Accessory function of human mononuclear phagocytes for lymphocyte responses to the superantigen staphylococcal enterotoxin B.

The role of the cytokines IL-1 alpha, IL-1 beta, and IL-6 and the cell adhesion molecules ICAM-1, LFA-1 (alpha and beta), and Mac-1 as accessory molecules for stimulation of T cells by the superantigen staphylococcal enterotoxin B (SEB) was examined. Both blood monocytes and alveolar macrophages were used as accessory cells because these cells differ in patterns of cytokine expression and thus potentially in accessory cell function for superantigens. The blastogenic response of highly purified T cells to SEB was reconstituted with either monocytes or alveolar macrophages. IL-1 secretion was increased comparably in monocytes and alveolar macrophages by SEB, but IL-6 was not stimulated by SEB. IL-1 alpha plus IL-1 beta reconstituted the response of T cells to SEB but required the addition of accessory cells. The cell adhesion molecules ICAM-1 and LFA-1 but not Mac-1 also functioned as accessory molecules for SEB-induced cluster formation and lymphocyte blastogenesis. Thus, not only must this superantigen bind to Class II MHC on accessory cells as is well known, but also SEB requires at least certain cytokines (IL-1 alpha and IL-1 beta) produced by accessory cells and cell adhesion molecules (ICAM-1 and LFA-1) for activation of T lymphocytes.     

Interleukin-2-activated murine cell lines with macrophage- and B-lymphoblast-lytic activity.

Interleukin-2 (IL-2)-activated murine killer cell lines with macrophage- and B-lymphoblastic-lytic activity were established, and their target specificity, surface markers, recognition-related structures, and requirements for optimal cell growth were characterized. Sustained growth of IL-2-activated lymphocytes was supported by the combination of IL-2 and IL-4-enriched T cell conditioned medium (CM), but was not supported by IL-2 alone or the combination of IL-2 and IL-3-containing CM in the presence of macrophages (M phi). The established line required continuous contact with M phi to maintain anti-M phi cytolytic activity. Flow cytometric analysis showed that the original line isolated by the first cloning was Thyl+, CD4-, and weakly CD8+, FcR+. The majority of these cells were CD3+ and TCR-V beta 8+. From this line, the CD3+, TCR-V beta 8+ and CD3-, TCR-V beta 8- clones were isolated by subcloning. The former clone showed Thyl+, CD3+, CD4-, CD8-, TCR-V beta 8+, FcR(+)-phenotype, and the latter clone showed Thyl+, CD3-, CD4-, CD8-, TCR-V beta 8-, FcR- phenotype. The original line and subclones showed a similar target specificity and killed resident or thioglycollate (TG)-induced peritoneal M phi and B-lymphoblasts, but did not kill T-lymphoblasts. Allogeneic M phi, M phi-like cell line P388D1, and B cell hybridoma were sensitive, whereas fresh lymphocytes, T cell lymphoma BW5147, natural killer (NK)-sensitive YAC-1, and NK-resistant P815 tumor cells were resistant to lysis by these cytotoxic lines. The addition of anti-H-2 heteroserum, anti-MHC class 1, anti-MHC class II, anti-CD3, or anti-TCR-V beta 8 monoclonal antibody (mAb) to assay cultures did not inhibit the anti-M phi cytolysis by these killer cells. In addition, the CD3- TCR-V beta 8- clone killed M phi and B lymphoblasts better than the CD3+, TCR-V beta 8+ clone. These results suggest that cytotoxic lines established in this study do not use the T cell receptor (TCR) molecules to recognize target cells and the MHC molecules are not involved in recognition. Anti-LFA-1 mAb partially inhibited anti-M phi-lysis, suggesting that the cell contact between targets and effectors is important in cytolysis. Our present data suggest that the culture condition containing IL-2, IL-4, and M phi may support the continuous growth of non-MHC-restricted killer cells with relative target specificity against M phi and B-lymphoblasts.     

Ligand binding to the LFA-3 cell adhesion molecule induces IL-1 production by human thymic epithelial cells.

