Growth inhibition of a colonic adenocarcinoma cell line (HT29) by T cells specific for mutant p21 ras

Interleukin-2 (IL-2)-activated killer cells, also referred to as lymphokine-activated killer (LAK) cells, are stimulated by tumor cells to express cytotoxic activity and to also secrete cytokines such as interferon (IFN) and tumor necrosis factor (TNF ). We previously reported that secretion of cytokines by IL-2-activated T cells (LAK-T cells) is dependent on the initial cross-linking of the T cell receptor (TCR)-CD3-molecular complex, but the cross-linking of accessory molecules, such as LFA-1, CD2, CD44 and CD45, on LAK-T cells can enhance this cytokine production. We have developed an approach involving interspecific gene transfer to define further the contributions of LFA-1 and CD2 to the activation of LAK-T cells. The genes for huICAM-1 (a ligand for LFA-1) and huLFA-3 (a ligand for CD2) were transfected singly and in combination into a null mouse melanoma background, and clonal populations of cells that stably express ICAM-1 and/or LFA-3 were derived. Expression of the introduced ICAM-1 and/or LFA-3 by transfected cells enhanced their ability to bind LAK-T cells; the LFA-1/ICAM-1-mediated binding was not further enhanced by activation with phorbol 12-myristate 13-acetate. ICAM-1- and/or LFA-3-transfected cells, in the presence of immobilized anti-CD3, exhibited a greater ability to stimulate IFN secretion by LAK-T cells compared to the untransfected parental lines. This experimental system, which allows ICAM-1/LFA-1 and CD2/LFA-3 interactions to occur on the LAK-T cell at a site distal from the anti-CD3 signal, extends our understanding of LAK-T cell activation by establishing that both LFA-1/ICAM-1 and CD2/LFA-3 can mediate co-stimulation via adhesion and signaling events.     

Stimulator cell-dependent requirement for CD2- and LFA-1-mediated adhesions in T lymphocyte activation by superantigenic toxins.

The staphylococcal enterotoxins and related microbial T cell mitogens stimulate T cells by cross-linking variable parts of the T cell receptor (TCR) with MHC class II molecules on accessory or target cells. We have used cloned human T cells and defined tumor cells as accessory cells (AC) to study the requirements for T cell activation by these toxins. On AC expressing high levels of CD54 (intercellular adhesion molecule-1, ICAM-1) and CD58 (lymphocyte function-associated antigen-3, LFA-3), mAb to CD2 were relatively ineffective in inhibiting the response to the toxins and antibodies to the lymphocyte function-associated antigen-1 (LFA-1) did not inhibit at all. If added together, however, these mAb inhibited the response completely. Similar results were obtained using antibodies to the target structures of CD2 and LFA-1. In contrast, on cells expressing low levels of LFA-3, mAb to LFA-1 but not to CD2 were strongly inhibitory. The same pattern of inhibition was found when these same cells were used as presenters of specific antigen to the T cells. These data show that adhesions via CD2 or LFA-1 are alternatively required for the stimulation of the T cells by superantigenic toxins and demonstrate another similarity between T cell stimulation by superantigens and by specific antigen recognition.     

Ability of cell-sized beads bearing tumor cell membrane proteins to stimulate LAK cells to secrete interferon-gamma and tumor necrosis factor-alpha

We recently reported that lymphokine activated killer (LAK) cells were stimulated to release both interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) when stimulated by a variety of tumor cells. We proposed then that the released cytokines may play a role in mediating tumor cell regression in vivo. In this paper, we provide further information on the nature of the signals, provided by the tumor cells (K562 erythroleukemia), that stimulate LAK cells to secrete IFN-gamma and TNF-alpha. Using a previously published protocol for coating tumor-membrane molecules onto cell-sized hydrophobic beads (also called pseudocytes), we demonstrate that the signal provided by the tumor cell is membrane associated. Beads coated with K562 membranes stimulated LAK cells to release IFN-gamma and TNF-alpha. The pretreatment of these beads with trypsin and sodium periodate eliminated the ability of these pseudocytes to stimulate cytokine release in LAK cells. The glycoproteins that stimulate LAK cells to secrete IFN-gamma and TNF-alpha were further enriched by their ability to bind concanavalin A (Con A, Jack Bean). To determine if the tumor-associated molecules that stimulate LAK cells to release IFN-gamma and TNF-alpha are also the molecules involved in mediating tumor cell lysis, we tested the ability of the Con A binding and nonbinding proteins to inhibit the LAK cell-mediated lysis of K562 cells. Our results demonstrate that molecules that inhibited LAK cell-mediated cytotoxicity were not enriched by Con A. These results are therefore consistent with the conclusion that different sets of tumor-associated molecules are involved in the stimulation of LAK cells to secrete cytokine and in the induction of LAK cells to mediate tumor cell cytolysis.     

