Suppression of lymphocyte proliferation by a nonspecific factor produced by the Burkitt lymphoma-derived lymphoblast cell line.

The DAUDI lymphoblast cell line derived from a patient with Burkitt lymphoma was obtained from two different sources. One of these (DAUDI-I) produced a factor that inhibited lymphocyte proliferation in both human and mouse regardless of the stimulator, i.e. allogeneic lymphocytes or mitogens. Glutaraldehyde treatment eliminated production of the factor and demonstrated that DAUDI-I was capable of stimulating normal lymphocytes in MLR. A second DAUDI cell line (DAUDI-S) did not produce the inhibitory factor and was capable of MLR stimulation.     

Surface immunoglobulins as targets for anti-immunoglobulin-dependent cell-mediated lysis of B cells

Human K cells are able to lyse human lymphoblastoid B cells in the presence of specific anti-human immunoglobulin isotype antibodies. The human lymphoblastoid B-cell line DAUDI (surface mu- and kappa-chains positive) was lysed in the presence of anti-mu and anti-kappa antisera, or IgG fraction of these antisera, and B-cell-depleted human lymphocytes. Lysis was not induced by anti-isotypic antisera alone or human lymphocytes alone. Lysis was not induced by antisera directed at isotypes which were not expressed on the DAUDI cells. The human lymphoblastoid B-cell line RAJI, which does not express surface membrane isotypes, could not be lysed by the anti-isotype-dependent cell-mediated mechanism. Lysis of DAUDI cells by this mechanism was mediated by Fc gamma receptor-bearing human non-B lymphocytes and required an intact Fc piece in the inducing anti-isotypic antibody. These observations are discussed as a possible role for K cells in the regulation of immune responses by interaction between anti-idiotypic antibodies and idiotype-bearing cells.     

Soluble suppressor of T cell proliferation in primary in vitro response to murine minor histocompatibility antigens

Normal mouse lymphocytes are not capable of mounting a primary cytotoxic T cell response to Mls encoded, non H-2, allodeterminants, although a strong lymphoproliferative response is observed in primary MLR between Mls incompatible cells. In this study it is reported that in the supernatant of primary cultures between AKR macrophages and CBA/H lymphocytes (H-2 identical, incompatible for Mls and other minor antigens) a suppressor of T cell proliferation in MLR is detected. By contrast, a suppressor is not detected in supernatants from primary cultures between BALB/C macrophages and CBA/H lymphocytes (H-2 incompatible, Mls identical), B10.BR macrophages and CBA/H macrophages and CBA/H lymphocytes (syngeneic) suggesting that the production of the suppressor factor occurs only when an Mls incompatibility exists. The suppressive activity of the Mls incompatible culture supernatant upon MLR between incompatible macrophages and lymphocytes is neither antigen specific nor Mls or H-2 restricted, nor is it due to an irreversible toxic effect on T lymphocytes or macrophages. The inhibition of T cell proliferation could be explained by inhibition of IL 2 production, by blocking its union to T cells or by a combination of both effects. Our findings could help explain previous observations that lymphocytes from mice preimmunized with Mls incompatible cells have a depressed proliferative response as well as depressed cytotoxicity against alloantigens.     

Mixed lymphocyte reactions: absence of stimulation by irradiated allogeneic cells

¹³⁷Cs-irradiated mouse spleen cells, in contrast to mitomycin-blocked cells, do not stimulate a mixed lymphocyte reaction (MLR) when cocultured with normal allogeneic lymphocytes. Attempts to find an irradiation dose which blocks the DNA-synthetic capability of alloantigenic cells, but which does not also render them unstimulatory in mixed cultures, have not been successful. Low-level irradiation (100–500 rad) does not completely block mitogen (PHA) reactivity, hence the target cells may be capable of participating in a two-way MLR. High-level irradiation (> 1000 rad), however, thoroughly blocks PHA-stimulated DNA synthesis and eliminates the capacity of these cells to stimulate an MLR. Potentiation of MLR by irradiated cells syngeneic with the reacting cell population was not possible in these experiments. Cell death and lysis in irradiated suspensions occurred during the 72-hr culture period. It is believed that a combination of cell lysis and irradiation-blocked metabolic events normally necessary for the MLR sufficies to explain the poor or nonexistent MLR's obtained in these experiments.     

