Bcl-2 inhibition to overcome memory cell barriers in transplantation.

Memory T cells (Tm) represent a major barrier for immunosuppression and tolerance induction after solid organ transplantation. Taking into consideration the critical role of the intrinsic apoptosis pathway in the generation and maintenance of Tm, we developed a new concept to deplete alloreactive Tm by targeting Bcl-2 proteins. The small-molecule Bcl-2/Bcl-XL inhibitor ABT-737 efficiently induced apoptosis in alloreactive Tm in vitro and in vivo and prolonged skin graft survival in sensitized recipients. A short course of ABT-737 induction therapy prevented Tm-mediated resistance in a donor-specific transfusion model and allowed mixed chimerism induction across Tm barriers. Since Bcl-2 inhibitors yielded encouraging safety results in cancer trials, this novel approach might represent a substantial advance to prevent allograft rejection and induce tolerance in sensitized recipients.

Resistance to ABT-737 in activated T lymphocytes: molecular mechanisms and reversibility by inhibition of the calcineurin-NFAT pathway.

Dynamic regulation of the intrinsic apoptosis pathway controls central and peripheral lymphocyte deletion, and may interfere with the pro-apoptotic potency of B-cell lymphoma 2 inhibitors such as ABT-737. By following a T-cell receptor (TCR) transgenic population of alloantigen-specific T cells, we found that sensitivity to ABT-737 radically changed during the course of allo-specific immune responses. Particularly, activated T cells were fully resistant to ABT-737 during the first days after antigen recognition. This phenomenon was caused by a TCR-calcineurin-nuclear factor of activated T cells-dependent upregulation of A1, and was therefore prevented by cyclosporine A (CsA). As a result, exposure to ABT-737 after alloantigen recognition induced selection of alloreactive T cells in vivo, whereas in combination with low-dose CsA, ABT-737 efficiently depleted alloreactive T cells in murine host-versus-graft and graft-versus-host models. Thus, ABT-737 resistance is not a prerogative of neoplastic cells, but it physiologically occurs in T cells after antigen recognition. Reversibility of this process by calcineurin inhibitors opens new pharmacological opportunities to modulate this process in the context of cancer, autoimmunity and transplantation.

Effects of noninherited maternal antigens on allotransplant rejection in a transgenic mouse model.

We studied the influence of noninherited maternal antigen (NIMA) on allotransplant rejection using a mouse transgenic model. CBK transgenic (CBA [H-2k] expressing K(b) MHC class I transgene) mice were used as donors in heart transplantation experiments. Offspring of BM3.3 (CBA anti-K(b) TCR transgenic) male mice and (CBA x CBK)F1 females were used as NIMA (offspring that did not inherit K(b)) and IMA (offspring that inherited K(b) maternal antigen) recipient mice. Survival of allografts was monitored and the alloimmune response evaluated using an ELISPOT assay. IMA mice accepted CBK heart allografts and displayed no alloresponse to K(b+) cells. In contrast, mice never exposed to K(b) (offspring of BM3.3 males and CBA females) acutely rejected their grafts within 18 days posttransplantation and exhibited potent inflammatory alloresponses to K(b+) cells. NIMA mice displayed prolonged survival of allotransplants (MST >60 days). Although no deletion of anti-K(b) TCR transgenic cells was detected in these mice, they had a marked reduction in the frequency of activated alloreactive T cells producing type 1 (IFN-gamma and IL-2) cytokines and concomitant expansion of type 2 (IL-4) cytokine-secreting cells. Finally, depletion of CD4+ T cells from NIMA mice restored acute rejection of CBK hearts. This study is the first demonstration of the tolerogenic effects of NIMA on alloimmunity and allotransplant rejection in a transgenic model. It is shown that, although the NIMA tolerogenic effect is not due to deletion of alloreactive T cells, it is mediated by CD4+ T cells producing type 2 cytokines.

Identification of endogenous peptides recognized by in vivo or in vitro generated alloreactive cytotoxic T lymphocytes: distinct characteristics correlated with CD8 dependence.

