Functional analysis of cloned macrophage hybridomas. V. Induction of suppressor T cell responses

It has been suggested that macrophage-like accessory cells are involved in suppressor T cell (Ts) induction. To further analyze this issue, we obtained several cloned macrophage hybridoma cell lines by somatic cell fusion of the macrophage tumor P388D1 of DBA/2 (H-2d) origin with splenic adherent cells of CKB mice (H-2k). Several cloned lines displayed the serological and functional characteristics of macrophages. We evaluated the ability of these hybridomas to induce third order or effector Ts (Ts3) to suppress the contact sensitivity response against the hapten 4-hydroxy-3-nitrophenyl acetyl (NP). In contrast to the parental P388D1 and two other macrophage hybridomas, one macrophage hybridoma clone, termed 63, when conjugated with NP, induced Ts3, which suppressed contact sensitivity responses against NP but not DNFB, showing that the Ts3 were antigen specific. Macrophage hybridoma 63 could specifically induce Ts3 activity in either H-2k, H-2d, or H-2k/H-2d heterozygous hosts. Thus, macrophage hybridoma 63 functionally expressed major histocompatibility complex-related restricting determinants, and the fusion with cells from a H-2k macrophage donor caused a functional complementation of H-2d-related, Ts-inducing elements. The genetic restriction governing induction of Ts3 was controlled by genes that mapped to I-J region. Furthermore, NP-conjugated macrophage hybridoma 63 could serve as a target for elicitation of suppressor responses after administration of I-Jk, but not I-Jb, restricted suppressor factor. The data suggest that macrophage hybridomas represent a means to dissect heterogeneity within the macrophage population. The data also imply that the I-J determinants expressed on macrophages represent a ligand for the antigen receptor of Ts.     

Expression of functional alpha beta T cell receptor gene rearrangements in suppressor T cell hybridomas correlates with antigen binding, but not with suppressor cell function

We have examined the expression of TCR genes in 4-hydroxy-3-nitrophenyl-acetyl (NP)-specific Ts cell hybridomas. Each of three independently isolated hybridomas expressed in-frame TCR alpha-chain rearrangements derived from the original suppressor Ts cell. Different V alpha and J alpha gene segments were rearranged and expressed in each Ts cell line. The only TCR beta-chain expressed in these cells was derived from the BW5147 fusion partner. Expression of the BW5147 beta-chain was found to correlate with cell surface Ag binding, inasmuch as subclones derived from one of the original Ts lines expressed greatly reduced levels of beta-chain mRNA and no longer bound to NP-coupled RBC. Subclones that continued to express beta-chain mRNA did bind to NP-coupled RBC. This suggests that the Ag receptor on Ts hybridomas is a TCR-alpha beta dimer composed of a unique alpha-chain and the BW5147 beta-chain. Ag binding could be modulated by preincubation of Ts hybridoma cells with anti-TCR-alpha beta antibody, thereby supporting this conclusion. Suppressor factor activity was measured in the conditioned media of Ts subclones that differed by 250-fold in levels of beta-chain mRNA expression. No difference in suppressor factor activity was found; conditioned media from these subclones suppressed both plaque-forming cell responses and delayed-type hypersensitivity responses at approximately equivalent dilutions. Suppressor factor activity in the conditioned media of both a beta-chain negative subclone and a beta-chain positive subclone could be absorbed with an antibody that recognizes the TCR alpha-chain, but not with an antibody that recognizes the TCR beta-chain. We conclude that suppressor factor activity in the conditioned media of these Ts hybridomas is not derived from surface TCR-alpha beta receptors, although it does share TCR alpha-chain determinants.     

