Soluble suppressor of T cell proliferation in primary in vitro response to murine minor histocompatibility antigens

Normal mouse lymphocytes are not capable of mounting a primary cytotoxic T cell response to Mls encoded, non H-2, allodeterminants, although a strong lymphoproliferative response is observed in primary MLR between Mls incompatible cells. In this study it is reported that in the supernatant of primary cultures between AKR macrophages and CBA/H lymphocytes (H-2 identical, incompatible for Mls and other minor antigens) a suppressor of T cell proliferation in MLR is detected. By contrast, a suppressor is not detected in supernatants from primary cultures between BALB/C macrophages and CBA/H lymphocytes (H-2 incompatible, Mls identical), B10.BR macrophages and CBA/H macrophages and CBA/H lymphocytes (syngeneic) suggesting that the production of the suppressor factor occurs only when an Mls incompatibility exists. The suppressive activity of the Mls incompatible culture supernatant upon MLR between incompatible macrophages and lymphocytes is neither antigen specific nor Mls or H-2 restricted, nor is it due to an irreversible toxic effect on T lymphocytes or macrophages. The inhibition of T cell proliferation could be explained by inhibition of IL 2 production, by blocking its union to T cells or by a combination of both effects. Our findings could help explain previous observations that lymphocytes from mice preimmunized with Mls incompatible cells have a depressed proliferative response as well as depressed cytotoxicity against alloantigens.     

Induction by an immunogenic immunomodulating agent of nonspecific T cell suppression of lymphocyte responsiveness in MLR but not of antibody production

Summary Spleen cells derived from BALB/c mice that had been repeatedly immunized with the methanol extraction residue (MER) fraction of tubercle bacilli exhibited a depressed capacity to act as responder cells in allogeneic and syngeneic mixed lymphocyte reactions (MLR). Previously reported studies revealed that such spleen cells are also defective in the in vitro generation of antibodies. In order to determine the nature of the cells responsible for the depressed MLR reactivity, purified populations of splenic macrophages, B lymphocytes, T lymphocytes originating from normal and from MER-immunized mice, and cell culture supernatants were added to MLR mixtures consisting of normal mouse splenocytes. Macrophages originating from MER-immunized mice and their culture supernatants exerted a significantly higher suppressive effect on MLR than that of corresponding preparation from normal mice. Splenic T cells originating from MER-immunized mice and their supernatants also significantly suppressed the MLR response. However, the same T cell populations that were inhibitory in MLR failed to suppress the in vitro generation of antibodies against sheep red blood cells in the presence of either MER or 2-mercaptoethanol. These and previously reported findings indicate that a nonspecific immunomodulating agent, MER, can, under certain conditions of treatment, elicit the induction of nonspecific suppressor T cells for MLR but not for antibody production, and, accordingly, can inhibit cellular and humoral immunological responsiveness by different mechanisms.     

Characterization of a suppressor cell-activating factor (SCAF) released by adherent cells treated with M. tuberculosis

Peripheral blood adherent cells ingesting killed Mycobacterium tuberculosis release a suppressor cell-activating factor (SCAF) into their culture supernatants. When adherent cells ingested 125I-labeled M. tuberculosis, radioactivity could be detected in the supernatant within 2 hr. When this supernatant was fractionated on a Sepharose 2B column, the fraction with suppressor cell-activating activity was also found to contain the majority of the radiolabel, which suggests that the macrophage processed bacteria (or bacterial product) constituted the major portion of the SCAF. This fraction also contained a high proportion of lipid, and the fraction with suppressor activity resided purely within the phospholipid fraction. By employing thin layer chromatography, the phospholipids responsible were identified as phosphatidylethanolamine and phosphatidylinositol. These results indicate that when macrophages ingest mycobacteria, they release phosphatidylethanolamine and phosphatidylinositol of bacterial origin into their culture supernatants, which are responsible for activating suppressor T cells.     

Mixed lymphocyte reactivity in spleen cells from F-9 tumor-bearing ice. I. Suppressor cell induction and characterization

The immune capacity of spleen cells from F-9 tumor-bearing mice was assessed in a one-way mixed leukocyte culture (MLR) and in MLR mixing experiments. Alloreactivity was found to be significantly depressed in mice bearing palpable tumors. Mixing experiments demonstrated that the reduced alloreactivity was due to suppressor cell activity in the spleens of the tumorous mice. Suppressor cells were characterized as adherent, Ly2⁺ T cells. This study provides the basis for an analysis of the cell surface antigens on F-9 embryonal carcinoma cells which lead to the induction of suppressor T cells in these mice.     

