The fine specificity of antigen and Ia determinant recognition by T cell hybridoma clones specific for pigeon cytochrome c

The activation of proliferative T lymphocytes normally involves the simultaneous recognition of a particular foreign antigen and a particular Ia molecule on the surface of antigen-presenting cells, the phenomenon of major histocompatibility complex (MHC) restriction. An analysis of T cell clones specific for pigeon cytochrome c, from B10.A and B10.S(9R) strains of mice, revealed the unusual finding that several of the clones could respond to antigen in association with Ia molecules from either strain. Using these cross-reactive clones, we performed experiments which demonstrated that both the Ia molecule and the T cell receptor contribute to the specificity of antigen recognition; however, MHC-linked low responsiveness to tuna cytochrome c (an immune response gene defect) could not be attributed solely to the efficacy with which the Ia molecules associated with the antigen. These results imply that antigen and Ia molecules are not recognized independently, but must interact at least during the process of T cell activation.     

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

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

Proteins synthesized by inducer T cells: evidence for a mitogenic peptide shared by inducer molecules that stimulate different cell types

Inducer T lymphocytes synthesize and secrete peptides that stimulate growth and differentiation of many cell types, including lymphocytes and monocytes that kill foreign organisms, B lymphocytes, mast cells and hematopoietic precursor cells. To define these inducer molecules more precisely, we have generated clones of these T cells as a source of homogeneous material for biochemical analysis. These clones synthesize peptides that stimulate T and B cells to divide and that also induce the latter cells to secrete immunoglobulin. Inducer cells synthesize a 14 kilodalton growth polypeptide that stimulates T and B lymphocytes, as well as other cell types, to divide. This 14 kilodalton peptide is normally associated with different, larger peptides that appear to focus its mitogenic activity to one or another target cell.     

Fine specificity of antigen recognition by T cell hybridoma clones specific for poly-18: a synthetic polypeptide antigen of defined sequence and conformation

Antigen-specific T cell blasts to poly-18, a polypeptide antigen of defined sequence and conformation, were generated from lymph nodes of antigen-primed BALB/cCr mice. These blasts were fused with the BW5147 thymoma to obtain anti-poly-18-reactive T cell hybridomas. All of the hybridomas were IAd-restricted and secreted IL2 in the presence of IAd/poly-18. On the basis of fine specificity analysis, these hybridomas were classified into two groups. Group A hybridomas recognized a minimal peptide sequence of Glu-Tyr-Lys-(Glu-Tyr-Ala)3-Glu-Tyr-Lys, whereas Group B needed the sequence Glu-Tyr-Ala-(Glu-Tyr-Ala)3-Glu-Tyr-Lys/Ala for activation. Three critical residues were identified in Group A hybridomas: the alanine residue at position 9, the carboxy terminal lysine, and the lysine at position 3. In Group B hybridomas, the alanine at position 3 was found to be the critical residue. We suggest that the amino acid residue at position 3 (lysine/alanine) is the T cell receptor contact residue on the poly-18 antigen in BALB/cCr mice.     

T cell clones specific for IgG2a of the a allotype: direct evidence for presentation of endogenous antigen

T cell clones specific for IgG2a of the alpha allotype have been isolated from C57BL/6J mice. Antigenic determinants recognized by these clones were localized by using a panel of hybrid IgG2b-IgG2a myeloma proteins. These experiments provide evidence for two distinct antigenic sites, one located in a segment encompassing the hinge region and most of the CH2 domain, and the other in a segment spanning the CH3 domain and the C-terminal eight residues of the CH2 domain. As judged by their failure to respond in the presence of B6.C-H-2bm12 spleen cells, all the clones recognize determinants created, in part, by the I-A beta chain. A strong proliferative response was observed in the presence of spleen cells from several H-2b strains, including C3H.SW, A.BY, D1.LP, and BALB.B. Experiments testing reactivity directed toward spleen cells from appropriate allotype-congenic mouse strains demonstrated that this response was controlled by Igh-linked genes. These results clearly indicated 1) that Igh-1a-specific T cell clones are stimulated by endogenously synthesized IgG2a, and 2) these T cells recognize shared determinants expressed on IgG2a molecules of various strains. These experiments thus provide strong evidence for presentation of self antigens under normal physiological conditions.     

