The T lymphocyte glycoprotein CD2 binds the cell surface ligand LFA-3

CD2 (known also as T11 (ref. 1), LFA-2 (ref. 2) and the erythrocyte rosette receptor (ref. 3] is a functionally important T lymphocyte surface glycoprotein of relative molecular mass 50,000 to 58,000 (Mr 50-58 K) which appears early in thymocyte ontogeny and is present on all mature T cells. Monoclonal antibodies to CD2 inhibit cytotoxic T-lymphocyte (CTL)-mediated killing by binding to the T lymphocyte and blocking adhesion to the target cell. Such antibodies also inhibit T helper cell responses including antigen-stimulated proliferation, interleukin-2 (IL-2) secretion, and IL-2 receptor expression. Certain combinations of monoclonal antibodies to CD2 epitopes trigger proliferation of peripheral blood T lymphocytes, cytotoxic effector function and expression of IL-2 receptors by thymocytes, resulting in thymocyte proliferation in the presence of exogenous IL-2 (ref. 11). These findings suggest that CD2 can function in signalling as well as being an adhesion molecule. To understand the role of CD2 in T-cell adhesion and activation, it is essential to define its natural ligand. Our previous observation that purified CD2 inhibits rosetting of T lymphocytes with sheep erythrocytes and can be absorbed by sheep erythrocytes suggested it also might bind with detectable affinity to human cells. We now report that CD2 binds to a cell-surface antigen known as lymphocyte function-associated antigen-3 (LFA-3) with high affinity, and can mediate adhesion of lymphoid cells via interaction with LFA-3.     

CD2-mediated adhesion facilitates T lymphocyte antigen recognition function

The CD2 T lymphocyte-surface glycoprotein serves to mediate adhesion between T lymphocytes and their cognate cellular partners which express the specific ligand LFA-3. In addition, CD2 by itself or in conjunction with T-cell receptor stimulation, transduces signals resulting in T-lymphocyte activation. One or both of these functions seems to be physiologically important, given that certain anti-CD2 monoclonal antibodies block T-cell activation and that antigen-responsive memory T cells express a high level of CD2 relative to virgin T cells, which are largely antigen-unresponsive. Nevertheless, the contribution of the individual CD2 functions in T-cell responses has not been independently examined. To this end, human CD2 complementary DNAs encoding an intact LFA-3-binding adhesion domain, but lacking a functional cytoplasmic signal transduction element (CD2trans-), were introduced into an ovalbumin-specific, I-Ad restricted murine T-cell hybridoma. The antigen-specific response of T hybridoma cells expressing human CD2trans- protein was enhanced up to 400% when the human LFA-3 ligand was introduced into the I-Ad expressing murine antigen-presenting cells. In contrast, no augmentation was observed if human LFA-3 was absent or expressed on a third-party cell lacking the I-Ad restriction element. These results directly demonstrate the functional significance of adhesion events mediated between CD2 on the antigen-responsive T lymphocyte and LFA-3 on the presenting cell in optimizing antigen-specific T-cell activation.     

Association of CD2 and CD45 on human T lymphocytes

At least two membrane receptors have been defined through which human T lymphocytes can be induced to proliferate and differentiate, namely the CD3-Ti antigen receptor complex and the CD2 molecule. Monoclonal antibodies directed at either CD2 or CD3 induce intracellular second messenger production and subsequent protein phosphorylation. On most human non-B lymphocytes, CD3-Ti and CD2 are coexpressed and seem to be functionally interrelated. But there are minor subpopulations in which these receptor systems can transduce signals despite a mutually exclusive expression, indicating that CD3-Ti and CD2 can act independently of each other. This view is supported by the finding that most monoclonal antibodies directed at the CD45 molecules are strongly co-mitogenic with CD2 but not CD3 monoclonal antibodies. As the intracytoplasmic domains of CD45 have tyrosine phosphatase activity these functional effects could be explained by a physical association between CD2 and CD45. Using chemical crosslinking techniques, we now show that CD45 is linked to CD2 on the surface of human T lymphocytes.     

