Alpha and beta subunits of the LFA-1 membrane molecule are involved in human monocyte-endothelial cell adhesion

Human monocyte adhesion to vascular endothelium is an important transitional event in mononuclear phagocyte development. The molecular mechanism involved in monocyte adhesion to endothelial cells was studied using purified human monocytes and a panel of monoclonal antibodies (MAb). The purified human monocytes were phenotypically characterized and expressed relatively low levels of HLA class II antigens. The monocytes were labeled with Indium-111 to provide high specific activity and a sensitive measure of adhesion. Using this radionuclide adhesion assay, monocytes demonstrated consistent and reproducible adhesion to a confluent monolayer of human umbilical vein-derived endothelial cells. To identify the cell surface molecules involved in human monocyte-endothelial cell adhesion, 15 MAb to 11 monocyte surface structures were used to attempt to inhibit adhesion. MAb recognizing 10 monocyte cell surface molecules did not inhibit adhesion. In contrast, MAb recognizing the alpha and beta subunits of LFA-1 (lymphocyte function-associated) significantly inhibited monocyte adhesion to endothelial cells. Monocyte adhesion was comparably inhibited by F(ab')2 and intact MAb. Significant inhibition was observed at 5 micrograms/ml of anti-LFA-1 MAb. These results indicate that the alpha and beta subunits of the LFA-1 membrane molecule are involved in human monocyte-endothelial cell adhesions.     

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 role of CD18 in phorbol ester-induced human monocyte-mediated cytotoxicity

Monocyte cell surface molecules play an important role in the regulation of monocyte function. To investigate the molecular basis of monocyte-mediated cytotoxicity, we tested the ability of a variety of mediators to stimulate human monocyte-mediated cytotoxicity. Phorbol myristic acetate (PMA) stimulated significant monocyte-mediated killing of tumor cells in an 18-hr indium-111 release assay. Five other cytoactive substances did not induce monocyte-mediated cytotoxicity. The acquisition of monocyte cytotoxicity was associated with nearly a twofold increase in surface expression of three CD18-bearing cell surface molecules (CD11a, CD11b, CD11c). The direct involvement of the CD18-bearing molecules in monocyte-mediated cytotoxicity was investigated using monoclonal antibody (MAb) inhibition. MAb recognizing the CD18 subunit significantly inhibited monocyte-mediated killing. The inhibition by anti-CD18 MAb could not be attributed to LFA-1 (CD11a) alone, suggesting that CR3 (CD11b) and p150,95 (CD11c) may also participate in monocyte-mediated cytotoxicity. In contrast, seven of eight other cell surface structures were not affected by PMA treatment, and MAb to all eight cell surface structures did not inhibit killing. These findings suggest that CD18-bearing molecules are upregulated with monocyte activation and may play a functional role in monocyte-mediated killing.     

Viral-restricted cytolytic T lymphocyte recognition of hybrid human-murine class I histocompatibility antigens

Hybrid human-murine major histocompatibility antigens have been constructed and expressed on the surface of both human RD and murine L cell lines after DNA mediated gene transfer. These antigens linked the polymorphic domains (alpha 1 and alpha 2) of H-2Kb and the carboxy-terminal domains (alpha 3, transmembrane, and intracellular) of HLA-A2. Previously we demonstrated that these antigens were serologically intact and were recognized by allospecific cytolytic T lymphocytes. However, the cell lines expressing the hybrid antigen were less well lysed than the native H-2Kb expressing cell lines. In this study, we extend these observations and demonstrate that virally restricted cytolytic T lymphocytes specific for vesicular stomatitis virus and for Sendai virus can recognize cell lines expressing the hybrid antigen, whether expressed on murine (L cell) or human (RD cell) lines. Furthermore, the data show a profound influence by the carboxy-terminal domains upon the polymorphic T-cell restricting epitopes.     

Requirement for three distinct lymphokines for the induction of cytotoxic T lymphocytes from thymocytes

The induction of cytotoxic T lymphocytes (CTL) from CTL precursors requires a combination of antigen and lymphokine signals. To investigate lymphokine requirements for CTL generation, we used an assay in which helper T cell and accessory cell-depleted spleen cells or whole thymocytes were cultured with lectin (Con A) and lymphokines. This culture was followed by assessment of lectin-dependent cytolysis. High concentrations of recombinant interleukin 2 (R-IL 2) (100 U/ml) alone were not sufficient for lectin-mediated CTL induction from thymocytes, whereas 20 to 100 U/ml of R-IL 2 alone could induce a significant lectin-mediated CTL response from accessory cell-depleted spleen cells. Using thymocytes as responders, we found purified or recombinant interferon-gamma (IFN-gamma) did not cause cytolytic activity either in the absence of or in the presence of R-IL 2. However, supernatant from Con A-stimulated rat spleen cells (rat Con A SN) in combination with R-IL 2 could induce cytolytic activity, suggesting that several factors are required for CTL induction. Con A SN was fractionated by gel filtration and the fractions were tested for ability to induce CTL. In the presence of a low level of R-IL 2 (5 U/ml), fractions with a Mr of approximately 31,000 could induce CTL, and this activity was referred to as CTL differentiation factor (CDF). The peak fractions containing CDF activity did not have detectable IL 1, IL 2, IFN-gamma, or CSF activity. However, by add-back experiments and the use of blocking antibodies, a monoclonal antibody against the IL 2 receptor or antibodies against murine IFN-gamma, we demonstrated that CTL induction from mature thymocytes (L3T4-, Lyt-2+) requires CDF activity in addition to IL 2 and IFN-gamma.     

