Ischemic Preconditioning Enhances Muscle Endurance during Sustained Isometric Exercise.

Ischemic preconditioning (IPC) enhances whole-body exercise endurance. However, it is poorly understood whether the beneficial effects originate from systemic (e. g., cardiovascular system) or peripheral (e. g., skeletal muscle) adaptations. The present study examined the effects of IPC on local muscle endurance during fatiguing isometric exercise. 12 male subjects performed sustained isometric unilateral knee-extension exercise at 20% of maximal voluntary contraction until failure. Prior to the exercise, subjects completed IPC or control (CON) treatments. During exercise trial, electromyography activity and near-infrared spectroscopy-derived deoxygenation in skeletal muscle were continuously recorded. Endurance time to task failure was significantly longer in IPC than in CON (mean±SE; 233±9 vs. 198±9 s, P<0.001). Quadriceps electromyography activity was not significantly different between IPC and CON. In contrast, deoxygenation dynamics in the quadriceps vastus lateralis muscle was significantly faster in IPC than in CON (27.1±3.4 vs. 35.0±3.6 s, P<0.01). The present study found that IPC can enhance muscular endurance during fatiguing isometric exercise. Moreover, IPC accelerated muscle deoxygenation dynamics during the exercise. Therefore, we suggest that the origin of beneficial effects of IPC on exercise performance may be the enhanced mitochondrial metabolism in skeletal muscle.

Planned Overreaching and Subsequent Short-term Detraining Enhance Cycle Sprint Performance.

We investigated the effect of a training program consisting of planned overreaching and subsequent short-term detraining on sprint performance. 24 physically active men participated in an 18-day sprint-training program. They were divided into 2 groups: the overreaching-detraining (OR-DT) and the control (CON) groups. Subjects in the OR-DT group performed 12 consecutive days of maximal cycle sprint training followed by 6 days of detraining, whereas a rest day was provided after every 2 successive training days for the CON group. Peak power output during maximal pedaling increased significantly after 6 days of detraining in the OR-DT group compared with the baseline (P<0.05), whereas no change was observed in CON group. Intramuscular phosphocreatine concentration increased significantly after 12 days of daily training in the OR-DT group (69.3±45.8% increase vs. baseline, P<0.05), and it was maintained after the detraining period (46.6±33.6% increase vs. baseline, P<0.05). However, no change was observed in CON group. No significant changes in blood variables were observed after the training period except significant reduction of serum cortisol in the CON group. Daily sprint training and subsequent short-term detraining enhanced peak power output after the detraining period.

Immunological tolerance in bone marrow-derived lymphocytes. II. Effects of allogeneic cell interactions and enzymatic digestion with trypsin or inactivated hapten-specific precursors of antibody-forming cells.

The studies presented here have focused on the important question of reversibility of inactivation of DNP-specific B lymphocytes induced by the DNP derivative of the copolymer of D-glutamic acid and D-lysine (D-GL). In so doing, we have analyzed the capacity of a strong T-cell stimulus, such as that provided by the allogeneic effect, and of gentle enzymatic treatment with trypsin to alter, prevent, or reverse the tolerance induced by DNP-D-GL. Under experimental conditions in which DNP-specific B lymphocytes were exposed first to the tolerogenic molecule, and rendered markedly unresponsive by such exposure either in vitro or in vivo, subsequent exposure to an allogeneic effect failed to appreciably reverse or alter the tolerant state. This contrasts directly with the capacity of DNP-D-GL to serve as a stimulus for DNP-specific B lymphocytes when the critical moment of specific binding occurs subsequent to the development of an allogeneic effect. In another series of experiments, the effects of enzymatic treatment with trypsin on the tolerant B-cell population were found to vary depending on the stage of tolerance at which such treatment was performed. Thus, when exposure of cells to DNP-D-GL for a relatively short time in vitro is carried out at low temperature (4 degrees C), the development of tolerance can be interceded by immediate trypsinization. In contrast, cells exposed to DNP-D-GL for longer periods of time and/or at 37 degrees C were not reversed to responsiveness by trypsinization. These data were interpreted to indicate that: (a) the effect(s) of trypsin in reversing (or preventing) tolerance at the cellular level does not depend necessarily on the susceptibility of the tolerogenic moiety to the action of the enzyme, and (b) the generation of the tolerance-inducing signal involves metabolic cellular processes that can be delayed somewhat by low temperature leaving such cells relatively more susceptible to intercedent manipulations such as trypsinization. Taken collectively, therefore, the evidence obtained in these studies reinforces the concept of central tolerance in B cells induced by DNP-D-GL as reflecting sub- or intracellular inactivating events.

