Alloantigen presentation by B cells: two types of alloreactive T cell hybridomas, B cell-reactive and B cell-nonreactive

T cell-depleted, Sephadex G-10-passed unstimulated splenic B cells from C57BL/6 mice stimulated splenic T cells from CKB mice to produce IL 2 and to proliferate. The stimulatory ability of the unstimulated B cells was eliminated by 4000 rad irradiation of the unstimulated stimulator B cells. LPS-activated B cells could stimulate responder T cells more efficiently than unstimulated B cells. For further analysis of allostimulation by B cells, we established a series of alloreactive T cell hybridomas. Forty-five percent of these alloreactive T cell hybridomas could be stimulated to produce IL 2 by either macrophage-dendritic cells or unstimulated B cells. Fifty-five percent of these alloreactive T cell hybridomas could be stimulated by macrophage-dendritic cells but not by unstimulated B cells. T cell hybridomas that were not reactive with unstimulated B cells were also nonreactive to LPS-activated B cells. Analysis of two representative I-Ab-reactive T cell hybridoma clones, B cell-reactive clone CB-11.4 and B cell-nonreactive clone HTB-9.3, revealed again that the stimulatory ability of unstimulated B cells was sensitive to 4000 rad irradiation in the activation of CB-11.4 clone and that CB-11.4 could be stimulated more efficiently by LPS-activated B cells than by unstimulated B cells, but HTB-9.3 could not be stimulated by LPS-activated B cells. Thus, there may be two distinct types of T cells in the alloreaction: B-cell-reactive and B cell-nonreactive.     

The role of class II MHC molecules in the activation of class I-reactive T cell hybridomas

To examine the role of Ia molecules in T cell responses to allo-class I major histocompatibility antigens, a series of allo-class I-reactive T cell hybridomas was established. Of 134 T cell hybridomas obtained from the fusion of C3H/HeJm or B10.HTT T cells stimulated with C57BL/6 splenocytes, nine T cell hybridomas were reactive to class I antigens and 126 T cell hybridomas were reactive to class II antigens. Six of the nine IL 2-producing T cell hybridomas were further analyzed: five mapped to H-2Kb and the other mapped to H-2Db. Three of these T cell hybridomas, HTB-157.7, HTB-176.10, and HTB-177.2, could react to the EL-4 cell line that expresses H-2Kb and H-2Db class I antigens but lacks class II I-Ab molecules. Furthermore, the activation of these three T cell hybridomas with C57BL/6-derived splenocytes was not blocked by either anti-I-A or anti-L3T4 antibody. In contrast, the other three T cell hybridomas, CB-127.6, CB-221.7, and HTB-102.7, failed to react with EL-4 but reacted with the LB cell line which expresses class I (H-2Kb, H-2Db) and class II (I-Ab) molecules. Although class II molecules were required for activation of the latter clones, there was no apparent I-A allele specificity, suggesting that a relatively nonpolymorphic Ia determinant was involved. The activation of the three latter T cell hybridoma clones with C57BL/6 splenocytes could be blocked completely by either anti-I-A or anti-L3T4 antibody. The data are interpreted in terms of possible T cell receptor models for recognition of class I with nonpolymorphic class II determinants.     

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.     

Activation requirements for normal T cells: accessory cell-dependent and -independent stimulation by anti-receptor antibodies

We have examined the requirements for the activation of normal T cells by two anti-T cell receptor antibody preparations, including a rabbit antiserum, R3497, which binds to all normal T cells, and a rat monoclonal antibody, KJ16-133, which binds to about 20% of T cells. The requirements for stimulation of T cells by both antibodies were similar. Soluble antibodies in the absence of accessory cells (AC) failed to induce either proliferation or the expression of IL 2 receptors, and the addition of either IL 2 or PMA failed to synergize with these soluble antibodies for an AC-independent proliferative response. Activation could only be achieved in the presence of Fc receptor-positive AC, although Fc receptor expression alone appeared not to be sufficient for AC activity because some Fc receptor-positive cells did not function in this capacity. Activation with anti-receptor antibody conjugated to Sepharose 4B beads could be demonstrated in the presence of some exogenous cofactors, such as IL 2 and PMA, but not in the presence of recombinant IL 1. When activation by soluble antibody plus AC was compared to activation by bead-conjugated antibody + recombinant IL 2, it was found that the former favored the stimulation of Lyt-2+ cells. The effects of the addition of anti-L3T4 monoclonal antibody was also examined in this system. Anti-L3T4 inhibited the response of L3T4+ cells when used in the presence of Ia+ as well as Ia- AC, and it also inhibited activation in a system in which KJ16-133 conjugated to Sepharose was used in the absence of AC. Because anti-L3T4 had an inhibitory effect in the presence of Ia- AC as well as in the absence of any AC, it is concluded that L3T4 does not necessarily function by interacting with Ia on the surface of AC, and may directly transmit down-regulatory signals when bound by anti-L3T4.     

