Recombinant human interferon-inducible protein 10 is a chemoattractant for human monocytes and T lymphocytes and promotes T cell adhesion to endothelial cells.

The human cytokine interferon-inducible protein 10 (IP-10) is a small glycoprotein secreted by activated T cells, monocytes, endothelial cells, and keratinocytes, and is structurally related to a family of chemotactic cytokines called chemokines. Although this protein is present in sites of delayed-type hypersensitivity reactions and lepromatous leprosy lesions, the biological activity of IP-10 remains unknown. We report here that recombinant human IP-10 stimulated significant in vitro chemotaxis of human peripheral blood monocytes but not neutrophils. Recombinant human IP-10 also stimulated chemotaxis of stimulated, but not unstimulated, human peripheral blood T lymphocytes. Phenotypic analysis of the stimulated T cell population responsive to IP-10 demonstrated that stimulated CD4+ and CD29+ T cells migrated in response to IP-10. This resembles the biological activity of the previously described T cell chemoattractant RANTES. Using an endothelial cell adhesion assay, we demonstrated that stimulated T cells pretreated with optimal doses of IP-10 exhibited a greatly enhanced ability to bind to an interleukin 1-treated endothelial cell monolayer. These results demonstrate that the IP-10 gene encodes for an inflammatory mediator that specifically stimulates the directional migration of T cells and monocytes as well as potentiates T cell adhesion to endothelium.

Human interferon-inducible protein 10 is a potent inhibitor of angiogenesis in vivo.

Human interferon-inducible protein 10 (IP-10), a member of the alpha chemokine family, inhibits bone marrow colony formation, has antitumor activity in vivo, is chemoattractant for human monocytes and T cells, and promotes T cell adhesion to endothelial cells. Here we report that IP-10 is a potent inhibitor of angiogenesis in vivo. IP-10 profoundly inhibited basic fibroblast growth factor-induced neovascularization of Matrigel (prepared by H. K. Kleinman) injected subcutaneously into athymic mice. In addition, IP-10, in a dose-dependent fashion, suppressed endothelial cell differentiation into tubular capillary structures in vitro. IP-10 had no effect on endothelial cell growth, attachment, and migration as assayed in vitro. These results document an important biological property of IP-10 and raise the possibility that IP-10 may participate in the regulation of angiogenesis during inflammation and tumorigenesis.

Opiates transdeactivate chemokine receptors: delta and mu opiate receptor-mediated heterologous desensitization.

An intact chemotactic response is vital for leukocyte trafficking and host defense. Opiates are known to exert a number of immunomodulating effects in vitro and in vivo, and we sought to determine whether they were capable of inhibiting chemokine-induced directional migration of human leukocytes, and if so, to ascertain the mechanism involved. The endogenous opioid met-enkephalin induced monocyte chemotaxis in a pertussis toxin-sensitive manner. Met-enkephalin, as well as morphine, inhibited IL-8-induced chemotaxis of human neutrophils and macrophage inflammatory protein (MIP)-1alpha, regulated upon activation, normal T expressed and secreted (RANTES), and monocyte chemoattractant protein 1, but not MIP-1beta-induced chemotaxis of human monocytes. This inhibition of chemotaxis was mediated by delta and micro but not kappa G protein-coupled opiate receptors. Calcium flux induced by chemokines was unaffected by met-enkephalin pretreatment. Unlike other opiate-induced changes in leukocyte function, the inhibition of chemotaxis was not mediated by nitric oxide. Opiates induced phosphorylation of the chemokine receptors CXCR1 and CXCR2, but neither induced internalization of chemokine receptors nor perturbed chemokine binding. Thus, inhibition of chemokine-induced chemotaxis by opiates is due to heterologous desensitization through phosphorylation of chemokine receptors. This may contribute to the defects in host defense seen with opiate abuse and has important implications for immunomodulation induced by several endogenous neuropeptides which act through G protein-coupled receptors.

Preferential migration of activated CD4+ and CD8+ T cells in response to MIP-1 alpha and MIP-1 beta.

