A suppressor T cell in the human mixed lymphocyte reaction

Lymphocytes from an HLA-B7 DW2 homozygous multiparous woman, J.H., failed to respond in the mixed lymphocyte reaction to lymphocytes from her DW1 homozygous husband, W.H., and certain other homozygous typing cells. J.H. lymphocytes could suppress the response of HLA matched responders to W.H. This effect was shown to be radiosensitive and due to a T cell. The suppressor cell showed antigen specificity.     

Antibodies to membrane structures that distinguish suppressor/cytotoxic and helper T lymphocyte subpopulations block the mixed leukocyte reaction in man

Two major subsets of human T lymphocytes that are functionally analogous to the mouse Lyt-2+ and Lyt-2- subsets have been defined by their expression of two thymus-dependent membrane antigens, Leu-2 and Leu-3. Leu-2+,3- cells have suppressor/cytotoxic functions and Leu-2- ,3+ cells have helper functions. These studies were designed to determine the effects of adding IgG1 monoclonal anti-Leu-2 and anti-Leu- 3 antibodies to the mixed leukocyte reaction (MLR). At high concentrations, each antibody partially inhibited the proliferative response of unseparated T cells and abolished the response of the isolated subset having the appropriate phenotype. An IgG1 monoclonal antibody to HLA-A2 and an IgG2a antibody to Leu-1, a pan-T antigen, failed to inhibited the MLR. These results suggest that the Leu-2 and Leu-3 antigens may have a direct role in the mechanism whereby T cells recognize and respond to alloantigen.     

Correlation between stable E-rosette formation and induction of alloantigen specific MLR suppressor cells in human mixed lymphocyte culture

The induction kinetics of human suppressor cells in unidirectional mixed lymphocyte culture (MLC) and the E-receptor of the suppressor cells were studied. Suppressor cell function was measured by the degree of suppression of mixed lymphocyte reaction (MLR). MLR suppressor cells appeared 3 days after priming MLC was started, and maintained the same suppressive effect until the 11th day. These suppressor cells were allo-antigen specific and resistant to x-irradiation up to 1000 rad, but susceptible to 1500 rad or to mitomycin C treatment. MLR suppressor cells which were obtained from priming MLC after 3 to 8 days formed rosettes with sheep erythrocytes at 37 degrees C.     

Suppression of the mixed lymphocyte reaction in man by a soluble T-cell factor. Specificity of the factor for both responder and stimulator

J.H., an HLA-Dw2 homozygous multiparous woman, fails to respond to her husband, W.H. (HLA Dw1,-) in the unidirectional mixed lymphocyte reaction. T cells from J.H. were previously shown to suppress the responses of Dw2-positive cells but not Dw2-negative cells to W.H. We now report that a soluble factor released into the supernate of the mixed lymphocyte reaction by J.H. T cells, mediates this suppression. Like the cell from which it is derived, the factor is highly specific for HLA Dw2 in the responder cell and partially specific for the stimulatory alloantigen.     

Deficient autologous mixed lymphocyte reaction in Kaposi's sarcoma associated with deficiency of Leu-3+ responder T cells.

Autologous mixed lymphocyte reaction (AMLR) and T cell subsets defined with monoclonal antibodies were analyzed in the peripheral blood of homosexual males with Kaposi's sarcoma (KS). All seven patients demonstrated decreased AMLR (P less than 0.001) when compared with age- and sex-matched simultaneously studied controls. These patients also showed decreased proportions of Leu-3+ (helper/inducer phenotype) and an increase in the proportion of Leu-2+ (suppressor/cytotoxic phenotype) T cells. Leu-3+ T cells were purified from two patients by depleting Leu-2+ T cells in complement-dependent cytotoxicity. Leu-3+ T cells from both patients demonstrated poor proliferative response in the AMLR. In allogeneic MLR, patients' T cells were poor responders and their non-T cells were poor stimulators against healthy controls. This study demonstrates deficiency of both AMLR and allogeneic MLR in patients with KS. The decreased AMLR is associated with qualitative and functional deficiency of Leu-3+ responder T cells. Whether the functional deficiency of Leu-3+ responder T cells in the AMLR is a general phenomena or a feature of a subset of patients with KS remains to be determined.     

