xenogeneic with regard to the target cells) but not by foetal calf serum.

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.