Reversal of the inhibitory effects of HIV-gp120 on CD4+ T cells by stimulation through the CD28 pathway

The effects of exposure to HIV-gp120 on proliferation and cytokine production by T cell lines were investigated. T cell lines were generated by stimulation of peripheral blood mononuclear cells from several healthy donors with cross-linked anti-CD3 antibodies and IL-2. These T cell lines exhibited the characteristics of Th1 cells, producing IL-2 and interferon-gamma (IFN-γ), but not IL-4, on stimulation with anti-CD3 antibodies. In the presence of gp120, stimulation with anti-CD3 antibodies was inhibited in terms of both proliferative responses and the secretion of IL-2 and IFN-γ. Similar effects were observed when a T cell line was stimulated in the presence of a synthetic peptide representing the CD4-binding region of gp120. Neither gp120 nor the CD4-binding region peptide had any effect on IL-4 production by the T cell lines. Stimulation through the CD28 pathway partially restored both the proliferative effect and cytokine production by T cell lines in response to anti-CD3 antibodies in the presence of gp120. Anti-CD28 antibodies also partially restored cytokine production when purified CD4⁺ T cells from a T cell line were stimulated with anti-CD3 antibodies in the presence of gp120. Anti-gp120 antibodies partially or completely reversed the inhibitory effects of gp120 on T cell proliferation. These results indicate that stimulation through the CD28 pathway may restore defective CD4⁺ T cell responses in HIV-infected individuals.     

Clonal analysis of CD4 mediated accessory function on the effector activity of human CD4+ T cell subsets

Background: It has been reported for the peripheral T cell repertoire that CD4 molecules may enhance adhesion between T cells and antigen presenting cells and, through their physical association with T cell antigen receptors, contribute to signal transduction. Objective: The aims of this study were to determine if the modulation of CD4 molecules had differential effects on T cell recognition, antigen induced cytokine (IL-4 and IFNγ), release and the induction of specific anergy for human TH-0, TH-1 and TH-2 cells. Methods: A panel of anti-CD4 antibodies was examined for its ability to modulate T cell proliferation, cytokine production and tolerance induction in house dust mite (TH-0 and TH-2) and influenza haemagglutinin (TH-1) specific human CD4+ T cell clones all restricted by DRB1^*1101 and isolated from dust mite allergic individuals. Results: We observed that anti-CD4 antibodies may inhibit or enhance antigen mediated T cell proliferation, which may reflect the differential requirements of T cells for selective functions of CD4. Furthermore, IFNγ and IL-4 production was differentially modulated depending on the specificity of the anti-CD4 antibody and the clone of T cells. However, pretreatment of T cells with anti-CD4 antibody alone neither induced nor enhanced the susceptibility of T cells to peptide mediated anergy. Conclusion: Antigen recognition by different subsets of human CD4+ T cells has differential requirements on CD4, whereas the induction of specific anergy appeared to be independent of the functions of CD4 molecules. Antigen induced IFNγ production was more susceptible than IL-4 to the inhibitory effects of anti-CD4 antibodies. Furthermore, it appeared that certain anti-CD4 antibodies can dissociate antigen induced IFNγ and IL-4 production, and may downregulate IFNγ synthesis without inhibiting antigen dependent proliferation.     

HIV-1 glycoprotein gp120 disrupts CD4-p56lck/CD3-T cell receptor interactions and inhibits CD3 signaling

