The extracellular domain of CD4 regulates the initiation of T cell activation

To assess the respective contribution of the extracellular and intracellular domains of CD4 in regulating early T cell activation events, we have used a CD4-independent murine T cell clone transfected with human CD4. Stimulation of CD4 positive clones could only be observed if CD4 molecules associated to lck were co-aggregated with the TCR complex, confirming that the simultaneous interaction of MHC class II molecules with the CD4/lck complex and the TCR is required to initiate T cell activation. To assess the involvement of the extracellular portion of CD4 in this process, we transfected a chimeric molecule (EGFRCD4) consisting of the extracellular portion of the epidermal growth factor receptor (EGFR), and of the transmembrane and cytoplasmic domains of human CD4. Although this chimeric molecule associates with lck, transfected clones were induced to proliferate by mAb specific for TCR in the absence of co-aggregation. A new regulatory role for the extracellular domain of CD4 which is independent of its interaction with MHC class II molecules is thus revealed in these experiments. Taken together, our results demonstrate that, in a CD4-independent cell line, two domains of CD4 regulate early T cell activation events: (1) its association with lck and (2) its extracellular domain, independently of its interaction with MHC class II molecules.     

The cytoplasmic domain of CD4 is required for stable association with the lymphocyte-specific tyrosine protein kinase p56lck

The CD4 T cell surface molecule binds MHC class II determinants expressed on antigen-presenting cells. CD4 is thought to enhance T cell activation by serving as an adhesion molecule as well as possibly by transducing an independent intracellular signal during the process of antigen stimulation. The recent observation that CD4 is physically associated with the Src-related tyrosine protein kinase p56lck suggests that tyrosine phosphorylation might be involved in these CD4 "signaling" events. The results presented in this report demonstrate that deletion of the cytoplasmic domain of CD4 significantly diminishes its ability to stably associate with p56lck. This observation provides a biochemical basis for the decreased ability of this mutant CD4 molecule to enhance T cell activation during suboptimal antigen stimulation.     

Role of the CD1a molecule in the superantigen-induced activation of MHC class II negative human thymocytes

Bacterial superantigens (Sag) are potent activators of T cells. This T-cell activation has been described as an MHC class II dependent phenomenon. We have observed that human thymocytes depleted of MHC class II positive cells are still able to proliferate in response to the staphylococcal enterotoxin A (SEA). This proliferation was clearly inhibited by the addition of monoclonal antibodies directed against the CD1a molecule. In contrast, monoclonal antibodies directed against the CD1b and CD1c molecules have no effect on the Sag-induced activation of the CD2 (+) MHC class II (-) thymocytes. We next examined the ability of the CD1a molecule to transmit transmembrane signals. Results obtained indicate that CD1a ligation on these thymocytes induced tyrosine phosphorylation of the p56(lck) tyrosine kinase. Signal transduction via CD1a is further confirmed by the observation of a significant intracellular calcium flux (Ca(i)(++)) in thymocytes following CD1a engagement. These data demonstrate that CD1a ligation induces a signal transduction pathway which has a potential role in the bacterial superantigen-induced activation of human CD2 (+) MHC class II (-) thymocytes.     

Binding of foreign DNA to mouse sperm mediated by its MHC class II structure

By means of radioimmunoassay, the expression of the major histocompatibility complex (MHC) class II molecules on murine sperm cells was clearly demonstrated as well as by our previous enzyme immunoassay (Mori T, et al. The expression of class II major histocompatibility antigen on mouse sperm and its role in fertilization. Am J Reprod Immunol. 1990; 24:9-14). The present study revealed that the site of sperm for binding foreign DNA was mediated by the complex structure of the MHC class II molecules localized at the posterior region of sperm head. This binding activity of sperm was time-, temperature-, and viability-dependent and completely inhibited by the treatment of sperm cells with mouse anti Iak serum, but not with mouse normal serum. Scatchard analysis of this binding activity also showed a single receptor type on sperm cells. These results were directly confirmed morphologically by taking autoradiography of sperm cells binding foreign DNA.     

Interactions between the tyrosine kinases p56lck,p59fyn and p50csk in CD4 signaling in T cells

Interaction of the CD4 co-receptor with major histocompatibility complex (MHC) class II molecules during antigen presentation results in enhancement of antigen receptor signaling. The synergism between the two receptors is believed to result from the juxtaposition of the CD4-associated tyrosine kinase p56lck with the cytoplasmic domains of CD3 complex components. Here, we report that cross-linking of CD4 on the surface of Jurkat cells using monoclonal antibodies results in activation of the CD3-associated kinase p59fyn. Co-cross-linking of CD4 and CD3 results in synergistic activation of p59fyn. The p59fyn kinase is also hyperactive in a Jurkat cell line stably transfected with a constitutively active p56lck mutant, indicating that p56lck mediates CD4 activation of p59fyn. In support of this hypothesis, expression of a dominant inhibitory mutant of p59fyn blocks CD4 signals involved in gene activation. In addition, the p59fyn dominant inhibitor mutant blocks gene-activating signals induced by expression of a constitutively active mutant of p56lck. Overexpression of the regulatory kinase p50csk, which attenuates TcR signaling by inactivation of p59fyn, inhibits signaling from the constitutively active form of p56lck. Taken together, these data suggest that CD4/p56lck enhancement of TcR signaling is, at least in part, mediated by activation of p59fyn, and may be regulated by p50csk.     