Monoclonal antibodies anti-CD3, anti-TCRαβ and anti-CD2 act synergistically with tumor cells to stimulate lymphokine-activated killer cells and tumor-infiltrating lymphocytes to secrete interferon γ

Summary Peripheral blood lymphocytes cultured in interleukin-2 IL-2 acquire the ability to recognize and kill a wide range of tumor cells. Such promiscuous killer cells are termed lymphokine-activated killer (LAK) cells. We recently reported that the interaction of LAK cells with tumor cells stimulated the LAK cells to release interferon (IFN). Here, we report that the release of IFN by LAK cells can be further enhanced by addition of the monoclonal antibodies (mAbs), anti-CD3, anti-(T cell receptor ) (TCR) and a mitogenic combination of anti-CD2 (T11₂+T11₃). Other antibodies, including a non-mitogenic anti-CD2 mAb (Leu5b), that recognize T cell-associated antigens were not stimulatory. The same stimulatory mAbs also synergized with tumor cells to stimulate tumor-infiltrating lymphocytes (TIL) to secrete IFN. Additional experiments indicated that it was the T cell subset of LAK cells (LAK-T cells) that was stimulated by tumor cells and mAbs to release IFN. Inhibition studies with specific mAbs suggest that the stimulation of IFN release by LAK-T cells was dependent both on the aggregation of TCR-CD3 complexes on the LAK-T cell, and on the interaction of accessory molecules with their ligands. The accessory molecules we have identified as critical are LFA 1 and CD2/LFA-2 on LAK-T cells interacting with their respective ligands ICAM-1 and LFA3. Thus our data suggest that cytokine production in LAK-T cells can be regulated by multiple molecular interactions, involving the TCR-CD3 complex and adhesion molecules.     

Human memory T lymphocytes express increased levels of three cell adhesion molecules (LFA-3, CD2, and LFA-1) and three other molecules (UCHL1, CDw29, and Pgp-1) and have enhanced IFN-gamma production

Studies of cell-surface molecules involved in human T cell interaction reveal that differential expression of each of three adhesion molecules (LFA-3, CD2, and LFA-1) subdivides human peripheral blood T cells into major subpopulations. Systematic analysis of the relationship between expression of these and other markers of T cell subsets demonstrates a single major subset of human peripheral blood T lymphocytes distinguished by enhanced expression of LFA-3, CD2, LFA-1, and three other markers (CDw29 [4B4], UCHL1, and Pgp-1). Large differences in relative expression are observed for UCHL1 (29-fold) and LFA-3 (greater than 8-fold), and smaller differences (2- to 4-fold) are seen for CDw29, CD2, LFA-1, and Pgp-1. Bimodal distribution of LFA-3 is found on both CD4+ cells and on CD8+ cells as well as on B lymphocytes (CD19+). Neonatal T cells (CD3+) are comprised almost exclusively of the subset expressing low LFA-3, CD2, LFA-1, CDw29, and UCHL1. Activation of cord peripheral blood mononuclear leukocytes with PHA leads to uniform enhanced expression of each of these molecules on CD3+ cells. Functional analyses of these T cell subsets were performed after sorting of adult T cells based on differential LFA-3 expression. Only the LFA-3+ subset proliferated in response to the Ag tetanus toxoid, even though the LFA-3- subset proliferated more strongly to PHA. Furthermore, the LFA-3+ subset made greater than fivefold more IFN-gamma than the LFA-3- subset in response to PHA, despite the fact that both subsets made equivalent amounts of IL-2. This phenotypic and functional analysis of resting and activated newborn and adult T cells indicates that human memory T cells express enhanced levels of LFA-3, CD2, LFA-1, UCHL1, CDw29, and Pgp-1; we speculate that the increase in expression of T cell adhesion molecules LFA-3, CD2, and LFA-1 on memory cells is functionally important in their enhanced responsiveness.     