Fractionation of lymphocyte subpopulations which regulate mixed lymphocyte reactions.

Four days after injection of allogeneic lymphocytes BALB/c splenic T cells suppress proliferation of syngeneic cells in mixed lymphocyte reactions (MLR). Conversely, lymph node cells from the same mice amplify MLR responses. To further characterize these functional subpopulations, alloantigen-primed lymphocyte suspensions from both organs were fractionated by velocity sedimentation at unit-gravity. After fractionation MLR suppressor cells from spleens localized exclusively in rapidlly sedimenting fractions of large cells. MLR suppressor activity of cells from these fractions, as well as that of unfractionated spleen cell suspensions, was abolished by treatment with anti-Thy-1.2 serum and complement. Spleen cell fractions of similar sedimentation velocity also secreted a soluble MLR suppressor into culture supernatants. Although inhibitory of MLR, spleen cells of rapid sedimentation velocity did not suppress responses to T cell mitogens. In marked contrast with the effects of spleen cells, large 4-day-alloantigen-primed lymph node cells had no suppressive activity in MLR. MLR amplifier cells of uncertain derivation were found in fractions of medium sedimentation velocity from both spleens and lymph nodes. Fractionation of alloantigen-primed lymph node cell suspensions did reveal, however, a subpopulation of small cells with MLR suppressor acitivty which was unaffected by treatment with anti-Thy-1 serum and complement. The data thus indicate that large alloantigen-activated lymphocytes are not intrinsically suppressive nor are cells which suppress MLR necessarily large. We consequently conclude that regulation of MLR responses by alloantigen-primed lymphocytes involves a complex interaction between distinct functional subpopulations of cells which are separable both by physical and biologic properties.     

Hormonal regulation of soluble immune response suppressor (SIRS): a possible role of SIRS in the maintenance of pregnancy

This study was conducted to investigate the effects of sex hormones upon the nature of soluble immune response suppressor (SIRS) produced by concanavalin A-stimulated Lyt-2+ T cells. Conventional SIRS affected IgM PFC only. However, SIRS made with progesterone (20-400 ng/ml or Prog-SIRS) suppressed IgM PFC, one-way MLR, and generation CTL; and SIRS made with estrogen (0.2-50 ng/ml or Est-SIRS) enhanced these responses. The factor(s) (MW 40,000-55,000) to stimulate macrophages to produce the second soluble factor (M phi-SF) was isolated from all preparations by gel filtration. Furthermore, Est-SIRS contained a factor(s) (MW 10,000-30,000) to enhance IgM PFC, MLR, and mitogen-induced blastogenesis of both T and B cells; and Prog-SIRS possessed the suppressive factor(s) to IgM PFC, MLR, and mitogen-induced T-cell proliferation. These activities were not impaired by 2-mercaptoethanol. Moreover, the suppressive activity of Prog-SIRS was completely absorbed by T cells only, but the enhancing activity of Est-SIRS was not completely absorbed by a single-cell population. These data suggest that progesterone can contribute to the suppression of allograft rejection through soluble factors, and estrogen can enhance host responses which may be affected by several soluble factors during pregnancy.     

Suppression of cytolytic T-cell activity by staphylococcal enterotoxin B-induced suppressor cells: Role of interleukin 2

We have previously shown that staphylococcal enterotoxin B (SEB) is a potent inducer of suppressor T cells which function to inhibit antibody production in vitro. In the present paper we extend these studies and show that the SEB-induced suppressor cells also inhibit the development of cytotoxic lymphocytes in mixed-lymphocyte reaction (MLR) cultures. Since further analysis also showed that the level of interleukin 2 (IL-2) in cultures of SEB-primed cells was significantly reduced, experiments were carried out to determine the role of IL-2 in the inhibition of cytotoxic cell activity. Attempts to neutralize the suppression by supplementing MLR cocultures with delectinated supernatants from concanavalin A (Con A)-stimulated rat splenocytes were not successful. In addition, MLR cocultures supplemented on Day 0 with 50 units of affinity-purified IL-2 were also suppressed. Further analysis showed that the IL-2 activity in the supplemented MLR cocultures containing suppressor cells were significantly reduced by Day 3. However, repeated supplementation of the MLR cocultures with exogenous IL-2 was successful in achieving (and maintaining) “normal” levels of IL-2. The cytotoxic cell activity in these MLR cocultures remained suppressed. These results suggest that the inhibition of cytotoxic cell activity by SEB-induced suppressor cells is independent of IL-2 levels in the MLR coculture.     

