Minu K. Srivastava

Myeloid-Derived Suppressor Cells Inhibit T-Cell Activation by Depleting Cystine and Cysteine

Myeloid-derived suppressor cells (MDSC) are present in most cancer patients and are potent inhibitors of T-cell-mediated antitumor immunity. Their inhibitory activity is attributed to production of arginase, reactive oxygen species, inducible nitric oxide synthase, and interleukin-10. Here we show that MDSCs also block T-cell activation by sequestering cystine and limiting the availability of cysteine. Cysteine is an essential amino acid for T-cell activation because T cells lack cystathionase, which converts methionine to cysteine, and because they do not have an intact xc- transporter and therefore cannot import cystine and reduce it intracellularly to cysteine. T cells depend on antigen-presenting cells (APC), such as macrophages and dendritic cells, to export cysteine, which is imported by T cells via their ASC neutral amino acid transporter. MDSCs express the xc- transporter and import cystine; however, they do not express the ASC transporter and do not export cysteine. MDSCs compete with APC for extracellular cystine, and in the presence of MDSCs, APC release of cysteine is reduced, thereby limiting the extracellular pool of cysteine. In summary, MDSCs consume cystine and do not return cysteine to their microenvironment, thereby depriving T cells of the cysteine they require for activation and function.

Myeloid Suppressor Cell Depletion Augments Antitumor Activity in Lung Cancer

Background Myeloid derived suppressor cells (MDSC) are important regulators of immune responses. We evaluated the mechanistic role of MDSC depletion on antigen presenting cell (APC), NK, T cell activities and therapeutic vaccination responses in murine models of lung cancer. Principal Findings Individual antibody mediated depletion of MDSC (anti-Gr1 or anti-Ly6G) enhanced the antitumor activity against lung cancer. In comparison to controls, MDSC depletion enhanced the APC activity and increased the frequency and activity of the NK and T cell effectors in the tumor. Compared to controls, the anti-Gr1 or anti-Ly6G treatment led to increased: (i) CD8 T cells, (ii) NK cells, (iii) CD8 T or NK intracytoplasmic expression of IFNγ, perforin and granzyme (iv) CD3 T cells expressing the activation marker CD107a and CXCR3, (v) reduced CD8 T cell IL-10 production in the tumors (vi) reduced tumor angiogenic (VEGF, CXCL2, CXCL5, and Angiopoietin1&2) but enhanced anti-angiogenic (CXCL9 and CXCL10) expression and (vii) reduced tumor staining of endothelial marker Meca 32. Immunocytochemistry of tumor sections showed reduced Gr1 expressing cells with increased CD3 T cell infiltrates in the anti-Gr1 or anti-Ly6G groups. MDSC depletion led to a marked inhibition in tumor growth, enhanced tumor cell apoptosis and reduced migration of the tumors from the primary site to the lung compared to controls. Therapeutic vaccination responses were enhanced in vivo following MDSC depletion with 50% of treated mice completely eradicating established tumors. Treated mice that rejected their primary tumors acquired immunological memory against a secondary tumor challenge. The remaining 50% of mice in this group had 20 fold reductions in tumor burden compared to controls. Significance Our data demonstrate that targeting MDSC can improve antitumor immune responses suggesting a broad applicability of combined immune based approaches against cancer. This multifaceted approach may prove useful against tumors where MDSC play a role in tumor immune evasion.

Lung cancer patients’ CD4+ T cells are activated in vitro by MHC II cell-based vaccines despite the presence of myeloid-derived suppressor cells

