Properties of ultraviolet light-induced suppressor lymphocytes within a syngeneic tumor system

It has been reported that ultraviolet light (uv) irradiation of normal C3Hf mice generates theta-positive suppressor lymphocytes which, as assayed by adoptive transfer studies, mediates susceptibility to transplanted syngeneic uv-induced tumors. We now report that the cells with suppressor activity can be recovered and enriched for using nylon wool columns. These suppressor cells are found in the nylon nonadherent fraction. Further, while uv irradiated mice are susceptible to supporting the growth of transplanted syngeneic uv tumors long after termination of the uv exposures, adoptively transferred suppression wanes and normal mice exhibit time-dependent recovery.     

Cell-mediated immune response to syngeneic UV induced tumors: I. The presence of tumor associated macrophages and their possible role in the in Vitro generation of cytotoxic lymphocytes

A primary in vitro sensitization system employing a chromium release assay was utilized to investigate reactivity of murine spleen cells toward syngeneic ultraviolet (uv) light induced fibrosarcomas. These tumors are immunologically rejected in vivo when implanted into normal syngeneic mice but grow progressivly when implanted into syngeneic mice that had previously been irradiated with subcarcinogenic levels of uv light. Following appropriate sensitization, spleen cells from both normal and uv irradiated mice are capable of developing cytotoxic lymphocytes in vitro against the uv induced tumors. It was subsequently discovered that in situ uv induced tumors all contained macrophages of host origin that became demonstrable only after enzymatic dissociation of the tumor tissue. These macrophages were immunologically active in vitro as their presence in the stimulator cell population was necessary to achieve an optimum anti-tumor cytotoxic response following in vitro sensitization. Anti-tumor reactivity generated by mixing spleen cells and tumor cells in the absence of tumor derived macrophages could be greatly enhanced by the addition of normal syngeneic peritoneal macrophages. When in vitro anti-tumor reactivity of spleen cells from normal and uv treated mice was compared under these conditions we again found no significant difference in the magnitude of the responses. In addition, the cytotoxic cells generated in response to uv induced tumors appeared to be highly cross reactive with respect to their killing potential. Cross reactive killing was observed between all uv induced tumors tested as well as with a syngeneic benz[a]pyrene (BP) induced fibrosarcoma. No cytotoxicity was observed against normal syngeneic PEC's even through these cells were shown to be susceptible to lysis by anti-H-2^k effector cells. It was concluded that: (a) A significant number of host-derived macrophages are present in uv tumor tissue. (b) These macrophages are important for the in vitro generation of tumor specific cytotoxicity. (c) Spleen cells from uv treated mice are capable of recognizing and responding against uv tumor associated antigens in vitro. Cytotoxic effector cells generated in response to uv induced tumors appear to have specificity for tumor associated antigens (TAA) present on all uv tumors tested as well as a syngeneic BP induced tumor. The relationship between in vivo and in vitro reactivity against uv tumors is discussed.     

Analysis of certain mechanisms of antigen-specific suppression of the immune response

CBA mice were immunized with sheep red blood cells (SRBC) to obtain immune spleen cells (ISc) which were used to suppressor cells. Administration of ISC to intact syngeneic recipients on the immunization day led to a more powerful suppression of the immune response as compared to that seen one day after antigen injection. Four days after immunization the animals' immune response was not liable to be suppressed. ISC extract possessed similar effects with respect to the immune response of normal spleen cells which were transplanted to the cyclophosphamide-treated recipients. The immune response of spleen cells from mice immunized with SRBC in a dose of 10(6) was less liable to be suppressed. Hyperimmune spleen cells from donors immunized with SRC in a dose of 10(9) were insensitive to ISC or to the extract. Experiments with the use of adoptive transfer of a mixture of immune and intact T- and B-cells have disclosed that B-cells from hyperimmune donors were resistant to suppression. Therefore, B-lymphocytes are the most probable target cells exposed to T-suppressors in the given system. The mechanism is discussed of the selective effect of T-suppressors on B-cells in the course of the immune response development during immunization with high doses of antigen.     

Regualtion of the immune response to tumor antigens. I. Immunosuppressor cells in tumor-bearing hosts.