Using the CD4⁺ human T cell clone P28, we demonstrated that the HIV-1 glycoprotein gp120 inhibited CD3-induced inositol trisphosphate production, calcium influx and T cell proliferation. Additionally, gp120 was shown to dissociate the tyrosine kinase p56^lck from CD4 in CEM cells, with a concommittant inhibition of CD4-linked kinase activity. We have addressed the question whether disruption of CD4/p56^lck or CD4/CD3-T cell receptor interactions, or both, could account for the inhibitory effect of gp120 in P28 cells. By comparing the effects of various anti-CD4 monoclonal antibodies (mAb) with those of gp120, we show that gp120 and IOT4a modulate CD4 expression, and decrease CD4-associated p56^lck and CD4-linked kinase activity at the plasma membrane. In contrast, OKT4A and OKT4 anti-CD4 mAb have no inhibitory effect. Interestingly, gp120 also inhibits CD3-induced Lck activation and cellular tyrosine phosphorylation, particularly of phosphoinositide-specific phospholipase C-γ-1. Kinetic experiments reveal that the inhibitory effect of gp120 on CD3-induced tyrosine phosphorylation appears as early as 30 min, but culminate when CD4-p56^lck complexes disappear from the cell surface after 4 h. These results suggest that a negative signal is triggered by gp120 that results, after a few hours, in down-modulation of CD4-p56^lck complexes and the impairment of CD3 signaling. Supporting this hypothesis, gp120 inhibits CD3-linked kinase activity as shown by the inhibition of the phosphorylation of CD3 chains, leading to the inhibition of subsequent signal transduction.     

CD28 activation promotes Th2 subset differentiation by human CD4+ cells

Ligation of CD28 provides a costimulatory signal to T cells necessary for their activation resulting in increased interleukin (IL)-2 production in vitro, but its role in IL-4 and other cytokine production and functional differentiation of T helper (Th) cells remains uncertain. We studied the pattern of cytokine production by highly purified human adult and neonatal CD4⁺ T cells activated with anti-CD3, phorbol 12-myristate 13-acetate (PMA) and ionomycin, or phytohemagglutinin (PHA) in the presence or absence of anti-CD28 in repetitive stimulation-rest cycles. Initial stimulation of CD4⁺ cells with anti-CD3 (or the mitogens PHA or PMA+ionomycin) and anti-CD28 monoclonal antibodies induced IL-4, IL-5 and interferon-γ (IFN-γ) production and augmented IL-2 production (6- to 11-fold) compared to cells stimulated with anti-CD3 or mitogen alone. The anti-CD28-induced cytokine production corresponded with augmented IL-4 and IL-5 mRNA levels suggesting increased gene expression and/or mRNA stabilization. Most striking, however, was the progressively enhanced IL-4 and IL-5 production and diminished IL-2 and IFN-γ production with repetitive consecutive cycles of CD28 stimulation. The enhanced Th2-like response correlated with an increased frequency of IL-4-secreting cells; up to 70% of the cells produced IL-4 on the third round of stimulation compared to only 5% after the first stimulation as determined by ELISPOT. CD28 activation also promoted a Th2 response in naive neonatal CD4⁺ cells, indicating that Th cells are induced to express a Th2 response rather than preferential expansion of already established Th2-type cells. This CD28-mediated response was IL-4 independent, since enhanced IL-5 production with repetitive stimulation cycles was not affected in the presence of neutralizing anti-IL-4 antibodies. These results indicate that CD28 activation may play an important role in the differentiation of the Th2 subset in humans.     

Differential susceptibility to monomeric HIV gp120-mediated apoptosis in antigen-activated CD4+ T cell populations