We studied the molecular basis for CD8 independence of in vivo generated (BM3.3) versus CD8 dependence of in vitro sensitized (KB5.C20/Des) alloreactive H-2K(b)-specific cytotoxic T lymphocytes (CTL). Using microcapillary high-performance liquid chromatography fractionation of H-2K(b) eluates, mass spectrometry and CTL reconstitution assays, we determined that BM3.3 and KB5.C20 recognize, respectively, a single peptide (pBM1) expressed on 8,000 H-2K(b) molecules per allogeneic cell, and three distinct peptides (pKB1, 2, 3), each expressed on around 200 H-2K(b) molecules per allogeneic cell. CD8 (in)dependence was intrinsic to the respective TCR/H-2K(b)-peptide interactions. KB5.C20 and BM3.3 TCR illustrate the correlation that appears to exist between CD8 dependence/low affinity and in vitro sensitization as opposed to low dependency on CD8 and high TCR affinity observed after in vivo sensitization. The results suggest that CD8-dependent alloreactive CTL obtained in vitro with high frequency correspond to low-affinity TCR from the MHC-biased TCR repertoire unpurged by negative selection and have implications for cellular immunotherapeutic approaches.

Crystal structure of a T cell receptor bound to an allogeneic MHC molecule.

Many T cell receptors (TCRs) that are selected to respond to foreign peptide antigens bound to self major histocompatibility complex (MHC) molecules are also reactive with allelic variants of self-MHC molecules. This property, termed alloreactivity, causes graft rejection and graft-versus-host disease. The structural features of alloreactivity have yet to be defined. We now present a basis for this cross-reactivity, elucidated by the crystal structure of a complex involving the BM3.3 TCR and a naturally processed octapeptide bound to the H-2Kb allogeneic MHC class I molecule. A distinguishing feature of this complex is that the eleven-residue-long complementarity-determining region 3 (CDR3) found in the BM3.3 TCR alpha chain folds away from the peptide binding groove and makes no contact with the bound peptide, the latter being exclusively contacted by the BM3.3 CDR3 beta. Our results formally establish that peptide-specific, alloreactive TCRs interact with allo-MHC in a register similar to the one they use to contact self-MHC molecules.

Antigen-induced TCR-CD3 down-modulation does not require CD3delta or CD3gamma cytoplasmic domains, necessary in response to anti-CD3 antibody.

We studied cytotoxic T lymphocyte (CTL) clones expressing cytoplasmic domain-deleted CD3delta and CD3gamma chains. These cells retained efficient antigen-specific cytolysis. Because the cytoplasmic domains of native CD3delta and CD3gamma chains contain a dileucine-based and a tyrosine-based motif thought to be important for receptor endocytosis, we compared TCR-CD3 down-modulation on the CTL clones expressing or not these domains. We found that antigen-induced TCR-CD3 down-modulation was not dependent on either the CD3delta or CD3gamma cytoplasmic domains. This contrasts with phorbol ester- and anti-CD3 mAb (soluble or plastic-coated)-induced TCR-CD3 down-modulation, that are respectively dependent on CD3gamma and on either CD3delta or CD3gamma cytoplasmic domains, suggesting that differences may exist between the mechanisms of TCR-CD3 down-modulation in response to the three stimuli. TCR-CD3 down-modulation in response to antigen was demonstrated by confocal microscopy to be associated with TCRbeta chain internalization, whether CD3delta and CD3gamma were native or truncated. Inhibition by the protein tyrosine kinase inhibitor PP1 of TCR-CD3 down-modulation in response to antigen was also similar whether CD3delta and CD3gamma cytoplasmic domains were present or not. These properties of receptor down-modulation are discussed with respect to the requirements for TCR engagement on antigen-presenting cells.

Class I- and class II-reactive TCRs coexpressed on CD4+ T cells both trigger CD4/CD8-shared and CD4-unique functions.