A/Jax mice were rendered immune to the syngeneic and transplantable methylcholanthrene-induced Sarcoma 1509a by the surgical removal of the tumor 7 days after implantation; subsequent injection i.v. transfer of 10(7) to 10(8) washed thymus or spleen cells of tumor-bearing animals (TBA) to immune animals significantly inhibited the rejection of the tumor; this suppressive effect was entirely abolished by the treatment of these lymphocytes with anti-theta serum or anti-thymocyte serum (ATS) and complement before adoptive transfer. On the other hand, an equal number of thymus or spleen cells of normal animals or of animals bearing an unrelated tumor had no suppressive effect. Treatment of normal syngeneic animals with ATS after tumor cell inoculation or splenectomy of TBA resulted in the suppression of the tumor growth. The serum of TBA had no effect on tumor growth in immune syngeneic mice. Together these results suggest that TBA possess immunosuppressor T cells regulating negatively their immune response to the tumor.     

Studies of the mechanisms for the induction of in vivo tumor immunity. I. Induction of primary and secondary cell-mediated cytotoxic responses by adoptive transfer of lymphocytes.

Cell-mediated immunity to FBL-3, a syngeneic Friend virus-induced leukemia in C57BL/6 mice, could be adoptively transferred. Characteristic primary and secondary cytotoxic responses could be induced by adoptive transfer of normal and presensitized lymphocytes, respectively. In vivo tumor immunity could also be produced by adoptive transfer of presensitized lymphocytes. Both the primary and secondary cell-mediated cytotoxic reactions were T-cell dependent. The specificity of these reactions was primarily directed against F (Friend) type-specific antigen and FMR (Friend, Moloney, Rauscher) common antigen. The cytotoxic responses produced by adoptive transfer experiments gave better correlation to in vivo tumor immunity than those generated by in vitro mixed lymphocyte tumor cell culture reactions.     

Reversal of CD8+ T cell ignorance and induction of anti-tumor immunity by peptide-pulsed APC

In the present report, we have studied the potential of naive and activated effector CD8(+) T cells to function as anti-tumor T cells to a solid tumor using OVA-specific T cells from TCR-transgenic OT-I mice. Adoptive transfer of naive OT-I T cells into tumor-bearing syngeneic mice did not inhibit tumor cell growth. The adoptively transferred OT-I T cells did not proliferate in lymphoid tissue of tumor-bearing mice and were not anergized by the tumor. In contrast, adoptive transfer of preactivated OT-I CTL inhibited tumor growth in a dose-dependent manner, indicating that E.G7 was susceptible to immune effector cells. Importantly, naive OT-I T cells proliferated and elicited an anti-tumor response if they were adoptively transferred into normal or CD4-deficient mice that were then vaccinated with GM-CSF-induced bone marrow-derived OVA-pulsed APC. Collectively, these data indicate that even though naive tumor-specific T cells are present at a relatively high fraction they remain ignorant of the tumor and demonstrate that a CD8-mediated anti-tumor response can be induced by Ag-pulsed APC without CD4 T cell help.     

Characterization of CD8+ cytotoxic T lymphocyte/tumor cell interactions reflecting recognition of an endogenously expressed murine wild-type p53 determinant

p53 mutations are frequently found in human cancers and are often associated with the overexpression of wild-type (WT) protein or peptide sequences, supporting the notion that WT p53 epitopes may serve as potential targets for tumor immunotherapy. We have developed a cytotoxic T lymphocyte (CTL)/p53 tumor-associated antigen (TAA) model, based on immune recognition of a WT p53 determinant. WT p53-peptide-specific, major histocompatibility complex (MHC) classI-restricted CTL were produced from immunocompetent C57BL/6 (H-2b) mice after immunization with a previously defined WT p53 peptide (p53(232-240)) Epitope-specific CTL were then employed to identify syngeneic tumor cell populations expressing that antigenic determinant. Two syngeneic tumor cell lines, MC38 colon carcinoma and MC57G fibrosarcoma, were demonstrated to express the endogenous WT p53(232-240) determinant naturally, as defined by CD8 + CTL recognition. Cold-target inhibition assays confirmed that CTL-mediated lysis was due to immune recognition of the p53(232-240) peptide epitope. The p53(232-240)-specific CTL line did not lyse syngeneic normal cells (i.e., mitogen-activated splenocytes) in the absence of exogenous peptide, suggesting that the WT-p53-specific CTL could distinguish between tumor cells expressing self-TAA and normal host cells. We have demonstrated, for the first time, that the adoptive transfer of WT-p53-specific CTL to mice with established pulmonary metastasis resulted in antitumor activity in vivo. The ability to generate MHC-class-I-restricted CD8- CTL lines specific for a non-mutated p53 determinant from normal, immunocompetent mice, which display antitumor activity both in vitro and in vivo (by adoptive transfer), may have implications for the immunotherapy of certain p53-expressing malignancies.     