BackgroundAdvanced non-small cell lung cancer (NSCLC) remains an incurable disease. Immunotherapies that activate patients’ T cells against resident tumor cells are being developed; however, these approaches may not be effective in NSCLC patients due to tumor-induced immune suppression. A major cause of immune suppression is myeloid-derived suppressor cells (MDSC). Because of the strategic role of CD4+ T lymphocytes in the activation of cytotoxic CD8+ T cells and immune memory, we are developing cell-based vaccines that activate tumor-specific CD4+ T cells in the presence of MDSC. The vaccines are NSCLC cell lines transfected with costimulatory (CD80) plus major histocompatibility complex class II (MHC II) genes that are syngeneic to the recipient. The absence of invariant chain promotes the presentation of endogenously synthesized tumor antigens, and the activation of MHC II-restricted, tumor-antigen-specific CD4+ T cells.MethodsPotential vaccine efficacy was tested in vitro by priming and boosting peripheral blood mononuclear cells from ten NSCLC patients who had varying levels of MDSC. CD4+ T cell activation was quantified by measuring Type 1 and Type 2 cytokine release.ResultsThe vaccines activated CD4+ T cells from all ten patients, despite the presence of CD33+CD11b+ MDSC. Activated CD4+ T cells were specific for NSCLC and did not cross-react with tumor cells derived from non-lung tissue or normal lung fibroblasts.ConclusionsThe NSCLC vaccines activate tumor-specific CD4+ T cells in the presence of potent immune suppression, and may be useful for the treatment of patients with NSCLC.

Myeloid suppressor cells and immune modulation in lung cancer

Many tumors, including lung cancers, promote immune tolerance to escape host immune surveillance and facilitate tumor growth. Tumors utilize numerous pathways to inhibit immune responses, including the elaboration of immune-suppressive mediators such as PGE2, TGF-β, IL-10, VEGF, GM-CSF, IL-6, S100A8/A9 and SCF, which recruit and/or activate myeloid-derived suppressor cells (MDSCs). MDSCs, a subset of heterogeneous bone marrow-derived hematopoietic cells, are found in the peripheral blood of cancer patients and positively correlate to malignancy. Solid tumors contain MDSCs that maintain an immune-suppressive network in the tumor microenvironment. This review will focus on the interaction of tumors with MDSCs that lead to dysregulation of antigen presentation and T-cell activities in murine tumor models. Specific genetic signatures in lung cancer modulate the activities of MDSCs and impact tumor progression. Targeting MDSCs may have a long-term antitumor benefit and is at the forefront of anticancer therapeutic strategies.

Early intervention with a small molecule inhibitor for tumor nefosis factor-α prevents cognitive deficits in a triple transgenic mouse model of Alzheimer’s disease

BackgroundChronic neuroinflammation is an important component of Alzheimer’s disease and could contribute to neuronal dysfunction, injury and loss that lead to disease progression. Multiple clinical studies implicate tumor necrosis factor-α as an inflammatory mediator of neurodegeneration in patients with Alzheimer’s because of elevated levels of this cytokine in the cerebrospinal fluid, hippocampus and cortex. Current Alzheimer’s disease interventions are symptomatic treatments with limited efficacy that do not address etiology. Thus, a critical need exists for novel treatments directed towards modifying the pathophysiology and progression.MethodsTo investigate the effect of early immune modulation on neuroinflammation and cognitive outcome, we treated triple transgenic Alzheimer’s disease mice (harboring PS1M146V, APPSwe, and tauP301L transgenes) with the small molecule tumor necrosis factor-α inhibitors, 3,6′-dithiothalidomide and thalidomide, beginning at four months of age. At this young age, mice do not exhibit plaque or tau pathology but do show mild intraneuronal amyloid beta protein staining and a robust increase in tumor necrosis factor-α. After 10 weeks of treatment, cognitive performance was assessed using radial arm maze and neuroinflammation was assessed using biochemical, stereological and flow cytometric endpoints.Results3,6′-dithiothalidomide reduced tumor necrosis factor-α mRNA and protein levels in the brain and improved working memory performance and the ratio of resting to reactive microglia in the hippocampus of triple transgenic mice.In comparison to non-transgenic controls, triple transgenic Alzheimer’s disease mice had increased total numbers of infiltrating peripheral monomyelocytic/granulocytic leukocytes with enhanced intracytoplasmic tumor necrosis factor-α, which was reduced after treatment with 3,6′-dithiothalidomide.ConclusionsThese results suggest that modulation of tumor necrosis factor-α with small molecule inhibitors is safe and effective with potential for the long-term prevention and treatment of Alzheimer’s disease.