CD4+ and CD8+ T cells emerge from thymic selection expressing a TCR restricted by MHC class II (TCRII) and MHC class I (TCRI), and upon Ag stimulation develop respectively into Th and CTL effector cells. The influence of thymic differentiation and antigenic stimulation on the determination of T cell functions was studied, with CD4+ T cells expressing a transgenic TCRI that reacts with the class I alloantigen H-2K(b) in a CD8-independent fashion. Such T cells additionally express a TCR, probably TCRII, in which the transgenic TCR beta-chain is associated with endogenously rearranged TCR alpha-chains. Upon in vitro stimulation with H-2K(b)-expressing cells, both CD8+ and CD4+ transgenic TCR+ T cells developed into CTL capable of killing Ag-expressing target cells through a perforin-dependent mechanism, and secreted IL-2 and IFN-gamma. Fas ligand-dependent killing could also be induced in both CD8+ and CD4+ in vitro stimulated T cells. The capacity to secrete IL-4 was restricted to the CD4+ T cells, however, suggesting that both CD8/CD4-shared and CD4-unique programs can be elicited by stimulation of CD4 T cells through a TCRI. Acquisition of CTL function was also induced upon class II alloantigen stimulation through the endogenously rearranged TCRII, which represents a polyclonal set of TCRs. IL-2, IFN-gamma, and after restimulation, IL-4, were also produced. Thus: 1) events associated with intrathymic selection influence the gene program activated in response to the same TCRI/APC interaction; and 2) CD4+ T cells expressing a TCRI and a TCRII can activate the same gene program after engagement of either one of these TCRs.

The degree of CD8 dependence of cytolytic T cell precursors is determined by the nature of the T cell receptor (TCR) and influences negative selection in TCR-transgenic mice.

Although much has been learned about CD8 structure-function properties, it has so far not been tested whether the nature of the TCR is sufficient to transfer the property of CD8 dependence versus non-dependence to CD8+ cytotoxic T lymphocytes (CTL) and their precursors differentiating in T cell receptor (TCR)-transgenic (Tg) mice. In the present study, we compared the characteristics of dependence on CD8 for stimulation of CTL precursors and antigen-specific cytolysis by CD8+ T cells from two TCR-Tg mice expressing respectively the TCR (Tg) from a "CD8-dependent" and from a "CD8-independent" CTL clone, which were both reactive against the H-2Kb alloantigen and originated from H-2k mice. The results indicate that the property of the Tg+CD8+ cells from H-2k TCR-Tg mice corresponds to that of the CTL clone of origin, demonstrating that it is linked to the nature of the TCR. Consistent with this property, Tg+CD4+ cells could also differentiate into H-2Kb-specific CTL when originating from the "CD8-independent", but not from the "CD8-dependent" Tg-TCR. The influence of the property of "CD8 dependence" on negative selection occurring in TCR-Tg H-2k/b mice was apparent at two levels: (i) in the thymus, the extent of deletion was much more pronounced for the "CD8-independent" TCR-Tg mice; (ii) in the periphery, Tg+(hi) cells with low to negative CD8 expression were present for the "CD8-dependent" Tg-TCR, whereas only Tg+CD4-CD8- cells with low surface Tg-TCR and CD3 expression were found for the "CD8-independent" Tg-TCR, indicating that Tg+CD4-CD8- cells are susceptible to tolerance induction involving TCR/CD3 surface down-modulation. Furthermore, different in vitro conditions led to H-2Kb-induced stimulation of Tg+CD4-CD8- cells to differentiate into CTL detected in an anti-TCR clonotypic monoclonal antibody redirected cytolysis assay. Culture in interleukin-2 of H-2k/b Tg+CD4-CD8- cells was sufficient to induced CTL activity in the "CD8-independent" model, whereas stimulation with cells which overexpressed H-2Kb was required in addition to interleukin-2 to induce CTL differentiation in the "CD8-dependent" model. These data suggest that peripheral Tg+CD4-CD8- cells present in a situation of in vivo tolerance to H-2Kb can still be triggered by H-2Kb with a sensitivity correlated with the degree of CD8 dependence.

CD8 requirements for negative selection events are directly related to the TCR-antigen interaction.

