Expression of CD4 can confer major histocompatibility complex class II-associated superantigen reactivity upon a T cell receptor derived from a CD8-dependent cytotoxic T lymphocyte clone

It has been shown that reactivity against major histocompatibility complex (MHC) class II-associated Mlsa determinants is mainly mediated by CD4+ V beta 6+ T cells. 3F9 is a CD8+ CTL clone which is specific for the alloantigen H-2Db. While 3F9 is V beta 6+, it is not Mlsa reactive, presumably because it does not express CD4. 3F9 utilizes the same T cell receptor (TcR) V alpha V beta combination as LB2, a CD4+ T helper clone specific for chicken red blood cells (cRBC)/I-Ab and yet differs from LB2 in the junctional sequences in both TcR chains. CD4+ CD8- and CD4-CD8- hybridomas expressing the 3F9 TcR were tested for reactivity against Mlsa and cRBC/I-Ab. Only the CD4+CD8- hybridomas were Mlsa reactive, and antibody inhibition studies revealed that this reactivity was both CD4 and MHC class II dependent. Therefore the expression of the CD4 molecule can make an MHC class I-restricted TcR Mlsa reactive. Neither type of hybridoma reacted against cRBC, thus the main difference in the antigen reactivity between 3F9 and LB2 lies in the TcR junctional regions.     

Anti-leucocyte function-associated antigen-1 antibodies inhibit T-cell activation following low-avidity and adhesion-independent interactions.

Anti-leucocyte function-associated antigen-1 (LFA-1) antibodies can provide either stimulatory or inhibitory signals to T cells, depending on the epitope they recognize, type and stage of activation of the T cells, and nature of the activation stimulus. Because of the low affinity of interaction between the T-cell receptor (TcR) and the antigen/major histocompatibility complex (MHC), it was proposed that the LFA-1 molecule strengthens the adhesion between the interacting cells, thus contributing in an additive manner to TcR-specific interactions. To check if high-avidity, TcR-specific interactions still require the accessory function of the adhesion molecule, we studied the effect of anti-LFA-1 antibodies on T-cell triggering mediated through chimeric receptors composed of an Fv of an antibody and a constant region of the TcR. Such chimeric TcR (cTcR) confer on T cells antibody-type specificity and affinity. We made use of transfected T-cell hybridomas expressing various amounts of either one cTcR chain (composed of VH linked to C beta) or double-chain cTcR (VHC beta + VLC alpha). When such transfectants were stimulated with hapten-modified cells, anti-LFA-1 antibodies inhibited activation predominantly mediated through cTcR composed of a single chimeric chain and did not inhibit stimulation of the double-chain transfectants. Moreover, these anti-LFA-1 antibodies blocked antigen-specific T-cell activation regardless of whether the stimulus was adhesion dependent or not, such as in the case of stimulation by immobilized hapten-protein conjugates. These studies show that the 'off-signal' provided by anti-LFA-1 antibodies is adhesion independent and affects mainly low-avidity TcR-antigen interactions.     

Generation of monoclonal antibodies against a human T cell receptor beta chain expressed in transgenic mice.

The generation of a panel of monoclonal antibodies specific for different variable (V) regions of human T cell receptors will be of great importance in the study of T cell-mediated diseases. However, relatively few such reagents exist, due in part to the poor immunogenicity of TcRs on the surface of human T cells. We have employed a strategy in which T cells from a transgenic mouse line expressing a human V beta 3 C beta 1 TcR were used to immunise syngeneic conventional mice to generate two monoclonal antibodies specific for human T cell receptors. Binding of antibody JOVI.3, which stained approximately 5% of human peripheral blood CD3 positive T cells, correlated with the expression of the human TcR V beta 3 gene segment. Antibody JOVI.1 recognised a determinant on the majority of TcRs, staining 50-75% of peripheral blood T cells and T cell lines expressing different V beta regions. Some TcRs, however, failed to react with this antibody. Both antibodies immunoprecipitated detergent-solubilised TcR molecules and were capable of inducing proliferation of peripheral blood T cells.     

Preferential positive selection of V alpha 2+ CD8+ T cells in mouse strains expressing both H-2k and T cell receptor V alpha a haplotypes: determination with a V alpha 2-specific monoclonal antibody.

