Autologous mixed lymphocyte reaction in man. III. Regulation of autologous MLR by theophylline-resistant and -sensitive human T-lymphocyte subpopulations

Human peripheral blood T lymphocytes were separated into theophylline-resistant (TR) and -sensitive (TS) subpopulations. Proliferative responses TR, TS and unfractionated T cells were studied, using irradiated autologous or allogeneic non-T cells as stimulators. TR cells proliferated vigorously in both autologous and allogeneic mixed lymphocyte cultures (MLC). TS cells. which constitute about 20% of unfractionated T cells, exhibited poor proliferative responses to autologous and allogeneic stimulation. The magnitude of proliferation in autologous and in allogeneic MLC was found to be directly dependent on the number of TR cells in the culture. Mitomycin-C (MMC)-treated TR cells augmented and MMC-treated TS cells suppressed (P less than 0.05) the autologous and allogeneic MLC responses of unfractionated T cells. However, the response of TS cells did not increase in autologous or allogeneic MLC when co-cultured with MMC-treated TR cells. MMC-treated TS cells, when co-cultured with TR cells, suppressed the responses of TR cells (P less than 0.05). The enhancing effect of TR cells was radiation-resistant. Suppressor influence of TS cells, in contrast, was abolished by irradiation (P less than 0.05). These findings demonstrate that TR cells are the responding cell in autologous MLR and augment the MLC responses of unfractionated T cells. TS cells, on the other hand, respond poorly in autologous or allogeneic MLC and suppress the response of TR cells.     

Plaque-Forming Cells in Man

When the immunoglobulin secretion of 172 normal healthy individuals was investigated with the protein-A plaque assay, 12 persons (7%) did not develop any plaque-forming cells (PFC) in cultures of pokeweed mitogen (PWM)-activated unfractionated peripheral blood lymphocytes (PBL), incubated for 6 days. The effect of irradiation on normal PFC responders and non-responders was also investigated; 2000-rad-irradiated non-responder T lymphocytes co-cultured with autologous untreated B lymphocytes restored the PFC response to normal levels. The evidence of a high level of suppressor activity in non-responder T lymphocytes was further demonstrated by the decreased PFC response of normal B lymphocytes co-cultured with untreated non-responder T lymphocytes.     

Synergy in antigen presentation by thyroid epithelial cells and monocytes from patients with graves'' disease

The present study was undertaken to examine the ability of thyrocytes from Graves' patients to present purified protein derivative (PPD) to autologous peripheral blood T cells. Normal human thyrocytes which were pre-cultured with interferon-gamma were able to induce the proliferation of T cells in response to PPD antigen, but unstimulated thyrocytes failed to do. Thyrocytes from Graves' patients on which HLA-DR antigens were expressed have an ability to induce the proliferation of T cells. Thyrocytes from Graves' patients which were pulsed with PPD antigen for 4 h were capable of stimulating proliferation of the T cells. However, the stimulation index of T cells co-cultured with thyrocytes and PPD were significantly lower than that of T cells co-cultured with monocytes and PPD. Sub-optimal numbers of monocytes which by themselves were unable to support T-cell proliferation synergistically augmented antigen presentation by thyrocytes. These results suggest that cellular interactions among thyrocytes, monocytes and T cells may perpetuate immune or autoimmune responses in thyroid tissues from Graves' patients.     

Autologous Mixed Lymphocyte Reaction in Man

Human peripheral blood T lymphocytes were separated into theophylline-resistant (T_R) and -sensitive (T_S) subpopulations. Proliferative responses of T_R, T_S and unfractionated T cells were studied, using irradiated autologous or allogeneic non-T cells as stimulators. T_R cells proliferated vigorously in both autologous and allogeneic mixed lymphocyte cultures (MLC). T_S cells, which constitute about 20% of unfractionated T cells, exhibited poor proliferative responses to autologous and allogeneic stimulation. The magnitude of proliferation in autologous and in allogeneic MLC was found to be directly dependent on the number of T_R cells in the culture. Mitomycin-C (MMC)-treated T_R cells augmented and MMC-treated T_S cells suppressed ( P <0.05) the autologous and allogeneic MLC responses of unfractionated T cells. However, the response of T_S cells did not increase in autologous or allogeneic MLC when co-cultured with T_R cells. MMC-treated T_S cells, when co-cultured with T_R cells, suppressed the responses of T_R cells ( P <0.05). The enhancing effect of T_R cells was radiation-resistant. Suppressor influence of T_S cells, in contrast, was abolished by irradiation ( P <0.05). These findings demonstrate that T_R cells are the responding cells in autologous MLR and augment the MLC responses of unfractionated T cells. T_S cells, on the other hand, respond poorly in autologous or allogeneic MLC and suppress the response of T_R cells.     