Immunological tolerance in bone marrow-derived lymphocytes. I. Evidence for an intracellular mechanism of inactivation of hapten-specific precursors of antibody-forming cells.

Administration of the 2,4-dinitrophenyl (DNP) derivative of the copolymer of D-glutamic acid and D-lysine (D-GL) to inbred mice induces a state of DNP-specific tolerance in such animals irrespective of their immune status at the time of treatment. Taking advantage of the relative ease with which DNP-D-GL can induce tolerance in an animal previously primed with an immunogenic DNP-carrier conjugate, we have established conditions for tolerance induction in an adoptive cell transfer system. Thus, the adoptive secondary anti-DNP antibody response of DNP-keyhole limpet hemocyanin (KLH)-primed spleen cells was completely, or almost completely, abolished by exposure of such cells to DMP-D-GL either in vivo or in vitro. Tolerance induction in vivo occurred irrespective of whether the DNP-primed cells were exposed to DNP-D-GL in the donor animal before adoptive transfer or in recipient mice after transfer. In the latter situation, it was possible to show that tolerance induction in this model occurs very rapidly (1 hr) and with relatively low doses of tolerogen (50 microg). Incubation of DNP-KLH-primed cells with DNP-D-GL in vitro under varying culture conditions also resulted in depression of the adoptive secondary response of such cells, although the kinetics and degree of tolerance induction in this way were slightly different from that obtained by in vivo tolerization. Utilizing the adoptive transfer tolerance system, it was possible to approach certain questions concerning the mechanism of tolerance induction and fate of tolerant bone marrow-derived (B) lymphocytes in the DNP-D-GL model. The possibility that suppression of anti-DNP antibody from the DNP-D-GL reflects blocking of surface receptor molecules on B lymphocytes has been ruled out by several experimental observations. The most conclusive evidence on this point derives from the failure of enzymatic treatment with trypsin to reverse the tolerant state induced by in vitro exposure of primed cells to DNP-D-GL, whereas trypsinization completely restored the immunocompetence of DNP-KLH-primed cells rendered unresponsive by exposure to DNP-ovalbumin in vitro. The present studies also demonstrate that the tolerant state induced by DNP-D-GL represents a predominantly irreversible inactivation of specific B lymphocytes. This conclusion is derived from experiments in which it was found that tolerance was maintained through as many as two serial adoptive transfers performed over a period of time of at least 24 days from the single exposure of such cells to the tolerogen. Moreover, the possibility that maintenance of tolerance through such serial transfers was due to inadvertent transfer of tolerogenic doses of DNP-D-GL was definitively ruled out. It appears, therefore, that DNP-specific tolerance induced by DNP-D-GL is an example of irreversible inhibition of cell reactivity to antigen reflecting yet-to-be-determined events at the intra- and subcellular levels.

Cell interactions between histoincompatible T and B lymphocytes. I. Allogeneic effect by irradiated host T cells on adoptively transferred histoincompatible B lymphocytes.

The adoptive transfer of 2,4-dinitrophenyl(DNP)-keyhole limpet hemocyanin(KLH)-primed lymphocytes into a heavily irradiated allogeneic recipient permits the development of a secondary anti-DNP antibody response to DNP-bovine gamma globulin(BGG) whether or not the irradiated allogeneic host possesses BGG-specific helper T cells. This "allogeneic effect" has been demonstrated to result from the capacity of residual, apparently radioresistant, T cells in the irradiated host to exert an active effect on the transferred histoincompatible B lymphocytes. This conclusion derives from two corroborative experiments. In the first, an allogeneic effect was shown to occur on DNP-primed F(1) spleen cells that had been adoptively transferred to irradiated parental recipients; the second experiment demonstrated the development of an allogeneic effect on anti-theta-treated, DNP-specific donor cells transferred to irradiated allogeneic hosts. These results emphasize the extreme caution required in designing and interpreting experiments that may involve adoptive cell transfers into histoincompatible hosts, and illustrate why such models are unsuitable for investigation of the question of physiologic cooperative interactions between T and B lymphocytes. Suitable approaches are described in the accompanying paper.