Long-lasting deficit of functional T cell precursors in human bone marrow transplant recipients revealed by limiting dilution methods

We have applied limiting dilution methods suitable for the estimation of mitogen-reactive helper (pHTL) and cytotoxic (pCTL) T cell frequencies to the analysis of immune function in patients 1 mo to 6 yr after allogeneic bone marrow transplantation (BMT). Although the majority of these patients have regained normal levels of Leu-3+ (helper) and Leu-2+ (killer/suppressor) cells by 6 to 12 mo after BMT as assessed by cytofluorimetry, the fraction of these cells that can function in limiting dilution cultures is substantially below normal levels in nearly all patients. Although some BMT patients eventually recover normal frequencies of pCTL and pHTL, values typically remain greatly depressed even in patients transplanted as many as 4 to 6 yr previously. In contrast, recovery of precursors able to proliferate (without expressing either helper or cytotoxic function) in response to phytohemagglutinin (PHA) and interleukin 2 occurs in many patients by 1 yr after transplant. In spite of the decreased frequency of functional precursor cells found after BMT, each precursor is capable of giving rise to the same amount of function at limiting dilutions as that produced by cells from normal controls. In many BMT patients, proliferation in conventional PHA-stimulated cultures returns to near-normal levels even though precursor frequencies remain low. The limiting dilution method is sensitive to residual immune dysfunction in BMT recipients not easily quantitated by other, more conventional techniques.     

The immune response to Moloney murine leukemia virus-induced tumors: induction of cytolytic T lymphocytes specific for both viral and tumor-associated antigens

The specificity of CTL generated against tumors induced by murine leukemia viruses (MuLV) has been reported to parallel the expression of two serologically defined tumor cell surface antigens--the cross-reactive FMR antigen expressed on the surface of tumors induced by Friend, Moloney, and Rauscher MuLV, and the Gross cell surface antigen (GCSA) expressed on tumors induced by AKV/Gross MuLV. We examined the specificity of CTL generated against MuLV-induced tumors and identified two distinct patterns of reactivity. The first follows the traditional pattern of FMR vs GCSA reactivity as assessed on a panel of established MuLV-induced lymphomas. However, CTL exhibiting this pattern of reactivity are incapable of lysing MuLV-infected fibroblasts. CTL exhibiting the second pattern of reactivity are capable of lysing MuLV-induced lymphomas as well as MuLV-infected fibroblasts. In addition, these CTL exhibit extensive cross-reactivity between lymphomas and fibroblasts infected by both groups of MuLV. Our results suggest that CTL exhibiting the traditional FMR vs GCSA pattern of reactivity are directed against a tumor-associated antigen and not against virus-encoded antigens, and that CTL directed against MuLV-encoded antigens demonstrate extensive cross-reactivity, including the ability to lyse AKV-infected cells.     

Inhibition of normal lymphocyte responses by cell membranes

Cell membranes bearing the appropriate antigen are known to stimulate a variety of cell-mediated immune responses. This report confirms that tumor cell membranes at doses of 2-5 micrograms protein/ml will stimulate in vitro generation of allogeneic cytotoxic T lymphocytes (CTL). However, higher doses (50-100 micrograms protein/ml) of the same membranes completely abrogate the generation of lytic activity. Responding lymphocytes are inhibited by membranes from either syngeneic or allogeneic cells. The inhibition appears to act at a proliferative or differentiation step in the generation of the CTL response, since membranes are known to have little direct effect on the lytic phase of CTL activity. Similar doses of membranes also inhibit LPS-induced B-cell proliferation. B-Cell proliferation is inhibited equally well by allogeneic and syngeneic membranes, and membranes from normal spleen cells are as inhibitory as tumor cell membranes. The inhibitory activity copurifies with the plasma membrane. The results raise important considerations regarding the use of subcellular forms of antigen in studies of lymphocyte recognition. In addition, these data suggest that cell-cell contacts might provide signals regulating the proliferation of lymphocytes.     

Thymectomized, irradiated, and bone marrow-reconstituted chimeras have normal cytolytic T lymphocyte precursors but a defect in lymphokine production

A model system has been developed to study extrathymic T cell differentiation; mice have been thymectomized, lethally irradiated, and reconstituted with bone marrow cells depleted of Thy-1+ cells. After 8 wk, the spleen cells of these athymic, bone marrow-reconstituted chimeras contain Thy-1+ precytolytic T lymphocytes (CTL) that are able to respond to antigen only if supernatant from Con A-activated T cells is added to culture. The phenotype of these pre-CTL is similar to that of thymocytes, suggesting that they may be immature T cells. Initial evaluation of the CTL repertoire of these athymic mice demonstrated that the CTL generated to trinitrophenyl-modified syngeneic cells are H-2-restricted, and that the CTL generated to alloantigens have many of the cross-reactivities observed in normal mice but not in nude mice. In this report, we demonstrate a helper T cell defect in these thymectomized chimeras. These chimeras lack an Ly-1+ helper cell required for thymocytes to differentiate to CTL. Further studies revealed that when spleen cells from these thymectomized chimeras were stimulated with Con A, they produced normal levels of interleukin 2. However, these splenocytes were defective in the production of another factor needed for CTL differentiation.