The role of suppressor T cells in BCNU-mediated rejection of a syngeneic tumor

The present investigation was initiated to determine the mechanism by which 1,3-bis(2-chloro-ethyl)-1-nitrosourea (BCNU) treatment of tumor-bearing mice results in a high percentage of surviving mice which are resistant to subsequent homologous tumor challenge. Spleen cells from C57BL/6 mice bearing the syngeneic LSA ascites tumor failed to demonstrate significant tumor-specific cytotoxic T lymphocyte (CTL) activity when stimulated in vitro with irradiated tumor cells. This lack of CTL activity correlated with the presence and high activity of two types of CTL-regulatory suppressor T cells (Ts), tumor-specific Thy-1+, Lyt-1-2+ and tumor-nonspecific Thy-1+, Lyt-1+2+ cells, as demonstrated by a double-positive selection technique. In contrast, spleen cells from BCNU-treated tumor-bearing mice generated high tumor-specific CTL activity when stimulated in vitro with irradiated tumor cells. This CTL activity correlated with the lack of demonstrable tumor-specific Ts and greatly diminished tumor-nonspecific Ts activity. The tumor-specific helper activity of Thy-1+, Lyt-1+,2- cells was found to be similar in both BCNU-treated and untreated tumor-bearing mice. BCNU-treated mice that survived a primary LSA tumor challenge (referred to as BCNU-cured mice) resisted subsequent challenge with the homologous (LSA) but not with a heterologous syngeneic tumor (EL-4). However, rejection of a secondary challenge with LSA tumor by BCNU-cured mice was inhibited by adoptive transfer of spleen cells from either normal mice or mice bearing LSA tumors. Furthermore, LSA tumor cells that failed to evoke tumor-specific CTL activity in normal mice could induce high CTL activity in BCNU-cured mice. The present study suggests that, in addition to its direct tumoricidal activity, BCNU inhibits the induction of tumor-specific Ts, thereby explaining why a high percentage of mice survive a primary syngeneic tumor challenge after treatment with BCNU, and also resist subsequent rechallenge with the homologous tumor.     

Regression of extensive pulmonary metastases in mice by adoptive transfer of antigen-specific CD8(+) CTL reactive against tumor cells expressing a naturally occurring rejection epitope

In this study, we developed a mouse model of adoptive immunotherapy reflecting immune recognition of syngeneic tumor cells naturally expressing an endogenous rejection Ag. Specifically, in a pulmonary metastases model, we examined the potency and maintenance of an antitumor CD8(+) CTL response in vivo, as well as its effectiveness against an "extensive" tumor burden. The approach taken was to first generate tumor-specific CTL from mice challenged with the CMS4 sarcoma coadministered with anti-CTLA4 mAb, which has been shown to facilitate the induction of Ag-specific T cell responses in vivo. An H-2L(d)-restricted nonamer peptide, derived from an endogenous murine leukemia provirus was identified as a CMS4-reactive CTL epitope based upon the following: CTL cross-recognition of another syngeneic tumor cell line (CT26 colon carcinoma) previously characterized to express that gene product; sensitization of Ag-negative lymphoblasts or P815 targets with the peptide; and by cold target inhibition assays. In vivo, the adoptive transfer of CMS4-reactive CTL (> or =1 x 10(6)) resulted in nearly the complete regression of 3-day established lung metastases. Furthermore, mice that rejected CMS4 following a single adoptive transfer of CTL displayed antitumor activity to a rechallenge 45 days later, not only in the lung, but also at a s.c. distal site. Lastly, the adoptive transfer of CTL to mice harboring extensive pulmonary metastases (> 150 nodules) led to a substantial reduction in tumor burden. Overall, these data suggest that the adoptive transfer of tumor-specific CTL may have therapeutic potential for malignancies that proliferate in or metastasize to the lung.     