Functional cloning of ICAM-2, a cell adhesion ligand for LFA-1 homologous to ICAM-1

The leukocyte adhesion molecule LFA-1 mediates a wide range of lymphocyte, monocyte, natural killer cell, and granulocyte interactions with other cells in immunity and inflammation. LFA-1 (CD11a/CD18) is a receptor for intercellular adhesion molecule 1 (ICAM-1, CD54), a surface molecule which is constitutively expressed on some tissues and induced on other in inflammation. Induction of ICAM-1 on epithelial cells, endothelial cells and fibroblasts mediates LFA-1-dependent adhesion of lymphocytes. Several lines of evidence have suggested the existence of a second LFA-1 ligand: homotypic adhesion of one cell line was inhibited by a monoclonal antibody to LFA-1, but not by one to ICAM-1; there exists an LFA-1-dependent, ICAM-1-independent pathway of adhesion to endothelial cells; and also, there are some types of target cells in which LFA-1-dependent T-lymphocyte adhesion and lysis are independent of ICAM-1. We have cloned this second ligand, designated ICAM-2, using a novel method for identifying ligands of adhesion molecules. ICAM-2 is an integral membrane protein with two immunoglobulin-like domains, whereas ICAM-1 has five. Remarkably, ICAM-2 is much more closely related to the two most N-terminal domains of ICAM-1 (34% identity) than either ICAM-1 or ICAM-2 is to other members of the immunoglobulin superfamily, demonstrating the existence of a subfamily of immunoglobulin-like ligands that bind the same integrin receptor.     

ICAM-1 a ligand for LFA-1-dependent adhesion of B, T and myeloid cells

Cell-cell adhesion is essential for many immunological functions. The LFA-1 molecule, a member of a superfamily of adhesion molecules, participates in adhesion which is critical to the function of each of the three major subsets of leukocytes: lymphocytes, monocytes and granulocytes. Putative LFA-1 ligands have been identified functionally in different laboratories using three different monoclonal antibodies that inhibit LFA-1-mediated leukocyte adhesion in particular model systems; however, there may be more than one LFA-1 ligand. We have directly compared the three relevant monoclonal antibodies, and show that each binds to the same molecule, intercellular-adhesion molecule-1 (ICAM-1). Most important, B, T and myeloid cells adhere specifically to purified ICAM-1-coated surfaces; such adhesion has distinctive requirements for Mg2+ and Ca2+. This constitutes biochemical evidence that ICAM-1 functions as a ligand for LFA-1-dependent adhesion by a variety of leukocytes.     

Enhancement of T cell immune response to lymphoma cells by CD28/CD80 alternative pathway

The aim of this study is to determine whether myeloma and lymphoma tumor cells can function as efficient antigen presenting cells (APC) to enhance the co-stimulation of T cells. The expression and function of T cell activation-related molecules, especially CD80, CD28, CD40 and CD40 ligand (CD40L), were studied on nine human myeloma cell lines (HMCL) and two B lymphoma cell lines. In the case of myeloma cell lines, the cells generally lacked CD80 antigen and expressed a heterogeneous CD40, and the expressions of CD40 and CD80 molecules could not be induced by either CD28 stimulation or CD40 ligation. Conversely, in the two B lymphoma cell lines, tumor cells expressed both CD80 and CD40 to some extent. CD28 stimulation could obviously increase the expression of CD80, CD40 and some adhesion molecules, and therefore generate a more efficient anti- tumor cell immunity. In conclusion, CD28 stimulation combined with CD40 antibody or soluble CD40 ligand may be a promising immunotherapeutic approach to B lymphoma.     

ICAM, an adhesion ligand of LFA-1, is homologous to the neural cell adhesion molecule NCAM

Antigen-specific cell contacts in the immune system are strengthened by antigen-nonspecific interactions, mediated in part by lymphocyte-function associated (LFA) antigens. The LFA-1 antigen is widely expressed on cells of haematopoietic origin and is a major receptor of T cells, B cells and granulocytes. LFA-1 mediates the leukocyte adhesion reactions underlying cytolytic conjugate formation, helper T-cell interactions, and antibody-dependent killing by natural killer cells and granulocytes. Recently, ICAM-1 (intercellular adhesion molecule-1) has been defined as a ligand for LFA-1. Monoclonal antibodies to ICAM-1 block T lymphocyte adhesion to fibroblasts and endothelial cells and disrupt the interaction between cytotoxic T cells and target cells. In addition, purified ICAM-1 reconstituted into artificial membranes binds LFA-1+ cells. ICAM-1 is found on leukocytes, fibroblasts, epithelial cells and endothelial cells and its expression is regulated by inflammatory cytokines. LFA-1 has been placed in the integrin family of cell surface receptors by virtue of the high sequence similarity between the LFA-1 and integrin beta chains. The adhesion ligands of the integrin family are glycoproteins bearing the Arg-Gly-Asp (RGD) sequence motif, for example, fibronectin, fibrinogen, vitronectin and von Willebrand factor. Here we show that a complementary DNA clone ICAM-1 contains no RGD motifs, but instead is homologous to the neural cell adhesion molecule NCAM.     