Glucocorticosteroid modification of lymphocyte blastogenesis in tumor-bearing hosts

The present study was done to evaluate glucocorticosteroid modulation of the individual cells contributing to tumor-induced dualistic (macrophage and suppressor T-cell) suppression in mitogen-induced lymphocyte proliferation. Steroid-sensitive T cells from normal mice showed a characteristic decrease in phytohemagglutinin responsiveness in the in vitro presence of 0.1 μg hydrocortisone sodium-21 succinate (HC), while tumor-bearing host (TBH) T cells demonstrated little or no decrease. This correlates well with kinetic study results demonstrating decreased HC-mediated suppression of mitogen-induced TBH T-cell DNA synthesis at 8 to 10 days posttumor cell inoculation. Hydrocortisone-treated cells from mice with advanced tumors had significantly higher degrees of blastogenesis than untreated TBH cells. In the optimal presence of both macrophages (Mφ) and HC, TBH cells had significantly greater blastogenesis than their normal counterparts. Alone, Mφ or HC depressed normal host PHA responsiveness. Experiments were carried out to determine if the increased blastogenesis was due to the additional Mφ presence in TBH. In vivo administration of methylprednisolone acetate demonstrated that steroids can inhibit tumor growth. A significant delay in tumor appearance occurred when the steroid was administered 4 to 7 days after tumor transplant perhaps due to the measurable lack of suppressor T cells. These studies indicated that steroid suppression was directed against a sensitive suppressor T cell and possibly acts by preventing its differentiation. The role of the Mφ in the afferent limb of the cell-mediated immune response seems less clear, but the results suggest nonspecific inhibition of TBH T-cell response to blastogenic stimulus.     

Tumors induce the formation of suppressor endothelial cells in vivo

Patients with solid tumors have defects in immune effector cells, which have been associated with a poorer prognosis. Previous studies by our laboratory have shown that exposure to Lewis lung carcinoma (LLC)-secreted products induces the formation of suppressor endothelial cells in vitro. The current studies examined if tumors could induce the formation of suppressor endothelial cells in vivo. Endothelial cells were immunomagnetically isolated from the lungs of tumor-bearing mice or normal controls and examined for their ability to modulate NK cell, T-cell and macrophage functions. Compared to normal controls, supernatants from endothelial cells isolated from tumor-bearing lungs had elevated secretion of PGE₂, IL-6, IL-10 and VEGF. Conditioned media from endothelial cells isolated from normal lungs increased CD8⁺ T-cell IFN-γ and CD4⁺ T-cell IL-2 production in response to anti-CD3 stimulation, while media conditioned by endothelial cells from tumor-bearing lungs had a diminished stimulatory capacity. Examination of NK cell functions showed that supernatants from endothelial cells isolated from normal lungs were potent activators of NK cells, as indicated by their secretion of TNF-α and IFN-γ. Endothelial cells isolated from tumor-bearing lungs had a significantly diminished capacity to activate NK cells. Finally, supernatants from endothelial cells of tumor-bearing lungs diminished macrophage phagocytosis compared to either treatment with supernatants of normal endothelial cells or treatment with media alone. The results of these studies demonstrate that tumors induce the formation of suppressor endothelial cells in vivo and provide support for the role of endothelial cells in tumor-induced immune suppression.     

Immunoregulatory function of T cells activated in the autologous mixed lymphocyte reaction

This study was undertaken to define the functional properties of T cells stimulated in the autologous mixed lymphocyte reaction (MLR) by purified B cells or macrophages. In preliminary experiments, it was found that T cells that had been cultured with autologous non-T cells inhibited pokeweed mitogen- (PWM) stimulated immunoglobulin synthesis by autologous B cells. In addition, the T cell-mediated suppression was eliminated by x-irradiation and hydrocortisone treatment, was mediated by a mechanism that occurred early in the PWM-stimulated cultures, and did not involve killing of mature immunoglobulin-secreting cells. T cells were then cultured with either autologous B cells or macrophages in order to determine whether such autoreactive T cells had a similar capacity to regulate PWM-induced immunoglobulin synthesis. Although T cell populations stimulated either by B cells or by macrophages suppressed proliferative responses and immunoglobulin synthesis, both these populations of autoreactive T cells provided help for immunoglobulin synthesis that was not significantly different from that provided by fresh T cells. These results suggest that the predominant functional consequence of activation of T cells in the autologous MLR is the generation of suppressor T cells capable of inhibiting immunoglobulin synthesis. Thus, the autologous MLR may represent a negative feedback mechanism for the regulation of the immune response.     