Recombinant human macrophage inflammatory protein-1 alpha (rhMIP-1 alpha) and rhMIP-1 beta were potent chemoattractants of human T lymphocytes. These rhMIP-1 cytokines attracted only T cells activated by monoclonal antibody to CD3 and did not attract unstimulated lymphocytes. Phenotypic analysis revealed that CD4+ T cells were capable of migrating in response to rhMIP-1 beta, whereas rhMIP-1 alpha induced chemotaxis of predominantly CD8+ T lymphocytes. Activated naïve and memory T cells also migrated in response to rhMIP-1 cytokines. Furthermore, these cytokines enhanced the ability of T cells to bind to an endothelial cell monolayer. These results suggest that rhMIP-1 cytokines preferentially recruit specific T cell subsets during the evolution of the immune response.

Claudin-1 overexpression in melanoma is regulated by PKC and contributes to melanoma cell motility.

Serial analysis of gene expression followed by pathway analysis implicated the tight junction protein claudin-1 (CLDN1) in melanoma progression. Tight junction proteins regulate the paracellular transport of molecules, but staining of a tissue microarray revealed that claudin-1 was overexpressed in melanoma, and aberrantly expressed in the cytoplasm of malignant cells, suggesting a role other than transport. Indeed, melanoma cells in culture demonstrate no tight junction function. It has been shown that protein kinase C (PKC) can affect expression of claudin-1 in rat choroid plexus cells, and we observed a correlation between levels of activated PKC and claudin expression in our melanoma cells. To determine if PKC could affect the expression of CLDN1 in human melanoma, cells lacking endogenous claudin-1 were treated with 200 nM phorbol myristic acid (PMA). PKC activation by PMA caused an increase in CLDN1 transcription in 30 min, and an increase in claudin-1 protein by 12 h. Inhibition of PKC signaling in cells with high claudin-1 expression resulted in decreased claudin-1 expression. CLDN1 appears to contribute to melanoma cell invasion, as transient transfection of melanoma cells with CLDN1 increased metalloproteinase 2 (MMP-2) secretion and activation, and subsequently, motility of melanoma cells as demonstrated by wound-healing assays. Conversely, knockdown of CLDN1 by siRNA resulted in the inhibition of motility, as well as decreases in MMP-2 secretion and activation. These data implicate claudin-1 in melanoma progression.

Monocyte chemotactic protein-1 (MCP-1), -2, and -3 are chemotactic for human T lymphocytes.

Monocyte chemotactic protein (MCP)-1, -2, and -3 all have been shown to induce monocyte/macrophage migration in vitro and MCP-1, also known as MCAF, chemoattracts basophils and mast cells. We report here that natural MCP-1 as well as synthetic preparations of MCP-2 and MCP-3 stimulate significant in vitro chemotaxis of human peripheral blood T lymphocytes. This MCP-induced migration was dose-dependent and directional, but not chemokinetic. Phenotypic analysis of the T cell population responsive to MCP-1, MCP-2, and MCP-3 demonstrates that both CD4+ and CD8+ T cells migrated in response to these chemokines. Similar results were observed using human CD4+ and CD8+ T cell clones. Neutralizing antisera to MCAF or MCP-2 abrogated T cell migration in response to MCP-1 and MCP-2, respectively, but not to RANTES. Subcutaneous administration of purified MCP-1 into the hind flanks of SCID mice engrafted with human peripheral blood lymphocytes (PBL) induced significant human CD3+ T cell infiltration into the site of injection at 4 h. These results demonstrate that MCP-1, MCP-2, and MCP-3 are inflammatory mediators that specifically stimulate the directional migration of T cells as well as monocytes and may play an important role in immune cell recruitment into sites of antigenic challenge.

T lymphocyte recruitment by interleukin-8 (IL-8). IL-8-induced degranulation of neutrophils releases potent chemoattractants for human T lymphocytes both in vitro and in vivo.