A monoclonal antibody, B20.1, was generated by fusing spleen cells from a Lou rat immunized with a soluble alpha/beta T cell receptor (TcR; V alpha 2/V beta 2) to mouse myeloma cells. Analysis of a panel of V alpha 2 mRNA-expressing T cell lines, hybridomas and transfectants revealed that the B20.1 antibody was specific for murine TcR V alpha 2 chains. The V alpha 2+ T cell population was examined in various inbred strains by two-color immunofluorescence using B20.1 and CD4- and CD8-specific antibodies with the following results: (a) the B20.1 antibody detected most members of the TcR V alpha 2 subfamily in the four TcR V alpha haplotypes tested; (b) in most strains examined, TcR V alpha 2 expression was biased to the CD4 subset (7.4%-17.4% V alpha 2+ T cells) as compared to the CD8 compartment (3.8%-13.3%); (c) TcR V alpha 2 expression was not influenced by Mls gene products and (d) increased positive selection of V alpha 2+ CD8+ T cells by H-2k major histocompatibility complex molecules occurred in all murine strains tested of the TcR V alpha a, but not in those bearing the TcR V alpha b haplotype.     

Reactivity of mouse T-cell hybridomas expressing human Vbeta gene segments with staphylococcal and streptococcal superantigens.

A panel of 15 mouse T-cell hybridomas, each expressing a different human Vbeta gene segment (hVbeta) in an otherwise mouse T-cell receptor (i.e., mouse alpha chain and CD3 complex), was constructed by transfection of hVbeta/mouse Cbeta chimeric T-cell receptor (TCR)-beta genes into a mouse T-cell hybridoma recipient lacking the endogenous TCR-beta chain. Several qualities that are conferred by the hVbeta chain of the TCR are retained in the chimeric human-mouse TCR complex: a large panel of hVbeta-specific antibodies specifically stained the hVbeta expressed by the mouse T-cell hybridomas. Moreover, hVbeta-transfected mouse cells could readily produce interleukin 2 when stimulated by superantigens presented by antigen-presenting cells. These characteristics made it possible to refine the reactivity of 17 superantigen preparations with the available transfected Vbetas. Each superantigen gave a characteristic pattern of reactivity on the transfectants. Positive reactivities with some of these transfectants, which differ only by the expressed hVbeta, demonstrate unambiguously the superantigenic character of a protein or fraction and its potential to react with the corresponding Vbetas. Therefore, these hVbeta-transfected cells constituted a valuable tool for determining "specificity fingerprints" of known or putative superantigens. First, commonly used, commercially available superantigens such as staphylococcal enterotoxin B and toxic shock syndrome toxin-1 (TSST-1) showed additional Vbeta reactivities, compared with those of their recombinant counterparts. This stresses the importance of using defined preparations of superantigens for the definition of Vbeta specificities. Second, the stimulatory pattern of a strain of Streptococcus pyogenes demonstrated that this strain, unlike others, produces a potent Vbeta 8-specific superantigen that is an yet undefined at the molecular level.     

T Lymphocytes in Human Gut Epithelium Preferentially Express the α/β Antigen Receptor and are often CD45/UCHL1-Positive

A revived interest in intraepithelial lymphocytes (IEL) has been elicited by several recent reports suggesting that murine and avian intestinal epithelium contains mainly CD3+CD8+ cells expressing the gamma/delta T-cell receptor (TcR) for antigen; this contrasts with systemically distributed T cells which preferentially employ the TcR alpha/beta. An anatomical dichotomy in the distribution of these two T-cell lineages has hence been proposed. Here we report that this concept does not hold true in man. In situ studies with monoclonal TcR-framework antibodies showed that most (70-90%) human intestinal IEL (which are mainly CD3+CD8+) expressed TcR alpha/beta. Moreover, almost half of the intraepithelial CD3+ cells were positive for the smallest (180 kDa) CD45 molecule (UCHL1); this probably reflected that they are antigen-primed and thus represent traditional CD3+CD8+ alpha/beta+ memory T cells.     

Membranes of activated CD4+ T cells expressing T cell receptor (TcR) alpha beta or TcR gamma delta induce IgE synthesis by human B cells in the presence of interleukin-4.

