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Some tumor cells have deficits in class I MHC antigen processing, suggesting that T cells exert selective pressure on tumor cells. Previous studies have not revealed increased tumor incidence in mice with deficits in T-cell immunity, including mice lacking TAP1 (a subunit of the transporter for antigen presentation) or LMP2 (a regulated subunit of the 20S proteasome). The incidence of spontaneous tumors in these mice, however, is too low to assess differences in host resistance to tumors. To increase tumor incidence and better assess the role of systemic expression of TAP1 and LMP2 in responses to tumors, TAP1-/- and LMP2-/- mice were bred with p53-/- mice to create TAP1-/-p53-/- and LMP2-/-p53-/- double knockout mice. Lymphomas and sarcomas (malignant fibrous histiocytoma and angiosarcoma) occurred with high incidence in all p53-deficient populations. Tumor incidence and death rate were similar in TAP1-/-p53-/- mice and closely matched control TAP1+/+p53-/- mice. Tumor incidence and death rate were slightly accelerated in LMP2-/-p53-/- mice relative to control LMP2+/+p53-/- mice, but the biological significance of this difference was unclear. The relative incidence of lymphomas vs. sarcomas was not significantly altered by variation in TAP1 or LMP2. In conclusion, systemic absence of TAP1 did not alter tumor incidence, while absence of LMP2 was associated with only a slight acceleration of tumor incidence of uncertain significance. These observations are consistent with other evidence that normal T-cell responses do not effectively limit tumorigenesis. Even though T cells can attack some tumor cells, the ability of tumors to alter their immunogenicity and evade T-cell surveillance may render the native immune system ineffective at providing a rate-limiting barrier to tumorigenesis and preventing cancer.

Collagen-induced arthritis (CIA) in susceptible strains of mice is an animal model of T cell-mediated inflammatory polyarthritis. Analysis of T cell receptor (TCR) V beta gene usage in cells isolated from arthritic joints of BUB/BnJ (BUB) mice (H-2q, TCR V beta a) showed that TCR V beta chain gene usage was limited to TCR V beta 3 and V beta 10 gene families. All of the BUB mice immunized with a mixture of TCR V beta 3 and TCR V beta 10 peptides, but not with control TCR V beta 14 peptide, were refractory to the induction of CIA. Immunization with TCR V beta 3 and V beta 10 peptides completely blocked the development of clinical and subclinical inflammation, formation of pannus and synovial hyperplasia, and the erosion of cartilage and bone. Further studies revealed that preimmunization of BUB mice with V beta 10 peptide alone was sufficient to render the mice resistant to CIA. Analysis of TCR V beta chain gene expression in lymph node cells from arthritic and arthritis-protected mice showed the expression of TCR V beta 10 subfamily in all of the arthritic mice, but not in arthritis-protected mice. Immunization with TCR V beta peptides did not diminish the humoral responses to chicken type-II collagen and also elicited significant levels of anti-V beta 3 and anti-V beta 10 peptide antibodies. Antibodies cross-reactive with mouse chicken type-II collagen were detected in both the arthritic and arthritis-protected mice. Adoptive transfer of serum from arthritis-protected BUB mice significantly delayed the onset (P < 0.005) of arthritis in recipient BUB mice. In contrast, mice injected with serum from arthritic mice had early onset of arthritis. These results demonstrate that immunization of BUB mice with TCR V beta chain peptides elicited antibodies reactive with the self-TCR and prevented the induction of collagen-induced arthritis by eliminating or downregulating pathogenic T cells and consequently blocking the development of humoral immune response. These findings may have clinical applications in treating human autoimmune diseases characterized by common TCR gene usage.

T cell hybridomas that are deathless upon TCR crosslinking were generated from lpr mice. The deathless hybridomas (1.4 and 5D5) expressed extremely low Fas even after anti-CD3 activation, whereas activation-induced cell death (AICD) was observed for Fas-expressing hybridomas. The deathless hybridomas were activated to produce FasL and IL-2, indicating that the intrinsic defect in Fas expression or up-regulation resulted in AICD blockade. The deathless hybridoma cells expressed longer and stronger FasL cytotoxicity than AICD-sensitive hybridomas. Although deathless, activated 5D5 cells were arrested at the G1/S border. Growth arrest lasted for at least 5 days, but some cells eventually recovered and proliferated. The deathless 5D5 cells were used to demonstrate that AICD includes a fratricidal mechanism that kills AICD-sensitive bystanders. The deathless T cell hybridomas are useful tools for studying T cell activation-dependent functions sensitive to AICD.

N-methylnitrosourea (MNU) induces thymic lymphoma in a high proportion of susceptible C57BL/6xSJL (C57/SJL) mice. Expression of the human DNA repair gene, MGMT cDNA, which encodes O6-methylguanine-DNA-methyltransferase, in transgenic mice effectively prevents MNU-induced thymic lymphomas. In this study, we determined the phenotype of thymocytes expressing the transgene and defined whether the target cell population for MNU induced lymphomas were actually those that expressed the transgene. Transgene expression was characterized by in situ hybridization for MGMT mRNA and immunohistochemistry for the human alkyltransferase protein and was compared to the phenotype of the MNU induced lymphomas. The MGMT transgene was expressed uniformly in immature cortical thymocytes that were CD4+CD8+J11d+ and to a lesser extent in the medullary thymocyte. Lymphomas were induced by single [50 or 80 mg/kg] or multiple doses [30 mg/kg x 5] of MNU to evaluate the dose response of tumor induction and protection by the MGMT-CD2 transgene. Forty-seven of the 108 treated mice developed lymphomas: 38 of 58 nontransgenic and 9 of 50 MGMT+ mice. The T-cell phenotype of thymic lymphomas was established by immunohistochemistry and FACS analysis. Most of the lymphomas were J11d+ (98%), 70% of the tumors were CD4+CD8+, 21% were CD4-CD8+, 9% were CD4-CD8-, and none were CD4-CD8-. All lymphomas in MGMT+ transgenic mice were CD4+CD8+. Since the main phenotype of MNU induced lymphomas in these mice, CD4+CD8+J11d+, is also the cell phenotype which expresses the MGMT-CD2 transgene at high levels, it appears that MGMT-induced protection has occurred in the cell target for MNU induced transformation.

