Rat natural killer cells synthesize fibronectin. Possible involvement in the cytotoxic function

The ability of NK cells to synthesize and secrete fibronectin (FN), an extracellular matrix glycoprotein which plays a key role in many biologic processes including cellular adhesion, morphology, cytoskeletal organization, cell migration, and invasiveness, was studied. By using affinity-purified polyclonal antibodies directed against human cellular or plasma FN, the presence of FN was evidentiated on Percoll-purified rat large granular lymphocyte or on a large granular lymphocyte tumor cell line (CRC) by flow cytometry and immunoelectron microscopy. Its expression increased after NK cell activation by poly I:C administration. Biochemical analysis by immunoprecipitation and SDS-PAGE indicated that FN was associated to cell surface and secreted in the supernatant in a molecular form similar to that of FN from L929 fibroblasts. In an attempt to understand the role of FN in the NK cell function, we found that an antibody against human plasma FN and its F(ab')2 fragment inhibited NK cytotoxicity against YAC-1 target at the effector cell level. Inhibition occurred at the postbinding level, because F(ab')2 anti-FN inhibited induction of phosphatidylinositol hydrolysis by YAC-1 target cells, whereas binding to target cells was not affected. The possible role of FN in the NK cytotoxic function is suggested.     

CTL adhesion and antigen recognition are discrete steps in the human CTL-target cell interaction

Th initial step in cytolytic T lymphocyte (CTL)-mediated cytolysis involves target cell adhesion and antigen recognition. To investigate these initial events in the CTL-target interaction, we used HLA-A2- and HLA-B7-specific human CTL clones and HLA-typed B lymphoblastoid target cells. By using two different adhesion assays, we demonstrated antigen nonspecific CTL-target cell adhesion. To more precisely define the contribution of the antigen-specific receptor to CTL-target cell adhesion, we used the HLA-A2, HLA-B7, and mock transfected RD target cells. Consistent with the results when using B lymphoblastoid target cells, the CTL clones demonstrated equivalent adhesions to the RD target cells whether or not they expressed HLA-A2 or HLA-B7. These results suggested that CTL-target cell adhesion occurred independent of the T cell receptor. By using the calcium-sensitive dye Indo-1 and flow cytometry, we assessed CTL-target cell adhesion and CTL activation. Simultaneous measurement of adhesion and intracellular free calcium demonstrated that CTL-target cell adhesion alone did not activate CTL clones. Both CTL-target cell adhesion and the presence of the appropriate HLA target molecule were necessary for the efficient activation of human CTL. MAb inhibition studies indicated that antigen nonspecific adhesion is largely regulated by the LFA-1, CD2 (LFA-2/T11), and LFA-3 cell surface molecules. These antigen nonspecific cell-cell interaction molecules appear to play an important role in facilitating antigen recognition and subsequent target cell lysis.     

IL-15-mediated induction of LFA-1 is a late step required for cytotoxic differentiation of human NK cells from CD34+Lin- bone marrow cells

Optimal differentiation of cytotoxic NK cells is important to provide protective innate immunity to patients after bone marrow transplantation. In vitro differentiation of CD56(+)CD3(-) NK cells takes weeks and is supported by several cytokines, including IL-2, IL-7, and IL-15, and thus can be useful for immunotherapy. However, IL-2 therapy is problematic in vivo, and NK cells differentiated in vitro with only IL-7 lack cytotoxicity. We assessed whether human NK cells initially differentiated in vitro from CD34(+)Lin(-) bone marrow cells with IL-7 could acquire cytotoxicity after exposure to additional cytokines and what changes promoted cytotoxicity. The cells cultured with IL-7 already had granzyme B as well as perforin, as previously reported, the proteins of cytotoxic granules. The cells also lacked LFA-1. After 1 wk of secondary culture with either IL-2 or IL-15, but not with IL-12 or IL-18, the IL-7-cultured cells acquired cytotoxicity. IL-2 or IL-15 also induced LFA-1. Ab to the LFA-1 subunits CD11a and CD18 blocked lysis by the NK cells, indicating that the new LFA-1 correlated with, and was essential for, the cytotoxic function of the in vitro generated cells. The LFA-1 also participated in target cell binding by the in vitro differentiated cells. In this study, we demonstrated a new function for IL-15, the induction of LFA-1 in NK progenitor cells, and that IL-15 does more than merely support NK progenitor cell proliferation. The efficacy after only 1 wk of IL-15 administration is a positive practical feature that may apply to human therapy.     