The roles of CD4 and CD8 in T cell activation

CD4 and CD8 T cell surface molecules play a role in T cell recognition and activation by binding to their respective class II and class I major histocompatibility complex (MHC) ligands on an antigen presenting cell (APC). Though CD4 and CD8 are capable of binding to MHC molecules in the absence of the T cell receptor (TCR), increasing evidence suggests that they may primarily function by complexing with the TCR to form a 'co-receptor' for recognition of antigen-bound MHC. Using gene transfer studies we have demonstrated that CD4 and CD8 can augment antigen-induced IL-2 production through different mechanisms dependent on whether or not they can bind MHC independently of the TCR or complexed with the TCR. Under circumstances where CD4 and CD8 can bind to the same MHC ligand as the TCR, they potentiate antigen-induced IL-2 production maximally by a mechanism in large part dependent on their cytoplasmic tails. Enhancement of antigen-induced IL-2 production can also occur under circumstances where CD4 and CD8 bind on MHC ligand distinct from that recognized by the TCR. In this instance, the magnitude of this enhancement is not as great and appears (at least for CD8) to be independent of the cytoplasmic tail and the associated p56lck. The dependence of co-receptor function on the cytoplasmic tail of CD4 or CD8 may reflect the activity of the associated intracellular tyrosine kinase p56lck.(ABSTRACT TRUNCATED AT 250 WORDS)     

CD4/major histocompatibility complex class II interaction analyzed with CD4- and lymphocyte activation gene-3 (LAG-3)-Ig fusion proteins

We analyzed CD4 major histocompatibility complex (MHC) class II interactions with CD4 and lymphocyte activation gene (LAG)-3 recombinant fusion proteins termed CD4Ig and LAG-3Ig. CD4Ig bound MHC class II molecules expressed on the cell surface only when used in the micromolar range. This weak CD4Ig binding was specific, since it was inhibited by anti-CD4 and anti-MHC class II mAb. LAG-3Ig bound MHC class II molecules with intermediate avidity (K_d = 60 nM at 37°C). Using LAG-3Ig as a competitor in a CD4/MHC class II-dependent cellular adhesion assay, we showed that this recombinant molecule was able to block CD4/MHC class II interaction. In contrast, no inhibition was observed in a CD4/MHC class II-dependent T cell cytotoxicity assay. Together, these results suggest that co-engagement of the TcR with CD4 alters the CD4/MHC class II molecular interaction to become insensitive to LAG-3Ig competition.     

CD20 is physically and functionally coupled to MHC class II and CD40 on human B cell lines

Engagement of MHC class II and CD40 on B cell lines triggers intracellular signals that activates cell surface adhesion receptors, resulting in LFA‐1‐dependent and ‐independent cell‐cell adhesion. In this study, a murine monoclonal antibody (mAb R21) has been produced against a LFA‐1‐negative human B cell line and proven to completely block MHC class II‐and CD40‐induced LFA‐1‐independent homotypic adhesion. However, this mAb failed to prevent MHC class II‐ or CD40‐induced homotypic adhesion in LFA‐1‐positive Raji B cells, and alone, it triggered LFA‐1‐dependent cell‐cell adhesion. Biochemical characterization indicated that the CD20 molecule, a tetraspan phosphoprotein expressed on B cells that functions as a Ca²⁺ ‐conductive ion channel, is the target of mAb R21. Interestingly, further biochemical analysis demonstrated that CD20 is physically associated with MHC class II and CD40 molecules on the cell surface of LFA‐1‐negative and LFA‐1‐positive B cell lines. Although these three molecules are associated with each other, the complex formation between any two of them is not dependent on the simultaneous expression of the three molecules. Altogether, these results indicate that CD20 is physically and probably functionally coupled to the MHC class II and CD40 molecules; thereby it may have certain modulatory effects on their functions.     

