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.     

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.     

Allogeneic non-T spleen cells restore the responsiveness of normal T cell clones stimulated with antigen and chemically modified antigen-presenting cells

Stimulation of IL-2-producing T cell clones with chemically modified APC and Ag induces a state of proliferative unresponsiveness, i.e., subsequent stimulation with normal APC and Ag fails to elicit IL-2 production. One possible effect of chemical modification on the APC is the destruction of its ability to provide costimulatory signals. To test this, various potential costimulators were added to T cells at the time of exposure to Ag and chemically modified APC. None of the cytokines tested, including IL-1, had a positive effect; however, addition of allogeneic spleen cells allowed a T cell proliferative response and prevented the induction of subsequent unresponsiveness. Fractionation of the spleen cells showed that low density B cells and macrophages were the best source of costimulatory activity. Allogeneic resting B cells provided some costimulatory activity and resting T cells, none at all. Attempts to mimic costimulatory signals with the phorbol ester PMA were only partially successful. PMA prevented the induction of T cell unresponsiveness but failed to allow T cell proliferation in response to Ag plus chemically modified APC. Our results suggest that IL-2 production by normal T cell clones is dependent not only on T cell receptor occupancy, but also on short range costimulatory signals that are provided to different degrees by various non-T accessory cells.     

Th1 and Th2 clones differ in their response to a tolerogenic signal.

Th1 and Th2 clones specific for human gamma globulin (HGG) were compared and shown to differ in terms of the effects of tolerance induction on Ag-induced proliferation and helper activity. In developing a method to induce tolerance, splenic APC that had been pulsed with HGG and then fixed with 0.15% paraformaldehyde (HGG-FAPC) were used as a means to present Ag to the Th clones in the absence of costimulatory signals. Both Th1 and Th2 clones recognized HGG-FAPC as evidenced by their ability to proliferate to HGG-FAPC. Unlike Th2, Th1 proliferated to HGG-FAPC only in the presence of T cell-depleted allogeneic spleen cells as a source of accessory cell signals. The inability of Th1 cells to proliferate in the absence of costimulatory signals was due to Ag-specific inactivation: Th1 clones preincubated with HGG-FAPC were unable to proliferate when recultured with HGG and irradiated APC. In contrast to Th1 clones, Th2 clones showed no decrease in their Ag-induced proliferative capacity after exposure to any concentration of HGG-FAPC. However, when examined by using a second assay system, that of providing help for anti-HGG antibody production by primed B cells, Th2 preincubated with HGG-FAPC were markedly inhibited (up to 90%) in their ability to provide help. Preincubation with HGG-FAPC also inhibited the helper activity of the one Th1 clone that was found to induce a significant secondary antibody response. Taken together, the results suggest that exposure of Th1 to tolerogen in the form of HGG-pulsed fixed APC inactivates Th1 proliferative capacity, and possibly Th1 helper activity as well. Exposure of Th2 cells to a tolerogen suppresses the mechanism by which the Th2 cells provide Ag-induced B cell help, but does not inhibit the mechanism by which they proliferate to HGG. Furthermore, the results define a model that incorporates Ag processing as well as Ag presentation in the induction of tolerance in vitro.     

CD4+ T cell-mediated killing of MHC class II-positive antigen-presenting cells. I. Characterization of target cell recognition by in vivo or in vitro activated CD4+ killer T cells

Ag-specific as well as Ia-restricted killing of certain APC by CD4+ T cells was investigated. The CD4-mediated killing is not only a characteristic of in vitro long term cultured T cell lines or clones, but is also manifest after in vivo priming. Thus, CD4+ killer T cells are generated in vivo as well. CD4+ killer T cells are detected in the Th1, but not in the Th2 subset, and they do not appear to lyse Ia+ APC or bystander cells by a pathway mediated by secreted T cell factors. The latter observation is demonstrated by cold target inhibition experiments as well as by the failure of puromycin to inhibit killing, if applied in doses which completely block lymphokine secretion. Ia+ APC differ in their susceptibility to lysis. Transformed APC are usually better lysed than nontransformed APC. Unstimulated B cells are not killed, while LPS-stimulated B cell blasts are killed. The results of cold target inhibition and bystander killing experiments suggest that CD4+ killer T cells are activated by the common pathway, i.e., by Ag presented in the context of Ia, but killing requires the recognition of additional determinant(s) on APC. It is proposed that these killing-inducing determinants are continuously expressed on most transformed Ia+ cells and on nontransformed but stimulated APC.     

