Ravikumar Muthuswamy

Positive feedback between PGE2 and COX2 redirects the differentiation of human dendritic cells toward stable myeloid-derived suppressor cells

Dendritic cells (DCs) and myeloid-derived suppressor cells (MDSCs) show opposing roles in the immune system. In the present study, we report that the establishment of a positive feedback loop between prostaglandin E(2) (PGE(2)) and cyclooxygenase 2 (COX2), the key regulator of PGE(2) synthesis, represents the determining factor in redirecting the development of CD1a(+) DCs to CD14(+)CD33(+)CD34(+) monocytic MDSCs. Exogenous PGE(2) and such diverse COX2 activators as lipopolysaccharide, IL-1β, and IFNγ all induce monocyte expression of COX2, blocking their differentiation into CD1a(+) DCs and inducing endogenous PGE(2), IDO1, IL-4Rα, NOS2, and IL-10, typical MDSC-associated suppressive factors. The addition of PGE(2) to GM-CSF/IL-4-supplemented monocyte cultures is sufficient to induce the MDSC phenotype and cytotoxic T lymphocyte (CTL)-suppressive function. In accordance with the key role of PGE(2) in the physiologic induction of human MDSCs, the frequencies of CD11b(+)CD33(+) MDSCs in ovarian cancer are closely correlated with local PGE(2) production, whereas the cancer-promoted induction of MDSCs is strictly COX2 dependent. The disruption of COX2-PGE(2) feedback using COX2 inhibitors or EP2 and EP4 antagonists suppresses the production of MDSC-associated suppressive factors and the CTL-inhibitory function of fully developed MDSCs from cancer patients. The central role of COX2-PGE(2) feedback in the induction and persistence of MDSCs highlights the potential for its manipulation to enhance or suppress immune responses in cancer, autoimmunity, or transplantation.

PGE(2)-induced CXCL12 production and CXCR4 expression controls the accumulation of human MDSCs in ovarian cancer environment.

Signals mediated by CXCL12 (SDF1) and its receptor CXCR4 are centrally involved in cancer progression, both directly by activating cancer cells and indirectly by inducing angiogenesis plus recruiting T regulatory and plasmacytoid dendritic immune cells. Here, we show that in ascites isolated from ovarian cancer patients, both CXCL12 and CXCR4 are controlled by the tumor-associated inflammatory mediator prostaglandin E(2) (PGE(2)), which attracts myeloid-derived suppressor cells (MDSC) into the ascites microenvironment. In this setting, PGE(2) was essential both for expression of functional CXCR4 in cancer-associated MDSCs and for production of its ligand CXCL12. Frequencies of CD11b(+)CD14(+)CD33(+)CXCR4(+) MDSCs closely correlated with CXCL12 and PGE(2) levels in patient ascites. MDSCs migrated toward ovarian cancer ascites in a CXCR4-dependent manner that required COX2 activity and autocrine PGE(2) production. Inhibition of COX2 or the PGE(2) receptors EP2/EP4 in MDSCs suppressed expression of CXCR4 and MDSC responsiveness to CXCL12 or ovarian cancer ascites. Similarly, COX2 inhibition also blocked CXCL12 production in the ovarian cancer environment and its ability to attract MDSCs. Together, our findings elucidate a central role for PGE(2) in MDSC accumulation triggered by the CXCL12-CXCR4 pathway, providing a powerful rationale to target PGE(2) signaling in ovarian cancer therapy.