Positive selection of class I-restricted T cells has been suggested to always require the surface expression of CD8 molecules on CD4+8+ thymocytes, whilst negative selection was found to be differentially dependent, relating to the antigen being engaged by the T cell receptor (TCR). We have studied the CD8-dependency of positive and negative selection using two TCR-transgenic (Tg) mice models, which both react against the same allo-antigen, H-2kb, back crossed with mice which are deficient for the expression of CD8. Whilst CD8 expression was always required for positive selection of cells expressing high levels of the Tg-TCR, events of negative selection were differentially dependent upon CD8, reflecting the CD8-dependency of the original CTL clones. For one TCR-Tg model (derived from a CD8-dependent CTL clone), deletion of CD4+8+ thymocytes was partially dependent upon the expression of CD8, though there was still selection against Tg-TCR positive CD4+ cells, which normally exit to the periphery expressing low levels of Tg-TCR. For the other model (derived from a CD8-independent CTL clone) negative selection was unaffected by the absence of CD8. For both models the CD4-8- Tg-TCR positive population was unaffected by the absence of CD8, including, for the CD8-independent model, reduced expression of the Tg-TCR/CD3 complex. These results suggest that CD8 expression may be a prerequisite for positive selection, whilst negative selection events can occur in the absence of CD8, depending directly upon the TCR-antigen interaction.

Evaluation of functional heterogeneity in the CD8 subset with T cells from T cell receptor-transgenic mice.

The question of functional differentiation within the CD8 subset has been addressed in a model of TcR-transgenic (TcR-tg) mice expressing a TcR specific for H-2Kb (Ti). CD8+ Ti+ T cells present in the periphery of these mice have no cytotoxic T lymphocyte (CTL) activity unless they are stimulated with H-2Kb-expressing cells. In contrast to T cells from normal H-2k littermates, alloantigen induction of CTL from TcR-tg mice is independent of CD4+ T helper (Th) cells and is accompanied by high level secretion of interleukin-(IL)-2 by Ti+ CD8+ T cells. Precursor frequency analysis performed on CD8+ cells from TcR-tg mice revealed a high frequency of Th as compared to CTL precursors. This raised the possibility of the existence of distinct subpopulations within CD8+ precursors with different requirements for differentiation to functional CTL. FACS analyses (performed on resting and on in vitro stimulated T cells from normal and TcR-tg mice) demonstrated a heterogeneous expression of Ly-6C on CD8+ cells with a large enrichment of Ly-6C- cells among the Ti+ cells which persisted after stimulation with H-2b cells in conditions that led to a homogeneous expression of the activation markers pgp-1 and CD69. The possibility that Ly-6C expression could mark functionally different subpopulations in CD8+ T cells was investigated. Stimulation of sorted populations of Ly-6C- and Ly-6C+ cells allowed detection of CTL precursors in both these subsets and the majority of limiting dilution wells containing one pCTL also scored positive for IL-2 secretion. Thus, for CD8+ T cells expressing the same TcR, differentiation led to acquisition of both IL-2 secretion and CTL function and there was no evidence for the existence of a distinct population of helper-dependent CTL precursors.

A viral peptide can mimic an endogenous peptide for allorecognition of a major histocompatibility complex class I product.

Alloreactive class I-restricted T cells may recognize the class I structure alone, in association with a specific peptide, or with any stabilizing peptide. We have tested the role of endogenous peptides in the recognition of H-2Kb molecules by two alloreactive cytolytic T lymphocyte (CTL) clones using the mutant tumor line RMA-S, which expresses its surface H-2b molecules devoid of peptides and is not lysed by these two CTL clones. Empty H-2b molecules on RMA-S cells can be stabilized by binding exogenously added peptides. H-2Kb-specific recognition of the RMA-S cells by one of the CTL clones was restored by endogenous peptide extracts which only minimally stabilized H-2Kb on the surface of RMA-S cells, indicating the requirement for a specific peptide on a limited number of H-2Kb molecules. In addition, one out of three peptides which greatly enhance the expression of H-2Kb, the nucleoprotein peptide 52-59 from vesicular stomatitis virus (VSV), was also able to restore the lysis of RMA-S cells by the clone. The recognition of a common motif by an alloreactive clone (H-2k anti-H-2Kb) and virus-specific Kb-restricted clones suggests that both H-2k and H-2b thymic environments allow selection of T cells capable of recognizing H-2Kb+VSV and that tolerance to self, as would be the case in the (H-2k x H-2b)F1 mice, would partially delete the repertoire of antiviral T cells.