Antigen receptor-mediated anergy in resting T lymphocytes and T cell clones. Correlation with lymphokine secretion patterns.

The goal of these studies was to define the stimuli and factors that control the induction of anergy in unimmunized resting T lymphocytes. Initial experiments, aimed at establishing the system, showed that exposure of Th1 but not Th2 clones to immobilized anti-CD3 leads to a block in autocrine growth factor production and proliferation upon subsequent restimulation with Ag+APC. Anergy is not prevented by accessory cells, suggesting that this model of T cell tolerance may be due to receptor-mediated inhibitory signals, independent of costimulatory molecules. Culture of small (resting) unimmunized T lymphocytes with anti-CD3 +/- IL-2 induces unresponsiveness to restimulation with anti-CD3, but culture with anti-CD3+IL-4, which stimulates the differentiation of resting cells into IL-4 producers, does not induce anergy. Thus, IL-4-producing clones and bulk populations of IL-4-producing T cells are resistant to Ag receptor-mediated inhibitory stimuli. These results provide experimental models for studying the mechanisms of anergy in normal, unselected, mature T cells, and demonstrate fundamental similarities between cloned cell lines and unimmunized T lymphocytes in the induction of anergy.     

Activation requirements of cloned inducer T cells. II. The failure of some clones to respond to antigen presented by activated B cells

Inducer/helper T cells recognize nominal antigen in association with Ia on the surface of the antigen-presenting cell (APC). Recent studies have shown that B cells can effectively function as APC. In the present study we have assessed the ability of cloned inducer T cells to discriminate between activated B cells or splenic macrophages as APC. We found that most of the clones tested demonstrated an equivalent response to antigen presented by activated B cells or splenic adherent cells. Some clones were very efficiently stimulated by antigen presented by activated B cells, whereas other clones failed to respond or responded very poorly when activated B cells were used to present antigen. We attempted to determine the mechanism responsible for the inability of certain clones to proliferate in response to antigen presented by activated B cells.     

Antigen-specific, MHC-restricted B cell activation by cell-free Th2 cell products. Synergy between antigen-specific helper factors and IL-4

Ag-specific and MHC-restricted Th clones of different Ag specificities and MHC haplotypes were tested for their ability to produce soluble factors capable of providing the signals required for B cell activation and IgG antibody production. Each of five Th clones tested generated significant helper activity in supernatants derived from coculture of the T cell clone with specific Ag and syngeneic APC. The same helper activity was detected in supernatants of clones stimulated with immobilized anti-CD3 antibody in the absence of Ag or APC. The secreted helper activity resembled the activity of the intact Th cells in that it was Ag-specific, carrier-hapten-linked and MHC-restricted. These T cell products functioned to activate only those B cells expressing MHC products which corresponded to the specificity of each Th clone. Thus, the specificity of the cell-free T cell product mimicked precisely that expressed by the intact Th cell and presumably mediated by the cell surface TcR. In addition to the apparent presence of specific helper factor in Th clone supernatants, a role for nonspecific lymphokines was also identified in these preparations. Although recombinant or purified IL-4 alone was not sufficient to stimulate hapten-primed B cells to secrete hapten-specific IgG antibodies, mAb specific for IL-4 blocked the induction of antibody secretion by Th cell supernatant. These results indicate that stimulation of B cells to produce hapten-specific IgG antibody requires at least two distinct signals: an Ag-specific T cell signal which is restricted by MHC products expressed on the B cells, and a nonspecific signal mediated at least in part by the lymphokine IL-4.     