Regulatory mechanisms in cell-mediated immune responses. III. I-region control of suppressor cell interaction with responder cells in mixed lymphocyte reactions

Active suppression of mixed lymphocyte reaction (MLR) response is mediated by a soluble factor released by alloantigen-activated murine suppressor cells. Genetic restrictions controlling suppressor factor interaction with MLR responder cells were elucidated in this study. Non-H-2 genetic background was irrelevant to effective interaction. Using congenic strains and strains with intra-H-2 recombinants the genetic locus controlling suppressor T-cell-responder cell interaction was mapped in the I-C or S regions of the H-2 complex. Similarly, recombinant strains were used to exclude the presence of another suppressor cell-responder cell interaction locus in K,I-A, and I-B regions. It thus appears that the I-C subregion of the H-2 complex controls suppressive cell interactions in this T-cell-mediated immune response.     

Alterations of both responder T cells and stimulator non-T cells are responsible for abnormal mixed lymphocyte reaction in aged humans

Summary The autologous and allogeneic mixed lymphocyte reactions of 15 young and 15 aged human adults were compared. Both autologous and allogeneic mixed lymphocyte reactions were significantly reduced in the aged group. T cells from aged adults displayed a reduced proliferative response to non-T cells of either aged or young adults. T cells from young adults also showed a reduced proliferative response to non-T cells from aged adults. Sera from aged adults, showing depression of autologous and allogeneic mixed lymphocyte reaction, did not exert any inhibitory effect on the autologous and allogeneic mixed reaction of lymphocytes from young donors. These data suggest that depression of mixed lymphocyte reaction in aged humans probably reflects intrinsic abnormalities of both responder T cells and stimulatory non-T cells. This work was supported by a grant from theConsiglio Nazionale delle Ricerche (CNR), Roma, Italy ‘Progetto Finalizzato Medicina Preventiva, Sottoprogetto Meccanissmi di Invecchiamento’.     

Induction of suppressor activity in the autologous mixed lymphocyte reaction and in cultures with concanavalin A.

T lymphocytes that are activated in the autologous mixed lymphocyte reaction (MLR) have suppressor activity. Concanavalin A (Con A) augments the suppressor activity generated in cultures containing both T and non-T lymphocytes and can induce suppressor activity in T-lymphocyte preparations that contain too few (10%) non-T cells to generate a significant autologous MLR. However, when such T-lymphocyte preparations are further depleted of adherent cells and contain less than 2% non-T cells, Con A fails to induce suppressor activity. These findings support the concept that an autologous MLR may play an important role in generation of suppressor cells by Con A.     

Concanavalin A induced suppression of lymphocyte proliferation in chronic liver disease. A study of suppressor and responder populations in autologous and allogeneic systems

Abnormal T-cell regulation of lymphocyte proliferation may contribute towards tissue damaging mechanisms in chronic liver disease. We therefore studied Concanavalin A induced suppressor cell activity in T-T interaction in 47 patients with chronic liver disease, using both an autologous and an allogeneic system. In the autologous system, no differences were found between those with auto-immune chronic active hepatitis, HBsAg positive chronic active hepatitis, primary biliary cirrhosis, alcoholic liver disease and normal controls. However, several abnormalities were identified in allogeneic cultures with normal lymphocytes which allowed separate analysis of the influence of suppressor and responder cells from patients with chronic liver disease. An abnormality of the suppressor population was found in those with autoimmune chronic active hepatitis, primary biliary cirrhosis and alcoholic liver disease, while the responder population was abnormal in those with autoimmune or HBsAg positive CAH. Failure to demonstrate an abnormality in an autologous system may reflect a combined defect of suppressor and responder populations, and in this study the allogeneic system was a more sensitive index of abnormal cellular T-T interaction.     

Regulatory mechanisms in cell-mediated immune responses. IV. Expression of a receptor for mixed lymphocyte reaction suppressor factor on activated T lymphocytes

Suppression of the mixed lymphocyte reaction (MLR) by a soluble factor produced by alloantigen-activated spleen cells requires genetic homology between the factor-producing cells and responder cells in MLR. The ability of lymphocytes used as MLR responder cells to adsorb MLR suppressor factor was tested to investigate the expression of a receptor structure for suppressor molecules. Normal spleen or thymus cells had no effect on suppressor activity. Concanavalin A (Con A)-activated thymocytes, however, effectively removed suppressor activity, suggesting that the receptor is expressed only after activation and is not present or not functional on resting cells. Significantly neither phytohemagglutinin- nor lipopolysaccharide-activated lymphoid cells absorbed the factor. Furthermore, only Con A-activated thymocytes demonstrating genetic homology with the cell producing suppressor factor for H-2 regions to the right of I-E were effective absorbants. Alloantigen-stimulated spleen cells syngeneic to the suppressor cell also removed suppressor activity. These data support an hypothesis that subsequent to stimulation in MLR, T lymphocytes express a receptor, either through synthesis or alteration of an existing molecular structure, which then provides the appropriate site for interaction with suppressor molecules.     

