The evolution of immunosuppressive cell populations in experimental mycobacterial infection.

Immunosuppressor activity of considerable potency and complexity was generated during the course of chronic, progressive infection of C3H/Anf mice by Mycobacterium lepraemurium. From the 5th through 10th week after inoculation, spleen cells from infected mice mildly but reproducibly suppressed the direct plaque-forming cell response of normal spleen cell cultures to sheep erythrocytes. Suppression at this stage of infection was mediated by cells with macrophage-like characteristics. A marked increase in splenic suppressor activity at 10 to 11 weeks was associated with the appearance of a second suppressor cell subpopulation composed of T lymphocytes. The appearance of these cells was closely related in time to the onset of rapid splenic enlargement and a loss of cutaneous delayed type hypersensitivity to antigens of M. lepraemurium in mice at 10 to 11 weeks of infection. Suppressor cells were not present in peripheral lymph nodes until terminal infection at 22 to 25 weeks. Suppressor spleen cells depressed the T-dependent antibody response most severely, but there was also a direct effect upon B cells as shown by moderate suppression of responses to TNP-LPS and DNP-Ficoll. Spleen cells from 14-week-infected mice generated a soluble suppressor factor(s) that induces depression of moderate severity, however, the immunosuppression by intact cells was far greater.     

Suppressor cell activity of intestinal mucosal leucocytes from buffalo (Bubalus bubalis arni)

Suppressor activity of buffalo intestinal intraepithelial leucocytes and lamina propria leucocytes was induced by Concanavalin A, and was assayed against the mitogenic response of autologous and allogenic leucocytes to mitogens. Appreciable suppression was observed with 25 micrograms ConA/ml on the proliferative activity of the responder cells cocultured at a ratio of < 2:1 (suppressor:responder cell). Mitomycin C treatment of intestinal leucocytes did not totally vanish the viability and functionality of leucocytes.     

Control of human B lymphocyte responsiveness: enhanced suppressor T cell activity after in vitro incubation.

Human peripheral blood mononuclear cells (PBM) lost the capacity to generate immunoglobulin-secreting cells (ISC) in response to pokeweed mitogen (PWM) after a period of preincubation in vitro. When fresh PBM were co-cultured with preincubated PBM their response to PWM was inhibited, indicating that enhanced suppressor activity developed in the aged PBM concomitant with the loss of PWM responsiveness. Suppressor cell activity of aged PBM was present within the T lymphocyte population. The suppressor T cell inhibited PWM responsiveness of autologous and homologous PBM to an equivalent degree. The action of the suppressor cell was abrogated by inhibitors of DNA synthesis or by hydrocortisone. A suppressor T cell population with similar characteristics was found in freshly prepared PBM before in vitro incubation. Expansion of this suppressor T cell population during preincubation required cell division. There was no change in the functional capability of the helper T cell population as a result of similar in vitro culture. These observations indicate that a T cell population capable of suppressing PWM-induced generation of ISC can be selectively expanded by in vitro incubation of normal human PBM without additional mitogenic stimulation. Moreover, these data emphasize that control of B lymphocyte differentiation involves a critical interrelationship between T lymphocyte subpopulations exerting both positive and negative influences.     

Modulation of the human in vitro antibody response by human leukocyte interferon preparations

The ability of human leukocyte Interferon to modulate the plaque-forming-cell response of human peripheral blood leukocytes to horse red blood cells was examined. Human peripheral blood mononuclear cells were cultured in vitro with the addition of varying doses of human leukocyte interferon 24 hr prior to, simultaneously with, and 24 hr after sensitization of the cultures with horse red blood cells. Plaque-forming-cell responses were measured 5 days after sensitization with antigen using poly-L-lysine-coupled horse red blood cell monolayers. When human leukocyte interferon preparations were added 24 hr prior to sensitization with antigen, a significant enhancement of the plaque-forming-cell response was observed. When the interferon was added simultaneously with antigen, the plaque-forming-cell response was significantly suppressed. Therefore, human leukocyte interferon appears to have a time-dependent immunomodulatory activity. The kinetics of immunomodulation appear to be different from those of previously described mouse models.     

Antibody-dependent cytolytically active human leukocytes: an analysis of inactivation following in vitro interaction with antibody-coated target cells.

