Responder cells in the human autologous mixed lymphocyte reaction.

Isolated human T4+ cells proliferate in the autologous mixed lymphocyte reaction (AMLR), whereas isolated T8+ cells do not. However, in the presence of Interleukin 2 or T4+ cells, the T8+ cells demonstrated substantial proliferation. These studies suggest that T8+ cells recognize signals from autologous non-T cells, but require an additional factor for the subsequent proliferative response. Since this stimulus can be provided by T4+ cells, the AMLR appears to constitute an inducer circuit. Different defects in this circuit may be responsible for the common abnormality of the AMLR in different diseases.     

Autologous mixed lymphocyte reaction in man. X. T-T autologous mixed lymphocyte reaction in young and aging humans

T cells upon activation with mitogens or autologous non T cells express surface HLA-DR antigens and are capable of stimulating autologous T cells in the autologous mixed lymphocyte reaction (T-T AMLR). We have examined T-TA AMLR, using T-non T AMLR activated-(TA) T cells as stimulators in young (21-32 yr) and aging humans (62-84 yr). In aging subjects a significantly (p less than 0 . 01) higher proliferative response was observed in T-TA AMLR as compared to simultaneously studied young subjects. In allogeneic MLR, no significant difference was observed between young and aging subjects. The increased T-TA AMLR could be a mechanism responsible for deficient T-non T AMLR reported in aging humans.     

Impairment of the autologous mixed lymphocyte reaction in atopic dermatitis.

The T cell proliferative response to autologous non-T cells is termed the autologous mixed lymphocyte reaction (AMLR). Recent studies have suggested that the AMLR represents an inducer circuit for the activation of T8+ suppressor/cytotoxic effector cells. Since atopic dermatitis (AD) patients are deficient in T8+ cytolytic T cell function, we investigated the AMLR in AD. When sheep erythrocytes were used to separate T cells from non-T cells, the AMLR was found to be significantly decreased (P less than 0.001) in AD patients (n = 11; delta cpm = 1,550 +/- 393) when compared with normal control subjects (n = 13; delta cpm = 25,819 +/- 4,609). To exclude the possibility that these results were an artifact of the sheep erythrocyte separation, T cells were also separated on a fluorescence-activated cell sorter after treatment of peripheral blood lymphocytes with the OKT3 monoclonal antibody. AD T cells separated by the latter method were also found to have a significantly reduced AMLR response when compared with similarly treated normal T cells. Co-culture studies using cells from AD patients and their HLA identical siblings indicated that the defect resided at the responder T cell level rather than at the stimulator non-T cell level. Co-culture studies revealed no evidence for excessive suppressor cell activity resulting in the decreased AMLR. However, enumeration of T cells reactive with the monoclonal antibody T29, which recognizes a subset of T cells proliferating in the AMLR, demonstrated that AD patients (n = 8; % T29 = 2.5 +/- 0.7) had a significantly decreased (P less than 0.001) number of circulating T29+ T cells when compared with normal controls (n = 8; % T29 = 10.4 +/- 0.8). These studies suggest that a deficiency of T4+ T29+ cells contributes to the deficient AMLR in AD and possibly underlies the abnormalities of T8+ effector cells present in this disease.     

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.     

Decreased autologous mixed lymphocyte reaction in myasthenia gravis

Myasthenia gravis is an autoimmune disease in which the autoantigen, the acetylcholine receptor at the neuromuscular junction, is well characterized. As with many other autoimmune diseases, however, the basic cause of immune malfunction is unknown. The autologous mixed lymphocyte reaction (AMLR) involves the proliferation of T lymphocytes when co-cultured with autologous non-T cells and may reflect in vivo mechanisms of immune control. We have measured the AMLR in 22 patients with myasthenia gravis and found the magnitudes of the peak responses to be significantly depressed compared to those of 41 normal healthy controls. Proliferative responses of T cells from myasthenic patients to the mitogens, Concanavalin A and Phytohaemagglutinin-P, were also found to be significantly depressed relative to controls. These abnormal immune cell responses can be, in part, interpreted in terms of defective suppressor cell functions in myasthenia gravis.     