Suppressor T lymphocyte induction by a factor released from cultured blastocysts

For the analysis of immunologic escape mechanisms of embryos during the implantation period in mice, the effects of culture supernatant of blastocysts on in vitro responsiveness to alloantigen of mice was investigated. Blastocyst-cultured conditioned medium was prepared by culturing late blastocysts of outbred ICR mice for 5 days. The addition of culture supernatant containing four or eight blastocysts to allogeneic mixed lymphocyte culture inhibited both the MLR responses and the generation of cytotoxic T lymphocytes (CTL). Preincubation of the culture supernatant with lymphocytes syngeneic to the responder cells of MLR induced potent suppressor cell activity in the MLR. The supernatant did not inhibit the activity of CTL at the effector phase, but preinduced suppressor cells obtained by incubation of splenocytes with the supernatant showed almost complete suppression of CTL activity at the effector phase. Both of the suppressor cells, active on MLR and at the generation phase of CTL as well as active at the effector phase, had a surface phenotype of Thy-1+ and Ig-. The suppressive material could be extracted from the eight-cell stage of fertilized ova or blastocysts but not from unfertilized ova, indicating that the production of the factor(s) is dependent on the stages of early embryogenesis. These results suggest that the active induction of suppressor T lymphocytes by the factor(s) released from implanted embryos is one of the protective mechanisms from maternal immunologic attack.     

B-cell-derived interleukin 1 (IL-1)-like factor. I. Relationship of production of IL-1-like factor to accessory cell function of Epstein-Barr virus-transformed human B-lymphoblast lines

The relationship of production of interleukin 1 (IL-1)-like factor to accessory function of Epstein-Barr virus (EBV)-transformed B lymphocytes was examined. Six of eight human EBV-B cell lines spontaneously produced and released detectable levels of thymocyte comitogenic factor in vitro, but no interleukin 2 (IL-2) activity. Eight of eight produced fibroblast proliferation activity. Culture supernatants from the two apparent nonproducers of thymocyte comitogenic activity induced the proliferation of the IL-1-dependent murine helper-T-cell clone D10G4.1 in the presence of concanavalin A (Con A). One of the EBV-B cell lines produced a potent inhibitory factor in addition to IL-1-like thymocyte comitogenic and fibroblast proliferation factors. The inhibitory factor inhibited mouse thymocyte proliferative response to Con A, and the proliferation of the IL-2-dependent CT6 cell line, but not human fibroblast growth. All but one of the eight EBV-B cell lines tested, the exception being the line that produced an inhibitory factor, were able to serve as antigen-presenting cells that enabled purified human T lymphocytes to proliferate in one-way mixed lymphocyte reactions (MLR) and in response to Con A. The supernatants of 14 of 16 clones derived from two of the EBV-B cell line cells contained thymocyte comitogenic activity and all 16 stimulated fibroblast proliferation. The phenotypic characteristics of the EBV-B cell lines were heterogeneous, but there was no clear-cut relationship between the cell surface phenotypes of either the cloned or uncloned EBV-B cells and their ability to produce these factors. These studies show that all of the EBV-B cell lines that can function as accessory cells have the capacity to produce an IL-1-like factor.     

Preliminary characterization of human T cell suppressor factor (HTsF) isolated from tonsil cells by monoclonal antibody immunoadsorbence