Target specificity and cell surface structures involved in the human cytolytic T lymphocyte response to endothelial cells

Two long-term cytolytic T lymphocyte (CTL) lines derived from the peripheral blood lymphocytes (PBL) of a single donor were analyzed for target specificity and involvement of cell surface molecules in CTL-target interactions. One line, AH2, was generated after stimulation with B lymphoblastoid cells. Cytolysis by these cells was restricted to targets expressing the appropriate HLA-A2 specificity and was blocked by mAb recognizing CD2, CD3, CD8, LFA-1, and LFA-3. The second line, AE1, was generated after stimulation with cultured endothelial cells derived from human newborn preputial microvessels. These CTL lysed all human target cells tested, except autologous cells and the Class I negative cell line Daudi. In addition, mAb specific for CD2, CD3, and CD8 did not affect cytolysis. Anti-LFA-1 and -LFA-3 mAb blocked cytolysis of B lymphoblastoid targets but not endothelial targets. These results indicate that some CTL utilize as yet uncharacterized cell surface structures for CTL-target interactions.     

Relative contribution of CD2 and LFA-1 to murine T and natural killer cell functions.

We have examined the functional property of murine CD2 as an intercellular adhesion molecule by using five anti-murine CD2 mAb which were classified into two groups according to their mutual competition in binding to cell surface CD2. Hamster fibroblasts transfected with murine CD2 cDNA exhibited increased conjugate formation with a murine mastocytoma P815 which expresses the putative murine LFA-3 mRNA detected by cross-hybridization with human LFA-3 cDNA under conditions of low stringency. This increase in conjugate formation was abrogated by both groups of anti-CD2 mAb, although some differences in the extent of inhibition were observed at lower concentrations of the mAb. We then examined the involvement of CD2 in several murine T cell responses by using these mAb to abrogate CD2-mediated cellular interactions. Anti-CD2 mAb significantly inhibited mitogenic T cell responses induced by suboptimal doses of Con A and PHA. In the allogenic MLR response and in the Ag response of two KLH/I-Ak-specific Th cell clones, the inhibitory effect of anti-CD2 mAb was also greatest under suboptimal conditions, i.e., with lesser doses of the Ag. These results indicate that the contribution of CD2 as an accessory molecule is variable, depending on the Ag dose used for stimulation, and they suggest that CD2 is involved in the Ag response of murine T cells under the physiologic conditions where only a limited amount of Ag is available. We next examined the contribution of CD2 to MHC-restricted cytotoxicity by CTL and to MHC-unrestricted cytotoxicity by NK and lymphokine-activated killer cells. Only a marginal inhibition by anti-CD2 mAb alone was observed. Anti-lymphocyte function-associated Ag (LFA)-1 mAb alone exhibited greater inhibitory effects than anti-CD2 mAb in all of the cases tested. In most cases, however, substantial levels of cytotoxicity remained, even in the presence of both anti-CD2 and anti-LFA-1 mAb. These results indicate a minor contribution of CD2, as compared with LFA-1, to cytotoxicity by murine CTL, NK cells, and lymphokine-activated killer cells, and they reveal the presence of undefined cellular interaction pathways other than those mediated by CD2 and LFA-1.     

