Interleukin-2 activity in chronic active liver diseases: response by T cells and in the autologous mixed lymphocyte reaction.

The T cell growth factor, interleukin-2 (IL-2), is a lymphokine which supports the immunoregulatory function of T cells. We measured the production of and response to IL-2 of peripheral blood T cell subsets from patients with chronic active liver diseases (CALD) and other liver diseases (Others) by the proliferative response of the cells activated with phytohaemogglutin P. Both production of and response to IL-2 of T cells from 24 patients with CALD were markedly decreased (P less than 0.001) in comparison with 13 controls. T cells from 10 patients with Others yielded low IL-2 titre (P less than 0.05) and responded to IL-2 in a depressed manner (P less than 0.05). OKT4+ and OKT8+ cells from five CALD patients as well as five controls equally produced IL-2 and responded to it. However, IL-2 production (P less than 0.05) and response to IL-2 (P less than 0.01) of OKT4+ cells from CALD patients were decreased in contrast to those of OKT8+ cells. We also examined the effect of IL-2 on the autologous mixed lymphocyte reaction. A highly significant increase (P less than 0.001) in the proliferative response of OKT8+ cells and unseparated T cells from 15 patients with CALD occurred with the addition of IL-2 although the values were still lower (P less than 0.01) than those of OKT8+ and unseparated T cells from 12 controls. Addition of IL-2 did not result in a significant increase of the reactivity of OKT4+ cells from patients with CALD. These results further delineate the nature of the immunoregulatory aberration in CALD.     

Mouse rosette positive B cells stimulate poorly in the autologous and allogeneic mixed lymphocyte reaction.

Mouse erythrocytes form spontaneous rosettes with a population of B lymphocytes from normal individuals and in the majority of lymphocytes from patients with B cell chronic lymphocytic leukaemia (CLL). We have compared the ability of mouse rosette positive (MR+) cells with mouse rosette negative (MR-) cells and monocytes to act as stimulators in the autologous mixed lymphocyte reaction (AMLR) and allogeneic mixed lymphocyte reaction (MLR). Mononuclear cells from the peripheral blood of healthy individuals were fractionated into T cells, MR+ cells, MR- cells and monocytes. Lymphocyte cultures were harvested on days 6, 8 and 10 and the incorporation of tritiated thymidine was determined. MR- cells and monocytes were potent stimulators in the AMLR and MLR. In contrast MR+ cells, like B cells from patients with CLL, stimulated less in the AMLR and MLR. We conclude that MR+ cells from normal individuals function similarly to cells from CLL in the AMLR and MLR.     

Impaired autologous mixed lymphocyte reaction (AMLR) reactivity of peripheral blood T cell subsets in rheumatoid arthritis

We examined AMLR reactivity of unseparated T cells and CD4+ and CD8+ T cell subsets in peripheral blood from 11 rheumatoid arthritis (RA) patients and 10 healthy controls. T cell subsets were isolated by negative selection using complement mediated cytotoxicity. AMLR reactivity of six patients (designated RA-L was reduced below the range of the controls' responses. Five patients (designated RA-N) exhibited normal AMLR reactivity. We observed impaired AMLR reactivity of CD4+ T cells from RA-L relative to RA-N and healthy controls (P < 0.05). CD4+ T cell reactivity of RA-L was reconstituted to normal with pharmacological doses of recombinant interleukin-2 (IL-2) (100 U/ml). In contrast, CD8+ T cells from RA-L in the presence of 100 U/ml IL-2 exhibited markedly impaired AMLR reactivity relative to RA-N and healthy controls (P < 0.05). Dose-response studies revealed partial reconstitution of CD4 T cells with physiological concentrations of IL-2 (10 U/ml). To examine the possibility that in vivo pre-activation of T cells in RA accounted for the findings, T cells or subsets were cultured alone for 7 days in the presence of 100 U/ml IL-2. A trend toward enhanced reactivity of CD4+ and CD8+ T cells in L-RA relative to N-RA and healthy controls was observed, but the differences were not statistically significant. There was no correlation between reactivity of T cells alone in the presence of IL-2 and AMLR reactivity. The results suggest the possibility that abnormal AMLR reactivity of CD4+ and CD8+ T cell subsets in RA may arise as a consequence of different pathophysiological mechanisms.     