Induction of lymphocyte proliferation by antigen-pulsed human neutrophils.

We have investigated whether purified antigen-pulsed human neutrophils can induce a proliferative response in purified resting blood lymphocytes. Neutrophils were pulsed with soluble tetanus toxoid (dose range 25-250 Lf/ml) and co-cultured with autologous lymphocytes that had been depleted of MHC class II expressing cells. The antigen-pulsed neutrophils induced an increase of lymphocyte proliferation which was dependent on the antigen dose and the neutrophil/lymphocyte ratios. Neutrophils were less potent than autologous monocytes in stimulating lymphocyte proliferation. Blocking with a monoclonal antibody to a common determinant of the human MHC class II complex failed to reduce the lymphoproliferative effects and allogenic antigen-pulsed neutrophils were also able to elicit lymphocyte proliferation similar to autologous neutrophils. We conclude that antigen-pulsed neutrophils are able to induce lymphocyte proliferation in a non-MHC-restricted fashion.     

Cellular Immunity in Human Milk

ABSTRACT: The responses of human milk lymphocytes (MIL) to a variety of immunogenic stimuli were studied and compared to those of peripheral blood lymphocytes (PBL) from the milk donors. MIL showed a decreased proliferative response to mitogens and allogeneic leukocytes in vitro but displayed the ability to stimulate alloreactivity equivalent to PBL. Neither pretreatment with cell-free autologous milk nor co-cultured MIL were capable of suppressing the proliferative responses of PBL. Moreover, macrophages isolated from milk and pulsed with soluble antigen or allogeneic cells effectively induced proliferation by peripheral blood T cells whereas the response of milk nonadherent cells to antigen presented by peripheral macrophages was very low. MIL respond better to pathogenic enteric E. coli than PBL but not as well as PBL to Yersinia enterocolitica. Treatment of MIL with monoclonal antibodies cytotoxic for T cells abolished their response to bacterial antigens. Application of an anti HLA class II antigen monoclonal antibody to mixed lymphocyte or lymphocyte-bacteria cultures resulted in substantial inhibition of the MIL response similarly to that of PBL. The relevance of these data to the immunological needs of the neonate are discussed.     

Early events during primary activation of T cells: antigen receptor cross-linking and interleukin 1 initiate proliferative response of human T cells

To study early events during primary activation of human T cells, a simple method was developed which simultaneously allows positive selection of T cells from peripheral blood lymphocytes (PBL) and their polyclonal, antigen receptor-mediated stimulation with anti-T3 monoclonal antibodies. In the absence of accessory cells, T cells activated with matrix-bound OKT3 express high levels of the Tac antigen within 15 h and produce interleukin 2 (IL2). Tac expression was further enhanced by addition of exogenous IL2. However, under these conditions purified T cells were unable to mount a proliferative response, whereas unfractionated PBL proliferated already after 24 h of culture. This unresponsiveness of purified T cells could be overcome by either re-addition of low numbers of autologous accessory cells or semipurified human IL1. As IL1 had no significant effect on Tac expression of T3-stimulated T cells, we conclude from these data that IL1 exerts in addition to its influence on IL2 production an effect, which allows antigen receptor-triggered T cells to enter the cell cycle.     

In vitro generation of adherent mononuclear suppressor cells to Toxoplasma antigen.