Novel CCL21-Vault Nanocapsule Intratumoral Delivery Inhibits Lung Cancer Growth

Background Based on our preclinical findings, we are assessing the efficacy of intratumoral injection of dendritic cells (DC) transduced with an adenoviral vector expressing the secondary lymphoid chemokine (CCL21) gene (Ad-CCL21-DC) in a phase I trial in advanced non-small cell lung cancer (NSCLC). While this approach shows immune enhancement, the preparation of autologous DC for CCL21 genetic modification is cumbersome, expensive and time consuming. We are evaluating a non-DC based approach which utilizes vault nanoparticles for intratumoral CCL21 delivery to mediate antitumor activity in lung cancer. Principal Findings Here we describe that vault nanocapsule platform for CCL21 delivery elicits antitumor activity with inhibition of lung cancer growth. Vault nanocapsule packaged CCL21 (CCL21-vaults) demonstrated functional activity in chemotactic and antigen presenting activity assays. Recombinant vaults impacted chemotactic migration of T cells and this effect was predominantly CCL21 dependent as CCL21 neutralization abrogated the CCL21 mediated enhancement in chemotaxis. Intratumoral administration of CCL21-vaults in mice bearing lung cancer enhanced leukocytic infiltrates (CXCR3+T, CCR7+T, IFNγ+T lymphocytes, DEC205+ DC), inhibited lung cancer tumor growth and reduced the frequencies of immune suppressive cells [myeloid derived suppressor cells (MDSC), T regulatory cells (Treg), IL-10 T cells]. CCL21-vaults induced systemic antitumor responses by augmenting splenic T cell lytic activity against parental tumor cells. Significance This study demonstrates that the vault nanocapsule can efficiently deliver CCL21 to sustain antitumor activity and inhibit lung cancer growth. The vault nanocapsule can serve as an “off the shelf” approach to deliver antitumor cytokines to treat a broad range of malignancies.

IL-27 inhibits epithelial-mesenchymal transition and angiogenic factor production in a STAT1-dominant pathway in human non-small cell lung cancer

BackgroundInterleukin-27 signaling is mediated by the JAK-STAT pathway via activation of STAT1 and STAT3, which have tumor suppressive and oncogenic activities, respectively. Epithelial–mesenchymal transition (EMT) and angiogenesis are key processes in carcinogenesis. Although IL-27 has been shown to have potent anti-tumor activity in various cancer models, the role of IL-27 in EMT and angiogenesis is poorly understood. In this study, we investigated the role of IL-27 in regulating EMT and angiogenesis through modulation of the STAT pathways in human non-small cell lung carcinoma (NSCLC) cells.MethodsSTAT activation following IL-27 exposure was measured in human NSCLC cell lines. Expression of epithelial (E-cadherin, γ-catenin) and mesenchymal (N-cadherin, vimentin) markers were assessed by Western blot analysis. Production of pro-angiogenic factors (VEGF, IL-8/CXCL8, CXCL5) were examined by ELISA. Cell motility was examined by an in vitro scratch and transwell migration assays. Selective inhibitors of STAT1 (STAT1 siRNAs) and STAT3 (Stattic) were used to determine whether both STAT1 and STAT3 are required for IL-27 mediated inhibition of EMT and secretion of angiogenic factors.ResultsOur results demonstrate that IL-27 stimulation in NSCLC resulted in 1) STAT1 and STAT3 activation in a JAK-dependent manner, 2) development of epithelial phenotypes, including a decrease in the expression of a transcriptional repressor for E-cadherin (SNAIL), and mesenchymal marker (vimentin) with a reciprocal increase in the expression of epithelial markers, 3) inhibition of cell migration, and 4) reduced production of pro-angiogenic factors. STAT1 inhibition in IL-27–treated cells reversed the IL-27 effect with resultant increased expression of Snail, vimentin and the pro-angiogenic factors. The inhibition of STAT3 activation had no effect on the development of the epithelial phenotype.ConclusionIL-27 induces mesenchymal to epithelial transition and inhibits the production of pro-angiogenic factors in a STAT1–dominant pathway. These findings highlight the importance of STAT1 in repressing lung carcinogenesis and describe a new anti-tumor mechanism of IL-27.

Tumor cell programmed death ligand 1-mediated T cell suppression is overcome by coexpression of CD80.