A leukocyte population consisting of approximately 85% lymphocytes, prepared from human peripheral blood by centrifugation through a Ficoll-Hypaque gradient, was studied for its capacity to destroy antibody-coated human liver (Chang) cells in vitro. Cytolysis was a rapid event: increased ionic flux (86Rb) from the target cell occurred within 10 min of the addition of effector cells. Kinetic analysis of target cell destruction (51 Cr release) was compatible with a "one hit" hypothesis, thereby indicating that cytolysis resulted from a single collision was an effector cell. The initial rate of cytolysis was linear and related to the number of leukocytes added, but lysis at all of the leukocyte to target cell ratios tested ceased after 5 hr. The number of target cells killed at that time was directly proportional to the number of leukocytes added. While the lytic capacity of the effector population was totally depleted after incubation with antibody-coated target cells, cytotoxicity was not affected by co-culturing leukocytes with Chang cells treated with pre-immune serum. The cytotoxic effector cells functioning in this antibody-dependent lytic system are thus to be contrasted with killer T cells, whose lytic activity is not compromised by interaction with homologous target cells. It was estimated that approximately 4% of the leukocyte population employed could kill antibody-coated Chang cells, a figure consistent with the estimated frequency of "null" cells within human peripheral lymphocytes.     

Activated suppressor cell dysfunction in progressive multiple sclerosis

Concanavalin A (Con A)-induced suppressor activity has previously been shown to be reduced in multiple sclerosis (MS) patients with active clinical disease. In this study, we demonstrate that OKT3, as well as Con A induced suppressor activity mediated by unfractionated peripheral blood mononuclear cells is reduced in patients with the progressive form of MS. By performing reconstitution experiments involving E+, T4+, or T8+ cells derived from either MS patients or controls, and normal allogeneic macrophages or E- cells, we sought to define the cellular basis for this suppressor defect. In both MS and control groups, E+ cells were required to obtain measurable levels of suppression. Suppressor levels induced by Con A-activated cultures containing E+ cells from MS patients were lower than those induced by those containing control donor E+ cells. Suppression mediated by T8+ cells from MS patients was also lower than for controls. In the control group, suppression mediated by T8+ cells exceeded that mediated by T4+ cells; such differences were not apparent in the MS group. These results suggest that although Con A-induced suppression can be mediated by a number of T and non-T cell subsets, the functional suppressor defect measured in the MS population does involve the T8+ cell subset.     

Characteristics and mechanisms of action of the concanavalin A-activated suppressor cell in man.

Human peripheral blood lymphocytes treated for 24 to 48 hr with optimally mitogenic doses of concanavalin A suppressed the proliferative response of autologous T cells to mitogens and antigens. Con A-treated cells also suppressed the proliferative response and the immunoglobulin synthetic response of autologous B cells stimulated in vitro by T cell helper factor. The human Con A suppressor cell was sensitive to treatment with mitomycin C and to exposure to radiation doses exceeding 1000 rads. The Con A suppressor cell was shown to reside in the nylon wool-nonadherent, sheep red cell rosette-forming, histamine receptor-bearing population of lymphocytes and to lack surface DRW antigens. One mechanism of action of Con A suppressor cells was shown to be the inactivation of nonspecific T cell helper factor.     

Suppression of a pokeweed mitogen-stimulated plaque-forming cell response by a human B lymphocyte-derived aggregated IgG-stimulated suppressor factor: suppressive B cell factor (SBF)

The mechanisms whereby formed immune complexes (IC) or immunoglobulin aggregates can suppress further antibody production were explored by culturing normal human peripheral blood mononuclear leukocytes (PBL) with heat-aggregated IgG (HAIgG) and collecting the culture supernatants at 24 hr. These supernatants were found to suppress a pokeweed mitogen (PWM)-induced rheumatoid factor plaque-forming cell (RF-PFC) response in normal individuals. PWM-induced anti-trinitrophenylated sheep red blood cell (TNP-SRBC) PFC were also inhibited by suppressor supernatants from HAIgG-stimulated PBL, suggesting that the polyclonal PFC response was inhibited by a suppressor factor. The suppressor factor inhibited PWM stimulated RF-PFC throughout the culture period, but suppression was maximal at the peak of the RF-PFC response. Suppressor factor was only effective at the initiation of cultures, suggesting that it inhibited early events in the PWM-stimulated RF-PFC response. Molecular weight determination of the suppressor factor by differential membrane fractionation suggested a m.w. range of 30,000 to 50,000, and chromatography on Sephadex G-100 showed a peak activity at an approximate m.w. of 32,000. Studies suggested the factor was not an interferon. Depletion of T lymphocytes by E rosetting and macrophages/monocytes by G-10 adherence did not affect the generation of suppressor factor. Depletion of T lymphocytes (OKT4, OKT8) and NK cells (Leu-11b) by antibody-dependent, complement-mediated cytotoxicity also did not affect the generation of suppressor factor. Depletion of B lymphocytes with OKB7 resulted in the generation of significantly less suppressor factor. Suppression produced by unstimulated purified B lymphocytes was approximately one-half that seen when B lymphocytes were stimulated with HAIgG. Differential membrane fractionation studies suggested that only HAIgG-stimulated B cell cultures contained peak activity in the 30,000 to 50,000 m.w. fraction. Supernatants from unstimulated purified T cells also generated suppression, which was approximately one-half of that seen with HAIgG-stimulated B cells, but no increase in suppressor activity was seen in T cell cultures after incubation with HAIgG. These studies demonstrate that HAIgG is capable of stimulating B lymphocytes to produce a lymphokine, suppressive B cell factor (SBF), which is capable of suppressing a polyclonal PFC response. SBF may be important in feedback control of human immunoglobulin production.     

