The processing and presentation of the self-antigen hemoglobin. Self-reactivity can be limited by antigen availability and costimulator expression.

APC do not distinguish between self- and foreign proteins. Previous studies from our laboratory demonstrated that most endogenous host APC constitutively processed and presented the self-Ag, hemoglobin (Hb), as detected by the Hb-specific T cell hybridoma, YO1.6. We have now examined APC in organs known to be involved in RBC degradation (liver Kupffer cells and splenic small resting B cells) for the presence of Hb/Ia complexes and for the expression of the costimulation necessary to trigger proliferation of T cell clones. We detected Hb/Ia complexes not only on splenic small resting B cells, but also on liver Kupffer cells. Interestingly, complexes were not present on lymph node small resting B cells. Splenic small resting B cells expressed costimulatory activity and efficiently stimulated the Th2 clones only. The opposite pattern was observed with liver Kupffer cells, which expressed costimulatory activity for Th1 clones only. However, if costimulatory activity was provided for the Th2 clones (IL-1 beta) and Th1 clones (allogenic spleen cells), the clones did proliferate in response to Kupffer cells and small resting B cells, respectively. In this report we have demonstrated that 1) endogenously formed self Hb/Ia complexes are expressed on splenic small resting B cells and liver Kupffer cells but not on lymph node small resting B cells and 2) these APC are also able to limit the expression of costimulatory activity for Th2 and Th1 T cell clones. Thus, endogenous APC not only constitutively process and present the self-Ag Hb, but also limit expression of the costimulatory activity necessary to trigger T cell proliferation against a self-Ag. The constitutive processing and presentation of self-Ag, as well as the regulation of costimulatory activity on APC, is likely an important feature of the maintenance of self-tolerance.     

Antigen-presenting B cells: efficient uptake and presentation by activated B cells for induction of cytotoxic T lymphocytes against vesicular stomatitis virus

We have evaluated the efficacy of mitogen (LPS/DxSO4)-activated B cells (B lymphoblasts) to function as antigen-presenting cells (APC) for vesicular stomatitis virus (VSV). Our studies revealed that B lymphoblasts induced potent cytotoxic thymus (T)-derived lymphocyte (CTL) activity in VSV-immune splenic T cells depleted of adherent accessory cells. Dose-response curves indicated that B lymphoblasts were approximately 15-20 times more efficient APC than spleen cells for CTL induction against VSV. There was little evidence of reprocessing of viral antigens by the responder population because only CTL activity restricted to the parental haplotype of the B lymphoblast was generated following stimulation of VSV-immune F1 T cells. B lymphoblasts activated VSV-specific memory CTL which expressed the Lyt-1-23+, AsGM1+ phenotype without activating natural killer and/or lymphokine-activated killer cells. The ability of B lymphoblasts to function as efficient APC was not related to enhanced viral replication in these cells because potent VSV-specific proliferative and class I-restricted CTL responses were induced by B lymphoblasts infected with VSV rendered noninfectious by exposure to ultraviolet (uv) light. This indicates that activated B cells can efficiently process and present input virion protein. Purified splenic B cells that were not activated by mitogen stimulation did not function as APC for VSV even at high multiplicities of infection. The failure of B cells to function as APC for VSV was related to inefficient uptake of VSV and their inability to provide accessory cell signals required for T-cell proliferation; both these functions developed following mitogen stimulation. These data suggest that activated B cells may function as a potent APC population for virus independent of the specificity of their immunoglobulin antigen receptor.     