Roles of deletion and regulation in creating mixed chimerism and allograft tolerance using a nonlymphoablative irradiation-free protocol

The induction of mixed chimerism (MC) is a powerful and effective means to achieve transplantation tolerance in rodent models. Host conditioning with irradiation or cytotoxic drugs has been used in many protocols for chimeric induction across allogeneic barriers. The deletion of alloreactive T cell clones has been described as the main mechanism responsible for the induction of a stable MC. In this study, we demonstrate that a stable MC and skin allograft tolerance can be established across MHC barriers by a noncytotoxic, irradiation-free approach using costimulation blockade plus rapamycin treatment. By using an adoptive transfer model of skin allograft and using specific Vbeta TCR probes, we demonstrated that deletion of donor-reactive cytopathic T cell clones is indeed profound in tolerant hosts. Nonetheless, the challenge of tolerant mixed chimeras with 5 million mononuclear leukocytes (MNL) from naive syngeneic mice was neither able to abolish the stable MC nor to trigger skin allograft rejection, a hallmark of peripheral, not central tolerance. Furthermore, in an adoptive transfer model, MNLs harvested from tolerant hosts significantly inhibited the capacity of naive MNLs to reject same donor, but not third-party, skin allografts. Moreover, when we transplanted skin allografts from stable tolerant chimeras onto syngeneic immune-incompetent mice, graft-infiltrating T cells migrated from the graft site, expanded in the new host, and protected allografts from acute rejection by naive syngeneic MNLs. In this model, both deletional and immunoregulatory mechanisms are active during the induction and/or maintenance of allograft tolerance through creation of MC using a potentially clinically applicable regimen.     

Mechanism of loss of natural killer activity in P815 ascites tumor bearing DBA/2 mice

P815 tumor cells (10(7] were administered intraperitoneally to DBA/2 mice. As the ascites tumor grew in the syngeneic host, a decline leading to a total loss of host spleen natural killer (NK) activity could be demonstrated. Removal of T and B cells or macrophages from the tumor-bearing (TB) mouse spleen cells did not raise the level of NK activity. Spleen cells from TB mice did not inhibit the NK activity of normal spleen cells. Comparable target (YAC cells) binding capacity could be demonstrated in spleen cells derived from normal or TB mice, but interferon failed to significantly stimulate the NK activity of TB mouse spleen cells. In adoptive transfer experiments, transfer of spleen or bone marrow cells from TB mice resulted in the development of significant levels of spleen NK activity in lethally X-irradiated recipient DBA/2 mice. These results indicate that the impairment of NK cell differentiation pathway rather than active suppression at the level of effector cells may be the mechanism of loss of NK activity in P815 TB DBA/2 mice.     

Mechanisms of depressed reactivity to dinitrochlorobenzene and ultraviolet-induced tumors during ultraviolet carcinogenesis in BALB/c mice

Chronic treatment of BALB/c mice with ultraviolet (uv) radiation produces two distinct immunologic deficiencies. These deficiencies are apparent long before visible skin tumors are induced by the uv irradiation. One is reflected in a transient inability to develop delayed hypersensitivity to dinitrochlorobenzene and appears to be due to a defect in antigen processing. The other is expressed by the failure of mice to reject syngeneic uv-induced tumors, which are highly antigenic. This lack of tumor rejection can be passively transferred with lymphoid cells and seems to be due to the presence of specific suppressor lymphocytes.     

Immunologic memory for Staphylococcus studied by adoptive transfer

The basic regularities of the formation and realization of immunological memory to staphylococcal corpuscular antigen were studied in adoptive transfer experiments on CBA mice. The capacity of spleen cells for generating anamnestic response to staphylococci in the body of irradiated syngeneic recipients appeared on day 3 after the immunization of donors. The formation of immunological memory to staphylococci in mice was shown to be directly related to the dose of the antigen. The study also revealed that intact splenocytes did not suppress the realization of immunological memory to staphylococci in the system of adoptive transfer. The conclusion of the absence of the "isogeneic barrier" for memory cells specific to staphylococcal corpuscular antigen was made.     

NK cell adoptive transfer combined with Ontak-mediated regulatory T cell elimination induces effective adaptive antitumor immune responses

Previous work from our laboratory showed that hydrocortisone (HC) combined with IL-15 induces expansion of activated human NK cells. We set up an experimental tumor model to evaluate the use of adoptively transferred, HC plus IL-15 (HC/IL-15)-activated and -expanded murine NK cells in the treatment of syngeneic mice carrying established lung metastases of the CT26 transplantable tumor. We also examined the effect of denileukin diftitox (Ontak) on the depletion of regulatory T cells to enhance the in vivo antitumor immunity induced by the adoptively transferred NK cells. Our results clearly demonstrate that murine DX5(+) NK cells are largely expanded in the presence of IL-15 plus HC while retaining intact their functional status. Moreover, when intravenously infused, they mediated significant antitumor responses against CT26 lung tumors in syngeneic BALB/c animals that were further enhanced upon pretreatment of the tumor-bearing animals with Ontak. Total splenocytes and isolated splenic T cells from NK-treated mice responded in vitro against CT26 tumor cells as evidenced by IFN-γ-based ELISPOT, proliferation, and cytotoxicity assays. Importantly, animals treated with Ontak plus adoptive transfer of HC/IL-15-expanded NK cells significantly retarded CT26 tumor growth after a rechallenge with the same tumor s.c. in their flanks. Taken altogether, our data suggest that NK cell adoptive transfer can trigger adaptive antitumor T cell responses, and regulatory T cell depletion by Ontak is mandatory for enabling HC/IL-15-activated NK cells to promote long-lasting adaptive antitumor immunity.     