CD44 cross-linking induces integrin-mediated adhesion and transendothelial migration in breast cancer cell line by up-regulation of LFA-1 (alpha L beta2) and VLA-4 (alpha4beta1)

CD44, a widely expressed cell surface glycoprotein, plays a major role in cell-cell adhesion, cell-substrate interaction, lymphocyte homing, and tumor metastasis. For tumor metastasis to occur through the blood vessel and lymphatic vessel pathway, the tumor cells must first adhere to endothelial cells. Recent studies have shown that high expression of CD44 in certain types of tumors is associated with the hematogenic spread of cancer cells. However, the functional relevance of CD44 to tumor cell metastasis remains unknown. In this study, we investigated the mechanisms of CD44 cross-linking-induced adhesion and transendothelial migration of tumor cells using MDA-MB-435S breast cancer cell line. Breast cancer cells were found to express high levels of CD44. Using flow cytometric analysis and immunofluorescence staining, we demonstrated that cross-linking of CD44 resulted in a marked induction of the expression of lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) by exocytosis. These results were also observed with the Hs578T breast cancer cell line. Furthermore, LFA-1- and VLA-4-mediated adhesion and transendothelial cancer cell migration were also studied. Anti-LFA-1 mAb or anti-VLA-4 mAb alone had no effect on adhesion or transendothelial cancer cell migration, but were able to inhibit both of these functions when added together. This shows that CD44 cross-linking induces LFA-1 and VLA-4 expression in MDA-MB-435S cells and increases integrin-mediated adhesion to endothelial cells, resulting in the transendothelial migration of breast cancer cells. These observations provide direct evidence of a new function for CD44 that is involved in the induction of LFA-1 and VLA-4 expression by exocytosis in MDA-MB-435S cells. Because these induced integrins promote tumor cell migration into the target tissue, it may be possible to suppress this by pharmacological means, and thus potentially cause a reduction in invasive capability and metastasis.     

Effect of interleukin 2, interferon-gamma, and mitogens on the production of tumor necrosis factors alpha and beta

Human peripheral blood mononuclear cells (PBMC) were induced by recombinant interleukin 2 and mitogens to secrete two distinct cytotoxic polypeptides, tumor necrosis factor-alpha (TNF-alpha) and tumor necrosis factor-beta (TNF-beta), previously called lymphotoxin. Treatment of PBMC with recombinant human interleukin 2 (rIL 2) or mitogens in combination with recombinant human interferon-gamma (rIFN-gamma) resulted in augmented production of both TNF-alpha and TNF-beta. rIFN-gamma alone had no effect on production of either cytotoxic polypeptide. TNF-alpha was produced within 2 to 3 hr after induction and was the major cytotoxin produced by PBMC during the first 48 hr of culture, after which time TNF-beta became the predominant species. TNF-beta was first secreted into the media after 8 hr of induction. Enhanced levels of both TNF-alpha and TNF-beta were seen when the PBMC were separated into adherent and nonadherent cells. Both TNF-alpha and TNF-beta were induced in different tumor cell lines of hematopoietic origin. The results demonstrate that the production of TNF-alpha and TNF-beta can be enhanced by two lymphokines, IL 2 and IFN-gamma.     

Role of LFA-1 and VLA-4 in the adhesion of cloned normal and LFA-1 (CD11/CD18)-deficient T cells to cultured endothelial cells. Indication for a new adhesion pathway.