Two roles for Ia in antigen-specific T lymphocyte activation

In this study we examined the mechanism by which a PPD-specific murine T cell hybridoma, 8B2, recognized PPD associated with antigen-presenting cells (APC) in a manner genetically restricted by I-Ad. It was found that PPD-pulsed APC that were glutaraldehyde-fixed and treated with anti-Ia monoclonal antibody (abbreviated as PGM) were unable to stimulate the 8B2 T cells, as expected, due to inhibition caused by antibody binding to the Ia. However, addition of non-antigen-treated, glutaraldehyde-fixed APC (abbreviated as G) to cultures containing 8B2 T cells and PGM restored T cell activation, as determined by IL 2 production. This second non-antigen-specific function provided by the additional APC, G, was attributed to Ia and could be substituted by APC plasma membranes and by soluble membrane extracts. Genetic restriction analysis in which a variety of Ia-positive and Ia-negative cell lines and B cell blasts from different mouse strains were used as PGM or as G showed that each APC provided different Ia determinants that were specifically recognized by the T cells. PGM cells had to express I-Ad in order to present the PPD determinant, whereas the non-antigen-specific function was specific for I-Ad or I-Ab. These results suggest that the anti-Ia antibody does not interfere with the PPD/I-Ad-specific determinant bound by the antigen-specific T cell receptor, but prevents a second non-antigen-specific interaction with another region of the Ia molecule, which is provided by G. These two roles for Ia (antigen-specific and non-antigen-specific) were also found for activation of normal polyclonal PPD-specific T cell responses; thus they are not unique to the 8B2 T cell, but are generally applicable. In addition, T cell interactions with PGM and with G each provide different intracellular activation signals. This was determined by substituting the PGM or the G with either the tumor promoter phorbol 12-myristate 13-acetate (PMA) or the Ca++ ionophore, ionomycin. It was found that 8B2 T cells cultured with PGM and ionomycin, but not with PGM and PMA, were activated for IL 2 production. Neither PMA nor ionomycin in conjunction with G resulted in T cell activation. Taken together, these results indicate that 8B2 T cell activation involves APC Ia antigens in two different ways: one is to contribute to the presentation of the foreign PPD antigen, and a second is a non-antigen-specific Ia-T cell interaction necessary to provide additional intracellular activation signals.(ABSTRACT TRUNCATED AT 400 WORDS)     

T lymphocyte heterogeneity in the rat. III. Autoreactive T cells are activated by B cells

Evidence has been presented to show that CD4+ autoreactive T cell lines (ATs)2 in the rat require periodic stimulation with syngeneic spleen cells for in vitro proliferation. This proliferation can be blocked by treatment of the stimulator (spleen) cells with mAb to Ia antigens. Although ATs are Ia+ and can activate the allogeneic MLR, they fail to be autostimulatory. Fractionation of the spleen cells revealed that ATs can be stimulated with B cells and not by macrophages, although the latter were efficient in several accessory cell functions, including antigen presentation, lectin-dependent T cell activation and allogenic MLR response. Moreover, B cells proliferated and differentiated in response to AT cells. These data are compatible with a model in which ATs respond to hitherto undetermined B cell membrane antigen(s) in association with MHC class II antigens. These results may have important implications in understanding autoimmune responses.     

The regulatory role of sialic acids in the response of class II reactive T cell hybridomas to allogeneic B cells

