CD4 and CD8 accessory molecules function through interactions with major histocompatibility complex molecules which are not directly associated with the T cell receptor-antigen complex

Both the subset-specific, CD4 and CD8 T cell accessory molecules and the antigen-specific T cell receptor (TcR) interact with major histocompatibility complex (MHC) class I and class II molecules on the surface of antigen-presenting cells. We analyzed whether the CD4/CD8 molecules exert their accessory function through binding with the same MHC molecules which participate in the TcR-antigen-MHC complex. We utilized a CD4-, CD8-, class I-allospecific T cell hybridoma which functionally manifests both cytotoxic T lymphocyte (CTL) and T helper1 (Th1) phenotypes, and rendered it bispecific by transfecting it with genes encoding either a class II-restricted, 2,4,6-trinitrophenyl (TNP)-I-Ad-specific TcR or a non-MHC-restricted chimeric TcR, composed of a variable part of an anti-TNP antibody. Expression of either CD4 or CD8 transgenes in these hybridomas enhanced and augmented their reactivity towards the appropriate target cells regardless of the type of TcR-MHC interaction. Thus, class I-specific responses could be enhanced through CD4-class II interactions, and class II-restricted responses could be augmented through CD8-class I interactions. Furthermore, these accessory molecules also potentiated TNP-specific responses by the chimeric TcR which is MHC unrestricted. The accessory molecules facilitated both interleukin 2 (IL2) production and cytolytic activity by shortening the activation time and rendering the cells responsive to lower antigenic stimuli. The degree of activity of the T cell hybridomas correlated with the level of accessory molecule expression and was not related to the effector function mediated by the cells. Anti-CD4 or -CD8 antibodies completely inhibited the activity of transfectants expressing the corresponding accessory molecule, regardless of the MHC type of the TcR interaction. Such antibodies blocked direct TcR stimulation provided by either anti-T3/Ti antibodies or lectins, but could not inhibit the activation through agents that bypass the TcR such as phorbol 12-myristate 13-acetate plus ionophore. Taken together, these studies demonstrate that the CD8/CD4 molecules can exert their accessory function through interactions with MHC molecules which are not directly associated with the TcR-Ag-MHC complex, and that this accessory effect is associated with TcR-mediated triggering at an early stage of the signaling process and is not related to the effector mechanism assigned to the CD4 and CD8 T cell subsets.     

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.     

Cyclosporin A and anti-Ia antibody cause a maturation defect of CD4+8- cells in organ-cultured fetal thymus

The effects of anti-Ia antibodies and cyclosporin A (CsA) on phenotypic differentiation of murine thymocytes were assessed in organ-cultured fetal thymuses. Both agents specifically abrogated the generation of CD4+8- thymocytes. Immunohistochemical studies revealed that Ia antigen on the organ-cultured thymic epithelial cells did not disappear with the addition of the agents, although anti-Ia antibody was proved to bind to Ia antigen during the culture. On the other hand, CsA neither changed the expression of Ia nor bound to it. As CsA is known to block the signalling cascade initiated by perturbation of T-cell receptor (TcR), it is suggested that both the Ia expression in the thymus and the signalling via receptors on thymocytes, the signals presumably generated by TcR binding to class II MHC molecules, might be necessary for phenotypic differentiation of class II MHC-restricted T cells (CD4+8- cells), but not for class I MHC-restricted T cells (CD4-8+).     

Existence of mature human CD4+ T cells with genuine class I restriction.

A human T cell receptor (TcR) alpha/beta CD4+CD8-T cell clone (R416) is reactive with the minor histocompatibility antigen H-Y in the context of major histocompatibility complex (MHC) class I and not class II molecules. Therewith clone R416 violates the so-called specificity association of mature TcR alpha/beta+ T cells. R416 displays H-Y-specific, HLA-A2-restricted proliferation as well as cytotoxicity in vitro. Its fine specificity is identical to that of a classical H-Y-reactive CD4-CD8+ MHC class I-restricted CTL clone, showing that CTL expressing either CD4 or CD8 can display identical antigenic specificities. Exploiting the MHC class I restriction of this CD4+ T cells clone, it was found that interaction of CD4 with non-TcR-bound MHC class II molecules does not contribute to antigen specific activation of these CD4+ T cells. This coreceptor-mismatched T cell clone was not generated in vitro but obtained by expansion of CD8-depleted peripheral blood mononuclear cells of a female who had been immunized against H-Y. The existence of such MHC class I-restricted mature TcR alpha/beta+ T cells expressing CD4 and not CD8 is relevant because it indicates that the generally accepted model for thymic selection, in which the TcR specificity alone determines CD4/CD8 expression of mature thymocytes, may not be absolute.     

