Cytotoxic T cells generated in the autologous mixed lymphocyte reaction. I. Primary autologous mixed lymphocyte reaction

In the course of the culture of an autologous mixed lymphocyte reaction (AMLR), T cells proliferated in response to autologous non-T cells, and differentiated to cytotoxic T cells (AMLR killers). DNA synthesis was necessary to generate AMLR killers, as the elimination of autoreactive proliferating cells with BUdR and UV light completely abrogated AMLR killer cytolysis. Amlr killers lysed various lymphoid cell lines, including autologous B cell lines, autologous or allogeneic mitogen blasts stimulated by Con A, PHA, or pokeweed mitogen, variious nonlymphoid cell lines derived from human, mouse, or rat, and weakly normal autologous or allogeneic non-T cells. KMT-17, methylcholanthrene-induced rat fibrosarcoma, was the only resistant cell line to have been tested. AMLR killers had characteristics similar to NK cells, Major histocompatibility antigens were not the target antigens for AMLR killers. AMLR killers distinguished the blasts stimulated by alloantigens as self from the blasts stimulated by mitogens as non-self.     

Suppression of a polyclonal B-cell response by supernatants from the murine autologous mixed lymphocyte reaction

Previously, we have demonstrated that supernatants from autologous mixed lymphocyte (AMLR) cultures contain helper factors which can mediate the development of a cytotoxic T-cell response to hapten modified self. In the current study, the effect of AMLR supernatants on the humoral response was explored. BALB/C splenic non-T cells produced a large polyclonal antibody response to lipopolysaccharide (LPS), as measured in a Protein A SRBC plaque assay. Surprisingly, syngeneic AMLR supernatants suppressed the LPS-induced generation of plaque-forming cells. The presence of T cells in the stimulated cultures did not affect suppressor activity. The decreased response was not the result of a shift in kinetics, as maximal activity was observed on Day 4, whether or not AMLR supernatants were added. The AMLR culture supernatants were most effective in suppressing the plaque-forming cell response when added at the initiation of culture. AMLR supernatants added after 24 hr of culture resulted in only about 50% of maximum suppression. Supernatants added at 48 or 72 hr had no effect. Interferon-gamma (IFN-gamma) has been detected in AMLR culture supernatant and has been reported to suppress the development of plaque-forming cells in response to LPS. However, it is unlikely the suppressive activity observed in these studies is due to IFN-gamma. Dialysis of the AMLR culture supernatant against a pH 2 buffer for 24 hr or incubation at 70, 80, or 90 degrees C for 10 min, treatments that inactivate IFN-gamma, enhanced suppression. These results suggest that in addition to cytotoxic-T-cell helper factors, the cellular interactions in the AMLR induces the production of a stable mediator(s) which is able to directly suppress B cells at an early stage of their development into plasma cells.     

In vivo regulation of the murine syngeneic mixed lymphocyte reaction

Previous work from this laboratory has suggested that a CD8+ T suppressor (Ts) cell network regulated the murine syngeneic mixed lymphocyte reaction (SMLR). We have attempted to disrupt this network by the inoculation of anti-CD8 monoclonal antibodies (mAb) in vivo. Intraperitoneal inoculation of three mAbs resulted in a marked increase in the proliferation of CD4+, self-Ia-reactive splenic T cells in vitro to syngeneic, but not to allogeneic, spleen cells. Suppression was not limited to a specific mouse strain as the enhanced SMLR was reproducible following anti-CD8 treatment of three strains of mice. In vivo depletion of CD8+ T cells was not a prerequisite for enhancement of the SMLR as several mAb to CD8 augmented the SMLR independent of their capacity to cause CD8 T cell depletion. Moreover, enhancement of the SMLR could be mimicked in vitro by inclusion of anti-CD8 mAb in in vitro cultures of responder T cells and irradiated Ia+ syngeneic stimulators. Since the in vitro SMLR was enhanced following mAb treatment, it was expected that the in vivo SMLR would also be increased. However, no evidence of increased in vivo autoreactivity could be detected following in vivo treatment with anti-CD8 mAb, indicating that other mechanisms in addition to CD8+ regulatory T cells acted to regulate the in vivo activity of autoreactive T cells.     

