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.     

Antigen-specific and antibody-mediated growth inhibition of suppressor T cell hybridomas. Roles of H-2 and the CD3-T cell receptor-alpha/beta complex

Eight different Ts cell hybridomas (including inducer (Ts1) and effector (Ts3) suppressor cells) specific for the 4-hydroxy-3-nitrophenyl acetyl (NP) hapten were tested for their ability to respond to Ag or anti-CD3 antibody in a growth-inhibition assay. Results suggest that the expression of the TCR-CD3 complex on Ts hybridomas is required for the Ag or anti-CD3-mediated growth inhibition. One of the CD3+, Ts hybridomas (CKB-Ts3-9.H3) was tested in detail; this CD4- effector suppressor cell hybridoma showed specific inhibition of growth in the presence of NP or NIP-coupled protein conjugates but not in the presence of other irrelevant hapten-protein conjugates. In addition, growth of this hybridoma was specifically inhibited by anti-CD3 and anti-TCR-alpha/beta antibodies but not by control hamster antibodies. In order to study the role of MHC molecules in Ag-mediated growth inhibition, Ts cell hybridomas were incubated with Ag (NP-keyhole limpet hemocyanin) in the presence of spleen cells from various H-2 congenic strains. The results suggest that the Ts hybridomas that express donor Ts-derived TCR beta-chain recognize Ag in an MHC-restricted manner, whereas the two Ts3 hybridomas that utilize BW5147-derived TCR-beta recognize Ag in H-2 unrestricted way. Co-incubation of anti-CD3 and anti-TCR-alpha/beta antibodies with specific Ag enhanced the Ag-mediated growth inhibition, whereas anti-LFA-1 antibody completely blocked the Ag-mediated effect. The combined data suggest that, like Th hybridomas, expression of CD3-associated-TCR complex is essential for the Ag responsiveness of Ts cell hybridomas.     

An IL 2-secreting T cell hybridoma that responds to a self class I histocompatibility antigen in the H-2D region

A self-reactive T cell hybridoma that secretes IL-2 in response to H-2d haplotype cells resulted from a fusion of BALB/cBy lymph node cells with the AKR thymoma BW5147. The lymph node cells used had been enriched for cells reactive to (TG)-A--L, but neither this antigen nor fetal calf serum were required for stimulation of the hybridoma designated 3DT52.5. The gene product responsible for stimulation mapped to the H-2D region. Allogeneic cells of the b, f, k, q, and s haplotypes failed to stimulate. Not all H-2d haplotype cells were effective stimulators of 3DT52.5. Peritoneal cells and splenic B cells were much more stimulatory than splenic T cells. Most tumor cell lines of H-2d derivation and of B cell or macrophage/monocyte lineage were stimulatory, whereas H-2d T cell lines were not. The capacity to stimulate 3DT52.5 did not correlate with the ability to stimulate I region-restricted hybridomas, or with the ability to be induced to stimulate such hybridomas. Stimulatory cell lines did not apparently produce a soluble factor required for stimulation, and negative cell lines were not inhibitory. The monoclonal antibody 27-11-13, which reacts with H-2D of the b, d, and q haplotypes, inhibited stimulation of 3DT52.5 but did not inhibit stimulation of the sibling hybridoma 3DT18.11, which responds to (TG)-A--L plus I-Ad. Conversely, the monoclonal anti-I-Ad antibody MK-D6 inhibited stimulation of 3DT18.11 but not 3DT52.5. Although it is clear that 3DT52.5 recognizes a class I antigen coded for in the H-2D region, the precise molecular nature of the antigen is unknown. The structure of the antigen receptor on this hybridoma may prove to be of interest when it can be compared with receptors found on T cell hybridomas restricted by class II histocompatibility antigens.     

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.     