Immunological tolerance in bone marrow-derived lymphocytes. 3. Tolerance induction in primed B cells by hapten conjugates of unrelated immunogenic or "nonimmunogenic" carriers.

The present studies were designed to probe the role(s) of T cells in preventing or altering tolerance induction in hapten-specific B cells. This was accomplished by using hapten conjugates of normally immunogenic heterologous carriers to selectively inhibit 2,4-dinitrophenyl (DNP)-primed B cells in adoptive transfer experiments in vivo. The data provide strong indications that one critical role of T-cell participation in humoral responses to antigens is to circumvent the development of a tolerogenic signal that, in the absence of such T-cell function, might otherwise ensue after binding of the antigenic determinants by specific precursor B lymphocytes.

Cell interactions between histoincompatible T and B lymphocytes. II. Failure of physiologic cooperative interactions between T and B lymphocytes from allogeneic donor strains in humoral response to hapten-protein conjugates.

Several experimental approaches, designed specifically to circumvent the possible contribution of a complicating "allogeneic effect," have been successfully used to answer the question of physiologic cooperative interactions between histoincompatible T and B lymphocytes in antibody responses to hapten-protein conjugates. This was accomplished for in vivo cell transfer studies by using an F(1) hybrid host as the recipient of irradiated, carrier-primed T lymphocytes from one parent and 2,4-dinitrophenyl (DNP)-primed B lymphocytes from the opposite strain. Under these conditions, very good T-B cell cooperative interactions were observed to occur between T and B lymphocyte populations derived from syngeneic donors, whereas no cooperative response was obtained when T cells were derived from one parental strain and B cells from the other. Corroborative experiments were performed in a totally in vitro system in which DNP-primed B cells developed good secondary anti-DNP antibody responses in vitro to soluble DNP-keyhole limpet hemocyanin (KLH) when cultured in the presence of irradiated KLH-primed T cells derived from syngenic donors but not from allogeneic donors. The failure of histoincompatible T and B lymphocytes to effect physiologic cooperative interactions has important implications for our understanding of how such interactions normally occur. The possibility that these results reflect the existence of a "block" of some sort to cell-cell interaction by virtue of the presence of a foreign major histocompatibility antigen on the surface of either cell has been definitively ruled out in the present studies. These observations demonstrate that the gene(s) that conditions the capability for physiologic T-B cell cooperation must be shared in common by the respective cell types, and suggest, furthermore, that this gene (or genes) belongs to the major histocompatibility system of the mouse. These findings, together with other relevant phenomena described previously, have led us to postulate that there exists on the B lymphocyte surface an "acceptor" molecule either for the putative active T cell product or for the T cell itself. The important genetic considerations and the possible sequence of events surrounding the actual T-B cell interaction implied by these postulates are discussed in detail.

Hapten-specific IgE antibody responses in mice. II. Cooperative interactions between adoptively transferred T and B lymphocytes in the development of IgE response.