Patients with the leukocyte adhesion deficiency (LAD) syndrome have a genetic defect in the common beta 2-chain (CD18) of the leukocyte integrins. This defect can result in the absence of cell surface expression of all three members of the leukocyte integrins. We investigated the capacity of T cell clones obtained from the blood of an LAD patient and of normal T cell clones to adhere to human umbilical vein endothelial cells (EC). Adhesion of the number of LAD T cells to unstimulated EC was approximately half of that of leukocyte function-associated antigen (LFA)-1+ T cells. Stimulation of EC with human rTNF-alpha resulted in an average 2- and 2.5-fold increase in adhesion of LFA-1+ and LFA-1- cells, respectively. This effect was maximal after 24 h and lasted for 48 to 72 h. The involvement of surface structures known to participate in cell adhesion (integrins, CD44) was tested by blocking studies with mAb directed against these structures. Adhesion of LFA-1+ T cells to unstimulated EC was inhibited (average inhibition of 58%) with mAb to CD11a or CD18. Considerably less inhibition of adhesion occurred with mAb to CD11a or CD18 (average inhibition, 20%) when LFA-1+ T cells were incubated with rTNF-alpha-stimulated EC. The adhesion of LFA-1- T cells to EC stimulated with rTNF-alpha, but not to unstimulated EC, was inhibited (average inhibition, 56%) by incubation with a mAb directed to very late antigen (VLA)-4 (CDw49d). In contrast to LAD T cell clones and the LFA-1+ T cell line Jurkat, mAb to VLA-4 did not inhibit adhesion of normal LFA-1+ T cell clones to EC, whether or not the EC had been stimulated with rTNF-alpha. We conclude that the adhesion molecule pair LFA-1/intercellular adhesion molecule (ICAM)-1 plays a major role in the adhesion of LFA-1+ T cell clones derived from normal individuals to unstimulated EC. Adhesion of LFA-1-T cells to TNF-alpha-stimulated EC is mediated by VLA-4/vascular cell adhesion molecule (VCAM)-1 interactions. Since we were unable to reduce significantly the adhesion of cultured normal LFA-1+ T cells to 24 h with TNF-alpha-stimulated endothelium with antibodies that block LFA-1/ICAM-1 or VLA-4/VCAM-1 interactions, and lectin adhesion molecule-1 and endothelial leukocyte adhesion molecule-1 appeared not to be implicated, other as yet undefined cell surface structures are likely to participate in T cell/EC interactions.     

Cytokine secretion by C3H-lpr and -gld T cells. Hypersecretion of IFN-gamma and tumor necrosis factor-alpha by stimulated CD4+ T cells.

Mice homozygous for lpr and gld develop profound lymphadenopathy characterized by the expansion of two unusual T cell subsets, a predominant Ly-5(B220)+ CD4- CD8- double negative (DN) population and a minor CD4 dull+ Ly-5(B220)+ population. The mechanisms promoting lymphoproliferation are unknown, but one possibility is a abnormality in the production of cytokines that regulate T cell growth. In the present report, unfractionated LN cells and sorted T cell subsets from C3H-lpr, -gld, and -+/+ mice were compared for spontaneous and induced secretion of a spectrum of lymphokines. In addition, CD4+, CD4 dull+ Ly-5(B220)+, and DN T cells were examined for expression of CD3 epsilon, TCR-alpha/beta heterodimers, Ly-6C, and CD44 and for proliferative responses to immobilized anti-TCR mAb and cofactors. These studies revealed that sorted DN T cells did not secrete IL-3, IL-4, IL-5, IL-6, GM-CSF, TNF-alpha, or IFN-gamma spontaneously or after TCR-alpha/beta cross-linking. In contrast, stimulated unfractionated lpr and gld LN cells proliferated strongly and secreted high levels of IFN-gamma and TNF-alpha and low levels of IL-3, IL-4, and IL-6. Despite a 5- to 10-fold deficit in the frequency of CD4+ and CD8+ T cells, cytokine secretion by lpr and gld LN generally exceeded that of +/+ LN. Comparisons of cytokine secretion by stimulated CD4+ T cells revealed that +/+, lpr, and gld CD4+ Ly-5(B220)- T cells proliferated strongly, but only lpr and gld cells produced significant levels of IFN-gamma. The lpr and gld CD4+ T cells also produced higher levels of TNF-alpha and IL-2 than +/+ cells. In contrast to normal CD4+ T cells, lpr and gld CD4+ Ly-5(B220)+ T cells proliferated weakly and did not secrete TNF-alpha, IL-2, or, in most experiments, IFN-gamma after stimulation. Phenotypic studies of T cell subsets revealed that unstimulated lpr and gld CD4+ Ly-5(B220)- T cells express significantly higher levels of CD44 than +/+ CD4+ T cells. In addition, CD4 dull+ Ly-5(B220)+ cells closely resembled DN T cells in size and expression of TCR-alpha/beta, CD3epsilon, CD44, and Ly-6C. Since elevated CD44 expression is generally associated with T cell activation and only previously activated normal CD4+ T cells produce high levels of IFN-gamma in vitro, our data suggest that lpr and gld CD4+ Ly-5(B220)- T cells contain a higher than normal proportion of primed or memory T cells and thus may be polyclonally activated in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)     