Lymphocyte transformation induced by autologous cells. XI. The effect of age on the autologous mixed lymphocyte reaction

The autologous mixed lymphocyte reaction (MLR) was lower in newborn infants and healthy subjects over 65 years of age than in adults between the ages of 20 and 32. In contrast, the allogeneic MLR, although impaired in newborn infants, was normal in elderly subjects. The degree of impairment of the autologous MLR in elderly subjects was correlated with the impairment in the response of lymphocytes from elderly subjects to phytohaemagglutinin (PHA) and Staphylococcus aureus proteins A (SPA). The percentage of autorosetting T cells and of T cells with the OKT4 phenotype was increased in elderly subjects. These findings are paradoxical as autoreactive T cells in young adults have been reported to be drawn from these two T-cell subpopulations.     

The effect of arginine vasopressin on the autologous mixed lymphocyte reaction.

Arginine vasopressin (AVP) is a nonapeptide that has been shown to be released from the supraoptic and paraventricular nuclei of the hypothalamus during stress. Although noted primarily for its hemodynamic as well as homeostatic properties, AVP also appears to have an effect on the immune system. It may modulate cellular immunity via its enhancement of the autologous mixed lymphocyte response (AMLR), an effect which we have demonstrated to occur over a wide dose range with a maximum at 10(-7) M. The increase in proliferation following a single addition of AVP in a 6-day culture appears to be augmented when the peptide is added daily throughout the same culture period. Enhanced proliferation appears to be a specific response that is influenced by arginine residues in position 8 of this nonapeptide. Having provided evidence for the existence of receptors with moderate affinity for AVP, we suggest a potential modulatory role for AVP in support of the concept of a communication between the neuroendocrine and immune systems. Since various autoimmune conditions may be aggravated by stress, stress-induced release of neuropeptides such as AVP may play an important role in modulating immune regulation of these disease states.     

CD8+/DR+ T gamma cells inhibit the autologous mixed lymphocyte reaction.

The proliferative response of T cells during autologous mixed lymphocyte reactions (AMLR) was affected by depletion of IgG Fc receptor+ T lymphocytes (Tg). Removal of Tg cells resulted in enhanced proliferation, and EA-rosette isolated Tg cells, when added to AMLR cultures as irradiated third components, reduced the uptake of 3H-thymidine by 63-87% in a dose-dependent manner. Negative selection using an avidin-biotin affinity chromatography technique demonstrated that the suppression was mediated by DR+ Tg cells; the major proportion of which also expressed the CD8 antigen. By comparing AMLR supernatants collected from control (lacking Tg) and suppressed (containing Tg) cultures on days 2, 3, and 4, it was established that supernatants from suppressed cultures had significantly reduced levels of cytokine activity. These data indicate that the CD8+/DR+ Tg cells function as suppressor cells during an AMLR and reduce the proliferative response by inhibiting AMLR responder T cells from producing the cytokines necessary for in vitro growth.     

Suppressor cells induced in the human autologous mixed lymphocyte reaction

Theophylline, a phosphodiesterase inhibitor, also reversibly inhibits the SRBC receptor of a subpopulation of human T lymphocytes. We have previously reported that the precursor for the Concanavalin A (Con A) inducible suppressor cell (SC) is found predominantly in the theophylline-sensitive (Ts) population; whereas, the precursor of the mitomycin resistant suppressor cell induced in the allogeneic mixed lymphocyte reaction (MLR) is predominantly in the theophylline-resistant (TR) subset. This study examines the theophylline sensitivity and functional characteristics of suppressor cells induced in autologous MLR (AMLR). Both TS and TR proliferate moderately in AMLR but differ in their ability to form suppressors. AMLR activated Ts and TR cells demonstrate early blastogenesis and weak suppression in the Con A assay system, but suppression in an MLR assay system appeared to be confined largely to the TR subset. AMLR suppressors were mitomycin sensitive. These data illustrate that suppressors generated in allogeneic and autologous MLR have significant functional and phenotypic similarities, and strengthen the association between these two immunologic phenomena.     