IL-8 has been shown to be a human neutrophil and T cell chemoattractant in vitro. In an effort to assess the in vivo effects of IL-8 on human leukocyte migration, we examined the ability of rhIL-8 to induce human T cell infiltration using a human/mouse model in which SCID mice were administered human peripheral blood lymphocytes intraperitoneally, followed by subcutaneous injections of rhIL-8. rhIL-8 induced predominantly murine neutrophil accumulation by 4 h after administration while recombinant human macrophage inflammatory protein-1beta (rhMIP-1beta) induced both murine monocytes and human T cell infiltration during the same time period as determined by immunohistology. Interestingly, 72 h after chemokine administration, a marked human T cell infiltrate was observed in the IL-8 injection site suggesting that rhIL-8 may be acting indirectly possibly through a murine neutrophil-derived T cell chemoattractant. This hypothesis was confirmed using granulocyte-depleted SCID mice. Moreover, human neutrophils stimulated in vitro with IL-8 were found to release granule-derived factor(s) that induce in vitro T cell and monocyte chemotaxis and chemokinesis. This T cell and monocyte chemotactic activity was detected in extracts of both azurophilic and specific granules. Together, these results demonstrate that neutrophils store and release, upon stimulation with IL-8 or other neutrophil activators, chemoattractants that mediate T cell and monocyte accumulation at sites of inflammation.

Growth hormone promotes human T cell adhesion and migration to both human and murine matrix proteins in vitro and directly promotes xenogeneic engraftment.

Recombinant human growth hormone (rhGH) promotes human T cell engraftment in mice with severe combined immunodeficiency, suggesting that rhGH may have effects on T cell adhesion and migration in vivo. The ability of rhGH to directly affect the adhesion capacity of human T cells to a variety of human or murine adhesion molecules and extracellular matrix proteins was examined. rhGH induced significant human T cell adherence to both human and murine substrates via either beta 1 or beta 2 integrin molecules. rhGH was capable of inducing significant migration of resting and activated human T cells and their subsets. Most of the migratory response to rhGH was chemokinetic rather than chemotactic. In vivo engraftment studies in severe combined immunodeficiency mice receiving human T cells revealed that treatment with rhGH resulted in improved thymic engraftment, whereas treatment with non-human-reactive ovine GH demonstrated no significant effects. These data demonstrate that rhGH directly augments human T cell trafficking to peripheral murine lymphoid tissues. rhGH appears to be capable of directly altering the adhesive and migratory capacity of human T cells to molecules of either murine or human origin. Therefore, GH may, under either isogeneic or xenogeneic conditions, play a role in normal lymphocyte recirculation.

Immunologic and hematopoietic effects of CD40 stimulation after syngeneic bone marrow transplantation in mice.

CD40 is a molecule present on multiple cell types including B lymphocyte lineage cells. CD40 has been shown to play an important role in B cell differentiation and activation in vitro, although little is known concerning the effects of CD40 stimulation in vivo. We therefore examined the effects of CD40 stimulation in mice using a syngeneic bone marrow transplantation (BMT) model in an effort to augment B cell recovery after high dose therapy with hematopoietic reconstitution. After the BMT, mice were treated with or without 2-6 microg of a soluble recombinant murine CD40 ligand (srmCD40L) given intraperitoneally twice a week. A significant increase in B cell progenitors (B220+/ surface IgM-) was observed in the bone marrow of mice receiving the srmCD40L. The treated recipients also demonstrated improved B-cell function with increases in total serum immunoglobulin and increased splenic mitogen responsiveness to LPS being noted. Additionally, srmCD40L treatment promoted secondary lymphoid organ repopulation, accelerating germinal center formation in the lymph nodes. Total B cell numbers in the periphery were not significantly affected even with continuous srmCD40L administration. Lymphocytes obtained from mice treated with the ligand also had increases in T cell mitogen and anti-CD3 mAb responsiveness and acquired the capability to produce IL-4. Surprisingly, treatment with srmCD40L also produced hematopoietic effects in mice, resulting in an increase of BM and splenic hematopoietic progenitor cells in the mice after BMT. Treatment with srmCD40L significantly increased granulocyte and platelet recovery in the peripheral blood. Incubation of BMC with srmCD40L in vitro also resulted in increased progenitor proliferation, demonstrating that the hematopoietic effects of the ligand may be direct. Thus, stimulation of CD40 by its ligand may be beneficial in accelerating both immune and hematopoietic recovery in the setting of bone marrow transplantation.