Subpopulations of the T4+ inducer T cell subset in man: evidence for an amplifier population preferentially expressing Ia antigen upon activation

Prior studies demonstrated that Ia molecules were expressed on a fraction of human peripheral T4+ inducer cells upon stimulation by soluble antigen. In the present study, we utilized a fluorescence-activated cell sorter to separate antigen-activated T4+,Ia+ and T4+,Ia- populations and characterized their function. It was found that the T4+,Ia+ population contained the majority of proliferating T cells as assessed by tritiated thymidine incorporation. This proliferation largely appeared to be nonspecific. Despite macrophage repletion, elimination of the Ia+ subset of T cells with monoclonal anti-Ia antibody and complement treatment equally diminished subsequent proliferation to both the triggering antigen and an unrelated antigen. Moreover, the antigen-induced Ia+ subset of T cells alone produced a nonspecific helper factor, LMF. In contrast, the the T4+,Ia- population showed minimal proliferation to soluble antigen and did not generate LMF. Nevertheless, both T4+,Ia+ and T4+,Ia- inducer T cells were required to generate maximal immunoglobulin production by B cells in an antigen-driven system. We conclude that the human T4+ inducer T cell subset is comprised of at least 2 functionally distinct subpopulations, which are capable of acting in a synergistic fashion to provide help to B cells.     

Role of I-region gene products in T cell activation. I. Stimulation of T lymphocyte proliferative responses by subcellular membrane preparations containing Ia alloantigens

A model system has been developed for exploring the requirements for activation of T cells by subcellular forms of Ia alloantigen. Lymph node cells from mice recently primed subcutaneously with viable allogeneic cells show strong proliferative responses in vitro to membrane preparations derived from cells bearing the appropriate I-region-encoded glycoproteins. This stimulation shows kinetics characteristic of a secondary response, with a peak at 24 to 48 hr. Primary responses to alloantigen-bearing membranes are weak or absent under these conditions. The predominant cell type involved in the secondary response is the Lyt-1+ T lymphocyte, and the major antigenic stimulus is the I-A subregion-encoded Ia glycoprotein. Syngeneic Ia+ accessory cells do not appear necessary for activation to occur. Detergent solubilized reconstituted membrane vesicles also will stimulate primed T lymphocytes to respond by proliferation. The applications of this approach to the study of T cell recognition of antigen and the role of nonspecific lymphokines in T cell triggering are discussed.     

Ia antigen: a murine B lymphocyte receptor for transformed cell induction of interferon-alpha/beta

A receptor on the surface of nonsensitized mouse spleen cells that recognizes a glycoprotein from transformed mouse L-929 cells is described. The interaction of the receptor and glycoprotein inducer results in the production of MoIFN alpha/beta. An assay was developed to assess certain biologic and physicochemical characteristics of the receptor. The receptor and glycoprotein inducer bound in a concentration-dependent manner, which tends to indicate a direct interaction between the two. The receptor was not ubiquitous; spleen cells but not normal mouse embryo cells appeared to be the source. It was specific for MoIFN alpha/beta inducers from transformed cells, but not from other MoIFN alpha/beta or gamma inducers such as NDV, LPS, PWM, or SEA. The receptor appeared to be a cell surface protein in that its activity was abolished by trypsinization of whole spleen cells. Previous studies indicated that the receptor was probably located on B cells. Gel filtration indicated that the receptor had a m.w. of 30,000 to 60,000. Because the receptor appeared to be: 1) B lymphocyte associated, 2) a surface protein, and 3) 30,000 to 60,000 daltons, a similarity to Ia antigen was suggested. This possibility was confirmed by showing binding of the receptor to an anti-IaK antibody-Sepharose affinity column. PAGE analysis of the affinity-purified receptor revealed a single protein band with a m.w. of approximately 60,000. ELISA of the above gel slices with anti-Ia antibody further confirmed the specificity of the column. A physical association of the receptor and inducer was demonstrated by showing binding of the glycoprotein inducer to a receptor (Ia antigen)-Sepharose affinity column. Furthermore, the receptor (Ia antigen) was highly purified by a glycoprotein inducer-Sepharose affinity column.     