In the present study it is demonstrated that human B cells can be induced to switch to IgE production following a contact-mediated signal provided by activated T cell receptor (TcR) gamma delta+, CD4+ and TcR alpha beta+, CD4+ T cell clones and interleukin (IL)-4. The signal provided by these T cell clones was antigen nonspecific, indicating that the TcR alpha beta/CD3 or TcR gamma delta/CD3 complexes were not involved in these T-B cell interactions. Activated TcR alpha beta+, CD8+, and TcR gamma delta+, CD4-CD8-, or resting CD4+ T cell clones were ineffective. Intact TcR alpha beta+ or TcR gamma delta+, CD4+ T cell clones could be replaced by plasma membrane-enriched fractions isolated from these activated CD4+ T cell clones. In contrast, membranes isolated from resting TcR alpha beta+, CD4+, TcR gamma delta+, CD4+ T cell clones or an Epstein-Barr virus (EBV)-transformed B cell line (EBV-LCL) failed to provide the costimulatory signal that, in addition to IL-4, is required for induction of IgE synthesis. As described for intact CD4+ T cells, CD4+ T cell membranes induced purified surface IgM+ B cells to switch to IgG4- and IgE- but not to IgA-producing cells, excluding the possibility of a preferential outgrowth of IgG4- and IgE-committed B cells. The membrane activity was inhibited by protease or heat treatment. Induction of IgE synthesis by B cells co-cultured with both TcR alpha beta+, CD4+ and TcR gamma delta+, CD4+ T cell clones and membrane preparations of these cells was blocked by anti-class II major histocompatibility complex (MHC) monoclonal antibodies (mAb), whereas various anti-CD4 mAb had differential blocking effects. Murine L cells, or EBV-LCL transfected with CD4 could not replace CD4+ T cell clones. These results indicate that, although CD4 and class II MHC antigens are required for productive CD4+ T cell clone-B cell interactions, an additional signal, provided by a membrane associated (glyco)protein that is induced by activation of both TcR alpha beta and TcR gamma delta, CD4+ T cells, is needed for induction of IgE production in the presence of IL-4.     

5株鼠抗人CD28单克隆抗体的研制及生物学特性的研究

目的：制备鼠抗人CD28分子功能性单克隆抗体，研究其对T细胞活化、增殖及信号转导等方面的生物学效应。方法：以天然高表达CD28分子的人多发性骨髓瘤细胞株U266和小鼠淋巴瘤细胞转人CD28基因细胞株CD28-T分别作为免疫原和检测细胞株，采用B淋巴细胞杂交瘤技术进行单抗的研制；以快速定性试纸法鉴定单抗亚类；腹水诱生法和免疫亲和层析法进行单抗的制备和纯化；经间接免疫荧光法分析单抗对不同细胞膜表面CD28分子的识别；采用竞争抑制法分析单抗识别的抗原位点；利用^3H-TdR掺入法分析单抗对PBTC的刺激效应和免疫荧光法分析PBTC活化前后的表型变化。结果：成功获得5株鼠抗人CD28功能性单克隆抗体，分别命名为2D5、2F5、3136、3F8和8G8，其中2D5为IgG2a亚类，其余均为IgG1亚类；流式细胞仪分析结果显示，5株单抗均能良好识别CD28-T、U266、XGI和Jurkat细胞表面的CD28分子；竞争抑制试验表明，2D5和8G8能完全阻断标准抗人CD28单抗与U266膜CD28分子的结合，其余3株为部分阻断；^3H-TdR掺入法实验结果表明，单抗8G8联合激发型CD3单抗能明显促进PBTC的增殖，刺激指数为7．4，活化细胞CD4、CD25、ICOS、4IBB及OX40分子的表达上调。结论：5株单抗均为抗人CD28单克隆抗体，具有重要的基础研究及潜在的临床应用价值。     

A novel surface molecule expressed by long-term cultured T and natural killer cells is involved in cell activation.