Activation of T lymphocytes and autologous mixed lymphocyte reaction in allergic patients

In allergic patients the authors previously observed high proportions of circulating T lymphocytes bearing Ia antigens, assumed to be "activated" T cells. In the present investigation they employed other T cell activation markers (4F2, insulin receptor, MLR4) which differ in the kinetics of appearance upon the surface of stimulated T cells. They report high proportions of Ia and 4F2-positive T cells, normal levels of MLR4-positive T lymphocytes and no insulin binding on T cells. However, T cells of allergic subjects are able to express insulin receptors in PHA-induced culture, such as normal subjects do. The authors conclude that these data, supported by similar observations in autoimmune diseases, indicates differences between in vivo and in vitro features of expression of T cell activation markers. In addition the autologous mixed lymphocyte reaction (AMLR) in atopic patients was studied. The results indicate that AMLR responsiveness is defective in allergic patients.     

Autologous mixed lymphocyte reaction in man. XIII. Characterization of the T-T autologous mixed lymphocyte reaction

In this study we have demonstrated that in the T-TA autologous mixed lymphocyte reaction (AMLR), OKT4⁺ T cells are the major responders; however, in the presence of additional interleukin-2 (IL-2), OKT8⁺ T cells also respond by proliferation. Both OKT4⁺ and OKT8⁺ T cells, activated in the T-non-T AMLR, act as stimulators in the T-TA AMLR. OKT4⁺ T cells activated in the T-TA AMLR suppress the proliferative response of the fresh T-non-T AMLR; control OKT4⁺ cells show no immunoregulatory activity in this system. In contrast, control OKT8⁺ T cells spontaneously suppress the proliferation of the T-non-T AMLR, but activation of OKT8⁺ T cells in the T-TA AMLR does not result in a further increase in the suppressor activity of OKT8⁺ T cells. In summary, in the T-non-T and T-TA AMLR phenotypically similar T-cell subpopulations proliferate but express distinct immunoregulatory functions and perhaps regulate the tempo of the AMLR.     

Peripheral blood lymphocyte populations in chronic liver disease.

Mature T lymphocyte concentrations are reduced, null cell concentrations are increased, and Fc receptor bearing (B and K) lymphocyte concentrations are normal, in the peripheral blood of patients with chronic hepatocellular or cholestatic liver disease. Some null cells can be stimulated by either thymosin or levamisole to form rosettes with sheep erythrocytes. These changes are present in viral, alcohol associated and 'autoimmune' liver disease and are therefore probably secondary phenomena relating to liver damage.     

Lymphocyte proliferation induced by autologous cells. XV. Relationships between the human autologous mixed lymphocyte reaction stimulated by non-T and activated T cells

The human {T:T act} AMLR was characterized in its relationship to the {T:Non-T} AMLR and its validity as a nonxenogeneic antigen induced response was extended. Human T cell lines, established from responding T cells in an autologous mixed lymphocyte reaction (MLR), were maintained in medium containing human serum and interleukin-2 (IL-2). These cells stimulated ³H-thymidine incorporation by autologous T cells and by autologous unfractionated blood mononuclear cells. Freshly activated T cells isolated from an autologous MLR stimulated autologous T cells to a lesser extent could be enhanced by adding IL-2. Twenty-five to 50% of T cells stimulated by activated T cells express the T8 determinant. In contrast, we have previously shown that less than 10% of T cells activated after 6 days in culture with non-T cells express the T8 determinant. The number of T8 bearing cells were increased significantly after 10 days in culture with non-T cells. This suggested that two types of reactions, the {T:Non-T} and {T:T act} AMLR, might occur in sequence when T cells and autologous non-T cells are cocultured: first, the activation of T4 cells by non-T cells, then by the activation of T8 cells by activated T4 cells. Finally, activated T cells can stimulate unfractionated autologous mononuclear cells without prior exposure to sheep erythrocytes or fetal calf serum.     

