Immune response in experimentally induced uremia. II. Suppression of PHA response in uremia is mediated by an adherent, Ia-negative and indomethacin-insensitive suppressor cell

The characteristics of the adherent suppressor cell in uremic rats were examined. We found that: 1) adherent spleen cells from uremic rats display a more potent suppressor activity than do control adherent cells; 2) the suppression that is mediated by uremic adherent spleen cells cannot be eliminated by pretreatment with indomethacin, whereas the suppression that is mediated by naturally present adherent cells in the control rat is reversed by pretreatment with indomethacin; 3) the uremic adherent suppressor cell does not have Ia antigens that can be detected by the monoclonal antibodies OXL, whereas control adherent cells have Ia antigens on their surface; 4) both the control and uremic adherent suppressor cells are insensitive to mitomycin C and do not have any detectable levels of Thy 1 antigens on their surface. It appears that immune suppression in uremic rats is mediated by an adherent cell that differs from adherent cells present in control animals. The suppression in uremic rats is either not mediated by prostaglandins or may be mediated by preformed prostaglandin synthetase products.     

Modulation of the rats' immune status by monoassociation with anaerobic bacteria

The capacity of a pure culture of anaerobic intestinal bacteria to influence the host's cellular and humoral immune systems was investigated with germfree, monoassociated, and conventionally reared rats. Monoassociation of germfree rats with Bacteroides fragilis stimulated the production of serum gamma globulin, agglutinating antibodies, and an apparent IgG (immunoelectrophoresis) band. A comparison of the in vitro blastogenic potential of lymphocytes (spleen cells and mesenteric lymph node cells) from germfree, monoassociated, and conventionally reared rats indicated the following: (1) the microbial flora had no obvious effect on the capacity of nonstimulated lymphocytes to incorporate [3H]thymidine; (2) spleen cells from conventionally reared rats responded to phytohemagglutinin, concanavalin A, or pokeweed mitogen better than splenocytes from germfree rats; (3) colonization of germfree rats with Fusobacterium necrophorum increased the responsiveness of splenocytes to photohemagglutinin and concanavalin A; and (4) monoassociation of germfree rats with B. fragilis, but not with F. necrophorum or propionibacterium acnes, increased splenocyte blastogenesis to homologous (i.e., colonizing) bacterial antigens. This study indicated that some intestinal bacteria can modulate the immune status of the host; the extent and nature of this modulation depended on the particular species of colonizing bacteria.     

Natural suppressor-like cells in local graft-vs-host disease

We examined the generation of suppressor cells in the popliteal lymph nodes (PLN) of F1 recipients of parental spleen cells in rats, i.e., a local form of graft-vs-host disease (GVHD). PLN cells of F1 recipients, or subpopulations of these cells, were tested for their ability to suppress the mixed lymphocyte culture and cell-mediated lympholysis. Suppressor cells were consistently generated in this local GVHD reaction. In contrast to others, we found no evidence that suppression was mediated by T cells. Instead, the suppressor cells had the CD5-, CD8+, asialo-GM1+ phenotype characteristic of rat NK cells. The suppression exerted by these cells was nonspecific and was not mediated by a veto effect. The suppressor cells did not kill alloreactive T cell blasts, but had strong NK activity. These cells appear to be of donor origin. These local-GVHD-associated NK-like suppressor cells are similar, but not identical, to the natural suppressor cells described by other investigators in various experimental systems.     

Lack of optimal activation of natural killer levels by interleukin-2 in rat spleen cells: evidence for suppression

The effect of human recombinant IL-2 on the levels of natural killer (NK) activity in spleen cells derived from BALB/c mice and different strains of rats was studied. Enhancement of murine NK activity in response to IL-2 was readily demonstrable. Levels of NK activity in control as well as IL-2-treated spleen cells from Wistar, Sprague-Dawley, Fischer, and Long-Evans rats increased initially and peaked on Day 1 or 2 of culture. No significant differences between the control and the IL-2-treated cultures was found in this duration. The subsequent fall in NK activity was slower in IL-2-treated cultures of Fischer and Long-Evans rat spleen cells resulting in a significant difference between the NK levels in control and IL-2-treated cells on Day 5 and Day 7 of the culture. In the case of Wistar and Sprague-Dawley rats, the boosting effect of IL-2 on NK levels was either poor or nonexistent. Even though NK activation was poor, spleen cells of all strains of rats did proliferate in response to IL-2, indicating that the IL-2 preparation used was biologically active on rat spleen cells. Rat spleen cells cultured alone or with IL-2 released a factor(s) in the culture supernatant which could suppress the IL-2-induced NK activation in mouse spleen cells. Indomethacin could block the release of suppressor factor by cultured rat spleen cells. Moreover the NK levels in rat spleen cells could be augmented by IL-2 in the presence of indomethacin. These results indicate that the subdued IL-2-induced NK activation in bulk cultures of rat spleen cells could be due to spontaneous release of prostaglandins by the cultured cells.     