Ability of Mature Dendritic Cells to Interact with Regulatory T Cells Is Imprinted during Maturation

Preferential activation of regulatory T (Treg) cells limits autoimmune tissue damage during chronic immune responses but can also facilitate tumor growth. Here, we show that tissue-produced inflammatory mediators prime maturing dendritic cells (DC) for the differential ability of attracting anti-inflammatory Treg cells. Our data show that prostaglandin E(2) (PGE(2)), a factor overproduced in chronic inflammation and cancer, induces stable Treg-attracting properties in maturing DC, mediated by CCL22. The elevated production of CCL22 by PGE(2)-matured DC persists after the removal of PGE(2) and is further elevated after secondary stimulation of DC in a neutral environment. This PGE(2)-induced overproduction of CCL22 and the resulting attraction of FOXP3(+) Tregs are counteracted by IFN alpha, a mediator of acute inflammation, which also restores the ability of the PGE(2)-exposed DC to secrete the Th1-attracting chemokines: CXCL9, CXCL10, CXCL11, and CCL5. In accordance with these observations, different DCs clinically used as cancer vaccines show different Treg-recruiting abilities, with PGE(2)-matured DC, but not type 1-polarized DC, generated in the presence of type I and type II IFNs, showing high Treg-attracting activity. The current data, showing that the ability of mature DC to interact with Treg cells is predetermined at the stage of DC maturation, pave the way to preferentially target the regulatory versus proinflammatory T cells in autoimmunity and transplantation, as opposed to intracellular infections and cancer.

PGE2 transiently enhances DC expression of CCR7 but inhibits the ability of DCs to produce CCL19 and attract naive T cells

Prostaglandin E(2) (PGE(2)) is an inflammatory mediator often used to increase CCR7 expression in the dendritic cells (DCs) used as cancer vaccines and to enhance their responsiveness to lymph node-associated chemokines. Here, we show that high surface expression of CCR7 on PGE(2)-matured DCs is associated with their suppressed production of the endogenous CCR7 ligand, CCL19, and is reversible by exogenous CCL19. In contrast to the PGE(2)-matured DCs, DCs matured in the presence of toll-like receptor (TLR) ligands and interferons produce high levels of both CCL19 and CCR7 mRNA/protein, but show selectively reduced expression of surface CCR7, which is compensated after DC removal from the CCL19-rich maturation environment. In accordance with these findings, PGE(2)-matured DCs show significantly higher in vitro migratory responsiveness to lymph node-associated chemokines directly after DC generation, but not after additional short-term culture in vitro, nor in vivo in patients injected with (111)indium-labeled DCs. The differences in CCL19-producing ability imprinted during DC maturation result in their different abilities to attract CCR7(+) naive T cells. Our data help to explain the impact of PGE(2) on CCR7 expression in maturing DCs and demonstrate a novel mechanism of regulatory activity of PGE(2), mediated by the inhibition of DCs ability to attract naive T cells.

Effective Immunotherapy against Murine Gliomas Using Type 1 Polarizing Dendritic Cells―Significant Roles of CXCL10

In an attempt to develop effective vaccines against central nervous system (CNS) tumors, we evaluated the ability of vaccines with standard dendritic cells (DC) versus type 1 polarizing DCs (DC1) to induce glioma-specific type 1 CTLs with CNS tumor-relevant homing properties and the mechanism of their action. C57BL/6 mouse-derived bone marrow cells were cultured with mouse granulocyte/macrophage colony-stimulating factor (GM-CSF) for 6 days, and CD11c(+) cells were subsequently cultured with GM-CSF, rmIFN-gamma, rmIFN-alpha, rmIL-4, and polyinosinic-polycytidylic acid stabilized by lysine and carboxymethylcellulose for 24 hours to generate DC1s. In analogy to their human counterparts, mouse DC1s exhibited surface marker profiles of mature DCs and produced high levels of IL-12 and CXCL10. Importantly for their application as cancer vaccines, such DC1s stably retained their type 1 phenotype even when exposed to type 2-promoting or regulatory T cell (Treg)-promoting environments. Consistently, mouse DC1s induced antigen-specific type 1 CTLs more efficiently than nonpolarized DCs in vitro. DC1s given s.c. migrated into draining lymph nodes, induced antigen-specific CTLs, and suppressed Treg accumulation. In addition, s.c. immunization with DC1s loaded with glioma-associated antigen (GAA)-derived CTL epitope peptides prolonged the survival of CNS GL261 glioma-bearing mice, which was associated with efficient CNS glioma homing of antigen-specific CTLs. Intratumoral injections of GAA peptide-loaded DC1s further enhanced the anti-CNS glioma effects of DC1-based s.c. immunization. Interestingly, the antitumor functions were abrogated with CXCL10(-/-) mouse-derived DC1s. Collectively, these findings show the anti-CNS glioma effects of DC1-based therapy and a novel role of CXCL10 in the immunologic and therapeutic activity of DC-based cancer vaccines.