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.     

Qa-1/Tla region alloantigen-specific CTL with alpha beta receptor

Recent studies involving T cells that express gamma delta T-cell receptor (gamma delta TcR) have raised the possibility that Qa-1/Tla region class I major histocompatibility complex (MHC)-like molecules are antigen-presenting molecules for gamma delta TcR. In this report, cytotoxic T lymphocyte (CTL) clones specific for a Qa-1/Tla region gene product were isolated from a bulk B10. QBR (Kb, Ib, Dq Qa-1/Tlab) anti-B10.MBR (Kb, Ik, Dq, Qa/Tlaa) CTL line. These CTL lysed blasts from all Qa-1a strains regardless of the H-2 haplotype, indicating that the recognition of the Qa-1 antigen by these CTL is not restricted by other class I molecules. In bulk populations, CTL activity of this specificity was found only in the CD8+CD4- subpopulation. Accordingly, all established CTL clones were phenotyped as Thy-1+, CD8+CD4-. Furthermore, these clones were shown to express alpha beta TcR rather than gamma delta TcR. Thus, the results indicate that Qa-1 antigen can be recognized by alpha beta TcR T cells in a manner similar to recognition of classical class I molecules.     

The structural basis of T-cell allorecognition

Foreign allogeneic major histocompatibility complex (MHC) class I and class II molecules elicit an exceptionally vigorous T-cell response. A small component of the alloresponse comprises CD4+ T cells that recognize allogeneic MHC indirectly after processing into peptide fragments that are bound and presented by self-MHC class II. The majority of alloreactive T cells directly recognize intact allogeneic MHC molecules expressed on foreign cells. Some alloreactive T-cell interactions with allogeneic MHC molecules are indifferent to the bound peptide, but evidence suggests that most show specificity to peptide. The vigor and diversity of the direct alloreactive T-cell response can therefore be explained by summation of numerous responses to each of the peptides in the novel set bound by allogeneic MHC molecules. Structural studies definitively show that the overall mechanism of T-cell receptor (TCR) recognition of self-MHC and allogeneic MHC molecules is similar. Many alloreactive T cells recognize several different combinations of MHC and bound peptide that do not necessarily possess structural homology. Flexibility within the TCR structure allows adaptation to the different contact surfaces. Crossreactivity seems to be an intrinsic property of the TCR required, because a single TCR must possess the ability to interact with both self-MHC during positive selection and at least one combination of foreign antigenic peptide presented by self-MHC. Recognition of allogeneic MHC molecules is an inadvertent consequence of the need for TCR crossreactivity.     

Mls determinants and anti-Mls receptors

We review evidence from this laboratory that T cell recognition of Mlsa determinants is not controlled solely by the alpha-beta T cell receptor (TcR) molecule. We propose a model in which Mlsa recognition reflects a receptor-ligand interaction between two sets of complementary accessory molecules, one molecule (Mlsa) being expressed on B cells and the other (the anti-Mlsa receptor) on T cells; this interaction augments recognition of self class II molecules by the TcR. The biological role of Mls molecules might be to facilitate physiological T-B interaction.     