Autologous mixed lymphocyte reaction in man. XII. Deficiency of the autologous mixed lymphocyte reaction in patients undergoing allogeneic bone marrow transplantation. Relationship with chronic graft-versus-host disease

Peripheral blood mononuclear cells from 14 patients with acute leukemia or aplastic anemia undergoing allogeneic bone marrow transplantation were examined for the autologous mixed lymphocyte reaction (AMLR) between T and non-T cells and its relationship with chronic graft-versus-host disease (GVHD). Five of 6 patients with GVHD demonstrated deficiency of the AMLR, whereas only three of 8 patients with no evidence of chronic GVHD had deficient AMLR. The nature of underlying disease had no effect on the AMLR. The significance of these results is discussed.     

Adoptive transfer of suppression of arthritis in the mouse model of collagen-induced arthritis. Evidence for a type II collagen-specific suppressor T cell.

This study details the suppressive mechanism involved in the antigen-specific suppression of collagen-induced arthritis. Intravenous injection of 500 micrograms of soluble native type II collagen 3 d before immunization with native type II collagen emulsified in complete Freund's adjuvant resulted in animals with decreased in vitro cellular and humoral immune response to native and denatured type II collagen compared with control groups. Control groups were composed of animals preinoculated with saline and type I collagen and established the antigen-specific nature of the observed suppression. Mice with reduced immune responses to type II collagen also were observed to portray little or no erythema and edema associated with collagen-induced arthritis. Adoptive transfer experiments established the requirement of T cells for the suppression of collagen-induced arthritis. Analysis of the phenotype of responding splenic cells in chronic immunotherapeutically suppressed mice in vitro revealed that responding cells were Ly1-2+ (suppressor/cytotoxic) T cells. On the other hand, the cellular phenotype of T cells responding to type II collagen in nonsuppressed collagen-induced arthritic mice was Ly1+2- (helper/inducer T cells). The data indicate that type II collagen-specific T cells are generated on intravenous inoculation of soluble native type II collagen. These cells are observed in type II collagen-immune animals, which are nonarthritic and portray reduced humoral and in vitro cellular immune response to type II collagen. This study suggests that specific suppression of immune responses to type II collagen by T-suppressor cells can be immunotherapeutic in certain forms of arthritis.     

T lymphocyte-mediated suppression of myeloma function in vitro. IV. Generation of effector suppressor cells specific for myeloma idiotypes

BALB/c mice immunized intravenously with syngeneic splenocytes, to which affinity-purified IgA produced by the MOPC 315 myeloma is covalently coupled, develop suppressor T cells (Ts1) that inhibit IgA secretion by MOPC 315 cells after 3-4 d of co-culture. Immunization with M315-coupled splenocytes subcutaneously, followed by administration of a soluble extract of Ts1 cells, leads to the generation of effector Ts that are also idiotype specific and inhibit myeloma function within 1 d. Moreover, effector Ts are Lyt-1-2+, whereas Ts1 are either Lyt-1+2+ or require Lyt-1+ and Lyt-2+ cells to mature into effector Ts in vitro. Such a protocol should be useful for analyzing the interactions that result in the maturation of Ts and in defining the mechanisms of action of Ts, whose effect can be measured on a homogeneous target population and that are specific for a well-characterized myeloma idiotype.     

Evidence for genetic restriction in the suppression of erythropoiesis by a unique subset of T lymphocytes in man.