Evidence for quantitative and qualitative differences in functional activation of Mls-reactive T cell clones and hybridomas by antigen or TcR/CD3 antibodies.

In this study, we demonstrated that some V beta 6+, CD4+, Mls-1a-specific T cell clones had cytolytic activity when stimulated with anti-T cell receptor(TcR)/CD3 monoclonal antibodies (mAb), but not with targets expressing Mls-1a, although they produced lymphokines (interleukin 2 and interferon-gamma) in response to both types of stimuli. To examine the possibility that lack of cytolysis resulted from expression of the Mls-1a antigen on merely a fraction of splenic B blasts, we (a) used the B cell lymphoma LBB.3.4.16 and (b) measured esterase secretion which is generally concurrent with cytotoxic T lymphocyte (CTL) activity. The B cell lymphoma maximally stimulated the T cell clone for interferon-gamma production when responding and stimulating cells were incubated at a 1:1 ratio, but it was never killed by the Mls-1a-specific T cell clone unless TcR/CD3-specific mAb were added. Furthermore, a fivefold excess of the Mls-1a B cell lymphoma did not induce any secretion of esterase, which was observed only in the presence of the TcR/CD3-specific mAb. Comparison of the reactivity of two Mls-1a-specific T cell hybridomas expressing the same TcR at similar surface density, revealed both quantitative and qualitative differences between CD3-specific mAb and Mls stimulation of the hybridomas. A small quantitative difference in the sensitivity of hybridoma FJ22.5 to stimulation with V beta 6 or CD3-specific mAb resulted in a marked decrease in efficiency of stimulation by Mls-1a for interleukin 2 production and to inability to detect growth inhibition by Mls-expressing cells. A qualitative difference was observed when analyses of inositol phosphate production were performed under optimal conditions of stimulation of the highly responsive T cell hybridoma (FJ8.1): only stimulation with CD3-specific mAb, but not Mls-expressing cells, could induce detectable inositol phosphate production. Lack of cytolysis of Mls-1a class II-expressing B cells may have evolutionary significance in view of the recent mapping of Mls to mouse mammary tumor virus genes.

Conditions of anti-Lyt-2-mediated inhibition of TcR/CD3-induced IFN-gamma secretion by a CTL clone.

The relationship between the T cell receptor (TcR) for antigen (Ag) and the Lyt-2/3 molecule during T cell activation was studied using the T cell clone KB5.C20, which is dependent upon Lyt-2 for target cell killing. This cytolytic T cell clone can be activated to secrete IFN-gamma by stimulation with H-2Kb expressing cells or with monoclonal antibodies directed against a clonotypic structure of the TcR or against associated CD3 molecules. IFN-gamma production induced by H-2Kb can be inhibited by anti-Lyt-2mAb. In addition, TcR-mediated activation using the anticlonotypic mAb Désiré-1 in soluble form can be inhibited by anti-Lyt-2 mAb in soluble form either as a divalent IgG or as its monovalent Fab fragment. Anti-Lyt-2 mAb immobilized on plastic wells was also inhibitory. Stimulation induced by the anti-TcR mAb or by anti-CD3 mAb immobilized on plastic can be inhibited only with plastic immobilized and not with soluble anti-Lyt-2mAb, however. These results are discussed in terms of local interactions between TcR and Lyt-2 molecules.

Interactions between the Thy-1 and T-cell antigen receptor pathways in the activation of cytotoxic T cells: evidence from synergistic effects, loss variants, and anti-CD8 antibody-mediated inhibition.

