Requirement for an Ia-bearing accessory cell in Con A-induced T cell proliferation.

Pretreatment of murine lymphoid cells with anti-Ia and C abrogated the proliferative response of these cells to Con A, but not to PHA. Reconstitution experiments demonstrated that T cell-enriched populations failed to restore Con A responsiveness and that T cell-depleted populations were more effective in restoring responsiveness to Con A. In particular, a population of 1000 R resistant, glass-adherent, non-T spleen cells was capable of completely restoring responsiveness to Con A when added in numbers as low as 4% of cultured cells. These splenic adherent cells were found to express Ia determinants encoded by at least two genes: one in I-A and the other in I-B, I-J, and/or I-E/C, and it was demonstrated that determinants encoded in these two regions were expressed on the same cell. These results demonstrate that non-T accessory cells may be the Ia+ cells entirely responsible for the anti-Ia and C-induced abrogation of T cell proliferative responses to Con A.     

Utilization of soluble fusion proteins for induction of T cell proliferation

A peptide display library was evaluated as a means to identify peptide binding motifs for class II molecules. Peptides expressed as part of a soluble fusion protein with a maltose binding protein (malE) were produced by Escherichia coli. Constructs containing the high-affinity binding influenza hemagglutinin peptide 307W–319 (mal-HA) or the low-affinity binding tetanus toxoid peptide 830–843 (mal-TT) were used as controls. mal-HA, but not mal-TT, inhibited synthetic biotinylated-HA peptide from binding to purified DR4 Dw4 molecules in a dose-dependent manner. The fusion-peptide presentation system was also evaluated for its ability to induce antigen-specific T cell proliferation. DR4 Dw4⁺ B cells pulsed with mal-HA, but not mal-TT, induced dose-dependent proliferation of an HA-specific DR4 Dw4-restricted T cell line to the same extent as synthetic HA peptide. Using this type of peptide display library, it may be possible to determine the antigenic specificity of T cell clones isolated from patients with autoimmune diseases.     

Antigen presentation by resting B cells. Effectiveness at inducing T cell proliferation is determined by costimulatory signals, not T cell receptor occupancy

Resting B cells stimulated the proliferation of two T cell clones much less efficiently than T cell-depleted low-density APC. In contrast, low-density cells and resting B cells stimulated the clones to produce similar levels of inositol phosphates, a rapid biochemical event dependent only on occupancy of the TCR. The inefficient stimulation of T cell proliferation by resting B cell APC was dramatically improved by the addition of allogeneic low-density accessory cells incapable of being recognized by the TCR on the responding T cells. The results are most consistent with a model where low-density and resting B cell APC display similar amounts of Ag/Ia molecule complexes capable of being recognized by the TCR on the responding T cells but differ in the provision of costimulatory signals that, together with TCR occupancy, are required for IL-2 production.     

Characterization of the accessory cells involved in suppressor T cell induction

The ability of UV-treated splenic adherent cells (SAC) to induce T cell-mediated immunity and suppressor T cells was analyzed in the 4-hydroxy-3-nitrophenyl acetyl (NP) system. UV irradiation of 0.88 KJ/m2 decreased the capacity of NP-coupled SAC to induce delayed-type hypersensitivity (DTH) responses by about 50%. The ability of uncoupled UV-treated SAC to induce allogeneic DTH response was also imparied, indicating that UV-treated SAC are inefficient at inducing DTH in these systems. TS1 induction by UV-treated NP-SAC was evaluated TS1 induction by UV-treated NP-SAC was evaluated by using adherent cells that were subjected to the same dose of UV irradiation that impaired DTH induction. Intravenous administration of 10(3) or 10(4) UV-treated NP-coupled SAC induced TS1 cells with the same efficiency as non-UV-irradiated cells. The TS1 cells induced in this fashion were antigen specific. Furthermore, to establish that the antigen was not reprocessed by the host, I-J-mismatched, UV-treated NP-SAC were unable to induce TS1 cells. The population of antigen-presenting cells responsible for TS1 induction appear to express both I-A and I-J determinants. TS2 induction by UV-treated accessory cells was also analyzed. TSF1 inducer suppressor factor was pulsed onto graded numbers of either normal or UV-treated adherent cells. The same levels of antigen-specific suppression were induced with normal and UV-treated cells. Finally, TS3 induction by UV-treated NP-SAC was analyzed. UV-treated and normal NP-SAC (3 X 10(3] induced antigen-specific suppression of NP DTH responses. I-J-mismatched, UV-treated NP-SAC failed to induce suppression, suggesting that the hapten was not reprocessed by the host under these experimental conditions. The accessory cell population responsible for TS3 induction appears to express both I-A and I-J determinants. Thus, there are at least two functional distinctions between the antigen-presenting cells that induce immunity vs those that induce suppressor cells. First, UV treatment selectively impairs the antigen-presenting cells, which activate the positive limb of the immune response. Second, I-J determinants appear to be specifically associated with the SAC, which induce suppressor T cells. Although these criteria can be used to distinguish the accessory cells involved in suppressor cell pathways from those controlling helper T cell induction, there were no discernible phenotypic differences among the accessory cells that induce the TS1, TS2, and TS3 subsets.     