Adherent mononuclear cells have been found to suppress the lymphocyte proliferation, of T lymphocytes of patients with various chronic infections, to pathogen-specific antigens. To explore mechanisms involved in the generation of these suppressor cells, we established an in vitro method for the generation of suppressor-adherent mononuclear cells. Adherent mononuclear cells separated from mononuclear cells from subjects with serological evidence of chronic Toxoplasma infection could be induced, by preincubation with Toxoplasma antigen for 8 days, to suppress the proliferative response to autologous mononuclear cells to Toxoplasma antigen (TA) (mean suppression = 47%) and tetanus toxoid (TT) (mean suppression = 39%) compared to the proliferative response of autologous mononuclear cells co-cultured with no antigen. When adherent cells were removed after 1-day culture there was no significant suppression of the lympho-proliferative response to TA or TT. Induction of the adherent suppressor cell depended on the presence of CD4-positive T cells and not CD8-positive T cells. Adherent suppressor cells acted directly on the proliferative response of CD4 cells to antigen. The adherent cells contained 90 +/- 5% esterase-positive cells. In cell-mixing experiments, equal numbers of CD8-positive T cells pretreated in a similar manner did not have a suppressive effect. However, pretreated CD4-positive cells did have a suppressive effect at higher concentrations of cells than found in the adherent cells. Indomethacin did not alter the suppressive effect. These studies demonstrate the induction of adherent suppressor cells in vitro and implicate the macrophage and CD4-positive T cells as the suppressor cells.     

Nonspecific recruitment of lymphocytes in purified protein derivative-induced lymphocyte proliferative response of patients with tuberculosis.

Peripheral blood lymphocytes (PBL) from tuberculosis patients were studied for their in vitro proliferative response stimulated with purified protein derivative (PPD)-tuberculin. Studies were designed to characterize the lymphocytes involved in the PPD-induced proliferation. PPD-responsive lymphocytes were eliminated in PBL by the procedure of cultivation of PBL with PPD in the presence of 5-bromodeoxyuridine and light illumination of the cultured cells. These PBL which lost PPD reactivity were no longer able to proliferate to PPD stimulation but were still capable of proliferation in the presence of both PPD and X-irradiated, autologous fresh PBL or upon addition of culture fluids from PPD-stimulated PBL. In addition, these nonspecifically activated lymphocytes released a soluble factor into the culture fluids which inhibited the migration of leukocytes. It was likely that large numbers of nonspecific T cells were induced to proliferate as a result of the presentation of specific T cells with the antigen PPD. It is suggested that a similar recruitment of lymphocytes by PPD-stimulated T cells takes place in vivo during the establishment of tuberculosis or antituberculous immunity or both.     

Analysis of antigen uptake and presentation by Epstein-Barr virus-transformed human lymphoblastoid B cells

Epstein-Barr virus-transformed human B cells (EBV-B cells), but not resting B cells or B cells activated by T cell-derived factors, have been shown to support the proliferation of tetanus toxoid (TT)-specific autologous T cell clones in response to TT antigen. The accessory cell function of EBV-B cells was compared to that of monocytes with regard to antigen uptake and processing. After an 18-h incubation period with 125I-labeled TT, the amount of radioactivity associated with the cells (approximately 50 ng/10(7) cells) and the percentage of cells containing radiolabeled material (approximately 50%) were equivalent for EBV-B cells and monocytes. Like with monocytes, EBV-B cells pulsed with TT for 18 h or more were equivalent in their capacity to induce T cell proliferation to EBV-B cells to which soluble TT was added for the duration of the culture period. The requirements for antigen uptake and presentation to T cells were similar for both EBV-B cells and monocytes. Both processes were energy dependent, inhibited by cold (4 degrees C), 2-deoxyglucose, and azide, and both required no de novo protein synthesis as they were not affected by pretreatment of the cells with the irreversible protein inhibitor pactamycin . Trypsin treatment of antigen-pulsed EBV-B cells and monocytes followed by fixation for 1 min in 0.03% paraformaldehyde completely abolished the capacity of both cell types to induce T cell proliferation. In both EBV-B cells and monocytes, antigen presentation, but not antigen uptake, was inhibited by the addition of the lysosomotropic agent chloroquine during the antigen-pulse period suggesting that the mechanisms of antigen processing are similar for both cell types. Vacuoles positive for acid phosphatase with an electron microscopic structure similar to that of lysosomes were found in EBV-B cells but not in resting B cells or B cells activated by T cell-derived factors. The present observations indicate that EBV-B cells take up antigen and process it in a fashion similar to monocytes. The presence of lysosomes appears to correlate with the capacity of B cells to present antigen.     