MHC Class II-Dependent Peptide Antigen Versus Superantigen Presentation to T Cells

T lymphocytes expressing the CD4 coreceptor can be activated by two classes of major histocompatibility complex (MHC) class II-bound ligands. The elaboration of a conventional T-cell mediated immune response involves recognition of an antigenic peptide bound to the MHC class II molecules by a T-cell receptor (TCR) specific to that particular antigen. Conversely, superantigens (SAgs) also bind to MHC class II molecules and activate T cells, leading to a completely different functional outcome; indeed, SAg-responsive T cells die through apoptosis following stimulation. Superantigens are proteins that are secreted by various bacteria. They interact with the TCR using molecular determinants that are distinct from the residues involved in the recognition of nominal antigenic peptides. Despite the similarities between the recognition of the two classes of ligands by the TCR, considerable structural difference is observed. Here, we discuss the current knowledge on the presentation of SAgs to T cells and compare the different aspects of the SAg response with the recognition of antigenic peptide/MHC complexes.     

CD4-class II major histocompatibility complex interaction does not enhance killing by a class I-restricted CD4+CD8+ cytotoxic T cell clone.

As unusual tumor-specific cytotoxic T lymphocyte (CTL) clone was isolated which expressed both CD4 and CD8 molecules. The target cells for this CTL can be induced to express either class I major histocompatibility complex (MHC) alone (with dimethylsulfoxide) or both class I and class II MHC (with interferon-gamma). Lysis of the tumor target depends on expression of class I MHC molecules, but does not require expression of class II MHC molecules. Furthermore, the lysis of target cells expressing both class I and class II is inhibited only by antibodies to class I (Kd), and not by antibodies to class II, demonstrating that the T cell receptor is class I restricted. We have used this CTL to assess the role of the interaction between CD4 and class II MHC in the absence of a class II-restricted T cell receptor. Our data indicate that CD4-class II interaction does not contribute to recognition by T cells in the absence of binding of the T cell receptor to class II molecules.     

Evidence for T cell receptor-HLA class II molecule interaction in the response to superantigenic bacterial toxins

The staphylococcal enterotoxins and related microbial T cell mitogens stimulate T cells by cross-linking variable parts of the T cell receptor (TcR) with MHC class II molecules on accessory or target cells. In this report we describe that a given combination of T cell, accessory cell (AC) and toxin can be non-stimulatory. However, the same T cell can respond to the same toxin on another AC and the same AC can present the same toxin to another T cell. This indicates that in the complex formed between TcR, toxin and class II molecule an interaction between TcR and class II molecule takes place.     

T cell receptor targeting to thymic cortical epithelial cells in vivo induces survival,activation and differentiation of immature thymocytes

We report that targeting of T cell receptors (TcR) to non-major histocompatibility complex (MHC) molecules on thymic cortical epithelial cells by hybrid antibodies in vivo and in fetal thymic organ cultures results in phenotypic and functional differentiation of thymocytes. A single pulse with hybrid antibodies rescues immature, CD4/8 double-positive thymocytes from their programmed death in vivo, induces expression of the early activation antigen CD69 followed by TcR up-regulation, concomitant down-regulation of CD8 or CD4 and their conversion to functional mature T cells by day 3. This temporal sequence of maturation only affects small thymocytes without co-induction of blastogenesis. TcR targeting to MHC class II-positive epithelial cells predominantly induces CD4-positive T cells. This generation of CD4 single-positive T cells occurs also in MHC class II-deficient mice and thus is independent of CD4-MHC class II interactions. Moreover, in the presence of a specific deleting antigen (Mls 1^a),TcR targeting results in transient activation of immature thymocytes, however, not in subsequent TcR (Vβ6) up-regulation and development of single-positive T cells. Our findings imply that TcR cross-linking to cortical epithelial cells is sufficient to confer a differentiation signal to immature thymocytes. Futhermore, this approach distinguishes two independent TcR-mediated intrathymic events: activation and subsequent deletion of the same thymocyte subset.     