Protein antigens must be taken up, processed, and displayed on the surface of antigen-presenting cells in association with major histocompatibility complex molecules before they can be recognized by T cells. Whether recognition of the haptens used to study allergic contact hypersensitivity in murine models similarly requires processing has not been determined. We analyzed whether presentation of trinitrophenol to trinitrophenol reactive T-cell hybridomas requires antigen processing by studying the effects of inhibitors of antigen processing and presentation on the ability of a syngeneic B-cell tumor (A20) to present trinitrophenol to a series of interleukin-2 producing, trinitrophenol specific, major histocompatibility complex class II-restricted T-cell hybridomas. The ability of trinitrophenol modified A20 cells to stimulate the hybridomas was completely inhibited by monoclonal, anti-trinitrophenol, or anti-Ia antibodies and was significantly reduced by paraformaldehyde fixation immediately after trinitrophenol modification. Trinitrophenol-modified A20 cultured at 37 degrees C for 2 h prior to fixation was significantly more effective at stimulating the hybridomas than trinitrophenol-modified A20 fixed immediately. The ability of A20 to present trinitrophenol was inhibited by chloroquine. Paraformaldehyde fixation and chloroquine treatment had similar effects on the ability of trinitrophenol modified lymph node dendritic cells to stimulate the trinitrophenol specific hybridomas. Paraformaldehyde fixation and chloroquine treatment had similar effects on the ability of A20 cells to present ovalbumin to ovalbumin-specific hybridomas as they had on the ability of trinitrophenol modified A20 cells to present trinitrophenol to the trinitrophenol specific hybridomas. One of seven T-cell hybridomas responded to trinitrophenol modified ovalbumin but not other trinitrophenol modified proteins. These results suggest that, at least in part, T cells in the contact hypersensitivity response to trinitrophenol recognize antigens that require processing and that trinitrophenol modified proteins can be recognized.

We used polyclonal rabbit anti-Thy-1 antiserum to eliminate T lymphocytes in the peripheral blood, lymph nodes, and spleens but not in the thymus of normal mice. T lymphocytes began to reappear in the peripheral lymphoid compartments 2 weeks after termination of the treatment. By 4 weeks, the numbers of peripheral T lymphocytes had recovered to normal levels. We found that injection of recombinant soluble forms of human CD44 or L-selectin but not human CD8 molecules delayed the reappearance of peripheral T lymphocytes in anti-Thy-1-treated mice. This delay in recovery most likely reflects the ability of these soluble receptors to interfere with lymphocyte migration in vivo. Our results provide in vivo evidence that CD44 and L-selectin regulates lymphocyte trafficking and suggest that soluble forms of both molecules provide useful tools for the study of lymphocyte migration in vivo.

We studied the interactions between CD44 and four different monoclonal anti-CD44 antibodies. All four monoclonal anti-CD44 antibodies studied (P3H9, Bu52, IM.7, and GKW.A3) act in synergy with human anti-CD2 antibodies in stimulating normal human peripheral blood lymphocytes to proliferate. GKW.A3 and IM.7 but not P3H9 or Bu52 inhibited the proliferation of normal human peripheral blood lymphocytes stimulated by anti-CD3. Interestingly, only GKW.A3 and IM.7 stimulated the incorporation of [3H]palmitic acid and palmitoylation of CD44 molecules by normal human peripheral blood lymphocytes. The two monoclonal anti-CD44 antibodies (P3H9 and Bu52) that failed to inhibit anti-CD3 induced proliferation also failed to induce the incorporation of [3H]palmitic acid. More importantly, the inhibitory effects of GKW.A3 were reversed in the presence of cerulenin, an inhibitor of protein palmitoylation. Therefore, palmitoylation of CD44 may interfere with anti-CD3 mediated signaling pathways. These data support the hypothesis that palmitoylation of cell surface receptors may play an active role in receptor and receptor interactions and signal transduction in normal human T lymphocytes.

T cells are major mediators of graft rejection and many autoimmune diseases. During the Ag recognition process, T cells often become activated. We tested the hypothesis that an anti-CD3:anti-CD25 (CD3,25) bispecific mAb (BSMAB) can effectively and selectively target activated T cells. By flow cytometric analysis, the CD3,25 BSMAB was shown to bind avidly to activated T cells that coexpress CD3 and CD25 (p55 chain of the IL-2R), achieving higher levels than the parent anti-CD3 and anti-CD25 mAb. It bound only weakly to unstimulated T cells. The CD3,25 BSMAB effectively redirected CTL to lyse CD25-bearing PHA-stimulated T lymphoblasts and the IL2-dependent CTLL tumor cell line in chromium release assays. It was highly effective in blocking MLR as shown by inhibition of [3H]TdR incorporation. However, the CD3,25 BSMAB has a low potential to activate resting T cells, as it induced only minimal [3H]TdR incorporation even in the presence of exogenous IL-2. In the absence of exogenous IL-2, the CD3,25 BSMAB was unable to induce [3H]TdR incorporation. In contrast, the parent anti-CD3 mAb induced a high degree of incorporation. In summary, the CD3,25 BSMAB selectively targets activated CD25-expressing T cells and lymphomas although maintaining a low activation potential for unstimulated T cells, potentially advantageous properties that can be exploited for immunotherapy.

Functional maturation of B lymphocytes correlates with expression of the B lineage-specific cell surface glycoprotein CD22. Two CD22 polypeptides have been characterized and suggested to play a role in B cell-B cell interaction as well as in B cell adhesion to monocytes. In this work we provide evidence that CD22 is directly involved in the cognate interaction between B and T cells. One of the two CD22 polypeptides, CD22 beta, interacts with a specific ligand on a subpopulation of CD4+ T cells. Our results suggest that the T cell ligand of CD22 is CD45RO, an isoform of the leukocyte common antigen class of phosphotyrosine phosphatases associated with the helper T cell phenotype. We further demonstrate that CD22 recognizes a second ligand, CD75, expressed predominantly on activated B cells and shown to be a cell surface alpha 2-6 sialyltransferase.