Relationships between antigen-specific helper and inducer suppressor T cell hybridomas

Ts1, or inducer suppressor T cells, share many phenotypic and functional characteristics with helper/inducer subset of T cells. In order to evaluate the relationship between these cell types, we made a series of new Ts1 hybridomas by the fusion of Ts1 cells with the functionally TCR alpha/beta-negative BW thymoma (BW 1100). Three Ts1 hybridomas (CKB-Ts1-38, CKB-Ts1-53, and CKB-Ts1-81) were established that express TCR and produce Ag-specific suppressor factors constitutively, thus making it possible to study the nature and specificity of Ag receptors, MHC restriction, and lymphokine production by the Ts1 hybridomas. Results presented in this report demonstrate that all the Ts1 hybridomas described here express CD3-associated TCR-alpha beta. These three Ts1 hybridomas recognize Ag (NP-KLH) specifically in a growth inhibition assay and this recognition is restricted by IE molecules. Two of the hybridomas also produce IL-2 or IL-2 and IL-4 upon Ag-specific activation. Thus, by these three criteria the Ts1 hybridomas appear indistinguishable from Th cells. These three Ts1 hybridomas, however, release suppressor factors (TsF1) in the supernatant that suppress both in vivo DTH and in vitro PFC responses in an Ag-specific manner. Like the TsF1 factors characterized previously, the suppression mediated by these factors are Igh restricted and lack H-2 restriction. These factors mediate suppression when given in the induction phase but not during the effector phase of the immune response. The TsF1 factors are absorbed by Ag (NP-BSA), and anti-TCR affinity columns and the suppressor activity can be recovered by elution. The data are consistent with the interpretation that Ts1 inducer-suppressor T cells are related to Th cells; the feature that distinguishes these cells is the ability to produce Ag-binding factors that specifically suppress immune responses.     

Relationship between T cell receptors and antigen-binding factors. I. Specificity of functional T cell receptors on mouse T cell hybridomas that produce antigen-binding T cell factors

Upon antigenic stimulation with OVA-pulsed syngeneic macrophages, the mouse T cell hybridoma 231F1 produced glycosylation inhibiting factor (GIF) having affinity for OVA and IgE-suppressive factors, whereas another T cell hybridoma, 12H5, cells produced OVA-binding glycosylation enhancing factor (GEF) and IgE-potentiating factor. The OVA-binding GIF from the 231F1 cells is an Ag-specific Ts cell factor, whereas OVA-binding GEF from the 12H5 cells is an Ag-specific augmenting factor. Both hybridomas express CD3 complex and functional TCR-alpha beta. Cross-linking of TCR-alpha beta or CD3 molecules on the hybridomas by anti-TCR-alpha beta mAb or anti-CD3 mAb and protein A resulted in the formation of the same factors as those obtained by the stimulation of the cells with OVA-pulsed syngeneic macrophages. It was also found that both the 231F1 cells and 12H5 cells formed IgE-binding factors upon incubation with H-2d and H-2b APC, respectively, with a synthetic peptide corresponding to residues 307-317 in the OVA molecules (P307-317). Six other synthetic peptides, including those containing the major immunogenic epitope, i.e., P323-339, failed to stimulate the hybridomas in the presence of APC. Indeed, all of the 10 T cell hybridoma clones, which could produce either OVA-binding GIF or OVA-binding GEF, responded to P307-317 and APC for the formation of IgE-binding factors. In contrast, GIF/GEF derived from six other hybridoma clones, whose TCR recognized P323-339 in the context of a MHC product, failed to bind to OVA-coupled Sepharose. The results indicate the correlation between the fine specificity of TCR and the affinity of GIF/GEF to the nominal Ag. The amino acid sequence of P307-317 suggested that TCR on the cell sources of Ag-binding factors are specific for an external structure of the Ag molecules.     