Two monoclonal antibodies (mAb), termed ED6 and LD6, were obtained by immunizing mice with cytotoxic T cell lines expressing the T cell receptor (TcR) gamma/delta. These mAb were selected according to their ability to trigger the cytolytic program of the immunizing cell lines in a redirected killing assay. Both mAb recognized molecule(s) expressed on the surface of most long-term cultured TcR gamma/delta +, TcR alpha/beta + and CD3-CD16+ lymphocytes, while it was absent on resting peripheral blood lymphocytes. In addition both mAb reacted with neoplastic B cell lines, Epstein-Barr virus-transformed B cell lines, small cell lung cancer and glioma cell lines, while no surface reactivity was detected on ovarian, breast, colon and non-small cell lung cancer lines. The functional activity of these mAb was studied by two cytolytic assays. Both mAb were able to trigger the cytolytic program of CD3+TcR gamma/delta + polyclonal cell lines and of a CD3-CD16+ NK cell clone against the murine mastocytoma target cell line P815 (Fc receptor+) in a 4-h 51Cr-release assay. In addition, ED6 and LD6 hybridomas were lysed by TcR gamma/delta + effector cells while other hybridomas (obtained from the same fusion) were not lysed. ED6 and LD6 mAb (in the presence of submitogenic doses of the phorbol 12-myristate 13-acetate) also induced the secretion of interleukin 2 by ED6/LD6+ T cell clones expressing TcR gamma/delta or alpha/beta. mAb-induced surface antigen modulation experiments showed that the antigenic determinant recognized by ED6 and LD6 co-modulated, thus indicating that the two mAb probably recognize the same or closely associated molecules. The molecular characteristics of the antigen recognized by the mAb were investigated by Western blot analysis. The LD6 mAb recognized a major band of approximately 65 kDa, both under nonreducing and reducing conditions. These data indicate that ED6 and LD6 mAb recognize a novel non-lineage-specific activation antigen which is involved in the induction of the functional program of long-term cultured T or natural killer cells.     

Differential expression and regulation of the human CD8α and CD8β chains

The CD8 glycoprotein is expressed by thymocytes, mature T cells and natural killer (NK) cells and has been implicated in the recognition of monomorphic determinants on major histocompatibility complex (MHC) Class I antigens, and in signal transduction during the course of T-cell activation. Both human and rodent CD8 antigens are comprised of two distinct polypeptide chains, alpha and beta. The majority of monoclonal antibodies (mAb) reactive with the human CD8 antigen bind the CD8 alpha chain, while a single mAb, T8/2T8-5H7, has been identified which binds to the CD8 alpha/beta heterodimer. While the two chains of CD8 have been presumed to be coordinately expressed in normal T cells, this is not always the case. Northern blot analysis of a panel of T-cell leukemias and normal cells demonstrate that CD8 alpha and CD8 beta are not invariably co-transcribed and phenotypic analysis of fresh and interleukin 2 (IL-2) expanded peripheral blood mononuclear cells (PBMC) confirm that the CD8 alpha and CD8 beta chains are differentially expressed at the cell surface. Four distinct subpopulations of CD8+ cells have been identified based on the expression of CD8 alpha/alpha or CD8 alpha/beta complexes: (1) T-cell receptor (TcR) alpha beta+ T cells which are CD8 alpha+/beta+; (2) TcR alpha beta+ T cells which are CD8 alpha+/beta-; (3) TcR gamma delta+ T cells which are CD8 alpha+/beta- and (4) natural killer (NK) cells which are CD8 alpha+/beta-. We also demonstrate the down-regulation of the CD8 alpha/beta heterodimers from the surface of a CD8+ T-cell clone following treatment with phorbol myristate acetate (PMA) while CD8 alpha/alpha homodimers remain on the cell surface. This observation demonstrates that a) a CD8+ T-cell clone can express both CD8 alpha/alpha homodimers and CD8 alpha/beta heterodimers and b) these two complexes do not have identical biological properties. Together, these data suggest that CD8 alpha/alpha and CD8 alpha/beta dimers may not subserve identical functions. The differential contribution of these two CD8 complexes should be considered in models of T-cell-mediated cytotoxicity and T-cell activation.     

Antibodies to soluble human T cell receptor beta chain recognize multiple epitopes on cell surface TcR.

The T cell receptor (TcR) is an integral membrane protein occurring as a disulfide linked heterodimer, non-covalently associated with CD3 on the surface of T lymphocytes. Antibodies to the TcR have been shown to be effective for treating autoimmune disorders in animals. We describe here a method for producing antibodies to cell surface determinants of the human TcR, using a soluble form of the receptor as antigen. Soluble V alpha 1.2, V beta 8.1, V beta 11 TcR chains are expressed from a construct in which the extracellular domains of the TcR are fused to the mouse gamma 2a heavy chain constant region lacking the CH1 domain. These chimeric molecules contain both immunoglobulin and TcR determinants, as revealed by antibody probes. Amino-terminal sequence analysis of a chimeric V beta 8.1 molecule indicates that the TcR leader peptide is correctly processed from the soluble form. Antibodies raised against the soluble human V beta 8.1 molecule recognize the native determinants on Jurkat cells, and on natural T cells derived from resting human peripheral blood lymphocytes. Epitope mapping studies using competitive binding assays suggest that the anti-V beta 8 antibodies produced using soluble antigen recognize multiple overlapping determinants on the cell surface form of the TcR.     

