Interaction of CD2 with its ligand, LFA-3, in human T cell proliferation

Recently, it has been demonstrated that lymphocyte function-associated Ag (LFA-3) is a natural ligand for CD2 and that this receptor-ligand interaction functions in cell-cell adhesion. In this report, we demonstrate that LFA-3 plays a role in T cell activation. L cells were transfected with human genomic DNA and sorted for expression of LFA-3. We demonstrate that LFA-3+ L cells, together with anti-CD3 mAb or with suboptimal doses of PHA, stimulate proliferation of human peripheral blood T cells. Furthermore, thymocyte proliferation was induced by LFA-3+ L cells and suboptimal doses of PHA. Proliferation was inhibited by mAb directed against either CD2 or LFA-3. Stimulation of thymocytes by the combination of PHA and LFA-3+ L cells resulted in the increased expression of the IL-2R, as well as of the surface Ag 4F2, transferrin receptor, and HLA-DR. These data support the conclusion that LFA-3 plays a role in CD2-dependent T cell activation. LFA-3 is widely distributed and is expressed on all APC and target cells. Thus, the ability of the CD2/LFA-3 interaction to costimulate with an anti-CD3 mAb suggests that the CD2/LFA-3 interaction may be involved not only in an Ag-independent alternate pathway of T cell activation but also in Ag-specific T cell activation.     

The potentiation of B lymphocyte responses through CD2/LFA-3 interactions involving erythrocytes is IL2 independent

The response of human B cells to pokeweed mitogen (PWM) stimulation is potentiated when autologous erythrocytes (E) are added to peripheral blood mononuclear cell (PBMC) cultures. This potentiation has been previously shown to be dependent on interactions between the CD2 molecule on T cells and the lymphocyte function-associated antigen 3 (LFA-3) expressed by autologous erythrocytes. Since in other experimental systems the activation of T cells by CD2/LFA-3 interactions has resulted in increased secretion of interleukin 2 (IL2), we were interested in studying the role of IL2 in PBMC cultures stimulated with PWM and autologous E. The addition of autologous E significantly depressed IL2 levels in PWM-stimulated PBMC cultures. This effect was not secondary to increased expression of IL2 receptors by activated cells, since the addition of anti-TAC antibodies did not result in a significant increase in measurable levels of IL2. The addition of anti-IL2 to PBMC failed to abrogate the potentiating effect of E and it actually further enhanced the production of IgM and IgG from cultures stimulated with PWM + E. These results suggest that the potentiation of B cell function induced by autologous E is not mediated by IL2, either directly or indirectly. It is possible that the effect of autologous E either is mediated by other interleukins or is dependent on cell-to-cell contact with directed release of IL2 and/or other lymphokines without detectable secretion to the extracellular compartment.     

Human B cell activation by autologous NK cells is regulated by CD40-CD40 ligand interaction: role of memory B cells and CD5+ B cells.

NK cells are a subpopulation of lymphocytes characterized primarily by their cytolytic activity. They are recognized as an important component of the immune response against virus infection and tumors. In addition to their cytolytic activity, NK cells also participate either directly or indirectly in the regulation of the ongoing Ab response. More recently, it has been suggested that NK cells have an important role in the outcome of autoimmune diseases. Here, we demonstrate that human NK cells can induce autologous resting B cells to synthesize Ig, including switching to IgG and IgA, reminiscent of a secondary Ab response. B cell activation by the NK cell is contact-dependent and rapid, suggesting an autocrine B cell-regulated process. This NK cell function is T cell-independent, requires an active cytoplasmic membrane, and is blocked by anti-CD40 ligand (anti-CD154) or CD40-mIg fusion protein, indicating a critical role for CD40-CD40 ligand interaction. Depletion studies also demonstrate that CD5+ B cells (autoreactive B-1 cells) and a heterogeneous population of CD27+ memory B cells play a critical role in the Ig response induced by NK cells. The existence of this novel mechanism of B cell activation has important implications in innate immunity, B cell-mediated autoimmunity, and B cell neoplasia.     