We have shown that a murine monoclonal antibody, B16G, recognizes a constant region determinant of a T cell suppressor factor (TsF) in DBA/2 mice. The molecule recognized by B16G was shown to be a heterodimer with a native m.w. in the region of 80,000. We now show that B16G also reacts with a similar molecule derived from human lymphoid tissue. Yields of about 100 micrograms could be obtained from the solubilized membranes and cytosol from about 10(10) tonsillar cells by elution of adsorbed materials from B16G immunoadsorbent columns. As with the murine system, the human TsF (HTsF) thus derived was capable of suppressing the mixed leukocyte reaction (MLR) of homologous effector T lymphocytes. However, this same material was not suppressive across the HL-A barrier, that is, when allogeneic effector cells were used in the MLR. Preliminary characterization of the HTsF showed it to have a native m.w. of 80,000 to 90,000 to be composed of a heterodimer with subunit m.w. in the region of 45,000 to 50,000, and to have an associated peptide of approximately 25,000. These observations provide evidence for the conserved nature of genes encoding TsF and correlate with observations of other investigators on the considerable homology between genes encoding the murine and human T cell receptor.     

Establishment of a human T cell hybridoma cell line producing suppressor factor specific for anti-thyroglobulin antibody production

Thyroglobulin (Tg)-binding peripheral blood T cells from a normal individual were fused with a T cell leukemia cell line (Jurkat-AG9) treated by emetine and actinomycin D. Several cell lines were established from thus-prepared human T cell hybridomas. The culture supernatant from one of these lines (Tg-Ts47) whose phenotype was OKT3- 11+ 4+ 8- suppressed the generation of Tg-specific antibody-forming cells from the lymphocytes of patients with Hashimotos' chronic thyroiditis, but not anti-SRBC and anti-ovalbumin antibody production from both autologous and patient lymphocytes. Tg-Ts47-derived factors also bore Tg antigen-binding sites. The suppressive activity of the supernatants was shown in almost all patients lymphocytes tested. This indicated that the supernatants of Tg-Ts47 line contain a suppressive factor specific for Tg antigen and capable of acting across allogeneic barriers.     

Functional differences between cells from MLR colonies grown in soft agar cultures

This report describes a method of growing soft agar colonies of human T lymphocytes activated in the MLR. Two types of colonies were demonstrated: lower colonies grew within the agar layer, and upper colonies grew on the surface of the agar layer. Three days of priming the lymphocytes in the MLR and the use of supernatants of day-3 MLR cultures to provide T cell colony growth factor were necessary for optimal colony formation. Lymphocytes obtained from colonies were grown in long-term (2 to 4 weeks) cultures to generate sufficient numbers of cells to be tested in different functional assays. Cells from both types of colonies exhibited PLT activity. Upper colony cells showed considerably higher CML activity than lower colony cells (mean percent cytotoxicity 37 +/- 5 vs 6 +/- 3). Cells from both types of colonies contained radiosensitive suppressor cell activity that inhibited the primary MLR. The suppressor cell effect of lower colony cells was specific for the original stimulator, but upper colony cells displayed nonspecific suppressive effects. For both types of colony cells, it appeared that suppressive effects were unrelated to the CML activity of these cells. These data suggest that the soft agar colony assay offers a promising approach to separate subpopulations of lymphocytes activated in the MLR.     

Immunotoxins containing recombinant anti-CTLA-4 single-chain fragment variable antibodies and saporin: in vitro results and in vivo effects in an acute rejection model

Immunotoxins containing recombinant human-derived single-chain fragment variable (scFv) reagents (83 and 40) against CTLA-4 (CD152) linked to saporin, a ribosome-inactivating protein, were prepared and tested on CD3/CD28-activated T lymphocytes, MLRs, CTLA-4-positive cell lines, and hemopoietic precursors. Immunotoxins induced apoptosis in activated T lymphocytes and were able to specifically inhibit MLR between T lymphocytes and dendritic cells. The 83-saporin immunotoxin also inhibited the T cell activation in an MLR between T lymphocytes and an EBV-positive lymphoblastoid B cell line. Toxicity tests on hemopoietic precursors showed little or no effects in inhibiting colonies' growth. As the 83 scFv Ab was reactive also with activated mouse T lymphocytes, 83-saporin was tested in a model of tumor rejection consisting of C57BL/6 mice bearing a murine H.end endothelioma cell line, derived from DBA/2 mice. The lymphoid infiltration due to the presence of the tumor was reduced to a high extent, demonstrating that the immunotoxin was actually available and active in vivo. Thus, taking the results altogether, this study might represent a new breakthrough for immunotherapy, showing the possibility of targeting CTLA-4 to kill activated T cells, using conjugates containing scFv Abs and type 1 ribosome-inactivating protein.     