To support the hypothesis that indirect mechanisms mediated by viral products like the HIV envelope glycoprotein gp120 could be responsible for T lymphocyte depletion in HIV infection, we developed a system in which the impairment of T cell functions could be investigated in vitro. In particular, we characterized the conditions that allow T lymphocytes repeatedly stimulated with an antigen to be sensitive or resistant to gp120-mediated apoptotic signals. To achieve this goal, a panel of antigen-specific CD4⁺ T cell clones and primary CD4⁺ T lymphocytes were treated for 2 and 18 h with saturating amounts of monomeric gp120 (without cross-linking with specific antibodies) and antigen-driven T cell proliferation and apoptosis were analyzed. We show that monomeric gp120 induces apoptosis only in T lymphocytes repeatedly stimulated with the antigen, that primary T lymphocytes are resistant to programmed cell death mediated by monomeric gp120, but are sensitive to anti-CD4 antibodies, and that gp120-mediated apoptosis is dependent on the period of time between the binding of gp120 to CD4 and the encounter with antigen. To investigate the different susceptibility to gp120 induced apoptosis of primary CD4⁺ and T cell clones further, the number of membrane CD4 molecules and their affinity for gp120, together with Bcl-2 and Fas expression, were studied. Our data suggest that a down-modulation of membrane CD4 together with high expression of the Bcl-2 gene and protein characterizes the susceptibility to apoptosis of gp120-treated cells. In conclusion, our results define the phenotypic features of T cells susceptible to HIV gp120-induced apoptosis and demonstrate that the same clonotype, depending on the activation state, may present a differential sensitivity to apoptosis induction.     

Proliferative response of human CD4+ T lymphocytes stimulated by the lectin jacalin

The Galβ(1–3)GalNAc-binding lectin jacalin is known to specifically induce the proliferation of human CD4⁺ T lymphocytes in the presence of autologous monocytes and to interact with the CD4 molecule and block HIV-1 infection of CD4⁺ cells. We further show that jacalin-induced proliferation is characterized by an unusual pattern of T cell activation and cytokine production by human peripheral blood mononuclear cells (PBMC). A cognate interaction between T cells and monocytes was critical for jacalin-induced proliferation, and human recombinant interleukin (IL)-1 and IL-6 did not replace the co-stimulatory activity of monocytes. Blocking studies using monoclonal antibodies (mAb) point out the possible importance of two molecular pathways of interaction, the CD2/LFA-3 and LFA-1/ICAM-1 pathways. One out of two anti-CD4 mAb abolished jacalin responsiveness. Jacalin induced interferon-γ and high IL-6 secretion, mostly by monocytes, and no detectable IL-2 synthesis or secretion by PBMC. In contrast, jacalin-stimulated Jurkat T cells secreted IL-2. CD3^? Jurkat cell variants failed to secrete IL-2, suggesting the involvement of the T cell receptor/CD3 complex pathway in jacalin signaling. IL-2 secretion by CD4^? Jurkat variant cells was delayed and lowered. In addition to CD4, jacalin interacts with the CD5 molecule. Jacalin-CD4 interaction and the proliferation of PBMC, as well as IL-2 secretion by Jurkat cells were inhibited by specific jacalin-competitive sugars.     

Ligands of CD4 inhibit the association of phospholipase Cγ1 with phosphoinositide 3 kinase in T cells: regulation of this association by the phosphoinositide 3 kinase activity

We have previously shown that CD4 ligands inhibit interleukin-2 (IL-2) production and T cell proliferation in human peripheral CD4⁺ T lymphocytes, in an MHC-independent way. Two major pathways implicated in T cell activation are inhibited by binding of CD4 ligands to the CD4 molecule, i. e. Ca²⁺ signaling by phospholipase Cγ1 (PLCγ1), and ERK-2 activation, suggesting a p21^ras inhibition. We have correlated these inhibitions with the disruption of multifunctional complexes containing PLCγ1, p120GAP and Sam68, induced by T cell activation. We report here that T cell activation through the TCR/CD3 induces an association of the phosphoinositide 3 kinase (PI3 kinase) with PLCγ1, both in peripheral CD4⁺ T lymphocytes and the HUT-78 CD4⁺ T cell line. PI3 kinase is present in the multifunctional complexes that we have described previously. Preincubation of human peripheral CD4⁺ T cells and HUT-78 CD4⁺ T cells with gp160 or a peptide analogue of the HLA class II DR molecule precludes the association of PLCγ1 with PI3 kinase. We also demonstrate, using two specific inhibitors of PI3 kinase activity (LY294002 and wortmannin), that this activity plays a key role in the association of PLCγ1 with PI3 kinase. Moreover, we demonstrate the implication of the PI3 kinase activity in the negative signal mediated by HIV gp160 binding to CD4 molecules. We propose that the products of the PI3 kinase are important mediators of the negative signaling induced by the binding of CD4 ligands to the CD4 molecule implicated in the regulation of the formation of multifunctional complexes.     