Mechanism of action of Cyclosporin A: inhibition of lymphokine secretion studied with antigen-stimulated T cell hybridomas

We have employed bifunctional T cell hybridomas, which can be stimulated to secrete lymphokine(s) and lyse specific target cells, to analyze the effect of Cyclosporin A (CsA) on T cell helper and effector functions. We report here the effects of CsA on antigen- and lectin-induced lymphokine secretion. We have found that a pharmacologic level of CsA (10 ng/ml) blocks antigen- and lectin-driven interleukin 2 (IL 2) secretion without affecting cell proliferation. In addition, one monoclonal hybridoma that is induced by concanavalin A to secrete colony stimulating factors (CSF) as well as IL 2 is concomitantly blocked by CsA for production of IL 2 and CSF. Because the hybridomas grow constitutively and are devoid of functional IL 2 receptors, they permit analysis of the kinetics of the inhibitory response. We have shown that CsA blocks not only stimulation of lymphokine secretion but also ongoing IL 2 production, probably by interfering with the effective interaction of receptor and antigen. Thus, blocking of IL 2 secretion from preactivated cells by CsA occurs by 1 to 2 hr, the time required to stop IL 2 production by removal of Ag/Lectin stimulator. The results are consistent with a mechanism of action of CsA on T cells that involves a direct interference of CsA with binding of Ag to Ag-receptor and results in blocking of induction and active secretion of multiple lymphokines.     

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.     

Two roles for Ia in antigen-specific T cell activation. II. Toward a Velcro model of antigen recognition

It has been previously reported that Ia Ag on APC seems to be involved in Ag-specific T cell activation in at least two different ways: one is to associate with foreign Ag to form a neoantigenic determinant (the Ag-specific Ia function), and the second is to interact with T cells in a non-Ag-specific manner. Both Ia functions are required for T cell activation. In the present study we examined whether the T cell structures responsible for the non-Ag-specific Ia interaction were separable from the Ag-specific alpha/beta TCR. Purified protein derivative of tuberculin (PPD)-specific murine hybridoma T cells and polyclonal lymph node T cells were stimulated for IL-2 production by APC pulsed with PPD, glutaraldehyde fixed, and anti-Ia antibody treated, to provide the antigenic PPD/Ia determinant, in the presence of glutaraldehyde-fixed non-Ag-pulsed APC, to provide the non-Ag-specific Ia interactions. However, in several different approaches the T cell structures or activation signals responsible for the Ag-specific recognition and non-Ag-specific Ia interactions seemed to be associated with each other in this experimental system. First, the Ag-specific and non-Ag-specific Ia interactions with T cells were both required simultaneously to initiate T cell activation, and it was not possible to activate T cells by providing either Ia signal subsequent to the other. Second, the T cell structures responsible for the non-Ag-specific Ia interactions appeared to be clonally distributed in PPD-specific lymph node T cells. Third, another T cell hybridoma specific for bovine insulin also showed dual Ia interactions, but the specificity of the non-Ag-specific Ia function was different than that for the PPD-specific T cell response. Fourth, all subclones of PPD-specific T hybridomas that had lost Ag-specific responsiveness also lost functional non-Ag-specific Ia interactions. Taken together, these observations suggest that a single species of TCR may mediate both the Ag-specific and non-Ag-specific Ia interactions. In addition, the non-Ag-specific Ia interaction with T cells augmented the Ag-specific Ia interaction for T cell activation, indicating that both types of interactions may be involved in some T cell responses. Based on these observations, a Velcromodel depicting the synergy between the two Ia functions is proposed in which a matrix of interactions consisting of higher affinity Ag binding and lower affinity Ia-TCR associations provides cooperative sets of signals necessary for cellular activation.     

Production of T-T hybrids from T cell clones. Direct comparison between cloned T cells and T hybridoma cells derived from them