Induced suppression of the efferent phase of contact sensitivity in rats

A study of the immunosuppressive systems of rats has been conducted with special attention to whether suppressor cells can be induced to down-regulate the efferent limb of contact sensitivity. Contact sensitivity (CS)1 was induced in DA rats 5 days after immunization with trinitrochlorobenzene (TNCB). Intravenous pretreatment of naive rats with TNP-coupled syngeneic spleen cells 7 days before sensitization suppressed the induction of CS by 60%. Suppression of the inductive phase of CS could be transferred adoptively into syngeneic rats with spleen cells of such tolerized animals. Cell fractionation studies showed the OX8+ (CD8) T cell population (cytotoxic/suppressor) was responsible for the suppression in the afferent phase of CS. Such cells were incapable of suppressing preexisting CS. To investigate whether suppression could be induced for the efferent phase, spleen and peritoneal exudate cells (PEC) from rats tolerized by administering TNP-spleen cells iv plus epidermal paintings with TNCB were adoptively transferred into recipients sensitized 4 days earlier. Both spleen cells and PEC suppressed the efferent phase of CS but PEC did so more efficiently. Separation of splenic cells revealed the suppressors to be CD8+ T cells. Furthermore, separation of PEC into plastic adherent and nonadherent cells showed the nonadherent (T cell enriched) cells to be noneffective alone. The adherent subpopulation conveyed suppression but did so more effectively upon addition of the T cells. Thus, T cells and macrophages may operate in concert to achieve suppression of the efferent limb of CS. PEC from tolerized rats suppressed performed CS of any specificity but only after the suppressor cells were triggered with the same antigen that induced them. Since both the afferent and efferent phases of CS have now been shown to be suppressable, two separate suppressor mechanisms may be operable in rats.     

Studies on the mechanism of suppression of experimental allergic encephalomyelitis induced by myelin basic protein-cell conjugates

The mechanism of suppression of experimental allergic encephalomyelitis (EAE) induced in Lewis rats by pretreatment with myelin basic protein (MBP) coupled to syngeneic spleen leukocytes (SL) was examined. Studies on the kinetics of the tolerance induction showed that pretreatment with MBP-SL suppressed EAE if given 7 but not 3 days before the disease-inducing injection of MBP in Freund's complete adjuvant. Treatment with cyclophosphamide 48 hr before administration of MBP-SL completely abolished the suppression of EAE. Transfer of lymph node and spleen cells from MBP-syngeneic erythrocyte conjugate (MBP-RBC) but not MBP-SL-pretreated rats resulted in suppression of disease in recipients subsequently given a disease-inducing injection of MBP. Administration of MBP coupled to SL from the histocompatible rat strain F344 resulted in suppression of the MBP-induced proliferative response of spleen cells from Lewis rats which had been given a disease-inducing injection of MBP. Taken together these results are consistent with the suppression of EAE induced by MBP-SL being mediated by suppressor T cells.     

Are Actinomyces viscosus antigens B cell mitogens?

An extracellular heteroglycan (ECHG) and a sonicated cell supernatant (SCS) of Actinomyces viscosus Ny 1 induced strong lymphocyte proliferation. This was shown with spleen and thoracic duct cells form germfree rats and confirmed with cells from conventional "nude" mouse spleens. Spleen cells developed direct plaque-forming cells against densely coupled TNP-SRBC. The mitogenic property of ECHG was diminished considerably after mild alkaline hydrolysis for lymphocytes form rat spleens and was totally abolished for cells from "nude" mouse spleens. These results suggest that ECHG and SCS have B cell mitogenicity.     