Interleukin-2 receptor β and γ chain dysregulation during the inhibition of CD4 T cell activation by human immunodeficiency virus-1 gp120

We have observed that CD4 T lymphocytes from human immunodeficiency virus (HIV)-infected patients marginally express interleukin-2 receptor (IL-2R)β and IL-2Rγ chains which are essential for IL-2 signal transduction. To analyze this observation further, we studied the influence of gp120 on the cell surface expression of IL-2Rβ and IL-2Rγ by purified CD4 lymphocytes in vitro. Cross-linking of the T cell receptors of these lymphocytes initiates entry into the cell cycle as measured by CD69 and CD71 cell surface expression and [³H]thymidine incorporation. It also induces the cell surface expression of IL-2Rβ and IL-2Rγ. We have shown that treatment of the CD4 T lymphocytes with HIV-1 gp120 before anti-CD3 stimulation impedes cell cycle progression as measured by reduced CD71 expression and inhibition of [³H]thymidine incorporation. Furthermore, cell surface expression of IL-2Rβ and IL-2Rγ subunits, which form the functional intermediate-affinity IL-2R, are significantly inhibited. More importantly, addition of exogenous IL-2 does not restore the proliferation of the CD4 T cells treated with gp120, suggesting that cells are anergic and/or that the remaining IL-2R are not functional. This is the first study of IL-2Rβ and IL-2Rγ dysregulation in the context of HIV infection and shows that CD4 is also involved in IL-2R expression.     

Inhibitory effect of interleukin-16 on interleukin-2 production by CD4+ T cells

Signalling through CD4 by human immunodeficiency virus (HIV)-1 envelope glycoprotein (gpl20) and/or anti-CD4 antibodies can promote T-cell activation and anergy. Interleukin (IL)-16 is a competence growth factor for CD4+ T cells that can induce a G0 to G1 cell cycle transition but cannot induce cell division. The receptor of this cytokine is thought to be the CD4 molecule, although the binding epitope of IL-16 differs from that of HIV. We have demonstrated that both HIV-1/gp120 and IL-16 induced CD4+ T-cell dysfunction, as indicated by suppression of mitogen-induced IL-2 production. Two anti-CD4 antibodies with different binding sites on CD4 also showed an inhibitory effect on IL-2 production. These results indicate that promotion of CD4+ T-cell anergy via the CD4 molecule does not depend on the binding sites of the CD4 ligands.     

Engagement of the CD4 receptor inhibits the interleukin-2-dependent proliferation of human T cells transformed by Herpesvirus saimiri

Infection with Herpesvirus saimiri, a tumor virus of non-human primates, transformed human CD4⁺ T cell clones to permanent interleukin (IL)-2-dependent growth without need for restimulation with antigen and accessory cells. The IL-2-dependent proliferation of these cells was dramatically inhibited by soluble anti-CD4 whole antibodies, F(ab′)₂ and Fab fragments, and also by gp 120 of human immunodeficiency virus. The inhibition was not due to cell death and could be overcome by high concentrations of exogenous IL-2. Cell surface expression of CD4, and to a lesser degree the density of the IL-2 receptor α chain, were reduced upon anti-CD4 treatment. After long lasting (>12h) incubation with anti-CD4, abundance and activity of CD4-bound p56^lck were diminished while the free fraction of p56^lck remained unchanged. Since IL-2 binding to its receptor activated only the CD4-bound fraction of p56^lck, the IL-2-induced p56^lck activity was diminished after long-term CD4 ligation. Taken together, our results suggest a cross talk between CD4- and IL-2 receptor-mediated signaling via p56^lck.     