鼠抗人CD28分子单克隆抗体的研制及生物学特性研究

目的 制备鼠抗人CD28分子的单克隆抗体（mAb),研究其在T细胞的活化、增殖及信号传导中的作用。方法 以小鼠淋巴瘤细胞转染人CD28基因的细胞株（CD28-T）为免疫原,采用B淋巴细胞杂交瘤技术,获取分泌特异性mAb的杂交瘤细胞株,以体内诱生法生产腹水,并以免疫亲和层析法对其纯化,以快速定性试纸法鉴定mAb的Ig亚类,竞争抑制法分析mAb识别的抗原表位,3H-TdR掺入法研究mAb对T细胞的刺激效应,结果 成功地获得了5株分泌特异性抗入CD28mAb的杂交瘤细胞株,鉴定的1株（克隆18G8）属IgG2a,腹水效价（流式细胞仪分析）达1：2400以上,该mAb能60%阻断标准鼠抗入CD28抗体与相应抗原的结合,提示其识别的抗原表位与标准mAb不完全相同,mAb18G8可取代B7-1分子介导的协同刺激信号,促进人外周血T细胞增殖（ST=7）。结论 18G8是12株功能性mAb,具有重要的研究和应用价值。     

Clonal analysis of human T cell activation by the Mycoplasma arthritidis mitogen (MAS)

Mycoplasma arthritidis produces an as yet undefined soluble molecule (MAS) that has a potent mitogenic effect on T cells of several species. We have used cloned human cytotoxic and proliferative T lymphocytes to dissect the molecular mechanism of T cell activation by this mitogen. Reactivity to MAS is clonally expressed among T cell receptor (TcR) alpha/beta chain-expressing T cell clones of CD4+ or CD8+ phenotype, as well as CD4-8- TcR alpha/beta chain-negative T lymphocyte clones expressing the CD3-associated TcR gamma chain. MAS is able to induce cytotoxicity and/or proliferation in these T cell clones. For triggering of these T cells, regardless of their phenotype of specificity, the presence of autologous, allogeneic or xenogeneic major histocompatibility complex (MHC) class II molecules on accessory cells or target cells is necessary. However, T cells do not immunologically recognize MAS on class II molecules, since a direct action of MAS on the T cells themselves can be demonstrated. Triggering of T cells by MAS can be blocked by monoclonal antibodies against CD2, CD3 and the TcR alpha/beta chain dimer. We discuss as a possible explanation that MAS is a functionally bivalent molecule cross-linking TcR and MHC class II molecules. Thus, the mechanism of T cell activation by MAS has striking similarities to the mechanisms by which Staphylococcal enterotoxins activate T cells. It is intriguing that a similar mitogenic principle has been developed by two evolutionary distinct pathogenic microorganisms.     

Positive signal transduction via surface CD4 molecules does not need coexpression of the CD3/TcR complex.

We previously reported that the human CD4 molecule is capable of transducing a positive signal when activated by an anti-CD4 mAb B66. This antibody, in contrast to many other anti-CD4 mAb, induced IL2 production and proliferation of resting CD4+ peripheral blood T lymphocytes in the absence of any other signal. We further reported that anti-CD4 mAb B66 was able to induce IL2 production in murine T-cell hybridoma cells transfected with full-length human CD4 cDNA. In the present study, we extend these findings by demonstrating that anti-CD4 mAb B66 was able to induce Ca2+ mobilization and IL2 production in a CD3/TcR- variant 31-13, of the CD3/TcR+ Jurkat cell line. We further showed that anti-CD4 mAb B66 was able to activate CD4+ cells from the promonocytic cell line U937. In these cells, mAb B66 induced Ca2+ mobilization when cross-linked with a second antibody and, in addition, the production of large quantities of IL1 beta was measured. In essence, our findings provide direct evidence that cross-linking of CD4 may cause T-cell activation in the absence of the coexpression of the CD3/TcR molecular complex and that, in addition, CD4 might transduce a positive signal in CD4+ cells of the myeloid lineage.     

Genetic reconstitution of the T cell receptor (TcR) alpha/beta heterodimer restores the association of CD3 zeta 2 with the TcR/CD3 complex.