The suppressor pathway that regulates the T cell response to p-azobenzenearsonate (ABA)-coupled cells has been studied. It has been found that the ability of anti-idiotypic second-order T suppressor cells (Ts2) to inhibit T cell-dependent delayed-type hypersensitivity (DTH) responses depended upon the presence of cross-reactive-idiotype (CRI)-bearing T cells present in ABA-primed mice. This suppressor T cell subset, termed Ts2, so exists with CRI-negative T cells that mediate DTH in vivo. It appears that antigen-activated CRI+ Ts3 require signals from the anti-CRI Ts2 subset to suppress DTH reactions in an idiotype-nonspecific manner. The relevance of these observations to a comprehensive scheme of T and B cell regulation is discussed.

UV light irradiation of BALB/c mice was found to result in impairment of antigen-presenting cell function. Adherent trinitrophenyl-derivatized cells from the peritoneal exudate cell population or the spleen of UV-treated donors could not induce hapten-specific delayed hypersensitivity responses in UV-irradiated syngeneic mice, whereas adherent trinitrophenyl-derivatized cells from normal mice were able to do so. The failure to induce immunity in UV-treated mice by utilizing UV-treated adherent antigen-presenting cells was associated with the development of antigen-specific suppressor T cells. The implication of these results for UV-induced carcinogenesis is discussed.

Biologic activities and immunochemical characteristics of an azobenzenearsonate- (ABA)specific suppressor T cell factor produced by a longterm T cell hybridoma, F12, were studied. In vivo administration of F12 culture supernatant resulted in the suppression of ABA-specific delayed-type hypersensitivity (DTH) responses and the inhibition of priming for ABA-specific cytotoxic T lymphocyte responses. Moreover, F12 induced a second set of suppressor cells that act in the efferent phase of DTH. The active material in the F12 culture supernatant expressed major cross-reactive idiotypic (CRI) determinants of anti-ABA antibodies of A/J mice and I-J subregion-coded specificities but not express determinants of immunoglobulin constant regions. These results demonstrated that F12 is a functioning hybrid cell line of the first-order suppressor T cell subset.

Anti-p-azobenzenearsonate (ABA) antibodies, coupled covalently to normal syngeneic spleen cells and then given intravenously to normal animals, were found to be potent tolerogens for delayed-type hypersensitivity (DTH) to ABA. The ability of the antibody-coupled cells to induce tolerance was determined to be a result of the cross-reactive idiotype (CRI+) fraction of the antibodies, because anti-ABA antibodies lacking the CRI+ components when coupled to spleen cells were unable to cause any significant inhibition. Furthermore, genetic analysis revealed that the ability of CRI-coupled cells to inhibit ABA-specific DTH is linked to Igh-1 heavy chain allotype, in as much animals which possess heavy chain allotypes similar to that of A/J were sensitive to this inhibition. Adoptive transfer experiments provided evidence that CRI-coupled cells induce suppressor cells, and spleen cells or thymocytes from animals received CRI-coupled cells were able to transfer suppression to naive recipients. In addition, treatment with anti-Thy1.2 serum plus complement completely abrogated their ability to transfer suppression. Thus, this active suppression is a T-cell-dependent phenomenon. In investigating the specificity of these suppressor T cells, it was found that they functioned in an antigen-specific manner and were unable to suppress the development of DTH to an unrelated hapten 2,4-dinitro-1-fluorobenzene.

Infection of mice with vesicular stomatitis virus (VSV) at the time of immunization failed to enhance T cell-mediated immune response to azobenzenearsonate-(ABA) conjugated spleen cells as measured by delayed-type hypersensitivity and by in vitro proliferation and in vitro generation of ABA-specific cytotoxic T cells. However, mice infected with VSV are incapable of responding to signals from suppressor T cells or their soluble factors. Further analysis revealed that VSV infection does not interfere with the induction of Ts-1 or Ts-2 cells. Because infection of Ts-1 or Ts-2 donors had no effect on the subsequent response seen in the recipients of antigen and suppressor T cells, the most likely candidate for the target of VSV infection is therefore the Ts-3 cell or another T cell interacting with Ts-3. This is supported by our observation that it is possible to bypass the VSV effect by providing the recipients of VSV with normal Lyt-2+-bearing T cells.

B cell-deficient, rabbit anti-mouse IgM-treated mice were compared with normal or normal rabbit immunoglobulin-treated controls in their ability to develop proliferative T cell responses, delayed hypersensitivity, and primary or secondary cytotoxic T cell responses. Immunization with hapten-coupled autologous spleen cells resulted in anti-mu-treated mice generating only marginal T cell responses. This decreased responsiveness was shown to be attributable not to an intrinsic T cell defect or to changes in the ability of macrophages from anti-mu-treated mice to present soluble antigen, but rather to the greatly diminished capacity of B cell-deficient spleen cells to present antigen. The results support the concept that B cells play a significant role in antigen presentation required for T cell activation.

We generated mAb from an unimmunized autoimmune MRL/Mp-lpr/lpr mouse. One of these mAb A108, reacted with cell surface Ag present on abnormal T cells from MRL/Mp-lpr/lpr, C3H-lpr/lpr, and C57BL/6-lpr/lpr mice. We failed to detect significant numbers of A108 bearing cells in the lymph nodes of MRL-Mp/+/+, normal C3H or normal C57BL/6 mice. Therefore, the expression of A108 correlates with the presence of the lpr/lpr gene. A108 binds to a variety of murine T cell tumor lines (e.g., EL4, BW5147, and YAC-1) and human T cell tumor lines (e.g., MOLT-3, Sup T1, and Jurkat). A108 does not bind to normal human PBL. Immunoprecipitation of surface iodinated EL-4 and BW5147 with A108 identified one major protein with a Mr of about 17.5-kDa. The significance of these findings with respect to the development of lymphoid proliferation and autoimmune disease in mice bearing the lpr/lpr gene will be discussed.