Programmed death ligand 1 (PDL1, or B7-H1) is expressed constitutively or is induced by IFN-γ on the cell surface of most human cancer cells and acts as a “molecular shield” by protecting tumor cells from T cell-mediated destruction. Using seven cell lines representing four histologically distinct solid tumors (lung adenocarcinoma, mammary carcinoma, cutaneous melanoma, and uveal melanoma), we demonstrate that transfection of human tumor cells with the gene encoding the costimulatory molecule CD80 prevents PDL1-mediated immune suppression by tumor cells and restores T cell activation. Mechanistically, CD80 mediates its effects through its extracellular domain, which blocks the cell surface expression of PDL1 but does not prevent intracellular expression of PDL1 protein. These studies demonstrate a new role for CD80 in facilitating antitumor immunity and suggest new therapeutic avenues for preventing tumor cell PDL1-induced immune suppression.

The absence of invariant chain in MHC II cancer vaccines enhances the activation of tumor-reactive type 1 CD4+ T lymphocytes

Activation of tumor-reactive T lymphocytes is a promising approach for the prevention and treatment of patients with metastatic cancers. Strategies that activate CD8+ T cells are particularly promising because of the cytotoxicity and specificity of CD8+ T cells for tumor cells. Optimal CD8+ T cell activity requires the co-activation of CD4+ T cells, which are critical for immune memory and protection against latent metastatic disease. Therefore, we are developing “MHC II” vaccines that activate tumor-reactive CD4+ T cells. MHC II vaccines are MHC class I+ tumor cells that are transduced with costimulatory molecules and MHC II alleles syngeneic to the prospective recipient. Because the vaccine cells do not express the MHC II-associated invariant chain (Ii), we hypothesized that they will present endogenously synthesized tumor peptides that are not presented by professional Ii+ antigen presenting cells (APC) and will therefore overcome tolerance to activate CD4+ T cells. We now report that MHC II vaccines prepared from human MCF10 mammary carcinoma cells are more efficient than Ii+ APC for priming and boosting Type 1 CD4+ T cells. MHC II vaccines consistently induce greater expansion of CD4+ T cells which secrete more IFNγ and they activate an overlapping, but distinct repertoire of CD4+ T cells as measured by T cell receptor Vβ usage, compared to Ii+ APC. Therefore, the absence of Ii facilitates a robust CD4+ T cell response that includes the presentation of peptides that are presented by traditional APC, as well as peptides that are uniquely presented by the Ii− vaccine cells.

MHC Class II–Transduced Tumor Cells Originating in the Immune-Privileged Eye Prime and Boost CD4+ T Lymphocytes that Cross-react with Primary and Metastatic Uveal Melanoma Cells

Uveal melanoma, the most common malignancy of the eye, has a 50% rate of liver metastases among patients with large primary tumors. Several therapies prolong survival of metastatic patients; however, none are curative and no patients survive. Therefore, we are exploring immunotherapy as an alternative or adjunctive treatment. Uveal melanoma may be particularly appropriate for immunotherapy because primary tumors arise in an immune-privileged site and may express antigens to which the host is not tolerized. We are developing MHC class II (MHC II)-matched allogeneic, cell-based uveal melanoma vaccines that activate CD4(+) T lymphocytes, which are key cells for optimizing CD8(+) T-cell immunity, facilitating immune memory, and preventing tolerance. Our previous studies showed that tumor cells genetically modified to express costimulatory and MHC II molecules syngeneic to the recipient are potent inducers of antitumor immunity. Because the MHC II-matched allogeneic vaccines do not express the accessory molecule, Invariant chain, they present MHC II-restricted peptides derived from endogenously encoded tumor antigens. We now report that MHC II-matched allogeneic vaccines, prepared from primary uveal melanomas that arise in the immune-privileged eye, prime and boost IFNgamma-secreting CD4(+) T cells from the peripheral blood of either healthy donors or uveal melanoma patients that cross-react with primary uveal melanomas from other patients and metastatic tumors. In contrast, vaccines prepared from metastatic cells in the liver are less effective at activating CD4(+) T cells, suggesting that tumor cells originating in immune-privileged sites may have enhanced capacity for inducing antitumor immunity and for serving as immunotherapeutic agents.