The suppression of erythropoiesis by lymphocytes from patients with a T cell lymphoproliferative syndrome and pure erythrocyte aplasia has been previously demonstrated. To study the nature of the suppressor cell and possible genetic restriction of this suppression, we investigated a patient with severe anemia, splenomegaly, lymphocytosis, and erythroid aplasia. A 3-mo course of low-dose daily oral cyclophosphamide achieved a complete remission for over 12 mo. The surface phenotype of his lymphocytes was analyzed by means of antibodies to lineage, differentiation, and activation-specific surface antigens. The cells expressed mature T cell antigens T3, T8, and T11, while lacking T1. Immature T cell, B cell, and the monocyte-specific antigen Mo2 were absent, while Mo1, a monocyte-associated antigen not normally expressed on T cells, was present. T10 and Ia expressed as activation antigens were also present. The cells, cryopreserved at diagnosis, were thawed and co-cultured in plasma clot with patient remission marrow samples at T cell/bone marrow ratios of 1:1 and 2:1. There was nearly 90% suppression of erythroid colony-forming unit expression and 60% suppression of erythroid burst-forming unit expression at 2:1 T cell to bone marrow ratios and somewhat less suppression at 1:1. Granulocyte/macrophage progenitor expression was unaffected. Erythroid progenitor differentiation in the marrows of two HLA identical siblings was similarly suppressed. The cells were co-cultured with the marrows of nine nonrelated donors to investigate the potential genetic restriction of this suppression. Colony suppression equal to that observed in the marrow of the patient and his siblings was found in studies of two partially HLA identical individuals. No suppression was detected in marrow co-cultures of two entirely HLA dissimilar individuals. These results show that suppression of erythropoiesis by a unique subset of T8, Mo1, Ia-positive lymphocytes isolated from a patient with lymphocytosis and erythrocyte aplasia is genetically restricted.     

An Ia-like antigen positive null cell leukemia cell line (THP-5) lacking in the stimulating capacity in autologous and allogeneic mixed lymphocyte reaction

A null cell leukemia cell line, THP-5, was established from the peripheral blood of a patient with acute null cell leukemia. THP-5 cells were characterized by the presence of Ia-like antigen and the absence of other cell surface markers examined. Despite the presence of Ia like antigen, THP-5 cells had no stimulating capacity in autologous or allogeneic mixed lymphocyte reaction.     

Macrophage heterogeneity in man. A subpopulation of HLA-DR-bearing macrophages required for antigen-induced T cell activation also contains stimulators for autologous-reactive T cells

Utilizing somatic cell hybridization, we have developed a monoclonal antibody that interacts only with cells of the monocyte/macrophage (M phi) line and not with other myeloid or lymphoid cells. This antibody detects a 120,000-dalton determinant present on 37 +/- 2.8% of the peripheral blood M phi from several (HLA-DR)-disparate individuals and only depicts a subpopulation (approximately 30%) of HLA-DR-bearing M phi from any single subject. Cytolytic removal of this subpopulation of HLA-DR-bearing cells markedly diminished antigen-induced T cell reactivity, a deficiency that can be reconstituted with autologous M phi but not with either their soluble products containing lymphocyte- activating factor or with intact HLA-DR-disparate M phi. Whereas M phi bearing both the 120,000-dalton determinant and HLA-DR serve as effective stimulators for autologous mixed lymphocyte reactions. M phi bearing only HLA-DR determinants do not. However, this latter population of M phi can stimulate proliferation among alloreactive T cells. These studies indicate that the Mac-120 monoclonal antibody detects a subpopulation of HLA-DR-bearing M phi that is required for the genetically restricted presentation of conventional antigen to reactive T cells. Within the M phi population, these Mac-120+ cells constitute the most effective stimulators for autologous mixed lymphocyte reactions.     

Inhibition of antigen-specific T lymphocyte activation by structurally related Ir gene-controlled polymers. Evidence of specific competition for accessory cell antigen presentation

The interaction of nominal Ag with major histocompatibility complex (MHC)-restricted T cells and accessory cells was studied by analyzing the effect of structurally related antigens on the response of antigen- specific MHC-restricted T cell hybridomas. The copolymer L-glutamic acid50-L-tyrosine50 (GT) completely inhibits the response of L-glutamic acid60-L-alanine40-L-tyrosine10 (GAT)-specific, I-Ad-restricted T cell hybridomas to GAT plus accessory cells. This inhibition is specific, as hybridomas of other specificities are not inhibited under identical conditions, and is unique to the GT antigen, as other similar copolymers are not inhibitory. The inhibitory effect is reversible by adding increasing amounts of GAT. Antigen-pulsing experiments localized the inhibition to the level of antigen-presenting cell (APC). GT- prepulsed APC are not inhibitory in cell-mixing experiments and can present other antigens. GT only inhibits the nominal antigen-directed component of a GAT-specific, autoreactive hybrid's response. Together these findings suggest that GT causes inhibition by competing for GAT association at the accessory cell. GT interferes with GAT presentation by an I-Adxb F1 APC to a BALB/c, I-Ad-restricted, but not B10, I-Ab- restricted, T cell hybridoma, and GT inhibits presentation by GAT- prepulsed APC. The implications of these findings for MHC-restricted presentation of antigen are discussed.     