Distinct proliferative T cell clonotypes are generated in response to a murine retrovirus-induced syngeneic T cell leukemia: viral gp70 antigen-specific MT4+ clones and Lyt-2+ cytolytic clones which recognize a tumor-specific cell surface antigen

After immunization of B6 mice with the syngeneic retrovirus-induced T cell leukemia/lymphoma FBL-3, two major tumor-specific proliferative T cell clonotypes were derived. T cell clones derived from long-term lines propagated by in vitro culture with irradiated tumor cells and syngeneic spleen cells were exclusively of the Lyt-2+ phenotype. Such clones were cytolytic, retained their proliferative phenotype indefinitely when expanded by repeated cycles of reactivation and rest, and recognized a tumor-specific cell surface antigen in association with class I MHC molecules. This tumor cell antigen was not present on nontransformed virus-infected cells. Class II MHC-restricted MT4+ clones specific for the viral antigen gp70 were derived from lymph node T cells of FBL-3 tumor-immune mice only by in vitro culture with purified Friend virus in the presence of syngeneic splenic APC. Once derived, however, such clones could be stimulated in the presence of FBL-3 tumor cells and syngeneic spleen cells, demonstrating the reprocessing of tumor-derived gp70 antigen by APC in the spleen cell population. In contrast, no reprocessing of the tumor cell surface antigen by splenic APC for presentation to the class I MHC-restricted T cell clones could be demonstrated. Evidence is presented that FBL-3 T leukemia/lymphoma cells function as APC for Lyt-2+ class I MHC-restricted clones, and that no concomitant recognition of Ia molecules is required to activate these clones. Both Lyt-2+ and MT4+ clones were induced to proliferate in the presence of exogenous IL2 alone, but this stimulus failed to result in significant release of immune interferon. In contrast, antigen stimulation of both clones resulted in proliferation as well as significant immune interferon release. Immune interferon production is not required for the generation of MHC-restricted cell-mediated cytolytic function.     

Adherent Ia+ murine cell lines with characteristics of dendritic cells. II. Characteristics of I region-restricted antigen presentation

The ability of an adherent Ia+, interleukin 1+ (IL-1) tumor cell line (P388AD) to present turkey gamma-globulin (TGG) to primed T lymphocytes was demonstrated and compared with normal antigen-presenting cells (APC) found in mouse spleen. P388AD tumor cells presented TGG to long-term cultures of TGG-reactive T cells (LTTC) and to lymph node-derived T cells which were enriched on nylon wool columns and subsequently depleted of endogenous antigen-presenting cells with anti-Ia antisera and complement. MHC-restricted antigen presentation by P388AD was observed when long-term cultures of TGG-reactive T cells were used as the responding T-cell population. Furthermore, antisera directed against I-region determinants expressed on the P388AD tumor cells inhibited TGG-specific T-cell proliferation in a dose-related fashion, suggesting a functional role for the tumor cell-associated Ia molecules. The kinetics of antigen presentation to LTTC by P388AD were similar to the kinetics observed for splenic APC, although the magnitude of the proliferative response to LTTC to TGG was generally lower when antigen (Ag) was presented by the tumor cells compared to splenic antigen-presenting cells (APC). However, the magnitude of T-cell proliferation of immune lymph node (LN) T cells was comparable when Ag was presented on tumor cells or splenic APC. Several experiments suggested that Ag uptake and/or processing may be less effective in P388AD tumor cells as compared to normal splenic APC. A nonadherent Ia+, IL-1- tumor cell line (P388NA), which was isolated from the same parental tumor as P388AD, was also tested for the ability to present Ag to primed T lymphocytes and Ag-reactive LTTC. In contrast, to P388AD, the nonadherent tumor cell failed to present TGG under identical culture conditions even though Ia molecules were expressed on the tumor cells and Ag uptake had occurred. However, the defect in Ag presentation by P388NA could be corrected if an exogenous source of purified interleukin 1 was supplied to the cultures. A unique opportunity thus exists with both the P388AD and P388NA tumor cell lines to decipher some of the molecular interactions leading to T-cell proliferation during antigen presentation.     

Can resting B cells present antigen to T cells?