The CD2 antigen associates with the T-cell antigen receptor CD3 antigen complex on the surface of human T lymphocytes

T lymphocytes can be activated by various stimuli directed either against the T-cell antigen receptor-CD3 antigen complex (Ti-CD3) or the CD2 molecule; see ref. 1 for a review. Activation signals generated by antigen binding to the antigen-specific alpha/beta heterodimer (Ti) are thought to be transduced via the invariant CD3 gamma, epsilon and delta chains, and the associated zeta and eta subunits. The physiological role of the interaction of CD2 with its homologous cell-surface associated ligand LFA-3 remains to be fully elucidated. It has been suggested that CD2 regulates an antigen-independent pathway of activation or that signals delivered via CD2 are an integral part of the antigen-specific pathway. Several recent studies have indicated a requirement for the Ti-CD3 complex in CD2 signalling. Thus, mutant T-cell lines expressing CD2, but not Ti-CD3, on the cell surface cannot be activated via the CD2 molecules. Functional interaction between the Ti-CD3 complex and the CD2 antigen suggests that these T-lymphocyte cell-surface structures are physically associated. Here we use a digitonin-based solubilization procedure to explore this possibility and show that 40% of the cell-surface CD2 molecules can be specifically co-precipitated in association with the Ti-CD3 complex.     

Hax1-mediated processing of HtrA2 by Parl allows survival of lymphocytes and neurons

Cytokines affect a variety of cellular functions, including regulation of cell numbers by suppression of programmed cell death. Suppression of apoptosis requires receptor signalling through the activation of Janus kinases and the subsequent regulation of members of the B-cell lymphoma 2 (Bcl-2) family. Here we demonstrate that a Bcl-2-family-related protein, Hax1, is required to suppress apoptosis in lymphocytes and neurons. Suppression requires the interaction of Hax1 with the mitochondrial proteases Parl (presenilin-associated, rhomboid-like) and HtrA2 (high-temperature-regulated A2, also known as Omi). These interactions allow Hax1 to present HtrA2 to Parl, and thereby facilitates the processing of HtrA2 to the active protease localized in the mitochondrial intermembrane space. In mouse lymphocytes, the presence of processed HtrA2 prevents the accumulation of mitochondrial-outer-membrane-associated activated Bax, an event that initiates apoptosis. Together, the results identify a previously unknown sequence of interactions involving a Bcl-2-family-related protein and mitochondrial proteases in the ability to resist the induction of apoptosis when cytokines are limiting.     

Recognition of cluster of differentiation 1 antigens by human CD4-CD8-cytolytic T lymphocytes

Human cluster-of-differentiation 1 (CD1) is a family of cell surface glycoproteins of unknown function expressed on immature thymocytes, epidermal Langerhans cells and a subset of B lymphocytes. Three homologous proteins, CD1a, b and c, have been defined serologically, and the CD1 gene locus on human chromosome 1 contains five potential CD1 genes. Analysis of the predicted amino-acid sequences of CD1 molecules reveals a low but significant level of homology to major histocompatibility complex (MHC) class I and class II molecules, and, like MHC class I molecules, CD1 molecules are associated non-covalently with beta 2-microglobulin. These structural similarities to known antigen-presenting molecules, together with the expression of CD1 on cells capable of antigen presentation, suggest a role for CD1 molecules in antigen recognition by T cells. Here we demonstrate the specific recognition of CD1a by a CD4-CD8- alpha beta T-cell receptor (TCR) expressing cytolytic T lymphocyte (CTL) line and the specific recognition of CD1c by a CD4-CD8- gamma delta TCR CTL line. The interaction of CD1-specific CTLs with CD1+ target cells appeared to involve the CD3-TCR complex, and did not show evidence of MHC restriction. These results suggest that for a subset of T cells, CD1 molecules serve a function analogous to that of MHC class I and II molecules.     