Two different kinds of alloreactive T cell hybridomas were established in previous experiments. One is reactive and the other is nonreactive to allogeneic I-A region-associated membrane antigen (mIa) on B cells. In the present experiments the difference between these hybridomas were analyzed by using representative clones, B cell mIa-reactive clone CB-11.4, and nonreactive clone HTB-9.3. Unresponsiveness of HTB-9.3 clone to allogeneic B cells could not be due to the inability of B cells in interleukin 1 production or the density of mIa molecules on B cells. HTB-9.3 clone could respond to C57BL/6 mouse B cells treated with neuraminidase (Nase), and Nase-treated HTB-9.3 clone could respond to normal B cells from C57BL/6 mouse, indicating that sialic acid on both B cells and HTB-9.3 clone plays a regulatory role in the alloreactivity of the clone. In response to B cells from C57BL/6 mouse, T cells from C3H/He mouse spleen showed similar reactivity to HTB-9.3 clone; that is, T cells could respond to Nase-treated B cells, and Nase-treated T cells to B cells, and T cells primed with C57BL/6 spleen cells in vitro showed similar reactivity to CB-11.4 clone. These results suggest that HTB-9.3 clone represents virgin T cells and CB-11.4 clone-primed T cells at least in alloreactivity. Anti-L3T4a was shown to block alloreactivities of both T cell hybridomas and splenic T cells against B cells more efficiently than against splenic adherent cells. These results suggest that L3T4a on T cell plays more important role in allogeneic response to B cells than to splenic adherent cells.     

Influence of accessory cell and T cell surface antigens on mitogen-induced IL 2 receptor expression

We have assessed the inhibitory effects of various monoclonal antibodies on the expression of the IL 2 receptor. Anti-LFA-1, but not anti-Ly-2, markedly inhibited the induction of the IL 2 receptor on the Ly-2+ subset. T-depleted spleen cells, L cells, and B lymphoma cells all functioned as potent accessory cells (AC) for the induction of the IL 2 receptor on L3T4+ T cells. Anti-LFA-1 inhibited the induction of the IL 2 receptor irrespective of the type of AC used. Anti-L3T4 only inhibited the induction of IL 2 receptor expression when L cells were the source of AC. The inhibitory capacity of anti-L3T4 was not related to the expression of Ia on the AC population, because the magnitude of inhibition was comparable in cultures containing either Ia+ or Ia- L cells, whereas no inhibition was seen with either Ia+ or Ia-B lymphoma cells. We conclude from these studies that LFA-1 plays a critical role in mitogen-induced activation of both T cell subsets by promoting both T-AC and T-T interactions. Although anti-L3T4 can inhibit T cell activation in the absence of the recognition of Ia, the mechanism of inhibition and the proposed target molecule for L3T4 on the AC or the T cell have not been determined in our studies. A number of different models for the function of this cell surface antigen are discussed.     

Characterization and antigen-presenting function of a murine thyroid-derived epithelial cell line

We have developed and evaluated the antigen-presenting function of a murine thyroid-derived epithelial cell line M.5 in order to further investigate the possible role of the thyroid follicular epithelium in the inductive phase of autoimmune thyroiditis. M.5 cells did not express class II major histocompatibility complex encoded (Ia) antigens constitutively, but these could be readily induced with interferon-gamma. We found that Ia expressing M.5 cells were ineffective in stimulating T cell proliferation when tested in a 4-day primary mixed leukocyte reaction (MLR). However, significant T cell stimulation was obtained when phorbol myristate acetate (PMA) was added either to the M.5-T cell co-cultures, or for a brief period to the M.5 cells prior to adding the responder T cells. Cytofluorographic analysis of M.5 cells disclosed that PMA did not significantly alter the expression of Ia antigens. Additional experiments indicated that interleukin 1 (IL-1) was unlikely to represent the co-stimulatory factor generated by PMA. This was based on the observations that M.5 cells failed to secrete significant IL-1 either spontaneously, or in the presence of various stimuli, and that murine recombinant IL-1 failed to substitute for PMA in the activation of T cells. The nature of the co-stimulatory signal is as yet unknown. We conclude from these experiments that a pure population of thyroid-derived epithelial cells may be able to function, under the described circumstances, as antigen presenting cells.     

Antigen-presenting cells that induce anti-H-2K T-cell responses: Differences in stimulator-cell requirements for induction of proliferation and cell-mediated lympholysis