Epitope-specific regulation. IV. In vitro studies with suppressor T cells induced by carrier/hapten-carrier immunization

Sequential immunization with a carrier molecule and a new epitope (hapten) conjugated to the carrier (carrier/hapten-carrier immunization) induces specific suppression for IgG antibody production to the new epitope (hapten) on the carrier. Once induced, this "epitope-specific" suppression persists and specifically suppresses subsequent in vivo IgG antibody responses to the hapten presented on the same or on an unrelated carrier molecule. In vitro studies presented here characterize the surface markers and specificity of suppressor T cells generated in carrier/hapten-carrier-immunized animals. Thus we show (1) that spleen cells from these donors suppress in vitro IgG anti-hapten antibody production by cocultured hapten-primed spleen cells; (2) that some but not all of the suppressor cells carry surface Lyt-2; (3) that at least some of the suppressor cells have receptors for the inducing hapten (DNP); and (4) that, unlike the suppression obtained in vivo, the in vitro suppression extends to IgG responses to unrelated carrier protein epitopes presented in association with the inducing hapten.     

Molecular basis of "suppressor" macrophages. Arginine metabolism via the nitric oxide synthetase pathway.

A molecular explanation for "suppressor" macrophage inhibition of lymphocyte proliferation is described. NG-monomethyl-L-arginine (NGMMA), a specific inhibitor of the nitric oxide synthetase pathway, markedly augments Con A-induced proliferation of rat splenic leukocytes. Macrophages are necessary and sufficient for NGMMA-releasable-suppression, as indicated by a loss of suppression after either pretreatment of isolated splenic macrophages with NGMMA or their depletion by plastic adherence or L-leucine methyl ester. L- (but not D-) arginine overrides NGMMA-releasable suppression, and suppression is blocked by RBC as would be expected if nitric oxide were the effector molecule. Unlike rats, NGMMA did not augment Con A-induced proliferation of normal mouse splenic leukocytes. However, NGMMA did augment Con A-induced proliferation of mouse splenic leukocytes induced to contain suppressor macrophages by intravenous injection of Corynebacterium parvum, which suggests a quantitative, not qualitative, difference in suppressor macrophages between rats and mice. Nitrite production, as an indicator of nitric oxide synthesis, correlated with suppressor macrophage activity in rats and mice and was inhibited by NGMMA. Finally, NGMMA also markedly enhanced proliferation with every other mitogen examined (PHA, protein A, PWM, and LPS). It is concluded that immunoregulation of lymphocyte proliferation by suppressor macrophages is mediated, in part, directly or indirectly by products of the nitric oxide synthetase pathway.     

An in vitro model for induction of immunologic unresponsiveness to turkey gamma-globulin in primed mouse spleen cells, II. Antigen-specific suppressor cells.

Unresponsiveness induced to turkey γ-globulin (TGG) in cultures of TGG-primed spleen cells by incubation with high concentrations of soluble TGG (sTGG) was shown to involve a state of active suppression. Upon transfer to secondary cultures of primed spleen cells stimulated with an optimal dose of TGG-conjugated erythrocytes, such tolerant spleen cells were able to actively inhibit a secondary plaque-forming cell response to TGG in these cultures. Almost complete inhibition was observed with a tolerant cell to primed cell ratio of as low as 0.1. The suppression was antigen specific in that tolerant spleen cells which were suppressive for the secondary TGG response were unable to inhibit a primary response to sheep erythrocytes. T cells were shown to be required for the suppressor effect, in that (i) suppressor activity could be removed by complement-mediated lysis with an anti-Thy 1.2 antiserum and (ii) suppressor activity was retained in the effluent fraction after passage of suppressor spleen cells over a nylon wool column. Induction of the T-cell suppressor activity was found to be associated with a loss of T-cell helper activity within the TGG-pulsed cell population. The presence of adherent cells was not required for induction of suppressor activity. Furthermore, the suppressor effect was found to be resistant to 1000 R of γ irradiation.     

The autoimmune response in active Heymann's nephritis in Lewis rats is regulated by T-lymphocyte subsets

In this study the cellular events which are responsible for the induction and suppression of active Heymann's nephritis (HN) in Lewis rats were investigated. Using an enzyme-linked short-term culture assay specific autoantibody production in vitro by lymphoid cells directed against the nephritogenic renal tubular epithelial glycoprotein (RTE-Gp) was measured. By this method it was shown that only the lymph nodes that drain the site of immunization contained autoreactive B cells. Pretreatment with cyclosporine A (Cy-A) or with multiple injections of high doses of antigen in Freund's incomplete adjuvant markedly inhibited the development of disease to a subsequent nephritogenic challenge. In challenged high-dose-tolerant (HDT) rats the autoimmune response was only 5-10% of immunized nontolerant rats. This tolerance could not be transferred by lymphoid cells from Cy-A-treated rats, but could be transferred by lymphoid cells derived from the thymus or spleen of HDT rats. Thus a suppressor cell of thymic origin may be responsible for HDT. Transfer of affinity column-fractionated splenic T cells from HDT rats demonstrated that OX8- helper and OX8+ suppressor T cells are involved in the induction and suppression, respectively, of the autoimmune response in this experimental nephropathy.     