We have shown that human thymic epithelial (TE) cells produce IL-1 alpha, IL-1 beta, and TE cells bind to thymocytes by CD2 and LFA-1 molecules on thymocytes and LFA-3, ICAM-1 on TE cells. We investigated whether ligand binding to LFA-3 on human TE cells can modulate TE cell IL-1 production. First, we investigated the ability of human thymocytes to regulate IL-1 release by TE cells. Both autologous and allogenic emetine-treated thymocytes when cultured with TE cells augmented IL-1 release by TE cells. The augmentation of IL-1 release was cell density dependent. Inasmuch as the interaction between thymocytes and TE cells is mediated in part by CD2 molecules on thymocytes and LFA-3 molecules on TE cells we next determined the effect on IL-1 release of ligand binding (anti-LFA-3 mAb TS2/9) to TE cell surface LFA-3. Purified anti-LFA-3 mAb augmented IL-1 release in a concentration-dependent fashion. The anti-LFA-3-mediated augmentation of IL-1 release required both new protein and RNA synthesis as shown by the ability of cycloheximide and actinomycin-D to inhibit augmentation of IL-1 production by TE cells, and by direct quantitation of IL-1 alpha and IL-1 beta mRNA by Northern blot analysis. Both F(ab)'2 and Fab' fragments of anti-LFA-3 mAb augmented IL-1 alpha and IL-1 beta mRNA production, indicating that monovalent binding to cell surface LFA-3 was sufficient to provide the inducing signal. The identification of LFA-3, the cell surface ligand for thymocyte CD2 molecules, as a molecule via which TE cell-derived cytokine production may be regulated suggests a mechanism at the cell surface by which direct TE cell-thymocyte interaction might result in the triggering of local IL-1 release within the human thymic microenvironment.     

Cultured human follicular dendritic cells. Growth characteristics and interactions with B lymphocytes.

Minced human tonsils were digested with DNase and collagenase, and lymphoid cell-depleted low density cells were cultured and grown in granulocyte-macrophage-CSF. Large, morphologically homogenous adherent cells with elongated extensions grew continuously in culture. These nonphagocytic cells appear to be related to follicular dendritic cell (FDC) as they do not have properties of monocytic lineage cells or dendritic cells and because, like FDC, 1) they express CD11b, CD14, CD29, CD40, CD54, CD73, CD74, and VCAM-1, and do not express CD11c, CD22, T cell markers, CD18, CD25 and CD45; and 2) they bind human B lymphocytes and B cell lines, but not T lymphocytes by an adhesion blocked in part by mAb to VLA-4 (CD49d). The cultured FDC also augmented B cell proliferation stimulated by anti-mu sera and/or CD40 mAb. Cultured FDC spontaneously produced low levels of IL-6, but did not produce IL-1 alpha or TNF-alpha; however, after treatment with either IFN-gamma or LPS, they produced more IL-6. The expression of CD54 (ICAM-1) was elevated by treating the cultured FDC with either TNF-alpha, IL-1 beta, IFN-gamma or granulocyte-macrophage-CSF; in contrast, IL-4 had no effect on CD54 but rather up-regulated expression of VCAM-1. IFN-gamma, unlike the other cytokines tested, increased expression of a set of markers on cultured FDC (CD54, VCAM-1, and CD14) and converted these class II-negative cells into class II+ cells. The fact that various T cell-derived cytokines have different effects on FDC suggests that the T cell products may influence the manner by which FDC stimulate B cell proliferation and maturation.     

On the species specificity of the interaction of LFA-1 with intercellular adhesion molecules

Species restrictions in immune cell interactions have been demonstrated both in Ag-specific responses of T lymphocytes and the phenomenon of natural attachment. To determine the possible contribution of adhesion receptors to these restrictions, we have studied binding between the murine and human homologues of LFA-1 (CD11a/CD18) and ICAM employing purified human LFA-1 and ICAM-1 (CD54) bound to solid substrates. Murine cell lines bind to purified human LFA-1 through ICAM-1 and at least one other counter-receptor. This provides evidence for multiple counter-receptors for LFA-1 in the mouse as well as in the human. In contrast to binding of murine ICAM-1 to human LFA-1, murine LFA-1 does not bind to human ICAM-1. The species specificity maps to the LFA-1 alpha subunit, because mouse x human hybrid cells expressing the human alpha subunit associated with a mouse beta subunit bind to human ICAM-1, whereas those with a human beta subunit associated with a murine alpha subunit do not. Increased adhesiveness for ICAM-1 stimulated by phorbol esters could be demonstrated for hybrid LFA-1 molecules with human alpha and murine beta subunits.     