Decreased autologous mixed lymphocyte reaction in Sj?gren's syndrome.

The autologous mixed lymphocyte reaction (AMLR) measures the response of peripheral blood T cells to antigens present on the surface of non-T cells. The AMLR was studied in 25 patients with Sjögren's syndrome (SS). The AMLR was decreased in 15 of 25 (60%) of patients with SS (5,272 +/- 6,738 cpm vs. 14,396 +/- 10,092 cpm for the normal controls, P < 0.001). The AMLR was decreased in 8 of 15 patients with only glandular disease who were not on any systemic medications. Patients with SS and associated disease had lower responses than patients with SS alone. Two patients with pseudolymphoma had absent response. The decreased AMLR correlated with a decreased response to concanavalin A, suggesting a possible abnormality of a T cell subpopulation. There was no correlation between the decreased AMLR and age, focus score, serum immunoglobulin concentration, the titer of antilymphocyte antibody, or phytohemagglutinin response. In allogeneic MLR, SS non-T cells and macrophages stimulated normal allogeneic T cells less well than normal non-T cells and macrophages, suggesting a possible abnormality in the cells that stimulate in the cells that stimulate in the allogeneic MLR.     

Autologous mixed lymphocyte reaction in man. VI. Deficiency of autologous mixed lymphocyte reaction in type I (insulin-dependent) diabetes mellitus

Autologous mixed lymphocyte reaction (AMLR) was examined in the peripheral blood from 20 patients with type I (insulin-dependent) diabetes mellitus. Six of 20 patients demonstrated deficient AMLR when compared to the range for simultaneously studied age and sex matched healthy controls. The kinetics of AMLR with regard to duration of the peak proliferative response was similar to controls, the peak response being on day 6. In allogeneic MLR. T cells from patients responded normally. However, non-T cells from patients were poor stimulators against responder T cells from healthy controls. This study demonstrates a deficiency of AMLR in a subset of patients with type I diabetes that further supports an abnormal immune regulation and might be an important mechanism in the pathogenesis and autoimmune manifestations of type I diabetes.     

Deficiency of the autologous mixed lymphocyte reaction in patients with primary biliary cirrhosis.

In this study we show that patients with primary biliary cirrhosis (PCB) have a marked deficiency in the ability to generate an autologous mixed lymphocyte reaction (AMLR) but have a normal ability to generate an allogeneic mixed lymphocyte reaction (MLR). This deficiency is not due to differences in the time-course of the proliferative response or to an altered response to variable numbers of stimulator cells. The deficiency was consistently found irrespective of the methods used to isolate autologous stimulator cells. Both responder cells and stimulator cells obtained from patients with PBC were similar to normal cells in their ability to generate an MLR in allogeneic normal human serum. In addition, serum from patients with PBC inhibited the ability of normal lymphocytes to generate both the AMLR and MLR to a similar degree, suggesting that the defect of the AMLR in PBC is not due to a serum factor. It has been shown that the responder cell population in the AMLR contains a subpopulation of cells that mediate suppression. Therefore, it is possible that the deficiency of the AMLR may be related to previously described abnormalities of suppressor function in patients with PBC.     

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.     

Lymphocyte transformation induced by autologous cells. V. Generation of immunologic memory and specificity during the autologous mixed lymphocyte reaction.

Lymphocyte proliferation in vitro may follow antigen recognition and serve as a correlate of cell-mediated immunity. Lymphocyte proliferation can also be simulated by nonimmune mechanisms as, for example, following culture with plant lectin, lipopolysaccharides, or staphylococcal protein A (1). The autologous mixed lymphocyte reaction (MLR) refers to the proliferation of T lymphocytes cultured with autologous mon-T lymphocytes (2,3). The purpose of this study was to determine whether lymphocyte proliferation in the autologous MLR results from immune or nonimmune mechanisms. We have shown that the autologous MLR has two classical attributes of an immune phenomenon: memory and specificity.     