Th2-dependent B cell responses in the absence of CD40-CD40 ligand interactions

CD40 is thought to play a central role in T cell-dependent humoral responses through two distinct mechanisms. CD4+ T helper cells are activated via CD40-dependent Ag presentation in which CD80/CD86 provides costimulation through CD28. In addition, engagement of CD40 on B cells provides a direct pathway for activation of humoral responses. We used a model of adenovirus-mediated gene transfer of beta-galactosidase (lacZ) into murine lung to evaluate the specific CD40-dependent pathways required for humoral immunity at mucosal surfaces of the lung. Animals deficient in CD40L failed to develop T and B cell responses to vector. Activation of Th2 cells, which normally requires CD40-dependent stimulation of APCs, was selectively reconstituted in CD40 ligand-deficient mice by systemic administration of an Ab that is agonistic to CD28. Surprisingly, this resulted in the development of a functional humoral response to vector as evidenced by formation of germinal centers and production of antiadenovirus IgG1 and IgA that neutralized and prevented effective readministration of vector. The CD28-dependent B cell response required CD4+ T cells and was mediated via IL-4. These studies indicate that CD40 signals to the B cells are not necessary for CD4+ Th2 cell-dependent humoral responses to be generated.     

CD36 is differentially expressed on B cell subsets during development and in responses to antigen

Of a number of mAbs made by immunization with sort-purified marginal zone (MZ) B cells, one was shown to recognize the mouse scavenger receptor CD36. Although CD36 is expressed by most resting MZ B cells and not by follicular and B1 B cells, it is rapidly induced on follicular B cells in vitro following TLR and CD40 stimulation. In response to T-independent and T-dependent Ag challenge, we found that CD36 was expressed on IgM+ plasma cells, but down-regulated on isotype-switched plasma cells in vivo. Although development, localization, and phenotype of MZ B cells in CD36-/- mice appeared normal, there was a minor block in the transitional stages of mature B cell development. In both primary and secondary Ab responses to heat-killed Streptococcus pneumoniae (R36A strain), both phosphoryl choline (PC)-specific IgM and IgG levels in CD36-/- mice were slightly reduced compared with wild-type mice. In addition, mice deficient in both TLR2 and CD36 produced significantly reduced levels of anti-PC IgG titers than those of single gene-deficient mice, suggesting that they may cooperate in an anti-PC Ab response. Collectively, these results show that CD36 does not affect the development of B cells, but modulates both primary and secondary anti-PC Ab responses during S. pneumoniae infection similarly to TLR2.     

The CD40-CD154 interaction in B cell-T cell liaisons

The CD40 receptor is expressed constitutively on B lymphocytes, for which it provides important signals regulating clonal expansion, antibody production and isotype switching, as well as the development of humoral memory. The major source of CD154, the ligand for CD40, is activated T lymphocytes. Interactions between CD40 and CD154 provide a number of signals that play important roles in regulating the complex and multifactorial interactions between these two major cell types of the adaptive immune response. Understanding both the biological effects of this receptor-ligand interaction, as well as how CD40 signaling pathways are controlled, adds to our detailed picture of the complex interplay between B and T cells.     

Monoclonal antibodies against LFA-1 or its ligand ICAM-1 accelerate CD2 (T11.1 + T11.2)-mediated T cell proliferation

Activation of human-purified T cells can be mediated by pairwise combinations of monoclonal antibodies directed against T11.1 and T11.2 epitopes on the CD2 molecule. Monoclonal antibodies (mAbs) reactive with either the alpha and beta chains of the lymphocyte-function-associated antigen-1 (LFA-1) molecule or one of its ligands, intercellular adhesion molecule-1 (ICAM-1), were found to accelerate anti-CD2-induced proliferation. This effect was seen on thymocytes and resting or preactivated T cells (phytohemagglutinin blasts and alloproliferative T cell clones) and could be observed, following the introduction of anti-LFA-1 or -ICAM-1 mAbs, up to 50 hr after the CD2 stimulatory signal. This effect was equally abrogated by 55 kDa anti-interleukin-2 (IL-2) receptor mAb, but neither the expression of IL-2 receptor nor the production of IL-2 was modified. The effects of anti-LFA-1 or anti-ICAM-1 on T cell activation through the CD2 pathway were therefore opposite to those observed in the CD3 pathway, where both mAbs strongly delayed T cell proliferation.     