The role of the T cell receptor, CD8, and LFA-1 in different stages of the cytolytic reaction mediated by alloreactive T lymphocyte clones

The cytotoxic reaction mediated by cytotoxic T lymphocytes (CTL) consists of three phases: first, the CTL binds to the target cell; next, the CTL is triggered to lyse the target cell; and in the third phase, the CTL detaches from the target cell which is lysed in the absence of the CTL. Recently, we obtained evidence that human alloreactive CTL clones initially adhere to target cells without the involvement of the interaction between the T cell receptor (Tcr) and its specific target antigen. In the present study, we investigated the effect of monoclonal antibodies specific for the Tcr on the cytotoxic reaction of three CD8+ HLA-A2-specific CTL clones, using a single cell assay in which the binding event can be distinguished from the post-binding (lytic) phase of the cytolytic reaction. It was found that monoclonal antibodies directed at a variable part of the Tcr do not affect the binding phase but strongly block the lytic phase of the cytotoxic reaction. An anti-constant region Tcr antibody and an anti-CD3 reagent had a similar effect on the two phases of the reaction as the anti-variable part Tcr antibodies. In contrast, antibodies specific for LFA-1 strongly blocked the adhesion phase but did not affect the lytic phase. Antibodies specific for CD-8 had intermediate effects. They could block both the adhesion as well as the lytic phase. The effect of anti-CD8 appeared to be dependent on the CTL clone tested. One clone was found to be inhibited in the adhesion phase, but not in the lytic phase, whereas anti-CD8 hardly blocked the adhesion phase of two other CTL clones, but affected the lytic step of those clones. Our data indicate that LFA-1 is a major adhesion molecule in the CTL reaction, whereas the Tcr/CD3 complex is implicated in a phase after the initial formation of conjugates. CD8 is associated with both steps in the cytolytic reaction. In addition to its minor role in the adhesion phase, our data suggest strongly that CD-8 is involved in the triggering phase of the cytolytic reaction.     

Stimulation of IFN-gamma, TNF-alpha, and TNF-beta secretion in IL-2-activated T cells: costimulatory roles for LFA-1, LFA-2, CD44, and CD45 molecules.

Lymphokine-activated killer (LAK) cells are peripheral blood lymphocytes (PBLs) that possess the ability to kill target cells in a non-major histocompatibility complex (MHC)-restricted manner. Both NK and T cells can be stimulated with interleukin-2 (IL-2) to become LAK cells. We previously reported that the interaction of LAK cells with tumor cells also induces the secretion of interferon-gamma (IFN-gamma). The NK subset of LAK (LAK-NK) cells is stimulated by tumor cells to secrete IFN-gamma in a non-MHC-restricted manner while the T cell subset of LAK (LAK-T) cells is stimulated to secrete IFN-gamma upon cross-linking of the T cell receptor (TCR)-CD3 complex. We here report that LAK-T cells stimulated with anti-CD3 mAbs and tumor cells secrete two additional cytokines, tumor necrosis factor-alpha (TNF-alpha) and TNF-beta/lymphotoxin (TNF-beta). In addition, we demonstrate that at least four other structurally unrelated molecules, in addition to the TCR-CD3 complex, on LAK-T cells participate in the stimulation of IFN-gamma, TNF-alpha, and TNF-beta production. These molecules are the lymphocyte function associated antigen-1 (LFA-1), lymphocyte function associated antigen-2 (LFA-2), CD44, and CD45. LFA-1 is an integrin, LFA-2 is a member of the immunoglobulin supergene family, CD44 is homologous to the cartilage link proteins, and CD45 is a tyrosine phosphatase. Ligands to three of these molecules have been identified; ICAM-1, LFA-3, and hyaluronic acid binding to LFA-1, LFA-2, and CD44, respectively. LFA-1, LFA-2, and CD44 are reported to function both as adhesion molecules and as costimulators in resting T cells. Our data suggest that these three molecules enhance IFN-gamma, TNF-alpha, and TNF-beta production by augmenting LAK-T cell to tumor cell adhesion and also by functioning as costimulators.     