The present studies have established conditions for the demonstration of cooperative interactions between specific T and B lymphocyte populations in the development of IgE antibody responses in vivo in mice. This has been accomplished by utilizing a system which permits the successful adoptive transfer to irradiated recipients of DNP-specific secondary IgE responses with spleen cells from suitably primed syngeneic donor mice. Thus, adoptively transferred DNP-KLH or DNP-ASC-primed spleen cells produced high levels of anti-DNP antibodies of both IgE and IgG antibody classes in response to challenge with the appropriate homologous priming conjugate but failed to develop more than meager responses to the reciprocal heterologous conjugate. However, when spleen cells from donors primed to the second carrier were concomitantly transferred with hapten-primed lymphocytes, secondary IgE ant-DNP responses were consistently obtained upon challenge with the heterologous conjugate. Moreover, we have been able to elicit augmented primary IgE anti-DNP antibody responses to either DNP-ASC or DNP-KLH after adoptive transfer of spleen cells from donors primed only to the carrier, ASC or KLH, respectively. This adoptive transfer system has enabled us to provide direct proof for the participation of theta-bearing T lymphocytes in antibody responses of the IgE class. Thus, the capacity of ASC-primed spleen cells to effectively cooperate with the DNP-KLH-primed lymphocytes in the adoptive secondary response to DNP-ASC could be abolished by in vitro treatment of such cells with anti-theta serum plus complement. This was true not only for the anti-DNP response of the IgG antibody class, but for the IgE antibody class as well. These studies have, furthermore, demonstrated the capacity to stimulate secondary anti-DNP antibody production in vivo by the concomitant administration of the DNP and relevant carrier determinants on separate molecules. This was more readily seen in the IgE than in the IgG antibody class. Thus, DNP-ASC-primed cells developed significant IgE, but more variable IgG, anti-DNP responses upon challenge with DNP-KLH plus unconjugated ASC. Antibody responses of both classes elicited in this manner were appreciably improved by the transfer of additional carrier (ASC)-primed cells. These and other results presented herein suggest that IgE B lymphocyte precursors may be inherently more sensitive than IgG B cells to at least certain of the functions of T lymphocytes concerned with regulatory mechanisms involved in antibody production.

The mechanism of tolerance induction in thymus-derived lymphocytes; I. intracellular inactivation of hapten-reactive helper T lymphocytes by hapten-nonimmunogenic copolymer of D-amino acids.

Treatment of a p-azobenzoate (PAB) derivative of a copolymer of D-glutamic acid and D-lysine (D-GL) induced a profound state of unresponsiveness to PAB-reactive helper T lymphocytes generated in PAB-mouse gamma globulin (MGG)-primed mice. This unresponsiveness in T lymphocytes was specific for PAB-reactive cells, since the bacterial alpha-amylase-, keyhole limpet hemocyanin-, or ovalbumin-primed helper T lymphocytes were not suppressed by PAB-D-GL treatment. Taking advantage of the relative ease with which PAB-D-GL can induce specific unresponsiveness to helper T lymphocytes in an animal previously primed with PAB-MGG, it was possible to approach certain questions concerning the mechanisms of tolerance-induction and the fate of tolerant helper T lymphocytes in the PAB-D-GL model by utilizing a classical adoptive cell transfer systemmelimination of the possibility of carry-over of the tolerogen with cells or of the generation of suppressor cells as the result of PAB-D-GL treatment as an explanation of the suppression of helper T-cell activity strongly inplicates the existence of a central intracellular mechanism of specific tolerance on the helper T-cell level. The possibility that suppression of the activity of PAB-reactive helper T lymphocytes by PAB-D-GL reflects simple blocking of surface receptor molecules on T lymphocytes was ruled out as it was found that the helper activity of PAB-reactive cells was minimally suppressed even when PAB-D-GL was directly exposed in vitro to helper T lymphocytesmmoreover, the most conclusive evidence on te the tolerant state induced by in vivo exposure of primed T cells to PAB-D-GL. It appears, therefore, that specific tolerance induced by PAB-D-GL' TO PAB-reactive helper T lymphocytes is an example of irreversible inhibition of T-cell reactivity to antigen, reflecting yet to be determined events at the intra- and subcellular levels.

Induction of anti-allo-class I H-2 tolerance by inactivation of CD8+ helper T cells, and reversal of tolerance through introduction of third-party helper T cells.

The intravenous sensitization of C57BL/6 (B6) mice with class I H-2-disparate B6-C-H-2bm1 (bm1) spleen cells resulted in the abrogation of CD8+ T cell-mediated anti-bm1 (proliferative and interleukin 2-producing) T helper (Th) cell activities. In vitro stimulation of lymphoid cells from these mice with bm1 cells, however, generated a reduced, but appreciable, anti-bm1 cytotoxic T lymphocyte (CTL) response. Moreover, the anti-bm1 CTL response, upon stimulation with [bm1 x B6-C-H-2bm12 (bm12)]F1 spleen cells, was enhanced when compared with the response induced upon stimulation with bm1 cells. These in vitro results were reflected on in vivo graft rejection responses; bm1 skin grafts engrafted in the bm1-presensitized B6 mice exhibited prolonged survival, whereas (bm1 x bm12)F1 grafts placed collateral to bm1 grafts (dual engrafted mice) inhibited the tolerance to bm1. In the B6 mice 1-2 d after rejecting the bm1 grafts, anti-bm1 Th activities remained marginal, whereas potent anti-bm1 CTL responses were found to be generated from their spleen cells. Administration in vivo of anti-CD4 antibody into bm1-presensitized, dual graft-engrafted mice prolonged bm1 graft survival and interfered with enhanced induction of anti-bm1 CTL activity. These results indicate that anti-class I alloantigen (bm1) tolerance as induced by intravenous presensitization with the relevant antigens is not ascribed to the elimination of CD8+ CTL precursors, but to the specific inactivation of CD8+ Th cells, whose function can be bypassed by activating third-party Th cells.