The cell surface expression of the T cell receptor (TcR)/CD3 complex and, consequently, the functional competence of the cell is partly dependent on CD3 zeta. In its absence, a pentameric complex (TcR alpha/beta/CD3 gamma delta epsilon) is formed which is inefficiently transported to the cell surface. Reconstitution of CD3 zeta by transfection, in turn, restores the cell surface expression and function of the complex. Through the use of transfection experiments, we here provide direct evidence that the association of CD3 zeta 2 with the TcR/CD3 complex is dependent on the presence of both the TcR alpha and beta polypeptide chains. Despite wild-type levels of the CD3 zeta protein in a TcR alpha-negative mutant human T cell line, a complex was formed intracellularly which lacked CD3 zeta 2 and consisted of beta gamma delta epsilon and beta 2 gamma delta epsilon. Upon transfection of the mutant with a TcR alpha cDNA, a TcR/CD3 complex which contained CD3 zeta 2 was observed intracellularly. In contrast to the partial subcomplex on the cell surface of the untransfected cell line, the TcR/CD3 complex on the transfectant was functional as demonstrated by its ability to mobilize intracellular calcium after stimulation with a mitogenic CD3 epsilon-specific monoclonal antibody. Transient transfection studies performed in COS cell fibroblasts indicated that CD3 zeta 2 was not interacting with the TcR alpha protein alone, implying that a conformation provided by either the TcR alpha/beta heterodimer or the TcR alpha/beta/CD3 gamma delta epsilon complex was necessary for the association of CD3 zeta 2. Transfection studies performed in a TcR alpha/beta-negative murine T-T hybridoma confirmed the requirement of both the TcR alpha and beta proteins in CD3 zeta 2 binding. We conclude that the TcR alpha and beta chains harbor polypeptide sequences essential for the association of CD3 zeta 2 with the TcR/CD3 complex.     

抗人CD100单克隆抗体的制备与初步鉴定

目的：研制可识别天然CD100分子的单克隆抗体（mAb)。方法：采用真核表达的CD100分子免疫BALB/c小鼠，以间接ELISA法筛选阳性杂交瘤，并分别用人急性T淋巴细胞性白血病细胞系MOLT-4，猿T淋巴细胞系MLA-144及猴EB病毒转化的B淋巴细胞系（BLCL），进行间接免疫荧光染色和流式细胞术鉴定mAb的特异性。结果：共获得7株可稳定分泌mAb的杂交瘤细胞株。所有mAb均可识别人和猴细胞系的膜表面天然CD100分子，其中6株可识别猿细胞系膜表面的天然CD100分子，结论：成功地制备了7株抗CD100分子的特异性mAb。为研究人和猿，猴CD100分子的结构与功能提供了新的手段。     

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

In this study, we demonstrated that some Vp6⁺, CD4⁺, Mls-l^a-specific T cell clones had cytolytic activity when stimulated with anti-T cell receptor(TcR)/CD3 monoclonal antibodies (mAb), but not with targets expressing Mls-1^a, although they produced lymphokines (interleukin 2 and interferon-y) in response to both types of stimuli. To examine the possibility that lack of cytolysis resulted from expression of the Mls-l^a antigen on merely a fraction of splenic B blasts, we (a) used the B cell lymphoma LBB.3.4.16 and (b) measured esterase secretion which is generally concurrent with cytotoxic T lymphocyte (CTL) activity. The B cell lymphoma maximally stimulated the T cell clone for interferon-y production when responding and stimulating cells were incubated at a 1:1 ratio, but it was never killed by the Mls-1^a-specific T cell clone unless TcR/CD3-specific mAb were added. Furthermore, a fivefold excess of the Mls-1^a B cell lymphoma did not induce any secretion of esterase, which was observed only in the presence of the TcR/CD3-specific mAb. Comparison of the reactivity of two Mls-1^a-specific T cell hybridomas expressing the same TcR at similar surface density, revealed both quantitative and qualitative differences between CD3-specific mAb and Mls stimulation of the hybridomas. A small quantitative difference in the sensitivity of hybridoma FJ22.5 to stimulation with V_β6 or CD3-specific mAb resulted in a marked decrease in efficiency of stimulation by Mls-1^a for interleukin 2 production and to inability to detect growth inhibition by Mls-expressing cells. A qualitative difference was observed when analyses of inositol phosphate production were performed under optimal conditions of stimulation of the highly responsive T cell hybridoma (FJ8.1): only stimulation with CD3-specific mAb, but not Mls-expressing cells, could induce detectable inositol phosphate production. Lack of cytolysis of Mls-1^a class II-expressing B cells may have evolutionary significance in view of the recent mapping of Mls to mouse mammary tumor virus genes.