Signals involved in T cell activation. I. Phorbol esters enhance responsiveness but cannot replace intact accessory cells in the induction of mitogen-stimulated T cell proliferation

The role of accessory cells (AC) in the initiation of mitogen-induced T cell proliferation was examined by comparing the effect of intact macrophages (M phi) with that of 4-beta-phorbol 12-myristate 13-acetate (PMA). In high-density cultures, purified guinea pig T cells failed to proliferate in response to stimulation with phytohemagglutinin (PHA), concanavalin A (Con A), or PMA alone. The addition of M phi to PHA or Con A but not PMA-stimulated cultures restored T cell proliferation. The addition of PMA to high-density T cell cultures stimulated with PHA or Con A also permitted [3H]thymidine incorporation, but was less effective than intact M phi in this regard. This action of PMA was dependent on the small number of AC contaminating the T cell cultures as evidenced by the finding that PMA could not support mitogen responsiveness of T cells that had been depleted of Ia-bearing cells by planning, even when these cells were cultured at high density. When PMA was added to T cell cultures supported by optimal numbers of M phi, catalase-reversible suppression of responses was noted. Even in cultures containing catalase, PMA failed to enhance responsiveness above that supported by optimal numbers of M phi. A low-density culture system was used to examine in greater detail the possibility that PMA could completely substitute for M phi in promoting T cells activation. In low-density cultures, mitogen-induced T cell proliferation required intact M phi. PMA could not support responses even in cultures supplemented with interleukin 1-containing M phi supernatants or purified interleukin 2 alone or in combination. Similar results were found in high-density cultures of T cells depleted of Ia-bearing cells. These results support a model of T cell activation in which AC play at least two distinct roles. The initiation of the response requires a signal conveyed by an intact M phi, which cannot be provided by either a M phi supernatant factor or PMA. The response can be amplified by additional M phi or M phi supernatant factors. PMA can substitute for M phi in this regard and can provide the signal necessary for amplification of T cell proliferation supported by small numbers of intact AC.     

Requirement for noncognate interaction with T cells for the activation of B cell immunoglobulin secretion by IL-2

The mechanism whereby noncognate contact with activated IL-2-producing Type 1 helper T cells (TH1) induces B cell activation was examined. Small resting B cells from C57B1/6 mice were cultured, in the absence of any ligand for surface Ig, with irradiated cells of the hapten-specific, CBA-derived, F23.1+ TH1 clone E9.D4 in F23.1 (anti-T cell receptor V-beta 8)-coated microwells. This induced polyclonal B cell activation to enter cell cycle (thymidine incorporation) at 2 days and to secrete immunoglobulin at 5 days. An anti-IL-2 mAb (S4B6) inhibited antibody production completely. Anti-IL-2 did not inhibit either LPS-induced B cell responses, or T cell activation (measured as IL-3 secretion). Anti-IL-2 receptor (anti-Tac) mAbs also inhibited T-dependent B cell responses, without affecting LPS responses. An anti-IFN-gamma mAb partially inhibited Ig secretion, without affecting entry into cycle. LPS responses or T cell activation. Other antibodies (anti-IL-3, IL-4, IL-5, Thy-1.2, CD5) were not inhibitory. After 2 days of culture with F23.1-activated T cells, B cells appeared to have become responsive to IL-2, in that they could be driven to immunoglobulin production by the addition of IL-2. Flow cytometry showed no expression by these B cells of 55-kDa (Tac) IL-2 receptors. Also, rigorous removal of T cells from 2-day cocultures prevented the response to IL-2, and readdition of T cells restored it. Because the reconstituted responses were inhibited both by anti-IL-2 and by anti-Tac, IL-2 must have acted indirectly, via the T cells that were present in these cultures. Continued contact with T cells was therefore necessary for the progression of B cells to antibody secretion.     