Characterization of a CD4(L3T4)-positive cytotoxic T cell clone that is restricted by class I major histocompatibility complex antigen on FBL-3 tumor cell

An L3T4(CD4)+ CTL clone specific for Friend virus-induced tumor FBL-3 was isolated, characterized and compared with a conventional Lyt-2(CD8)+ CTL clone. This clone L3.1 was obtained from the limiting dilution culture of splenic MLTC cells from a CB6F1 mouse whose CD8+ T cells had been suppressed by an in vivo injection of anti-Lyt-2.2 mAb. The phenotype of clone L3.1 was sIg-, Thy-1.2+, L3T4(CD4)+, Lyt-2 (CD8)-, and Ia- as determined by flow-cytometry. Northern blot analysis also confirmed that mRNA for L3T4(CD4), but not for Lyt-2 (CD8) was present in the total RNA of L3.1. The FBL-3-specific killing activity of L3.1 was inhibited by anti-H-2D6 mAb, and the tumor cells did not express class II MHC antigen, indicating that the recognition of tumor antigen by this CD4+ CTL clone was restricted by the class I MHC molecule on the tumor cells. Furthermore, the finding that anti-L3T4(CD4) mAb GK1.5 inhibited the specific and lectin-dependent non-specific cytotoxicity of L3.1 suggested that CD4 molecules on this CTL clone are not ligand (MHC class II)-binding proteins, but are involved in signal transduction.     

BY55/CD160 acts as a co-receptor in TCR signal transduction of a human circulating cytotoxic effector T lymphocyte subset lacking CD28 expression

In the present study, we examined the role of the recently identified glycosylphosphatidylinositol (GPI)-anchored cell surface molecule BY55, assigned as CD160, in TCR signaling. CD160 is expressed by most intestinal intraepithelial lymphocytes and by a minor subset of circulating lymphocytes including NK, TCRgammadelta and cytotoxic effector CD8bright+CD28- T lymphocytes. We report that CD160, which has a broad specificity for MHC class Ia and Ib molecules, behaves as a co-receptor upon T cell activation. Anti-CD160 mAb enhance the CD3-induced proliferation of freshly isolated CD160-enriched peripheral blood lymphocytes and CD160+ T cell clones. Further, the engagement of CD160 receptors on normal clonal T lymphocyte populations lacking CD4, CD8 and CD28 molecules by MHC class I molecules results in an increased CD3-induced cell proliferation. Further, we found that CD160 co-precipitates with the protein tyrosine kinase p56lck and tyrosine phosphorylated zeta chains upon TCR-CD3 cell activation. Thus, we demonstrate that CD160 provides co-stimulatory signals leading to the expansion of a minor subset of circulating lymphocytes including double-negative CD4/CD8 T lymphocytes and CD8bright+ cytotoxic effector T lymphocytes lacking CD28 expression.     

Lack of evidence for aggregation-dependent enhancement of p56lck in the signal transduction upon major histocompatibility complex recognition by mature T cells

The kinase activity of lymphocyte-specific tyrosine kinase p56lck (Lck) upon physiological major histocompatibility complex (MHC) recognition by normal mature T cells was examined. Recognition of the target MHC molecules by T cells induced phosphorylation of the zeta-chain without obvious enhancement of the background Lck activity. There was no sign of enhancement of Lck through putative T-cell receptor (TCR)-independent class II MHC/CD4 interactions either. As has been reported, cross-linking of CD4 molecules by antibodies induced a marked enhancement of Lck activity. However, it did not have an immediate relevance to TCR-mediated signal transduction, as judged from the lack of detectable de novo phosphorylation of zeta-chain and the absence of functional responses of T cells. These results strongly favour the model in which TCR-mediated signal transduction does not involve aggregation-dependent enhancement of Lck, suggesting that the signal can be triggered simply by the recruitment of already active Lck with basal kinase activity through the formation of a TCR/MHC/CD4 ternary complex.     

The lymphocyte-specific tyrosine protein kinase p56lck

The CD4 and CD8 T cell surface antigens are physically associated with the tyrosine protein kinase p56lck. Accumulating data indicate that p56lck transduces intracellular tyrosine protein phosphorylation signals upon engagement of CD4 and CD8 by major histocompatibility complex (MHC) determinants expressed on antigen-presenting cells (APCs). Recent studies show that these p56lck-related phosphorylation events enhance T cell receptor (TCR)-mediated functions and are critical for the proposed co-receptor roles of CD4 and CD8. p56lck is also capable of enhancing antigen receptor responsiveness in the absence of CD4 or CD8 expression, suggesting that it can directly contribute to the TCR-induced tyrosine phosphorylation signal.     