Productive dual infection of human CD4+ T lymphocytes by HIV-1 and HHV-6

Although infection by HIV-1 has been implicated as the primary cause of AIDS and related disorders, cofactorial mechanisms may be involved in the pathogenesis of the disease. For example, several viruses commonly detected in AIDS patients and capable of transactivating the long terminal repeat of HIV-1, such as herpesviruses, papovaviruses, adenoviruses and HTLV-I have been suggested as potential cofactors. Another candidate is human herpesvirus-6 (HHV-6, originally designated human B-lymphotropic virus), which has not only been identified in most patients with AIDS by virus isolation, DNA amplification techniques and serological analysis, but is also predominantly tropic and cytopathic in vitro for CD4+ T lymphocytes. Here we demonstrate that HHV-6 and HIV-1 can productively co-infect individual human CD4+ T lymphocytes, resulting in accelerated HIV-1 expression and cellular death. We also present evidence that HHV-6 transactivates the HIV-1 long terminal repeat (LTR). These observations indicate that HHV-6 might contribute directly or indirectly to the depletion of CD4+ T cells in AIDS.     

T3-Ti receptor triggering of T8+ suppressor T cells leads to unresponsiveness to interleukin-2

T3-associated disulphide linked heterodimers (Tin) comprised of clonally unique alpha-chains of molecular weight (MW) 49,000-54,000 and beta chains of MW 43,000 have been identified as the antigen receptors on human cytotoxic effector and inducer T-lymphocytes. Crosslinking of Ti molecules by either the appropriate nominal antigen/MHC specificity or anti-clonotypic monoclonal antibody results in clonal expansion of such cells via induction of IL-2 receptor expression, endogenous IL-2 release and IL-2-IL-2 receptor interaction. To determine whether analogous antigen receptor molecules and autocrine growth mechanisms are utilized by suppressor T-cells, we produced an anti-clonotypic monoclonal antibody against a non-cytotoxic T8+ suppressor T-cell, T8AC6, which defines a T3-associated disulphide-linked heterodimer of similar molecular weight to the above clonotypes. We find that Te-Ti triggering of suppressor clones (T8AC6, T8AC7 or T8RW) does not result in IL-2 production or T-cell proliferation and in contrast to inducer clones, also leads to a transient IL-2 unresponsive state. We suggest that such T3-Ti receptor mediated autoregulation of suppressor T-cell growth is necessary in the facilitation of initial inducer T-cell activation following antigenic perturbation.     

Thy-1-mediated T-cell activation requires co-expression of CD3/Ti complex

In addition to monoclonal antibodies against the CD3 (T3)-T-cell antigen receptor (CD3/Ti) complex, several other monoclonals directed towards distinct cell surface structures on human (CD2 (T11) and Tp44) and murine (Thy-1, TAP, and Ly-6) T lymphocytes are capable of activating T cells. It has been proposed that such structures may function as alternative pathways of stimulation. To examine directly whether any relationship exists between Thy-1-dependent activation phenomena and T-cell activation mediated through the CD3/Ti complex, we have transfected several CD3/Ti- variants of the human T-cell line Jurkat with the murine Thy-1.2 gene. Our data indicate that in CD3/Ti-, Thy-1.2+ transfectants, monoclonal antibodies against Thy-1.2 can induce a rise in cytoplasmic free calcium ([Ca2+]i), but fail to stimulate interleukin-2 (IL-2) production. The only defect in these variant cell lines responsible for the inability to produce IL-2 in response to Thy-1 stimulation was in the expression of the CD3/Ti complex, because replacement of defective Ti alpha- or beta-chain genes reconstributed both surface expression of CD3/Ti and responsiveness to Thy-1 in the IL-2 production assay.     

Recognition of H-2 domains by cytotoxic T lymphocytes

The polymorphic major histocompatibility antigens (H-2) have a crucial role in the activation of antigen-specific T lymphocytes. Thus, H-2 antigens are not only recognized by allogenic lymphocytes leading to generation of cytotoxic T lymphocytes (CTLs), but it has also been demonstrated that in syngeneic systems most T cells are only able to recognize foreign antigens in conjunction with their own MHC (major histocompatibility complex) antigens. This phenomenon, termed H-2 restriction, may be the key to our understanding to the biological function of MHC antigens. It is not clear whether recognition by T cells of H-2 on a molecular level is confined to particular domains on the H-2 molecule, nor whether the same polymorphic H-2 sites, which are characterized by antibodies, are recognized by allogeneic as well as by H-2 restricted syngeneic CTLs. Previous findings indicate the existence of at least two major polymorphic domains on the H-2Kk molecule as defined by antibodies. Here we show the existence of CTLs with specifity for these polymorphic domains, and the preferential recognition of a particular domain by both alloreactive as well as H-2 restricted CTLs.     