Requirements for suppressor cell activation. Role of accessory cells

In the 4-hydroxy-3-nitrophenyl acetyl (NP) suppressor system, third order suppressor cells (Ts3) subset of suppressor cells is generated after Ag priming, but, in order to express suppressor activity, these cells need to be further activated or triggered with a specific second order suppressor factor. By in vitro activation of Ts3-containing lymph node cells or a pTs3 hybridoma we now show that macrophages are also required for Ts3 activation. In addition, we demonstrate that IJ genetic restrictions control this activation process. Furthermore, we directly demonstrate Ts3 activation using cloned macrophage hybridoma cells. To further investigate the interactions between Ts3 cells and the accessory cells involved in their activation, we attempted to block the second order suppressor factor mediated activation of Ts3 cells with antibodies. The activation of Ts3 cells can be blocked by the addition of anti-IJ, anti-IJ idiotype or anti-NPb idiotype antibodies, but not by anti-CD8, anti-IA, or anti-IE antibodies. Anti-IJ mAb blocked Ts3 activation at the lymphocyte level whereas anti-IJ idiotype blocked activation at the accessory cell level. Finally we tested, whether these antibodies can also directly activate primed Ts3 cells. We demonstrate that cross-linked anti-IJ, anti-NPb and anti-CD3 antibodies can activate Ts3 cells. The results are discussed in terms of receptor-ligand structures on Ts and accessory cells which are required for the activation of Ts3 cells.     

Functional analysis of cloned macrophage hybridomas. VI. Differential ability to induce immunity or suppression

We previously screened a series of macrophage hybridomas derived from fusion of P388D1 (H-2d) tumor cells with CKB (H-2k) splenic adherent cells for their ability to induce I-J restricted Ts cell responses. One Ia+ macrophage clone (63) consistently induced Ag-specific, I-J-restricted Ts. To evaluate whether macrophage hybridoma 63 also induced delayed-type hypersensitivity (DTH) immunity, mice were immunized with hapten-coupled macrophage hybridoma cells. Hapten-coupled splenic adherent cells and control macrophage hybridomas induced significant primary DTH responses, whereas hapten-coupled macrophage 63 induced little or no immunity when injected into H-2 compatible hosts. However, macrophage hybridoma 63 specifically activated I-Ak, I-Ad, or I-Ed restricted T cell hybridomas/clones, in vitro in the presence of appropriate Ag. Three different strategies designed to eliminate suppressor cell activity were successfully used to demonstrate that hapten-coupled macrophage 63 could also induce in vivo immunity. First, after immunization with hapten-coupled macrophages, mice were treated with cyclophosphamide. Second, macrophage 63 was treated with anti-IJ idiotype antibody before 4-hydroxy-3-nitrophenyl acetyl hapten (NP) coupling. Finally, haptenated macrophages were injected into I-A compatible but I-J incompatible recipients. These protocols are known to inhibit the induction of Ts activity, thus these results indirectly suggest that there is stimultaneous generation of Ts activity in vivo. The latter hypothesis was tested in adoptive transfer experiments. Transfer of lymph node cells from NP-63 primed B10.BR (H-2k) mice induced immunity in naive 4R animals, whereas the same number of immune cells suppressed NP-induced DTH responses in 5R mice. The combined results indicate that a cloned macrophage line can activate both Th and Ts cells. Macrophages which induce Ts activity may be responsible for maintaining the balance of immunity vs suppression. The data support the hypothesis that IJ interacting molecules (IJ-IM) expressed on macrophages are critical for induction of suppressor cell activity.     

Functional and biochemical analysis of CD16 antigen on natural killer cells and granulocytes

CD16 Ag is associated with the low affinity FcR for IgG expressed on human NK cells and granulocytes. In this study, we demonstrate that NK cells specifically lyse murine anti-CD16 hybridoma cell lines, but do not lyse hybridomas against other cell surface differentiation Ag expressed on NK cells. Moreover, the CD18 structure is involved in the CD16-specific xenogeneic interaction between human effector cells and murine hybridoma target cells. Although interaction with anti-CD16 hybridomas or antibodies triggers the cytolytic mechanism of NK cells, this interaction does not induce cellular proliferation. In contrast to NK cells, CD16+ granulocytes do not lyse anti-CD16 hybridoma cell targets and do not mediate ADCC against antibody-coated human tumor cell targets. These findings indicate a fundamental difference in the antibody-dependent cellular cytotoxicity mechanisms of NK cells and granulocytes. Comparative biochemical analysis of CD16 on NK cells and granulocytes revealed significant differences in the size of the polypeptides obtained after removal of N-linked carbohydrate residues with endo-F and N-glycanase digestion.     