Splenic T or B cells, which have been depleted of adherent cells by passage through Sephadex G10 columns, fail to stimulate allogeneic lymph-node cells (LN) in primary mixed lymphocyte reactions (MLR) both when the stimulating antigens are H-2 plus Ia and H-2K only. This failure cannot be ascribed to lack of viability of G10-passed cells, since by dye exclusion they are 95 percent viable and can be induced to proliferate in vitro by exposure to LPS or allogeneic cells. Stimulation of MLR activity could be restored by addition of small numbers of plastic-adherent spleen cells (SAC) which had to be syngeneic with the G10-passed stimulator cells. Further, SAC alone without G10-passed cells induced MLR activity which was, on a cell-for-cell basis, 40 times more effective than that induced by unfractionated spleen cells. If the SAC were first depleted of Ia⁺ cells, no stimulation was obtained. This result was observed both in cases where responder and stimulator strains differed across the entireH-2-gene complex and in a mutant-wild type combination (CBA and H-2^km1) in which the difference between the two strains has been mapped to theK region only. These results indicate that Ia⁺ SAC contain a subset(s) of cells which are responsible for stimulation in MLR, regardless of whether the alloantigenic differences involve either Ia or H-2K. In contrast to the inability of G10-passed splenic cells to stimulate MLR activity, these cells were able to stimulate CTL from cytotoxic T lymphocyte precursors (CTL.P) in combinations where the antigenic differences between responder and stimulator were at the entireH-2 complex or atH-2K only. However, SAC were more potent stimulators of cell-mediated lympholysis (CML) activity on a cell-for-cell basis. Thus, either CTL.P can be stimulated by nonadherent spleen cells or they are specifically sensitive to a small subpopulation of contaminating cells that cannot readily be removed by G10 passage.     

Stimulator requirements for primed alloreactive T cells: macrophages and dendritic cells activate T cells across all genetic disparities

The cellular requirements for stimulating primed alloreactive T cells have been investigated. In vitro-primed secondary alloreactive cells, long-term lines, and Ly 1+2- noncytolytic clones which reacted with allo-H-2K, D, or Mls (M locus) antigens were tested. The data indicated that a specialized antigen-presenting cell such as a macrophage or a dendritic cell was required for stimulating primed alloreactive cells across all the genetic disparities tested. B and T lymphocytes were ineffective stimulators. The stimulator requirement for secondary and Ly 1+2- clone responses was heterogeneous, since both macrophages and dendritic cells were effective stimulators. Thus, the allostimulator requirement for inducing proliferation and mediator secretion by the primed T-cell populations closely paralleled the requirement for stimulating unprimed populations. The only exception found was the peritoneal washout population, which did not stimulate a primary response but did stimulate secondary responses. The failure of peritoneal macrophages to stimulate a primary response was shown to be due to an inhibitory pathway which did not occur when the responding population was alloantigen primed.     

The CD28 ligand, B7, enhances IL-2 production by providing a costimulatory signal to T cells.

Previous studies demonstrated that a human pre-B acute lymphoblastic leukemia cell line, NALM-6, failed to stimulate a primary MLR, despite expression of class II MHC and adhesion molecules. Here we demonstrate that this is the result of the fact that NALM-6 cells do not express the ligand for CD28, namely B7. NALM-6 transfectants that expressed high levels of B7 gained the capacity to stimulate IL-2 production by class II MHC molecule-specific alloreactive T cells and to costimulate a polyclonal population of purified T cells cultured with immobilized anti-CD3 mAb. In the presence of PMA, NALM-6 cells transfected with B7 polyclonally stimulated T cells in a cyclosporine A-resistant fashion, a property previously attributed only to agonistic anti-CD28 mAb. The gain of these functions could not be explained solely by an increased capacity of the transfectants to form conjugates with T cells, suggesting that the CD28/B7 interaction transduces a costimulatory signal in T cells.     

The role of Ia molecules in the activation of T lymphocytes. II. Ia-restricted recognition of allo K/D antigens is required for class I MHC-stimulated mixed lymphocyte responses

The role of Ia molecules in the T cell proliferative response to class I (H2K/D) MHC alloantigens was examined. Proliferation in response to allo-K/D antigenic stimulation, but not to allo-Ia, was markedly inhibited by the addition of monoclonal anti-responder Ia antibodies to cultures in the absence of C. This anti-Ia blocking was observed in responses against both allelic and mutant class I antigens. Partial blocking was observed by using an anti-I-A or anti-I-E monoclonal antibody alone, whereas marked inhibition was seen with these two reagents together when the proliferating cells derived from a responder strain expressing both IA and IE gene products. Syngeneic Ia molecules appear to function as restriction elements, because they are required even in the presence of a source of exogenous second signal, phorbol myristic acetate or IL 1. The K/D-specific response required a responding cell that bears both Lyt-1 and -2 antigens, whereas responses generated to alloantigenic differences, including the I region, require only an Ly-1+ cell. The implications of these data with respect to the repertoire of the alloreactive proliferating T cell and the expression of the Lyt-2 antigen by such cells are discussed.     