It has been assumed, without direct evidence, that T cell hybridomas and non-transformed T cell clones are both good models of normal Ag-specific T cells. To compare directly the difference in activation of cloned normal T cells and T hybridoma cells with the same TCR, cloned T hybridoma cells were obtained by fusing pre-established, myoglobin-specific, Iad-restricted T cell clones (14.5 and 9.27) with BW5147 cells. T cell clones were pre-activated with IL-2 as well as specific Ag before fusion. Cloned T hybridoma A3.4C6 was derived from Lys 140-specific and I-Ed-restricted clone 14.5. The other cloned T hybridoma, C7R14, was a fusion product of Glu 109-specific and I-Ad-restricted clone 9.27. Both T hybridomas showed the same Ag specificity and Ia restriction as the parental cloned T cells. However, C7R14 showed higher apparent affinity and broader cross-reactivity than 9.27. Clone 14.5, but not hybridoma A3.4C6, appeared to stimulate splenic cells to secrete cytokines inhibiting HT-2A cell proliferation. The most striking difference between the clones and hybridomas was that both clones, but neither of the matched hybridomas, were induced to synthesize IL-1 on stimulation with Ag. Finally, both cloned T cells and T hybridomas killed Ag-pulsed Iad-bearing B lymphoma target cells. This evidence suggests that killing function can be inherited from clones to hybridomas. However, the clones were much more efficient at killing than the hybridomas, and the hybridomas were more efficient at IL-2 production than the clones. Thus, matched pairs of clones and hybridomas differ in their capacity to mediate the two functions or may tend to be selected differently during cloning. Thus, although our results generally support the validity of T cell hybridomas as faithful models of the corresponding T cell clones, a number of subtle and not-so-subtle differences indicate that caution must be used in such an extrapolation.     

Use of I region-restricted, antigen-specific T cell hybridomas to produce idiotypically specific anti-receptor antibodies

Murine T cell hybridomas bearing receptors for antigen plus I region gene products were used as immunogens in mice in an effort to raise anti-receptor antisera. The antisera were assayed for anti-receptor activity by the ability to inhibit interleukin 2 production by the T cell hybridomas stimulated by antigen and I region expressing antigen-presenting cells. The T cell hybridomas used in these experiments were made by fusing antigen-specific, I region-restricted BALB/c T cell blasts to the AKR thymoma, BW5147. Three groups of mice were immunized with the T cell hybridomas: (BALB/c X AKR)F1 animals, syngeneic to the hybridoma; (BALB.B X aKR)F1 animals, differing from the hybridomas at H2; and (C.B20 X AKR)F1 animals, differing from the hybridomas at Igh. Mice were immunized multiple times and sera from individual animals were assayed for anti-receptor antibodies. In all groups, some mice produced anti-receptor antibodies by the criterion that they were inhibitory in the assay mentioned above. The frequency of mice producing these inhibitory antibodies varied considerably between groups, with the (BALB.B X AKR)F1 animals producing these antibodies most frequently, and the (BALB/c X AKR)F1 animals producing them least often. All inhibitory antisera were idiotypically specific; they inhibited the response of the immunizing T cell hybridomas, but not the responses of closely related hybridomas with different specificities. Moreover, when they could be absorbed, the inhibitory antibodies could only be absorbed by the immunizing hybridoma. It is hoped that these antisera, and B cell hybridomas prepared from the immunized animals, will be useful in the elucidation of the structure of the receptors for antigen plus I region products on T cells.     

Structure and expression of Fc gamma receptors on mouse suppressor T cell hybridomas

Antigen-specific and idiotype-specific mouse suppressor T cell hybridomas were analyzed for the presence and specificity of Fc gamma receptors (Fc gamma R) by EA rosetting and by flow microfluorometry with the use of monoclonal antibodies. We found that four hybridomas expressed Fc gamma R specific for IgG1 and IgG2b, one of which became Fc gamma R- during prolonged culture. Four other hybridomas and the fusion parent, BW5147, consistently lacked Fc gamma R. The 125I-labeled Fc gamma R were isolated from surface radioiodinated hybridoma cells solubilized with 1% Nonidet P-40, were purified by using single or repetitive chromatography on mouse IgG-Sepharose columns, and were analyzed by SDS-PAGE. An 125I-labeled 56,000 to 61,000 Mr macromolecule was isolated from each of the Fc gamma R+ hybridomas, but from none of the Fc gamma R- hybridomas nor from BW5147 cells. This macromolecule rebound to insolubilized mouse IgG1, IgG2b, and human Fc fragments, but not to insolubilized mouse IgG2a, IgG3, or IgA or human F(ab')2 fragments, consistent with the specificity observed for Fc gamma R on intact hybridoma cells. The mouse suppressor T cell Fc gamma R differs in size and specificity from mouse B cell Fc gamma R. A 70,000 Mr protein expressed on all hybridomas and on BW5147 cells was radiolabeled and, despite preclearing with ovalbumin-Sepharose, bound to the mouse IgG-Sepharose columns, presumably due to mouse antibodies to gp-70. This macromolecule was completely and specifically removed by using goat antiserum to gp-70.     