A recombinant vector expressing transgenes for four T-cell costimulatory molecules (OX40L,B7-1, ICAM-1, LFA-3) induces sustained CD4+ and CD8+ T-cell activation,protection from apoptosis,and enhanced cytokine production

The role of OX40L on the activation of T cells was investigated using poxvirus vectors expressing OX40L alone or in combination with three other T-cell costimulatory molecules: B7-1, ICAM-1, and LFA-3. Poxvirus vector-infected cells were used to stimulate nai;ve or activated CD4(+) and CD8(+) T cells. These studies demonstrate that (a) OX40L plays a role in sustaining the long-term proliferation of CD8(+) T cells in addition to the known effect on CD4(+) T cells following activation, (b) OX40L enhances the production of Th1 cytokines (IL-2, IFN-gamma, and TNF-alpha) from both CD4(+) and CD8(+) while no change in IL-4 expression was observed, and (c) the anti-apoptotic effect of OX40L on T cells is likely the result of sustained expression of anti-apoptotic genes while genes involved in apoptosis are inhibited. In addition, these are the first studies to demonstrate that the combined use of a vector driving the expression of OX40L with three other costimulatory molecules (B7-1, ICAM-1, and LFA-3) both enhances initial activation and then further potentiates sustained activation of nai;ve and effector T cells.     

Production of TNF-alpha and TNF-beta by staphylococcal enterotoxin A activated human T cells

Staphylococcal enterotoxin at concentrations of less than 1 pg/ml induces significant TNF activity in human peripheral blood T cells and monocytes. Maximal TNF activity is routinely detected after 48 to 72 h of culture. IL-2 and IL-4 were both growth promoting for human T cells but only IL-2 could efficiently induce TNF production. The production of TNF-alpha and TNF-beta differed greatly in kinetics. An early intracytoplasmatic production of TNF-alpha after 6 h was detected in both monocytes and T cells whereas a late production of TNF-beta (lymphotoxin) after 48 h, occurred in the T cell population. Induction of TNF-alpha and TNF-beta production by Staphylococcal enterotoxin requires the presence of both monocytes and T cells. The CD4+45R- but not CD4+45R+ and CD8+ cells supported TNF-alpha production in monocytes. The main lytic component from Staphylococcal enterotoxin-activated mononuclear cells is TNF-beta. CD4+ and CD8+ T cells produced about equal amounts of biologically active TNF into the culture supernatants but a fourfold higher frequency of TNF-beta producing cells was demonstrated among CD4+ vs CD8+ cells. The CD4+45R- T cell subset was an efficient producer of TNF-beta and IFN-gamma whereas the CD4+45R+ T cell subset produced significant amounts of TNF-beta but only marginal amounts of IFN-gamma.     

Role of sulfation in CD44-mediated hyaluronan binding induced by inflammatory mediators in human CD14(+) peripheral blood monocytes.