Regulation of macrophage Ia expression in mice with severe combined immunodeficiency: induction of Ia expression by a T cell-independent mechanism

We have studied the expression of Ia molecules by macrophages from mice with severe combined immunodeficiency (CB-17 scid) that lack demonstrable T cell and B cell functions. CB-17 scid mice had approximately normal numbers of Ia-bearing macrophages in the peritoneal cavity, spleen, and liver. Peritoneal macrophages responded in culture to T cell-derived lymphokines with enhanced expression of Ia molecules. However, unlike immunocompetent controls, SCID mice could not enhance Ia expression in an antigen-specific T cell-dependent manner after secondary challenge in vivo with a conventional protein antigen such as hemocyanin. Further demonstration of their T cell deficiency was the failure of CB-17 scid spleen cells to proliferate and produce IL 2 in response to the T cell mitogen, concanavalin A. Upon infection with Listeria monocytogenes, CB-17 scid mice developed chronically high loads of bacteria, whereas CB-17 control mice eliminated all viable bacteria and became resistant to secondary infection. However, Listeria-infected CB-17 scid mice did show, in parallel with the CB-17 controls, an unexpected and striking increase of Ia-positive macrophages. These data indicate that induction of Ia expression in macrophages can occur via a mechanism that is independent of mature T cells.     

Interaction of charged lipid vesicles with planar bilayer lipid membranes: Detection by antibiotic membrane probes

A technique has been developed for monitoring the interaction of charged phospholipid vesicles with planar bilayer lipid membranes (BLM) by use of the antibiotics Valinomycin, Nonactin, and Monazomycin as surface-charge probes. Anionic phosphatidylserine vesicles, when added to one aqueous compartment of a BLM, are shown to impart negative surface charge to zwitterionic phosphatidylocholine and phosphatidylethanolamine bilayers. The surface charge is distributed asymmertically, mainly on the vesicular side of the BLM, and is not removed by exchange of the vesicular aqueous solution. Possible mechanisms for the vesicle-BLM interactions are discussed.     

Distinct proliferative T cell clonotypes are generated in response to a murine retrovirus-induced syngeneic T cell leukemia: viral gp70 antigen-specific MT4+ clones and Lyt-2+ cytolytic clones which recognize a tumor-specific cell surface antigen

After immunization of B6 mice with the syngeneic retrovirus-induced T cell leukemia/lymphoma FBL-3, two major tumor-specific proliferative T cell clonotypes were derived. T cell clones derived from long-term lines propagated by in vitro culture with irradiated tumor cells and syngeneic spleen cells were exclusively of the Lyt-2+ phenotype. Such clones were cytolytic, retained their proliferative phenotype indefinitely when expanded by repeated cycles of reactivation and rest, and recognized a tumor-specific cell surface antigen in association with class I MHC molecules. This tumor cell antigen was not present on nontransformed virus-infected cells. Class II MHC-restricted MT4+ clones specific for the viral antigen gp70 were derived from lymph node T cells of FBL-3 tumor-immune mice only by in vitro culture with purified Friend virus in the presence of syngeneic splenic APC. Once derived, however, such clones could be stimulated in the presence of FBL-3 tumor cells and syngeneic spleen cells, demonstrating the reprocessing of tumor-derived gp70 antigen by APC in the spleen cell population. In contrast, no reprocessing of the tumor cell surface antigen by splenic APC for presentation to the class I MHC-restricted T cell clones could be demonstrated. Evidence is presented that FBL-3 T leukemia/lymphoma cells function as APC for Lyt-2+ class I MHC-restricted clones, and that no concomitant recognition of Ia molecules is required to activate these clones. Both Lyt-2+ and MT4+ clones were induced to proliferate in the presence of exogenous IL2 alone, but this stimulus failed to result in significant release of immune interferon. In contrast, antigen stimulation of both clones resulted in proliferation as well as significant immune interferon release. Immune interferon production is not required for the generation of MHC-restricted cell-mediated cytolytic function.     

