Chapoval, Andrei I.

Abstract Bifunctional Abs (BFA) with specificity for the TCR/CD3 complex of T cells and tumor Ag can bridge T lymphocytes and tumor cells and, thereby, trigger activation events. The ability of intact and F(ab')2 anti-CD3 (500A2) x anti-p97 (96.5) BFA to induce activation of T lymphocytes in the presence of murine melanoma tumor cells (CL-62) expressing human melanoma-associated Ag (p97) was investigated in vitro and in vivo. Intact and F(ab')2 BFA induced significant proliferation of T lymphocytes in the presence of p97+ tumor cells. Incubation of splenocytes with intact or F(ab')2 BFA and p97+ tumor cells increased BFA-mediated cytotoxicity against relevant tumor cells. Intact BFA, in contrast to F(ab')2 BFA, induced some activation of T cells in vitro even in the absence of p97+ target cells. In nontumor-bearing mice, administration of F(ab')2 BFA, in contrast to intact BFA, did not increase cytotoxic activity of lymph node (LN) cells and splenocytes. However, when F(ab')2 BFA was administrated into CL-62-bearing mice, an increase of BFA-mediated cytotoxicity of tumor-infiltrating lymphocytes, but not splenocytes nor LN cells, was observed. Moreover, in D-galactosamine-sensitized mice, injection of intact BFA (1 microgram/mice) induced 100% lethality, whereas the same dose of F(ab')2 BFA was not toxic. These results demonstrate that F(ab')2 BFA can induce activation of cytotoxic lymphocytes only in the presence of relevant tumor cells, both in vitro and in vivo. That these activated lymphocytes can be redirected to lyse relevant tumor cells by the same BFA has important implications for the clinical application of BFA anti-tumor therapies.

AbstractDendritic cells (DCs) are critical subsets of leukocytes providing antigen presentation for initiation of humoral and cellular immune responses. Their role as effector cells in tumor resistance, however, is less known. We report here that human DCs generated by culturing plastic-adherent peripheral blood monocytes in the presence of granulocyte-monocyte colony–stimulating factor (GM-CSF) and interleukin-4 have potent growth-inhibition activity in vitro on a wide spectrum of human tumor lines of different tissue origin. Proinflammatory stimuli lipopolysaccharide (LPS) and interferon-γ, but not tumor necrosis factor– and CD40 signaling, can further enhance DC-mediated inhibition of tumor growth. The growth inhibition requires contact between DCs and tumor cells while LPS treatment enhances the antitumor activity in DC culture supernatants. Our results suggest that in addition to their predominant role as regulatory cells, activated DCs are also potential effector cells in tumor immunity.

Abstract This report describes a new human B7-like gene designatedB7-H2. Cell surface expression of B7-H2 protein is detected in monocyte-derived immature dendritic cells. Soluble B7-H2 and immunoglobulin (Ig) fusion protein, B7-H2Ig, binds activated but not resting T cells and the binding is abrogated by inducible costimulator Ig (ICOSIg), but not CTLA4Ig. In addition, ICOSIg stains Chinese hamster ovary cells transfected with B7-H2 gene. By suboptimal cross-linking of CD3, costimulation of T-cell proliferation by B7-H2Ig is dose-dependent and correlates with secretion of interleukin (IL)-2, whereas optimal CD3 ligation preferentially stimulates IL-10 production. The results indicate that B7-H2 is a putative ligand for the ICOS T-cell molecule.

Dendritic cells (DCs) are critical subsets of leukocytes providing antigen presentation for initiation of humoral and cellular immune responses. Their role as effector cells in tumor resistance, however, is less known. We report here that human DCs generated by culturing plastic-adherent peripheral blood monocytes in the presence of granulocyte-monocyte colony–stimulating factor (GM-CSF) and interleukin-4 have potent growth-inhibition activity in vitro on a wide spectrum of human tumor lines of different tissue origin. Proinflammatory stimuli lipopolysaccharide (LPS) and interferon-γ, but not tumor necrosis factor– and CD40 signaling, can further enhance DC-mediated inhibition of tumor growth. The growth inhibition requires contact between DCs and tumor cells while LPS treatment enhances the antitumor activity in DC culture supernatants. Our results suggest that in addition to their predominant role as regulatory cells, activated DCs are also potential effector cells in tumor immunity.

This report describes a new human B7-like gene designatedB7-H2. Cell surface expression of B7-H2 protein is detected in monocyte-derived immature dendritic cells. Soluble B7-H2 and immunoglobulin (Ig) fusion protein, B7-H2Ig, binds activated but not resting T cells and the binding is abrogated by inducible costimulator Ig (ICOSIg), but not CTLA4Ig. In addition, ICOSIg stains Chinese hamster ovary cells transfected with B7-H2 gene. By suboptimal cross-linking of CD3, costimulation of T-cell proliferation by B7-H2Ig is dose-dependent and correlates with secretion of interleukin (IL)-2, whereas optimal CD3 ligation preferentially stimulates IL-10 production. The results indicate that B7-H2 is a putative ligand for the ICOS T-cell molecule.