Priming in the presence of IL-10 results in direct enhancement of CD8+ T cell primary responses and inhibition of secondary responses

Although IL-10 acts as an inhibitory cytokine for APC and CD4(+) T cell function, its effects on CD8(+) T cells are unclear. Additionally, little is known about whether initial priming in the presence of IL-10 can have long-lasting effects and influence subsequent CD8(+) T cell responses that occur in the absence of the cytokine. In the present study, we clarified the role of IL-10 during primary responses and examined whether exposure to IL-10 during initial priming of CD8(+) T cells impacted secondary responses. To determine the effect of IL-10 on Ag-specific T cell responses, peptide-pulsed IL-10R2(-/-) splenic dendritic cells were used to prime T cells from OT-I CD8(+) TCR transgenic mice. During the primary response, the presence of IL-10 resulted in enhancement of CD8(+) T cell numbers without detectable alterations in the kinetics or percentage of cells that underwent proliferation. A modest increase in survival, not attributable to Bcl-2 or Bcl-x(L), was also observed with IL-10 treatment. Other parameters of CD8(+) T cell function, including IL-2, IFN-gamma, TNF-alpha, and granzyme production, were unaltered. In contrast, initial exposure to IL-10 during the primary response resulted in decreased OT-I expansion during secondary stimulation. This was accompanied by lowered IL-2 levels and reduced percentages of proliferating BrdU(+) cells and OT-I cells that were CD25(high). IFN-gamma, TNF-alpha, and granzyme production were unaltered. These data suggest that initial exposure of CD8(+) T cells to IL-10 may be temporarily stimulatory; however, programming of the cells may be altered, resulting in diminished overall responses.     

T cell surface molecules regulating noncognate B lymphocyte activation. Role of CD2 and LFA-1.

A central event in humoral responses is the Ag-mediated interaction of Th cells and B cells. This interaction leads to the activation of both cell types and results in cytokine secretion by the T cells and proliferation and secretion of Ig by the B cells. The proliferative and differentiative responses of B cells are dependent on contact-mediated signals and cytokines provided by the activated Th cells. Although the role of cytokines in B cell activation and differentiation is understood, the nature of the signals delivered by the activated Th cells and the molecules involved in this process are not known. In this study we have examined Ag-mediated "cognate" T-B cell interactions as well as B cell activation induced by contact with preactivated and fixed Th lymphocytes. Our results indicate that both the T cell surface molecules lymphocyte function associated Ag-1 and CD2 are important in the activation of T cells by Ag presented by B lymphocytes. This indicates that B cells have similar characteristics as other APC. However, once the T cells are activated, contact-mediated stimulation of resting B lymphocytes (the noncognate phase) is dependent on CD2 but not lymphocyte function associated Ag-1. Two lines of evidence indicate this; first, it is inhibited by blocking of CD2 on the T cells and, second, such stimulation is not efficiently mediated by a CD2- Th cell line. Thus, CD2 plays an obligatory role at several discrete stages of T cell-mediated activation of resting B lymphocytes.     

Mechanism of induction of NK activation by 2B4 (CD244) via its cognate ligand

We have previously shown that coincubation of purified B cells with IL-2-propagated NK cells can result in the induction of IL-13 mRNA and that the induction requires the presence of CD48 on B cells and 2B4 on NK cells. Because both of these molecules are expressed on NK cells, it is surprising that very little IL-13 mRNA can be detected in the absence of B cells. We have now found that incubation of NK cells on plates containing immobilized anti-CD48 Abs results in the clustering of CD48 and colocalization with 2B4 on the same cell. This colocalization, together with the requirement for SAP, the signal transducer for 2B4, is necessary for the induction of IL-13 mRNA expression. Activation of NK cell via CD48 on another cell may require a similar ability to alter the configuration of 2B4 to activate downstream signaling. By the use of double CD2/2B4 knockout mice, we have also shown that the induction of NK cell activation by anti-CD48 or by B cells is not due to the release of inhibitory effects of 2B4.     