HLA antigen expression at the single cell level on a K562 X B cell hybrid: an analysis with monoclonal antibodies using bacterial binding assays

The influence of different genetic environments on the expression of HLA complex-controlled antigens has been investigated using cell lines with various defects in the synthesis of these molecules and a somatic cell hybrid derived from them. A very sensitive bacterial binding assay allowing simultaneous evaluation of the morphology of a given cell and the quantity of a surface molecule has been developed for these studies. The fetal erythroid cell line K562, the Burkitt's lymphoma-derived cell line DAUDI, and their hybrid DUTKO1 have been employed. K562 and the hybrid, but not DAUDI, expressed HLA-A,B,C heavy chains as detected by the monoclonal antibody W6/32.HL, while two monoclonal antibodies (TU48 and 2BC4) against the supertypic specificities HLA-Bw4 and Bw6 showed no reactivity. The presence of human Ia-like antigens on the cell surfaces was investigated with a panel of eight monoclonal antibodies. K562 cells were completely unreactive, and DAUDI cells gave the expected positive reaction, but about 1% or less of the cells in the DUTKO1 population appeared to express these antigens as well. We discuss possible reasons for the failure to detect HLA-B antigens with monoclonal antibodies and the lack of complete "dominance" of the K562 genome in the hybrid cell line.     

T-rosettes in hemodialysis patients and renal allograft recipients

Subcellular fractions, isolated from the lymphoid cell line IM-1, are capable of stimulating a weak proliferative response in allogeneic lymphocytes. They also stimulate the generation of cytotoxic effector lymphocytes. The proliferative response to subcellular fractions, as measured by ³H-thymidine incorporation, is only one-fourth to one-sixth as great as that to intact IM-1 cells, suggesting that a component(s) synthesized during the mixed lymphocyte reaction (MLR), or a short-lived cellular constituent, may be responsible for the ability of intact cells to stimulate a lymphocyte proliferative response. This component appears to be lacking or in limiting quantity in subcellular fractions, including the soluble fractions. In contrast to the decreased proliferative response to subcellular fractions, the cytotoxic capacity of the stimulated lymphocytes is comparable to that after stimulation by intact IM-1 cells. The data demonstrate that, in this system, cytotoxic effector lymphocytes can be generated in the absence of the extensive proliferative response normally observed in the MLR. The antigenic stimulus responsible for the generation of cytotoxic effector cells appears to reside on intracellular components as well as on plasma membrane. In these reactions, specificity is shown by the failure of the cytotoxic cells to release ⁵¹Cr from autologous target cells. In fact, reactivity of lymphocytes stimulated by subcellular fractions is more specific than the reactivity of cells stimulated by intact IM-1 as judged by their lytic capacity for another target cell, RPMI 4265.     

Suppression of mixed lymphocyte reactions by alloantigen-activated spleen-localizing thymocytes.

Heavily irradiated BALB/c and C57BL/6 mice were injected intravenously with BALB/c thymocytes. At varying times thereafter spleen and lymph node cell suspensions from these animals were treated with mitomycin C and added as regulator populations to mixed lymphocyte reactions (MLR) with syngeneic responder cells. Alloantigen-activated spleen-localizing thymocytes suppressed MLR responses 40 to 95%. Suppressor activitity, manifested as a quantitative reduction in peak proliferative responses, was maximal 4 days after cell transfer. Antigenic specificity for the stimulator cell strain in MLR was not demonstrated. The effect of lymph nodelocalizing thymocytes as regulators in MLR was variable, but in most experiments these cells slightly enhanced responses. We conclude that splenic localization is an intrinsic property of the thymocyte subpopulation capable of suppressing MLR responses, and that the suppressor activity of this subpopulation is substantially enhanced by activation in allogeneic hosts.     

