Oxidized lipids block antigen cross-presentation by dendritic cells in cancer.

Cross-presentation is one of the main features of dendritic cells (DCs), which is critically important for the development of spontaneous and therapy-inducible antitumor immune responses. Patients, at early stages of cancer, have normal presence of DCs. However, the difficulties in the development of antitumor responses in patients with low tumor burden raised the question of the mechanisms of DC dysfunction. In this study, we found that, in differentiated DCs, tumor-derived factors blocked the cross-presentation of exogenous Ags without inhibiting the Ag presentation of endogenous protein or peptides. This effect was caused by intracellular accumulation of different types of oxidized neutral lipids: triglycerides, cholesterol esters, and fatty acids. In contrast, the accumulation of nonoxidized lipids did not affect cross-presentation. Oxidized lipids blocked cross-presentation by reducing the expression of peptide-MHC class I complexes on the cell surface. Thus, this study suggests the novel role of oxidized lipids in the regulation of cross-presentation.

Radiation-induced autophagy potentiates immunotherapy of cancer via up-regulation of mannose 6-phosphate receptor on tumor cells in mice.

There is a significant body of evidence demonstrating that radiation therapy (XRT) enhances the effect of immune therapy. However, the precise mechanisms by which XRT potentiates the immunotherapy of cancer remain elusive. Here, we report that XRT potentiates the effect of immune therapy via induction of autophagy and resultant trafficking of mannose-6-phopsphate receptor (MPR) to the cell surface. Irradiation of different tumor cells caused substantial up-regulation of MPR on the cell surface in vitro and in vivo. Down-regulation of MPR in tumor cells with shRNA completely abrogated the combined effect of XRT and immunotherapy (CTLA4 antibody) in B16F10-bearing mice without changes in the tumor-specific responses of T cells. Radiation-induced MPR up-regulation was the result of redistribution of the receptor to the cell surface. This effect was caused by autophagy with redirection of MPR to autophagosomes in a clathrin-dependent manner. In autophagosomes, MPR lost its natural ligands, which resulted in subsequent trafficking of empty receptor(s) back to the surface. Together, our data demonstrated a novel mechanism by which XRT can enhance the effect of immunotherapy and the molecular mechanism of this process.

Novel mechanism of synergistic effects of conventional chemotherapy and immune therapy of cancer.

There is mounting evidence to support the use of a combination of immunotherapy with chemotherapy in the treatment of various types of cancers. However, the mechanism(s), by which these modalities are synergized, are not fully understood. In this review, we discuss several possible mechanisms of the combined effect of immunotherapy and chemotherapy of cancer. We will examine various aspects of this issue such as the combination of different treatment options, the dosage for each arm of treatment, and, more importantly, the timing and sequence of the administration of these treatments.

Bryostatin-1, a naturally occurring antineoplastic agent, acts as a Toll-like receptor 4 (TLR-4) ligand and induces unique cytokines and chemokines in dendritic cells.

Bryostatin-1 (Bryo-1), a natural macrocyclic lactone, is clinically used as an anti-cancer agent. In this study, we demonstrate for the first time that Bryo-1 acts as a Toll-like receptor 4 (TLR4) ligand. Interestingly, activation of bone marrow-derived dendritic cells (in vitro with Bryo-1) led to a TLR4-dependent biphasic activation of nuclear factor-κB (NF-κB) and the unique induction of cytokines (IL-5, IL-6, and IL-10) and chemokines, including RANTES (regulated on activation normal T cell expressed and secreted) and macrophage inflammatory protein 1α (MIP1-α). In addition, EMSA demonstrated that Bryo-1-mediated induction of RANTES was regulated by NF-κB and the interferon regulatory factors (IRF)-1, IRF-3, and IRF-7 to the RANTES independently of myeloid differentiation primary response gene-88 (MyD88). Bryo-1 was able to induce the transcriptional activation of IRF-3 through the TLR4/MD2-dependent pathway. In vivo administration of Bryo-1 triggered a TLR-4-dependent T helper cell 2 (Th2) cytokine response and expanded a subset of myeloid dendritic cells that expressed a CD11c(high)CD8α(-) CD11b(+)CD4(+) phenotype. This study demonstrates that Bryo-1 can act as a TLR4 ligand and activate innate immunity. Moreover, the ability of Bryo-1 to trigger RANTES and MIP1-α suggests that Bryo-1 could potentially be used to prevent HIV-1 infection. Finally, induction of a Th2 response by Bryo-1 may help treat inflammatory diseases mediated by Th1 cells. Together, our studies have a major impact on the clinical use of Bryo-1 as an anti-cancer and immunopotentiating agent.