Costimulatory requirements of murine Th1 clones. The role of accessory cell-derived signals in responses to anti-CD3 antibody

We examined the role of accessory cell-derived signals in promoting growth and lymphokine production by murine Th1 clones. Five of six Th1 clones failed to proliferate to immobilized anti-CD3 antibody despite producing IL-2 and IFN-gamma. These clones became unresponsive to Ag after exposure to anti-CD3. With the addition of irradiated splenic accessory cells (SAC), Th1 clones proliferated to anti-CD3 and produced greater amounts of IL-2 and IFN-gamma. High doses of plate-bound anti-CD3 completely inhibited responses of these clones to IL-2 and diminished the growth-promoting activity of SAC. The costimulatory effects of SAC on growth of Th1 clones were also seen in the presence of exogenous IL-2, indicating that enhanced IL-2 production alone was not responsible for the costimulatory effect. Delivery of the costimulatory signal from SAC required their close proximity to the T cells. The costimulatory activity of SAC was not reproduced by the addition of IL-1, IL-6, or IL-1 plus IL-6. IL-7 induced weak proliferation of Th1 clones, but did not synergize with plate-bound anti-CD3. Our results suggest a model in which SAC-derived costimulatory signals regulate growth of Th1 cells primarily at the level of cell cycle progression rather than at the level of IL-2 production.     

CD28-independent costimulation of T cells by OX40 ligand and CD70 on activated B cells.

OX40 and its ligand (OX40L) have been implicated in T cell-dependent humoral immune responses. To further characterize the role of OX40/OX40L in T-B cell interaction, we newly generated an anti-mouse OX40L mAb (RM134L) that can inhibit the costimulatory activity of OX40L transfectants for anti-CD3-stimulated T cell proliferation. Flow cytometric analyses using RM134L and an anti-mouse OX40 mAb indicated that OX40 was inducible on splenic T cells by stimulation with immobilized anti-CD3 mAb in a CD28-independent manner, while OX40L was not expressed on resting or activated T cells. OX40L was inducible on splenic B cells by stimulation with anti-IgM Ab plus anti-CD40 mAb, but not by either alone. These activated B cells exhibited a potent costimulatory activity for anti-CD3-stimulated T cell proliferation and IL-2 production. Anti-CD80 and anti-CD86 mAbs partially inhibited the costimulatory activity, and further inhibition was obtained by their combination with RM134L and/or anti-CD70 mAb. We also found the anti-IgM Ab- plus anti-CD40 mAb-stimulated B cells exhibited a potent costimulatory activity for proliferation of and IL-2 production by anti-CD3-stimulated CD28- T cells from CD28-deficient mice, which was substantially inhibited by RM134L and/or anti-CD70 mAb. These results indicated that OX40L and CD70 expressed on surface Ig- and CD40-stimulated B cells can provide CD28-independent costimulatory signals to T cells.     

Characterization of rat OX40 ligand by monoclonal antibody

OX40 (CD134) is a member of the tumor necrosis factor (TNF) receptor superfamily first identified as a rat T cell activation marker. We previously identified the rat ligand for OX40 (OX40L) by molecular cloning. In the present study, we newly generated an anti-rat OX40L mAb (ATM-2) that can inhibit the binding of OX40 to rat OX40L and thus efficiently inhibits the T cell costimulatory activity of rat OX40L. Flow cytometric analyses using ATM-2 and an anti-rat OX40 mAb (MRC OX40) indicated that OX40 was inducible on splenic CD4(+) T cells by stimulation with immobilized anti-CD3 mAb, while OX40L was not expressed on resting or activated T cells. OX40L was expressed on splenic B cells after stimulation with lipopolysaccharide (LPS), but not on peritoneal macrophages. Interestingly, splenic dendritic cells (DC) expressed OX40L constitutively, which was further upregulated by LPS stimulation. The potent costimulatory activities of splenic DC for anti-CD3-stimulated rat CD4(+) T cell proliferation and cytokine (IL-2, IFN-gamma, IL-10, and IL-13) production were substantially inhibited by ATM-2. These results indicated that OX40L is expressed on professional antigen-presenting cells (APC), and may be involved in humoral immune responses via T-B interaction and in cellular immune responses via T-DC interaction in the rat system.     