Antibodies to adhesion molecules inhibit the lytic function of MHC-unrestricted cytotoxic cells by preventing their activation.

We evaluated the effect of the antibodies to adhesion molecules CD2, CD11a/CD18 (LFA-1), and CD56 (N-CAM) on MHC-unrestricted cytotoxicity mediated by polyclonal NK cells and LAK cells or by CD3+ or CD3- cytolytic cell clones against a panel of tumor cell targets selected according to expression or absence of the corresponding ligands. We show that (i) antibodies to CD11a/CD18 and, to a lesser extent, antibodies to CD2 inhibit target cell lysis, whereas anti-CD56 antibodies exert little if any effect; (ii) in a model system using polyclonal NK/LAK cells as effectors and K562 or HL60-R (NK-resistant) cells as targets, inhibition of cytotoxicity occurs without a significant impairment of effector to target cell binding; (iii) the cytotoxic function of CD3+ or CD3- cytotoxic cell clones is inhibited differentially by antibodies to adhesion molecules; (iv) conjugates formed in the presence of antibodies which inhibit target cell lysis display a significant reduction of target to effector cell contact surface; and (v) this may lead to defective activation of effector cells, as indicated by lack of redistribution of the microtubular apparatus. We conclude that (i) MHC-unrestricted cytotoxicity is regulated by a number of molecular interactions that span far beyond our present knowledge and that it is strictly dependent on the surface phenotype of the effector cell and of the target cell; (ii) in certain types of effector/target cell interactions, antibodies to adhesion molecules do not prevent conjugate formation but reduce the extent of cell-to-cell surface contact which, in turn, leads to defective activation of the effector cell and, therefore, to inhibition of target cell lysis.     

Intercellular adhesion molecule-1 (ICAM-1) is involved in the cytolytic T lymphocyte interaction with a human synovial cell line

The cell surface molecules involved in the human cytolytic T lymphocyte (CTL)-synovial cell interaction may play an important role in T cell interactions with connective tissue mesenchymal cells. To examine the molecular basis for the CTL-synovial cell interaction, we immortalized synovial cell explants to establish the cell line SYN.SPP. The SYN.SPP cell line was compared to the established B lymphoblastoid cell line JY. Cell surface immunofluorescence demonstrated significantly different levels of the immunologically relevant cell surface molecules ICAM-1 and LFA-3. Both cell lines were used to stimulate CTL precursors. After several months in culture, CTL lines stimulated by the SYN.SPP and JY cell lines demonstrated HLA class I-directed cytolytic activity. The cell surface molecules utilized by the anti-SYN.SPP and anti-JY CTL lines were identified by monoclonal antibody (MAb) inhibition. MAb recognizing the CTL cell surface molecules CD3, CD8 and LFA-1 (CD11a) significantly inhibited CTL-mediated lysis of both target cells. An interesting observation was that the anti-SYN.SPP CTL line appeared to utilize the ICAM-1 and not the LFA-3 target cell molecule. In contrast, the anti-JY CTL line utilized the LFA-3 and not the ICAM-1 membrane molecule. These results indicate that CTL interactions with connective tissue mesenchymal cells may be regulated by a unique pattern of antigen nonspecific cell-cell interaction molecules.     

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.     