Serological analysis of idiotypic specificities present in azobenzenearsonate (ABA)-specific first-order suppressor T cell factors (TsF1) from C.AL-20 and BALB/c mice revealed a significant difference between TsF from these two strains of mice. The idiotypic composition of TsF1 from BALB/c mice appears to be more heterogeneous, and at least two different fractions can be readily identified. One bears the characteristic BALB/c-associated CRI(C) (crossreactive idiotype) determinants, and the other is non-CRI(C)-bearing. Analysis of ABA-specific TsF1 from animals lacking B cells uncovered a fundamental change in the expression of their idiotypic specificities. TsF from rabbit anti-mouse IgM (anti-mu)-treated C.AL-20 mice failed to express the characteristic CRI(A) determinants. Instead, they express CRI(C) specificities. Similarly, TsF1 from anti-mu-treated BALB/c mice did not express their characteristic CRI(C) specificities, but rather express CRI(A) determinants. These experiments provide strong evidence that the Igh restriction specificity of TsF is dictated by the particular idiotypic specificities expressed. They also clearly demonstrate that B cells and their products play an important role in establishing the idiotypic composition and repertoire of suppressor T cells.

The administration of azobenzenearsonate-modified syngeneic spleen cells (ABA-SC) intravenously induces a population of first order hapten-specific inducer suppressor T cells (Ts1), which downregulate various aspects of T-cell-mediated immune responses via a well defined suppressor-T-cell pathway. In this study, we investigated the effects of these suppressor cells on the generation of ABA-specific cytolytic T lymphocytes (CTL) and helper T cells (Th) in vivo. We found evidence for functional impairment of ABA-activated Th and ABA-specific CTL precursors (CTLp) in the suppressed animals by a number of different in vitro criteria. Functional analysis of ABA-specific CTLp and ABA-activated Th in suppressed animals revealed that ABA-specific Ts inhibit the generation of CTL by impairing the antigen-specific activation of Th, which may in turn, prevent the clonal expansion of antigen-specific CTLp. The significance of these findings in relationship to our understanding of the cellular interactions necessary for the generation of CTL and the mode of action and mechanisms of suppressor T cells is discussed.

Delayed-type hypersensitivity (DTH) to the azobenzenearsonate (ABA) hapten can be readily induced in A/J mice injecting ABA-coupled syngeneic spleen cells subcutaneously. To further characterize this T-cell-dependent immunological phenomenon, the effect of passively administered anti-cross-reactive idiotype common to anti-ABA antibodies of A/J mice (CRI) antibodies on the development of ABA-specific DTH was investigated. Animals given daily injections (of minute amounts) of anti-CRI antibodies subsequent to immunization with ABA-coupled cells show significant reduction of ABA specific responses. This inhibition is antigen specific and requires the intact immunoglobulin molecule, as F(ab')2 treatments were ineffective in suppressing the reaction. Investigations of the mechanism of the anti-CRI-induced suppression of ABA DTH revealed that the observed suppression is a result of the activation of suppressor cells. Spleen cells taken from animals which received anti-CRI antibodies were able to adoptively transfer suppression to naive recipients. This suppression was shown to be mediated by T cells, as anti-Thy1.2 plus complement completely abrogated the transfer of suppression. In addition, animals pretreated with low doses of cyclophosphamide were not suppressed by the administration of anti-CRI antibodies. The genetic restriction of anti-CRI-induced suppression was demonstrated. Antibodies to the major cross-reactive idiotype, (CRI) associated with anti-ABA antibodies in A/J mice were unable to suppress the development of DTH to ABA in BALB/c mice (H-2d, Igh-1a). Such antibodies were, however, fully active in suppressing ABA DTH in the allotype-congenic C.AL-20 strain which has an allotype (Igh-1d) similar to that of A/J (Igh-1e) on a BALB/c background, and which produces humoral antibodies with the CRI.

We produced a series of T cell hybridomas that produce IL-2 when cultured with syngeneic APC coupled to FITC or TNP. These hybridomas are hapten specific and Ia restricted. The hybridomas were used to detect hapten-bearing APC in draining lymph nodes of mice sensitized with trinitrochlorobenzene or FITC in vivo. Hapten-bearing APC capable of stimulating the hybridomas were detectable in draining lymph nodes of hapten-painted mice within 3 h after sensitization. The ability of lymph node APC to stimulate the hybridomas peaked at 24 h and declined by 48 h. The dendritic cell subpopulation was the subpopulation of cells that were found in the regional lymph nodes of hapten-painted animals that were capable of stimulating the hybridomas to produce IL-2. Prior treatment of the skin with low dose UVB irradiation before epicutaneous application of contact sensitizers significantly reduced the capacity of hapten-bearing APC to stimulate the hybridomas. This observation was corroborated by results obtained from flow microfluorometry analysis of lymph node cells from FITC-sensitized mice. Lymph node dendritic cells obtained from FITC-painted mice contain a brightly staining group of cells by flow microfluorometry analysis. Lymph node dendritic cells from FITC-painted, UVB-irradiated mice did not contain this brightly staining population. These results indicate that low dose, local UVB irradiation may affect APC migration and/or function. We believe that these hybridomas will prove to be useful tools in the study of the development and regulation of contact hypersensitivity.

Intrathymic, Ia-bearing antigen-presenting cells (APC) are believed to play an important role in the development of a mature, functional T-cell repertoire. We used chronic in vivo treatment of neonatal mice with anti-I-A monoclonal Ab (MAb) to examine the expression of I-A and I-E antigens on intrathymic and peripheral APC. Three weeks after continuous treatment with anti-I-A MAb, FACS analysis of unfractionated spleen cells revealed a 75-90% reduction in the number of I-A bearing cells. Splenic antigen-presenting capacity measured by the ability of unseparated or density gradient-enriched APC to stimulate I-A- or I-E-reactive T-cell hybridomas was also greatly reduced. In contrast to the expression of I-A and I-E molecules in the splenic APC, anti-I-A MAb treatment resulted in decreased thymic APC I-A expression without significant changes in I-E as measured by FACS analysis. This was confirmed in functional studies in which allo-I-A- or auto-I-A-reactive T-cell hybridomas could not be stimulated by treated thymic APC. Unlike splenic APC, anti-I-A-treated thymic APC did not differ significantly from normals in their ability to stimulate allo-I-E-reactive T hybridomas. This lack of linkage or comodulation of I-A and I-E expression on thymic but not splenic APC may allow us to study the role of I-A molecules and I-E molecules on the development and expansion of functional, mature T-cell repertoires.