Antigen stimulation of T lymphocytes can occur only in the presence of an antigen-presenting cell (APC). An ever-increasing number of cell types have been found to act as APCs; these include macrophages, splenic and lymph node dendritic cells, and Langerhans' cells of the skin. Although activated B lymphocytes and B cell lymphomas are known to serve as APCs, it has been generally believed that resting B cells cannot perform this function. However, in recent studies we have found that resting B cells can indeed present soluble antigen to T cell clones as well as to antigen-primed T cells. The previous difficulty in demonstrating this activity can be explained by the finding that, in contrast to macrophages and dendritic cells, the antigen-presenting ability of resting B cells is very radiosensitive. Macrophages are usually irradiated with 2000-3300 rads to prevent them from incorporating [3H]thymidine in the T cell proliferation assay. Resting B cells, however, begin to lose presenting function at 1500 rads and have completely lost this activity at 3300 rads. It was also possible to distinguish two distinct T cell clonal phenotypes when resting B cells were used as APCs on the basis of two different assays (T cell proliferation, and B cell proliferation resulting from T cell activation). The majority of T cell clones tested were capable of both proliferating themselves and inducing the proliferation of B cells. Some T cells clones, however, could not proliferate in the presence of antigen and B cell APCs, although they were very good at inducing the proliferation of B cells. This suggests that there are two distinct pathways of T cell activation, one leading to T cell proliferation and the other leading only to the release of lymphokines (as measured by the polyclonal activation of B cells).     

B cell presentation of a tolerogenic signal to Th clones.

Lightly irradiated (950 R) splenic B cells were inefficient, in comparison to unseparated spleen cells, in stimulating antigen-specific proliferation of Th1 clones specific for human gamma globulin (HGG). This inefficiency was due to antigen-specific inactivation: Th1 clones preincubated with HGG and lightly irradiated B cells or mitomycin C-treated B cells were unable to proliferate to HGG in secondary cultures. In contrast to Th1 clones, Th2 clones proliferated well in response to B cell APC, and showed no decrease in their subsequent antigen-induced proliferative capacity after exposure to lightly irradiated B cells and HGG. However, preincubation of Th2 with lightly irradiated B cells and HGG did inactivate the capacity of Th2 to provide help for antibody production in secondary cultures. These results suggest that under certain conditions B cells may present antigen to Th1 and Th2 cells in a tolerogenic rather than an immunogenic manner.     

Presentation of autoantigen by human T cells.

Activated human T cells express MHC class II and have been shown to present foreign Ag to autologous T cells. We now demonstrate that MHC class II+ T cell clones can present myelin basic protein (MBP) peptide autoantigen in the absence of traditional APC to autologous MBP reactive T cell clones. MBP peptide-pulsed T cell clones specifically stimulated autologous MBP-reactive T cell clones to flux calcium and proliferate. Activation responses were peptide epitope specific and blocked by mAb to MHC class II, indicating a TCR-mediated response. In addition, mAb to the adhesion molecules LFA-3, CD2, LFA-1, CD29, and to the tyrosine phosphatase CD45 also inhibited proliferation, indicating the involvement of T to T cell interactions. In contrast to peptide Ag, T cell clones did not respond to autologous T cells pulsed with HPLC-purified MBP, suggesting that T cells are unable to process whole MBP. However, batch-purified MBP Ag preparations containing lower m.w. breakdown products were presented by T cells, indicating that naturally occurring breakdown products of autoantigens could be presented by activated T cells in vivo. These results raise the possibility that T cell presentation of autoantigen at inflammatory sites may be important in regulation of immune responses to self Ag.     