The relationship between activation of cytolytic T-lymphocyte (CTL) clones via the T-cell receptor (Ti) or the Thy-1 molecule was investigated. Anti-Ti and anti-Thy-1 monoclonal antibodies (mAb) can activate CTL clones to secrete interferon-gamma (IFN-gamma). Suboptimal doses of anti-Ti and anti-Thy-1 mAb, as well as suboptimal doses of two different anti-Thy-1 mAb, can synergize to activate T-cell clones. The addition of phorbol myristic acetate (PMA), which is not stimulatory by itself, can enhance the synergistic effect of mAb on IFN-gamma production. Although the Ti and Thy-1 molecules were not found associated at the cell surface, the results presented here indicate that these molecules are functionally associated. Use of Ti loss variants of a CTL clone confirms that Thy-1-mediated signaling is not an alternative to, but is dependent on the Ti-mediated activation pathway. Additionally, use of anti-Lyt-2/3 mAb, previously described as interfering with class I MHC-Ti binding and/or activation and, in some cases, with anti-Ti-mediated activation revealed that anti-Thy-1 mAb-mediated activation was also greatly reduced by the presence of Lyt-2/3-specific mAb. Thus the interaction between Thy-1 and Ti might also involve Lyt-2 (Lyt-3) molecules.

Pleiotropic loss of activation pathways in a T-cell receptor alpha-chain deletion variant of a cytolytic T-cell clone.

Untransformed T-cell clones maintained in culture are dependent on signals transmitted through their antigen receptors (Ti; alpha and beta chains associated with the CD3 molecules) for growth and effector function. For cytolytic T cells (CTL), Ti stimulation also activates the killing machinery and induces synthesis of gamma interferon (IFN-gamma) messenger RNA and IFN-gamma secretion. The Thy-1 molecule, expressed on all murine cells of the T-cell lineage, has been suggested to function in transmembrane signalling, based on the ability of some anti-Thy-1 monoclonal antibodies (mAb) to activate T cells. Recently, it was suggested that Thy-1 could function as a signal-transduction molecule when expressed in B-cell lymphomas after transfection of the gene, leading to speculation that the molecule was part of an activation pathway independent of the Ti/CD3 structures. Here we report the immunoselection of a variant CTL clone which has lost expression of mRNA for the alpha-chain of the Ti. The Ti- variant was defective in lectin-mediated activation whether measured by increase in intracytoplasmic Ca2+, CTL effector function or IFN-gamma synthesis. The variant, which expressed normal levels of Thy-1, was also unresponsive to Thy-1 mAb activation as measured by IFN-gamma secretion, whereas it responded to calcium ionophore plus phorbol ester. These results indicate that in a non-transformed, functional mature T-cell, Thy-1 mediated signalling is not an alternative to, but might depend on elements associated with the Ti/CD3-mediated T-cell activation pathway.

Phosphorylation of T-cell antigen receptor-associated proteins: correlation with activation for killing and/or for gamma-interferon production by a cytolytic T-cell clone.

The activation-induced phosphorylation of T-cell antigen receptor (Ti)-associated proteins was investigated in order to analyse possible signal-transduction mechanisms leading to two distinct effector functions of a mouse cytolytic T-cell clone (KB5.C20): target cell killing (independent of protein synthesis) and de novo production of gamma interferon (gIFN; dependent on gIFN gene expression). Ti-associated T3-like proteins were first identified by immunoprecipitation of 125I-labelled cell surface proteins from 1% digitonin lysates of clone KB5.C20 by 1- and 2-dimensional (non-reduced (NR)/reduced (R)) gel electrophoresis. In addition to the alpha and beta chains of the Ti (NR: 80-Kd; R: 43 and 40 Kd), two doublets of 35-37 Kd (NR) and 32-34 Kd (NR) leading to bands of 25, 16 and 14 Kd (R) were identified, as well as three bands (25, 23 and 22 Kd (NR)) leading to 27-, 25- and 21-Kd bands (R). Activation of clone KB5.C20 (prelabelled with 32P-orthophosphate) with either anti-Ti mAb or exposure to both ionomycin and phorbol myristic acetate (PMA) induced the phosphorylation of 21- and 25-27-Kd (R) Ti-associated proteins, whereas exposure to either ionomycin or PMA alone induced only weak phosphorylation of 21-Kd (R) components. A weak phosphorylation of 32- and 34-Kd Ti-associated proteins was sometimes observed after stimulation with anti-Ti mAb. Functional studies suggested that activation for gIFN production was observed only when both the 21- and 25-27-Kd proteins were phosphorylated, whereas activation for killing (when measured by PMA-induced non-specific killing) could occur in conditions where no phosphorylation of the 25-27-Kd protein was detected.