CD4-Ia interactions can occur in the absence of T-cell receptor/antigen-Ia recognition

The T-cell differentiation antigen, CD4, is expressed by major histocompatibility (MHC) class II restricted T lymphocytes. CD4+CD8- T cells use their T-cell receptor to recognize foreign antigens in association with MHC class II products (Ia). The association between CD4 expression and restriction by MHC class II products has led to the hypothesis that CD4 may interact with monomorphic determinants of MHC class II molecules. A large body of experimental evidence suggests that CD4 interaction with MHC class II molecules leads to an increase in the binding avidity of T cell-stimulator cell interactions. A direct test for a functional CD4-MHC class II interaction in T-cell activation requires a separate evaluation of CD4-Ia interactions from T-cell receptor (TcR)-antigen (Ag)/Ia recognition. However, a separate evaluation proves difficult since the T-cell receptor and CD4 may interact with the same MHC class II molecule. In this report, we use a T-cell activation protocol where TcR-Ag/Ia recognition is replaced by TcR complex-anti-CD3 antibody interactions. Therefore, the affinity of the TcR complex for its ligand (the anti-CD3 mAb) is independent from MHC expression on target cells and allows a separate evaluation of the role of accessory molecules in T-cell activation. We have analysed the effects of monoclonal anti-MHC class II antibodies on the activation of a CD4+ T-cell hybridoma in the absence of its TcR restricting MHC class II molecule (I-Ek) but in the presence of unrelated MHC class II molecules (I-Ed, I-Ad). The data obtained indicate a functional interaction between the CD4 molecule and a non-polymorphic region of the MHC class II product in T-cell triggering.     

Inhibition of CD28-mediated natural cytotoxicity by KIR2DL2 does not require p56(lck) in the NK cell line YT-Indy.

CD28 functions as a cytotoxicity activation receptor in the NK cell line YT-Indy. To analyze the requirement of p56(lck) kinase in the function of killer inhibitory receptors, we transfected the p56(lck) negative YT-Indy cell line with the cl43 gene encoding for KIR2DL2. Pervanadate treatment revealed KIR2DL2 phosphorylation in YT-Indy-cl43, as well as SHP1/SHP2 recruitment. YT-Indy-cl43 cells were inhibited in their ability to lyse target cells expressing HLA-Cw3, a ligand for KIR2DL2. This inhibition was blocked by anti-KIR2DL2 or anti-HLA class I mAb. CD28 crosslinking on YT-Indy-cl43 enhanced tyrosine phosphorylation of PLC-gamma1. The simultaneous ligation of KIR2DL2 with mAb resulted in a decrease in CD28-induced tyrosine phosphorylation of PLC-gamma1 confirming that dephosphorylation of this protein is involved in the KIR2DL2-induced inhibition of CD28-mediated cytotoxicity. As YT-Indy-cl43 did not express detectable levels of p56(lck), these results indicate that this kinase is not required for transmitting the negative signals generated by KIR2DL2 ligation.     

A non-class I MHC intestinal epithelial surface glycoprotein, gp180, binds to CD8

The activation of CD8(+) T cells by normal intestinal epithelial cells in antigen-specific or allogeneic mixed cell culture systems has significant implications for the modulation of mucosal immune responses due to the fact that these T cells appear to have regulatory rather than cytolytic activity. A 180-kDa glycoprotein (gp180) has been identified and shown to be important in CD8(+) T cell activation by intestinal epithelial cells. In this study, we examine, in further detail, the role that the CD8 molecule plays in this interaction. It has been previously shown that monoclonal antibodies against gp180 inhibited the activation of CD8-associated p56(lck) in T cells. Although indirectly suggested by these data, there was no evidence that the activation of this protein tyrosine kinase was a direct result of gp180 interacting with the CD8 molecule. In this study, we document that soluble gp180 is able to bind to CD8-Fc fusion proteins and is absorbed by human CD8 alpha but not CD4 transfected murine T cells and that this interaction is dependent upon carbohydrate on the gp180 molecule. Furthermore, the sites used for binding by gp180 are distinct from those used by the conventional CD8 ligand, class I MHC. Thus, gp180 appears to be a novel CD8 ligand that plays an important role in the activation of CD8-associated kinases and of CD8(+) T cells.     

Do CD4 and CD8 control T-cell activation via a specific tyrosine protein kinase?

The CD4 and CD8 glycoproteins play an important role in T-cell activation by binding to major histocompatibility complex (MHC) class II or class I molecules, respectively, and stabilizing their interactions with the T-cell receptor-CD3 complex during antigen presentation. Recent evidence suggesting that the cytoplasmic domains of CD4 and CD8 are physically, and perhaps functionally, linked to the T-cell specific tyrosine protein kinase, p56lck, adds a new dimension to our current understanding of their physiological function. Based on these and other recent findings, Tomas Mustelin and Amnon Altman present a working hypothesis that defines a novel role for CD4 or CD8 in regulating T-cell activation, and perhaps other processes, such as thymic repertoire selection and human immunodeficiency virus (HIV)-induced immunosuppression.