CD4+CD25+ T cells lyse antigen-presenting B cells by Fas-Fas ligand interaction in an epitope-specific manner

Suppression by regulatory T cells is now acknowledged to play a key role in the down-regulation of T cell responses to foreign and self Ags. In addition to the naturally occurring CD4(+)CD25(+) population, several subtypes of induced regulatory cells have been reported, but their mechanisms of action remain unclear. Conversely, cytotoxic CD4(+) cells that lyse cells presenting their cognate peptide have been described, but their potential role in immunoregulation remains to be delineated. A CD4(+) T cell line derived from BALB/c mice immunized with peptide 21-35, containing a major T cell epitope of a common allergen, Dermatophagoides pteronyssinus group 2 allergen, was found to lyse the Ag-presenting WEHI cell line via Fas-Fas ligand and only in the presence of the cognate peptide. Cytolytic activity was likewise shown for other T cell lines and occurred even after a single cycle of in vitro stimulation. Moreover, T cells that efficiently lysed WEHI cells were unresponsive to stimulation with their cognate Ag and were dependent on IL-2 for growth and survival, which was reflected in a constitutive expression of CD25 independently of activation status. Proliferating B cells were also killed by the CTLs. By lysing Ag-presenting B cells in an epitope-specific manner, the nonproliferating CTLs were shown to down-regulate the proliferation of bystander T cells. These data demonstrate that cytotoxic CD4(+)CD25(+) T cells that lack proliferation capacities have the potential to down-regulate an immune response by killing Ag-presenting B cells. This could represent an important and specific down-regulatory mechanism of secondary immune responses in vivo.     

The functional significance, distribution, and structure of LFA-1, LFA-2, and LFA-3: cell surface antigens associated with CTL-target interactions

Three cell surface antigens associated with the cytolytic T lymphocyte(CTL)-target cell interaction were identified by generation of monoclonal antibodies (MAb) against OKT4+, HLA-DR-specific CTL and selection for inhibition of cytolysis in a 51Cr-release assay. These MAb block cytolysis by both OKT4+ and OKT8+ CTL and the proliferative responses to PHA and the mixed lymphocyte response (MLR). LFA-1 is an antigen widely distributed on lymphoid tissues and is composed of two polypeptides of 177,000 and 95,000 Mr on all cell types studied. Anti-LFA-1 MAb block NK cell-mediated cytolysis in addition to T lymphocyte-mediated cytotoxicity and proliferation. LFA-2 (Mr = 55,000 to 47,000), a determinant on the sheep red blood cell receptor, is expressed by T cells but not B cells and appears specific for T cell functions. LFA-3 (Mr = 60,000) is a widely distributed antigen present on both hematopoietic and nonhematopoietic tissues and appears to only be involved in T cell functions. MAb to LFA-1 and LFA-2 inhibit function by binding to effector cell surface molecules, whereas anti-LFA-3 MAb appear to block by binding to the target cells. Together with previously described molecules, LFA-1, LFA-2, and LFA-3 demonstrate the complexity of CTL-mediated cytotoxicity at the molecular level.     

The human LFA-3 gene is located at the same chromosome band as the gene for its receptor CD2

Lymphocyte function-associated antigen 3 (LFA-3) is a widely distributed cell surface glycoprotein that has been assigned a role in cell-cell adhesion on the basis of its capacity to bind to the T-lymphocyte CD2 antigen. The amino acid sequences of the extracellular domains of these two antigens, predicted from their cDNA sequences, show significant similarities, and both are members of the immunoglobulin supergene family. In this communication, a probe prepared from LFA-3 cDNA has been used in Southern blot analyses of somatic cell hybrids and in in situ hybridization to assign the LFA-3 gene to the human chromosome band 1p13. This is the same location previously assigned to CD2. Thus the LFA-3 and CD2 genes have probably arisen by duplication of a common evolutionary precursor. These genes therefore represent a further instance in which related members of the immunoglobulin superfamily are located in adjacent regions of the genome.     

p53 interaction with JMJD3 results in its nuclear distribution during mouse neural stem cell differentiation