Inhibition of lymphokine-activated killer cell-mediated cytotoxicity by phorbol ester

As previously reported, the culture of mouse spleen cells in the presence of high amounts of human rIL-2 for 4 days caused proliferation and generation of lymphokine-activated killer (LAK) cells, which could lyse a variety of tumor cells. However, an addition of PMA to the culture resulted in a striking inhibition of the generation of LAK cells. In contrast, IL-2-induced cell proliferation, IL-2R expression, and LFA-1 expression were enhanced by the addition of PMA. Kinetic studies revealed that the addition of PMA during the final 24 h, but not 4 h, of the culture was sufficient to inhibit the generation of LAK cells. The same inhibition of LAK activity was observed when 4-day cultured LAK cells were pretreated with PMA for over 12 h before cytotoxicity assay. Flow cytometry analysis showed that PMA pretreatment had no effect on the binding of LAK cells to target cells. PMA pretreatment of LAK cells caused total disappearance of protein kinase C (PKC) activity from LAK cells concomitant with the loss of LAK activity. However, PMA-pretreated LAK cells cultured for another 24 h in the absence of PMA revealed levels of PKC activity and cytotoxicity identical with untreated LAK cells. These results strongly suggest that PMA-induced down-regulation of LAK cell-mediated cytotoxicity is due to the inactivation of PKC-dependent transduction systems that are essential post LAK cell-target cell binding.     

Species-restricted recognition of transfected HLA-A2 and HLA-B7 by human CTL clones

Human cytolytic T lymphocytes (CTL) clones and HLA-A2- and HLA-B7-transfected human, monkey, and mouse cell lines were used to investigate the basis for species-restricted antigen recognition. Most allospecific CTL clones obtained after stimulation with the human JY cell line (source of HLA-A2 and HLA-B7 genomic clones) recognized HLA antigens expressed in human and monkey cell lines but did not recognize HLA expressed in murine cells. By initially stimulating the responder cells with HLA-transfected mouse cells, two CTL clones were obtained that recognized HLA expressed in murine cells. Functional inhibition of these CTL clones with anti-class I monoclonal antibodies (MAb) indicated that clones reactive with HLA+ murine cells were of higher avidity than clones that did not recognize HLA+ murine target cells. MAb inhibition of accessory molecule interactions demonstrated that the LFA-1 and T8 surface molecules were involved in CTL-target cell interactions in all three species. In contrast, the LFA-2/CD2 molecule, previously shown to participate in a distinct activation pathway, was involved in the cytolysis of transfected human and monkey target cells, but not in the lysis of HLA+ murine cells. Thus transfection of HLA genes into different recipient species cell lines provides us with the ability to additionally delineate the functional requirements for allospecific CTL recognition and lysis.     

Involvement of fibronectin during epiboly and gastrulation in embryos of the common carp,Cyprinus carpio

Summary The present report firstly describes a pilot study in which, during early development of embryos of the common carp, Cyprinus carpio, the cellular adhesion to fibronectin (FN) was blocked by administration of GRGDS peptide (which binds to the FN-receptor). As this treatment resulted in developmental aberrations, suggesting a functional role for FN, the major part of the work was focussed on the distribution of reactivity of anti-FN antibodies during epiboly and gastrulation. GRGDS treatment had a concentration dependent effect on development. Incubation of embryos in 1.5 mg/ml from the 32-cell stage onwards caused a retardation of epiboly, which did not proceed beyond 60%. The embryos did not show involution, as was confirmed by histological study. These preliminary results suggest that FN is involved in both epiboly and gastrulation of carp embryos. During cleavage, no specific extracellular binding of anti-FN antiserum could be observed. However, binding to a number of cell membranes took place from early epiboly onwards. After the onset of gastrulation, we observed a gradually increasing number of the deepest epiblast cells, showing immunostaining on part of their surface, facing the yolk syncytial layer (YSL) or the involuted cells. During early epiboly, anti-FN binding was restricted to areas in front of the migratory hypoblast cells. Later on, binding was found at the border of hypoblast and epiblast cells. At 100% epiboly, some contact areas of epiblast and hypoblast showed a discontinuous lining of reactivity, whilst other areas appeared devoid of anti-FN binding sites. The results indicate that FN is involved in the migration and guidance of hypoblast cells during gastrulation in carp. Correspondence to: P. Gevers     

Human natural killer cell adhesion molecules. Differential expression after activation and participation in cytolysis.