Ligation of TAPA-1 (CD81) or major histocompatibility complex class II in co-cultures of human B and T lymphocytes enhances interleukin-4 synthesis by antigen-specific CD4+ T cells

We have previously shown that CD4⁺ T cells from allergic individuals are predisposed to producing interleukin (IL)-4 in response to allergens. IL-4 production could be modulated by antigen concentration as well as by the type of antigen-presenting cells (APC), with B lymphocytes inducing greater quantities of IL-4 than monocytes. Using this system we examined IL-4 synthesis after culture of CD4⁺ T cells with B cells, monocytes, or both, as APC in the presence of allergen and a monoclonal antibody against CD81 (TAPA-1), a member of the TM4 superfamily of proteins that regulates activation, proliferation and trafficking of B cells. Addition of anti-CD81 mAb during culture enhanced IL-4 synthesis by 2- to 70-fold over that using an isotype-matched control mAb. Furthermore, anti-CD81 mAb enhanced IL-4 synthesis in CD4⁺ T cells only when CD4⁺ T cells were cultured with B cells but not monocytes as APC, indicating that anti-CD81 mAb affected IL-4 synthesis in T cells via interactions with B cells. However, pretreatment of either population separately with anti-CD81 mAb prior to culture had no effect on subsequent IL-4 synthesis, suggesting a requirement for temporal or cooperative interactions between T and B lymphocytes. In addition, anti-CD81 mAb enhanced IL-4 production but reduced CD4⁺ T cell antigen-specific proliferation, demonstrating that IL-4 production and proliferation by CD4⁺ T cells were inversely related. Finally, mAb to major histocompatibility complex class II but not to anti-CD19 also enhanced IL-4 synthesis when B lymphocytes were used as APC. In all instances, enhancement of CD4⁺ IL-4 synthesis correlated with the presence of large cell aggregates in T-B lymphocyte cocultures. These results indicate that the capacity of B cells to induce IL-4 can be significantly enhanced by ligation of particular molecules on their surface and should aid in the design of treatments for diseases in which modulation of the cytokine profile would be beneficial.     

CD8+ T cells and not CD4+ T cells are hyporesponsive to CD28- and CD40L-mediated activation in HIV-infected subjects

T cell dysfunction in HIV-infected subjects could be the consequence of altered sensitivity of CD4⁺ or CD8⁺ T cells to various costimulatory signals. Therefore, we studied proliferation and cytokine production in highly purified CD8⁺ and CD4⁺ T cells from HIV-infected and HIV⁻ subjects, induced by co-activation via cell-bound CD80, CD86 and CD40 or by allo-activation. Regardless of the nature of the first and the costimulatory signal, CD8⁺ T cells from patients proliferated consistently less than controls, while responses from CD4⁺ T cells were similar in patients and controls. This phenomenon was observed after ligation of CD28 combined with anti-CD3 or phorbol myristate acetate (PMA), but also after allogeneic stimulation and after activation by CD40 and anti-CD3. Anti-CD3 combined with CD80 or CD86 induced a mixed Th1/Th2-type cytokine profile in both CD4⁺ and CD8⁺ T cells from controls, whereas anti-CD3 plus CD40 induced only low levels of Th2-type cytokines and no interferon-gamma (IFN-γ) in CD4⁺ T cells. Compared with controls, CD4⁺ T cells from patients produced slightly lower levels of IL-10 but equal amounts of IFN-γ, IL-4 and IL-5, while CD8⁺ T cells from patients produced less of all cytokines tested. In conclusion, responses of purified CD4⁺ T cells from HIV⁺ subjects to various costimulatory pathways are relatively intact, whereas CD8⁺ T cells are hyporesponsive at the level of proliferation and cytokine production. A generalized intrinsic CD8⁺ T cell failure might contribute to viral and neoplastic complications of HIV infection.     

