Detection of autologous mixed lymphocyte reaction responding cells and their precursor frequency in NZB mice

The autologous mixed lymphocyte reaction (AMLR) can be detected in older NZB mice after treatment of the responding cell population with monoclonal anti-I-A^d and complement and supplementation of the culture medium with T-cell growth factor (TCGF) from young animals. The addition of TCGF to cultures containing responding cells alone that had not been pretreated with anti-I-A plus complement resulted in high levels of background proliferation. This is indicative of a high number of preexisting I-A-positive, activated, TCGF-responsive T cells in these mice. These activated cells could also be removed by treatment with anti-I-A antibody and panning on anti-mouse Ig plates, or by BUdR and light killing of those cells proliferating in the presence of TCGF or purified IL-2. Prior treatment of the responding cells with anti-Lyt 2 and complement did not effect the AMLR. An NZB AMLR responding cell line was established using these methods. This line retained haplotype specificity in a proliferation assay. Limiting dilution analysis of the precursor frequency of AMLR responding cells in the nonautoimmune C58 and BALB/C strains in culture medium with TCGF gave a frequency of between 1 in 35,000 and 1 in 88,000. In young, AMLR-positive, NZB mice, supplementation with TCGF yielded precursor frequencies within the normal range. In older NZB mice, the addition of TCGF resulted in increased background proliferation of preactivated, IA⁺ T cells. After removal of these cells with anti-I-A plus complement, AMLR responding cells were found at normal frequency levels when stimulated in the presence of TCGF. In the oldest animals tested (greater than 18–20 weeks), normal precursor frequencies could not be demonstrated even after this treatment, representing a true decline in the AMLR responding cell number. AMLR deficiency in NZB mice appears therefore to be the result of the combined effects of decreased lymphokine production, excessive T-cell activation, and finally decreased numbers of AMLR responding cells.     

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.     

Syngeneic mixed lymphocyte reaction (SMLR) with dendritic cells: direct visualization of dividing T cell subsets in SMLR

Syngeneic mixed lymphocyte reaction (SMLR) has been considered to represent T cell response to self antigens. In this study using stimulator dendritic cells (DC), we analyzed cellular components responding to the syngeneic DC. It was shown that the predominant dividing cells were CD8(+) T cells although the response of CD4(+) T cells was essential for initiation of SMLR. In spite of the vigorous proliferation and expression of several activation markers, these SMLR-activated CD8(+) T cells hardly killed syngeneic targets and most of the CD8(+) T cells produced no interferon-gamma upon restimulation with DC. Furthermore, in SMLR where CD8(+) T cells were absent or inhibited, a considerable proliferation of CD4(-) CD8(-) double negative-T cells that included TCRalpha/beta(+) natural killer-T cells (NKT cells), TCRgamma/delta(+) NKT cells and TCRgamma/delta(+) T cells was observed.     

Surface phenotype of responder cells in the syngeneic mixed lymphocyte reaction in mice

The organ distribution and surface phenotype of SMLR responder cells has been investigated. Nylon-wool-passed spleen cells, which proliferate in response to mitomycin-C-treated syngeneic spleen cells, are Thy 1.2⁺ Ly 1⁺2^?3^?. SMLR responder cells are not confined to the spleen since nylon-wool-nonadherent lymph node cells as well as unfractionated thoracic duct lymphocytes show activity. Responder cells have characteristics of mature T cells since cortisone-resistant thymocytes, but not thymocytes from untreated mice, are capable of SMLR response. In addition, naturally occurring thymocytotoxic antibody (NTA), which in our experiments exhibits cytotoxicity only for thymocytes, does not appear to affect the subpopulation of the T cells which respond in the SMLR.     

Age-related changes, including synergy and suppression, in the mixed lymphocyte reaction in long-lived mice.

The mixed lymphocyte culture reaction represents the in vitro counterpart of the recognition phase of the graft-versus-host reaction, and of allograft rejection. The mixed lymphocyte culture reactivities of lymph node and spleen cells from all strains show a striking decline with advanced age. Furthermore, studies of "synergy" between subpopulations of T cells in the mixed lymphocyte culture reaction suggest that the cells of the recirculating lymphoid pool (T2 cells) in particular display a functional decline. Finally, spleen cells from old mice of appropriate strains inhibit or suppress the mixed lymphocyte culture reactivity of lymph node or spleen cells from young mice.     