NF-κB Hyperactivation in Tumor Tissues Allows Tumor-Selective Reprogramming of the Chemokine Microenvironment to Enhance the Recruitment of Cytolytic T Effector Cells

Tumor infiltration with effector CD8(+) T cells (T(eff)) predicts longer recurrence-free survival in many types of human cancer, illustrating the broad significance of T(eff) for effective immunosurveillance. Colorectal tumors with reduced accumulation of T(eff) express low levels of T(eff)-attracting chemokines such as CXCL10/IP10 and CCL5/RANTES. In this study, we investigated the feasibility of enhancing tumor production of T(eff)-attracting chemokines as a cancer therapeutic strategy using a tissue explant culture system to analyze chemokine induction in intact tumor tissues. In different tumor explants, we observed highly heterogeneous responses to IFNα or poly-I:C (a TLR3 ligand) when they were applied individually. In contrast, a combination of IFNα and poly-I:C uniformly enhanced the production of CXCL10 and CCL5 in all tumor lesions. Moreover, these effects could be optimized by the further addition of COX inhibitors. Applying this triple combination also uniformly suppressed the production of CCL22/MDC, a chemokine associated with infiltration of T regulatory cells (T(reg)). The T(eff)-enhancing effects of this treatment occurred selectively in tumor tissues, as compared with tissues derived from tumor margins. These effects relied on the increased propensity of tumor-associated cells (mostly fibroblasts and infiltrating inflammatory cells) to hyperactivate NF-κB and produce T(eff)-attracting chemokines in response to treatment, resulting in an enhanced ability of the treated tumors to attract T(eff) cells and reduced ability to attract T(reg) cells. Together, our findings suggest the feasibility of exploiting NF-κB hyperactivation in the tumor microenvironment to selectively enhance T(eff) entry into colon tumors.

Type 1-polarized dendritic cells loaded with autologous tumor are a potent immunogen against chronic lymphocytic leukemia

Induction of active tumor‐specific immunity in patients with chronic lymphocytic leukemia (CLL) and other hematologic malignancies is compromised by the deficit of endogenous dendritic cells (DCs). In attempt to develop improved vaccination strategies for patients with CLL and other tumors with poorly identified rejection antigens, we tested the ability of ex vivo‐generated DCs to cross‐present the antigens expressed by CLL cells and to induce CLL‐specific, functional CTL responses. Monocyte‐derived DCs from CLL patients were induced to mature using a “standard” cytokine cocktail (in IL‐1β, TNF‐α, IL‐6, and PGE2) or using an α‐type 1‐polarized DC (αDC1) cocktail (in IL‐1β, TNF‐α, IFN‐α, IFN‐γ, and polyinosinic:polycytidylic acid) and were loaded with γ‐irradiated, autologous CLL cells. αDC1 from CLL patients expressed substantially higher levels of multiple costimulatory molecules (CD83, CD86, CD80, CD11c, and CD40) than standard DCs (sDCs) and immature DCs, and their expression of CCR7 showed intermediate level. αDC1 secreted substantially higher (10–60 times) levels of IL‐12p70 than sDCs. Although αDC1 and sDCs showed similar uptake of CLL cells, αDC1 induced much higher numbers (range, 2.4–38 times) of functional CD8+ T cells against CLL cells. The current demonstration that autologous tumor‐loaded αDC1 are potent inducers of CLL‐specific T cells helps to develop improved immunotherapies of CLL.