Restricted recognition of beta 2-microglobulin by cytotoxic T lymphocytes

Recognition of foreign antigen by cytotoxic T lymphocytes (CTL) is restricted by class I major histocompatibility complex (MHC) products. Class I heavy chains (relative molecular mass (Mr) 45,000-48,000) are reversibly and noncovalently associated with beta 2-microglobulin (beta 2M, Mr = 12,000). Cells expressing human or murine class I heavy chains can exchange their native beta 2M for exogenously added free beta 2M, which is present in serum. Two allelic forms of beta 2M exist among the common laboratory mouse strains, beta 2M-A and beta 2M-B, which are represented in BALB and C57BL mice, respectively. The two forms differ at a single amino acid at position 85, the gene (beta 2m) is located on chromosome 2 linked to a minor histocompatibility (H) region, H-3. It has been proposed that one of the H-3 loci is identical with beta 2m, and that CTL raised across certain H-3 incompatibilities are actually specific for beta 2M. Here we describe CTL raised in such a combination which recognize endogenous as well as exogenous beta 2M-B in the context of H-2Kb. This represents a unique case of CTL recognition, as CTL usually recognize antigens inserted into the membrane, and it is the first molecular identification of the product of a minor H locus.     

CD1b restricts the response of human CD4-8- T lymphocytes to a microbial antigen.

Molecules encoded by the human CD1 locus on chromosome 1 (ref. 33) are recognized by selected CD4-8- T-cell clones expressing either alpha beta or gamma delta T-cell antigen receptors. The known structural resemblance of CD1 molecules to antigen-presenting molecules encoded by major histocompatibility complex (MHC) genes on human chromosome 6 (refs 3, 4, 34, 35), suggested that CD1 may represent a family of antigen-presenting molecules separate from those encoded in the MHC. Here we report that the proliferative and cytotoxic responses of human CD4-8- alpha beta TCR+ T cells specific for Mycobacterium tuberculosis can be restricted by CD1b, one of the four identified protein products of the CD1 locus. The responses of these T cells to M. tuberculosis seemed not to involve MHC encoded molecules, but were absolutely dependent on the expression of CD1b by the antigen-presenting cell and involved an antigen processing requirement similar to that seen in MHC class II-restricted antigen presentation. These results provide, to our knowledge, the first direct evidence for the proposed antigen-presenting function of CD1 molecules and suggest that the CD1 family plays a role in cell-mediated immunity to microbial pathogens.     

Peptide-dependent recognition of H-2Kb by alloreactive cytotoxic T lymphocytes

Antigen-specific T lymphocytes appear to recognize foreign antigens in the form of peptide fragments presented within the antigen-binding groove of class I or class II molecules encoded by the major histocompatibility complex (MHC). Alloreactive T cells also show specificity for MHC molecules, and various reports suggest that residues of the MHC molecules constitute at least part of the ligand to which alloreactive T-cell receptors bind. The X-ray crystal structure of the human MHC class I molecule, HLA-A2, has provided evidence to strengthen the argument that MHC-bound self-peptide might also contribute to such recognition. We now provide direct evidence for this, showing that at least some alloreactive cytotoxic T lymphocyte clones recognize peptide fragments derived from cytoplasmic proteins. We reasoned that if self-peptides were involved in allorecognition, then the sequence of some of these peptides could vary between species, resulting in species-restricted distribution of the relevant ligand(s). Several alloreactive cytotoxic T lymphocyte clones specific for H-2Kb, expressed by the murine cell line EL4, did not lyse a human-cell transfectant expressing the H-2Kb molecule (Jurkat-Kb cells). However, these clones were able to lyse Jurkat-Kb cells sensitized by preincubation with an EL4 cytoplasmic extract cleaved by cyanogen bromide. The sensitizing activity from this extract was destroyed by protease and appeared to be due to a peptide consisting of 10 to 15 amino acids.     

Na_2CO_3调控醇热法制备规则形貌Ag纳米晶

以乙二醇为溶剂、Na2S为成核剂、聚乙烯吡喏烷酮(PVP)和Na2CO3为修饰剂,采用醇热法制备不同形貌的Ag纳米晶。通过调节反应时间、Na2CO3浓度、滴加速率以及起始滴加时间等来调控Ag纳米晶的微结构。利用场发射扫描电子显微镜(FESEM)和紫外-可见光(UV-vis)吸收光谱对Ag纳米晶的形貌和尺寸以及局域表面等离子特性(LSPR)进行表征。结果表明:浓度为50μmol/L的Na2CO3,在AgNO3滴加完毕后5.5 min时以2 mL/min的速率滴加到体系中反应3 h,能够得到平均颗粒尺寸为60 nm、尺寸分布均匀的截角立方Ag纳米晶。