The mixed lymphocyte reaction: selective activation and inactivation of the stimulating cells.

Allogeneic cells pretreated for 48 hr with 2 × 10^?6M ouabain have lost the capacity to show the mixed lymphocyte reaction (MLR). Analysis of various combinations of cells in the one-way MLR revealed that this effect was on the stimulating cells and not on the responding cells. Pretreatment of cells from both donors with 10^?7M ouabain caused no change in incorporation of labeled thymidine into DNA during the first 5 days of mixed lymphocyte culture; thereafter, as incorporation by the controls declined, that of the pretreated cells continued to increase. This effect was also on the stimulating cells and not on the responders. The irreversible effects of ouabain are thus either to activate or inactivate the stimulating cells depending on the concentration of the drug; there is little or no effect on the responding cells.     

Induction kinetics of human suppressor cells in the mixed lymphocyte reaction and influence of prednisolone on their genesis

The induction kinetics of human suppressor cells in mixed lymphocyte cultures (MLC) and the influence of prednisolone on the genesis of these suppressor cells is reported. We induced over 1 to 6 days suppressor cells in one-way MLC (MLC-1), the inhibitory activity of which was tested on a secondary MLC (MLC-2), and on responder cells alone, where lymphocytes were obtained from the same lymphocyte donors as for the MLC-1. In four experiments the degree of inhibition ($\text{x}\text{?}\text{±}\text{SE}$) when suppressor cells were induced for 2, 4, or 6 days was 38.5 ± 11.8, 79.5 ± 7, and 85 ± 6%, respectively, compared to 50.5 ± 9.4, 83.3 ± 7.8, and 85.3 ± 9.8% when 500 ng/ml prednisolone was added to the MLC-1. A similar inhibition pattern was observed when the generated suppressor cells were incubated with responder cells only. The inhibitory activity of these MLC-induced suppressor cells was abrogated by irradiation with 3000 R. Suppressor cells apparently are generated in MLCs between Days 1 and 4; furthermore, their genesis is not affected by usual therapeutic concentrations of prednisolone.     

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.     

Autologous or syngeneic mixed lymphocyte reaction in bone marrow and thymic chimera mice

Unfractionated peripheral blood mononuclear (UM) cells from adult donors of known serological status wtih respect to Epstein-Barr (EB) virus were exposed to four or more successive in vitro stimulations with irradiated cells of the autologous EB virus-transformed cell line at a responder: stimulator ratio of 4:1, and effector UM and T cells were prepared after each stimulation. Ten out of fourteen seropositive donors and all four seronegative donors thus tested showed at best moderate cell proliferation over two or three stimulations only and a cytotoxic response which became dominated by non-E-rosette-forming cells active against the K562 cell line but not against EB virus-transformed lymphoblastoid lines. Cocultures from three other seropositive donors gave stronger proliferative responses and yielded effector cells dominated by a polyclonal E-rosette-forming population cytotoxic to the autologous and to certain allogeneic (both HLA-related and -unrelated) EB virus-transformed cell lines as well as to some but not all EB virus genome-negative hemopoietic cell lines of the kind sensitive to natural killer cells. With one other seropositive donor, this same repeated stimulation induced a quite different type of cytotoxic response, selectively amplifying an effector T-cell population which appeared on the basis of target cell specificity and of sensitivity to monoclonal antibody blocking to be both EB virus-specific and HLA-A and B antigen restricted in its function.     

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.     

The autologous mixed lymphocyte reaction in strains of mice with autoimmune disease

We have studied the autologous mixed lymphocyte reaction (AMLR) in 3 strains of mice with autoimmune disease. T cell proliferation to autologous non-T cells occurs in young mice of these 3 strains (as it does in normal mice) but is absent or greatly reduced in older mice of strains with autoimmune disease. Reciprocal mixing experiments revealed that the defect in the AMLR of the older mice resides in the responder T cell population. Further analysis of the cells participating in the AMLR of young mice of the B/W F1 strain revealed that: 1) a Thy 1- and Ly1-positive responder cell was necessary at the start of the culture to initiate the AMLR; 2) the cells present after 5 days of culture contained very few, if any, Ly123 cells in the B/W F1 strain compared with the normal C57BL/6 strain; and 3) the stimulating cell appear to be a macrophage, and an Ia-bearing cell must be present for the reaction to occur.     