Differential effects of the absence of interferon-gamma and IL-4 in acute graft-versus-host disease after allogeneic bone marrow transplantation in mice.

Graft-versus-host disease (GVHD), in which immunocompetent donor cells attack the host, remains a major cause of morbidity after allogeneic bone marrow transplantation (BMT). To understand the role of cytokines in the pathobiology of GVHD, we used cytokine knockout (KO) mice as a source of donor T cells. Two different MHC-disparate strain combinations were examined: BALB/c (H2(d)) donors into lethally irradiated C57BL/6 (H2(b)) recipients or C57BL/6 (H2(b)) donors into B10.BR (H2(k)) recipients. Donor cells were from mice in which either the interferon-gamma (IFN-gamma) or the IL-4 gene was selectively disrupted to understand the role of these cytokines in acute GVHD. In both strain combinations the same pattern was noted with regard to GVHD onset and morbidity. All mice exhibited the classic signs of acute GVHD: weight loss with skin, gut, and liver pathology resulting in morbidity and mortality. Surprisingly, donor cells obtained from mice lacking IFN-gamma gave rise to accelerated morbidity from GVHD when compared with cells from wild-type control donors. Similar results were obtained using normal donors when neutralizing antibodies to IFN-gamma were administered immediately after the BMT. These results suggest that IFN-gamma plays a role in protection from acute GVHD. In marked contrast, cells obtained from IL-4 KO mice resulted in protection from GVHD compared with control donors. Splenocytes from IFN KO mice stimulated with a mitogen proliferated to a significantly greater extent and produced more IL-2 compared with splenocytes obtained from IL-4 KO or control mice. Additionally, there was increased IL-2 production in the spleens of mice undergoing GVHD using IFN-gamma KO donors. These results therefore indicate, with regard to the TH1/ TH2 cytokine paradigm, the absence of a TH1-type cytokine can be deleterious in acute GVHD, whereas absence of a TH2 cytokine can be protective.

Suppression of graft-versus-host disease and amplification of graft-versus-tumor effects by activated natural killer cells after allogeneic bone marrow transplantation.

Bone marrow transplantation (BMT) is currently used for the treatment of a variety of neoplastic diseases. However, significant obstacles limiting the efficacy of allogeneic BMT are the occurrence of graft-versus-host disease (GvHD) and tumor relapse. Natural killer (NK) cells exert a variety of immunologic and homoeostatic functions. We examined whether adoptive transfer of activated NK cells of donor type would prevent GvHD after allogeneic BMT in mice. Lethally irradiated C57BL/6 (H-2(b)) mice, were transplanted with MHC incompatible BALB/c (H-2(d)) bone marrow cells and spleen cells and rapidly succumbed to acute GvHD. In contrast, mice that also received activated NK cells of donor type exhibited significant increases in survival. In determining the mechanism by which the NK cells prevented GvHD, mice were concurrently treated with a neutralizing antibodies to the immunosuppressive cytokine TGFbeta. Anti-TGFbeta completely abrogated the protective effects of the activated donor NK cells indicating that TGFbeta plays an important role in the prevention of GvHD by NK cells. We then examined whether activated NK cells of donor type after allogeneic BMT would induce graft-versus-tumor (GvT) effects without GvHD in mice bearing a murine colon adenocarcinoma (MCA-38). 10 d after receiving the tumor, in which the mice had demonstrable lung metastases, recipients received an allogeneic BMT with or without activated NK cells. Administration of activated NK cells resulted in significant GvT effects after allogeneic BMT as evidenced by increases in median survival and fewer lung metastasis. No evidence of GVHD was detected compared with recipients receiving spleen cells alone which also developed fewer lung metastases but in which all had succumbed to GVHD. Thus, our findings suggest that adoptive immunotherapy using activated donor NK cells combined with allogeneic BMT inhibits GvHD and promotes GvT in advanced tumor-bearing mice. These results also suggest that GvT and GvHD can be dissociable phenomena.