Interactions between CD8alphabeta and the TCRalphabeta/CD3-receptor complex

CD8+ T cells recognize antigenic peptides bound to major histocompatibility complex (MHC) class I molecules on normal antigen-presenting cells (APC), as well as on virus-infected cells or tumour cells (pMHC). At least two receptor types participate in recognition of these complexes: T-cell receptor (TCR) alphabeta heterodimers and CD8alphabeta molecules. The former molecules react with antigenic peptide and variable regions of MHC class I molecules, whereas the latter molecules react with constant alpha3 regions of MHC class I molecules. As the avidity of both receptor-MHC interactions is low, it is believed that TCRalphabeta and CD8alphabeta heterodimers collaborate in T-cell recognition. We have established a TCR/CD3-CD8 capture ELISA, which can measure the interaction of pMHC with CD8alphabeta molecules and with TCR/CD3 complexes. The major findings are: (1) TCR/CD3 complexes derived from in vitro activated T cells and captured by anti-CD3 MoAb, do bind specific pMHC and (2) CD8+ T cells express at least three forms of CD8alphabeta molecules: single CD8alphabeta, CD3-CD8 and TCR/CD3-CD8 complexes. Only the latter complexes are associated with CD3zeta homodimers, and the quantity of TCR/CD3-CD8 complexes relative to total CD8alphabeta molecules appears to increase and to be selected into sucrose-gradient microdomains as a function of TCRalphabeta-mediated T-cell activation.     

T cell major histocompatibility complex class II molecules down-regulate CD4+ T cell clone responses following LAG-3 binding

T cell response to its antigen requires recognition by the T cell receptor together with a co-receptor molecule, either CD4 or CD8. Additional molecules have been identified that are capable of delivering the co-stimulatory signals provided by APC. Following T cell priming, a number of T cell activation antigens are expressed that may play a role in the inactivation phase of the T cell response. The lymphocyte activation gene (LAG)-3 protein and its counter-receptors, the major histocompatibility complex (MHC) class II molecules, are such activation antigens whose interaction may result in the down-regulation of the ongoing immune response. To investigate the role of LAG-3/class II molecule interaction, we produced a soluble form of LAG-3 by fusing the extracellular Ig domains of this membrane protein to the constant region of human IgG1 (LAG-31g). Here, we show a direct and specific binding of LAG-3Ig to class II molecules on the cell surface. In addition, we show that LAG-3/class II molecule interaction leads to the down-regulation of CD4⁺ Ag-specific T cell clone proliferation and cytokine secretion. This inhibitory effect is observed at the level of the effector cells and not the APC and is also found with anti-CD3 mAb, PHA + PMA or low-dose IL-2 driven stimulation in the absence of APC. These functional studies indicate that T cell MHC class II molecules down-regulate T cell proliferation following LAG-3 binding and suggest a role for LAG-3 in the control of the CD4⁺ T cell response.     

Presentation of superantigen by human T cell clones: a model of T-T cell interaction.

Superantigens (SAg) interact with T lymphocytes bearing particular V beta sequences as part of their T cell receptor (TcR). The interaction, however, requires the presence of major histocompatibility complex (MHC) class II molecules on antigen-presenting cell (APC). In peculiar circumstances, MHC class II+ T cell clones (TCC) have been shown to present peptides and selected antigens interacting with antigen-specific TCC in the absence of APC. In this report we studied the capacity of SAg to mediate a T-T cell interaction, investigating the TCC ability to present a panel of staphylococcal enteroxins (SE) independently of the presence of added APC. Upon exposure to a broad range of SE concentrations, MHC class II+ TCC showed an intense proliferative response even in the absence of professional APC. Diverse SE optimally stimulated responder TCC at different concentrations. The proliferation was inhibited by anti-DR monoclonal antibodies, both in the presence and in the absence of APC. The SE activation of TCC in the absence of APC induced the same series of phenotypic variations as that observed following the TCC stimulation with APC. Irradiated TCC efficiently presented membrane-bound SE to responder TCC as well as professional APC. These results show that a single cell of a given clone effectively presents the SE to other cells of the same clone, and provide evidence that SAg can efficiently mediate T-T cell interaction. In addition, the possibility also exists that one cell of the clone can actually undergo an auto-stimulation via SAg-mediated interactions between its own TcR and MHC class II molecule. It has recently been suggested that the V beta-selective depletion of T cells observed in acquired immunodeficiency syndrome (AIDS) patients might be a consequence of the interaction between a human immunodeficiency virus (HIV)-encoded SAg and T cells expressing a SAg complementary V beta. We suggest that the hypothesized HIV-encoded SAg might mediate T-T cell interactions that could play a relevant role in the V beta-selective depletion of T lymphocytes observed in HIV-infected patients.     