Antibody inhibition of suppressor cell induction

The IJ genetic restrictions of suppressor T (Ts) cells are controlled by H-2-related determinants that are expressed on antigen-presenting cells. This has led to the hypothesis that Ts cells carry receptors for a self H-2-related ligand that is expressed on specialized antigen-presenting cells. We refer to this H-2-related ligand as the IJ interacting molecule. This report evaluates the ability of rabbit antibodies directed against idiotypes on monoclonal anti-IJ antibodies (the latter are presumably reactive with the Ts cell receptor) to bind IJ interacting molecule and to inhibit antigen presentation to Ts cells. Such anti-idiotypic reagents were prepared against T cell-reactive monoclonal anti-IJk and anti-IJd antibodies. The F(ab')2 fragments of these anti-idiotypic reagents blocked Ts cell induction. The inhibition was haplotype specific and mapped to the IJ region. The anti-idiotypic antibodies blocked the generation of Ts1, Ts2, and Ts3 cells. The cellular target of the blocking activity mediated by these anti-idiotypic antibodies is a macrophage. This was shown by using a cloned macrophage hybridoma line for both Ts induction and absorption of antibody activity. The combined data support the concept that macrophages express IJ interacting determinants that are responsible for Ts cell induction.     

Activation via TCR or IL-2 receptor of a CD8+ suppressor T cell clone: Effect on IL-10 production and on proliferation of the suppressor T cell

In a previous study, we established CD8⁺ suppressor T cell (Ts) clone 13G2 which produced the suppressive lymphokine, interleukin-10 (IL-10). In this study, we examined what physiological activator could induce both production of IL-10 from 13G2 and the proliferation of 13G2. Both the antigenic stimulation mimicked by the anti-CD3 antibody and the T cell growth factor interleukin-2 (IL-2) induced IL-10 production from the 13G2 clone equally well. 13G2 cells proliferated remarkably with IL-2 stimulation, while anti-CD3 only slightly induced proliferation of the clone. 13G2 cells also produced IL-10 in the presence of hydroxyurea which blocked transit of cells from G₁ to S phase. However, cycloheximide blocked the production of IL-10 from the Ts clone. The study demonstrates that both the anti-CD3 antibody and IL-2 induced IL-10 synthesis of the Ts clone equally well, and the proliferative response of Ts cells was induced more by IL-2 than by anti-CD3. IL-2 proved to be a good stimulator for Ts cells to produce suppressive lymphokine and to multiply their population.Abbreviation Ts suppressor T cell - Th helper T cell - Ag antigen - APC antigen presenting cell - IL interleukin - TCR T cell receptor - mAb monoclonal antibody     

A monoclonal antibody raised to tumor-specific T cell-derived suppressor factors also recognizes T suppressor inducer factors of the 4-hydroxy-3-nitrophenyl acetyl hapten suppressor network