Infection with human immunodeficiency virus type 1 upregulates DNA methyltransferase, resulting in de novo methylation of the gamma interferon (IFN-gamma) promoter and subsequent downregulation of IFN-gamma production.

The immune response to pathogens is regulated by a delicate balance of cytokines. The dysregulation of cytokine gene expression, including interleukin-12, tumor necrosis factor alpha, and gamma interferon (IFN-gamma), following human retrovirus infection is well documented. One process by which such gene expression may be modulated is altered DNA methylation. In subsets of T-helper cells, the expression of IFN-gamma, a cytokine important to the immune response to viral infection, is regulated in part by DNA methylation such that mRNA expression inversely correlates with the methylation status of the promoter. Of the many possible genes whose methylation status could be affected by viral infection, we examined the IFN-gamma gene as a candidate. We show here that acute infection of cells with human immunodeficiency virus type 1 (HIV-1) results in (i) increased DNA methyltransferase expression and activity, (ii) an overall increase in methylation of DNA in infected cells, and (iii) the de novo methylation of a CpG dinucleotide in the IFN-gamma gene promoter, resulting in the subsequent downregulation of expression of this cytokine. The introduction of an antisense methyltransferase construct into lymphoid cells resulted in markedly decreased methyltransferase expression, hypomethylation throughout the IFN-gamma gene, and increased IFN-gamma production, demonstrating a direct link between methyltransferase and IFN-gamma gene expression. The ability of increased DNA methyltransferase activity to downregulate the expression of genes like the IFN-gamma gene may be one of the mechanisms for dysfunction of T cells in HIV-1-infected individuals.

Chemokine-leukocyte interactions. The voodoo that they do so well.

Leukocyte recruitment from the circulation into inflammatory tissues requires a series of soluble and cell-bound signals between the responding leukocyte and vascular endothelial barrier. Chemotactic factors are believed to be responsible for this selective adhesion and transmigration. A superfamily of small, soluble, structurally-related molecules called 'chemokines' have been identified and shown to selectively promote the rapid adhesion and chemotaxis of a variety of leukocyte subtypes both in vivo and in vitro. Chemokines are produced by almost every cell type in the body in response to a number of inflammatory signals, in particular those which activate leukocyte-endothelial cell interactions. These molecules also appear to play important roles in hematopoesis, cellular activation, and leukocyte effector functions. In addition, chemokines have been found in the tissues of a variety of disease states characterized by distinct leukocytic infiltrates, including rheumatoid arthritis, sepsis, atherosclerosis, asthma, psoriasis, ischemia/reperfusion injury, HIV replication, and a variety of pulmonary disease states. This review will primarily focus on the role of chemokines in cell adhesion and trafficking as well as their role as effector molecules.

Induction of T-cell immunity against Ras oncoproteins by soluble protein or Ras-expressing Escherichia coli.