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.     

Structures on T cells and macrophages involved in interleukin 1 (Il-1) secretion by macrophages upon contact with syngeneic thymocytes

We demonstrate, using a new rosette method of determination of interleukin 1 activity that macrophages secrete Il-1 upon contact with syngeneic thymocytes or with thymocytes homologous in the Ia region of MHC complex. The phenomenon takes place in the absence of foreign antigen. Blocking of class II MHC antigens on macrophages with monoclonal antibodies against structures of the T-cell receptor complex (alpha/beta T-cell receptor-TCR, CD3 and L3T4) inhibits production of Il-1 to the background level. We conclude that secretion of Il-1 from macrophages, upon contact with syngeneic thymocytes, is triggered by a T-cell signal following interaction: TCR-Ia molecules.     

Probing the activation requirements for naive CD8+ T cells with Drosophila cell transfectants as antigen presenting cells

Summary: Activation of T cells involves multiple receptor-ligand interactions between T cells and antigen presenting cells (APC), At least two signals are required for T-cell activation: Signal 1 results from recognition of MHC/peptide complexes on the APC by cell surface T-cell receptors (TCR). whereas Signal 2 is induced by the interactions of co-stimulatory molecules on APC with their complementary receptors on T cells. This review focuses on our attempts to understand these various signals in a model system involving the 2C TCR. The structural basis of Signal 1 was investigated by determining the crystal structure of 2C TCR alone and in complex with MHC/peptide. Analysis of these structures has provided some basic rules for how TCR and MHC/peptide interact; however, the critical question of how this interaction transduces Signal I to T cells remains unclear. The effects of Signal 1 and Signal 2 on T-cell activation were examined with naive T cells from the 2C TCR transgenic mice, defined peptides as antigen and transfected Drosophila cells as APC. The results suggest that, except under extreme conditions, Signal I alone is unable to activate naive CD8 T cells despite the induction of marked TCR downregulation. Either B7 or intercellular adhesion molecule (ICAM)-l can provide the second signal for CD8 T-cell activation. However, especially at low MHC/peptide densities, optimal activation and differentiation of CD8 T cells required interaction with both B7 and [CAM-1 on the same APC. Thus, the data suggest that at least two qualitatively different co-stimulation signals are required for full activation of CD8 T cells under physiological conditions.     

The role of CD4 in T-cell activation: accessory molecule or co-receptor?

CD4 is an MHC class II binding protein found on T cells that recognize peptide fragments of protein antigens bound to MHC class II molecules. In this review, Charlie Janeway argues that CD4 is a physical component of the T-cell receptor, and that CD4 augments signalling via the receptor by about 100-fold. He proposes the term co-receptor to describe this molecule and its functional homologue, CD8.     

Increased density of class I major histocompatibility complex antigens and decreased density of T-cell differentiation antigens in the early stages of T-cell activation

Major histocompatibility complex (MHC) antigens and T-cell differentiation antigens on activated T cells play a central role in T-cell interactions. In the present study, we have analyzed time courses of both quantity and density of the T-cell differentiation antigens, CD3 (T3), CD4 (T4), and CD8 (T8), as well as MHC antigens, on the cell surface of T cells, and made correlated measurements of DNA content with the surface antigen quantity as well with RNA content and cell size following activation of T cells by phytohemagglutinin. We found that the quantity and density of class I MHC antigens increase within 24 hr following activation and then decrease, while the quantity and density of the T-cell differentiation antigens decrease within 24 hr following activation, which suggests that T-cell recognition involving class I MHC gene products occurs at an early stage of T-cell activation. Class II MHC antigens can be detected on more than 40% of T cells as the expression of the T-cell differentiation antigens increases much later in the response. Cell cycle studies demonstrated that the density of class I MHC, CD3, CD4, and CD8 antigens was greater in G0/G1 phase cells than G2 phase cells at all times tested during T-cell activation. Our findings suggest that T cells demonstrate a differential regulation in expression of MHC and T-cell differentiation antigens following activation which may reflect their role in cellular interactions.