Cell adhesion molecules (CAM) participate in interactions between lymphocytes, accessory cells, and target cells that are critical in the generation of effective immune responses. To characterize the involvement of CAM in NK and lymphokine activated killer (LAK) activities, we examined the expression of several CAM by freshly isolated human NK cells and by NK cells activated in vitro with IL-2, and compared this to CAM expression by T lymphocytes under similar conditions. Freshly isolated human NK cells were uniformly LFA-3 (CD58)+ and expressed two to three-fold higher surface levels of LFA-1 (CD11a/CD18) than resting T lymphocytes. More NK cells than T cells also expressed phenotypically detectable levels of intercellular adhesion molecule-1 (CD54). After in vitro incubation with IL-2, human NK cells demonstrated four- to sixfold increases in surface levels of CD11a/CD18, CD2, CD54, CD58, and the NK cell-associated Ag NKH-1 (CD56). Furthermore, essentially all NK cells became CD54+ within 3 days of exposure to IL-2. T cells did not demonstrate comparable up-regulation of CAM after incubation with IL-2. Increases in NK cell CAM expression were associated with enhanced formation of E:T cell conjugates, enhanced killing of NK-sensitive targets, and the induction of cytotoxicity for previously NK-resistant targets (LAK activity). The LAK activity induced by exogenous IL-2 could be partially inhibited by anti-CD2, anti-CD11a, or anti-CD54 antibodies and almost completely abrogated by anti-CD2 and anti-CD11a in combination. These studies suggest that CAM play a central role in the regulation of NK cytolysis, and that changes in CAM expression may alter the target cell specificity of activated NK effectors.     

Human neutrophil adherence to thrombospondin occurs through a CD11/CD18-independent mechanism.

Thrombospondin (TSP), a 450-kDa trimeric glycoprotein secreted by platelets and endothelial cells at sites of tissue injury or inflammation, may play an important role in polymorphonuclear leukocyte (PMN) adherence to blood vessel walls before diapedesis. We have examined the adherence of PMN to TSP and compared it to adherence to other extracellular matrix proteins. PMN adherence to TSP-coated plastic was complete by 60 min with spreading completed by 2 h. The kinetics of adhesion and spreading on TSP were similar to that of vitronectin (VN), laminin (LN), and fibronectin (FN). Activation of PMN with the calcium ionophore A23187 or the chemotactic peptide FMLP increased PMN adherence to LN and FN, but not to TSP or VN, suggesting that PMN activation may differentially regulate expression of TSP and VN receptors as compared to LN and FN receptors. The specificity of PMN adherence to TSP was confirmed by competition with saturating amounts of TSP and inhibition with anti-TSP antibodies. mAb A6.1, which binds to the protease-resistant core of TSP, was the most effective in blocking PMN adherence to TSP. Using TSP proteolytic fragments, we demonstrated that the primary interaction of PMN with TSP was mediated through the 140-kDa COOH-terminal domain. Inasmuch as the 140-kDa fragment of TSP contains an Arg-Gly-Asp sequence similar to the cell recognition site of FN and VN, we determined whether RGDS peptides would inhibit PMN adhesion. RGDS did not significantly inhibit PMN adhesion to TSP, VN, or LN, but reduced PMN adhesion to FN by 50%. To determine if PMN adhesion to TSP was mediated by a beta 2 integrin receptor such as LFA-1, MO-1, or p150,95, we performed adhesion assays using PMN isolated from patients with leukocyte adhesion deficiency that lack beta 2 receptors. Leukocyte adhesion deficiency PMN exhibited normal adherence to TSP. In contrast, adherence to VN, LN, and FN was reduced by 95%. Therefore, adherence to TSP is probably not mediated by a beta 2 integrin receptor. These data contribute to the accumulating evidence that PMN can interact with extracellular matrix proteins through a CD11/CD18-independent process.     