Analysis of T cell activation requirements with the use of alloantigens or an anti-clonotypic monoclonal antibody.

H-2Kb-specific alloreactive cytotoxic T lymphocyte (CTL) clones, which secrete macrophage-activating factor (MAF) in response to antigen, were used to study the antigenic and physiologic parameters for T cell-antigen receptor (Ti)-mediated activation. When H-2Kb was presented on untreated, formaldehyde (FOR)-fixed cells, or liposomes, optimal stimulation in the absence of co-factors was obtained only with the untreated cells. FOR cells, which were efficient inhibitors of CTL-target cell interactions for the CTL clones studied, had a greatly reduced activating capacity. Phorbol myristic acetate (PMA), which alone had no activating effect on the CTL clones, acted in synergy with FOR cells, restoring an unimpaired H-2Kb-dependent activation for some CTL clones. H-2Kb-liposomes were not stimulatory even in the presence of PMA and/or splenic feeder cells. For one H-2Kb-specific CTL clone (KB5-C20), activation was studied with the use of an anti-clonotypic (anti-Ti) monoclonal antibody (mAb), Désiré-1. By using this immunoglobulin (Ig) G2a mAb or its F(ab')2 or Fab fragments, it was observed that the association constant of the IgG2a (4.7 X 10(9) M-1) for KB5-C20 was 30-fold higher than that of the Fab fragment, whereas the molar concentration of mAb required to inhibit 50% of the H-2Kb-specific CTL activity of clone KB5-C20 was 70-fold to 90-fold higher for the Fab fragment than for the IgG2a (1 to 5 X 10(-10) M). It was also observed that activation as measured by MAF secretion of clone KB5-C20 was obtained by using the divalent IgG2a mAb or its F(ab')2 fragment at 10(-9) M to 10(-8) M in the absence of any added co-factor or feeder cells, whereas a 500-fold higher molar concentration of the Fab fragment only induced very low levels of MAF secretion. Also, in the presence of PMA, the mAb-mediated activation of MAF secretion by clone KB5-C20 was increased, but Fab-induced activation never resulted in high titers of MAF. We conclude that the intrinsic "affinity" of the H-2Kb-Ti interaction is probably so low for the Ti of clone KB5-C20 that efficient activation by H-2Kb is obtained only when multivalency is achieved on H-2Kb-expressing cells, and possibly when molecules on the T cells such as Lyt-2 and LFA-1 stabilize the interactions.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of helper function with anti-Lyt-2 or anti-L3T4 monoclonal antibodies depending on stimulating antigens.

The role of L3T4+ and Lyt-2+ cells in the induction of cytotoxic T cells (CTL) after stimulation with trinitrobenzene sulfonate (TNBS)-treated syngeneic cells, or with cells differing for the entire major histocompatibility complex or for class I molecules alone was investigated using anti-L3T4 and anti-Lyt-2 monoclonal antibodies (mAb). Anti-L3T4 mAb inhibited both induction of CTL and interleukin 2 (IL2) production by T cells stimulated with allogeneic (class I + class II differences) or by TNBS-treated syngeneic cells. In the same culture conditions, anti-Lyt-2 mAb inhibited CTL induction but had no effect on IL2 production. By contrast, only anti-Lyt-2 mAb inhibited both CTL induction and IL2 production when T cells were stimulated by allogeneic cells differing only for class I antigens. Anti-L3T4 mAb had no inhibitory effect in this situation. These results indicate that two helper pathways can be activated, depending on the type of T cell stimulation: one, implicating L3T4+ cells, is used when stimulation involves class I and class II alloantigens or hapten on syngeneic cells; the other, involving Lyt-2+ T cells, is preferentially stimulated when responding and stimulating cells differ only for class I major histocompatibility complex antigen.