Kevin W. Bruhn

LXR-Dependent Gene Expression Is Important for Macrophage Survival and the Innate Immune Response

The liver X receptors (LXRs) are nuclear receptors with established roles in the regulation of lipid metabolism. We now show that LXR signaling not only regulates macrophage cholesterol metabolism but also impacts antimicrobial responses. Mice lacking LXRs are highly susceptible to infection with the intracellular bacteria Listeria monocytogenes (LM). Bone marrow transplant studies point to altered macrophage function as the major determinant of susceptibility. LXR-null macrophages undergo accelerated apoptosis when challenged with LM and exhibit defective bacterial clearance in vivo. These defects result, at least in part, from loss of regulation of the antiapoptotic factor SPalpha, a direct target for regulation by LXRalpha. Expression of LXRalpha or SPalpha in macrophages inhibits apoptosis in the setting of LM infection. Our results demonstrate that LXR-dependent gene expression plays an unexpected role in innate immunity and suggest that common nuclear receptor pathways mediate macrophage responses to modified lipoproteins and intracellular pathogens.

Type I Interferon Production Enhances Susceptibility to Listeria monocytogenes Infection

Numerous bacterial products such as lipopolysaccharide potently induce type I interferons (IFNs); however, the contribution of this innate response to host defense against bacterial infection remains unclear. Although mice deficient in either IFN regulatory factor (IRF)3 or the type I IFN receptor (IFNAR)1 are highly susceptible to viral infection, we show that these mice exhibit a profound resistance to infection caused by the Gram-positive intracellular bacterium Listeria monocytogenes compared with wild-type controls. Furthermore, this enhanced bacterial clearance is accompanied by a block in L. monocytogenes–induced splenic apoptosis in IRF3- and IFNAR1-deficient mice. Thus, our results highlight the disparate roles of type I IFNs during bacterial versus viral infections and stress the importance of proper IFN modulation in host defense.

Involvement of receptor-interacting protein 2 in innate and adaptive immune responses

Host defences to microorganisms rely on a coordinated interplay between the innate and adaptive responses of immunity. Infection with intracellular bacteria triggers an immediate innate response requiring macrophages, neutrophils and natural killer cells, whereas subsequent activation of an adaptive response through development of T-helper subtype 1 cells (TH1) proceeds during persistent infection. To understand the physiological role of receptor-interacting protein 2 (Rip2), also known as RICK and CARDIAK, we generated mice with a targeted disruption of the gene coding for Rip2. Here we show that Rip2-deficient mice exhibit a profoundly decreased ability to defend against infection by the intracellular pathogen Listeria monocytogenes. Rip2-deficient macrophages infected with L. monocytogenes or treated with lipopolysaccharide (LPS) have decreased activation of NF-κB, whereas dominant negative Rip2 inhibited NF-κB activation mediated by Toll-like receptor 4 and Nod1. In vivo, Rip2-deficient mice were resistant to the lethal effects of LPS-induced endotoxic shock. Furthermore, Rip2 deficiency results in impaired interferon-γ production in both TH1 and natural killer cells, attributed in part to defective interleukin-12-induced Stat4 activation. Our data reflect requirements for Rip2 in multiple pathways regulating immune and inflammatory responses.

The TLR-7 agonist, imiquimod, enhances dendritic cell survival and promotes tumor antigen-specific T cell priming: Relation to central nervous system antitumor immunity

Immunotherapy represents an appealing option to specifically target CNS tumors using the immune system. In this report, we tested whether adjunctive treatment with the TLR-7 agonist imiquimod could augment antitumor immune responsiveness in CNS tumor-bearing mice treated with human gp100 + tyrosine-related protein-2 melanoma-associated Ag peptide-pulsed dendritic cell (DC) vaccination. Treatment of mice with 5% imiquimod resulted in synergistic reduction in CNS tumor growth compared with melanoma-associated Ag-pulsed DC vaccination alone. Continuous imiquimod administration in CNS tumor-bearing mice, however, was associated with the appearance of robust innate immune cell infiltration and hemorrhage into the brain and the tumor. To understand the immunological mechanisms by which imiquimod augmented antitumor immunity, we tested whether imiquimod treatment enhanced DC function or the priming of tumor-specific CD8+ T cells in vivo. With bioluminescent, in vivo imaging, we determined that imiquimod dramatically enhanced both the persistence and trafficking of DCs into the draining lymph nodes after vaccination. We additionally demonstrated that imiquimod administration significantly increased the accumulation of tumor-specific CD8+ T cells in the spleen and draining lymph nodes after DC vaccination. The results suggest that imiquimod positively influences DC trafficking and the priming of tumor-specific CD8+ T cells. However, inflammatory responses induced in the brain by TLR signaling must also take into account the local microenvironment in the context of antitumor immunity to induce clinical benefit. Nevertheless, immunotherapeutic targeting of malignant CNS tumors may be enhanced by the administration of the innate immune response modifier imiquimod.