Mouse T lymphocytes proliferative responses specific for human MHC products in mouse anti-human xenogeneic MLR

It has been reported that human T cells recognize the polymorphism of murine Ia antigens in the human anti-mouse xenogeneic mixed lymphocyte reactions (MLR). In this study, murine T cell recognition of human Class II antigens of the major histocompatibility complex (MHC) was analyzed in mouse anti-human xenogeneic MLR responses. The xenoreactive murine T cell proliferative response was blocked by adding anti-HLA-DR monoclonal antibody to the xenogeneic MLR culture. The specificity of xenoreactive murine T cells was examined with regard to the secondary and tertiary xenogeneic MLR system. The xenoreactive murine T cells were restimulated by distinct human stimulator cells that had no shared HLA antigens with the stimulator used in the primary MLR. The data presented here show that the murine xenoreactive T cells recognize the shared determinant(s) of HLA-DR antigen on non-T, non-B stimulator cells. The xenoreactive murine T cell proliferative responses were mediated by Thy-1+, Lyt-1+, and Lyt-2- cells. Furthermore, the xenoreactive T cell responses required Ia+ cells, and Ia antigen on accessory cells plays a crucial role in eliciting the xenoreactive responses against human stimulator cells, while Ia+ accessory cells in the responding cell population are not essential for the elicitation of allogeneic MLR responses, as reported previously.     

Accessory cell stimulation of T cell proliferation requires active antigen processing, Ia-restricted antigen presentation, and a separate nonspecific 2nd signal

The roles of Ia+ accessory cells in H-2-restricted stimulation of antigen-specific T cell proliferation were explored in an in vitro model. L-glutamic acid60-L-alanine30-L-tyrosine10-(GAT) primed BALB/c nylon wool-passed T cells were depleted of Ia+ antigen-presenting cells (APC) by treatment with monoclonal anti-Ia antibody plus complement. Such cells failed to respond to soluble GAT, or to soluble GAT in the presence of phorbol myristic acetate (PMA), which is known to stimulate production of, or replace, IL-1 in vitro. Addition of gamma-irradiated syngeneic spleen cells reconstituted the response to soluble GAT, but addition of ultraviolet (UV) light-irradiated spleen cells did not, even in the presence of PMA. Preincubation of cells with GAT for 24 hr, followed by washing, then gamma irradiation, generated a cell population able to stimulate GAT-primed T cells to proliferate. The same pulsed cells exposed to UV irradiation failed to stimulate T cell responses unless PMA was added to the cultures. The relevant cells in this UV-irradiated population are Ia+. It is concluded that a finite period of time for interaction of metabolically intact APC with antigen is required before creation of an appropriate (Ia + antigen) signal recognized by the T cell. In addition to such Ia-restricted antigen presentation, however, a 2nd nonspecific signal, again requiring metabolically active APC for elaboration, is necessary for detectable T cell activation. These studies thus define 3 separable activities of APC during the process of H-2 restricted T cell activation.     

Accessory cell function in the Con A response: role of Ia-positive and Ia-negative accessory cells

We have examined the role of Ia-positive and Ia-negative accessory cells (AC) and soluble factors in Con A-stimulated murine T cell activation. Supernatant fluids containing interleukin 1 (IL 1) derived from the P388D1 macrophage cell line and from a lipopolysaccharide (LPS)-stimulated macrophage hybridoma provided only partial reconstitution of the response of purified T cells (18 to 27%). The complete reconstitution obtained with gamma-irradiated spleen cells or LPS-activated B cells was inhibited by approximately 60 to 77% when anti-Ia antibody was included in the culture. Despite this apparent involvement of Ia+ spleen AC, Ia-negative L cell AC could also reconstitute the response of both Class I-restricted Lyt-2+ T cells and Class II-restricted L3T4+ T cells. When the Ia-negative AC were employed, the L3T4 antigen on L3T4+ T cells played a critical role because addition of anti-L3T4 antibody to the culture inhibited the response by 85 to 90%. In contrast, anti-L3T4 did not inhibit the response in the presence of spleen AC. These results suggest that the molecules involved in T cell-AC interactions may vary depending on the AC source. Moreover, at least one of the putative target ligands for L3T4 presumably is not Ia, because anti-L3T4 inhibited T cell stimulation when Ia-negative AC were used.     