Regulation on murine T cell responses to autologous antigens by alpha-fetoprotein

Murine α-fetoprotein (AFP), a major component of fetal and newborn sera, was shown to exert potent immunosuppressive effects on autologous mixed lymphocyte reactions (AMLR) in vitro. Thus, the relatively vigorous proliferative response of newborn CBA/J thymocytes reacting in mixed cultures against adult syngeneic spleen cells was almost totally abrogated by 200 and 100 μg/ml AFP over the 6-day time course studied, with significant suppression still evident in the presence of 10 μg/ml AFP. In contrast, the maximum achievable suppression of parellel allogeneic MLRs was only 40 to 60%. The newborn thymocyte anti-adult syngeneic spleen AMLR was shown to be mediated by an Lyt 1⁺23^? T-cell subset reacting against Ia⁺ adult non-T stimulator cells. Newborn and adult AMLRs resulting from autochthonous T responder/non-T stimulator cell mixtures from individual animals were also found to be highly sensitive to AFP-mediated suppression. The fact that fetal-derived AFP could be shown to efficiently inhibit neonatal thymocyte responses to autologous antigens when tested in vitro in amounts 20 to 50 times lower than the levels present in fetal and newborn sera suggests a potentially important role for endogenous AFP in the regulation of autosensitization during ontogeny.     

Selective suppression of the murine autologous mixed lymphocyte reaction by physiological concentrations of hydrocortisone: effects on cell-surface Ia antigens

Strong, adult (Type II) autologous mixed lymphocyte reactions (AMLR) were observed in cultures of lymphoid cells from both A.TH and A.TL mice. These were suppressed by more than 90% in the continuous presence of 7.5 × 10^?8M hydrocortisone-21-sodium succinate. This concentration of hormone had minimal effects on the allogeneic mixed lymphocyte response (MLR) and the mitogenic response to concanavalin A (Con A). Higher concentrations suppressed all three responses. Treatment of autologous cell mixtures for the first 30 hr with 7.5 × 10^?8M hydrocortisone resulted in a 78% suppression of the AMLR. This was not associated with a detectable decrease in the quantity of Ia antigens on the stimulator-cell surface, as evaluated by the susceptibility of treated cells to antibody dependent, complement-mediated lysis, using [A.TH × B.10M]F₁ anti-A.TL antiserum. Hence, this suppression did not appear to result from an alteration of the antigens putatively associated with stimulation of the AMLR. Separate pretreatment of stimulator and responder cells with 7.5 × 10^?8M hydrocortisone followed by culturing with appropriate companion cells had no major effect on the AMLR. Therefore, low-dose hydrocortisone did not appear to selectively eliminate or permanently inactivate subpopulations of responder or stimulator cells. Rather, it appeared to regulate active cellular processes that are initiated by the coculturing of these cells and are required for the early stages of autologous lymphocyte activation.     

Enhancement of the mixed lymphocyte response in the mouse by addition of thymocytes

Synthesis of DNA by mixtures of mouse lymph node and thymic cells was studied in vitro using mitomycin-treated allogeneic spleen cells as stimulator cells. The tests were performed to see whether there occurs a similar cell synergy during this reaction as has been reported during the in vivo graft-vs-host response.It was observed that mixtures of thymocytes and lymph node cells give higher incorporations of isotope-labelled thymidine than can be explained by a pure additive effect of the two cell populations tested separately. This enhancement of the reactivity was more pronounced using combinations of lymph node cells and medullary thymocytes obtained from cortisone-treated donors. Enhancement was also noted between lymph node cells and spleen cells. Blocking of the capacity of lymph node cells to synthesize DNA by treatment with mitomycin abolished this enhanced activity when mixed with thymic cells. On the contrary, mitomycin treatment of thymocytes did not abolish their capacity to increase the reactivity when mixed with normal lymph node cells. Thymocytes, which were unresponsive to the mitomycin-treated cells for genetic reasons, were also found to increase DNA synthesis when combined with lymph node cells. The mechanism by which thymocytes increase DNA synthesis of lymph node cells is not clear, but the results show that they have to be present during the reaction, since culture medium “conditioned” by thymocytes did not exhibit any enhanced capacity to promote a mixed lymphocyte reaction of lymph node cells.The results are thus in agreement with the findings obtained by others showing that mixtures of lymph node cells and thymic cells yield higher immunological reactivities in vivo against foreign transplantation, antigens than can be explained by a pure additive effect of the reactivities by the two cell populations tested separately. However, in contrast to these findings, the thymic cells do not have to be able to synthesize DNA or to react against the foreign cells in vitro to yield an enhanced response when mixed with lymph node cells.     