Protection from anti-TCR/CD3-induced apoptosis in immature thymocytes by a signal through thymic shared antigen-1/stem cell antigen-2.

During T cell development in the thymus, the expression of thymic shared antigen-1 (TSA-1)/stem cell antigen-2 (Sca-2), a glycosylphosphatidylinositol (GPI)-anchored differentiation antigen, is developmentally regulated. The expression level of TSA-1 is the highest in most immature CD4- CD8- thymocytes, high in CD4+ CD8+ thymocytes, but barely detectable in mature CD4+ CD8- or CD4- CD8- thymocytes and peripheral T cells. We have previously shown that surface TSA-1 expression in peripheral T cells is induced upon activation and that anti-TSA-1 mAb inhibits the T cell receptor (TCR) signaling pathway in activated T cells. In the present study, we have analyzed a role of TSA-1 in thymic selection events, especially in TCR-mediated apoptosis. In in vitro experiments, anti-TSA-1 blocked anti-CD3-induced cell death of T cell hybridomas. When anti-TSA-1 was injected into newborn mice in vivo together with anti-CD3 epsilon or anti-TCR-beta, TCR/CD3-mediated apoptosis of thymocytes was almost completely blocked. The blockade of apoptosis was defined by the inhibition of, first, the decrease in total number of thymocytes; second, the decrease in percentages of CD4+ CD8+ thymocytes; and third, the induction of DNA fragmentation. However, anti-TSA-1 did not block either steroid- or radiation-induced apoptosis, indicating that a signal via TSA-1 does not inhibit a common pathway of thymocyte apoptosis. Since TCR-mediated apoptosis is pivotal in thymic ontogeny, these results suggest that TSA-1/Sca-2 is an important cell surface molecule regulating the fate of a developing T cell.

Regulatory functions of hapten-reactive helper and suppressor T lymphocytes. I. Detection and characterization of hapten-reactive suppressor T-cell activity in mice immunized with hapten-isologous protein conjugate.

Helper and suppressor T-cell activities were detected simultaneously in the spleen cells of mice immunized with para-azobenzoate (PAB)-mouse gammaglobulin (MGG). Dinitrophenyl (DNP)-specific B cells were raised by immunization with DNP-keyhole limpet hemocyanin (KLH) and used as the indicator B-cell population. The helper and suppressor T-cell activities were determined after adoptively transferring spleen cells from PAB-MGG- primed donors and DNP-KLH-primed donors into X-irradiated recipients. Stimulation of these recipients with DNP-MGG-PAB detected helper T-cell activity, which was measured in terms of increased anti-DNP antibody responses of DNP-KLH-primed cells over these responses in the presence of unprimed cells. On the other hand, when DNP-KLH-primed cells were stimulated with DNP-KLH-PAB in the presence of PAB-MGG-primed cells, anti-DNP antibody responses were substantially lower than in unprimed normal cells. This suppressor cell population was (a) hapten-reactive, (b) present in B-cell-depleted spleen cells, (c) Thy-1 positive, (d) detectable earlier than the helper T-cell activities after priming (e) more radiosensitive than helper cells, and (f) found in the spleen but not the lymph nodes in contrast to helper T cells. These data indicate that these suppressor T cells are distinct from the helper T cells. PAB-reactive T cells clearly suppressed the antibody response by inhibiting KLH-reactive helper T-cell functions. The hapten-reactive T-lymphocyte system described here should be useful for analyzing and manipulating the immune response and for studying regulatory interactions of helper and suppressor T cells in the induction of antibody responses.