Activation of T cells by Ag or stimulation of monocytes with inflammatory cytokines induces CD44 to bind to hyaluronan (HA), an adhesion event implicated in leukocyte-leukocyte, leukocyte-endothelial cell, and leukocyte-stromal cell interactions. We have previously shown that TNF-alpha induces CD44 sulfation in a leukemic cell line, which correlated with the induction of HA binding and CD44-mediated adhesion. In this study, we establish that TNF-alpha and IFN-gamma induce HA binding and the sulfation of CD44 in CD14(+) PBMC, whereas no induced HA binding or CD44 sulfation was observed in CD14(-) PBMC stimulated with TNF-alpha. Treatment of cells with NaClO(3), an inhibitor of sulfation, prevented HA binding in a significant percentage of CD14(+) PBMC induced by TNF-alpha, LPS, IL-1beta, or IFN-gamma. Furthermore, stimulation with TNF-alpha or IFN-gamma in the presence of NaClO(3) reduced the ability of isolated CD44H to bind HA, demonstrating a direct effect of CD44H sulfation on HA binding. In contrast, the transient induction of HA binding in T cells by PHA was not affected by NaClO(3), suggesting that activated T cells do not use sulfation as a mechanism to regulate HA binding. Overall, these results demonstrate that inducible sulfation of CD44H is one mechanism used by CD14(+) peripheral blood monocytes to induce HA binding in response to inflammatory agents such as TNF-alpha and IFN-gamma.     

Differential regulation of IFN-gamma, TNF-alpha, and IL-10 production by CD4(+) alphabetaTCR+ T cells and vdelta2(+) gammadelta T cells in response to monocytes infected with Mycobacterium tuberculosis-H37Ra.

Mycobacterium tuberculosis bacilli readily activate CD4(+) and gammadelta T cells. CD4(+) and gammadelta T cells were compared for their ability to regulate IFN-gamma, TNF-alpha, and IL-10 production, cytokines with significant roles in the immune response to M. tuberculosis. PBMC from healthy tuberculin positive donors were stimulated with live M. tuberculosis-H37Ra. CD4(+) and gammadelta T cells were purified by negative selection and tested in response to autologous monocytes infected with M. tuberculosis. Both subsets produced equal amounts of secreted IFN-gamma. However, the precursor frequency of IFN-gamma secreting gammadelta T cells was half that of CD4(+) T cells, indicating that gammadelta T cells were more efficient producers of IFN-gamma than CD4(+) T cells. TNF-alpha production was markedly enhanced by addition of CD4(+) and gammadelta T cells to M. tuberculosis infected monocytes, and TNF-alpha was produced by both T cells and monocytes. No differences in TNF-alpha enhancement were noted between CD4(+) and gammadelta T cells. IL-10 production by M. tuberculosis infected monocytes was not modulated by CD4(+) or gammadelta T cells. Thus CD4(+) and gammadelta T cells had similar roles in differential regulation of IFN-gamma, TNF-alpha, and IL-10 secretion in response to M. tuberculosis infected monocytes. However, the interaction between T cells and infected monocytes differed for each cytokine. IFN-gamma production was dependent on antigen presentation and costimulators provided by monocytes. TNF-alpha levels were increased by addition of TNF-alpha produced by T cells and IL-10 production by monocytes was not modulated by CD4(+) or gammadelta T cells.     

Phosphorylation of the LFA-1 integrin beta2-chain on Thr-758 leads to adhesion, Rac-1/Cdc42 activation, and stimulation of CD69 expression in human T cells

Phosphorylation of the leukocyte function-associated antigen-1 (LFA-1) integrin beta2-chain on Thr-758 occurs after T cell receptor stimulation and leads to 14-3-3 recruitment to the integrin, actin cytoskeleton reorganization, and increased adhesion. Here, we have investigated the signaling effects of beta2 integrin Thr-758 phosphorylation. A penetratin-coupled phospho-Thr-758-beta2 peptide (mimicking the part of the integrin beta-chain surrounding Thr-758) stimulated adhesion of human T cells to the LFA-1 ligand intercellular adhesion molecule-1 (ICAM-1). Additionally, the peptide activated the small GTPases Rac-1 and Cdc42 in T cells. Constitutively active forms of Rac-1 and Cdc42, but not Rho, could compensate for the reduction of cell adhesion to ICAM-1 caused by the T758A mutation in the beta2 integrin. Additionally, the active GTPases salvaged the cell-spreading defect of T758A integrin-transfected cells on coated ICAM-1. A dominant negative form of Cdc42, on the other hand, significantly reduced wild-type beta2 integrin-mediated cell adhesion and spreading. In a T cell stimulation system, the pThr-758 penetratin peptide acted in a similar manner to coated ICAM-1 to increase T cell receptor-induced CD69 expression. These results show that Thr-758-phosphorylated LFA-1 is upstream of Rac-1/Cdc42, cell adhesion, and costimulatory activation of human T cells, thus identifying phosphorylation of Thr-758 in beta2 as a proximal element in LFA-1 signaling.     