The requirement for surface Ig signaling as a prerequisite for T cell:B cell interactions. A possible role for desialylation

Models for T cell:B cell collaboration suggest that activated B cells process and present Ag to Th cells which subsequently induce B cell proliferation and differentiation. In contrast to activated B cells, resting B cells have generally been shown to be less efficient APC. If this model of T:B collaboration is physiologically correct, then resting B cells must undergo a phenotypic change that permits effective interaction with T cells. In this report, the requirement for rapid signaling through surface Ig on resting B cells for the induction of T:B interaction was investigated with an in vitro clustering assay. Resting splenic B cells were unable to form specific conjugates with T cell clones, unless the B cells were first treated with neuraminidase to remove sialic acid. In contrast, LPS-activated B cells were able to form conjugates without prior treatment. The ability of antibody against LFA-1 or L3T4 to inhibit cluster formation depended on the state of B cell activation in that anti-LFA-1 and anti-L3T4 mAb inhibited cluster formation by neuraminidase-treated resting B cells, but not by LPS-activated B cells. In addition, Ag-specific B cells which were isolated by their capacity to bind specific Ag were able to form clusters without any additional treatment. Moreover, treatment of resting splenic B cells with anti-mu-antibody induced clustering potential in B cells in as little as 10 min, suggesting that signaling through surface Ig was sufficient to induce this phenotypic change in B cells. Furthermore, activation of protein kinase C and Ca2+ mobilization were shown to be involved in that PMA and ionomycin treatment were also able to induce clustering potential in resting B cells. The rapid induction of clustering potential in resting B cells after signaling through surface Ig may represent a fundamental change in B cell physiology which occurs after recognition of specific Ag and may be required for effective cognate recognition between resting hapten-specific B cells and carrier-specific T cells. The potential role of desialylation for the induction of T:B interaction is discussed.     

Ability of tolerized Th1 and Th2 clones to stimulate B cell activation and cell cycle progression.

Tolerant and nontolerant murine Th1 and Th2 clones, specific for human gamma-globulin (HGG), were compared for their ability to promote cell cycle entry and progression by B cells in vitro. When stimulated with HGG, nontolerant Th1 and Th2 clones induced similar increases in B cell membrane MHC class II levels--a phenomenon associated with early B cell activation. Nontolerant Th1 and Th2 clones also induced B cell DNA synthesis, an event associated with subsequent G1 phase traversal, although Th2 cells were more efficient than Th1 cells in stimulating this activity. Exposure of Th clones to tolerogen in the form of HGG-pulsed chemically fixed APC inhibited the ability of Th1 clones, but not Th2 clones to promote polyclonal B cell DNA synthesis in HGG-stimulated secondary cultures. However, Th1 clones exposed to tolerogen did not lose their ability to increase the expression of MHC class II molecules on B cells in these cultures. These results indicate that tolerance induction does not inhibit the ability of Th1 clones promote B cell cycle progression. In contrast, exposure of Th2 cells to tolerogen does not inhibit significantly the ability of these cells to stimulate B cell cycle entry or progression.     

Class II MHC/peptide complexes are released from APC and are acquired by T cell responders during specific antigen recognition.

T cell expression of class II MHC/peptide complexes may be important for maintenance of peripheral self-tolerance, but mechanisms underlying the genesis of class II MHC glycoproteins on T cells are not well resolved. T cell APC (T-APC) used herein were transformed IL-2-dependent clones that constitutively synthesized class II MHC glycoproteins. When pulsed with myelin basic protein (MBP) and injected into Lewis rats, these T-APC reduced the severity of experimental autoimmune encephalomyelitis, whereas unpulsed T-APC were without activity. Normal MBP-reactive clones cultured without APC did not express class II MHC even when activated with mitogens and exposed to IFN-gamma. However, during a 4-h culture with T-APC or macrophage APC, recognition of MBP or mitogenic activation of responder T cells elicited high levels of I-A and I-E expression on responders. Acquisition of class II MHC glycoproteins by responders was resistant to the protein synthesis inhibitor cycloheximide, coincided with transfer of a PKH26 lipophilic dye from APC to responders, and resulted in the expression of syngeneic and allogeneic MHC glycoproteins on responders. Unlike rested I-A- T cell clones, rat thymic and splenic T cells expressed readily detectable levels of class II MHC glycoproteins. When preactivated with mitogens, naive T cells acquired APC-derived MHC class II molecules and other membrane-associated proteins when cultured with xenogeneic APC in the absence of Ag. In conclusion, this study provides evidence that APC donate membrane-bound peptide/MHC complexes to Ag-specific T cell responders by a mechanism associated with the induction of tolerance.     