A/J anti-p-azobenzenearsonate (ABA) antibodies bearing cross-reactive idiotypic (CRI) determinants, when coupled to spleen cells and then injected intravenously into naive animals, stimulate suppressor T cell (Ts) responses. Moreover, previous studies have demonstrated that the ability of such idiotype-coupled spleen cells to induce immune unresponsiveness to subsequent immunization with ABA-coupled spleen cells is linked to Igh-1 genes. Thus, CRI bearing antibodies from A/J mice, when conjugated to normal BALB/c spleen cells in vitro and then injected intravenously to syngeneic BALB/c mice, failed to induce tolerance in these animals. However, spleen cells taken from these animals transferred significant degrees of suppression to Igh-1 congenic C.AL-20 but not to H-2 congenic, Igh-1 distinct B10.D2 mice. Therefore, the failure of CRI-coupled spleen cells to induce suppressor cell- mediated unresponsiveness in animals unable to express the appropriate VH genes (i.e. BALB/c and B10.D2) appears to be caused by the lack of idiotype- matched targets. The notion that the ability to express certain Vn genes in the recipient animal is a prerequisite for suppressor cell function was further supported by the observation that suppressor cells induced in C.AL-20 mice failed to transfer any degree of suppression to BALB/c mice. The ability to transfer suppression from BALB/c mice to C.AL-20 mice is a T cell- dependent phenomenon, since in vitro treatment with anti-Thy 1.2 antiserum and complement completely abrogated suppressor cell function. Furthermore, these suppressor T cells are antigen specific and can be enriched on idiotype-coated petri dishes, indicating they possess anti-idiotypic receptors. Therefore, appropriate anti-idiotype and idiotype interaction is essential for the manifestation of suppressor T cell function in ABA-specific suppressor pathways.

The genetic restrictions of the activation of third-order suppressor cells (Ts3) were studied in mice, using two different types of anti-azobenzenearsonate (ABA)-immune responses, namely delayed-type hypersensitivity (DTH) and cytotoxic T lymphocyte (CTL) generation. Ts2 cells were induced in several different strains of mice by injecting monoclonal T hybridoma molecules or first-order suppressor factors (TsF1) originating in A/J (H-2a, Igh-1e) mice and then testing the TsF2 molecules derived from these Ts2 in A/J and A.By (H-2b, Igh-1e) or (A/J X A.By)F1 (H-2a/b, Igh-1e) and (C57Bl/6 X A/J)F1 (H-2b/a, Igh-1e) mice. It was shown that the activity of TsF2 was restricted to the I-J of the strain in which Ts2 was induced. By genetic analysis, restriction was shown to be due to the requirement of H-2 identity between ABA-coupled cells used for Ts3 activation and the strain of the TsF2 origin. Moreover, by using H-2-congenic ABA-coupled cells, we were also able to precisely map and demonstrate that ABA-coupled cells I-J identical to TsF2 induced in various strains were necessary for effective suppression to occur. This selective activation of Ts3 suggested the existence of I-J-related antigen presentation for suppression as the counterpart of I-A or I-A-I-E-restricted antigen presentation for positive immune responses.

Treatment of responder cells with monoclonal anti-Ly-1,2 antibodies plus complement in vitro completely eliminated their ability to generate azobenzenearsonate (ABA)-specific cytolytic T lymphocytes (CTL). However, addition of the concanavalin A-stimulated supernatants of rat spleen cells (Con A-Sup) can fully reconstitute the response. Therefore, Lyt-1,2-bearing T cells are required for the generation of ABA-specific CTL, and such requirement can be replaced by factors present in the Con A- sup. Suppressor T cells (Ts), when adoptively transferred into naive recipients, will inhibit the in vivo priming of CTL. This inhibition can also be reversed by in vitro addition of Con A-Sup. furthermore, mice serving as donors of Ts also show profound unresponsiveness when primed and restimulated in vitro. In contrast to the Ts-mediated inhibition, in vitro addition of Con A-Sup was unable to abolish the unresponsiveness observed in these cultures. Thus, we identified two unresponsive states in a hapten-specific killing system that differ in their ability to be reconstituted by Con A-Sup.

Treatment of murine thymocytes, but not mature peripheral T cells, with the tumor promoter, phorbol 12-myristate 13-acetate (PMA), 3 results in a rapid disappearance of L3T4 molecules from the surface of thymocytes. The effect of PMA on L3T4 molecules persists in vitro for at least 72 hr. Down modulation of L3T4 molecules was PMA dose-dependent and temperature-dependent. L3T4 molecules on cortisone-resistant thymocytes were significantly less sensitive to the effect of PMA than were L3T4 molecules on cortisone-sensitive thymocytes. Down modulation of L3T4 molecules on thymocytes did not interfere with their capacity to respond to concanavalin A or activation signals delivered via their T cell receptors. The difference in the ability of thymocytes and peripheral T cells to respond to PMA cannot be explained by differences in the number of PMA receptors. Both thymocytes and peripheral T cells have PMA receptors in the range of 1 to 1.5 X 10(5) receptors/cell. However, there is a small difference in the affinity (Kd) of the receptors on thymocytes (Kd = 30 to 40 nM) and peripheral T cells (Kd = 10 to 15 nM). Immunofluorescent staining revealed that the down modulation of L3T4 molecules by PMA was a result of internalization of L3T4 molecules. After down modulation, L3T4 could be readily detected on the cytoplasm of thymocytes. These findings suggest that L3T4 molecules on thymocytes may be subject to different regulatory signals than L3T4 molecules on peripheral T cells.

Lymph node T cells from autoimmune MRL/Mp-lpr/lpr mice, but not from congeneic MRL/Mp-+/+ mice, spontaneously proliferate and produce IL 2 when cultured in vitro for 5 to 7 days. This autologous activation depends critically on the length of in vitro culture and the initial culture density, indicating that cell to cell interaction may be essential. Phenotypic characterization of cultured cells suggests that both L3T4+ and Lyt-2+ T cells proliferate. However, only L3T4+ T cells produce IL 2. Mixing experiments reveal that the inability of freshly isolated lymph node cells from MRL/Mp-lpr/lpr mice to proliferate is not due to the presence of suppressor cells. Supernatant from 7-day cultures failed to induce freshly isolated cells to proliferate. Thus, the failure of freshly isolated cells to spontaneously proliferate and secrete IL 2 is not due to the inability of the cells to produce soluble mediators. Similar to the inactivation of normal T lymphocytes, in vitro addition of monoclonal anti-L3T4 or anti-IL 2 receptor antibody significantly inhibits the activation of these cultured lymphocytes. Spontaneous proliferation and IL 2 production can be blocked by the addition of monoclonal anti-I-Ak but not by monoclonal anti-I-Ad. Spontaneous proliferation and IL 2 production can be detected in young (4-wk-old) MRL/Mp-lpr/lpr mice at a time when their lymphocyte composition and physiology appear to be normal. More interestingly, spontaneous proliferation and IL 2 production cannot be detected in C57BL/6J mice bearing the lpr/lpr gene. These experiments support the notion that aberrant syngeneic autoreactivity may act as an accelerating factor in the pathogenesis of lymphoproliferation and autoimmunity in MRL/Mp-lpr/lpr mice.