A murine very late activation antigen-like extracellular matrix receptor involved in CD2- and lymphocyte function-associated antigen-1-independent killer-target cell interaction

CD2 and lymphocyte function-associated antigen (LFA)-1 are well known as T cell adhesion molecules involved in killer-target cell interactions. However, our recent study revealed that molecule(s) other than CD2 and LFA-1 might be involved in the lymphokine-activated killer (LAK) cell cytotoxicity against certain target cells. In order to characterize such unknown molecules, we established a mAb (RMV-7) which could inhibit CD2/LFA-1-independent LAK cell cytotoxicity and binding to target cells at the effector site. The Ag identified by RMV-7 appeared on splenic T cells late after mitogenic stimulation and was a noncovalently linked heterodimer composed of a 140-kDa alpha-chain and a 95-kDa beta-chain. RMV-7 blocked LAK cell binding to fibronectin (FN), fibrinogen, and vitronectin but not that to laminin or type IV collagen, indicating that the RMV-7-defined molecule is a unique extracellular matrix receptor for FN, fibrinogen, and vitronectin. One of its ligand, FN, was found on the surface of several target cells, and LAK cell cytotoxicity against them was blocked by anti-FN antibody at the target site. Similarly, cytotoxicity of a H-2d-specific CTL clone was inhibited by RMV-7 and anti-FN antibody as well. These results indicate that a unique very late activation Ag-like extracellular matrix receptor on murine CTL and LAK cells contributes to target cell binding and cytotoxicity.     

LFA-1 on CD4+ T cells is required for optimal antigen-dependent activation in vivo

The leukocyte-specific integrin, LFA-1, plays a critical role in trafficking of T cells to both lymphoid and nonlymphoid tissues. However, the role of LFA-1 in T cell activation in vivo has been less well understood. Although there have been reports describing LFA-1-deficient T cell response defects in vivo, due to impaired migration to lymphoid structures and to sites of effector function in the absence of LFA-1, it has been difficult to assess whether T cells also have a specific activation defect in vivo. We examined the role of LFA-1 in CD4(+) T cell activation in vivo by using a system that allows for segregation of the migration and activation defects through the adoptive transfer of LFA-1-deficient (CD18(-/-)) CD4(+) T cells from DO11.10 Ag-specific TCR transgenic mice into wild-type BALB/c mice. We find that in addition to its role in trafficking to peripheral lymph nodes, LFA-1 is required for optimal CD4(+) T cell priming in vivo upon s.c. immunization. CD18(-/-) DO11.10 CD4(+) T cells primed in the lymph nodes demonstrate defects in IL-2 and IFN-gamma production. In addition, recipient mice adoptively transferred with CD18(-/-) DO11.10 CD4(+) T cells demonstrate a defect in OVA-specific IgG2a production after s.c. immunization. The defect in priming of CD18(-/-) CD4(+) T cells persists even in the presence of proliferating CD18(+/-) CD4(+) T cells and in lymphoid structures to which there is no migration defect. Taken together, these results demonstrate that LFA-1 is required for optimal CD4(+) T cell priming in vivo.     

Somatostatin down-regulates LFA-1 activation by modulating Rap1 expression in CD4+ and CD8+ T cells