Sex-associated differences in the regulation of immune responses controlled by the MHC of the mouse

The immunologic potential of T lymphocytes and antigen-presenting cells (APC) from male and female mice were compared. Lymphocytes from female mice or from male mice that cannot produce and respond to testosterone (Tfm/y) were more reactive than male lymphocytes to alloantigens in MLR. Spleen cells from Tfm/y mice equipped with estrogen implants showed a higher responsiveness than control Tfm/y to alloantigens. The removal of suppressive adherent cells or the addition of T cell growth factor (TCGF) enhanced the proliferative activity of the cells in the MLR. The responsiveness of female cells to alloantigens, however, remained superior to that observed in male cells. Similarly, in the presence of TCGF, thymocytes from female mice react more effectively than male cells in MLR. In addition, Con A-stimulated spleen cells from female mice produce more interleukin 2 (IL 2) than do spleen cells from males or female mice treated with testosterone. Lymphocytes from immunized mice were tested for their ability to respond to soluble antigens (KLH and OVA) in vitro. Again, female immunocompetent cells respond more vigorously than male cells or cells originating in female mice with testosterone implants. APC from female spleen were more efficient than male APC in initiating a secondary response in primed lymphocytes from either males or female mice. Moreover, castration of male mice enhanced, and treatment of female mice with androgen reduced, the efficiency of antigen presentation. In conclusion, these data suggest that female cells are superior to male cells in immunologic functions that are known to be associated with reactions to and recognition of histocompatibility antigens, i.e., antigen presentation and MLR. Furthermore, our present data indicate that the differential reactivity of immunocytes between male and female mice depends on the hormonal balance of the animal.     

Two distinct class II molecules encoded by the genes within HLA-DR subregion of HLA-Dw2 and Dw12 can act as stimulating and restriction molecules

By using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), we investigated the difference in the HLA class II molecule between HLA-Dw2 and Dw12, both of which are typed as HLA-DR2 serologically. The anti-HLA-DR framework monoclonal antibody (MoAb) HU-4 precipitated an alpha-chain and two beta-chains of human class II molecules from both Dw2 and Dw12 homozygous B lymphoblastoid cell lines. It was demonstrated clearly that an alpha-chain (alpha 1) and one of the beta-chains (beta 1) showed no difference in mobility in the 2D-PAGE between Dw2 and Dw12, but that another beta chain (beta 2) of Dw2 was distinct from that of Dw12 in the 2D-PAGE profile. Thus, MoAb HU-4 precipitated alpha 1 beta 1 and alpha 1 beta 2 molecules from Dw2 and Dw12, and the alpha 1 beta 1 molecule appears to be an HLA-DR2 molecule. The alpha 1 beta 2 molecule, on the other hand, is a class II molecule distinct from those precipitated with anti-DR2, anti-DQw1 (DC1, MB1, MT1), or anti-FA MoAbs. MoAb HU-4 completely inhibited the mixed lymphocyte culture reaction (MLR) between Dw2 and Dw12, but anti-DR2 MoAb HU-30, which reacts only with the alpha 1 beta 1 molecule, did not show an inhibitory effect on the MLR between Dw2 and Dw12. The alpha 1 beta 2 molecule is therefore the molecule which elicits MLR between Dw2 and Dw12. An IL 2-dependent T cell line established from an HLA-Dw12/D blank heterozygous high responder to the streptococcal cell wall antigen (SCW) clearly distinguished the Dw2 specificity from Dw12 specificity expressed on the antigen-presenting cell (APC). Moreover, MoAb HU-4 markedly inhibited the cooperation between the T cell line and APC to respond to SCW. These observations indicate that the alpha 1 beta 2 molecule is recognized as a restriction molecule by the T cell line at the antigen presentation of SCW through APC MoAb HU-30 on the other hand partially inhibited the MLR between Dw2 or Dw12 homozygous cell as a stimulator cell and non DR2 cell as a responder cell. It markedly inhibited the proliferative response of the Dw12/D- heterozygous T cell line to SCW, presented by Dw2+ but Dw12- allogeneic APC, and the peripheral response of Dw2 or Dw12 homozygous peripheral blood lymphocytes to SCW. Thus, two distinct class II molecules encoded by the genes within the HLA-DR subregion of HLA-Dw2 and Dw12 can act as stimulating molecules in the MLR and as restriction molecules in the antigen presentation by APC.     