The mechanism of antigen presentation by dendritic cells and splenic adherent cells in the induction of an allogeneic cytotoxic T-lymphocyte response to H-2Kk liposomes

Induction of an allogeneic cytotoxic T-lymphocyte (CTL) response to purified alloantigen is partially dependent on uptake and processing of the class I alloantigen by antigen-presenting cells (APC) followed by recognition of the alloantigen and self Ia by helper T cells (TH). The activated TH provides the helper signal(s) to the alloantigen-specific CTL for proliferation and differentiation into an active effector CTL. The role of antigen processing and presentation of major histocompatibility complex alloantigens was examined and the ability of different types of APC to present purified H-2Kk liposomes was investigated. Splenic adherent cells (SAC), splenic dendritic cells (DC), and B-cell lymphoblastoid lines were all shown to be effective in the presentation of H-2Kk liposomes. The relative ability of these cells to serve as APC was determined to be DC greater than B-cell tumors greater than SAC. The role of processing of H-2Kk liposomes by SAC and DC was examined by investigating the effect of weak bases on pulsing of the APC. These experiments suggest that presentation of alloantigen by both SAC and DC involves a step which is sensitive to inhibition by weak bases. We examined whether the TH were activated by similar mechanisms when stimulated by the various APC. The functional involvement of the T-cell surface marker L3T4 was demonstrated in the induction of TH. In contrast, L3T4 was not involved in the subsequent generation of CTL since monoclonal antibody (MAb) specific for L3T4 was not effective in blocking CTL function in the presence of nonspecific T helper factor (THF). Similarly, Ia on the APC was shown to be involved in the stimulation of the TH pathway but not directly in the differentiation of the CTL. Thus, DC and B cells in addition to SAC can present H-2Kk to TH. The presentation of alloantigen by both cell types may involve an intracellular route as demonstrated by the blocking of the TH response by weak bases. Both Ia and L3T4 are required on the APC for induction of the TH response. The minimal requirements for activation of the CTL were H-2Kk liposomes and a source of THF.     

Oxidative stress impairs intracellular events involved in antigen processing and presentation to T cells

For T cells to recognize foreign antigens, the latter must be processed into peptides and associated to major histocompatibility complex (MHC) class II molecules by antigen-presenting cells (APC). APCs frequently operate under stress conditions induced by tissue damage, antigens, or inflammatory reactions. We analyze the effects of oxidative stress on intracellular processing using APC B cell lines. Before being tested for APC function, B cells (IIA1.6) were exposed for 2 hours to hydrogen peroxide (H2O2), a treatment that impairs their capacity to stimulate specific T cell clones. Because paraformaldehyde-fixed H2O2-treated B cells can still present extracellular peptides to T cell clones, the intracellular events of processing were investigated. Purified lysosomes from H2O2-treated B cells show increased proteolytic activity and increased generation of antigenic peptides. In addition, H2O2 treatment targets antigens to compartments that express low levels of MHC II and proteins (H-2M, H-2O) required for peptide loading onto this molecule. Finally, we suggest that impairment of antigen processing by oxidative stress reduces the induction of a T cell's response because H2O2 decreases the activation of naive T lymphocytes by dendritic cells. Together, these data indicate that oxidative stress inhibits the capacity of APCs to process antigens and to initiate a primary T cell response. The role of such modifications on the outcome of the specific immune response is discussed.     

Studies on the capacity of B cells as well as T cells to serve as accessory cells for the activation of herpes simplex virus-specific cytolytic T cells

Experiments were conducted in an effort to determine the ability of B and T lymphocytes to serve as APC for the activation of HSV-primed splenic T cells to become class I-restricted, HSV-specific CTL. The results showed that both freshly isolated splenic B cells as well as LPS and dextran sulfate (L/D)-activated B cells were effective at stimulating the generation of CTL during a 5-day in vitro culture. There was no requirement for the addition of exogenous IL-2 to the culture and, since murine B cells do not appear to express either membrane or secreted IL-1, this lymphokine appears to either not be required for the activation of virus-specific CTL or to be provided by the T cells themselves. When normal B cells were separated into fractions enriched for resting vs activated cells and then tested for their ability to stimulate the generation of HSV-specific CTL, it was found that while the activated B cells were quite effective at stimulating the generation of CTL, resting B cells were ineffective at carrying out this function. In contrast to normal B cells, normal T cells were unable to act as APC. However, Con A-activated T lymphoblasts were equivalent to L/D B cells in their ability to mediate the generation of CTL activity. L/D B cells that had been pulsed with HSV and then incubated at 37 degrees C for greater than 1 h could be fixed with paraformaldehyde and were still able to function as APC. The finding that L/D B cells, that had been fixed at 1 h or less after exposure to HSV, were unable to function as APC suggested that either active Ag "processing" steps may be required for the presentation of Ag in the context of class I molecules or that there is a requirement for the synthesis of viral protein Ag before presentation.     