MHC-unrestricted transfer of antilisterial immunity by freshly isolated immune CD8 spleen cells

Immune CD8 cells, which play an essential role in the adoptive transfer of antilisterial immunity, can specifically lyse Listeria-bearing macrophages in vitro in an MHC-unrestricted manner. In contrast, the adoptive transfer of immunity by unseparated immune lymphocytes has been reported to be MHC-restricted. To address the restriction properties of CD8 effectors in vivo, we assessed their efficacy in protecting syngeneic and allogeneic recipients. Protection was determined by comparing the number of viable splenic Listeria in naive mice and in recipients of 60 million CD8-enriched, L3T4-depleted, Listeria-immune spleen cells, 2 days after the infusion of 10,000 Listeria. Donor cells from B6 (H-2b) mice transferred about 4 logs of protection in syngeneic recipients and more than 2 logs in allogeneic B10.A (H-2a) or B10.BR (H-2k) mice. Immune B10.A CD8 cells transferred equivalent protection to B6 mice. Protection was almost completely abrogated by the lysis or lethal irradiation of CD8 cells before transfer in vivo. On the other hand, the depletion of macrophages or NK cells did not impair adoptive transfer. By comparison, nonimmune CD8 cells from normal mice or from mice stimulated with an irrelevant Ag in vivo did not transfer substantial immunity to allogeneic recipients. We have noted previously that protective CD8 cells inhibit phagocyte accumulation in the spleen of Listeria-infected syngeneic recipients. In the present studies, we observed similar changes in adoptively immunized allogeneic mice. Reduced phagocyte accumulation may reflect Listeria-dependent lysis of infected phagocytes by immune CD8 cells. In support of this, we showed that Listeria-immune donor cells rapidly acquired the capacity to mediate Listeria-dependent, MHC-unrestricted lysis of macrophages after incubation with small amounts of IL-2 in vitro. In sum, our data establish that Listeria-immune CD8 cells can function in vivo in MHC incompatible hosts, and indirectly support the hypothesis that the destruction of infected phagocytes may be important in T cell-mediated immunity against Listeria and perhaps other intracellular pathogens.     

γ-Irradiation facilitates the expression of adoptive immunity against established tumors by eliminating suppressor T cells

It was found that sublethal (550 rad) whole-body gamma-irradiation of mice bearing established immunogenic tumors enabled tumor-sensitized spleen cells infused intravenously 1 h later to cause complete tumor regression in all mice. In contrast, gamma-irradiation alone caused only a temporary halt in tumor growth, and immune cells gave practically no therapeutic effect at all. This result was obtained with the SA1 sarcoma, Meth A fibrosarcoma, P815 mastocytoma, and P388 lymphoma. Additional experiments with the Meth A fibrosarcoma revealed that the spleen cells from tumor-immune donors that caused tumor regression in gamma-irradiated recipients were T cells, as evidenced by their functional elimination by treatment with anti-Thy-1.2 antibody and complement. It was shown next that adoptive T-cell-mediated regression of tumors in gamma-irradiated recipients was inhibited by an intravenous infusion of spleen cells from donors with established tumors, but not by spleen cells from normal donors. The spleen cells that suppressed the expression of adoptive immunity were functionally eliminated by treatment with anti-Thy-1.2 antibody and complement. Moreover, they were destroyed by exposing the tumor-bearing donors to 500 rad of gamma-radiation 24 h before harvesting their spleen cells. The results are consistent with the interpretation that gamma-radiation facilitates the expression of adoptive T-cell-mediated immunity against established tumors by eliminating a population of tumor-induced suppressor T cells from the tumor-bearing recipient.     