Listeria monocytogenes as a vaccine vector: virulence attenuation or existing antivector immunity does not diminish therapeutic efficacy.

The bacterium L. monocytogenes is a proposed vaccine carrier based upon the observation that this pathogen replicates within the intracytoplasmic environment facilitating delivery of Ag to the endogenous Ag processing and presentation pathway with subsequent stimulation of peptide specific MHC class I-restricted CD8+ effector cells. In this report, we evaluate virulence-attenuated strains of Listeria monocytogenes as vaccine vectors and examine whether existing antivector (antilisterial) immunity limits or alters its efficacy as a therapeutic cancer vaccine. Following immunization with virulence-attenuated mutants, we found that the effectiveness of L. monocytogenes as a recombinant cancer vaccine remains intact. In addition, we found that antibiotic treatment initiated 24 or 36 h following therapeutic immunization with recombinant L. monocytogenes allows full development of the antitumor response. We also demonstrate that the vaccine vector potential of L. monocytogenes is not limited in animals with existing antilisterial immunity. For these latter studies, mice previously immunized with wild-type L. monocytogenes were infused with melanoma cells and then 5 days later challenged with recombinant tumor Ag expressing L. monocytogenes. Collectively, these results add additional support for the use of L. monocytogenes as a vaccine vector and underscore its potential to be used repeatedly for stimulation of recall responses concomitant with primary cell-mediated responses to newly delivered heterologous tumor-associated epitopes.

A Critical Role for GRP78/BiP in the Tumor Microenvironment for Neovascularization during Tumor Growth and Metastasis

Glucose-regulated protein 78 (GRP78)/BiP is a multifunctional protein which plays a major role in endoplasmic reticulum (ER) protein processing, protein quality control, maintaining ER homeostasis, and controlling cell signaling and viability. Previously, using a transgene-induced mammary tumor model, we showed that Grp78 heterozygosity impeded cancer growth through suppression of tumor cell proliferation and promotion of apoptosis and the Grp78(+/-) mice exhibited dramatic reduction (70%) in the microvessel density (MVD) of the endogenous mammary tumors, while having no effect on the MVD of normal organs. This observation suggests that GRP78 may critically regulate the function of the host vasculature within the tumor microenvironment. In this article, we interrogated the role of GRP78 in the tumor microenvironment. In mouse tumor models in which wild-type (WT), syngeneic mammary tumor cells were injected into the host, we showed that Grp78(+/-) mice suppressed tumor growth and angiogenesis during the early phase but not during the late phase of tumor growth. Growth of metastatic lesions of WT, syngeneic melanoma cells in the Grp78(+/-) mice was potently suppressed. We created conditional heterozygous knockout of GRP78 in the host endothelial cells and showed severe reduction of tumor angiogenesis and metastatic growth, with minimal effect on normal tissue MVD. Furthermore, knockdown of GRP78 expression in immortalized human endothelial cells showed that GRP78 is a critical mediator of angiogenesis by regulating cell proliferation, survival, and migration. Our findings suggest that concomitant use of current chemotherapeutic agents and novel therapies against GRP78 may offer a powerful dual approach to arrest cancer initiation, progression, and metastasis.

Enhanced Antitumor Activity Induced by Adoptive T-Cell Transfer and Adjunctive Use of the Histone Deacetylase Inhibitor LAQ824