Resting B lymphocytes as APC for naive T lymphocytes: dependence on CD40 ligand/CD40

Although resting B cells as APC are tolerogenic for naive T cells in vivo, we show here that they can provide all the costimulatory signals necessary for naive T cell proliferation in vivo and in vitro. In the absence of an activating signal through the B cell Ag receptor, T cell proliferation after Ag recognition on resting B cells depends on CD40 expression on the B cells, implying that naive T cells use the membrane-bound cytokine, CD40 ligand (CD154), to induce the costimulatory signals that they need. Induction of B7-1 (CD80) and increased or sustained expression of CD44H, ICAM-1 (CD54), and B7-2 (CD86) are dependent on the interaction of CD40 ligand with CD40. Transient expression (12 h) of B7-2 is T cell- and peptide Ag-dependent, but CD40-independent. Only sustained (>/=24 h) expression of B7-2 and perhaps increased expression of ICAM-1 could be shown to be functionally important in this system. T cells cultured with CD40-deficient B cells and peptide remain about as responsive as fresh naive cells upon secondary culture with whole splenic APC. Therefore, B cells, and perhaps other APC, may be tolerogenic not because they fail to provide sufficient costimulation for T cell proliferation, but because they are deficient in some later functions necessary for a productive T cell response.     

IgM induction in murine B hybridomas with Ia-restricted T cell clones: a monoclonal model for T and B cell interactions in antibody response

The mechanism of MHC-restricted T and B cell interactions in antibody response was studied with IgM-inducible B hybridomas and antigen-specific helper T cell clones. B hybridomas were prepared by fusion between splenic B cells from (CBA/N (H-2k) X BALB/c (H-2d)) F1 (NBF1) male mice and a B lymphoma cell line, M12.4.5. A B hybridoma clone, 1M70, which expressed I-Ad but not I-Ak determinants was chosen in the present study. IgM secretion was induced in 1M70 when it was cocultured with a "resting" KLH-specific and H-2d restricted helper T cell clone in the presence of KLH. A "resting" KLH-specific and H-2k restricted T cell clone did not induce IgM secretion in 1M70 even in the presence of KLH. However, when these KLH-specific T cell clones were activated by KLH and appropriate antigen presenting cells, both H-2d and H-2k restricted T cell clones induced IgM secretion in 1M70 even in the absence of KLH. A monoclonal anti-I-Ad antibody inhibited IgM secretion induced by a "resting" H-2d restricted T cell clone, but not by an "activated" T cell clone. These results indicated that T cell clones recognized antigens in the context of Ia molecules on B hybridomas in a MHC-restricted manner and were activated to produce B cell stimulatory factors which in turn acted on B hybridomas in a non-MHC-restricted manner and induced differentiation of B hybridomas into IgM secreting cells.     

Heterogeneity of helper/inducer T lymphocytes. IV. Stimulation of resting and activated B cells by Th1 and Th2 clones

To test the hypothesis that resting and previously activated B lymphocytes differ in their proliferative and differentiative responses to various Th cell-derived stimuli, we have examined the interactions of purified small (resting) and large (activated) murine B cells with rabbit Ig-specific Th1 and Th2 clones in the presence of the Ag analogue, rabbit anti-mouse Ig antibody. Small numbers of Th2 cells induce strong Ag-dependent proliferation of and Ig secretion by both resting and activated B lymphocytes. In contrast, Th1 clones stimulate lower responses of activated B cells and fail to stimulate small resting B cells. An interaction with Th1 clones does make small B cells responsive to the Th2-derived cytokine, IL-4, indicating that Th1 clones are capable of delivering some but not all the stimuli necessary for the induction of humoral immunity. Finally, in order to compare the responses of small and large B cells to cognate interactions and secreted cytokines, we used an autoreactive I-Ak-specific Th2 line. This line induces proliferation of and Ig secretion by I-Ak expressing but not H-2d resting and activated B cells as a result of cognate interactions. However, when the H-2d B cells are bystanders in the presence of cytokine secretion by this Th2 line, or are directly exposed to Th2-derived cytokines, both small and large B cells are induced to proliferate but only the large B cells secrete antibody. These results indicate that the magnitude and nature of antibody responses depend on three principal factors: the cytokines produced by Th cells, the state of activation of the responding B lymphocytes, and whether the B cells are recipients of cognate help or are bystanders at the site of T cell stimulation. Our findings also confirm the view that cognate T-B interactions are most efficient for initiating B cell responses and may allow B cells to subsequently respond to a variety of T cell-derived cytokines.     