Tolerance to contact sensitization with DNFB may be induced by DNBSO3. This specific unresponsiveness may occur via one or both of two mechanisms--production of suppressor T cells or clone inhibition. We investigated the role of the spleen in this unresponsiveness. Splenectomized mice may be tolerized by i.v. injection of DNBSO3, but they are incapable of serving as donors of lymph node cells for transfer of tolerance to normal recipients. Kinetic studies indicated that the spleen must be present at least three days after tolerization in order to permit development of a significant number of suppressor cells in the peripheral lymph nodes. We interpret these results to indicate that 1) clone inhibition does not require the spleen, 2) the generation of suppressor T cells is dependent on the presence of the spleen, and 3) it is likely that tolerogens in this system induce suppressor cells in the spleen and some of these cells or their products leave the spleen to reach the peripheral lymph nodes.

Unresponsiveness to DNFB contact sensitivity induced by DNP-modified lymphoid cells (DNP-LC) is mediated by two separable pathways: a rapidly induced, long lasting inhibition of reactive T cell clones (donor tolerance), and a transient period of suppressor T cell (Ts) activity. The present report has examined the nature of the hapten-modified determinants responsible for the induction of these pathways by utilizing soluble DNP-LC cell lysate preparations as tolerogens. The results indicate that both DNP-modified MHC and non-MHC encoded determinants can mediate donor tolerance 7 days after tolerization. On the other hand, the induction of Ts requires DNP-modified MHC determinants, since DNP-LC lysates passed over lentil lectin or specific anti-H-2 immunoabsorbent columns lost their ability to induce Ts. Additional experiments showed that the injection of DNP-LC lysate compatible with the recipient strain at the H-2K and H-2D region of the MHC was sufficient for the induction of Ts. We propose that Ts induction involves the direct presentation of DNP-H-2 determinants to Ts precursors, whereas the induction of donor tolerance may involve host processing and presentation of DNP-modified membrane determinants in conjunction with host MHC structures.

Mice rendered B cell deficient by treatment with rabbit anti-mouse IgM (anti-mu) antibodies from birth fail to respond when primed with soluble protein antigens in CFA, as measured by T cell proliferation when challenged with antigen in vitro. The role of B cells in T cell priming in vivo was examined by adoptively transferring hapten-specific B cells into anti-mu mice, followed by immunization with haptenated Ag in CFA. The T cell proliferative response to OVA of anti-mu BALB/c mice was partially restored by the administration of TNP or FITC-specific B cells and immunization with TNP-OVA or FITC-OVA, respectively. This reconstitution was Ag-specific, inasmuch as hapten-binding B cells restored the T cell responses to OVA in mice immunized with the same hapten coupled to OVA. The mechanism of B cell reconstitution of T cell priming in anti-mu mice was addressed using parental to F1 B cell transfers. The Ia restriction pattern of the activated T cells from these mice indicated that both direct presentation of Ag by transferred B cells and antibody-mediated enhancement of Ag presentation by non-B, host Ag-presenting cells occurred. Thus, Ag-specific B lymphocytes play a critical role in priming of T cells in vivo.

Abnormal T cells from autoimmune MRL/Mp-lpr/lpr mice express T-cell-receptor complexes on their surfaces. These receptors are nonfunctional, since monoclonal anti-T-cell-receptor antibody-conjugated beads, which normally activate receptor-bearing T cells, were unable to activate these abnormal T cells. In addition, these abnormal T cells are unresponsive to the synergistic effect of phorbolmyristate acetate (PMA) and calcium ionophore A23187, as quantitated by proliferation, interleukin-2(IL-2) production, and the expression of IL-2 receptors. The failure of abnormal T cells to respond to PMA is not due to the absence of PMA receptors since Scatchard plot analysis reveals that there are 1-1.5 X 10(5) PMA-binding sites/cell with a Kd of 6-10 nM on these abnormal T cells. Similar to normal T lymphocytes, protein kinase c activity can be readily detected in the cytosolic fraction of these abnormal T cells. More importantly, in vitro culture of the abnormal T cells with PMA results in translocation of protein kinase c activity from the cytosolic fraction to the membrane fraction. From these experiments we concluded that the defect in the signal-transducing machinery in MRL/Mp-lpr/lpr T cells is not due to the lack of PMA receptors or the absence of protein kinase c activity, but may result from events which occur after the activation and translocation of protein kinase c. However, whether defects in response to a physiological stimulus (i.e., anti-receptor antibody) could occur in a step prior to protein kinase c activation remains to be determined.

Delayed-type hypersensitivity (DTH) to p-azobenzenearsonate (ABA) can be induced in A/J mice with intravenous injection of minute amounts of anti-cross-reactive idiotypic (CRI) antibodies, providing that the animals have been pretreated 2 d earlier with low doses of cyclophosphamide (50 mg/kg). However intravenous injection of the F(ab')2 fragments of the anti-CRI antibodies or subcutaneous administration with anti-CRI antibodies induces comparable immunity in both cyclophosphamide-pretreated and normal nontreated animals. Furthermore adoptive transfer experiments indicate that lymph node cells taken from animals sensitized with anti-CRI 4 d earlier can adoptively transfer immunity to naive recipients. Transfer of immunity is mediated by a population of thymus-dependent (T) cells, which express idiotypic structures on their surface. Treatment of effector cells with either anti-theta serum or anti-idiotypic antibodies plus complement completely abrogated their ability to transfer immunity. In addition idiotype-bearing suppressor T cells induced with ABA-coupled spleen cells inhibit the development of ABA-specific DTH induced with anti-CRI antibodies. Genetic analysis revealed that the ability of anti-CRI antibodies to induce ABA-specific DTH was linked to Igh-1 heavy-chain allotype. Anti-idiotypic antibodies to the major CRI associated with anti-ABA antibodies in A/J mice failed to induce significant immunity in BALB/c mice (H-2d, Igh-1a). Nevertheless, they were able to induce significant immunity in C.AL20 mice (H-2d, Igh-1d) which possess a heavy-chain allotype similar to that of A/J mice.