Constitutive expression of TGF-bêta1, interleukin-6 and interleukin-8 by tumor cells as a major component of immune escape in human ovarian carcinoma

Tumors could use several mechanisms to coexist with the host's immune system or to protect themselves from an immune response. Thus, insufficient expression of cell surface molecules on tumor cells, which are important for T cell recognition or activation, could lead to induction of a state of tolerance. Tumor cells could also produce cytokines that would inhibit the immune response and allow tumor progression. Here, we studied, in vitro, the cell surface expression of immunologically important molecules in seven ovarian carcinoma (OVCA) cell lines and the constitutive expression of cytokines. All OVCA cell lines expressed MHC class I molecules, ICAM-1 and LFA-3 adhesion molecules, necessary to induce a specific cytotoxic T-cell response, as well as the CD40 costimulatory molecules. Conversely, the lack of the dominant costimulatory molecules, CD80 (B7.1) and CD86 (B7.2) could be a possible explanation of poor immunogenicity of OVCA tumors. Immunosuppressive TGF-beta1 was detected at the mRNA level in all cell lines but was weakly secreted in supernatants. By contrast, IL-10 was never found. Most of them constitutively produced IL-8 and IL-6, two cytokines known as tumor promoting factors whereas the proinflammatory cytokines TNF-alpha, IL-1beta and GM-CSF were rarely produced. Data from this study could be useful for designing new strategies of immunotherapy to improve immunogenicity and/or limit protumor cytokine production.     

Adhesion of lymphocytes to bladder cancer cells: the role of the alpha(E)beta(7) integrin

The alpha(E)beta(7)integrin (defined by CD103) is expressed by most intra-epithelial lymphocytes (IEL) but by fewer than 2% peripheral blood lymphocytes (PBL). An important ligand for this molecule is the epithelial cell adhesion molecule E-cadherin. Loss of E-cadherin is associated with increased invasion and metastasis in bladder cancer. This study examines the role of the alpha(E)beta(7)-E-cadherin interaction in lymphocyte targeting of bladder cancer cells. Lymphocytes were activated in vitro by mixed lymphocyte reaction (MLR) and CD103 was upregulated by treatment with transforming growth factor beta (TGFbeta). The CD103(+) lymphocytes were used in a flow cytometric adhesion assay with bladder cancer cell lines, differing in expression of E-cadherin and intercellular adhesion molecule-1 (ICAM-1). Antibody blockade was used to confirm the relative importance of CD103 and ICAM-1 to intercellular adhesion. Lymphocytes with upregulated CD103 compared to control lymphocytes showed enhanced adhesion to an E-cadherin expressing bladder cancer cell line ( P=0.0003). This increased adhesion could be abrogated by anti-CD103 adhesion blockade. For ICAM-1 expressing bladder cells, adhesion of lymphocytes could be markedly reduced using anti-ICAM-1 blockade. In conclusion, the upregulation of CD103 by lymphocytes increases adhesion to E-cadherin expressing bladder cancer targets. Loss of E-cadherin in bladder cancer progression may provide a mechanism both for increased invasion and effective immune evasion.     

CD2 expression correlates with proliferative capacity of alpha beta + or gamma delta + CD4-CD8- T cells in lpr mice.

The T lymphocytes that accumulate in vast numbers in the lymphoid tissues of lpr/lpr (lpr) mice express a TCR-alpha beta that is polyclonally rearranged, and yet is devoid of surface CD4 or CD8 (CD4-8-) as well as CD2. lpr CD2- alpha beta + CD4-8- T cells exhibit an apparent block in signal transduction, in that when activated they produce little or no IL-2 and proliferate minimally in the absence of exogenous IL-2. In contrast to the predominant hyporesponsive alpha beta + CD4-8- T cells, we observe that a minor subset (1 to 2%) of lpr lymph node CD4-8- cells expresses a TCR-gamma delta and can proliferate upon activation with PMA and ionomycin in the absence of exogenous IL-2. Furthermore, these responsive gamma delta T cells express surface CD2. The functional and phenotypic distinctions of lpr gamma delta T cells led us to identify an analogous minor (4 to 10%) subset of alpha beta + CD4-8- cells in lpr thymus and lymph nodes that does express CD2. Similar to the gamma delta subset, these CD2+ alpha beta + CD4-8- cells are also capable of proliferation and IL-2 production. Thus the capacity for IL-2 production and proliferation by a small proportion of lpr CD4-8- T cells, either alpha beta + or gamma delta +, correlates with their expression of surface CD2. This correlation is supported by the observation that the lpr liver contains actively cycling alpha beta + CD4-8- lymphocytes that are strikingly enriched for CD2 expression. Consequently, unlike the vast proportion of abnormal lpr CD2- CD3+ CD4-8- cells, the CD2+ CD3+ CD4-8- T cells may not express the basic lpr defect, or else are not affected by its presence. These studies suggest that expression of the lpr abnormality may be restricted to a particular T cell lineage. This functional correlation with CD2 expression may be more broadly applicable to phenotypically similar subsets of normal thymocytes, and possibly peripheral tolerized T lymphocytes.