Regulatory functions of hapten-reactive helper and suppressor T lymphocytes. II. Selective inactivation of hapten-reactive suppressor T cells by hapten-nonimmunogenic copolymers of D-amino acids, and its application to the study of suppressor T-cell effect on helper T-cell development.

An experimental condition was established in vivo for selectively eliminating hapten-reactive suppressor T-cell activity generated in mice primed with a para-azobenzoate (PAB)-mouse gamma globulin (MGG)-conjugate and treated with PAB-nonimmunogenic copolymer of D-amino acids (D- glutamic acid and D-lysine; D-GL). The elimination of suppressor T-cell activity with PAB-D-GL treatment from the mixed populations of hapten- reactive suppressor and helper T cells substantially increased apparent helper T-cell activity. Moreover, the inhibition of PAB-reactive suppressor T-cell generation by the pretreatment with PAB-D-GL before the PAB-MGG-priming increased the development of PAB-reactive helper T-cell activity. The analysis of hapten-specificity of helper T cells revealed that the reactivity of helper cells developed in the absence of suppressor T cells was more specific for primed PAB-determinants and their cross-reactivities to structurally related determinants such as meta-azobenzoate (MAB) significantly decreased, as compared with the helper T-cell population developed in the presence of suppressor T lymphocytes. In addition, those helper T cells generated in the absence of suppressor T cells were highly susceptible to tolerogenesis by PAB-D- GL. Similarly, the elimination of suppressor T lymphocytes also enhanced helper T-cell activity in a polyclonal fashion in the T-T cell interactions between benzylpenicilloyl (BPO)-reactive T cells and PAB- reactive T cells after immunization of mice with BPO-MGG-PAB. Thus inhibition of BPO-reactive suppressor T-cell development by the BPO-v-GL- pretreatment resulted in augmented generation of PAB-reactive helper T cells with higher susceptibility of tolerogenesis to PAB-D-GL. Thus, these results support the notion that suppressor T cells eventually suppress helper T-cell activity and indicate that the function of suppressor T cells related to helper T-cell development is to inhibit the increase in the specificity and apparent affinity of helper T cells in the primary immune response. The hapten-reactive suppressor and helper T lymphocytes are considered as a model system of T cells that regulate the immune response, and the potential applicability of this system to manipulating various T cell-mediated immune responses is discussed in this context.

An in vitro model for cyclosporin A-induced interference of intrathymic clonal elimination.

The effects of cyclosporin A (CsA) on influencing the intrathymic clonal deletion were investigated by using our established thymic stromal cell clone with capacities to express Ia antigens and to produce a unique T cell growth factor. The following were revealed: (a) T cell clone with a given specificity was killed on the Ia+ stromal cell monolayer in the presence of the relevant antigens, a process depending on T cell receptor (TCR) stimulation; and (b) CsA allowed the T cell clone to continuously proliferate even during TCR stimulation by virtue of the stromal cell-derived T cell growth factor. This paper describes an in vitro model of a mechanism by which CsA is responsible for the generation of normally "forbidden" T cell clones.

Monoclonal antibodies to Pgp-1/CD44 block lympho-hemopoiesis in long-term bone marrow cultures.

A new panel of mAbs was prepared to a stromal cell line known to support lymphocytes in Whitlock-Witte type long-term bone marrow cultures. These antibodies were then screened with a cell adhesion assay and four were selected that inhibited the binding of B lineage cells to stromal cell monolayers. Immunofluorescent and biochemical analyses revealed that these new antibodies detected epitopes of the previously described Pgp-1/CD44 antigen complex. Addition of Pgp-1/CD44 antibodies to Dexter-type long-term bone marrow cultures completely prevented emergence of myeloid cells and they also blocked lymphocyte growth in Whitlock-Witte type cultures. mAbs MEL-14, LFA-1, and CD45R did not inhibit under the same conditions and there was no apparent relationship to Ig isotype. Adherent layers in treated cultures were not unusual in terms of morphology and the antibodies did not affect factor-dependent replication of lymphoid or myeloid progenitor cells. Therefore, the mechanism of inhibition may not involve direct toxicity to precursors or microenvironmental elements. Previous studies in humans and mice have implicated Pgp-1/CD44-related glycoproteins in the migration of peripheral lymphoid cells, as well as interactions of cells with the extracellular matrix. These findings suggest that they may also be critical for formation of lymphoid and myeloid cells within bone marrow.