Defective integration of activating signals derived from the T cell receptor (TCR) and costimulatory molecules in both CD4+ and CD8+ T lymphocytes of common variable immunodeficiency (CVID) patients

CVID is characterized by hypogammaglobulinaemia and impaired antibody production. Previous studies demonstrated defects at the T cell level. In the present study the response of purified CD4⁺ and CD8⁺ T lymphocytes to stimulation with anti-TCR monoclonal antibody (the first signal) in combination with anti-CD4 or anti-CD8, anti-CD2 and anti-CD28 MoAbs (the costimulatory signals) was investigated. Both CD4⁺ and CD8⁺ T cells from the patients showed significantly reduced IL-2 release following stimulation via TCR and costimulation via CD4 or CD8 and CD2, respectively. However, normal IL-2 production following TCR plus phorbol myristate acetate (PMA) costimulation and normal expression of an early activation marker, CD69, after TCR + CD28 stimulation indicated that TCR was able to transduce a signal. Furthermore, both IL-2 and IL-4 release were impaired in CD4⁺ lymphocytes following TCR + CD28 stimulation. In addition, stimulation via TCR + CD28 resulted in significantly decreased expression of CD40 ligand in the patients. These results suggest that the integration of activating signals derived from the TCR and costimulatory molecules is defective in CVID patients; the defect is not confined to costimulation via a single molecule, or restricted to cells producing Thl-type cytokines such as IL-2, and is expressed in both CD4⁺ and CD8⁺T cell subsets.     

Down-regulation of LFA-1-mediated T cell adhesion induced by the HIV envelope glycoprotein gp160 requires phosphatidylinositol-3-kinase activity

Human immunodeficiency virus binds to CD4⁺ T lymphocyte by the interaction, in part, between its gp120 envelope glycoprotein and the CD4 molecule. We and others have reported that the lipid kinase phosphatidylinositol-3-kinase (PI3-kinase) is associated with the CD4-p56^lck complex and can be activated by various CD4 ligands. In a previous report we showed that the gp160 envelope down-regulates lymphocyte function-associated antigen-1 (LFA-1)-dependent adhesion between CD4⁺ T cells and B cells. This down-regulation was shown to be p56^lck-dependent. Here we investigate the role of PI3-kinase in the inhibition of adhesion induced by gp160 binding to CD4. We found that gp160 activates the PI3-kinase of HUT78 CD4⁺ T cell lines in a way dependent on CD4-p56^lck association, since no activation was detected when the interaction between CD4 and p56^lck was disrupted. It was also shown, using different inhibitors of the PI3-kinase (wortmannin, Ly294002 and antisense oligonucleotides), that this lipid kinase was necessary for the down-regulation of LFA-1-mediated adhesion induced by gp160. These results strongly suggest that PI3-kinase activation induced by gp160 leads to down-regulation of LFA-1-mediated T cell adhesion to B cells. Inhibition by gp160 of cytoskeleton rearrangement-dependent, anti-CD3-mediated T cell adhesion to B cells was blocked by neutralization of PI3-kinase activity, while inhibition of cytoskeleton rearrangement-independent, Mg²⁺-induced T cell adhesion was not. These results emphasize the role of PI3-kinase in the regulation of cytoskeleton structure. It is proposed that gp160 activates both p56^lck and PI3-kinase which lead to a cytoskeleton organization unfavorable for LFA-1 function.     