Leukocyte function-associated antigen-1 (LFA-1) is one of the integrins that are expressed on the leukocytes, and has been shown to play an important role in leukocyte trafficking. The adhesive activity of LFA-1 is governed partially by the Rap1. This study examined that the relationship between LFA-1 and Rap1 mRNA expressions by anti-CD3 and anti-CD3+SOM treatment in the CD4+ and CD8+ T cells. The LFA-1 mRNA expression levels following the anti-CD3 and anti-CD3+SOM treatment for 30 min was greater on the CD8+ T cells, and the LFA-1 expression of the CD8+ T cells with anti-CD+SOM treatment was affected more severely than that of the CD4+ T cells. The Rap1 mRNA expression patterns following anti-CD3 and anti-CD3+SOM stimulation in the CD4+ and CD8+ T cells were similar to the LFA-1 expression patterns, and the expression level following anti-CD3+SOM treatment was suppressed more significantly in the CD8+ T cells. These results suggest that the difference in the Rap1 expression level after stimulation might explain the differences in the LFA-1 expression level on the T cell subsets, and that the down-regulation of Rap1 expression following SOM treatment is closely related to the diminished LFA-1 expression.     

Tumor targets stimulate IL-2 activated killer cells to produce interferon-gamma and tumor necrosis factor

Lymphokine-activated killer (LAK) cells are cytotoxic for a variety of autologous and allogeneic tumor cells as well as modified autologous cells. It is assumed that LAK cells lyse their targets solely by direct cell to cell contact, possibly involving the degranulation and exocytosis of pore-forming elements, similar to that observed with cytotoxic T lymphocytes and NK cells. Reported here are studies demonstrating that LAK cells release factor(s) that are cytotoxic for a human breast carcinoma cell line, MCF-7, when stimulated with tumor cells. The factor(s) are slow acting and maximum cytotoxicity is observed only in a 72-h cytotoxic assay. The ability of LAK cells to secrete cytotoxic factor(s) is dependent on both the ratio of LAK cells to stimulating tumor cells as well as the length of their coincubation. A number of similarly slow acting cytokines that are cytostatic and/or cytotoxic for tumor cells have been described. We tested the ability of specific polyclonal antibodies directed against TNF, IFN-alpha, IFN-beta, and IFN-gamma to neutralize the cytotoxic supernatant activity. Only antibodies specific for IFN-gamma and TNF were neutralizing. We measured the amounts of IFN-gamma and TNF in the cytotoxic supernatants and determined that increased amounts of IFN-gamma and TNF were released after LAK cell-tumor cell interactions compared to supernatants of LAK cells alone or tumor cell alone. Comparable concentrations of human rIFN-gamma and rTNF resulted in similar levels (50 to 90%) of MCF-7 cell cytotoxicity as those observed with the stimulated LAK cell supernatants. We thus concluded that the majority of the cytotoxic activity released by LAK cells when stimulated with tumor cells was attributed to the synergistic activities of IFN-gamma and TNF. The significance of these observations in relation to the possible mechanisms by which LAK cells mediate cytolysis is discussed.     

CD28, Ox-40, LFA-1, and CD4 modulation of Th1/Th2 differentiation is directly dependent on the dose of antigen

The involvement of specific accessory/costimulatory molecules in differentiation to Th1 and Th2 phenotypes is controversial. Reports suggest that molecules such as CD4, CD28, and Ox-40 support Th2 differentiation and suppress Th1 differentiation, whereas others such as LFA-1 support Th1 responses and suppress Th2 responses. We have previously defined an in vitro model of differentiation that is absolutely dependent on the initial dose and affinity of peptide presented to a naive CD4 cell. The dose and affinity of Ag regulate autocrine production of IL-2, IL-4, and IFN-gamma, which in turn govern differentiation to Th1 and Th2 phenotypes. We have used this system to confirm that CD4, CD28, and Ox-40 interactions can promote, and LFA-1 interactions can suppress, differentiation of cells secreting the Th2 cytokines IL-5 and IL-13. However, for CD4 and LFA-1, this is only seen over a certain range of peptide doses. In addition, CD28 and Ox-40 interactions also promote Th1 differentiation. In general, agonist Abs to accessory molecules shifted the response curves for IFN-gamma, IL-5, and IL-13 to lower doses, whereas antagonist reagents resulted in similar curves shifted toward the higher doses. We conclude that ligation of cell surface accessory receptors enables low doses of Ag to promote responses normally induced only by higher doses. Individual receptors do not intrinsically regulate one cytokine phenotype or another, suggesting that differentiation is controlled by the level of expression of multiple accessory molecule pairs integrated with the number and affinity of peptide/MHC complexes.