Heterogenous graft rejection pathways in class I major histocompatibility complex-disparate combinations and their differential susceptibility to immunomodulation induced by intravenous presensitization with relevant alloantigens.

The present study investigates the heterogeneity of graft rejection pathways in class I major histocompatibility complex (MHC)-disparate combinations and the susceptibility of each pathway to immunomodulation induced by intravenous presensitization with alloantigens. Depletion of CD8+ T cells was induced by repeated administration of anti-CD8 monoclonal antibody. CD8+ T cell-depleted mice failed to generate anti-allo class I MHC cytotoxic T cell (CTL) responses but exhibited anti-allo class I MHC T cell responses, such as mixed lymphocyte reaction (MLR)/IL-2 production, that were induced by CD4+ T cells. In contrast, donor-specific intravenous presensitization (DSP), as a model of donor-specific transfusion, induced almost complete elimination of CD4+ and CD8+ T cell-mediated MLR/IL-2 production, whereas this regimen did not affect the generation of CTL responses induced by DSP-resistant elements (CD8+ CTL precursors and CD4+ CTL helpers). Prolongation of skin graft survival was not induced by either of the above two regimens alone, but by the combination of these. Prolonged graft survival was obtained irrespective of whether the administration of anti-CD8 antibody capable of eliminating CTL was started before or after DSP. The combination of DSP with injection of anti-CD4 antibody also effectively prolonged graft survival. However, this was the case only when the injection of antibody was started before DSP, because such antibody administration was capable of inhibiting the generation of CTL responses by eliminating DSP-resistant CD4+ CTL helpers. These results indicate that (a) the graft rejection in class I-disparate combinations is induced by CD8+ CTL-involved and -independent pathways that are resistant and susceptible to DSP, respectively; (b) DSP contributes to, but is not sufficient for, the prolongation of graft survival; and (c) the suppression of graft rejection requires an additional treatment for reducing DSP-resistant CTL responses. The results are discussed in the context of potential clinical application in attempts to inhibit the generation of DSP-resistant CTL responses upon the prospective DSP.

Regulatory functions of hapten-reactive helper and suppressor T lymphocytes. III. Amplification of a generation of tumor-specific killer T-lymphocyte activities by suppressor T-cell-depleted hapten-reactive T lymphocytes.

2,4.6-trinitrophenyl (TNP)-reactive T-cell activities were raised in mice by immunization with TNP-isologous mouse gamma globulin. After establishing that TNP-reactive T lymphocytes can serve as amplifier cells for induction of killer T lymphocytes in allogeneic system, we explored the possibility of this hapten-reactive T-cell system to amplify tumor-specific killer T-lymphocyte activity in the syngeneic system. We utlized relatively weak immunogenic syngeneic plasmacytoma X5563 in C3H/He mice. Analysis of the TNP-reactive T-cell activities revealed that such T lymphocytes express the biological functions of both major subtypes of regulatory T cells, namely suppressors and helpers, and that TNP-reactive suppressor and helper T lymphocytes, respectively, differ in their relative susceptibility to specific inactivation by TNP conjugates of the nonimmunogenic D-amino acid copolymer, D-glutamic acid, and D-lysine (D-GL). By taking advantage of the relative susceptibility-difference to TNP-D-GL, selective inactivation of TNP-reactive suppressor T cells was induced by appropriate treatment with TNP-D-GL, and the generation of TNP-reactive helper T-cell activity was amplified. The supplement of augmented TNP-reactive helper T-cell activity to the system at the immunization with syngeneic X5563 with TNP-haptenation, resulted in a striking augmentation of induction of tumor-specific killer T-lymphocyte activity, and a considerable number of hosts survived after the challenge with lethal dose of viable tumor cells. Thus, appropriate manipulations designed to induce potent hapten-reactive helper T-lymphocytes provided the potential for a very effective mode of immunoprophylaxis against tumor.

Cell interactions between histoincompatible T and B lymphocytes. IV. Involvement of the immune response (Ir) gene in the control of lymphocyte interactions in responses controlled by the gene.