Tumors grow in the presence of antigen-specific T cells, suggesting the existence of intrinsic cancer cell escape mechanisms. We hypothesized that a histone deacetylase (HDAC) inhibitor could sensitize tumor cells to immunotherapy because this class of agents has been reported to increase tumor antigen expression and shift gene expression to a proapoptotic milieu in cancer cells. To test this question, we treated B16 murine melanoma with the combination of the HDAC inhibitor LAQ824 and the adoptive transfer of gp100 melanoma antigen-specific pmel-1 T cells. The combined therapy significantly improved antitumor activity through several mechanisms: (a) increase in MHC and tumor-associated antigen expression by tumor cells; (b) decrease in competing endogenous lymphocytes in recipient mice, resulting in a proliferative advantage for the adoptively transferred cells; and (c) improvement in the functional activity of the adoptively transferred lymphocytes. We confirmed the beneficial effects of this HDAC inhibitor as a sensitizer to immunotherapy in a different model of prophylactic prime-boost vaccination with the melanoma antigen tyrosinase-related protein 2, which also showed a significant improvement in antitumor activity against B16 melanoma. In conclusion, the HDAC inhibitor LAQ824 significantly enhances tumor immunotherapy through effects on target tumor cells as well as improving the antitumor activity of tumor antigen-specific lymphocytes.

The TLR7 Agonist Imiquimod Enhances the Anti-Melanoma Effects of a Recombinant Listeria monocytogenes Vaccine

Activation of innate immune cells through TLR triggers immunomodulating events that enhance cell-mediated immunity, raising the possibility that ligands to these receptors might act as adjuvants in conjunction with T cell activating vaccines. In this report, topical imiquimod, a synthetic TLR7 agonist, significantly enhanced the protective antitumor effects of a live, recombinant listeria vaccine against murine melanoma. This tumor protective effect was not dependent on direct application to the tumor and was associated with an increase in tumor-associated and splenic dendritic cells. Additionally, the combination of imiquimod treatment with prior vaccination led to development of localized vitiligo. These findings indicate that activation of the innate immune system with TLR ligands stimulates dendritic cell activity resulting in a bypass of peripheral tolerance and enhanced antitumor activity. The results of these studies have broad implications for future designs of immunotherapeutic vaccines against tumors and the treatment of metastatic melanoma.

Delineation of antigen-specific and antigen-nonspecific CD8+ memory T-cell responses after cytokine-based cancer immunotherapy

Memory T cells exhibit tremendous antigen specificity within the immune system and accumulate with age. Our studies reveal an antigen-independent expansion of memory, but not naive, CD8(+) T cells after several immunotherapeutic regimens for cancer resulting in a distinctive phenotype. Signaling through T-cell receptors (TCRs) or CD3 in both mouse and human memory CD8(+) T cells markedly up-regulated programmed death-1 (PD-1) and CD25 (IL-2 receptor α chain), and led to antigen-specific tumor cell killing. In contrast, exposure to cytokine alone in vitro or with immunotherapy in vivo did not up-regulate these markers but resulted in expanded memory CD8(+) T cells expressing NKG2D, granzyme B, and possessing broadly lytic capabilities. Blockade of NKG2D in mice also resulted in significantly diminished antitumor effects after immunotherapy. Treatment of TCR-transgenic mice bearing nonantigen expressing tumors with immunotherapy still resulted in significant antitumor effects. Human melanoma tissue biopsies obtained from patients after topically applied immunodulatory treatment resulted in increased numbers of these CD8(+) CD25(-) cells within the tumor site. These findings demonstrate that memory CD8(+) T cells can express differential phenotypes indicative of adaptive or innate effectors based on the nature of the stimuli in a process conserved across species.

Galectin-1 Tunes TCR Binding and Signal Transduction to Regulate CD8 Burst Size

T cell burst size is regulated by the duration of TCR engagement and balanced control of Ag-induced activation, expansion, and apoptosis. We found that galectin-1-deficient CD8 T cells undergo greater cell division in response to TCR stimulation, with fewer dividing cells undergoing apoptosis. TCR-induced ERK signaling was sustained in activated galectin-1-deficient CD8 T cells and antagonized by recombinant galectin-1, indicating galectin-1 modulates TCR feed-forward/feedback loops involved in signal discrimination and procession. Furthermore, recombinant galectin-1 antagonized binding of agonist tetramers to the TCR on activated OT-1 T cells. Finally, galectin-1 produced by activated Ag-specific CD8 T cells negatively regulated burst size and TCR avidity in vivo. Therefore, galectin-1, inducibly expressed by activated CD8 T cells, functions as an autocrine negative regulator of peripheral CD8 T cell TCR binding, signal transduction, and burst size. Together with recent findings demonstrating that gal-1 promotes binding of agonist tetramers to the TCR of OT-1 thymocytes, these studies identify galectin-1 as a tuner of TCR binding, signaling, and functional fate determination that can differentially specify outcome, depending on the developmental and activation stage of the T cell.