Conserved elements of apoptosis are also integral components of cellular differentiation. In this regard, p53 is involved in neurogenesis, being required for neurite outgrowth in primary neurons and for axonal regeneration in mice. Interestingly, demethylases regulate p53 activity and its interaction with co-activators by acting on non-histone proteins. In addition, the histone H3 lysine 27-specific demethylase JMJD3 induces ARF expression, thereby stabilizing p53 in mouse embryonic fibroblasts. We hypothesized that p53 interacts with key regulators of neurogenesis to redirect stem cells to differentiation, as an alternative to cell death. Specifically, we investigated the potential cross-talk between p53 and JMJD3 during mouse neural stem cell (NSC) differentiation. Our results demonstrated that JMJD3 mRNA and protein levels were increased early in mouse NSC differentiation, when JMJD3 activity was readily detected. Importantly, modulation of JMJD3 in NSCs resulted in changes of total p53 protein, coincident with increased ARF mRNA and protein expression. ChIP analysis revealed that JMJD3 was present at the promoter and exon 1 regions of ARF during neural differentiation, although without changes in H3K27me3. Immunoprecipitation assays demonstrated a direct interaction between p53 and JMJD3, independent of the C-terminal region of JMJD3, and modulation of p53 methylation by JMJD3-demethylase activity. Finally, transfection of mutant JMJD3 showed that the demethylase activity of JMJD3 was crucial in regulating p53 cellular distribution and function. In conclusion, JMJD3 induces p53 stabilization in mouse NSCs through ARF-dependent mechanisms, directly interacts with p53 and, importantly, causes nuclear accumulation of p53. This suggests that JMJD3 and p53 act in a common pathway during neurogenesis.     

The interaction of the retina cell surface N- acetylgalactosaminylphosphotransferase with an endogenous proteoglycan ligand results in inhibition of cadherin-mediated adhesion

We have previously shown that the binding to cells of a monoclonal antibody directed against the chick neural retina N- acetylgalactosaminylphosphotransferase (GalNAcPTase) results in inhibition of cadherin-mediated adhesion and neurite outgrowth. We hypothesized that the antibody mimics the action of an endogenous ligand. Chondroitin sulfate proteoglycans (CSPGs) are potential ligands because they inhibit adhesion and neurite outgrowth and are present in situ at barriers to neuronal growth. We therefore assayed purified CSPGs for their ability to inhibit homophilic cadherin-mediated adhesion and neurite outgrowth, as well as their ability to bind directly to the GalNAcPTase. A proteoglycan with a 250-kD core protein following removal of chondroitin sulfate chains (250-kD PG) inhibits cadherin-mediated adhesion and neurite outgrowth whether presented as the core protein or as a proteoglycan monomer bearing chondroitin sulfate. A proteoglycan with a 400-kD core protein is not inhibitory in either core protein or monomer form. Treatment of cells with phosphatidylinositol-specific phospholipase C, which removes cell surface GalNAcPTase, abolishes this inhibitory effect. Binding of the 250-kD core protein to cells is competed by the anti-GalNAcPTase antibody 1B11, suggesting that 1B11 and the 250-kD core protein bind to the same site or in close proximity. Moreover, soluble GalNAcPTase binds to the immobilized 250-kD core protein but not to the immobilized 400-kD core protein. Concomitant with inhibition of cadherin mediated adhesion, binding of the 250-kD core protein to the GalNAcPTase on cells results in the enhanced tyrosine phosphorylation of beta-catenin and the uncoupling of N-cadherin from its association with the cytoskeleton. Moreover, the 250-kD PG is present in embryonic chick retina and brain and is associated with the GalNAcPTase in situ. We conclude that the 250-kD PG is an endogenous ligand for the GalNAcPTase. Binding of the 250-kD PG to the GalNAcPTase initiates a signal cascade, involving the tyrosine phosphorylation of beta-catenin, which alters the association of cadherin with the actin-containing cytoskeleton and thereby inhibits adhesion and neurite outgrowth. Regulation of the temporal and spatial expression patterns of each member of the GalNacPTase/250-kD PG interactive pair may create opportunities for interaction that influence the course of development through effects on cadherin-based morphogenetic processes.