The human autologous mixed lymphocyte reaction. I. Suppression by macrophages and T cells

The autologous mixed lymphocyte reaction (AMLR) is a proliferative response of T cells to signals from autologous non-T cells. The AMLR has been an enigma to immunologists because spontaneous proliferation of cells removed from the body is usually substantially less than that observed with a strong AMLR. However, the AMLR is thought to represent an important in vitro function, since it has the attributes of other immune responses, and it is abnormal in a variety of disease states thought to have an immune basis. We reasoned that if the AMLR represented a fundamental immune phenomenon, it should be subject to regulation. In the present study, we present evidence for suppression of the AMLR by macrophages and by T cells. Macrophages inhibited the T cell proliferation to (B + null) cells in a dose-dependent fashion and throughout the time course of the AMLR. Elimination of suppressor T cells by a specific antiserum led to an increase in the AMLR, which was again suppressed in a dose-dependent way by addition of the suppressive T cells. It may be concluded that the AMLR itself is subject to immune regulation and that the suppressive influences observed probably strongly inhibit the AMLR in vivo. Removal of the suppressive principles allows the maximal expression of the AMLR in vitro. We believe that our demonstration of regulation of the AMLR should remove the enigma associated with it and lead to a better understanding of normal cell-cell interactions as well as the basis for abnormalities in a variety of immune-mediated diseases.     

T cell recognition in the mixed lymphocyte response. I. Non-T, radiation-resistant splenic adherent cells are the predominant stimulators in the murine mixed lymphocyte reaction.

The ability of subpopulations of murine spleen cells to stimulate a mixed lymphocyte response (MLR) was studied. It was found that T cells (nylon-nonadherent spleen cells) and B cells [G-10 passed and treated with rabbit anti-mouse brain serum (RAMB) and complement (C)] were poor stimulators of an MLR. In contrast, whole spleen cells or B cells plus adherent cells (RAMB +C-treated spleen cells) produced good stimulation. However, a non-T, radiation-resistant splenic adherent cell (SAC) population was up to 20 to 50 times more efficient as a stimulator of an MLR on a per cell basis than an unseparated spleen population. These SAC were shown to express Ia determinants encoded by genes in I-A and I-E/C. These results suggest that Ia+ SAC may be the predominant stimulating cells in spleen cell populations, and the preferential target for T cell recognition in cell interaction events.     

Altered lymphocyte functions in rats bearing syngeneic Moloney sarcoma tumors. I. Mitogen responses, mixed lymphocyte reactions (MLR) and mixed lymphocyte-tumor reactions (MLTR).

Impairment of mitogen responses to Con A and LPS and of MLR and MLTR was detected in the spleens of rats bearing syngeneic Moloney sarcoma tumors. Depressed responses of both T cell and Ig+ cell populations were observed. During the observation period of 6 to 10 days post-tumor inoculation when maximal T cell-mediated cytotoxicity was observed in spleen and draining lymph node cells, spleen cells showed marked impairment in response to stimuli mentioned above. By contrast, draining lymph node cell activity was either unaltered or somewhat elevated above the level of activity measured in normal control populations. Data presented in this and an accompanying paper strongly indicate that macrophages are activated as immunosuppressor cells in tumor-bearing rats.     

Lymphocyte responses of human neonates to bacterial antigens

Human lymphoid lines derived from normal or neoplastic B cells were assayed for insulin binding. ¹²⁵I-Labeled insulin was allowed to bind to cells. Bound radioactivity which was inhibited with unlabeled insulin was regarded as specific binding. Among 46 lines tested, 43 bound more insulin than normal peripheral B lymphocytes. The majority of the lines resembled activated lymphocytes, with regard to their insulin binding. More mature cells represented by EBV-transformed lines of normal origin, bound more insulin than the less differentiated Burkitt lymphoma lines. However, even the latter bound significantly more insulin than peripheral blood lymphocytes.     

Enhancement of enzymatic production of cyclodextrins by adding polyethylene glycol or polypropylene glycol

Enzymatic production of cyclodextrins (CDs) from soluble starch was studied using either Bacillus macerans or Bacillus ohbensis cyclomaltodextrin glucanotransferase (CGTase). The production yield of CDs was found to be increased up to 1.5–2 times by the addition of low molecular weight polyethylene glycol (PEG 400) or polypropylene glycol (PPG 425) to the reaction medium. Such results were interpreted as being due to a conformational change of the substrate as well as reduction of hydrolytic activity of the enzyme in the presence of these additives.     

The cytolytic T lymphocyte response to trinitrophenyl-modified syngeneic cells. I. Evidence for antigen-specific helper T cells.