Antigen presentation by human antigen-presenting cells to antigen-specific xenogeneic murine T cells

Successful antigen presentation by xenogeneic human antigen-presenting cells (APC) to stimulate the proliferation of antigen-specific, keyhole limpet hemocyanin (KLH)-specific, ovalbumin (OVA)-specific, and purified protein derivative of Mycobacterium tuberculosis (PPD)-specific murine T cells was observed. Evidence indicating a direct cell interaction between antigen-specific murine T cells and xenogeneic human APC was given by experiments using antigen-specific murine T cell clones. The OVA-specific B10.S(9R) T cell line (9-0-A1) and PPD-specific B10.A(4R) T cell line (4-P-1) were stimulated by both xenogeneic human APC and murine APC from syngeneic or I-A compatible strains, while the PPD-specific human T cell line (Y-P-5) was stimulated by autologous human APC but not by murine APC. Anti-HLA-DR monoclonal antibodies (MoAb) blocked the xenogeneic human APC-antigen-specific murine T cell clone interaction. Thus, human xenogeneic APC can stimulate antigen-specific murine T cells through HLA-DR molecules in the same manner as syngeneic murine APC do through Ia molecules coded for by the I region of the H-2 complex, while murine APC failed to present antigen to stimulate human antigen-specific T cells.     

IL-10 acts on the antigen-presenting cell to inhibit cytokine production by Th1 cells

Murine IL-10 (cytokine synthesis inhibitory factor) inhibits cytokine production by Th1 cell clones when they are activated under conditions requiring the presence of APC. By preincubating APC with IL-10, we demonstrate that IL-10 acts principally on APC to inhibit IFN-gamma production by Th1 clones. Moreover, IL-10 is not active when Th1 cells are stimulated with glutaraldehyde-fixed APC, which also indicates that its action involves regulation of APC function. Furthermore, IL-10 inhibits cytokine synthesis by Th1 cells stimulated with the super-antigen Staphylococcus enterotoxin B, which does not appear to require processing. Flow microfluorimetry purified splenic or peritoneal B cells and macrophages, and B cell and macrophage cell lines can present Ag to Th1 clones. However, IL-10 acts only on sorted macrophages and the macrophage cell line to suppress IFN-gamma production by Th1 clones. IL-10 does not show this effect when B cells are used as APC. In contrast, IL-10 does not impair the ability of APC to stimulate cytokine production by Th2 cells. IL-10 does not decrease IFN-gamma-induced I-Ad levels on a macrophage cell line. Inasmuch as IL-10 also inhibits IL-2-induced IFN-gamma production by Th1 cells in an Ag-free system requiring only the presence of accessory cells, these data suggest that IL-10 may inhibit macrophage accessory cell function which is independent of TCR-class II MHC interactions.     