Immunologic tolerance to HGG in mice. I. Suppression of the HGG response in normal mice with spleen cells or a spleen cell lysate from tolerant mice.

Adoptive transfer of spleen cells or spleen cell lysates from mice tolerant to human-gamma-globulin (HGG) specifically suppressed the response of normal syngeneic recipients to HGG. The suppressive activity could be transferred for over 100 days after tolerance induction. The suppression induced by both spleen cells and spleen cell lysate was found to be specific as evidenced by a normal response to a challenge with turkey-gamma-globulin or goat erythrocytes. The activity of the suppressive lysate could be removed by passing the material through an HGG immunoadsorbent column but not by passing it through an anti-HGG column or a BSA column. These results indicated that the factor had antigen specificity and was probably not antigen-antibody complexes. That this suppression was not due to a shifting of the kinetics of the antibody response has also been demonstrated. The antigen-specific suppressor factor in the tolerant spleen cell lysates was a protein with a m.w. of approximately 45,000 daltons. The kinetics of the appearance of both suppressor cells and suppressor factor were consistent with a mechanism of active suppression functioning in the maintenance of tolerance to HGG.     

Antigen-specific inhibition of immune interferon production by suppressor cells of autoimmune encephalomyelitis

Previous work from this laboratory has revealed that spleen and/or lymph node cells from Lewis rats, that have recovered from an acute episode of experimental autoimmune encephalomyelitis (EAE), suppress the development of EAE when injected into syngeneic recipients subsequently challenged with myelin basic protein (MBP) in CFA. In an effort to understand the mechanism of this suppression, we measured the production of immune IFN-gamma, which may be required for the induction of an immune response, by EAE effector T cells (which transfer disease) and EAE suppressor cells when cultured in vitro with MBP. We now report that EAE effector T cells produce IFN-gamma when cultured in vitro with MBP. In contrast, spleen cells from recovered rats (which manifest suppressor activity in vivo) do not produce IFN-gamma. Moreover, in cell mixing experiments, these suppressor spleen cells inhibited the production of IFN-gamma by EAE effector cells. This inhibition was not eliminated by the removal of macrophages nor by the inhibition of PG synthesis by indomethacin. Furthermore, the inhibition was shown to be Ag-specific and mediated by nylon-adherent, radiation-sensitive splenic T cells. The findings suggest that suppressor cells regulate EAE by inhibiting IFN-gamma production by effector cells. This inhibition may result in the down-regulation of IFN-gamma-induced expression of class II major histocompatibility Ag on cells of the central nervous system, thus reducing the presentation of tissue-specific Ag (i.e., MBP) to autoreactive lymphocytes.     

Failure of NZB spleen to respond to prethymic bone marrow suppressor cells.

Mouse bone marrow contains theta-negative lymphocytes that can suppress an in vitro plaque response by spleen cells primed in vivo with burro red blood cells (BRBC). These bone marrow cells are radiosensitive and can be induced with thymosin fraction 5 or alpha 1 thymic peptides to express the theta antigen. Enrichment for these suppressor pre-T lymphocytes can be achieved by a one-step density centrifugation, macrophage depletion, or a combination of both procedures. NZB mice, which spontaneously develop an autoimmune disorder, have a suppressor abnormality revealed by this assay system. Upon analysis, they have normal BM pre-T suppressor cells but their spleen cells are refractory to the BM suppressor signal. NZB BM suppressor cells inhibit the response by DBA/2 spleen cells, but DBA/2 BM suppressor cells do not inhibit NZB spleen. This resistance to suppression is a property of the B cell fraction recovered from NZB spleen.     