Interleukin 2 (IL-2) activity during tumor growth: IL-2 production kinetics,absorption of and responses to exogenous IL-2

A temporal study assessed the relationship between fibrosarcoma growth and immunologic encumberance due to the inability of BALB/c mouse splenocytes to elaborate the lymphokine Interleukin 2 (IL-2). Nylon-wool fractionation and antiserum treatments suggested the existence of a mildly nylon-wool-adherent, anti-Lyt 2-sensitive tumor-induced suppressor T (T_s,) cell which significantly decreased IL-2 activity. Absorption investigations indicated that ligand-activated tumor-bearing host (TBH) spleen cells were less receptive to IL-2 than their normal counterparts. When splenocytes were antiserum treated before absorption, removal of Lyt 2⁺ (suppressor T) cells resulted in greater IL-2 absorption by the remaining cells. Purified IL-2 only partially restored suppressed TBH spleen cell mitogen- or alloantigen-induced blastogenesis; whereas, normal host reactivity was significantly augmented. The collective data suggest that TBH spleen cells were capable of producing IL-2 and of responding to the IL-2 amplification signal when tumor-induced T_s cells were depleted.     

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 accessory cell function of murine Peyer's patches

The cellular composition and certain functional characteristics of murine Peyer's patches (PP) were examined and compared with other lymphoid tissues. The composition of PP resembled most closely that of the spleen with the exception of a significant decrease in the number of adherent and phagocytic cells. Very few cells with dendritic morphology could be identified in Peyer's patches. Whole PP (and the nonadherent population) were capable of presenting antigen ovalbumin, human gammaglobulin, and purified protein derivative in a T proliferative assay to sensitized lymph node cells and to an antigen-specific T-cell clone. The antigen-presenting cell in both the spleen and PP was concentrated in the low-density population which floated on 1.080 bovine plasma albumin. However, equal numbers of whole and PP floaters were deficient in their capacity to present antigen compared with similar populations from spleen. Moreover, in PP the antigen-presenting cell appeared in the nonadherent rather than the adherent population as found with other lymphoid tissues. Similar results were obtained with (B6A)F1, CBA, A.TFR-1 and B10.S (12R) mice, suggesting that the inability of adherent cells from PP to present antigen effectively was not genetically determined. Whole and nonadherent PP contained cells capable of stimulating an allogeneic MLR, although again they were generally inferior to those of the spleen when comparable numbers of cells were employed. The adherent population of PP did not elicit an MLR. However, whole PP contained accessory cells needed for mitogen-induced proliferation since passage over nylon-wool columns resulted in a nonadherent fraction which did not respond to concanavalin A or phytohemagglutinin and the addition of adherent peritoneal exudate cells restored the lectin response. The differences noted in the accessory cell function in PP and other lymphoid tissues suggest the possibility that quantitative or qualitative differences in the function of these cells may explain some of the previously observed characteristics of PP, such as the inability to detect a primary antibody response in this tissue. The possibility that the development of gut-associated suppressor cells and their migration to peripheral tissues may be involved in the systemic tolerance that follows oral immunization and that these may be related to numerical and/or functional differences in macrophages or accessory cells is discussed.     

Spontaneously Induced Suppressor Cells In Vitro: Nonspecific Suppression of In Vitro Antibody Formation