Fractionation of lymphocyte subpopulations which regulate mixed lymphocyte reactions.

Four days after injection of allogeneic lymphocytes BALB/c splenic T cells suppress proliferation of syngeneic cells in mixed lymphocyte reactions (MLR). Conversely, lymph node cells from the same mice amplify MLR responses. To further characterize these functional subpopulations, alloantigen-primed lymphocyte suspensions from both organs were fractionated by velocity sedimentation at unit-gravity. After fractionation MLR suppressor cells from spleens localized exclusively in rapidlly sedimenting fractions of large cells. MLR suppressor activity of cells from these fractions, as well as that of unfractionated spleen cell suspensions, was abolished by treatment with anti-Thy-1.2 serum and complement. Spleen cell fractions of similar sedimentation velocity also secreted a soluble MLR suppressor into culture supernatants. Although inhibitory of MLR, spleen cells of rapid sedimentation velocity did not suppress responses to T cell mitogens. In marked contrast with the effects of spleen cells, large 4-day-alloantigen-primed lymph node cells had no suppressive activity in MLR. MLR amplifier cells of uncertain derivation were found in fractions of medium sedimentation velocity from both spleens and lymph nodes. Fractionation of alloantigen-primed lymph node cell suspensions did reveal, however, a subpopulation of small cells with MLR suppressor acitivty which was unaffected by treatment with anti-Thy-1 serum and complement. The data thus indicate that large alloantigen-activated lymphocytes are not intrinsically suppressive nor are cells which suppress MLR necessarily large. We consequently conclude that regulation of MLR responses by alloantigen-primed lymphocytes involves a complex interaction between distinct functional subpopulations of cells which are separable both by physical and biologic properties.     

Intraepithelial lymphocytes modulate Ia expression by intestinal epithelial cells

We have demonstrated that although intestinal epithelial cells in fetuses and young rats do not express Ia antigens, in adult rats intestinal epithelial cells do express Ia antigens, as indicated by immunoperoxidase staining with monoclonal antibodies. Ia expression by intestinal epithelial cells appeared to be related to an increase in the number of intraepithelial lymphocytes (IEL). Most of the IEL were T cells and expressed the phenotype associated with cytotoxic/suppressor T cells, and a large number contained cytoplasmic granules. To directly study a possible modulating effect of IEL on intestinal epithelium, an Ia-negative intestinal epithelial cell line (IEC 17) of rat origin was cultured in the presence of supernatants obtained from Con A- or PHA-stimulated lymphocytes. IEL, as well as spleen cells but not bone marrow cells, were able to secrete a factor(s) capable of inducing Ia antigens on IEC 17 cells, as judged by immunoperoxidase staining and radioimmunoassay. Ia-positive IEC 17 cells were detectable after 12 hr and maximum Ia expression was obtained by 48-hr incubation. Persistence of Ia expression by intestinal epithelial cells required the continued presence of Ia-inducing factor in the medium. Lymphocyte proliferation was not essential for the secretion of the Ia-inducing factor(s). The characteristics and the kinetics of secretion of the Ia-inducing factor were similar to that of an interferon-like activity, but not of interleukin 2. Con A-induced supernatants from IEL and spleen cells were also capable of suppressing the growth of IEC 17 cells. The results of this study indicate that IEL, because of their close association with intestinal epithelial cells, may be involved in modulating a variety of epithelial cell functions, including the expression of Ia antigens. This leads us to speculate that Ia-positive epithelial cells, like Ia-positive macrophages and dendritic cells, may be involved in antigen presentation to T lymphocytes.     