In vivo treatment with anti-I-A antibodies: differential effects on Ia antigens and antigen-presenting cell function of spleen cells and epidermal Langerhans cells

The in vivo activation of T cells by a variety of antigens can be inhibited by the administration of anti-I-A antibodies (Ab) at the time of antigen priming. This inhibition can partially be explained by the temporary loss of Ia molecules from Ia-bearing antigen-presenting cells (APC) in the spleen. In this study, the effects of i.p. injected monoclonal Ab specific for I-A glycoproteins of different H-2 haplotypes on Ia antigen expression and APC function of spleen cells and epidermal Langerhans cells were compared. It was found that anti-I-A Ab quickly bound to both spleen cell and Langerhans cell Ia antigens. Although spleen cell Ia antigens were modulated and thus temporarily disappeared, Ia antigen expression by epidermal Langerhans cells was not modulated. In functional studies, the capacity of spleen cells and epidermal cells from anti-I-A Ab treated vs control animals to function as APC for antigen-specific, I-A- or I-E-restricted T cell clones was tested. A single injection of anti-I-A Ab completely abolished the APC function of spleen cells as shown in several inbred mouse strains, F1 animals, and with the use of several different Ab and T cell clones. In contrast, Langerhans cell-dependent APC function of epidermal cells remained completely unaltered. Even multiple injections of high doses of Ab never caused any inhibition of Langerhans cell function. Experiments with anti-I-Ak or anti-I-Ad Ab in an (H-2k X H-2d)F1 animal showed abrogation of APC function of spleen cells, but again not of Langerhans cells. Thus in vivo anti-I-A Ab administration appears to differentially affect Ia antigen expression and APC function from spleen and epidermis: Ia antigens are modulated from spleen cells but not from epidermis, and APC function disappears in the spleen but not in the epidermis. The abrogation of splenic but not of Langerhans cell APC function with anti-I-A Ab will facilitate the dissection of the relative contributions of Langerhans cells as compared with other APC in the generation of cutaneous immune responses.     

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.     

Effect of gamma radiation on resting B lymphocytes. II. Functional characterization of the antigen-presentation defect

The effect of radiation on three discrete Ag-presentation functions in resting B cells was examined: 1) Ag uptake and processing, 2) expression of processed Ag in the context of functional class II molecules, and 3) provision of necessary co-stimulatory, or "second," signals. Analysis of radiation's effect on B cell presentation of intact vs fragmented Ag or its effect on presentation by Ag-pulsed B cells indicated that damage to Ag uptake and processing could not account for the bulk of the radiation-induced Ag-presentation defect. Experiments with phosphatidylinositol hydrolysis as an indirect measure of TCR occupancy suggested that irradiation caused a fairly rapid (within 1 to 2 h) decrease in the ability of the B cell APC to display a stimulatory combination of Ag and class II molecule. Ag dose-response analyses demonstrated that when presenting a fragment of the Ag pigeon cytochrome c to a T cell clone, 3000 rad-treated B cell APC were able to stimulate approximately 50% as much phosphatidylinositol turnover as unirradiated B cells. It was also found that, in contrast to their inability to initiate T cell proliferation, and similarly to chemically cross-linked splenocytes, heavily irradiated resting B cells plus Ag induced a state of Ag hyporesponsiveness in T cell clones. This effect on T cells had the same Ag- and MHC-specificity as did receptor occupancy required for proliferation, indicating that heavily irradiated resting B cells bear functional class II molecules. Co-culture of T cells with allogeneic B cells and syngeneic heavily irradiated B cells or chemically cross-linked splenic APC plus Ag resulted in T cell proliferation and interfered with the induction of the hyporesponsive state. This co-stimulatory function was radiosensitive in resting allogeneic B cells. Together, these data support the hypothesis that the major functional consequences of radiation to resting B cell APC are a reduction in the effective display of Ag plus class II molecules and, probably what is more important, a loss in the ability to provide APC-derived co-stimulatory signals.