Complete remission of cancer in late-stage disease by radiation and transfer of allogeneic MHC-matched immune T cells: lessons from GvL studies in animals

Most immunotherapy studies in animal tumor models are performed in early stages of the disease. Reports on the studies of treatment in late stages of tumor growth and metastasis are much rarer. To guide future efforts for treatment in late-stage disease, a model of effective immune rejection of advanced metastasized cancer is reviewed and lessons therefrom are summarized. Already cachectic DBA/2 mice with a subcutaneously transplanted syngeneic tumor (ESb-MP lymphoma) of 1.5 cm diameter and with macroscopic liver and kidney metastases at 4 weeks could be successfully treated by a combination of sublethal (5 Gy) irradiation followed by a single transfer of 20 million anti-tumor immune spleen cells from tumor-resistant allogeneic MHC-B10.D2 mice. Following intravenous cell transfer, the primary tumors became encapsulated and were eventually rejected from the skin while visceral metastases gradually disappeared leaving behind only scar tissue. There was wound-healing at the site of the rejected primary tumor, and the animals survived long term without any tumor recurrence. The complete eradication of late-stage disease by adoptive cellular immunotherapy could be corroborated noninvasively by ³¹P-NMR spectroscopy of primary tumors and by ¹H-NMR microimaging of liver metastases. Conclusions from functional mechanistic studies in this model are summarized and clinical implications discussed.     

Treatment of disseminated leukemia with cyclophosphamide and immune cells: tumor immunity reflects long-term persistence of tumor-specific donor T cells

B6 mice bearing disseminated syngeneic FBL leukemia can be cured by treatment on day 5 with 180 mg/kg cyclophosphamide and 2 x 10(7) adoptively transferred syngeneic immune spleen cells. Complete tumor eradication in this model requires more than 30 days and is dependent upon the transfer of specifically immune T cells. To evaluate the relative contributions of host and donor T cells to tumor elimination and the maintenance of tumor immunity, donor cells obtained from Thy congenic mice were used for adoptive transfer. Thus, host and donor T cells could be readily distinguished by the expression of either Thy-1.2 or Thy-1.1 antigen. The results demonstrated that the majority of immunologically competent T cells present in hosts cured by adoptive therapy were of host origin. A small population of donor T cells, however, persisted long after transfer. At day 60, a time point shortly after tumor eradication had been completed, 5% of splenic T cells were of donor origin, and by day 120 this percentage had decreased to less than 2%. Functional studies performed at both time points revealed that this small number of residual donor T cells contained the subpopulation of tumor-reactive T cells present in the host. Thus, host T cells did not make a substantial contribution to the expression of the anti-tumor response and presumably have little role in either tumor eradication or the long-term maintenance of tumor immunity.     

Characteristics of the immunosuppressive activity of lymphoid organ cells in the process of sensitization with ram erythrocytes and exposure to antilymphocyte serum

In the adoptive transfer of cells obtained from the thymus, lymph nodes and the spleen to intact syngeneic animals the suppression of immune response was induced by lymph node cells. If the donors were previously sensitized, the cells of the thymus and lymph nodes showed suppressive activity in the adoptive transfer test. A single injection of antilymphocytic serum to the donors of lymphoid cells, previously sensitized with sheep red blood cells, enhanced the immunosuppressing action of thymocytes and lymph node cells.     

Host resistance to cyclosporine induced syngeneic graft-versus-host disease. Requirement for two distinct lymphocyte subsets

Cyclosporine is crucial for the prevention of organ allograft rejection and allogeneic graft-vs-host disease (GVHD). Despite its potent immunosuppressive activity, cyclosporine elicits a T cell-mediated autoimmune syndrome after autologous or syngeneic bone marrow transplantation, which has been termed syngeneic GVHD (SGVHD). Recent studies have shown that for disease manifestation, a cytoxan and radiation-sensitive T cell dependent host resistance mechanism must be eliminated, allowing the clonal expansion of autoreactive cells. This report characterizes the autoregulatory lymphocyte population, present in normal animals, capable of inhibiting the adoptive transfer of SGVHD. First, twice the number of unfractionated splenocytes from normal animals to those from autoimmune donors ensured complete inhibition of the adoptive transfer of immune reactivity. Second, the phenotype of this host resistance mechanism in normal splenocytes involves dual regulatory T cell subsets. A helper/inducer subset (W3/25+) must be cotransferred with a cytotoxic/suppressor subset (OX8+) in a ratio that approximates the normal ratio in normal unfractionated splenocytes in order to affect inhibition of the transfer of SGVHD. Moreover the specific inducer regulatory activity resides in the OX22-, W3/25+ subset of Th cells.