A role for Igh linked genes and the idiotypes they encode has been implicated in the activity of a variety of T cell subpopulations. Idiotype restricted T cell function has been observed for helper and suppressor cell populations. The finding that T cell receptor genes are distinct from B cell receptor (Igh) genes strongly argues against a direct role for immunoglobulin genes in the determination of the T cell repertoire. Nevertheless, idiotypic Ig determinants may play an indirect role in influencing the ultimate composition of the T cell repertoire. One approach to this question involves evaluation of T cell activity upon development in an immunoglobulin deficient environment. The availability of antigens which elicit T cell and antibody responses characterized by the expression of dominant crossreactive idiotypes under the control of Igh genes provides an ideal approach to investigate the basis for the expression of Igh-like structures on T cells and the concomitant functional genetic restrictions they determine. Thus, we have prepared B cell deficient mice by continuous treatment, beginning at birth, with rabbit anti-mouse IgM. The network which comprises the suppressor T cell response to azobenzenearsonate (ABA) was then examined in normal and anti-mu treated mice to assess what role, if any, immunoglobulin encoded determinants play in influencing the composition of the peripheral T cell pool. The results clearly demonstrate that the absence of Ig+ B cells leads to major alterations in the composition of the T cell repertoire. Anti-mu treated, but not normal rabbit Ig treated, mice produce TsF1 which fails to suppress cytotoxic T lymphocyte or helper T cell responses of normal syngeneic mice, yet efficiently suppresses those of syngeneic anti-mu treated recipients. Reciprocally, normal TsF1, though suppressive in normal Igh-1 syngeneic recipients, fails to affect the development of responses in anti-mu treated syngeneic mice. TsF1 obtained from anti-mu treated mice is antigen-specific. Testing of anti-mu TsF in a variety of normal or anti-mu treated recipients reveals no MHC restrictions. In marked contrast, anti-mu TsF reflects a novel pattern of Igh functional restrictions. The observed Igh restrictions were found to map to the idiotype encoding VH regions of the Ig heavy chain gene cluster (Igh-VH). The results demonstrate that T cell maturation in the virtual absence of environmental immunoglobulin can lead to profound changes in the composition of the T cell compartment. The means by which the absence of Ig encoded determinants leads to such changes is speculated upon.

Mice homozygous for the lpr gene develop a lymphoproliferative disorder due to expansion of a subset of CD4-CD8- T cells. Triggering of the T-cell receptor in these lpr T cells does not lead to translocation of protein kinase C or phosphorylation of CD3, interleukin-2 production, or proliferation, whereas a combination of phorbol ester and calcium ionophore does. Stimulation with concanavalin A or anti-CD3 induces phosphoinositide hydrolysis. The rise in inositol bisphosphate, inositol triphosphate, and inositol tetrakisphosphate, identified by HPLC, is similar in +/+ and lpr T cells. The concentration of cytoplasmic free calcium ([Ca2+]i), however, under basal and stimulated conditions is significantly lower in lpr T cells. The lower basal [Ca2+]i may explain why induction of proliferation with phorbol ester and calcium ionophore requires a higher concentration of ionophore in these cells than in normal T cells. The lower [Ca2+]i obtained on stimulation may contribute to the activation defect of CD4-CD8- lpr T cells.

We investigated the mechanism(s) by which MHC-restricted suppressor T cells (Ts) induced by i.v. injection of allogeneic DNP-modified lymphoid cells (alloinduced Ts) suppress the DNFB contact sensitivity response. It was shown that alloinduced Ts acted only during the early phases (afferent limb) of sensitization. They were incapable of suppressing previously sensitized recipients or of inhibiting the expression of DNFB-immune LN cells when co-transferred into normal recipients. The target of alloinduced Ts seems to be cell proliferation, i.e., inhibition of antigen-induced cell proliferation (DNA synthesis) in Ts recipient mice. The failure of recipients of alloinduced Ts to generate DNFB-immune LN cells capable of transferring contact sensitivity to normal recipients also suggests that these Ts act by preventing the development of an expanded clone of mature immune T cells. The suppressive effects of alloinduced Ts also were inhibited by prior in vitro treatment with anti-TNP serum. The data are discussed in terms of current models of suppression, and are compared to mechanisms of suppression in other contact sensitivity models.

Genetic restrictions governing the induction and expression of suppressor T cells (Ts) in tolerance to 1-fluoro-2,4-dinitrogenzene (DNFB) contract sensitivity were studied. Tolerance was induced by using 2,4-dinitrophenyl (DNP)-modified lymphoid cells (DNP-LC) as tolerogen. Two kinds of Ts were found-those produced by DNP-LC syngeneic to the donor of the Ts (syninduced Ts), and those produced by DNP-LC allogeneic to the donor of Ts (alloinduced Ts). Studies employing congenic resistant mouse strains indicated that recognition of DNP-modified-major histocompatibility region determinants on the tolerogenic DNP-LC was essential for the induction of both types of Ts. Non-H-2 genetic background was irrelevant to Ts induction. Mapping studies indicated that induction of both syninduced and alloinduced Ts was associated with recognition of DNP-modified-MHC region determinants which map to the right of the H-2G region (i.e., H-2D gene products). Tolerization of donor mice with DNP-LC which were H-2D region compatible, but not with H-2K or I region compatible DNP-LC, was both sufficient and required for the induction of hapten-specific syninduced Ts. Tolerization of donor mice with DNP-LC which were incompatible only at the H-2D region was sufficient for the induction of alloinduced Ts. These Ts were capable of suppressing recipient mice only if the recipients shared the H-2D region with the strain providing the DNP-LC tolerogen, and were not capable of suppressing recipients sharing all but the H-2D region with the tolerogen.