Functional heterogeneity within the human peripheral blood B cell pool engaged in IgE synthesis

Fractionation of human peripheral blood leukocytes (PBL) B cells by differential sedimentation on percoll gradients separates B cell subpopulations which vary markedly in rates of spontaneous IgE synthesis, in their response to identical populations of autologous T cells and in their response to soluble T cell factors. This B cell fractionation technique often reveals the presence of active IgE-secreting cells which are totally suppressed within unfractionated PBL B cell preparations and greatly improves the efficiency of detection of T cell-responsive, IgE-producing B cells in PBL of atopics.     

Macrophage-lymphocyte interaction in response to a bacterial antigen (E. coli).

The interactions of human lymphocytes with autologous macrophages in response to a clinically relevant particulate bacterial antigen (E coli) have been investigated. Macrophages were required as accessory cells for the E coli induced activation of both T and B lymphocytes. Lymphocyte activation was assayed by determining the degree of proliferation (measured by 3H-thymidine incorporation). The proliferative response was dependent on the number of macrophages and on the amount of antigen in the culture. Macrophages briefly exposed to E. coli acquired the ability to stimulate unfractionated lymphocytes, T and B cells in the absence of free antigen. The in vitro kinetics of this reaction has been determined and analysed with respect to polyclonal B cell response and the antigen specific T cell response.     

Pregnancy-associated growth factor. I. A proliferative agent which expands adult and cord T4 cells in unfractionated cultures

Experiments were designed to examine the effects of pregnancy-associated growth factor (PAGF), a substance found in commercial preparations of crude human chorionic gonadotropin (hCG), on unfractionated human cord blood cells (CBC) and adult peripheral blood lymphocytes (PBL) cultured in 5% fetal calf serum (FCS). Comparisons of PAGF-induced [3H]TdR incorporation in nine pairs of simultaneously cultured CBC and PBL with phytohemagglutinin (PHA) and tetanus toxoid (TT) showed that all CBC and PBL responded to PAGF and PHA whereas all PBL and one CBC responded to TT. The rank order of potency for CBC and PBL was PHA greater than PAGF greater than TT. To examine phenotypic changes induced by PAGF, flow cytometry was performed on precultured cells, control cultures, and PAGF-stimulated cultures at 2, 5, 7, and 9 days. The monoclonal antibodies (mAbs) included T3, T4, and T8 (T cells), T9 (transferrin receptor), Tac (IL-2 receptor), 12 (Ia or DR-framework antigen), and T10 (putative activation and/or maturation antigen). PAGF-stimulated cultures had statistically significant increased percentages of T3, T4, T9, T10, and Tac but not T8 when compared to precultured cells and control cultures. PAGF also increased PBL but not CBC Ia. In PAGF-stimulated cultures, CBC had more T3 and T4 cells with increased fluorescence intensity than PBL. Maximal expression of phenotypes usually occurred at Days 7 and 9, 2 days after maximal [3H]TdR incorporation. In comparison to PAGF, PHA-stimulated PBL had earlier expression of these phenotypes but included T8. These data indicate PAGF induces proliferation, activation antigens, and T3 expansion predominantly confined to the T4 subset in both CBC and PBL.     

Age-related impairment of the in vitro antibody response in the human.