BACKGROUND: Point mutations in the ras proto-oncogene that activate its oncogenic potential occur in approximately 30% of human cancers. Previous studies have demonstrated that T-cell immunity against some forms of mutant Ras proteins could be elicited, and some effectiveness against tumors expressing activated Ras has been reported. PURPOSE: The goal of this study was to determine if immunization of mice with two forms of mutant Ras protein can induce high levels of Ras mutation-specific T-cell immunity in vitro and tumor regression in vivo. METHODS: Mice (BALB/c or C3H/HeJ) were immunized subcutaneously at 2-week intervals with purified Ras oncoproteins mixed with the immunologic adjuvants Antigen Formulation or QS-21, both of which have been shown to enhance the induction of T-cell-mediated immunity when included as components of soluble protein vaccines. In some experiments, mice were immunized directly with heat-killed Escherichia coli that had been induced to express one of the mutant Ras proteins. Spleen cells plus lymph node cells from Ras-immunized mice were tested in vitro for lysis of syngeneic Ras-expressing tumor cells and proliferation in response to mutant Ras peptides. For some of the cytolytic activity experiments, the spleen cells were grown under TH1 conditions (growth in presence of interleukin 2, interferon gamma, and an antibody directed against interleukin 4 to stimulate a cell-mediated immune response) or TH2 conditions (growth in presence of interleukins 2 and 4 to stimulate a humoral immune response). The specificity of immunity was examined in vivo by challenge of Ras-immunized mice with syngeneic tumor cells expressing mutant Ras oncoproteins (HaBalb, i.e., BALB/c mouse cells expressing Ras with arginine substituted at amino acid position 12 [Arg 12 Ras]; C3HL61, i.e., C3H/HeJ mouse cells expressing Ras with leucine substituted at position 61 [Leu 61 Ras]). Ten mice per group were used in each experiment. RESULTS: Proliferative and cytolytic T-cell responses directed against the Arg 12 Ras protein were generated in BALB/c mice, resulting in protection against challenge with cells expressing Arg 12 Ras and therapeutic benefit in mice bearing established tumors expressing this protein. In C3H/HeJ mice, high levels of cytolytic and proliferative responses were induced against Leu 61 Ras. Immunization with heat-killed E. coli genetically engineered to express Leu 61 Ras also led to the induction of anti-Ras T-cell immunity. T cells grown under TH1 conditions were cytolytic against Ras-transformed tumor cells, whereas those grown under TH2 conditions were not. CONCLUSIONS: Immunization as described here leads to Ras mutation-specific antitumor immunity in vitro and in vivo, with therapeutic efficacy in an established tumor model.

Cytotoxic T-cell response and in vivo protection against tumor cells harboring activated ras proto-oncogenes.

BACKGROUND: Activated forms of the ras proto-oncogene have been found in approximately 30% of human malignancies, including pancreatic, colon, and lung adenocarcinomas. Ras oncoproteins arise by somatic mutation and contain amino acid changes at residues 12, 13, or 61, thus generating unique tumor-specific proteins that are attractive targets for cancer therapy. PURPOSE: The goal of this study was to determine whether vaccination with mutant Ras protein could lead to the generation of cytotoxic T lymphocytes (CTLs) specific for the mutant epitope and to protection against challenge with tumor cells expressing the mutant oncoprotein. METHODS: To determine a methodology for generating CTL responses following immunization with soluble protein, ovalbumin was used as a model tumor antigen. C57BL/6 mice were immunized with soluble ovalbumin administered intraperitoneally at 2-week intervals or with intravenous injection of ovalbumin or osmotically loaded splenocytes. Immunized mice were challenged with E.G7 cells (which express a transfected ovalbumin gene), and tumor growth was monitored. Generation of ovalbumin-specific CTLs was determined by 51Cr release assays. Purified wild-type or mutant H-Ras proteins (containing single amino acid substitutions at position 12 converting Gly to Arg or Val) were used to immunize BALB/c mice intraperitoneally. Ras-immunized mice were challenged with tumor cells containing Arg 12 or Val 12 mutations or not harboring mutant forms of Ras. Cytolytic and proliferative responses to mutant forms of Ras were studied, and the effects of in vivo depletion of CD4+ or CD8+ T lymphocytes were determined. RESULTS: In vivo challenge with E.G7 showed that intraperitoneal immunization with soluble ovalbumin was as effective as osmotic loading, resulting in long-term disease-free survival of some mice and the development of ovalbumin-specific CTLs. Immunization with Arg 12 Ras led to disease-free survival in nine of 10 animals challenged with tumor cells containing an Arg 12 mutation, while no protection was afforded against tumors expressing other forms of Ras or other oncogenes. Splenocytes from BALB/c mice immunized with Arg 12 Ras demonstrated cytolytic activity specific against tumor cells expressing Arg 12 Ras, with most of this activity residing in the CD8+ subset. Mutation-specific proliferation to Arg 12 Ras peptides was also observed. Immunization with Val 12 Ras did not elicit detectable Val 12-specific immunity. CONCLUSIONS: Antigen-specific CTLs can be induced following intraperitoneal immunization of mice with purified, soluble proteins. For both ovalbumin and Arg 12 Ras, specific in vivo protection against tumor cell challenge was observed.