The cellular basis of the impaired autologous mixed lymphocyte reaction in patients with systemic lupus erythematosus.

The autologous mixed lymphocyte reaction is impaired in patients with systemic lupus erythematosus. This is because nonthymus-derived (T)-lymphocyte preparations from such patients do not stimulate autologous T-lymphocyte proliferation normally. This defect may explain the impaired generation of suppressor activity in systemic lupus erythematosus and thereby the occurrence of autoantibodies in this disease.     

Defective expression of the 2H4 molecule after autologous mixed lymphocyte reaction activation in systemic lupus erythematosus patients.

Previous studies demonstrated that patients with active systemic lupus erythematosus (SLE), especially those with active renal disease, had a marked reduction in T4+2H4+ suppressor inducer cells in their peripheral blood. However, it was puzzling to find that active SLE patients without renal diseases often had normal percentages of T4+2H4+ cells. In the present study, we attempted to determine whether active SLE patients bearing normal percentages of T4+2H4+ cells had a defect in their expression of the 2H4 molecule on T4+ cells after autologous mixed lymphocyte reaction (AMLR) activation. The peripheral blood lymphocytes (PBL) from 50 SLE patients with normal percentages of T4+2H4+ cells (greater than or equal to 7% in PBL) were studied and the results were compared with those of 40 normal individuals. The density of the 2H4 molecule on T4 cells from normal controls increased during the 7-d AMLR; in contrast T4 cells from patients with SLE, especially those with active SLE, had defective expression of the 2H4 antigen after AMLR activation. Patients with inactive SLE, like normals, showed an increase in the 2H4 molecule after AMLR activation. Moreover, a strong correlation was observed between percent suppression of pokeweed mitogen (PWM)-driven IgG synthesis and the density of the 2H4 antigen on AMLR-activated T4 cells. Serial analysis of patients with SLE showed that the density of the 2H4 antigen expression and the suppressor inducer activity of AMLR-activated T4 cells were inversely correlated with disease activity. Thus, defective expression of the 2H4 antigen may be an important mechanism for the failure of active SLE patients with normal percentages of T4+2H4+ cells to generate suppression.     

Helper cells in the autologous mixed lymphocyte reaction. III. Production of helper factor(s) distinct from interleukin 2

In normal mice, the autologous mixed lymphocyte reaction (AMLR) can activate helper T cells that, in the presence of hapten-modified syngeneic cells, can induce a hapten-specific cytotoxic response. Supernatants from AMLR cultures contain a factor(s) that will mediate a cytotoxic T cell response to hapten-altered self. The AMLR factor is effective in facilitating the generation of cytotoxicity only in those cultures containing both T cells and hapten-altered, syngeneic, nonstimulatory cells. Factor production requires an interaction between Lyt-1+23- cells and non-T cells (the T cells synthesize it). The AMLR factor does not appear to be interleukin 2 (IL-2) because it does not activate thymocytes in the presence of antigen, nor does it maintain an IL-2-dependent cell line or function in co-stimulator assays. For the AMLR factor to facilitate the generation of cytotoxicity, thymic adherent cells are a necessary intermediate. These data suggest that the factor recoverable from AMLR cultures acts early in the cytotoxic pathway, before IL-1 production.     

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.     

A human lymphocyte chemotactic factor produced by the mixed lymphocyte reaction

Lymphocytes migrate to and concentrate at sites of antigenic challenge. Although these cells are seen in delayed cutaneous hypersensitivity reactions and graft rejections, the mechanism by which they are attracted to these sites is poorly understood. In the current study, human mononuclear cells from normal subjects were cultured for 7 days in a standard two-way MLR. Supernatants from the MLR were assayed at various times for the presence of lymphocyte chemotactic activity. Results indicate that a lymphocyte chemotactic factor is produced by the MLR, with maximal activity appearing after 24 to 48 hr of culture. This activity is diminished with longer incubation times. The observed factor is heat labile after a 56 degrees C, 30 min heat treatment and preferentially attracts T lymphocytes. Additional studies demonstrate that MLR supernatants stimulate both directional locomotion (chemotaxis) and enhanced random locomotion (chemokinesis) in T cells. The release of such a human T lymphocyte chemotactic factor during the MLR may represent an important mechanism of attracting lymphocytes to a site of antigenic challenge.     