Nonspecific suppressor T cells cause decreased mixed lymphocyte culture reactivity in bone marrow transplant patients

Decreased reactivity in mixed lymphocyte culture (MLC) was observed in patients within 1 yr after allogeneic and autologous bone marrow transplantation. Suppressor activity of peripheral blood mononuclear cells (PBMC) from transplant patients was studied by adding these cells as modulator cells to a bidirectional MLC with cells from normal individuals. PBMC from transplant patients markedly suppressed MLC reactivity in a dose-dependent manner. Suppressor activity was present in cells forming rosettes with sheep erythrocytes. Treatment of modulator cells with monoclonal antibodies against T cell differentiation antigens (OKT8, OKIa1) and complement completely abolished suppression of MLC. Suppressor activity was unaffected by 30 Gy irradiation. Suppressor activity declined gradually after transplantation and was inversely correlated with MLC reactivity of each patient at a significant level (p less than 0.01). These observations suggest that OKT8+ Ia+ radioresistant suppressor T cells play a role in the development of decreased MLC reactivity observed during the early post-transplant period.     

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.     

Cellular interactions in marrow-grafted patients. I. Impairment of cell-mediated lympholysis associated with graft-vs-host disease and the effect of interleukin 2

Forty patients with hematologic malignancy or aplastic anemia were given allogeneic marrow after conditioning with high-dose cyclophosphamide alone or in combination with total body irradiation. Between 28 and 3857 days after transplantation, their peripheral blood mononuclear leukocytes were tested for reactivity in indirect cell-mediated lympholysis against normal leukocytes from unrelated individuals, and the results were compared to those with cells from their healthy marrow donors. An impairment of cell-mediated lympholysis was found with cells from most patients with acute and chronic graft-vs-host disease (GVHD) whereas cells from most short-term and long-term patients without GVHD had cell-mediated lympholysis reactivity comparable to that of cells from the marrow donors. When interleukin 2 was added to the mixed leukocyte cultures during the sensitization phase, the impaired cell-mediated immunity of cells from most short-term patients with acute GVHD, but not that of cells from most patients with chronic GHVD, could be restored to normal levels. These results suggest the impairment of cell-mediated immunity seen in cells of short-term patients with acute GVHD is attributable to helper cell defects or to ineffective communication between antigen-presenting cells and helper T cells. The impairment in cell-mediated immunity seen in patients with chronic GVHD, however, may reside on the effector cells (or their precursors) or may be due to the presence of suppressor cell activity.     

Functional and morphologic characterization of human T cells continuously grown in vitro.

Long-term growth (now over 13 months) of thymus-derived lymphocytes from numerous normal human bone marrow and peripheral blood cell samples was accomplished by using a factor present in media obtained from mitogen-stimulated human peripheral blood lymphocytes. This long-term growth could neither be initiated nor maintained by mitogens alone. All cell cultures were greater than 90% E rosette-positive, whereas the tests for B cell markers, surface IgG and IgM, and EAC rosette were routinely negative. There was no evidence for the presence of granulocytes, monocytes, and their precursors in these cultures. The E rosette-positive cells were then tested to see if they had T cell functions. PHA, Con A, and pokeweed mitogens stimulated lymphproliferative responses in these cultures comparable to those of fresh peripheral blood cells. These proliferating cells were also able to release cell mediators, such as interferon and colony-stimulating activity. Further evidence for the T lymphocyte nature of these cultured cells was obtained from one-way mixed leukocyte cultures in which these cells responded to but were unable to stimulate allogeneic cells. The functional and morphologic characteristics of these cultured cells show that these cells are T cells that grow continuously in vitro.     