Fine specificity of murine class II-restricted T cell clones for synthetic peptides of influenza virus hemagglutinin. Heterogeneity of antigen interaction with the T cell and the Ia molecule

We have previously demonstrated diversity in the specificity of murine, H-2k class II-restricted, T cell clones for the hemagglutinin (HA) molecule of H3N2 influenza viruses and have mapped two T cell determinants, defined by synthetic peptides, to residues 48-68 and 118-138 of HA1. In this study we examine the nature of the determinant recognized by six distinct P48-68-specific T cell clones by using a panel of truncated synthetic peptides and substituted peptide analogs. From the peptides tested, the shortest recognized were the decapeptides, P53-62 and P54-63, which suggests that the determinant was formed from the 9 amino acids within the sequence 54-62. Asn54 was critical for recognition since P49-68 (54S) was not recognized by the T cell clones. Furthermore this peptide analog was capable of competing with P48-68 for Ag presentation, thereby suggesting that residue 54 is not involved in Ia interaction and may therefore be important for TCR interaction. Residue substitutions at position 63 also affected T cell recognition, but in a more heterogeneous fashion. Peptide analogs or mutant viruses with a single amino acid substitution at position 63 (Asp to Asn or Tyr) reduced the responses of the T cell clones to variable extents, suggesting that Asp63 may form part of overlapping T cell determinants. However since the truncated peptide P53-62 was weakly recognized, then Asp63 may not form part of the TCR or Ia interaction site, but may affect recognition through a steric or charge effect when substituted by Asn or Tyr. Ag competition experiments with the two unrelated HA peptides, P48-68 and P118-138, recognized by distinct T cell clones in the context of the same restriction element (I-Ak), showed that the peptides did not compete for Ag presentation to the relevant T cell clones, whereas a structural analog of P48-68 was a potent inhibitor. This finding is discussed in relation to the nature of the binding site for peptide Ag on the class II molecule.     

The role of three distinct Ia-like antigen molecules in human T cell proliferative responses: effect of monoclonal anti-Ia-like antibodies

Murine monoclonal antibodies (MoAb) to three distinct Ia-like molecules were studied for their inhibitory effects on antigen- and alloantigen-induced T cell proliferations. The MoAb were classified into three groups according to the molecules they recognized. Both the group I MoAb reacting with DR molecules and the group III MoAb were capable of inhibiting T cell proliferative responses to PPD- and HSV-Ag-pulsed APC, autologous B-LCL, and alloantigens. On th other hand, the group II MoAb, which reacted with a determinant on the molecule carrying MB1 determinants, was only capable of inhibiting T cell responses to alloantigens. These results suggest that the structure of the molecules correlates with the functional repertoire of the human Ia-like antigens.     

Contribution of antigen processing to the recognition of a synthetic peptide antigen by specific T cell hybridomas

Most antigens recognized by T cells require unfolding or partial degradation (processing) followed by association with Major Histocompatibility Complex (MHC) molecules. We examined the processing requirements for the presentation of antigen to two T cell hybridomas which recognize the alpha-helical synthetic polypeptide antigen Poly 18, Poly [EYK(EYA)5], in association with I-Ad. Hybridoma A.1.1 responds to EYK(EYA)4 as the minimum antigenic sequence while hybridoma B.1.1 recognizes (EYA)5 sequence. It was found that these hybridomas responded to Poly 18 and to minimum peptide sequences presented by glutaraldehyde and chloroquine treated antigen presenting cells (APC), suggesting that antigen processing is not a requirement for the activation of these cells. The reactivity pattern of hybridoma B.1.1 in the presence of glutaraldehyde fixed APC revealed that antigens containing lysine were presented with much less efficiency than antigens without lysine, suggesting an interaction of these residues with the antigen presenting cell surface. We discuss the possibility that alanine residues in the alpha-helical Poly 18 form a hydrophobic ridge which may be required for appropriate interaction between antigen, the T cell receptor, and MHC molecules.     