Studies on cytotoxicity generated in human mixed lymphocyte culture

Summary High levels of cytotoxic activity against the natural killer (NK) cell-sensitive target K562 and the NK-resistant target UCLA-SO-M14 (M14) can be generated in vitro either by mixed lymphocyte culture (MLC) or by culture of lymphocytes in interleukin 2 (IL2) (lymphokine activated killer (LAK) cells). The purpose of this study was to identify similarities and differences between MLC-LAK and IL2-LAK cells and allospecific cytotoxic T cells. Induction of cytotoxicity against K562 and M14 in both culture systems was inhibited by antibodies specific either for IL2 or the Tac IL2 receptor. Like NK effector cells, the precursors for the MLC-LAK cells were low density large lymphocytes. However these precursors differed from the large granular lymphocytes that mediated NK cytolysis in sensitivity to the toxic lysosomotropic agent L-leucine methyl ester (LME). The resistance of the MLC-LAK precursors to LME indicated that the precursors included large agranular lymphocytes. Although interferon-gamma (IFN-gamma) is produced in MLC and in IL2 containing cultures, it is not required for induction of either type of cytotoxic activity. Neutralization of IFN-gamma in MLC-and IL2-containing cultures with specific antibodies had no effect on the induction of cytotoxic activities. Both allospecific cytotoxic T lymphocyte (CTL) and LAK activities were enhanced by IL2 and IFN-gamma at the effector cell stage. However, the mechanism of cytolysis was different in the two systems. NK- and MLC-induced LAK activities were independent of CD3-T cell receptor complex while CTL activity was blocked by monoclonal antibodies specific for the CD3 antigen. These results suggest that NK and the in vitro induced LAK cytotoxicities are a family of related functions that differ from CTL. Furthermore, MLC-induced and IL2-induced cytotoxicities against K562 and M14 appear to be identical.This work was supported by NIH grant CA34442     

Functional distinctions between the LFA-1, LFA-2, and LFA-3 membrane proteins on human CTL are revealed with trypsin-pretreated target cells

We asked whether we could distinguish the roles of the human lymphocyte membrane proteins LFA-1, LFA-2, and LFA-3 in the function of CTL-mediated killing. Little is known about the functions of these molecularly distinct proteins beyond the facts that i) binding of a monoclonal antibody (MAb) to any one of them is sufficient to inhibit killing, ii) that in each case inhibition involves prevention of CTL-target cell conjugate formation, and iii) that MAb to LFA-1 and LFA-2 inhibit best when bound to the CTL, whereas anti-LFA-3 inhibits only when bound to the target cell. This latter is despite the fact that (in our test system) LFA-1 and LFA-3 are expressed both on the CTL and on the target. When the target cells were pretreated with trypsin, the sensitivity of CTL-mediated killing was affected in a different way for each site. Inhibition of anti-LFA-1 was increased by approximately 20-fold. Inhibition by anti-LFA-2 was unaffected. Inhibition by anti-LFA-3 was abolished. Trypsin did not remove the specific antigens recognized by the various CTL, HLA-A,B,C or HLA-DR. Nor did it remove LFA-1 from the target cell. It did, however, selectively remove LFA-3 from the target cell. These results indicate, for the first time, that LFA-1 and LFA-2 have functionally distinct roles. They suggest that an unidentified trypsin-sensitive target cell molecule, operationally designated the "trypsin-sensitive counter blocker" (TSCB), plays an important role in the function of LFA-1, possibly by providing a target cell binding site for LFA-1 on the CTL. The hypothesis that this TSCB is identical to LFA-3 (and the related possibility that LFA-1 and LFA-3 are mutual ligands) is not favored by our data, but is not excluded. Finally, the data indicate that the mechanisms by which MAb inhibit killing differ at the LFA-1 and LFA-3 sites. They are consistent with LFA-1 providing adhesion strengthening by binding to another site (the TSCB?) and with LFA-3 delivering an inhibitory signal when provoked with MAb.     