An in vitro model for Toxoplasma infection in man. Interaction between CD4+ monoclonal T cells and macrophages results in killing of trophozoites

The cell interactions that take place between Toxoplasma gondii trophozoites and the human immune system have been investigated by using an in vitro model of infection. PBMC were co-cultured with live, appropriately attenuated, trophozoites. When cells from immune (seropositive) donors were used, a proliferative response was observed. At the same time, the proliferating T cells proved capable of controlling the growth of live trophozoites. By contrast, cells from seronegative donors failed to mount a proliferative response and intracellular overgrowth of trophozoites with subsequent cell injury occurred. Actively proliferating T cells were expanded in continuous cell lines with IL-2 and periodical restimulation with Ag in the presence of autologous irradiated mononuclear cells. From some of the lines obtained, clones were also derived. Ten clones were selected for further studies. They proliferated in response to trophozoites but not to unrelated Ag. Their response required the presence of autologous monocytes-macrophages isolated from peripheral blood on Percoll density gradients. B cells that were obtained from the same donors and immortalized by EBV infection proved inefficient as APC. These data suggest that live trophozoites have to be processed by macrophages in order to be presented to T cells. Upon appropriate antigen stimulation, all of the clones produced IL-2 and IFN-gamma, a finding that was consistent with both their CD4+ surface phenotype and their helper capacity on B cell proliferation and differentiation in vitro. The supernatants of all of the stimulated clones released a factor that activated macrophages to kill intracellular trophozoites as well as an unrelated pathogen, Listeria monocytogenes. This factor was identified as IFN-gamma because it was neutralized by specific anti-IFN-gamma antibodies. The present in vitro model of response to live protozoa may prove suitable to assess the role of both T lymphocytes and macrophages in intracellular parasite infections in man. Furthermore, this experimental system may be applied to detect specific lesions of cell mediated immunity in a number of immunodeficiency syndromes.     

Heterogeneity of helper/inducer T lymphocytes. III. Responses of IL-2- and IL-4-producing (Th1 and Th2) clones to antigens presented by different accessory cells

Murine CD4+ T cell clones have been classified into at least two subsets, Th1 and Th2, on the basis of their distinct lymphokine secretion profiles and functions. In the present study, we compared the functional responses of Th1 and Th2 clones to Ag presentation by splenic B cells and peritoneal macrophages. Th2 clones secreted IL-4 in response to Ag presented by resting B cells, but their optimal proliferation required the addition of IL-1 or a source of IL-1. The degree of IL-1 dependence varied among the four Th2 clones examined. In contrast, Th1 clones secreted IL-2 and proliferated in response to Ag presented by both B cells and macrophages, without any requirement for exogenous IL-1. Furthermore, the proliferation of Th2 clones in response to Ag presented by splenocytes or macrophages was inhibited by an IL-1R antagonist. These results indicate that IL-1 is an important costimulator for the expansion of the Th2 subset of CD4+ T cells. The different requirements for the proliferation of Th1 and Th2 cells may be responsible for the preferential expansion of one or the other subset under different conditions of immunization.     

Analysis of CD4+ T cells that provide contact-dependent bystander help to B cells.

Although cognate, MHC-restricted interaction of Th cells with Ag-presenting B cells provides effective help to a resting B cell, substantial B cell responses have also been seen with preactivated T cell clones that cannot recognize Ag on the B cell but apparently interact in a noncognate fashion (the bystander response). Here, we have investigated the ability of distinct Th cell subsets and T cells activated by different stimuli to support such bystander B cell responses. We have also determined which cytokines are involved. We generated distinct CD4+ T cell subsets specific for both alloantigen (using normal mice) and cytochrome c (using TCR transgenic mice). To compare cognate and bystander help, we analyzed the response of allogeneic (cognate) vs syngeneic (bystander) resting B cells in the former case, and the response of syngeneic B cells in the presence vs absence of Ag, in the latter case. Both approaches gave similar results. T cells stimulated with Ag for 24 h (naive and memory cells) or generated from naive cells over 4 days in the presence of exogenous IL-2 ("Th1-like" effectors) induced B cells to secrete minimal amounts of bystander Ig (20 to 700 ng/ml), less than 6% of the Ig induced under cognate conditions. In contrast, effectors generated in IL-4 or IL-6 ("Th2-like" and "Th0-like") induced significantly more bystander Ig (4 to 9 micrograms/ml), which was 18 to 30% of the amount produced during a cognate response. Restimulation of Th cell populations with anti-CD3, instead of Ag/APC, enhanced their ability to induce bystander Ig to levels 40 to 100% of those produced through cognate interaction. The addition of anti-cytokine Ab to bystander responses indicated that the cytokines utilized were similar to those mediating response after cognate interaction. Addition of exogenous cytokines did not specifically enhance the extent of the bystander response as a function of the cognate response. These results suggest that most Th cells can efficiently activate only those B cells that present relevant Ag on class II MHC, but that highly activated/differentiated Th effectors also have the ability to induce significant bystander B cell responses through noncognate interactions. We also conclude that the mode of Th cell activation and the cytokines encountered during Th differentiation play a major role in the capacity of helper cells to initiate a bystander response.     