In the present study we have asked the question of whether F(1) carrier-primed T cells can serve as helper cells for either or both parental B cells when (a) the carrier molecule employed is under genetic control such that one parental strain is a responder and the other is a nonresponder, and (b) the determinant specificity of the parental B cells being assessed is not under genetic control and bears no relationship to the specificity of the carrier molecule. Utilizing the system of immune response gene control of responses to the terpolymer L-glutamic acid-L-lysine-L-tyrosine (GLT) to which A strain mice (H-2(a)) are nonresponders, whereas BALB/c (H-2(d)) and (BALB/c x A)F(1) hybrids (CAF(1)) are responders, these studies demonstrate that GLT-primed T cells of CAF(1) donors can provide for responder BALB/c, but not for nonresponder A/J, the required stimulus for the anti-DNP responses of DNP-specific B cells of these respective parental strains to the DNP conjugate of GLT. The implications of these findings for Ir gene function in physiologic T-B cell interactions are discussed in detail.

Identification of a T cell-derived b cell growth factor distinct from interleukin 2.

We report here a factor (B cell growth factor) found in induced supernatants of the mouse thymoma EL4 that co-stimulates with anti-IgM antibodies in short-term cultures of purified B lymphocytes to induce polyclonal B cell proliferation but not antibody-forming cell production. The factor is not mitogenic for resting B cells and interacts with anti-IgM-activated B cells in a non-H-2-restricted manner. Absorption studies and molecular weight analysis reveal the factor is distinct from interleukin 2. This factor synergises with antigen, interleukin 2, and an interleukin 2-free, B cell growth factor-free T cell supernatant that contains T cell-replacing factor to produce erythrocyte-specific plaque-forming cells in cultures of highly purified B cells.

Cell-cell interaction in graft rejection responses: induction of anti-allo-class I H-2 tolerance is prevented by immune responses against allo-class II H-2 antigens coexpressed on tolerogen.

The intravenous sensitization of C57BL/6 (B6) mice with class I H-2-disparate B6-C-H-2bm1 (bm1) spleen cells results in almost complete abrogation of anti-bm1 CD8+ helper (proliferative and interleukin 2-producing) T cell (Th) activities. Although an appreciable portion of CD8+ cytotoxic T lymphocyte (CTL) precursors themselves remained after this regimen, such a residual CTL activity was eliminated after the engrafting of bm1 grafts, and these grafts exhibited prolonged survival. In contrast, the intravenous sensitization with (bm1 x B6-C-H-2bm12 [bm12])F1 cells instead of bm1 cells failed to induce the prolongation of bm1 graft survival as well as bm12 and (bm1 x bm12)F1 graft survival. In the (bm1 x bm12)F1-presensitized B6 mice before as well as after the engrafting of bm1 grafts, anti-bm1 CTL responses that were comparable to or slightly stronger than those observed in unpresensitized mice were induced in the absence of anti-bm1 Th activities. bm1 graft survival was also prolonged by intravenous presensitization with a mixture of bm1 and bm12 cells but not with a mixture of bm1 and (bm1 x bm12)F1 cells. The capacity of CD4+ T cells to reject bm12 grafts was eliminated by intravenous presensitization with antigen-presenting cell (APC)-depleted bm12 spleen cells. However, intravenous presensitization with APC-depleted (bm1 x bm12)F1 cells failed to induce the prolongation of bm1 graft survival under conditions in which appreciably prolonged bm12 graft survival was induced. More surprisingly, bm1 graft survival was not prolonged even when the (bm1 x bm12)F1 cell presensitization was performed in CD4+ T cell-depleted B6 mice. This contrasted with the fact that conventional class I-disparate grafts capable of activating self Ia-restricted CD4+ as well as allo-class I-reactive CD8+ Th exhibited prolonged survival in CD4+ T cell-depleted, class I-disparate cell-presensitized mice. These results indicate that: (a) intravenous presensitization with class I- and II-disparate cells fails to reduce anti-allo-class I rejection responses that would otherwise be eliminated using only class I-disparate cells; (b) such failure is generated according to the coexpression of both classes of alloantigens on a single cell as tolerogen; and (c) allo-class II antigens coexpressed on tolerogen function to activate CD4+ as well as non-CD4+ Th leading to the generation of anti-class I effector T cell responses.