Requirement for BSF-1 in the induction of antigen-specific B cell proliferation by a thymus-dependent antigen and carrier-reactive T cell line

We report here a role of B cell stimulatory factor 1 (BSF-1) in the induction of antigen-specific proliferation of affinity-purified small B lymphocytes by a thymus-dependent antigen and a carrier-reactive T cell line. By using an ovalbumin-reactive T cell line (designated Hen-1), which does not produce BSF-1 following activation, it was possible to demonstrate that the antigen-specific proliferative response of trinitrophenyl (TNP)-binding B cells to TNP-ovalbumin required exogenous BSF-1 in addition to direct interaction with irradiated Hen-1 T cells. The activation obtained under these conditions was highly efficient, being sensitive to antigen doses as low as 0.001 microgram/ml. The addition of saturating amounts of BSF-1 did not alter the antigen-specificity or the requirements for hapten-carrier linkage or major histocompatibility complex-restricted T-B interaction in this system. The involvement of BSF-1 was confirmed by the ability of 11B11 anti-BSF-1 antibody to specifically suppress the response of TNP-binding B cells to TNP-ovalbumin, BSF-1, and irradiated Hen-1 T cells. Finally, this response was augmented by addition of the monokine interleukin 1. These data indicate that the proliferative response of small B cells to the thymus-dependent antigen and carrier-reactive T cell line used in our experiments can be regulated by the same factors that govern B cell proliferation induced by thymus-independent type 2 antigens or anti-IgM antibodies.     

Suppression of B cell MIF production by T cells and soluble T cell-derived factors.

Purified populations of guinea pig B cells from nonimmunized animals may be stimualted by PPD or LPS to produce MIF. Unfractionated lymphocyte suspensions from these animals do not produce MIF under these conditions. Reconstitution of B cells with T cells abolishes their ability to generate detectable MIF. A soluble factor obtained from stimulated T cell cultures (MIFIF) is also capable of suppressing this B cell activity. Thus suppressor T cells can interfere with lymphokine production by B cells and this effect is mediated at least in part by a soluble factor. This previously undescribed capacity of T cells may provide an explanation for the fact that B cells do not appear to play a role in reactions of cell-mediated immunity in vivo.     

A soluble inhibitor of B and T cell proliferation and antibody synthesis produced by dividing human T cells.

The increase in affinity and heterogeneity of antibody with respect to time after immunization to the 2,4,6-trinitrophenyl (TNP) determinant was studied using TNP-brucella (BA) and TNP-type III pneumococcal polysaccharide (SIII). Experimental evidence is presented in support of the maturation of 19S antibody affinity. The difficulties which have been encountered in some previous investigations in detecting such a maturation appear to be the tendency of the cells to switch from IgM to IgG synthesis early after the peak of the primary response. Data are presented indicating that this switch occurs in a non-antibody-secreting memory cell population prior to, or more likely very shortly after, boosting. We also present evidence that the use of an antigen that does not induce a massive switch from IgM to IgG antibody synthesis offers a way of unmasking maturation of the 19S response. Thus, with the T-independent antigen TNP-SIII, a definite increase in heterogeneity could be detected in the 19S response upon secondary boosting. A greater increase in heterogeneity was noted in nude mice and was possibly due to the absence of suppressor T cells.     

A soluble factor produced by lamina propria mononuclear cells is required for TNF-alpha enhancement of IFN-gamma production by T cells.

The role of TNF-alpha in the mucosal inflammation of Crohn's disease has been demonstrated by the prolonged clinical responses and/or remissions among patients receiving i.v. infusion of anti-TNF-alpha. A correlation between TNF-alpha and elevated IFN-gamma production is suggested by the reduction in the number of IFN-gamma producing lamina propria mononuclear cells (LPMC) found in colonic biopsies from anti-TNF-alpha-treated patients. The aim of this study was to define the mechanism of TNF-alpha-augmented mucosal T cell IFN-gamma production. In this paper we present evidence that cultured LPMC secrete a factor which acts on preactivated T cells in concert with TNF-alpha to augment IFN-gamma production. This activity is independent of IL-12 and IL-18, the well-documented potentiators of IFN-gamma expression, and is not produced by PBMC. Peripheral blood PHA-activated T cells incubated in supernatants from LPMC became responsive to TNF-alpha by increasing IFN-gamma output upon stimulation. These results are consistent with a model in which LPMC, but not PBMC, release an unidentified substance when cultured in vitro with low dose IL-2. This substance can act on preactivated peripheral T cells, as well as on lamina propria T cells, conditioning them to respond to TNF-alpha by increased IFN-gamma secretion upon stimulation. Expression of this factor in the gut mucosa could contribute to up-regulation of the Th1 response in the presence of TNF-alpha, and could be important for mucosal immunoregulation.     