Capacity of B cells to function as stimulators of a primary mixed leukocyte reaction

The capacity of B cells to serve as stimulator cells for a primary mixed leukocyte reaction (MLR) was evaluated. Percoll-fractionated B cells were stimulated with lipopolysaccharide and dextran sulfate (L/D) or a B cell stimulatory factor (BSF-1)-containing culture supernatant, and then were fixed before being used as stimulator cells to more precisely define the state of activation associated with MLR stimulatory capacity. It was found that unstimulated B cells or B cells stimulated for 1 day with L/D or BSF-1 were incapable of initiating a primary MLR, whereas B cells incubated for 3 days in L/D were potent stimulators. The differential activity of 1 day L/D- and BSF-1-activated B cells compared with 3 day L/D-activated B cells was not related to the amount of the relevant MHC class I or class II alloantigens on these cell populations, because all three groups had large increments in MHC class II expression in the following order: BSF-1 greater than 3 day L/D greater than 1 day L/D, and had little difference in MHC class I expression. Also, all three populations were capable of stimulating both MHC class I- and class II-specific T cell hybrids. It was concluded that the capacity of 3 day L/D-activated cells to stimulate a primary MLR was due to the elaboration of necessary co-stimulator molecules. We evaluated whether interleukin 1 (IL 1) was the co-stimulator involved. That this was not the case was indicated by two findings. First, 3 day-activated L/D cells failed to express IL 1 activity as measured by a highly sensitive IL 1 assay that utilizes the T cell line D10.G4.1. Second, recombinant IL 1 added to MLR cultures containing 1 day L/D- or BSF-1 activated B cells failed to function as a co-stimulator. In contrast, the phorbol ester PMA was a potent co-stimulator in this system. We conclude from these experiments that appropriately activated B cells can function as stimulators of a primary MLR, and that they elaborate critical co-stimulator molecules, distinct from IL 1, that enable them to function in this regard.     

Effect of anti-beta2-microglobulin on antigen and allogeneic lymphocyte-induced proliferation of human lymphocytes.

The effect of anti-beta2-microglobulin (beta2m) on the mixed lymphocyte reaction (MLR), and an antigen-induced proliferative response was studied. Anti-beta2m IgG and Fab' fragments completely inhibited the MLR. Preincubation of stimulator or responder cells with anti-beta2m suggested that the major effect of anti-beta2m may be on the responder cell population. A clear-cut effect on responder cells was demonstrated by showing that anti-beta2m completely inhibited a MLR in which the stimulator population was a beta2m negative lymphoblastoid cell line. Anti-beta2m also inhibited PPD-induced proliferation of sensitized lymphocytes. The kinetics of this inhibition indicated that anti-beta2m added within the first 18 hr of stimulation was effective in inhibiting the proliferative response. These data are discussed in light of the hypothesis that beta2m may be a subunit of an antigen receptor on T cells.     

Induction by an immunogenic immunomodulating agent of nonspecific T cell suppression of lymphocyte responsiveness in MLR but not of antibody production

Summary Spleen cells derived from BALB/c mice that had been repeatedly immunized with the methanol extraction residue (MER) fraction of tubercle bacilli exhibited a depressed capacity to act as responder cells in allogeneic and syngeneic mixed lymphocyte reactions (MLR). Previously reported studies revealed that such spleen cells are also defective in the in vitro generation of antibodies. In order to determine the nature of the cells responsible for the depressed MLR reactivity, purified populations of splenic macrophages, B lymphocytes, T lymphocytes originating from normal and from MER-immunized mice, and cell culture supernatants were added to MLR mixtures consisting of normal mouse splenocytes. Macrophages originating from MER-immunized mice and their culture supernatants exerted a significantly higher suppressive effect on MLR than that of corresponding preparation from normal mice. Splenic T cells originating from MER-immunized mice and their supernatants also significantly suppressed the MLR response. However, the same T cell populations that were inhibitory in MLR failed to suppress the in vitro generation of antibodies against sheep red blood cells in the presence of either MER or 2-mercaptoethanol. These and previously reported findings indicate that a nonspecific immunomodulating agent, MER, can, under certain conditions of treatment, elicit the induction of nonspecific suppressor T cells for MLR but not for antibody production, and, accordingly, can inhibit cellular and humoral immunological responsiveness by different mechanisms.