Establishment of T-cell hybridoma constitutively producing macrophage-dependent T-cell replacing factor

A T-cell hybridoma was established by the fusion of concanavalin A-stimulated splenic T cells with BW 5147. The hybridoma cells secrete a factor constitutively to support antibody formation of spleen cells depleted of T cells against TNP-Ficoll but not against horse red blood cells. The activity was indicated not to be due to interleukin 2, B-cell growth factor I, B-cell growth factor II, or interferon. The factor-mediated antibody response to TNP-Ficoll required the presence of adherent cells. The adherent cell function could be replaced by the macrophage culture supernatant containing interleukin 1. B cells responding to TNP-Ficoll in the culture with hybridoma factor were indicated to be Lyb 5+ and to bear receptors for third component of complement.     

Regulation of plasminogen receptor expression on human monocytes and monocytoid cell lines

The capacity of human monocytoid cell lines and peripheral blood monocytes to modulate their expression of plasminogen receptors has been assessed. After PMA stimulation, THP-1 or U937 monocytoid cells were separated into adherent and nonadherent populations. Plasminogen bound to adherent cells with similar capacity and affinity as to nonstimulated cells. In contrast, the nonadherent cells bound plasminogen with 5-17-fold higher capacity (without a change in affinity). This increase was selective as urokinase bound with similar affinity and capacity to the adherent and nonadherent populations. Upregulation of plasminogen receptors on the nonadherent monocytoid cells was rapid, detectable within 30 min, and reversible, adhesion of the nonadherent cells resulted in a sixfold decrease in plasminogen binding within 90 min. The increase in plasminogen binding to the nonadherent cells was associated with a marked increase in their capacity to generate plasmin activity from cell-bound plasminogen. PMA stimulation of human peripheral blood monocytes increased their expression of plasminogen receptors by two- to fourfold. This increase was observed in both adherent and nonadherent monocytes. Freshly isolated monocytes maximally bound 5.0 x 10(5) plasminogen molecules per cell, whereas monocytes cultured for 18 h or more maximally bound 1.7 x 10(7) molecules per cell, a 30-fold difference in receptor number. These results indicate that both monocytes and monocytoid cell lines can rapidly and markedly regulate their expression of plasminogen binding sites. As enhanced plasminogen binding is correlated with an increased capacity to generate plasmin, an enzyme with broad substrate recognition, modulation of plasminogen receptors may have profound functional consequences.     

Ia-independent binding of peptide antigen to the T cell receptor

We have investigated the potential for direct interaction between a peptide Ag, human fibrinopeptide B (hFPB), and the TCR on an hFPB-specific murine T hybridoma. Fluoresceinated hFPB binds specifically to hFPB-responsive T cells, but not to unrelated T hybrids. Among variant subclones of the original hybridoma, ability to bind hFPB correlates with hFPB-specific response and expression of the CD3/TCR complex, indicating that hFPB is binding to the TCR. This TCR-hFPB interaction has an affinity of approximately 6.6 microM, reflecting slow association and rapid dissociation of the Ag from its receptor. These findings confirm the potential for direct Ag-TCR interaction and indicate an Ag recognition mechanism that is not initiated by Ag-MHC interaction.     