Mice were primed subcutaneously with trinitrophenyl (TNP)-modified syngeneic spleen cells. Seven days later, spleen cells from these in vivo primed mice, or spleen cells from naive mice, were co-cultured with TNP-modified syngeneic cells. Spleen cells from the in vivo primed mice demonstrated augmented cytolytic T lymphocyte (CTL) activity. The spleens of these in vivo primed mice contained a population of radioresistant, antigen-specific, helper T cells. Specifically, spleen cells from these mice, after x-irradiation, were able to augment the in vitro CTL response of normal spleen cells to TNP-modified syngeneic cells.     

Stimulation of murine lymphocyte responses by cations

The capacity of the heavy cations Pb, Ni, and Zn to modulate murine in vitro lymphocyte responses was examined. Pb and Ni (100 microM) were shown to enhance the in vitro plaque-forming cell (PFC) response to sheep red blood cells while 100 microM Zn had inhibitory effects. Each metal was able to stimulate the proliferation of murine splenocytes as determined by [3H]thymidine incorporation and autoradiography. The enhancing effect of the metals on the PFC response was observed whether the results were expressed on a per culture or a per cell basis, indicating an actual increase in B-cell differentiation. Both the PFC response and the proliferative response were shown to be sensitive to the type of medium employed (M-199 gave optimum results) and to the presence or absence of 2-mercaptoethanol. As in autologous mixed-lymphocyte responses peak proliferation occurred after Day 5 in culture, was cell density dependent, and required the presence of both T cells and Ia+ cells. Treatment of splenocytes with anti-Thy-1.2, anti-Lyt-1, or anti-L3T4 plus complement completely abrogated the proliferative response, indicating that a Lyt-1+, Lyt-2-, L3T4+ T-cell was required for the induction of proliferation. The data are consistent with the hypothesis that the metals are capable of modifying the immune response directed at self either by directly altering self constituents (class II) or by modulating the autologous T-cell response.     

Suppressor T cells regulate the cytolytic T lymphocyte response to syngeneic tumors induced by murine sarcoma virus (MSV) in the mouse.

Studies were designed to analyze the immune activities of spleen cells from mice previously injected with murine sarcoma virus (MSV) and undergoing the processes of MSV tumor growth and rejection. Fractionation of MSV-primed spleen cells according to cell size by velocity sedimentation at unit gravity showed that MSV-specific cytolytic T lymphocytes (CTL) generated in vivo underwent an apparent transition in size from large to small cells as the tumor regressed. The majority of CTL precursors, however, were invariably recovered among small to medium-sized MSV-immune cells, as revealed to CTL generation in vitro in secondary mixed leukocyte-tumor cell cultures (MLTC). Evidence was obtained for the existence in MSV-immune spleens of two suppressor cell populations capable of inhibiting CTL generation in vitro: one population probably consisted of macrophages and could be removed by treatment with carbonyl iron; the second population was comprised of T cells and inhibited the differentiation of tumor-immune CTL precursors in a selective manner. These results provide a preliminary overview of the mechanisms regulating the generation, differentiation, and activity of tumor-specific CTL in a syngeneic model system.     

Ontogeny of murine B-cell responses to thymus-independent trinitrophenyl antigens.

The ontogeny of B-cell responsiveness to three thymus-independent trinitrophenyl (TNP) antigens has been examined in BALB/c mice in vivo and in vitro. When in vivo splenic plaque-forming cell (PFC) responses to TNP-conjugated lipopolysaccharide (TNP-LPS), Ficoll (TNP-Ficoll), and Brucella abortus (TNP-Brucella) were measured in neonatal and adult mice, a defined sequence of responsiveness was observed. Newborn mice responded well to TNP-LPS, but not to TNP-Ficoll or TNP-Brucella. Neonates injected at 1 day of age responded to TNP-LPS and TNP-Ficoll and mice 5 to 14 days of age responded to TNP-LPS, TNP-Ficoll, and TNP-Brucella. Furthermore, the antigen-reactive populations increased at different rates for the three antigens in the first 2 weeks of life. In vitro experiments confirmed the results obtained in vivo although slightly earlier responsiveness to TNP-Brucella was observed in vitro. PFC inhibition assays with free TNP hapten were performed so that avidity profiles could be examined in neonatal and adult anti-TNP PFC responses. The results clearly demonstrate that once a response becomes detectable in neonatal mice immunized with any of the three TI TNP antigens, fully heterogeneous or “adult-like” responses are found. In addition, experiments comparing avidity profiles in athymic (nu/nu) BALB/c mice and their normal (nu/+) littermates demonstrate that T cells are not required for the generation of fully heterogeneous anti-TNP PFC responses. These results indicate that B cells responsive to different TI TNP antigens mature at different times and at different rates during ontogeny. Late maturation events of such B cells do not include the acquisition of additional V-region specificities as detected in a PFC inhibition assay.