Gene mapping and somatic cell hybrid analysis of the role of human lymphocyte function-associated antigen-3 (LFA-3) in CTL-target cell interactions

LFA-3 is expressed on a wide variety of human cell lines, including those which have been used as recipients for gene transfer of human class I gene products, whereas a murine counterpart is either absent or significantly different such that the anti-LFA-3 monoclonal antibody (MAb) does not bind. By using a somatic cell genetic approach, we demonstrate that LFA-3 is not a major histocompatibility complex-encoded molecule, and that its gene locus maps to human chromosome 1. When LFA-3 and HLA-A2 are coexpressed on the mouse cell surface, anti-LFA-3 MAb interfered with specific recognition and lysis of these target cells by human CTL capable of lysing HLA-A2-expressing mouse transfectants. A significant contribution of the LFA-3 molecule to CTL reactivity was not observed, however, because the presence of LFA-3 did not restore recognition by CTL clones previously found incapable of lysing HLA-A2-expressing mouse transfectants, nor was it required by those human CTL that could lyse mouse cell transfectants. Thus, we have used genetic techniques to demonstrate that LFA-3 may serve a role in CTL-target cell interactions at the target cell level, but is not a molecule absolutely required for human allospecific CTL recognition of HLA antigens expressed on mouse cells. We suggest that LFA-3 may not participate directly in CTL function under normal circumstances, but delivers a more general inhibitory signal only when provoked by bound MAb.     

Opposite effects of thrombospondin-1 via CD36 and CD47 on homotypic aggregation of monocytic cells.

Thrombospondin-1 (TSP-1), an extracellular matrix protein, has a multimodular structure and each domain specifies a distinct biological function through interaction with a specific ligand. In this study we found that exogenously added TSP-1 inhibits phorbol myristate acetate (PMA)/LPS-induced homotypic aggregation of human monocytic U937 cells, whereas the 70-kDa fragment of TSP-1 generated by the proteolytic cleavage of the intact molecule promotes the homotypic aggregation. The aggregation was also inhibited by anti-CD47 mAb or the 4N1K peptide, of which sequence is derived from the CD47-binding site of TSP-1 and absent in the 70-kDa fragment. In contrast, the augmented cell aggregation by the 70-kDa fragment was hampered by anti-CD36 mAb or antibody against the CD36-binding site of TSP-1. The cell aggregation of U937 cells was completely blocked, even in the presence of the 70-kDa fragment, by mAb against leukocyte function associated antigen-1 (LFA-1) or intercellular adhesion molecule-1 (ICAM-1). We therefore propose that TSP-1 may regulate LFA-1/ICAM-1-mediated cell adhesion of monocytes/macrophages by either the inhibitory effect through CD47 or the promoting effect through CD36 depending on which domain/fragment is functional in a given biological setting.     

Lipid rafts mediate association of LFA-1 and CD3 and formation of the immunological synapse of CTL

Lipid rafts accumulate in the immunological synapse formed by an organized assembly of the TCR/CD3, LFA-1, and signaling molecules. However, the precise role of lipid rafts in the formation of the immunological synapse is unclear. In this study, we show that LFA-1 on CTL is constitutively active and mediates Ag-independent binding of CTL to target cells expressing its ligands. LFA-1 and CD3 on CTL, but not resting T cells, colocalize in lipid rafts. Binding of LFA-1 on CTL to targets initiates the formation of the immunological synapse, which is formed by LFA-1, CD3, and ganglioside GM1 distributed in the periphery of the cell contact site and cholesterol is more widely distributed. The formation of this synapse is Ag independent, but the recognition of Ag by the TCR induces accumulation of tyrosine phosphorylated proteins in the synapse as well as redistribution of the microtubule organization center toward the cell contact site. Our results suggest that LFA-1 recruits lipid rafts and the TCR/CD3 to the synapse, and facilitates efficient and rapid activation of CTL.