Suppression of antibody synthesis by CD4+ T cell clones and normal T cells stimulated with monoclonal anti-CD3 antibody

CD4+ve Th1 clones, as well as normal splenic T cells, were found to suppress LPS-driven antibody secretion in a non-Ag-specific and non-MHC-restricted manner when the T cells were activated with the anti-CD3 mAb, 145-2C11. Suppression was observed with both primed and naive B cells, as well as with purified hapten-specific B cells, a result that suggests a direct effect of anti-CD3-activated T cells on B cell differentiation. Th1 clones activated by cognate Ag also suppressed LPS-driven antibody secretion. Furthermore, suppression of LPS-driven antibody secretion could be achieved across a cell-impermeable porous membrane when T cells were activated with anti-CD3. Suppression by Th1 clones and by normal T cells could not be attributed to a concomitant decrease in B cell proliferation or to a shift in the kinetics or isotype of the antibody response. These data demonstrate that CD4+ve Th1 clones, as well as normal T cells, can effect suppression of polyclonal antibody formation.     

Regulation of antibody production by helper T cell clones in experimental autoimmune myasthenia gravis

Ten acetylcholine receptor (AChR)-specific T cell clones from Lewis rats were studied. These clones had various AChR subunit and peptide specificities, and proliferated in response to antigen on appropriate APC. All the T cell clones were CD4+CD8- and OX22-, helped anti-AChR antibody production by AChR-primed lymph node B cells, and could secrete IL-2. However, several lines of evidence suggested that IL-2 was not the lymphokine that mediated T cell help. B cells primed with native AChR and then exposed in culture to very low concentrations of native AChR effectively presented the Ag to the T cell lines, presumably due to uptake via Ag receptors, but primed B cells were no more effective than were non-specific APC at presenting a synthetic AChR peptide which is recognized by AChR-specific T cells but not by AChR-specific B cells. Increasing AChR doses produced an antibody production response that was bell shaped and low doses stimulated, whereas higher AChR concentrations suppressed the antibody production response. Evidence suggested that AChR exerted its inhibitory effect through the T cells, but not via IL-2.     

CTLA-4 and CD28 mRNA are coexpressed in most T cells after activation. Expression of CTLA-4 and CD28 mRNA does not correlate with the pattern of lymphokine production.