Production of a cytokine with interleukin 3-like properties and cytokine-dependent proliferation in human autologous mixed lymphocyte reaction

The autologous mixed lymphocyte reaction (AMLR) was assayed in a medium containing fresh autologous serum, by using nylon-adherent stimulator cells and nonadherent responder T cells, which were prepared from human peripheral blood mononuclear cells in the absence of fetal calf serum (FCS) to avoid any sensitization to xenogeneic protein antigens. DNA replication without a background proliferative response was induced by stimulator cells in the responder cells. The addition of monoclonal anti-HLA-DR antibody to the culture or treatment of the responder cells with complement plus anti-T4 but not anti-T8 monoclonal antibody suppressed the AMLR, suggesting that this specific AMLR involves an interaction between HLA-DR antigens and helper/inducer T cells. Regardless of this specific DNA replication, the AMLR generated no production of interleukin 2 (IL-2) and interferon gamma (IFN-gamma), both of which could be found in the allogeneic (allo) MLR. In addition, DNA replication in the AMLR was not inhibited by the addition of specific antisera for IL-2 and IFN-gamma, both of which significantly inhibited the DNA replication in allo-MLR. The AMLR was accompanied by production of a soluble factor, which could stimulate the proliferation of murine interleukin 3 (IL-3)-dependent cell line 32Dcl but not the proliferation of IL-2-dependent cell lines. This factor was also found to be responsible for proliferation of responder nonadherent cells in the AMLR. It strongly stimulated bone marrow cells, as did the murine IL-3. The factor had an Mr range, as determined by gel filtration, of 15,000-28,000, but it did not bind to fast protein liquid chromatography (FPLC)-MonoQ column. Thus, the factor is distinguishable from IL-2 in physicochemical or biological properties, but similar to murine IL-3. These results suggest that the human AMLR may be primarily a phenomenon in which non-T cells mediated by the HLA-DR antigens on the cell stimulate helper/inducer T cells to produce a lymphokine with IL-3-like properties, but no IL-2, which in turn stimulates the factor-dependent cells to proliferate.     

Lymphocyte transformation induced by autologous cells. VIII. Impaired autologous mixed lymphocyte reactivity in patients with acute infectious mononucleosis

The autologous mixed lymphocyte reaction (MLR) is severely impaired in patients with acute infectious mononucleosis. Reactivity returned during the course of convalescence. The allogeneic MLR was not impaired in these patients. B cells from patients with infectious mononucleosis do not stimulate autologous T-cell proliferation, and this observation appears to explain the cellular basis of the impaired autologous MLR in infection. Two explanations for the B-cell defect were considered: (a) the influence of serum factors on B-cell function and (b) the effect of Epstein-Barr virus infection.     

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.     

Characterization of the killer cell generated in the autologous mixed leukocyte reaction

Cytotoxic cells are produced in an autologous mixed leukocyte reaction (AMLR). At 1 wk in culture the AMLR killers are mainly IgG Fc- cells and can kill autologous lymphoblastoid cell lines and Raji and Daudi targets that are usually resistant to natural killer cell (NK) lysis. To define the phenotype of these cells, we have used complement (C')-mediated lysis with monoclonal antibodies (MAb). AMLR killer activity was virtually eliminated by treatment with C' and 9.6 or 4F2, but the cytotoxic cells did not express NK-specific antigens, OKM1 and Leu-7, nor cytolytic T lymphocyte-specific antigens, 9.3 and OKT8. None of the 10 MAb used could significantly block cytotoxicity at the final concentration of 1.5 mcg/ml which is generally sufficient to inhibit CTL. The majority of cells at 1 wk in AMLR cultures stained with T cell activation antigens Ia and 4F2; AMLR killing was proportional to the percentage of 4F2+ cells but unrelated to the expression of Ia antigen.