We investigated T-T cell interactions in the suppression of contact sensitivity. Suppressor cells that block the efferent limb of sensitivity (Ts-eff) can inhibit the passive transfer of contact sensitivity mediated by 1-fluoro-2,4-dinitrobenzene immune cells (T DH). But, Ts-eff cannot block the passive transfer of TDH which comes from cyclophosphamide (Cy) pretreated sensitized mice. We interpret these results to indicate that lymph node cells from sensitized mice contain not only TDH but also another intermediate cell which is required for the suppression of TDH by Ts-eff. This intermediate cell is sensitive to cyclophosphamide and requires antigen activation for its development. It is sensitive to adult thymectomy and anti-brain associated theta serum and is therefore designated as an auxiliary T-suppressor cell (Ts-aux). It is not sensitive to splenectomy and it carries I-J determinants. Ts-aux are required for the activity of suppressors of the efferent limb (Ts-eff) but not of suppressors of the afferent limb (Ts-aff). Thus, in the feedback loops in contact sensitivity, the generation of Tdh is coordinated with the development of auxiliary Ts which are essential for the suppression of those TDH.

Lymph node cells from 4-wk-old MRL/Mp-lpr/lpr mice, but not from MRL/Mp-+/+ mice, when cultured in vitro for 5 to 7 days, will spontaneously proliferate and produce IL-2. We examined the expression of several cell surface Ag on lymph node cells from MRL/Mp-lpr/lpr mice before and after in vitro culture. There is an increase in the expression of Thy-1, L3T4, IL-2R, T cell activating protein, T cell receptor, and T3 complex on the surface of cultured cells. Cultured cells produced IL-3, IFN-gamma, and small but detectable amounts of IL-1 in addition to IL-2. Gamma irradiation of APC from young MRL/Mp-lpr/lpr mice or treatment of APC with a mAb (J11D) and C, completely abrogated their stimulatory capacity. These experiments suggest that B cells are the predominant APC responsible in the activation of autoreactive T cells in MRL/Mp-lpr/lpr mice. Lymph node cells from C57BL/6-lpr/lpr or C3H-lpr/lpr mice were unable to spontaneously proliferate or produce IL-2. Lymph node cells from (MRL/Mp-lpr/lpr x C57BL/6-lpr/lpr) F1 mice or (C3H-lpr/lpr x MRL/Mp-lpr/lpr) F1 mice did proliferate and produced IL-2 after in vitro culture. Using T cells from these F1 animals and APC from each parental haplotype, we found that APC from MRL/Mp-lpr/lpr mice induced more proliferation and greater amounts of IL-2, when compared to APC from F1 animals. APC from C57BL6-lpr/lpr mice or C3H-lpr/lpr were unable to induce spontaneous proliferation and IL-2 production. Therefore, B cells from MRL/Mp-lpr/lpr mice appear to possess unique features that enable them to activate autoreactive T cells more effectively than B cells from other mice bearing the lpr/lpr gene.

The T cell receptor (TcR) for antigen, on the majority of T cells, is a disulfide-linked heterodimer composed of the alpha and beta chains, noncovalently associated with the CD3 complex of polypeptides (gamma, delta, epsilon and zeta). In this report, two murine thymoma cell lines are described which synthesized incomplete TcR/CD3 complexes and expressed low levels of CD3 on their surface in the absence of the TcR chains. The partial TcR/CD3 complexes were composed primarily of the inherently metabolically stable CD3 gamma and epsilon subunits. These results were in contrast to previous studies, which suggested that synthesis of all of the component chains of the TcR/CD3 complex is required for the successful transport of any of the chains to the cell surface. The efficiency of transport of the partial TcR/CD3 complexes from the endoplasmic reticulum (ER) to medial Golgi in the two thymomas was similar to complete complexes. However, the transport of the incomplete receptors was impaired at some point between the medial Golgi and the plasma membrane. Taken together with previous studies, these results suggested that T cells have mechanisms to retain partial TcR/CD3 complexes intracellularly both in the ER and in an undefined post-ER compartment. However, the transport of low levels of partial TcR/CD3 complexes to the cell surface in some T cell lines implied that the retention mechanisms may not always be completely efficient. Cross-linking of the surface, partial TcR/CD3 complexes with anti-CD3 epsilon antibodies did not stimulate interleukin 2 (IL 2) production. It is possible, however, that the partial TcR/CD3 complexes have some function which is unrelated to the stimulation of IL 2 production.

CD4 (T4) is a 60 kD glycoprotein expressed on a subset of T lymphocytes. CD4 augments T cell responses to suboptimal Ag stimulation. In addition, the CD4 molecule is the receptor for HIV-1. CD4 is phosphorylated on serine residues within the cytoplasmic domain and its cell surface expression is decreased in response to PMA, APC bearing the appropriate Ag or HIV infection. The kinetics of CD4 phosphorylation and modulation are similar, suggesting that the two events may be related. L3T4, the murine CD4 equivalent, is not modulated from the surface of mature, peripheral T cells in response to PMA. The difference in the ability to modulate L3T4 and CD4 in response to PMA may be due to differences between the two molecules or to differences between the cells in which they are expressed. To further define the requirements for CD4 modulation, we used retroviral vectors to transfer the cDNA for CD4 and various mutants of CD4 into two murine T cell hybridomas that express L3T4. One of these hybridomas, By155.16, does not modulate L3T4 in response to PMA and the other, 5D5.63, does modulate L3T4 in response to PMA. When expressed by these hybridomas CD4 is not modulated from the surface of By155.16 and is modulated from the surface of 5D5.63 in response to PMA. In both of these hybridomas, CD4 is phosphorylated on serine residues in response to PMA. A mutant form of CD4, CD4 delta, was constructed in which the majority of the cytoplasmic domain was deleted. When expressed in 5D5.63, CD4 delta was not modulated in response to PMA. Replacing the cytoplasmic domain of CD4 with that of the human IL-2 receptor did not reconstitute the ability of CD4 to be modulated. These results suggest that the inability to modulate L3T4 from the surface of murine peripheral T cells is due to features of the cell and not the molecule. Furthermore, the cytoplasmic domain of CD4 is required for its modulation from the cell surface in response to PMA.