Modulation of CD4 lateral interaction with lymphocyte surface molecules induced by HIV-1 gp120

CD4, a lymphocyte surface glycoprotein, serves as co-receptor for antigen with the T cell receptor (TCR). It is also the lymphocyte receptor for HIV by binding the gp120 viral envelope protein. Interaction of gp120 with CD4 is crucial for viral infection, but is not sufficient to allow viral entry into cells. Recombinant gp120 alters CD4⁺ T cell responsiveness to activation stimuli. To express its co-receptor function fully, CD4 must be laterally associated with the TCR and CD45 to form multi-receptor complexes competent to transduce potent activation signals. Here, we examine the possibility that gp120/CD4 binding alters lateral associations of CD4 with other lymphocyte surface molecules, and that assembly of abnormal multi-molecular complexes is involved in the gp120-induced CD4⁺ T cell dysfunction and in viral entry. In the absence of gp120, CD4 displayed high association with CD3, CD5, CD45RC, CD25, CD28, CD44, and CD53; weak association with CD2, CD38, CD45RB, CD62L, and CD26; and no association with CD45RA, CD45RO, CD11b, CD11a, CD54, CD7, CD48, CD98, CD59, CD55, HLA class I and class II molecules. Treatment with gp120 significantly increased CD4 association with CD3, CD45RA, CD45RB, CD59, CD38, CD26, and HLA class I, and decreased that with CD45RC. Specificity of these results were assessed at various levels. First, gp120 did not influence lateral associations displayed by other molecules, such as HLA class II. Second, the Leu3 mAb, which binds CD4 on a site overlapping the gp120 binding site, did not elicit the same CD4 lateral associations as gp120, and finally, a direct gp120/CD4 interaction was needed to induce the lateral associations, as shown by the observation that blocking the gp120/CD4 binding by the Leu3 mAb inhibited the gp120-induced associations. These results can be interpreted in several ways. gp120/CD4 interaction could trigger an inside-out signal responsible for the associations, or gp120 could induce steric modifications of CD4 that increase its affinity for the associating molecules. Alternatively, these molecules may interact directly with gp120, bridging them with CD4. It is also possible that the associations may be mediated by additional components, interacting with both gp120 and the associating surface molecule. The last hypothesis is likely for CD59, whose gp120-induced association with CD4 required the presence of serum in the co-capping assay. Since both CD59 and gp120 bind complement, the observed association could be mediated by complement components.     

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.     

CD40 ligand-positive CD8+ T cell clones allow B cell growth and differentiation

A fraction of activated CD8⁺ T cells expresses CD40 ligand (CD40L), a molecule that plays a key role in T cell-dependent B cell stimulation. CD8⁺ T cell clones were examined for CD40L expression and for their capacity to allow the growth and differentiation of B cells, upon activation with immobilized anti-CD3. According to CD40L expression, CD8⁺ clones could be grouped into three subsets. CD8⁺ T cell clones expressing high levels of CD40L (≥80% CD40L⁺ cells) were equivalent to CD4⁺ T cell clones with regard to induction of tonsil B cell proliferation and immunoglobulin (Ig) production, provided the combination of interleukin (IL)-2 and IL-10 was added to cultures. CD8⁺ T cell clones, with intermediate levels of CD40L expression (10 to 30% CD40L⁺ cells), also stimulated B cell proliferation and Ig secretion with IL-2 and IL-10. B cell responses induced by these CD8⁺ T cell clones were neutralized by blocking monoclonal antibodies specific for either CD40L or CD40. By contrast, CD40L^?? T cell clones (?5 % CD40L⁺ cells), only induced marginal B cell responses even with IL-2 and IL-10. All three clone types were able to activate B cells as shown by up-regulation of CD25, CD80 and CD86 expression. A neutralizing anti-CD40L antibody indicated that T cell-dependent B cell activation was only partly dependent on CD40-CD40L interaction. These CD40L^?? clones had no inhibitory effects on B cell proliferation induced by CD40L-expressing CD8⁺ T cell clones. Taken together, these results indicate that CD8⁺ T cells can induce B cell growth and differentiation in a CD40L-CD40-dependent fashion.     