Murine Th1 and Th2 cell clones differentially regulate macrophage nitric oxide production.

Previous studies have demonstrated that the combination of the T helper cell 1 (Th1)-derived cytokines interleukin (IL)-2 and interferon (IFN)-gamma induces nitric oxide (NO) production and tumor cytolysis by mouse peritoneal macrophages and the mouse macrophage cell line ANA-1 in vitro. Conversely, the Th2-derived cytokine IL-4 inhibits IL-2 and IFN-gamma-induced NO production and tumor cytolysis by ANA-1 macrophages. To examine the paracrine regulatory effects of Th1 and Th2 cells on macrophages, various mouse T cell clones were tested for their ability to regulate NO production by mouse peritoneal macrophages or ANA-1 macrophages. Antigen, superantigen, and mitogen stimulated Th1 cells but not Th2 cells induced NO production by macrophages. Supernatants from these activated Th1 clones also induced NO production by peritoneal macrophages and ANA-1 macrophages. Neutralization analysis using monoclonal anticytokine antibodies revealed that both IL-2 and IFN-gamma production by activated Th1 cells were required for the production of NO by macrophages. Co-culture studies using a panel of Th2 cell clones that share the same antigen specificity revealed that these cells suppressed Th1-mediated macrophage activation. The Th2-mediated impairment of Th1-induced NO production was primarily due to the secretion of IL-4. IL-4 appeared to have a direct effect on macrophage activation because neither mitogen-induced proliferation of Th1 cells nor cytokine production by Th1 cells were affected by IL-4. Overall, these results suggest that a potent paracrine regulatory network involving Th1 cells and Th2 cells may control the activation of macrophages for NO production and antitumor cytotoxicity.

Modulation of IL-8 receptor expression on purified human T lymphocytes is associated with changed chemotactic responses to IL-8.

Interleukin-8 is a member of the chemokine superfamily and is a major mediator of acute inflammation. Although IL-8 has been reported by some laboratories also to be a chemoattractant for T lymphocytes, this has been difficult to confirm and remains a controversial issue. By using freshly purified human T cells (90-95% CD3+), we could demonstrate consistent directional migration of T cells to recombinant human IL-8. IL-8 was as potent as RANTES, MIP1 alpha, and MIP1 beta in inducing T cell chemotaxis. Highly purified T cells, however, incubated at 37 degrees C for more than 12 h or cultured overnight with anti-CD3 antibody cross-linked to plastic dishes, showed a markedly reduced capacity to migrate in response to IL-8. This was associated with a decrease in binding of radioiodinated IL-8 to T cells. Northern blot and polymerase chain reaction analyses showed that freshly purified T cells expressed mRNA for both IL-8 receptor type A and type B. Steady-state levels of mRNA for the IL-8RA and IL-8RB genes were also reduced by incubation of the cells with or without anti-CD3 for 12 h at 37 degrees C. These results indicate that T cells are indeed one of the target cell populations for IL-8. The regulation of IL-8 receptor expression on T lymphocytes may contribute to the pathophysiological role of IL-8 in inducing the homing and infiltration of T cells.

Beta chemokines costimulate lymphocyte cytolysis, proliferation, and lymphokine production.