T cell activation by purified, soluble, class I MHC molecules. Requirement for polyvalency

To examine the nature of the interaction of the TCR with the MHC class I Ag, we have studied the stimulation requirements of an H-2Dd-reactive T cell hybridoma, using a homogeneous, purified preparation of a molecularly engineered soluble counterpart of the class I Ag, H-2Dd/Q10b. We demonstrate that this monovalent, soluble MHC Ag is incapable of stimulating the release of IL-2 from this T cell hybridoma. However, the same preparation of the purified protein can elicit a dose-dependent response when made multivalent either by covalent coupling to soluble, high m.w. dextran or to agarose beads, or by adsorption to polystyrene tissue culture plates.     

Role of antigen-specific B cells in the induction of SRBC-specific T cell proliferation

In this study, we ask whether antigen presentation can be effected by antigen-activated B cells. Antigen-dependent in vitro proliferation of T cells from mice primed with SRBC or HoRBC occurs in the presence of B cells primed to the relevant antigen. B cells prepared from lymph nodes of mice primed with irrelevant antigens are not effective antigen-presenting cells for RBC-specific T cell proliferation over a wide range of SRBC doses. This is true even when both RBC and the antigen to which the B cells are primed are included in the culture. In contrast, B cells specific for a hapten determinant coupled to SRBC are able to support proliferation of T cells specific for SRBC determinants. We conclude from these data that antigen-specific B cells play a role in the induction of T cell proliferative responses to SRBC and HoRBC antigens. Two models are proposed: either B cells, upon antigen interaction with surface antibody, are able to act as accessory cells to induce Ia-dependent proliferation of immune T cells; or B cells augment the T cell proliferative response by secretion of antibody, leading to opsonization of the antigen for macrophage uptake and presentation.     

The induction of TNP-altered, self-reactive human cytotoxic T cells by soluble factors: the role of Ia antigens.

This report demonstrates that human peripheral blood T lymphocytes, triggered by allogeneic cells or soluble antigens, elaborate helper factor(s) that promotes the in vitro differentiation of TNP altered-self reactive human CTL. Helper factor(s) alone is not sufficient for the generation of these killer cells, but requires the presence of TNP-derivatized autologous stimulators during sensitization. Additional experiments were performed with antisera to a human Ia-like antigen, p23, 30. These studies indicate that human Ia-like antigens play an important role in both the induction of helper factor(s) and in the functional activity of preformed helper factor(s) molecules.     

Activation of murine T cells by streptococcal pyrogenic exotoxin type A. Requirement for MHC class II molecules on accessory cells and identification of V beta elements in T cell receptor of toxin-reactive T cells

We investigated the mechanisms of murine T cell activation by streptococcal pyrogenic exotoxin type A (SPE A), focusing on the role of MHC class II molecules on accessory cells (AC) and V beta usage in alpha beta TCR of SPE A-reactive T cells in comparison with staphylococcal enterotoxin B-reactive T cells. L cells transfected with I-Ab genes functioned as effective AC for SPE A-induced responses by C57BL/6 T cells, proliferation, and IL-2 production, but control L cells were not effective AC. Anti-I-Ab mAb inhibited the SPE A-induced responses. Staphylococcal enterotoxin B-induced C57BL/6 T cell blasts were composed of cells bearing V beta 3, members of the V beta 8 family, and V beta 11. Most of the SPE A-induced T cell blasts (about 80%) bore V beta 8.2. mAb reactive to V beta 8.2 markedly inhibited SPE A-induced T cell responses. Apparently, SPE A activates mainly T cells bearing V beta 8.2 in physical association with MHC class II molecules expressed on AC. We also discuss the pathogenic activities of SPE A in relation to toxic shock syndrome.     

The murine Kupffer cell. I. Characterization of the cell serving accessory function in antigen-specific T cell proliferation.

Murine Kupffer cells, the tissue macrophages of the liver, were isolated by collagenase digestion, differential sedimentation over Metrizamide, and glass adherence. The resultant cell population was more than 86% phagocytic, and 95% of cells stained positively for alpha-naphthyl butyrate esterase activity. The cells also had cell surface receptors for complement (C) and the Fc portion of IgG. In addition, a large proportion of Kupffer cells was shown to bear Ia antigens: about half of the cells bore I-A subregion-encoded antigens and about half bore I-BJE or I-EC subregion-encoded antigens. Kupffer cell populations were capable of reconstituting antigen-stimulated proliferative responses of antigen-primed, macrophage-depleted, lymph node T cells. The ability to reconstitute proliferation was enriched in the adherent population and was resistant to radiation and treatment with an anti-Thy antiserum and C. We conclude that isolated murine Kupffer cells bear the Ia phenotype of accessory cells that function in antigen presentation and that Kupffer cells can participate in the induction of antigen-specific immune responses. These data suggest that Kupffer cells may play a role in modulating responses to enterically derived antigens.     