A monoclonal antibody (mAb), B16G, was raised from BALB/c mice immunized with affinity-purified T suppressor factors (TsF) specific for the murine mastocytoma P815. This mAb was found to bind to polyclonal TsF isolated from the spleens of tumor-bearing animals, and to the TsF released from a P815-specific T cell hybridoma. In this study, B16G was tested for its reactivity with TsF produced in the 4-hydroxy-3-nitrophenyl acetyl hapten system. The factors from three types of suppressor T cell hybridomas, each representing the immortalized analogues of the inducer T suppressor cell (Ts1), transducer suppressor cell (Ts2), and effector suppressor cell (Ts3) network populations, were tested. B16G was found to be reactive with two sources of TsF1 as assayed by enzyme-linked immunosorbent assay and delayed-type hypersensitivity bioassay. By contrast, TsF2 and TsF3 were nonreactive with B16G. These results indicate that B16G recognizes class-specific suppressor factor determinants, and that the transducer/effector factors of the network are apparently serologically distinct. Because the B16G mAb fails to recognize 4-hydroxy-3-nitro-phenyl acetyl-specific TsF3 that share idiotype-related determinants with TsF1 yet binds to TsF1 molecules that have interacted with antigen, the binding is apparently independent of the site of antigen recognition. Additionally, the results show that the tumor-specific TsF1 raised in one suppressor system share serologic determinants with anti-hapten TsF1 raised in another.     

In vitro generation of suppressor T cells. Induction of CD3+, IgH-restricted suppressor cells

Previous studies of the immune response of C57BL/6 mice to the 4-hydroxy-3-nitrophenyl acetyl (NP) hapten determined that challenge with antigenic forms of hapten induces both immunity and suppression. The anti-NP plaque-forming cell response can be down regulated by an Ag-induced cascade consisting of three suppressor T cell subsets. These three populations, termed Ts1, Ts2, and Ts3 have been characterized to have inducer, transducer and effector functions, respectively. Although the functions of each of these subsets have been examined in vivo, the cellular requirements for in vitro Ts induction have only been investigated for the Ts3 population. The present study characterizes the cellular events that lead to the induction of the Ts2, suppressor transducer population. Culture of naive C57BL/6 spleen cells with Ts1-derived suppressor factor in the absence of exogenous Ag leads to the generation of Ts2 cells that mediate Ag-specific suppression of NP plaque-forming cell responses. Phenotypic analyses demonstrate that a CD3+, CD4-, CD5+, CD8+, and I-J+ precursor population is stimulated by TsF1 to become mature Ts2 cells that express CD3, CD8, and I-J but not CD5. Although previous studies have reported an essential role for B cells in the induction of other Ts populations, depletion of B cells from Ts2 induction cultures had no effect on Ts2 generation. Despite the absence of B cells in these cultures, the mature Ts2 cells were functionally IgH restricted. Studies with IgH congenic B.C-8 mice suggest that this restriction specificity was imposed by the idiotype-related determinants expressed on the TsF1, not the T cell genotype.     

A monoclonal antibody raised to lipomodulin recognizes T suppressor factors in two independent hapten-specific suppressor networks

Five different Ag-binding suppressor factors from two types of hapten-specific Ts cell hybridomas (TsF1 inducer and TsF3 effector factors) were bound by an anti-lipomodulin mAb (141-B9), that crossreacts with rodent glycosylation inhibition factor (GIF). The Ag-specific suppressor activity in these hybridoma supernatants was bound by anti-lipomodulin columns and could be recovered by elution at acid pH. Additional evidence for the expression of lipomodulin/GIF activity on these TsF molecules was demonstrated by the ability of the eluted fractions to inhibit the glycosylation of IgE-binding peptides during their biosynthesis. The same biologic activity is associated with GIF and lipomodulin. The relationship between TsF and lipomodulin/GIF was confirmed in a serologic assay, which showed that TsF1 and TsF3 molecules, whether purified over Ag, anti-IJ or anti-TsF columns, are recognized by the mAb. 141-B9. The combined results indicate that Ag-binding Ts factors share a common antigenic determinant with phospholipase inhibitory proteins such as lipomodulin and GIF. In addition, the demonstration of glycosylation regulatory activity carried on these TsF molecules suggests a possible mode for their bioactivity.     

Characterization of suppressor T cell clones derived from a mouse tolerized with conjugates of ovalbumin and monomethoxypolyethylene glycol.