Regulation of in vitro cytotoxic T lymphocyte generation. I. Evidence that killer cell precursors differentiate to effector cells in two steps

The differentiation of cytotoxic T lymphocyte precursor cells (CTL-P) into CTL effector cells is a two-step process. In the first step, na?ve CTL-P (CTL-PN) become activated (CTL-PA) but do not yet have the capacity to kill target cells. CTL-PA can be distinguished from CTL-PN because the former are far less sensitive than the latter to the effects of in vitro-generated suppressor cells. Thus, the addition of suppressor T cells (Ts) to a fresh MLC can totally inhibit the production of CTL from CTL-PN, whereas the same Ts only minimally affect the generation of CTL from CTL-PA. It is not known whether these Ts act directly on CTL-PN or on a helper cell needed for activation to CTL-PA. The production of CTL-PA can take place in allogeneic mixed leukocyte cultures (MLC) treated with the drug pyrilamine, or when heat-inactivated stimulator cells are used. Each of these treatments inhibits the differentiation of CTL-PA to CTL. However, if pyrilamine is removed, a nonspecific MLC-derived signal can induce these CTL-PA to become CTL, even in the presence of significant numbers of Ts. This two step process of differentiation of CTL-P to CTL may be analogous to the way na?ve B cells become antibody-producing cells.     

A human T cell clone that mediates the monocyte procoagulant response to specific sensitizing antigen.

A panel of human purified protein derivative of the tubercle bacillus (PPD)-reactive T cell clones was derived by cloning out of soft agar followed by cultivation on inactivated feeder cells in the presence of interleukin-2. 1 of 4 clones tested was able to mediate an increase in monocyte procoagulant activity (PCA) in response to PPD. All four clones had identical surface marker phenotypes (T4+, T8-) and proliferated in response to antigen. The reactive T cell clone possessed no PCA of its own, but upon being presented with PPD was able to instruct monocytes to increase their expression of PCA. Antigen presentation could be performed only by autologous monocytes; allogeneic monocytes from donors unrelated to the donor of the reactive clone could not present antigen to cells of the clone in a way that would initiate the procoagulant response. Cells of the reactive clone did not mediate increased monocyte PCA in response to Candida, even though peripheral blood mononuclear cells from the donor demonstrated increased PCA to both Candida and PPD. Thus, the PCA response to specific antigen can be mediated by a single clone of cells that shows specificity in the recognition of both antigen and antigen presenting cell.     

Immunoregulatory T cell circuits in man. Alloantigen-primed inducer T cells activate alloantigen-specific suppressor T cells in the absence of the initial antigenic stimulus

Although alloantigen-specific suppressor T cells are generated in MLR, the cellular signals that lead to activation of suppressor T cells as opposed to cytotoxic T cells are unknown. The current study was undertaken to characterize interactions among T cell subsets involved in the generation of suppressor T cells in MLR. Human peripheral blood Leu-2+ (suppressor/cytotoxic) and Leu-3+ (helper/inducer) T cell subsets were activated with allogeneic non-T cells and then examined for their inductive effects on fresh autologous T cells. Fresh Leu-2+ cells proliferated in response to alloantigen-primed Leu-3+ cells and subsequently suppressed the response of fresh autologous Leu-3+ cells to the original, but not third party, allogeneic stimulator non-T cells. Moreover, only Leu-2+ cells that lacked the 9.3 marker, an antigen present on the majority of T cells including precursors of cytotoxic T cells, differentiated into suppressor cells. The alloantigen-specific suppressive effect of Leu-2+,9.3-cells was not mediated by cytolysis of allogeneic stimulator cells, nor could it be explained by alteration of MLR kinetics. Suppression was observed only when activated Leu-2+ cells were added to fresh MLRs within 24 h of initiation of cultures, suggesting that these cells block an early phase of the activation of Leu-3+ cells in MLR. These results indicate that alloantigen-primed inducer T cells can activate alloantigen- specific suppressor T cells in the absence of allogeneic stimulator cells.