Monoclonal antibody defined T cell subsets and autologous mixed lymphocyte reaction in Down''s syndrome.

Peripheral blood from 21 non-institutionalized children with Down's syndrome (DS) and 21 age and sex matched simultaneously studied healthy controls, was analysed for monoclonal antibody defined T cells and T cell subsets, using a fluorescence activated cell sorter, and the autologous (AMLR) and allogeneic mixed lymphocyte reaction (MLR). Total T cells (9.6+), OKT4+ (helper/inducer phenotype) and anti-Tac+ (activated T) cells were present in comparable proportions to that observed in the control group. In contrast, the proportion of OKT8+ (suppressor/cytotoxic phenotype) cells were significantly (P less than 0.05) decreased when compared to healthy controls. The proliferative response in the AMLR and MLR were comparable to control group. The significance of these results are discussed.     

Stimulation of rat autologous mixed lymphocyte reaction with xenogeneic sera and their protein preparations.

Autologous mixed lymphocyte cultures were set up from nylon non-adherent T-enriched lymphocytes and mitomycin C-treated spleen cells of individual ACI/N rats and the effect on the reaction (AMLR) of sera in the culture medium was studied with regard to the xenogeneic nature of the sera. Not only foetal calf serum, but also sera of adult human, horse and swine stimulated the AMLR response, but autologous or rat serum did not. Albumin fractions of these sera were also effective in inducing the AMLR response. Furthermore, the presence of xenogeneic serum in the culture medium was also required for the blastogenic response of AMLR-primed lymphocytes against the secondary stimulation with syngeneic spleen cells. Both autologous and xenogeneic sera, however, supported the Con-A response of the same responder cell population as used in the AMLR. These results substantiated our previous finding (Endho & Hashimoto, 1981) and suggest that two signals, one from autologous non-T cells and another from the xenogeneic factor in culture medium, are required to cause AMLR, at least in the rat system.     

Impaired autologous mixed lymphocyte reaction (AMLR) in patients with ataxia-telangiectasia and their family members.

We used the autologous mixed lymphocyte reaction (AMLR) to test T cell function in four patients with Ataxia-telangiectasia (AT), in 11 first-degree relatives and in 20 controls. There was a marked reduction of AMLR in the patients and in three relatives compared to the age-matched controls. In the AT patients the defect in AMLR was intrinsic to the CD4 subpopulation, since exogenous IL-2 did not improve the response of isolated CD4 cells. In contrast to normal controls, pre-incubation of autologous B cells with Epstein-Barr virus (EBV) did not enhance the reduced AMLR in the AT patients and the three first-degree relatives. We conclude that in both patients with AT and in some of their family members there is an intrinsic defect in CD4 T cells. This defect leads to diminished reactivity to EBV infected autologous B cells, and may explain in part the high incidence of malignancies observed in such families.     

Lymphocyte transformation induced by autologous cells: XVIII. Impaired autologous mixed lymphocyte reaction in subjects with AIDS-related complex.

T cells from patients with AIDS-related-complex (ARC) and normal controls were studied for their proliferative response in the autologous mixed lymphocyte reaction (AMLR). Cells from ARC subjects failed to proliferate in autologous MLR compared to normal controls. This defect was not attributable to an altered ratio of T4 to T8 cells, as purified T4 cells from ARC subjects also failed to proliferate in autologous mixed lymphocyte culture. In contrast, T cells from ARC subjects proliferated normally in response to allogeneic non-T cells, and non-T cells from ARC patients stimulated allogeneic T cells as well as did non-T cells from normal controls. Furthermore, there was no significant alteration in the 2H4 (suppressor-inducer) or 4B4 (helper-inducer) subsets of the T4 cell population in these patients. We conclude that an early immunologic defect that accompanies HTLV-III infection is a loss of the T cell response to autologous non-T cells.     

T cell interactions in active rheumatoid arthritis: insights from the human autologous mixed lymphocyte reaction as a model of T cell activation cascade.