We have used trinitrophenyl polyacrylamide beads (TNP-PAA) to induce a primary in vitro antibody response toward TNP in cultures of peripheral blood lymphocytes (PBL) from aged individuals. The response was virtually non-existent whereas PBL from young control individuals were able to respond. Recombination experiments showed that: (1) aged PBL did not suppress the response of young PBL; (2) young T cells enhanced the response of aged (unfractionated or T-depleted) cells; (3) aged T cells were unable to restore the response of young T-depleted cells, in contrast to young allogeneic T cells. Upon stimulation with Concanavalin A (Con A) aged PBL displayed a moderately diminished proliferative response and a normal ability to suppress the anti-TNP response of autologous PBL (in the few responders). However, contrasting with young PBL they could not suppress the anti-TNP response of young allogeneic PBL.     

Alteration of human accessory cell function by heat treatment: role of IL-1 and class II MHC antigens

Heat-treated monocytes (1 hr, 45 degrees C) cannot present soluble antigen or mitogen to purified autologous T cells. This is despite normal viability and normal expression of class II MHC antigens. They do not secrete IL-1 nor stimulate secretion of IL-2 by T cells. Addition of exogenous IL-1 or IL-2 does not, however, reconstitute the response to soluble antigen. Furthermore, even after overnight pulsing with antigen prior to heat treatment under circumstances in which the antigen is known to be appropriately processed, stimulation of T-cell proliferation still does not occur. Thus there appear to be at least two discrete lesions produced by heating: failure of IL-1 production, per se, and intrinsic failure to present previously processed antigen. It is also hypothesized that heat treatment may produce alterations in Ia molecules which specifically disallow transduction of the proliferation signal to T cells.     

Continuous presence of Th1 conditions is necessary for longer lasting tumor-specific CTL activity in stimulation cultures with PBL

The generation of tumor-associated, but self-antigen specific cytotoxic T lymphocytes (CTL) response is possible by vaccination even in patients at the advanced stages of the disease. The in vivo expansion of such CTLs is now the most important objective of the immunotherapy. In human melanoma, we show here that MART-1(27-35)-specific CTLs generated with purified CD8+ cells survive and maintain their activity longer in culture than those CTLs generated by using total peripheral blood lymphocytes (PBL) taken either from patients or from normal donors. When PBL are grown under Th1 conditioning the quantity and quality of CTL with total PBL are comparable with that of the CTLs generated with purified CD8+ cells. For patients either autologous melanoma tumor cells or MART-1(27-35) peptide pulsed autologous DC were used to generate CTL responses. For normal donors MART-1(27-35) peptide pulsed autologous DC were used. For both normal donors or patients, polarization of PBL with Th1 conditioning with interleukin (IL)-12 (250 U/ml) and anti IL-4 antibody 1 mug/ml for 7 days before CTL generation, induced better and longer living CTL response and prevented the expansion of CD4+ T cells that have downregulatory activity. We show that continuous presence of Th1 conditioning in cultures with total PBL generated significantly higher number of antigen-specific CTLs as detected by MART-1 HLA-A2 tetramer staining. The antigen specificity of such CTLs were determined by IFN-gamma secretion in response to target cells bearing the specific antigen. Our observations indicate that Th1 conditioning results in a longer lasting CTL response in vitro and points toward a newer approach for vaccine strategy.     

In-vitro cell-mediated cytotoxicity for autologous liver cells in chronic non-A, non-B hepatitis.

To investigate the possible mechanisms of liver cell injury in chronic non-A, non-B (NANB) hepatitis, peripheral blood lymphocytes (PBL) from 16 patients with chronic NANB hepatitis were incubated with autologous hepatocytes in a microcytotoxicity assay. Significant cytotoxicity was demonstrated in 11 patients. T-enriched lymphocytes exhibited significantly greater cytotoxicity than non-T enriched cells. No significant inhibition of cytotoxicity was observed following preincubation of the liver cells with either monoclonal or polyclonal anti-HBc, or monoclonal anti-HBs, or addition of either purified HBsAg or recombinant HBcAg to the culture, indicating that there was no detectable cross-reactivity in this system between hepatitis B virus (HBV) and NANB-associated antigen(s). Preincubation of the patients' hepatocytes with polyclonal IgG purified from a serum of a patient who recovered from an acute NANB hepatitis, did not significantly alter cytotoxicity. Liver cell surface-bound IgG was detected by immunofluorescence in only two of the patients, a finding consistent with existing evidence of poor antibody responses to both liver membrane and NANB-associated antigens. Control experiments using PBL from allogeneic normal donors exhibited normal cytotoxicity for the patients' hepatocytes supporting the hypothesis that antibody-dependent cell-mediated cytotoxicity (ADCC) is unlikely to play a significant role in this clinical setting.     