The induction of antigen-specific tolerance in mice by conjugates of ovalbumin (OVA) and monomethoxypolyethylene glycol (mPEG) previously had been shown to be associated with the generation of antigen-specific suppressor T (Ts) cells. For the elucidation of the nature of these Ts cells, five nonhybridized OVA-specific Ts cell clones were generated from the spleen cells of a BDF1 mouse which had been immunosuppressed by the tolerogenic conjugate, OVA(mPEG)12. The cloned Ts cells were maintained in vitro by periodic stimulation with OVA and feeder cells and were able to suppress the in vitro antibody production in an OVA-specific and MHC class I (H-2Kd or H-2Dd)-restricted manner. All these Ts cell clones were shown to be Thy1.2+, CD4-, CD5-, CD8+, and to express CD3 and the alpha beta heterodimer of the T cell receptor. The cell-free extracts of these cells contained soluble suppressor factors which could mimic in vitro the suppressive activity of the intact cells. In contrast to cytotoxic T lymphocytes (CTL), none of the cloned Ts cells were endowed with cytolytic activity as revealed in the perforin-mediated microhemolysis and in the 18-hr51Cr release assays. These results demonstrate that (i) OVA-specific Ts cell clones can be generated from mice pretreated with OVA(mPEG)12 by employing conventional T cell culture techniques, and (ii) these Ts cells are functionally different from conventional CD8+ CTL.     

The role of adherent accessory cells in the generation of effector suppressor T cells

The role of accessory cell populations in the generation of effector suppressor (Ts3) cells was studied. By using an in vitro culture system, it was previously determined that the induction of NP-specific effector suppressor activity requires T cells, antigen, and an anti-idiotypic B cell population. We now demonstrate that the generation of Ts3 cells in this system also requires accessory cells. The accessory population appears to play a role in the processing and presentation of antigen. These antigen-presenting accessory cells are required early in the induction phase of Ts3 generation. These accessory cells can present NP coupled to immunogenic or non-immunogenic polypeptide carriers, including polymers of L-amino acids. However, NP coupled to polymers of poorly metabolized D-amino acids fail to induce suppressor T cell generation. Furthermore, the data demonstrate that an H-2 homology must exist between the Ts3 precursors and the antigen-presenting cell population if suppressor activity is to be generated. We also characterize the differential genetic restrictions that govern the induction of Ts3 cells that control suppression of either T cell or B cell responses. The data suggest that although I-J region encoded gene products control the induction and effector phases of suppressor cell activity as measured on T cell responses, the suppression of B cell responses appear to be controlled by I-A gene products. Possible cellular mechanisms that might explain these findings are discussed.     

An in vitro system for the generation of suppressor cells and the requirement for B cells in their induction

An in vitro method for the generation of effector suppressor cells (Ts3) was developed. By utilizing this protocol, it was possible to investigate both the cellular and genetic requirements for suppressor cell induction. It was determined that populations containing Ts3 cells can be induced after a 4-day culture of spleen cells and antigen. These Ts3 cells are similar to Ts3 cells generated by in vivo immunization. Both populations are I-J+, bind NP hapten, bind NP hapten, bear receptors which share NPb idiotypic determinants with anti-NP antibodies, function during the effector phase of the immune response, and require activation with Ts2 cells. Generation of Ts3-containing populations required both nylon wool-nonadherent T cells and a nylon-adherent, B cell-enriched population from an Igh-identical donor. T cells cultured with antigen alone or with syngeneic macrophages and antigen did not develop suppressive activity. Lytic treatment of the nylon-adherent population with a B cell-specific monoclonal antibody (J11d) removed the ability to generate suppressor cells. These results imply that the induction of suppressor T cells requires B lymphocytes, and that this induction process is dependent on Igh-linked gene products.     