Macrophage-mediated suppression of immune responses in Toxoplasma-infected mice. I. Inhibition of proliferation of lymphocytes in primary antibody responses

The suppressor cells induced by Toxoplasma infection were shown to be macrophages, since they adhered to plastic, and their suppressive activity in anti-sheep erythrocytes (SRBC) antibody responses was abrogated by treatment with silica or carrageenan, which are selectively cytotoxic for macrophages. The suppressor macrophages strongly inhibited the uptake of tritiated thymidine ( [3H]TdR) by normal mouse spleen cells in the responses to SRBC and Toxoplasma antigens. Supernatant fluids from the suppressor macrophages could not passively transfer the suppressive effect on anti-SRBC antibody responses. Furthermore, when the suppressor macrophages were isolated by a cell-impermeable membrane from normal mouse spleen cells, the antibody responses of normal spleen cells were not suppressed. These results indicate that suppression of antibody responses in Toxoplasma-infected mice is caused by an inhibitory effect of the suppressor macrophages upon proliferation of lymphocytes via direct contact with responder target cells. The suppressive effect of the macrophages was not counteracted by indomethacin, a potent inhibitor of prostaglandin synthesis, or catalase, a catabolic enzyme for hydrogen peroxide (H2O2).     

Cyclophosphamide and abrogation of tumor-induced suppressor T cell activity

Summary Previously we have demonstrated that the in vitro generation of P815-specific anti-tumor cytotoxic T lymphocytes (CTL) was suppressed by splenic suppressor T cells from late tumor-bearing hosts (TBH). Suppression is not caused by in vitro growth of P815 from splenic metastases, since suppression was also seen with spleen cells from late TBH mice bearing a hypoxanthine/aminopterin/thymidine-sensitive subline (PHS-5) of P815 in the presence of HAT. Cyclophosphamide has been shown to inhibit theinduction of suppressor cells selectively in a number of immune responses, but evidence that it can inhibit active tumor-induced suppressor T cells is limited. We have found that suppressor T cells already induced by P815 in syngeneic late TBH are sensitive to low doses of cyclophosphamide (50 mg/kg) given 1 day before spleen harvest, but the in vitro CTL response of late TBH spleen cells could not be restored by pretreating the mice with cyclophosphamide, even when exogenous interleukin-2 was added to the cultures. Although 50 mg/kg cyclophosphamide did not inhibit the CTL response of spleen cells from mice immunized with P815 +Corynebacterium parvum, the same dose of cyclophosphamide eliminated the CTL response of spleen cells from early TBH. Interleukin-2 (IL-2) did not overcome this effect of cyclophosphamide, suggesting a direct effect on CTL. Ultra-low-dose cyclophosphamide (10 mg/kg) did not adversely effect early TBH CTL but was still able to eliminate suppressor T cell activity from late TBH. Nevertheless, late TBH CTL remained unresponsive after pretreatment of mice with ultra-low-dose cyclophosphamide, even when exogenous IL-2 was added in vitro. CTL precursor frequency analyses demonstrated that cyclophosphamide pretreatment had little or no effect on the numbers of CTL precursors from early TBH. Late TBH CTL precursor cells were not detectable in these studies, with or without suppressor T cell inhibition by cyclophosphamide pretreatment. Thus, it appears that most CTL precursor cells may be lost or irretrievably inactivated in the spleens of late TBH mice.This work was supported by grants CA42443, CA48075 and T32-CA09210 from the National Cancer Institute, Department of Health and Human Services, and an American Cancer Society Clinical Oncology Career Development Award (H. D. Bear)     

Peripheral tolerance induced in lymph nodes by syngeneic spleen cells inhibits generation of CTLs to hapten-altered self antigens but not to alloantigens.

The immunological tolerance that is induced in lymph nodes that have been exposed to syngeneic spleen cells has been examined. Development of cytotoxic T lymphocytes was used to assess the immunological status of the lymph node cells. The tolerance was studied from the viewpoint of its induction, its activation, and its specificity. We had already reported that injecting either T or B cells of splenic origin into a regional lymph node environment a week prior to immunization for CTL to hapten-altered self antigens prevents development of the CTL. Here, we confirm that syngeneic splenic cells but not lymph node cells will induce the suppression provided that spleen cells are not coupled with hapten. We now report that splenic cells that cannot replicate or synthesize and secrete protein are capable of inducing the suppression. The data suggest a preformed surface marker peculiar to spleen cells and perhaps on cells that traverse the thymus induces local tolerance that is mediated by suppressor cells. Triggering the induced suppressor T cells (previously identified as CD8-) was achieved by syngeneic spleen cells as well as by H-2-compatible, Mls-disparate spleen cells but not by syngeneic lymph node cells or apparently by allogeneic spleen cells. Furthermore, triggering suppression was achieved by hapten-coupled syngeneic spleen cells whereas such cells would not induce the suppression. Thus, activating the suppressor cells requires reexposure to splenic cells of the proper MHC haplotype, unaltered or coupled with either TNP or FITC. Once triggered, the suppression was manifested toward CTL generation against hapten-coupled syngeneic antigens on either spleen or lymph node cells but not against allogeneic antigens. Thus, the specificity of the tolerance was directed to altered self antigens despite its induction by unaltered spleen antigen. Furthermore, for suppression to be seen the spleen antigen was not required to be on the hapten-coupled syngeneic cells used for the CTL immunization. The relationship of the splenic cell "antigen" to hapten-altered self antigens and to other surface markers and its site of acquisition within the body and its significance for cell homing have become intriguing questions of importance. This information has been discussed from the viewpoint of its applicability to autoimmune diseases as well as to cessation of inflammatory reactions that may be mediated by lymph node cells.     