Ag-presenting cells provide at least two distinct signals for T cell activation. T cell receptor-dependent stimulation is provided by presentation of a specific peptide Ag in association with MHC molecules. In addition, APC also supply costimulatory signals required for T cell activation that are neither Ag- nor MHC restricted. One such costimulatory signal is mediated via the interaction of B7 on APC with the CD28 receptor on T cells. Recently, CTLA-4 has been shown to be a second B7 receptor on T cells. In the present report, we have examined the expression of CD28 and CTLA-4 on a panel of resting and activated normal T cell subsets and T cell clones by RNA blot analysis in an attempt to determine whether their expression defines reciprocal or overlapping subsets. CD28 was detected in resting T cells, whereas CTLA-4 was not. After stimulation with PHA and PMA for 24 h, CTLA-4 mRNA was expressed in both the CD4+ and CD8+ subsets as well as in CD28+ T cells. We examined 37 human and six murine T cell clones that had been previously characterized for their cytokine production. After activation, CTLA-4 and CD28 mRNA were coexpressed in 36 of 37 human T cell clones and all six murine T cell clones. These included T cells of CD4+8-, CD4-8+, and CD4-8- phenotypes as well as clones with Th1 and Th2 cytokine profiles. In contrast, CD28 but not CTLA-4 mRNA was detected in leukemic T cell lines and myelomas. CTLA-4 and B7 mRNA but not CD28 mRNA was detected in two long term HTLV-I-transformed T cell lines. These data demonstrate that CD28 and CTLA-4 mRNA are coexpressed in most activated T cells and T cell clones, providing evidence that they do not define reciprocal subsets. Moreover, they are consistent with the hypothesis that B7 transmits its signal through a single receptor, CD28, on resting T cells, and multiple receptors, CD28 and CTLA-4, on activated T cells.     

Inducer T-cell-mediated killing of antigen-presenting cells

L3T4+ inducer/helper T-cell clones, once activated by antigen-presenting cells (APC) expressing the appropriate Ia allele and antigen, autonomously kill their target APC. All 13 L3T4+ inducer T-cell clones tested demonstrated this cytolytic activity. In addition, 11 different target cells representing the three major APC types, namely, macrophages, B cells, and dendritic cells, were all sensitive to this cytolytic activity. Moreover, normal macrophages which were treated with interferon-gamma to increase Ia expression were also killed. These observations convincingly demonstrate that the cytolytic activity of L3T4+ inducer T-cell clones is a general phenomenon. In contrast to other reports, lysis of target APC could not be detected following 4-6 hr of incubation. Marginal lysis was observed after 9 hr and a 20-hr incubation period was required to achieve maximal killing. The kinetics of killing paralleled other parameters of T-cell activation such as IL-2 release and cell proliferation. Activation of T cells for cytolysis of APC requires the interaction of T-cell receptors with Ia and antigen. Monoclonal antibody to Ia, L3T4 and the T-cell receptor inhibited the cytolysis of APC. The ability to mediate nonspecific bystander killing was variable depending on both the T-cell clone and the target. The implications of these findings to immune regulation and autoimmunity are discussed.     

Heterogeneity in direct cytotoxic function of L3T4 T cells. TH1 clones express higher cytotoxic activity to antigen-presenting cells than TH2 clones

In the process of generating culture supernatant from T cell clones, with anti-CD3 antibodies and the B lymphoma A20 as APC, a striking difference in the stimulation of TH1 and TH2 clones was observed, i.e., TH2 clones produced higher levels of lymphokines than TH1 clones. This prompted us to test the hypothesis that differential killing of APC (thus the removal of stimuli) by T cells led to differential T cell activation. By studying a panel of five TH1 and seven TH2 clones, it was demonstrated that TH1 clones mediated significantly higher levels of cytotoxicity toward A20 cells in the presence of soluble anti-CD3 antibody (as opposed to immobilized anti-CD3). Although T cell clones could, when activated with immobilized anti-CD3, produce lymphokines cytotoxic to A20 cells, experiments in which lymphokine production was blocked indicated that T cell clones, in the presence of soluble anti-CD3, mediated killing of A20 through direct cytotoxicity. A higher level of cytotoxicity, by TH1 compared with TH2 clones, was not restricted to anti-CD3 or a particular target cell type, because it also occurred with Con A- or Ag-dependent killing (a monocyte-macrophage cell line), and LPS blasts. Furthermore, the higher cytotoxic activity of TH1 clones compared with TH2 clones was independent of the stage of T cell activation and was unlikely a result of the length of in vitro culture. High levels of killing of APC led to low levels of T cell activation, the significance of which may be as a negative feedback mechanism in the immune response. Other biologic relevancies of higher cytotoxic activity in TH1 vs TH2 cells were also discussed.