We report here the ability of the beta chemokines MIP-1 alpha, MIP-1 beta, RANTES, and MCP-1 to enhance some lymphocyte effector functions. Initial studies focused on the effects of chemokines on human and mouse cytotoxic T lymphocyte (CTL)- and natural killer (NK) cell-specific cytolytic responses. The results demonstrate that beta chemokines are capable of augmenting mouse and human CTL and human NK- but not lymphokine-activated killer cell- or antibody-dependent cell cytotoxicity-specific cytolytic responses. Neutralization analysis utilizing integrin-specific antibodies revealed that CTL/NK tumor cell conjugate formation is required for chemokine-induced killing. In addition, both CTLs and NK cells incubated with various beta chemokines were induced to degranulate and release granule-derived serine esterases, suggesting that chemokines may be important costimulators of CTL and NK cell degranulation and may thus augment local target cell destruction. Chemokines also modulate antigen-driven T cell proliferative responses as well as effects on lymphokine production. Many of the beta chemokines were found to potentiate human and mouse antigen-specific Th1 and Th2 clone activation promoting cellular proliferation and the release of various lymphokines. This chemokine-mediated T cell proliferation was chemokine and antigen dose dependent as well as clone dependent. Chemokine pretreatment analyses with T cells and antigen-presenting cells (APCs) revealed that chemokines up-regulate both T cells and antigen- presenting cells (APCs) revealed that chemokines up-regulate both T cell and APC functions. Costimulation assays using immobilized antiCD3 monoclonal antibody-coated plates and purified human and mouse T cells and T cell clones in the presence of various chemokines also exhibited enhanced proliferation and lymphokine secretion. This costimulation was interleukin-2 dependent and required the presence of free extracellular calcium. Examination of chemokine-treated APCs revealed that the T cell costimulatory molecule B7-1 was induced by various beta chemokines. Neutralization of endogenously produced chemokines, with specific antibodies during an antigen-specific T cell response blocked cellular proliferation, suggesting that the chemokines have an autocrine role in antigen-induced T cell proliferative responses. Together, these results suggest that chemokines play a significant role in the activation of polyclonal as well as antigen-specific helper and cytotoxic T cells during the genesis of an immune response.

Role for CD40-CD40 ligand interactions in the immune response to solid tumours.

CD40-mediated interactions play an important role in the response to infections, transplantation, and cancer by affecting the development, activation, proliferation and differentiation of a variety of immune cells. In the current study we examined the role of CD40-mediated interactions in immune responses to bladder, pancreatic and breast carcinomas as well as melanoma cell lines using soluble human CD40L (rhCD40L) or anti-CD40 mAb in vitro. CD40 expression was readily detected in a large proportion of the cell lines and was augmented but not induced de novo by treatment with IFNgamma. Treatment of CD40-positive cell lines with rhCD40L or anti-CD40mAb enhanced cell surface expression of ICAM-1 and FAS and stimulated the production of IL-6, IL-8, GROalpha, GM-CSF and TNFalpha but not IL-4, IL-10, TGFbeta, MCP-1, RANTES, MIP-1beta, or IP-10. In addition, incubation of CD40+ tumour cell lines with immobilised rhCD40L or anti-CD40 mAb in vitro resulted in significant inhibition of proliferation and a corresponding decrease in viability. This CD40-mediated inhibition of cell growth was due, at least in part, to alterations in cell cycle and the induction of apoptosis. Transfection of CD40-negative tumour cell lines with the cDNA for CD40 conferred responsiveness to rhCD40L and anti-CD40 antibody. Finally, the presence of CD40 on the surface of carcinoma lines was found to be an important factor in the generation of tumour-specific T cell responses.

A phase I trial of B7-transfected or parental lethally irradiated allogeneic melanoma cell lines to induce cell-mediated immunity against tumor-associated antigen presented by HLA-A2 or HLA-A1 in patients with stage IV melanoma. NCI protocol T93-0161. BRMP protocol 9401.

T cell lines generated by primary in vitro stimulation with B7-expressing HLA-A2+ melanoma cells lyse HLA-A2+ melanomas, but not non-melanomas that are HLA-A2+. Other data have demonstrated lack of response of these T cell lines against non-HLA-A2 melanomas. These concepts are verified by data from MALME MEL, which is killed, and MALME FIB, which is not. In no case was lysis directed at targets expressing potential allo-antigens (except for HLA-A2+ melanomas). A19 and Aw33 have not been excluded as possible allo-targets (but no data suggests they are). In total, it appears that much of the lytic activity observed in the two T cell lines is directed against HLA-A2-restricted, melanoma-specific antigens.