Abstract LIGHT is a recently identified member of the TNF superfamily and its receptors, herpesvirus entry mediator and lymphotoxin β receptor, are found in T cells and stromal cells. In this study, we demonstrate that LIGHT is selectively expressed on immature dendritic cells (DCs) generated from human PBMCs. In contrast, LIGHT is not detectable in DCs either freshly isolated from PBMCs or rendered mature in vitro by LPS treatment. Blockade of LIGHT by its soluble receptors, lymphotoxin β receptor-Ig or HVEM-Ig, inhibits the induction of DC-mediated primary allogeneic T cell response. Furthermore, engagement of LIGHT costimulates human T cell proliferation, amplifies the NF-κB signaling pathway, and preferentially induces the production of IFN-γ, but not IL-4, in the presence of an antigenic signal. Our results suggest that LIGHT is a costimulatory molecule involved in DC-mediated cellular immune responses.

Abstract B7-H3 is a B7 family molecule with T cell costimulatory function in vitro. The in vivo role of B7-H3 in the stimulation of tumor immunity is unclear. We report here that expression of B7-H3 by transfection of the mouse P815 tumor line enhances its immunogenicity, leading to the regression of tumors and amplification of a tumor-specific CD8+ CTL response in syngeneic mice. Tumor cells engineered to express B7-H3 elicit a rapid clonal expansion of P1A tumor Ag-specific CD8+ CTL in lymphoid organs in vivo and acquire the ability to directly stimulate T cell growth, division, and development of cytolytic activity in vitro. Our results thus establish a role for B7-H3 in the costimulation of T cell immune responses in vivo.

Abstract Interaction between dendritic cells (DCs) and T cells is a prerequisite for the initiation of a T cell response. The molecular nature of this interaction remains to be fully characterized. We report in this work that freshly isolated mouse splenic DCs and bone marrow-derived DCs express CD137 on the cell surface and in soluble form. Triggering CD137 increased the secretion of IL-6 and IL-12 from DCs. More importantly, infusion of an agonistic mAb to CD137 into naive mice enhanced the ability of DCs to stimulate T cell proliferation in response to both alloantigens and a nominal Ag in vitro. This enhancement of DC function is not mediated through activation of T cells, because the effect was also observed in RAG-1 knockout mice that lack T cells. Our findings implicate CD137 as an important receptor involved in the modulation of DC function.

Abstract To investigate the role of HLA-DQ molecules and/or CD4+ T cells in the pathogenesis of allergic asthma, we generated HLA-DQ6 and HLA-DQ8 transgenic mice lacking endogenous class II (Aβnull) and CD4 genes and challenged them intranasally with short ragweed allergenic extract (SRW). We found that DQ6/CD4null mice developed a strong eosinophilic infiltration into the bronchoalveolar lavage and lung tissue, while DQ8/CD4null mice were normal. However, neither cytokines nor eosinophil peroxidase in the bronchoalveolar lavage of DQ6/CD4null mice was found. In addition, the airway reactivity to methacholine was elevated moderately in DQ6/CD4null mice compared with the high response in DQ/CD4+ counterparts and was only partially augmented by CD4+ T cell transfer. The DQ6/CD4null mice showed Th1/Th2-type cytokines and SRW-specific Abs in the immune sera in contrast to a direct Th2 response observed in DQ6/CD4+ mice. The proliferative response of spleen mononuclear cells and peribronchial lymph node cells demonstrated that the response to SRW in DQ6/CD4null mice was mediated by HLA-DQ-restricted CD4−CD8−NK1.1− T cells. FACS analysis of PBMC and spleen mononuclear cells demonstrated an expansion of double-negative (DN) CD4−CD8−TCRαβ+ T cells in SRW-treated DQ6/CD4null mice. These cells produced IL-4, IL-5, IL-13, and IFN-γ when stimulated with immobilized anti-CD3. IL-5 ELISPOT assay revealed that DN T cells were the cellular origin of IL-5 in allergen-challenged DQ6/CD4null mice. Our study shows a role for HLA-DQ-restricted CD4+ and DN T cells in the allergic response.

AbstractT-cell anergy is a tolerance mechanism defined as a hyporesponsive status of antigen-specific T cells upon prior antigen encounter and is believed to play a critical role in the evasion of tumor immunity and the amelioration of allogeneic transplant rejection. Molecular mechanisms in controlling T-cell anergy are less known. We show here that administration of an agonistic monoclonal antibody (mAb) to CD137, a member of the tumor necrosis factor receptor superfamily, prevents the induction of CD8+ cytolytic T-lymphocyte (CTL) anergy by soluble antigens. More importantly, CD137 mAb restores the functions of established anergic CTLs upon reencountering their cognate antigen. As a result, infusion of CD137 mAb inhibits progressive tumor growth that is caused by soluble tumor antigen-induced tolerance in a P815R model. CD137 mAb also restores proliferation and effector functions of anergic alloreactive 2C T cells in a bone marrow transplantation model. Our results indicate that ligation of CD137 receptor delivers a regulatory signal for T-cell anergy and implicate manipulation of the CD137 pathway as a new approach to break T-cell tolerance.