Nonspecific suppressor cells were induced during in vitro culture of normal mouse spleen cells (SPC) using the Marbrook culture system. The suppressor cells inhibited both the primary and secondary antibody-formation responses antigen nonspecifically in vitro, and both IgM- and IgG-responses were inhibited. The supernatants from suppressive precultured cells were not suppressive. The suppressor cells also inhibited the response of allogeneic SPC beyond H-2 compatibility. The induction of the suppressor cells did not require the presence of antigen but required fetal calf serum (FCS) or both FCS and 2-mercaptoethanol (2-ME). The suppressor cells were generated from the nylon-wool adherent, radiation-sensitive T cell population. On the other hand, the suppressor cells were nylon-wool nonadherent, relatively radiation-sensitive T cells. Actively antibody-producing cells were not affected by the suppressor cells. The suppressor cells inhibited the mitogenic responses of normal SPC to phytohemagglutinin-P (PHA), bacterial lipopolysaccharide (LPS) and concanavalin A (Con A). The suppressor cells themselves inhibited the growth of EL4 cells (T-cell leukemia of C57BL/6 mouse origin) and MOPCll cells (B cells, plasmacytoma of BALB/c mouse origin) even at a low effector-to-target cell ratio (E:T ratio = 1:1), but did not kill these tumor cells. These results indicate that the target cells of the suppressor cells are both T and B cells, and that the mechanism of action of the suppression is either inhibition of proliferation or inhibition of early events in the course of the immune response.     

Cell interactions in alveolar macrophage-mediated suppression of the immune response: an unusual suppressor pathway involving a population of T-cells that express Lyt-1, L3T4, and I-J

Studies from this laboratory have demonstrated that incubation of murine alveolar macrophages (AM) with SRBC-primed spleen cells (SC) results in suppression of the in vitro plaque-forming cell (PFC) response and that suppression is mediated by a soluble factor contained in supernatants obtained from cultures of AM and SC. In the present study, immunological techniques employing monoclonal antibody (MoAb) were used to isolate various T-cell subsets in order to determine the phenotype of the cells which interact with AM to produce suppression. Spleen cell populations depleted of Thy-1+-, Lyt-1+-, L3T4+-, or I-J+-bearing cells failed to generate suppressive supernatants when cultured with AM. Depletion of Lyt-2+ T-cells (the classical suppressor/effector subset) did not alter the ability of the remaining cell population to cooperate with AM for generation of suppressive supernatants. Direct suppression of the PFC response in cultures containing AM was abrogated after treatment of the spleen cells with anti-I-J, but not anti-Lyt-2 MoAbs. Reconstitution of the AM-mediated suppressive response with enriched populations of SC required the presence of T-cells which expressed Lyt-1, L3T4, and I-J. These results suggest the existence of an unusual suppressor pathway involving I-J restriction but which appears to be mediated by the interaction of AM with a population of T-cells that expresses surface markers characteristic of T-helper cells.     

Analysis of responder cell-cell interactions in the syngeneic mixed leukocyte response

In a previous study we identified the target antigens on stimulator cells in the murine syngeneic mixed leukocyte response (MLR) as self Ia molecules. The experiments reported here utilized analysis of log-log cell number-response titration slopes to study the responder T-cell population in the syngeneic MLR. The data indicated that in the peripheral lymphoid population at least two cell populations interact to produce a murine syngeneic MLR. One of these cell types appeared to be a member of an Lyt 2- subset, another a member of an Lyt 2+ subset. A potential third cell type, which was detected in the presence of (PMA)-induced EL4 supernatants, was found in the thymus, an organ that did not appear to contain either of the other two subsets. Experiments with adult thymectomized and neonatal mice suggested that the cell populations involved in the syngeneic MLR were mature T cells and were not an early differentiation state of T lymphocytes whose ability to be stimulated by Ia antigens alone was a transient event.     

Suppressor T cells in thymus-reconstituted nude mice: regulation of mitogen-induced transformation

A population of glass-wool-adherent splenic cells which could suppress the response of other spleen cell populations to T-cell mitogens was isolated from thymus-reconstituted nude mice. Such adherent cells were characterized as sensitive to anti-Thy 1.2 and complement treatment. Glass-wool-adherent cells from athymic mice do not have suppressor activity to self or normal littermate NAC; however, these mice possess precursor suppressor cells as demonstrated by isolation of glass-wool-adherent T regulatory cells in thymus-grafted nude mice. Such cells are generated in either freshly obtained or in vitro cultured thymus. Evidence for suppressor T cells of host genotype was supported by their sensitivity to host-specific anti-Thy serum treatment as well as their generation in alymphoid thymus grafts. Prior anti-Thy 1.2 treatment of GAC partially removed the suppressor activity: however, macrophages and B lymphocytes were shown not to be secondary regulatory cells or suppressor mediators, thus mature T lymphocytes with low amounts of Thy 1.2 antigen may be responsible for this residual suppression. Further characterization of GAC indicates that active cell growth is required for their regulatory function, as irradiation removed the suppressor activity. This report provides evidence for the presence of a T-lymphocyte subpopulation which has a regulatory function and requires a thymus in the generation of these cells.     