Mitogen-stimulated glutaraldehyde-fixed spleen cells: ability to stimulate in the mixed lymphocyte reaction and generate effector cells in cell-mediated lympholysis.

Mouse spleen cells treated with glutaralde lose their stimulating ability in the MLR. If the spleen cells are first converted to a blastogenic state by lipopolysaccharide and subsequently fixed with glutaraldehyde, their stimulating capacity is maintained.     

Blocking of mixed lymphocyte reaction by spleen cells from total lymphoid-irradiated mice involves interruption of the IL-2 pathway.

Treatment with total lymphoid irradiation (TLI) before organ transplantation results in high incidences of donor-specific tolerance. However, the exact mechanism of how TLI induces or maintains tolerance is not known. In many experimental systems of tolerance, lack of IL-2 plays a central role in tolerance induction, as stimulation of immunocompetent cells with Ag in an insufficient IL-2 environment results in tolerance. To examine whether tolerance induction by TLI might involve the IL-2 pathway, we examined how TLI cells affect the ability of immunocompetent cells to produce IL-2 and express IL-2R in the MLR. Responder cells from MLR cultures in which cells from TLI-treated mice were added proliferated 50 to 70% less and produced 68 to 94% less IL-2 bioactivity than responder cells from control cultures. However, coculture of TLI cells into MLR did not alter IL-2R expression on responder cells, as measured by two-color FACS analysis. We found no evidence that TLI cells deleted MLR responder cells. Interestingly, exogenous IL-2 did not restore proliferation of MLR responder cells. These results suggest that TLI may induce tolerance by interrupting the IL-2 pathway in immunocompetent cells. Moreover, that exogenous IL-2 failed to restore immunocompetence suggests that tolerance in the TLI model may be easy to induce and very stable, and provides a rationale for the higher incidences of donor-specific tolerance after TLI treatment.     

Human leucocyte antigens and mixed lymphocyte reaction in severe pre-eclampsia.

Human leucocyte antigens (HLA) and mixed lymphocyte reactions (MLR) were studied in 38 women with severe pre-eclampsia and their husbands. Thirty-nine women with normal pregnancies and their husbands served as controls. Thirty-three of the control women were matched for age and parity with members of the study group. Infants were studied when possible. HLA compatibility was increased in the pre-eclamptic group compared with matched controls and with theoretical estimates for possible matings. The one-way MLR at delivery showed diminished response of maternal to paternal and cord cells in pre-eclamptic women. This reduced maternal reactivity in women with pre-eclampsia may have a role in the illness, and paternal/maternal histocompatibility may be a feature of the severe form.     

Immunoregulatory function of T cells activated in the autologous mixed lymphocyte reaction

This study was undertaken to define the functional properties of T cells stimulated in the autologous mixed lymphocyte reaction (MLR) by purified B cells or macrophages. In preliminary experiments, it was found that T cells that had been cultured with autologous non-T cells inhibited pokeweed mitogen- (PWM) stimulated immunoglobulin synthesis by autologous B cells. In addition, the T cell-mediated suppression was eliminated by x-irradiation and hydrocortisone treatment, was mediated by a mechanism that occurred early in the PWM-stimulated cultures, and did not involve killing of mature immunoglobulin-secreting cells. T cells were then cultured with either autologous B cells or macrophages in order to determine whether such autoreactive T cells had a similar capacity to regulate PWM-induced immunoglobulin synthesis. Although T cell populations stimulated either by B cells or by macrophages suppressed proliferative responses and immunoglobulin synthesis, both these populations of autoreactive T cells provided help for immunoglobulin synthesis that was not significantly different from that provided by fresh T cells. These results suggest that the predominant functional consequence of activation of T cells in the autologous MLR is the generation of suppressor T cells capable of inhibiting immunoglobulin synthesis. Thus, the autologous MLR may represent a negative feedback mechanism for the regulation of the immune response.