IL 1 induction by murine T cell clones: detection of an IL 1-inducing lymphokine

T cell activation is widely believed to depend on interleukin 1 (IL 1) provided by antigen (Ag)-presenting cells (APC). Because IL 1 is not a constitutive product of APC, we examined the features of its production during the interaction of murine T cell clones and APC. We observed that IL 1 was detectable in supernatants of most myoglobin-specific T cell clones grown with APC and Ag. Two of these T cell clones induced exceptionally high levels of IL 1 in their supernatants, and these same clones demonstrated the unusual restriction to I-Ek, which is a low responder type for sperm whale myoglobin. One of these clones was characterized additionally as to the mechanism of IL 1 induction. This clone rapidly stimulated IL 1 production in the APC population (detectable at 4 hr of co-culture) or in macrophages (M phi) or a M phi-like cell line. IL 1 induction was Ag dependent and H-2 restricted. Induction was radioresistant, both on the part of the T cell and of the IL 1 producer. The IL 1-induction process was attributable to a lymphokine produced by the T cell clone. This lymphokine was distinct from IFN-gamma, TNF and CSF-1 and may account for a principal mechanism of T----APC signalling. The induced IL 1 was the same in size, co-mitogenicity, and pyrogenicity as lipopolysaccharide-induced IL 1.     

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

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

Recognition of soluble Theileria parva antigen by bovine helper T cell clones: characterization and partial purification of the antigen

Theileria parva-specific bovine BoT4+ Th cell clones were used to characterize Ag associated with T. parva schizont-infected lymphoblastoid cells. All of the clones tested responded to cells infected with the immunizing (Muguga) as well as heterologous stocks of T. parva, indicating that the T cells are specific for an Ag shared by several geographically diverse parasites. The response was apparently MHC-restricted, and induced by Ag expressed on the infected cell surface. In the presence of autologous APC, the clones were also stimulated by a soluble high speed supernatant (HSS), but not by a schizont membrane-enriched, subcellular fraction prepared from homogenates of infected cells. The clones produced IFN-gamma and T cell growth factor in response to HSS. The soluble Ag was absent in cells from which schizonts had been eliminated by treatment with the anti-theilerial drug, parvaquone. Fractionation of HSS by hydroxylapatite chromatography revealed two antigenic peaks that separated from the majority of the protein. Fractionation of HSS by gel filtration with the use of HPLC revealed several peaks of activity ranging in Mr from 270 kDa to less than 5 kDa. Further fractionation of HSS by both hydroxylapatite chromatography and gel filtration yielded three major peaks of activity (Mr 43, 12, 4.2 kDa). We conclude that a T cell-dependent schizont-associated soluble Ag is also expressed on the surface of T. parva-infected cells.     

The mouse T cell receptor: structural heterogeneity of molecules of normal T cells defined by xenoantiserum

We have previously demonstrated that T lymphomas may express clonally specific epitopes that are carried by a T-cell-restricted, disulfide-bonded heterodimeric glycoprotein. We have used a monoclonal antibody, 124-40, to isolate the lymphoma-specific antigen and raise a xenoantiserum to the molecule. This antiserum immunoprecipitates a family of disulfide-bonded dimers from normal thymocytes and T cells, but is unreactive with B cells. Peptide maps prepared after limited proteolytic digestion indicate that the molecules from the different cell populations have homologous primary structures. Comparison of two-dimensional tryptic peptide maps indicate that, in addition to several common peptides, the molecules exhibit considerable structural heterogeneity. Taken together, these data indicate that the T-cell-specific heteroduplex has regions of constant and variable structure consistent with the properties expected for the T cell antigen receptor.     

Monoclonal antibodies specific for Ia glycoproteins raised by immunization with activated T cells: possible role of T cellbound Ia antigens as targets of immunoregulatory T cells

Two monoclonal antibodies, Y-3P and Y-8P, specific for conventional mouse Ia glycoprotein antigens are described. Both were raised by repeated immunization of primed mice with activated T cells over a period of 1 yr, and both detect a new and broad public Ia specificity. Both of the antibodies react with I-A subregion-controlled A alpha:A beta complexes of all mouse strains apart from the responder strain (H-2d), as well as the equivalent of A alpha:A beta complexes in rats carrying seven independent haplotypes. These antibodies have great utility as almost universal Ia reagents. On the basis of these results, we propose that Ia antigens presented to the immune system bound to activated T cells are immunogenic, and may induce the production of Ia antibodies of novel and broad specificity. In addition, we propose that such bound Ia glycoproteins could be a target for immunoregulatory T cells, and could account for the specificity of suppression of graft-vs-host reactions and Ia-restricted helper T cells observed by others in F1 animals injected with parental T cells.     

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

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