Modulation of the immunosuppressive effects of splenic macrophages in Fischer rats bearing adenocarcinoma 13762

Summary The nature of spleen cells in Fischer rats bearing a large size (>1 cm diameter) mammary adenocarcinoma 13762A (MAC) which block the immunostimulating capacities of MTP² (a synthetic immunomodulator) and suppress proliferation in vitro of splenic T and B lymphocytes by their respective mitogens was investigated. Splenic macrophages were recognized as the suppressor cells by (a) restoration of mitogenic responses by depletion of macrophages from spleen cell suspensions and (b) continued suppressor activity in spleen cell suspensions of tumor bearers devoid of viable T lymphocytes. Macrophage contact with T lymphocytes was required for the inhibition of T lymphocyte proliferation by concanavalin A as shown by (a) the absence of suppressor activity in supernatants derived from cultured suppressor macrophages, (b) lowering of the suppressor activity of intact macrophages after treatment with neuraminidase, (c) lowering of the suppressor activity of macrophages by addition of red cells to spleen cultures of tumor bearers indicating red cell interference with macrophage-T cell interaction and (d) lack of inhibiting action of suppressor macrophages on allogenic T lymphocyte proliferation showing macrophage T cell recognition for suppression.Animals bearing a large size tumor exhibited spleen hypertrophy and an increase in macrophage:lymphocyte ratio and a decrease in red cell:lymphocyte ratio. Splenic macrophages did not appear to be implicated in blocking antitumor immunity induction since (a) suppressor macrophages were absent in spleens during the inductive phase of the immune response and (b) MAC implanted in allogenic Wistar rats grew to about 2 cm diameter, induced splenic suppressor macrophages but the tumor was later rejected by the animals. Collectively the results suggest that suppressor macrophages are the result of increasing tumor volume rather than its cause.This study was supported by a grant from the National Cancer Institute of Canada Abbreviations used: Con A, Concanavalin A; LPS, lipopolysaccharide; PHA, phytohemagglutinin; MTP, maltose tetrapalmitate; MAC, mammary adenocarcinoma 13762; RPMI, Roswell Park Memorial Institute; TBR, tumor bearing rat; RBC, red blood cell     

Regulation of human cytotoxic T-lymphocyte responses by a soluble suppressor factor

We describe some aspects of the biology of a suppressor factor (SF) secreted by actively metabolizing and dividing alloantigen-primed T cells which functions by regulating human cytotoxic T-lymphocyte (CTL) activation. The SF functions most effectively during the first 24 hr of CTL activation, while it does not function at the CTL effector stage. Both T cells and adherent cells are capable of absorbing out the biological activity from suppressor factor supernatants. Experiments demonstrated that either fresh adherent cells or the addition of interleukin 2 (IL-2) into the test system could reverse the effects of the SF on CTL activation. These data suggest that the SF could be acting by either indirectly restricting IL-2 availability to proliferating CTLs by limiting adherent cell interleukin 1 (IL-1) secretion or, alternatively, SF acting directly on the IL-2-producing T cells.     

Functional differences between cells from MLR colonies grown in soft agar cultures

This report describes a method of growing soft agar colonies of human T lymphocytes activated in the MLR. Two types of colonies were demonstrated: lower colonies grew within the agar layer, and upper colonies grew on the surface of the agar layer. Three days of priming the lymphocytes in the MLR and the use of supernatants of day-3 MLR cultures to provide T cell colony growth factor were necessary for optimal colony formation. Lymphocytes obtained from colonies were grown in long-term (2 to 4 weeks) cultures to generate sufficient numbers of cells to be tested in different functional assays. Cells from both types of colonies exhibited PLT activity. Upper colony cells showed considerably higher CML activity than lower colony cells (mean percent cytotoxicity 37 +/- 5 vs 6 +/- 3). Cells from both types of colonies contained radiosensitive suppressor cell activity that inhibited the primary MLR. The suppressor cell effect of lower colony cells was specific for the original stimulator, but upper colony cells displayed nonspecific suppressive effects. For both types of colony cells, it appeared that suppressive effects were unrelated to the CML activity of these cells. These data suggest that the soft agar colony assay offers a promising approach to separate subpopulations of lymphocytes activated in the MLR.