Helper roles of NK and CD8+ T cells in the induction of tumor immunity. Polarized dendritic cells as cancer vaccines.

The work in our laboratory addresses two interrelated areas of dendritic cell (DC) biology: (1) the role of DCs as mediators of feedback interactions between NK cells, CD8+ and CD4+ T cells; and (2) the possibility to use such feedback and the paradigms derived from anti-viral responses, to promote the induction of therapeutic immunity against cancer. We observed that CD8+ T cells and NK cells, the classical “effector” cells, also play “helper” roles, regulating ability of DCs to induce type-1 immune immunity, critical for fighting tumors and intracellular pathogens. Our work aims to delineate which pathways of NK and CD8+ T cell activation result in their helper activity, and to identify the molecular mechanisms allowing them to induce type-1 polarized DCs (DC1s) with selectively enhanced ability to promote type-1 responses and anti-cancer immunity. The results of these studies allowed us and our colleagues to design phase I/II clinical trials incorporating the paradigms of DC polarization and helper activity of effector cells in cancer immunotherapy.

Independent Regulation of Chemokine Responsiveness and Cytolytic Function versus CD8+ T Cell Expansion by Dendritic Cells

The ability of cancer vaccines to induce tumor-specific CD8+ T cells in the circulation of cancer patients has been shown to poorly correlate with their clinical effectiveness. In this study, we report that although Ags presented by different types of mature dendritic cells (DCs) are similarly effective in inducing CD8+ T cell expansion, the acquisition of CTL function and peripheral-type chemokine receptors, CCR5 and CXCR3, requires Ag presentation by a select type of DCs. Both “standard” DCs (matured in the presence of PGE2) and type 1-polarized DCs (DC1s) (matured in the presence of IFNs and TLR ligands, which prevent DCs “exhaustion”) are similarly effective in inducing CD8+ T cell expansion and acquisition of CD45RO+IL-7R+IL-15R+ phenotype. However, granzyme B expression, acquisition of CTL activity, and peripheral tissue-type chemokine responsiveness are features exclusively exhibited by CD8+ T cells activated by DC1s. This advantage of DC1s was observed in polyclonally activated naive and memory CD8+ T cells and in blood-isolated melanoma-specific CTL precursors. Our data help to explain the dissociation between the ability of cancer vaccines to induce high numbers of tumor-specific CD8+ T cells in the blood of cancer patients and their ability to promote clinical responses, providing for new strategies of cancer immunotherapy.

Dendritic cells in cancer immunotherapy: vaccines and combination immunotherapies

Dendritic cells (DCs) are specialized immunostimulatory cells involved in the induction and regulation of immune responses. The feasibility of large-scale ex vivo generation of DCs from patients’ monocytes allows for therapeutic application of ex vivo-cultured DCs to bypass the dysfunction of endogenous DCs, restore immune surveillance, induce cancer regression or stabilization or delay or prevent its recurrence. While the most common paradigm of the therapeutic application of DCs reflects their use as cancer ‘vaccines’, additional and potentially more effective possibilities include the use of patients’ autologous DCs as parts of more comprehensive therapies involving in vivo or ex vivo induction of tumor-reactive T cells and the measures to counteract systemic and local immunosuppression in tumor-bearing hosts. Ex vivo-cultured DCs can be instructed to acquire distinct functions relevant for the induction of effective cancer immunity (DC polarization), such as the induction of different effector functions or different homing properties of tumor-specific T cells (delivery of ‘signal 3’ and ‘signal 4’). These considerations highlight the importance of the application of optimized conditions for the ex vivo culture of DCs and the potential combination of DC therapies with additional immune interventions to facilitate the entry of DC-induced T cells to tumor tissues and their local antitumor functions.