The autologous mixed lymphocyte reaction (AMLR) represents the activation, proliferation and differentiation of T cells in response to signals from autologous non-T cells. Using monoclonal anti-Leu8 antibody to isolate subpopulations of human CD4+ and CD8+ T cells, we have investigated the role of these subpopulations in the T cell activation cascade during the course of AMLR. In normal subjects, CD4+Leu8+ cells are necessary for the initiation of the AMLR response, and sequentially lead to activation and proliferation of both CD4+Leu8- cells and CD8+Leu8+ cells. The activated CD8+Leu8+ cells, in turn, induce CD8+Leu8- cells to generate proliferation of the latter cells. Soluble mediators could be involved in the T cell activation cascade induced by the AMLR. Patients with active rheumatoid arthritis have a profound defect in the AMLR. Further analysis indicates that rheumatoid arthritis CD8+ T cells are markedly defective as responding cells in the AMLR. The impaired AMLR response by CD8+ cells cannot be reconstituted with AMLR-derived supernatants from normal T cells. The data suggest that the defective CD8+ T cell function may contribute to the pathogenesis of the disease.     

An increase in peripheral blood Ia-positive T cells in Sjögren''s syndrome correlates with a decrease in the autologous mixed lymphocyte response.

The defective autologous MLR was studied in Sjögren''s syndrome (SS) in relation to Ia+ cells as determined by reactivity with a monoclonal anti-human Ia antibody. By indirect immunofluorescence, the percentage of Ia+ T lymphocytes was increased in nine of 15 patients. There was no correlation with clinical features or drugs. The percentage of Ia+ T cells in the non-T cell preparations was normal. An inverse correlation was found between the percentage of Ia+ T cells and the proliferative response to autologous non-T cells. Removal of Ia+ T cells enhanced both the autologous MLR and the allogeneic MLR. Thus Ia+ T cells contain suppressor cells in the MLR, but this may not be the sole explanation for the defective autologous MLR.     

Factors influencing the autologous mixed lymphocyte reaction in rats: effect of xenogeneic serum protein.

Mixed culture of nylon non-adherent (T-enriched) spleen cells or lymph node cells of ACI/N or F344 rats with mitomycin C-treated autologous spleen cells resulted in the increased DNA synthesis of the responder T cells when they were cultured in medium containing foetal calf serum (FCS) or bovine serum albumin. This autologous mixed lymphocyte reaction (AMLR), however, proceeds little when the cells were cultured in medium containing autologous or allogeneic rat serum, irrespective of the responder:stimulator cell ratio and to the culture period. Furthermore, the AMLR response in FCS was greatly reduced if a relatively large number of macrophages were present in the responder cell population. These results suggest that the AMLR response is unlikely to occur in a physiological condition or in vivo.     

The suppressive effects of monocytes in the autologous mixed lymphocyte reaction.

The response of isolated T cells to autologous non-T mononuclear cells is called the autologous mixed lymphocyte reaction (AMLR). The responding T cells show immunological memory and specificity. This reaction was present in all thirty normal individuals tested. Since the AMLR in the absence of evidence of in vivo excessive lymphoproliferation must somehow be controlled, we have studied the interactions of enriched T cells, B cells and monocytes in culture as possible means of regulation of this reaction. Increased rate of [3H] thymidine incorporation by T lymphocytes were observed when they were cultured with mitomycin-treated autologous non-T cells. This was increased when the stimulating cells were enriched for B lymphocytes and was significantly decreased when the stimulating cells were enriched for monocytes. Addition of monocyte-enriched cells to B-enriched cells in a 1:1 ratio, significantly suppressed the AMLR (P- less than 0.01). Equivalent numbers of monocytes did not suppress T-cell responses to phytohaemagglutinin (PHA). Monocyte-enriched cells were separated from stimulating B-enriched cells by nucleopore (0.6 mu) or dialysis tubing (0.12 mu) in Marbrook chambers. Soluble products released from monocyte-enriched cells also suppressed the AMLR. The significance of the AMLR in vivo is uncertain but it may be important in immunoregulation, monocytes playing an important role.