Costimulation of CD3/TcR complex with either integrin or nonintegrin ligands protects CD4+ allergen-specific T-cell clones from programmed cell death

An optimal stimulation of CD4⁺ cells in an immune response requires not only signals transduced via the TcR/CD3 complex, but also costimulatory signals delivered as a consequence of interactions between T-cell surface-associated costimulatory receptors and their counterparts on antigen-presenting cells ‘APC). The intercellular adhesion molecule-1 ‘ICAM-1, CD54) efficiently costimulates proliferation of resting, but not antigen-specific, T cells. In contrast, CD28 and CD2 support interleukin ‘IL)-2 synthesis and proliferation of antigen-specific T cells more efficiently than those of resting T cells. The molecular basis for this differential costimulation of T cells is poorly understood. Cypress-specific T-cell clones ‘TCC) were generated from four allergic subjects during in vivo seasonal exposure to the allergen. Purified cypress extract was produced directly from fresh collected pollen and incubated with the patients' mononuclear cells. Repeated allergen stimulation was performed in T-cell cultures supplemented with purified extract and autologous APC. The limiting-dilution technique was then adopted to generate allergen-specific TCC, which were also characterized by their cytokine secretion pattern as ThO ‘IL-4 plus interferon-gamma) or Th2 ‘IL-4). Costimulation-induced proliferation or apoptosis was measured by propidium iodide cytofluorometric assay. By cross-linking cypress-specific CD4⁺ and CD8⁺ T-cell clones with either anti-CD3 or anti-CD2, anti-CD28, and anti-CD54 monoclonal antibodies, we demonstrated that CD4+ clones ‘with ThO- or Th2-type cytokine production pattern) undergo programmed cell death only after anti-CD3 stimulation, whereas costimulation with either anti-CD54 or anti-CD28 protects target cells from apoptosis. The costimulation-induced protection from apoptotic death was associated with a significant rise in IL-4 secretion in both Th0 and Th2-type clones. In contrast, cypress-specific Th0 CD8⁺ clones were more susceptible to stimulation-induced apoptosis via either anti-CD3 or anti-CD2, alone or in combination with anti-CD54 or anti-CD28, thus displaying only slight but nonsignificant modifications in the pattern of IL-4 secretion. The death-promoting costimulatory effects were not observed with highly purified normal resting CD4⁺ or CD8⁺ lymphocytes. Taken together, these results suggest that TcR engagement by an allergen in the context of functionally active APC induces activation-dependent cell death of some, perhaps less specific, cells, and this may be an important homeostatic mechanism through which functional expansion of allergen-specific T cells is regulated during an ongoing immune response.     

High-frequency activation of single CD4+ and CD8+ T cells to proliferate and secrete cytokines using anti-receptor antibodies and IL-2(1).

A high cloning efficiency single-cell culture system was developed to define the activation requirements of isolated CD4+ and CD8+ T cells to proliferate and secrete cytokines. T cells were triggered using solid-phase anti-CD3 and anti-CD4 or anti-CD8 antibodies plus rIL-2. Activation was measured by microscopic scoring of proliferation and by measurement of cytokine production using the cytokine-responsive cell lines FDC-P1, which responds to GM-CSF, IL-3, IFN-gamma and IL-4, and 32D clone 3 which responds to IL-3 only. Whilst anti-CD3 plus rIL-2 triggered only 4% of peripheral T cells to proliferate, anti-CD3 plus anti-CD8 mAb triggered about 40% of CD8+ T cells; 80% of the resultant clones secreted cytokine and 90% of these were IL-3+. Anti-CD3 plus anti-CD4 mAb triggered proliferation in about 20% of CD4+ T cells, of which 34% formed cytokine-producing clones with 47% of these secreting IL-3. In addition to responding at higher frequency, CD8+ T cells formed larger clones which produced higher levels of cytokines than CD4+ cells. Cell separation on the basis of Pgp-1 expression suggested that this culture system did not select for previously activated cells. Whereas Pgp-1+ T cells from keyhole limpet haemocyanin (KLH)-primed mice were enriched in KLH-specific cells, no significant differences were observed in the clonogenicity or cytokine-secreting capacity of Pgp-1+ and Pgp-1- T cells from normal mice.