The role of class II MHC molecules in the activation of class I-reactive T cell hybridomas

To examine the role of Ia molecules in T cell responses to allo-class I major histocompatibility antigens, a series of allo-class I-reactive T cell hybridomas was established. Of 134 T cell hybridomas obtained from the fusion of C3H/HeJm or B10.HTT T cells stimulated with C57BL/6 splenocytes, nine T cell hybridomas were reactive to class I antigens and 126 T cell hybridomas were reactive to class II antigens. Six of the nine IL 2-producing T cell hybridomas were further analyzed: five mapped to H-2Kb and the other mapped to H-2Db. Three of these T cell hybridomas, HTB-157.7, HTB-176.10, and HTB-177.2, could react to the EL-4 cell line that expresses H-2Kb and H-2Db class I antigens but lacks class II I-Ab molecules. Furthermore, the activation of these three T cell hybridomas with C57BL/6-derived splenocytes was not blocked by either anti-I-A or anti-L3T4 antibody. In contrast, the other three T cell hybridomas, CB-127.6, CB-221.7, and HTB-102.7, failed to react with EL-4 but reacted with the LB cell line which expresses class I (H-2Kb, H-2Db) and class II (I-Ab) molecules. Although class II molecules were required for activation of the latter clones, there was no apparent I-A allele specificity, suggesting that a relatively nonpolymorphic Ia determinant was involved. The activation of the three latter T cell hybridoma clones with C57BL/6 splenocytes could be blocked completely by either anti-I-A or anti-L3T4 antibody. The data are interpreted in terms of possible T cell receptor models for recognition of class I with nonpolymorphic class II determinants.     

Functional CD2 mutants unable to bind to, or be stimulated by, LFA-3.

To define epitopes on the CD2 (T11, the T cell erythrocyte receptor) molecule that are necessary for interaction with lymphocyte function-associated Ag-3 (LFA-3), we have expressed the human wild-type CD2 cDNA and mutant CD2 cDNA in a murine Ag-specific T cell hybridoma that responds to human HLA-DR Ag. Here we have expressed mutations at amino acid 91 and 92 of CD2 in the T cell hybridoma. The mutated CD2 molecules were functional in that pairs of anti-CD2 mAb that continued to bind were able to stimulate IL-2 production by the hybridomas. However, CD2 mutants with either the 91 or 92 amino acid substitution had lost the ability to bind to or be activated by either SRBC, which bear an LFA-3 homologue, or by murine L cells expressing human LFA-3. Unlike hybridomas expressing the wild-type CD2 molecule, there was no enhanced response to Ag stimulation. Taken together, these data suggest that the mutated CD2 molecules were no longer able to bind to, or to utilize, LFA-3 for activation. We have previously demonstrated that a mutation at amino acid 51 of CD2 results in loss of binding to LFA-3. Whether these two regions of CD2, discrete and separable by amino acid sequence, form one or more binding sites for LFA-3 remains to be determined.     

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.     

Generation of cytotoxic T lymphocytes from thymocyte precursors to trinitrophenyl-modified self antigens. II. Establishment of a T cell hybrid clone constitutively producing killer-helper factor(s) (KHF) and functional analysis of released KHF

T cell hybridoma lines were constructed by fusion of Mycobacterium tuberculosis-primed and boosted BALB/c T cells with the AKR-derived T lymphoma cell line BW5147. Certain of the hybridomas prepared in this manner secreted constitutively into their culture supernatants biologically active molecules that displayed precursors of cytotoxic T cell activating properties characteristic of killer-helper factor (KHF). Cell surface analysis revealed that the hybridomas were indeed somatic cell hybrids between the two respective partner cells used for fusion. KHF properties of these hybridoma supernatants were verified by their capacity to stimulate peanut agglutinin-binding (PNA+) C3H/He thymocytes to respond in vitro to 2,4,6-trinitrophenyl(TNP)-modified syngeneic stimulator cells in conjunction with suboptimal doses (10 U/ml) of interleukin 2 (IL 2) for the generation of H-2-restricted, TNP-reactive cytotoxic T cells. The biologically active molecules secreted by a T cell hybrid clone (2Y4) were, like conventional KHF, distinct from IL 1, IL 2, or immune interferon (IFN-gamma). The partially purified KHF derived from 2Y4 cells shows activity at apparent m.w. range of 34,000 to 60,000 on gel permeation, and is relatively homogeneous with respect to isoelectric point, which was approximately 4.5 to 4.7. The partially purified 2Y4-KHF is able to augment proliferation of as well as the expression of IL 2 receptors on PNA+ thymocytes in conjunction with IL 2. Finally, addition of 2Y4-KHF on day 0, followed by the addition of IL 2 on day 2 for 7 days of culture was effective in generating potent CTL responses, whereas addition of IL 2 on day 0, followed by the addition of 2Y4-KHF on day 2 to the culture was ineffective.     