Binding of antigen-specific, H-2-restricted T cell hybridomas to antigen-pulsed adherent cell monolayers

Two methods were investigated for studying the binding of radiolabeled hybridoma T cells to antigen (Ag) and H-2 products for which they bore receptors. In both cases hybridoma T cells were labeled with tritiated thymidine. In one method labeled cells were added to adherent splenic cells prepulsed with antigen, and the mixture was incubated overnight at 37 degrees C before nonadherent cells were gently washed away. The percent of adherent hybridoma T cells was then estimated by harvesting the adherent monolayers and measuring tritium counts bound. In a second method radiolabeled hybridoma T cells were added to adherent antigen-pulsed B cell lymphomas or hybridomas for between 15 min and 1 hr at 37 degrees C before removal of nonadherent cells and harvesting of the adherent monolayers. In both cases binding was both antigen- and I-region specific. In the second case binding was also rapid; significant binding could be measured after 15 min incubation. These techniques were used to study subclones of one of our T cell hybridomas that were thought by a functional assay (interleukin 2 release) to have lost receptors for Ag/H-2. It was found that subclones of the hybridoma that no longer secreted interleukin 2 in response to Ag/H-2, even though they continued to secrete interleukin 2 in response to concanavalin A, also no longer bound specifically to Ag-pulsed monolayers of the appropriate H-2 type. This confirmed the idea that these subclones had lost the ability to synthesize receptors for Ag/H-2. It is hoped that assays of this type will be useful in the future for the study of Ag/H-2 receptors on T cells.     

Mechanism controlling the genetic restrictions of an NP-specific suppressor factor that inhibits B cell responses

The mechanism of B cell suppression by a T cell hybridoma-derived monoclonal effector suppressor factor (TsF3) was studied in the 4-hydroxy-3-nitrophenyl acetyl (NP) system. The NP-specific effector suppressor cells that produce TsF3 are Lyt-1-, 2+, I-J+, NP-binding T cells and are induced by immunization with NP conjugates. Monoclonal TsF3 inhibits both T cell activity as measured by suppression of contact sensitivity responses and B cell function as measured by suppression of antibody production to both T-independent and T-dependent antigens. The present studies were designed to specifically investigate the mechanisms and genetic restrictions that govern the interactions between TsF3 and its target cells in the plaque-forming cell (PFC) response. The results show that the target of TsF3 is a splenic adherent cell. Suppression will occur only if the restriction specificity of the TsF3 matches the H-2 genotype of the adherent population. Once this TsF3-adherent cell interaction has occurred, suppression of NP-specific B cells can occur across an H-2 barrier. The data also demonstrate that Igh-linked gene products do not appear to play a part in the TsF3-mediated suppression of in vitro PFC responses, which contrasts with the requirements for regulation of T cell-mediated contact sensitivity responses.     

Antibodies to adhesion molecules inhibit the lytic function of MHC-unrestricted cytotoxic cells by preventing their activation.

We evaluated the effect of the antibodies to adhesion molecules CD2, CD11a/CD18 (LFA-1), and CD56 (N-CAM) on MHC-unrestricted cytotoxicity mediated by polyclonal NK cells and LAK cells or by CD3+ or CD3- cytolytic cell clones against a panel of tumor cell targets selected according to expression or absence of the corresponding ligands. We show that (i) antibodies to CD11a/CD18 and, to a lesser extent, antibodies to CD2 inhibit target cell lysis, whereas anti-CD56 antibodies exert little if any effect; (ii) in a model system using polyclonal NK/LAK cells as effectors and K562 or HL60-R (NK-resistant) cells as targets, inhibition of cytotoxicity occurs without a significant impairment of effector to target cell binding; (iii) the cytotoxic function of CD3+ or CD3- cytotoxic cell clones is inhibited differentially by antibodies to adhesion molecules; (iv) conjugates formed in the presence of antibodies which inhibit target cell lysis display a significant reduction of target to effector cell contact surface; and (v) this may lead to defective activation of effector cells, as indicated by lack of redistribution of the microtubular apparatus. We conclude that (i) MHC-unrestricted cytotoxicity is regulated by a number of molecular interactions that span far beyond our present knowledge and that it is strictly dependent on the surface phenotype of the effector cell and of the target cell; (ii) in certain types of effector/target cell interactions, antibodies to adhesion molecules do not prevent conjugate formation but reduce the extent of cell-to-cell surface contact which, in turn, leads to defective activation of the effector cell and, therefore, to inhibition of target cell lysis.     