Defective handling of mannan by monocytes in patients with chronic mucocutaneous candidiasis resulting in a specific cellular unresponsiveness

Carbohydrate antigens from Candida albicans, essentially mannan, have previously been shown to persist in the serum of some patients with chronic mucocutaneous candidiasis, and to be able to inhibit specifically the candida antigen-induced proliferation of control lymphocytes. Lymphocytes from three out of six patients were shown to be hypersensitive to mannan inhibition. These data were explained by the demonstration of an apparently selective impairment of radiolabelled mannan handling by two patients' monocytes following a normal uptake. This defect was observed both in active and remission phases of the infection suggesting an intrinsic defect of patients' monocytes. In experiments performed with control lymphocytes, it was shown that mannan exerted its suppressive effect by interfering with candida antigen presentation by adherent cells to autologous T lymphocytes. Furthermore, mannan neither was cytotoxic nor induced suppressor T cells. Altogether, these data suggest that the in vivo persistance of mannan, in some patients, is secondary to a primary macrophage dysfunction leading to impairment of specific cellular immune responsiveness.     

Role of Autorosette Forming Cells in Antibody Synthesis in vitro: Suppressive Activity of ARFC in Humoral Immune Response

The role of autologous rosette forming cells (ARFC) in humoral immune responses was studied using an in vitro system. While depletion of ARFCs from PBL resulted in a significant increase of either total IgG or anti-TT IgG, addition of these cells to the system decreased the production of immunoglobulin to a level comparable to that of unfractionated PBL. The majority of the ARFCs reacted with anti-Leu2a and anti-Leu8. In contrast, the majority of non-ARFCs reacted with Leu3a and only 10 % with Leu8 monoclonal antibodies. Stimulation of unfractionated PBL with concanavalin A (ConA) resulted in an increase of the ARFC population. ConA stimulation also increased the number of cells reactive with anti-Leu2 and/ or anti-Leu8. The autorosette population had a higher purine nucleoside phosphorylase (PNP) content than the non-ARFC population. Although the ARFC suppressed synthesis of antibody by B cell in vitro when they were mixed with either autologous or allogeneic B cells, a marked proliferation of non-B cells was evident. We conclude that at least two different subpopulations of T cells are capable of forming rosettes with autologous red blood cells.     

Impaired autologous mixed lymphocyte reactivity in Hodgkin's disease

Summary In patients with Hodkin's disease, the impaired immune reactivity, especially of the thymus dependent system, is well established. This decreased immune response of the lymphocytes from the peripheral blood contrasts to an increased lymphocytopoiesis in the lymphatic organs with a hyperplasia of these tissues. We studied the reactivity of peripheral T lymphocytes from 20 patients with Hodgkin's disease and 26 healthy control persons against autologous and allogeneic non T cells respectively in the mixed lymphocyte culture (MLC). Our experiments show an extremely depressed autologous mixed lymphocyte reactivity (MLR) of T lymphocytes from patients with Hodgkin's disease compared to those from normal donors. In the allogeneic MLC, the proliferation of the patients' T cells was stronger than in the autologous MLC, but significantly lower than the proliferation of normal T lymphocytes when stimulated by normal non T cells. Patients' non T cells stimulated T lymphocytes from healthy donors as well as non T lymphocytes from normals did. Finally, the autologous MLR of normal lymphocytes was significantly suppressed by 18 of 23 sera from Hodgkin's patients when these sera were substituted for normal AB serum in the cultures. These results demonstrate an impaired function of T lymphocytes from patients with Hodgkin's disease in the autologous MLC and the presence of one or more factors in their serum which inhibit the proliferation of normal lymphocytes in the autologous MLC. The role of suppressor cells and their factors will be discussed.Supported by the Deutsche Forschungsgemeinschaft