Human B cell differentiation. II. Suppression by T cells of T-dependent and T-independent plasma cell maturation.

In vitro human plasma cell generation induced by both T-dependent (PWM) and T-independent (NWSM) mitogens was found to be suppressed by peripheral blood lymphocytes preincubated with human aggregated IgG. T cells, but not B lymphocytes, were able to mediate the suppressive activity; since aggregated (Fab)'2 fragments were found unable to generate suppressor cells, it was concluded that the suppressor cell was a T lymphocyte bearing Fcgamma receptors. These cells appeared to be largely radiosensitive. In most cases the proliferative responses remained unchanged. Since NWSM-induced activation is not dependent on the presence of T cells, these results show that, at least in this case, T cells exert their suppressor function directly on B lymphocytes. Whether PWM-induced B cell differentiation is suppressed by the same mechanism or/and by inactivation of T helper lymphocytes remains under investigation.     

Human suppressor lymphocytes: I. Induction and characterization

Human peripheral blood lymphocytes were induced to become suppressor cells by exposure to Concanavalin A or Phytohemagglutinin. These cells were able to suppress the cytotoxic responses but not the proliferative responses of alloantigen activated human lymphocytes. Suppressor cells were non-specific in their activity, suppressing both allogeneic and syngeneic responders. They were labile in short-term tissue culture and required a proliferative phase for full induction of suppressor activity. Removal of macrophages prior to mitogen exposure resulted in a small loss of suppressor activity. Suppression was not the result of too much help since enhancement could not be demonstrated upon the addition of small numbers of suppressor cells to MLC.     

Regulation of leukocyte glass adherence and tube leukocyte adherence inhibition (LAI) reactivity by serum factors in dogs with progressing or spontaneously regressing canine transmissible venereal sarcoma (CTVS)

Summary We examined the regulation of leukocyte glass adherence and tube leukocyte adherence inhibition (LAI) reactivity by serum factors in dogs with regressing or progressing canine transmissible venereal sarcomas (CTVS). Both regressor and progressor peripheral blood leukocytes (PBL), draining and nondraining lymph node cells (LNC), and splenic leukocytes were significantly responsive to CTVS antigen extract in tube LAI. In contrast, a significant decrease in basal glass adherence of progressor PBL, draining and nondraining LNC, and splenic leukocytes was observed. Normal glass adherence was restored to progressor leukocytes by extensive washing with warm serum-free media, while significant tube LAI responsiveness to CTVS antigen extract was maintained. Preincubation of regressor PBL and LNC with progressor sera in two-stage tube LAI decreased the basal glass adherence of treated leukocytes. This effect of progressor sera was heat labile, a characteristic of CTVS antigen. Collectively, these findings suggest that progessor leukocytes and progressor sera treated regressor leukocytes were activated by interaction with serum CTVS antigen and thus behaved in tube LAI as stimulated cells, even in the absence of CTVS antigen. Regressor but not progressor sera were shown to contain anti-CTVS IgG with specific arming activity for normal dog PBL, but not LNC in two-stage tube LAI. The nonadherent response of peripheral blood neutrophils in two-stage tube LAI was proportional to the concentration of arming IgG, whereas no change was observed in glass adherence of PBL. The results of this study define the role of progressor and regressor serum factors in the mechanism of tube LAI and demonstrate a relationship between leukocyte glass adherence and the clinical course of CTVS. These findings show that tube LAI is a simple and reproducible measure of active factors in the immune response to a tumor.This investigation was supported in part by grant, CA-23469, from the National Cancer Institute, DHHS, and is submitted as Scientific Contribution No. 1051, Storrs Agricultural Experiment Station, University of Connecticut, Storrs, CT 06268 USA     

Suppression of the mixed leukocyte response and of graft-vs-host disease by spleen cells following total lymphoid irradiation (TLI)

Total lymphoid irradiation (TLI) was administered to mice as 17 fractions of 200 rads delivered to the major lymphoid organs. Spleen cells capable of suppressing the in vitro mixed leukocyte response (MLR) and in vivo graft-vs-host disease (GVHD) were found in mice after treatment with TLI. Suppression was not antigen specific and was markedly reduced by treatment of the spleen cells with anti-Thy-1.2 antiserum and complement. Suppressor activity declined with time after irradiation and disappeared within 30 to 40 days. The evidence suggests that the suppressor cells may prevent initial BM rejection and acute GVHD in allogeneic BM transplant recipients prepared with TLI.     