Beta-chain broadens range of CD8 recognition for MHC class I molecule.

It is known that the alpha-chain of CD8 binds to a negatively charged loop composed of residues 223 to 229 on MHC class I Ag and that binding of CD8 alpha enhances Ag recognition of T cells. We have recently shown that the mouse CD8 alpha homodimer does not bind to either the HLA class I alpha 3 domain or a mutant of H-2Kb Ag containing a substitution of glutamine for methionine at residue 224, which brings this residue toward the human consensus. Here we report a complementary study of the CD8 beta-chain. The functional role of the CD8 beta-chain was analyzed by using four T cell hybridoma lines expressing mouse CD8 alpha and transfected with the mouse CD8 beta gene. As compared with the lines expressing only CD8 alpha, allorecognition of the chimeric H-2Kb Ag that contains the HLA class I alpha 3 domain was enhanced in lines expressing both CD8 alpha and -beta. This enhancement was blocked by either anti-CD8 mAb or anti-HLA class I alpha 3 domain mAb. In addition, we show that CD8 alpha beta binds the H-2Kb mutant Ag at residue 224. These results suggest that the beta-chain allows the CD8 alpha beta heterodimer to recognize the chimeric H-2Kb Ag. A model for the role of the beta-chain is presented.     

DNP-specific/class I MHC-restricted suppressor molecules bear determinants of the T cell receptor alpha- and beta-chains. The V beta 8+ chain dictates restriction to either K or D.

To examine in greater detail the relationship between DNP-specific/class I MHC-restricted suppressor molecules (SSF) that inhibit contact sensitivity to 2,4-dinitrofluorobenzene and the receptors on the T cells that produce them, we have generated two T cell hybridomas that can be induced to produce and secrete these molecules. In order to become activated to produce SSF, the Ts 15.15 and 15.31 cells required recognition of complexes of DNP/Dd on presenting cells. The suppressor molecules produced by each of the Ts hybrids had the same specificity, recognizing DNP/Dd on cells in the immune lymph node cell target population. The activation of the Ts hybrids was blocked when the cells were treated with the anti-V beta 8 antibody F23.1 before coculture with the DNP-presenting cells. Reduction of the 15.15 and 15.31 SSF followed by affinity chromatography on DNP-bovine-gamma-globulin-Sepharose beads indicated that these molecules are dimers and that one of the chains (Ag-binding(AgB] binds to cellfree DNP and one (non-Ag-binding (NAgB) chain) does not. The AgB chain was found to express an epitope bound by a mAb specific for a TCR alpha-chain-constant region determinant. Alternatively, the NAgB chain expressed an epitope bound by the anti-V beta 8 mAb F23.1. Active hybrid suppressor molecules were generated by combining the NAgB chain from a DNP-specific/H-2Kd-restricted SSF (produced by Ts hybridoma 3-10) with the AgB chain from Ts 15.31 and by combining the NAgB chain from Ts cell 15.15 with the 3-10 AgB chain. In each case, the class I MHC element (i.e., Kd or Dd) restricting the activity of these hybrid SSF correlated with the source of the V beta 8+, NAgB chain. Thus, these secreted immunoregulatory molecules have the Ag/MHC specificity of the T cells producing them and are structurally and serologically related to the TCR-alpha/beta. The results further suggest that for some hapten-specific/class I MHC-restricted TCR, the alpha-chain may have avidity for the hapten and the beta-chain may dictate the MHC restriction element (K or D) recognized by the receptor.