Effect of interferon-gamma on myelin basic protein-specific T cell line proliferation in response to antigen-pulsed accessory cells.

We have shown previously that treatment of SJL/J mice with anti-interferon-gamma monoclonal antibody (mAb) exacerbated experimental allergic encephalomyelitis (EAE) only if administered at the time of encephalitogenic challenge. Here we investigate the role of interferon-gamma (IFN-gamma) and anti-IFN-gamma mAb in the early events of T cell activation in vitro. Pretreatment of murine peritoneal exudate cells (PEC) with IFN-gamma led to a significant increase in their ability to activate myelin basic protein (MBP)-specific, short-term T cell lines. When exogenous IFN-gamma was added to cocultures of T cells and MBP-pulsed PEC, the antigen-specific T cell proliferation was considerably reduced. Anti-IFN-gamma mAb added to these cultures neutralized the inhibitory effect of the exogenous IFN-gamma on T cell proliferation but had no visible effect on class II MHC expression by the antigen-pulsed PEC present in the same cultures. A reduction in T cell proliferation was also observed when the T cells were treated with IFN-gamma prior to coculture with the MBP-pulsed PEC. These results demonstrate that, on one hand, IFN-gamma enhances the ability of PEC to induce antigen-specific T cell proliferation but, on the other hand, acts on the T cells themselves by inhibiting their proliferation in response to the antigen-pulsed PEC. This may explain why treatment with anti-IFN-gamma antibody in vivo induces EAE exacerbation.     

Enhanced proliferation of murine T cell lines following interaction of poly(Glu60,Phe40) (GPhe) and antigen-presenting accessory cells. II. The role of accessory cells and cytokines in the activity of GPhe on antigen-independent proliferation.

Our previous study (1) demonstrated the "cytokine-like" activity of poly(Glu60,Phe40)(GPhe) in augmenting the antigen-dependent proliferation of a variety of long-term murine T cell lines, particularly the bulk, BALB/c anti-poly (Glu36,Lys24,Ala40) (GLA), interleukin-4-producing, DCL-2 T cell line. GPhe was found to also augment the antigen-independent proliferation of DCL-2 in response to exogenous cytokines ([interleukin(IL)-2 +/- IL-1] in most experiments). Such exogenous cytokine-driven proliferative responses of DCL-2 were used to investigate further the role of accessory cells and of various soluble factors in the action of GPhe. GPhe did not act as a direct mitogen for T cells, rather it acted in a costimulatory fashion, requiring the presence of plastic-adherent accessory cells and a T cell growth factor (either IL-2 or IL-4). In the presence of accessory cells and exogenous IL-2, augmentation of antigen-independent DCL-2 proliferation by GPhe or by IL-1 depended upon the induction of autocrine IL-4 production. However, GPhe also augmented the response of these cells in the presence of exogenous IL-4 (+/- IL-2, +/- IL-1), and exogenous IL-4 added in combination with exogenous IL-2 (+/- IL-1) failed to mimic the GPhe effect, suggesting that another signal was involved in the mechanism of action of GPhe. The ability of allogeneic accessory cells to interact with GPhe to augment proliferative responses suggested that either a soluble factor or an unusual non-MHC-restricted cell-cell interaction provided this signal. In the presence of uv-irradiated accessory cells, DCL-2 proliferation was enhanced over that observed in the presence of non-uv-treated accessory cells, mimicking the GPhe effect, and interaction of GPhe with uv-irradiated accessory cells did not result in further enhancement of DCL-2 cytokine-driven proliferation. Using monoclonal antibodies which could block the function of IA or CD4 molecules, these cell-surface "adhesion" molecules were shown not to participate in the activity of GPhe. By the addition of recombinant cytokines, neutralizing antibodies, or indomethacin, the mechanism of action of GPhe was also shown not to be dependent upon IL-1, IL-6, IL-7, TNF alpha, or prostaglandin production by accessory cells. However, the presence, individually, of some of these factors (IL-1, IL-7, or prostaglandins) could influence to a variable degree the magnitude of the GPhe effect.