Immunoregulation of Heymann's nephritis. I. Induction of suppressor cells.

Pretreatment of Lewis rats with a series of injections of a renal tubular antigen (RTA) in IFA prevented induction of Heymann's nephritis (HN) when the rats were challenged with RTA in FCA. This absence of disease was confirmed by immunofluorescent staining for rat IgG and histologic examination of the kidneys as well as by lack of development of significant proteinuria. Passive transfer of spleen and lymph node cells from rats receiving such pretreatment into syngeneic recipients prevented induction of HN when these recipients were challenged with RTA in FCA. Passive transfer of serum obtained from pretreated rats was without effect. These results suggest that one of the mechanisms involved in preventing HN by this pretreatment regimen was the induction of suppressor cells. The results of spleen cell transformation indicated that the suppressor cells were specific for RTA as the immune response to a second antigen, PPD, was unaffected. When rats already had active early HN, the diseas course was unaffected by transfer of suppressor cells.     

Ontogeny of T-cell mitogen response in Lewis rats. III. Juvenile adherent suppressor cells block adult mitogen responses

Spleen cells from suckling female Lewis rats (4 to 20 days old) were able to suppress mitogenic responses to concanavalin A (Con A) and phytohemagglutinin (PHA) of spleen or thymus cells from adult female Lewis rats and thymus cells from suckling Lewis rats. Thymus cells from suckling rats were unable to suppress adult spleen cell mitogenic responses to Con A. Removal of carbonyl iron (cFe)-, plastic-, or nylon-wool-adherent cells removed the suppressive action of juvenile spleen cells, but irradiation did not. Separated plastic-adherent spleen cells from suckling animals suppressed adult mitogenic responses to Con A. at optimal Con A doses 2-mercaptoethanol (2-ME, 2 X 10(-5) M) abolished the suppressive effect of juvenile cells, however, at the hyperoptimal dose of Con A (125 micrograms/ml) even higher doses of 2-ME did not relieve suppression by juvenile cells. These suppressor cells in suckling pups were affected by early weaning which decreased suppression, resulting in enhanced mitogenic responses of juvenile cells and removal of the ability to suppress adult mitogenic response.     

Two phenotypically distinct populations of T cells have suppressor capabilities simultaneously in the maintenance phase of immunologic enhancement

The events leading to immunologic enhancement in LEW rats immunized actively with Brown Norway (BN) rat spleen cells and passively with LEW anti-BN hyperimmune serum 11 and 10 days before receiving (LEW X BN)F1 cardiac allografts, respectively, have been studied. Cellular suppressor mechanisms developing during the induction phase of this phenomenon have recently been shown to be mediated by W3/25+ T cells in an antigen-specific manner. The present study suggests that the late maintenance phase of immunologic enhancement is mediated in vivo by simultaneously present separate donor-specific T cell subpopulations of W3/25+ and OX8+ phenotypes. Splenocyte subsets from grafted recipients greater than 100 days after transplantation were adoptively transferred into unmodified syngeneic LEW rats that received a specific test allograft 24 hr later, or into B recipients bearing indefinitely surviving heart grafts. Test graft survival was prolonged significantly in the first group and not altered in the second. Indeed, nonoverlapping W3/25+ and OX8+ cell fractions were separately responsible for suppression. However, when suppressor activity was tested in vitro in a three-component coculture mixed lymphocyte reaction, no suppression by T cells was obtained; this lack of correlation between in vivo and in vitro results has also been noted by other investigators in different systems. Thus, in the maintenance phase of actively and passively induced immunologic enhancement, interplay between two phenotypically distinct T cells with suppressor characteristics, but not putative cell-surface blocking factors, seems to prevent development of an alloreactive response and mediate host unresponsiveness.