Ia-specific mixed leukocyte reactive T cell hybridomas: analysis of their specificity by using purified class II MHC molecules in synthetic membrane system

This study was undertaken to determine the nature of the antigens recognized in allogeneic and syngeneic mixed leukocyte reactions (MLR). Specifically, we wished to determine whether Ia antigens alone were recognized by MLR-reactive T cells, or whether the specificity was determined by the corecognition of non-MHC antigens together with syngeneic or allogeneic Ia. To do this we used 11 T cell hybrids that were characterized as being specific for Iad and were tested their capacity to respond to isolated I-Ad or I-Ed that had been incorporated into liposomes and had bound to the surface of glass beads. Of nine alloreactive T cell hybrids (five I-Ad-and four I-Ed-specific), seven were shown to be responsive to the relevant isolated Ia antigen on glass beads. Also, two of two syngeneic I-Ad-specific T cell hybrids responded to I-Ad on the glass beads. One of the two alloreactive T cell hybrids that failed to respond to the relevant Ia antigen on glass beads was shown to be specific for an antigen in fetal calf serum (FCS) that was recognized in the context of the allo-Ia antigen (I-Ed), because when intact accessory cells were used, a response by this hybrid was only observed when FCS was present in the assay culture medium or when the accessory cells were pre-pulsed with FCS. The possible involvement of FCS antigens and non-Ia accessory cell antigens in the stimulation of the nine T cell hybrids that responded to isolated Ia on glass beads was evaluated. T cell hybrids that were grown and were tested in serum free medium were still capable of reacting to Ia on beads. The isolated Ia preparations used were greater than 90% pure, and their capacity to stimulate the T cell hybrids did not correlate with the degree of contamination with non-Ia proteins. We conclude from these studies that the majority of T cells that respond to allogeneic or syngeneic Ia bearing stimulator cells are specific for the Ia antigens themselves, and do not require the co-recognition of other non-Ia antigens; nor is there any requirement for Ia antigen processing for this recognition.     

Functional analysis of cloned macrophage hybridomas. IV. Induction and inhibition of mixed lymphocyte responses

A series of macrophage (M phi) hybridomas were generated by fusion of drug-marked P388D1 (H-2d) tumor cells with CKB (H-2k) splenic adherent cells. The ability of this panel of cloned M phi hybridomas expressing various levels of surface Ia antigens to induce allogeneic mixed lymphocytes responses (MLR) was examined. All MLR stimulatory M phi hybridomas expressed surface Ia antigens. However, some Ia+ and all Ia- M phi hybridomas were unable to induce vigorous MLR responses. Furthermore, even after induction of surface Ia antigen expression with Con A supernatants (Con A Sn) or purified interferon-gamma, the nonstimulatory M phi hybridomas remained ineffective at inducing strong MLR proliferative responses. Furthermore, addition of the latter M phi hybridoma clones (both with and without Con A Sn treatment) to conventional MLR cultures resulted in inhibition of MLR responses. The series of inhibitory M phi hybridomas secreted normal levels of IL 1 upon stimulation with lipopolysaccharide. After surface Ia induction with Con A Sn, the inhibitory M phi hybridomas could stimulate secretion of IL 2 and expression of IL 2 receptors. Moreover, although they inhibited conventional MLR responses, IL 2 production and IL 2 receptor expression were not significantly inhibited. Addition of these M phi hybridomas 24 to 48 hr after initiation of MLR response also inhibited MLR proliferation. The results indicated that the group of inhibitory M phi hybridomas can inhibit MLR responses after IL 2 secretion and acquisition of IL 2 receptors. Finally, this inhibitory activity has been maintained during 1 yr of continuous in vitro culture, and the hybridomas represent a stable "homogeneous" subpopulation of inhibitory macrophages. Thus, the inhibitory phenotype appears to reflect arrest at a distinct differentiation stage.     

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.     

Characterization of murine T cell activation signals produced by B lymphoma cells

The activation requirements of alloreactive and antigen reactive murine T cells were examined by stimulating class II restricted T cell clones with monoclonal B lymphoma cells. One B lymphoma cell line (T27A) was found to stimulate IL 2 release from some alloreactive T cell clones without stimulating any significant T cell proliferation response. The same B lymphoma cells are capable of stimulating IL 2 release and proliferative responses from other T cell clones. Evidence is presented suggesting that B lymphoma cell stimulation of these T cell clones is largely IL 1 independent and that at least some T cell clones may require activation signals other than Ia, antigen, and IL 1. The addition of exogenous, purified IL 1 to the T cell activation assays was found to have a wide range of stimulatory effects on the proliferative responses of different T cell clones. The absence of comparable IL 1-induced stimulation of IL 2 secretion suggests that IL 1 primarily enhances antigen specific T cell proliferation through mechanisms other than acting as a co-stimulant for IL 2 release.