Growth and characterization of T cell colonies from human thymus

A semisolid microculture system was used to study T cell colonies grown from human thymocytes. Colony growth was absolutely dependent upon media conditioned by peripheral blood leukocytes (PBL) in the presence of phytohemagglutinin. Plating efficiency was further enhanced by the addition of a non-T, adherent, radiation-resistant (7500 rad) PBL subpopulation, but was not enhanced by culture supernatants of these cells. The T colony precursor cell in the thymus occurred with a frequency of 8.0 X 10(-3) and had a surface receptor for the OKT3 monoclonal antibody. Thymocyte colony cells were functionally distinct from PBL and the major thymocyte population. The colony cells proliferated in response to T cell mitogens, but only in the presence of exogenous growth factors. The cells stimulated normal PBL in mixed leukocyte culture (MLC), but did not respond to alloantigens in MLC or in assays of spontaneous cytotoxicity. This culture system should prove helpful in the study of human thymocyte differentiation.     

Neonatal infection with mouse thymic virus: effects on cells regulating the antibody response to type III pneumococcal polysaccharide.

Mice infected neonatally with mouse thymic virus (TA) were evaluated at different ages with respect to their ability to give a plaque-forming cell (PFC) response to type III pneumococcal polysaccharide (SSS-III), as well as the degree of amplifier and suppressor thymus-derived (T) cell activity present. B cell activity matured rapidly from 2 to 4 weeks of age and was not affected by TA infection. Amplifier T cell activity matured progressively over the first 8 weeks of life and was transiently suppressed in TA-infected mice at 4 weeks of age. Suppressor T cell activity measured at 2,4, and 6 weeks of age was unaffected by TA. The findings suggest that TA is highly tropic for T cells and has selective effects on subpopulations of T cells.     

Heterogeneity of suppressors of mitogen responsiveness in human malignancy

Summary Blood lymphocytes from cancer patients frequently showed reduced responsiveness to mitogens compared with those from healthy individuals. This impairment did not appear to be attributable to an intrinsic T cell deficit but to the involvement of suppressor cells. The role of monocytes as suppressors in cancer patients was supported by experiments in which they were depleted by removal of adherent cells or selectively eliminated in situ by treatment with macrophage toxic agents (carrageenan, silica). Phytomitogen responses were consistently elevated by these manoeuvres. Indomethacin elevated responses in unseparated but not adherent cell depleted populations, indicating an important role for prostaglandins in this phenomenon. None of these procedures markedly influenced the response of lymphocytes from healthy individuals.Following removal of adherents, response could be generated or increased in SRBC rosetting or non-rosetting fractions depending upon the initial responsiveness of the adherent depleted populations, an observation consistent with the coexistence of responder and suppressor populations. Tumour-draining lymph node cells differed from peripheral blood insofar as no evidence of monocyte-like suppressors was found, but SRBC rosetting cells depressed the mitogen stimulation of autologous blood lymphocytes.These data establish that suppressor cells are operative in the depression of T cell responses to mitogens and indicate that several cell types are involved.     

Suppressor factor secreted by T hybridoma established from peripheral blood lymphocytes of a bone marrow transplantation patient. I. Establishment of human T-cell hybridoma and partial characterization of suppressor factor

A stable human T-cell hybridoma was established by cell fusion between activated human peripheral blood lymphocytes from an allogeneic bone marrow transplantation patient and the JD1-17 cell line, a subclone of the human T leukemia Jurkat cell line. This hybrid clone 1-8, which bore the surface phenotype of suppressor cells (CD8+HNK1+), spontaneously secreted a factor which, at high dilutions, suppressed the responses of T and B cells induced by mitogens and alloantigens. This suppressor factor was found to be heat-resistant (56 degrees C, 30 min), stable at alkaline but not acid pH, unaffected by 2-mercaptoethanol, and sensitive to trypsin. Preparative isoelectric focusing revealed an isoelectric point of 5.35. The suppressor activity was selectively absorbed by blast T cells. By gel filtration on Sephacryl S-200 and HPLC, the suppressor activity was found in two peaks corresponding to 40-45 kDa (monomer) and 90-95 kDa (dimer).