Lymphokine production by human T cell hybridomas

Human T cell hybridomas were generated by hybridization of SH9 cells, the 6-thioguanine-resistant variant of human T lymphoma Hut102-B2, with concanavalin A-stimulated human peripheral blood lymphocytes. The hybrid nature of the established cell lines was documented by the difference in D14S1 restriction fragment length polymorphism between SH9 and the hybridoma cell DNA, and by the expression of OKT11 antigen on hybrid cells. T cell growth factor, macrophage growth factor (MGF), and interferon (IFN) activities have been demonstrated in the supernatants of different hybrid cultures, but not in SH9 cell cultures. Substantial quantities of MGF were secreted by several hybrids including the L23 line. MGF activity was dose-dependent, heat-labile, and synergistic with indomethacin. High titers of IFN activity were found in the cultures of hybridoma L415 and its subclones. Neutralization with specific antisera showed the IFN synthesized by L415 clones was immune interferon (IFN-gamma). Like the parental SH9 line, all of the hybridomas producing these lymphokines exhibited a cell surface phenotype typical for helper T cells. The hybridoma system therefore shows potential for the study of various lymphokines produced by human helper T lymphocytes.     

Specific lymphocyte-target cell conjugate formation between tumor-specific helper T-cell hybridomas and IA-bearing RCS tumors and IE-bearing allogeneic cells. I. Role of Ia and both L3T4 and LFA-1 antigens in recognition/binding

The studies reported here describe the feasibility of using single cell techniques with nonadherent target cells for the formation of T helper lymphocyte-target cell conjugates in an Ia recognition system. We have taken advantage of four tumor-specific T cell hybridomas lines, two of which respond only to IA-bearing RCS tumor cells of SJL/J (H-2s) origin, and the other two that respond to both RCS and IA- or IE-bearing allogeneic cells of H-2k,d haplotypes. The conjugate frequency between the T cell hybridomas and target cells was scored microscopically and was facilitated by labeling the lymphocyte with fluorescein. The frequency of conjugate formation ranged from 20 to 40% above background. Conjugate formation was antigen specific and correlated well with the hybridoma specificity determined by IL 2 responses after antigenic stimulation. The cross-reactive hybridomas formed conjugates with RCS and LPS blasts derived from CBA or DBA/2 origin, but not with cells of syngeneic or other allogeneic strains. Conjugate formation with RCS was inhibited greater than 50% with mAb directed against IAs determinants on the RCS tumor cells, and conjugate formation with allogeneic cells was blocked only with mAb directed to either IA/IEk or IA/IEd specificities directed against the alpha or beta polypeptide chain. Blocking of conjugate formation was also achieved by various mAb directed against surface membrane molecules associated with the T cell hybridomas. LFA-1 mAb inhibited significantly the formation of conjugates. However, L3T4 mAb blocked only partially the conjugates. Other antibodies directed against Lyt-1 or Thy-1.2 antigens were without blocking effect. The poor blocking observed with L3T4 mAb did not correlate with the almost complete blocking observed in the IL 2 response by the same hybridomas. These studies of the syngeneic anti-RCS tumor response directed against IA-bearing RCS showed that the conjugate assay permits mapping of tumor-associated Ia epitopes. In addition, the results of these studies demonstrate the feasibility of conjugate formation in determining the antigenic specificity of the T helper system. This assay system can be used to establish the minimal frequency of antigen-reactive cells and can divide the T helper response into multiple steps (i.e., recognition/binding, activation, proliferation, and lymphokine release) and determine the surface membrane molecules involved in recognition.