Therapeutic effect of intratumoral administration of DCs with conditional expression of combination of different cytokines.

In this study, we tested the effect of intratumoral administration of dendritic cells (DCs) with inducible expression of different cytokines, using the novel Rheoswitch Therapeutic System on the experimental models of renal cell cancer (RENCA) and MethA sarcoma. Intratumoral injection of DCs, engineered to express IL-12, IL-21, or IFN-α, showed potent therapeutic effect against established tumor. This effect was associated with the induction of potent tumor antigen-specific CD8+ T-cell responses, as well as the infiltration of tumors with CD4+ and CD8+ T cells but not with the cytotoxic activity of DCs. Combination of i.t. administration of DCs, producing different cytokines, did not enhance the antitumor effect of therapy with single cytokine. These results indicate that RTS can be a potent tool for conditional topical cytokine delivery, in combination with DC administration. However, combination of different cytokines may not necessarily improve the outcome of treatment.

Mechanism of synergistic effect of chemotherapy and immunotherapy of cancer.

In recent years, the combination of cancer immunotherapy with standard therapeutic modality is gaining credibility due to a number of clinical trials demonstrating therapeutic success of such combination therapies. However, the mechanism of this phenomenon is poorly understood. Here, we will discuss recent findings that suggest novel mechanisms of synergistic effect of cancer immunotherapy and chemotherapy.

Plumbagin-induced apoptosis in lymphocytes is mediated through increased reactive oxygen species production, upregulation of Fas, and activation of the caspase cascade.

Extracts from plants containing plumbagin (PLB) continue to be used as a treatment of a number of chronic immunologically-based diseases. However, most of these claims are supported only by anecdotal evidence with few scientific reports describing the mechanism of action or the efficacy of plumbagin in the suppression of the immune response. In the current study, we tested the hypothesis that plumbagin-induced suppression of the immune response was mediated through the induction of apoptosis. Splenocytes from C57BL/6 mice cultured in the presence of 0.5 microM or greater concentrations of PLB significantly reduced proliferative responses to mitogens, including anti-CD3 mAbs, concanavalin A (Con A), lipopolysaccharide (LPS) and staphylococcal enterotoxin B (SEB) in vitro. Exposure of naïve and activated splenocytes to PLB led to a significant increase in the levels of apoptosis. In addition, PLB treatment led to a significant increase in the levels of reactive oxygen species (ROS) in naïve and activated splenocytes. Furthermore, treatment with the ROS scavenger, N-acetylcysteine (NAC), prevented PLB-induced apoptosis, suggesting a role of ROS in PLB-induced apoptosis. PLB-induced apoptosis led to ROS-mediated activation of both the extrinsic and intrinsic apoptotic pathways. In addition, plumbagin led to increased expression of Fas. Finally, treatment of mice with PLB (5mg/kg) led to thymic and splenic atrophy as well as a significant suppression of the response to SEB and dinitrofluorobenzene (DNFB) in vivo. Together, these results suggest that plumbagin has significant immunosuppressive properties which are mediated by generation of ROS, upregulation of Fas, and the induction of apoptosis.

Decreased Suppression and Increased Phosphorylated STAT3 in Regulatory T Cells are Associated with Benefit from Adjuvant PD-1 Blockade in Resected Metastatic Melanoma.

PURPOSE: PD-1 blockade induces durable responses in patients with metastatic melanoma and prolongs relapse-free survival in patients with resected melanoma; however, current biomarkers do not consistently associate with patient responses. In this study, we investigated the impact of nivolumab therapy on peripheral blood regulatory T cells (Treg) and its relation to patient outcomes. EXPERIMENTAL DESIGN: Peripheral blood Tregs and conventional CD4+ T cells from patients with resected high-risk melanoma treated with adjuvant nivolumab were assessed for gene expression changes by RNA-seq. Percentages of circulating Tregs and phosphorylated-STAT3 (pSTAT3) expression levels were assessed by flow cytometry and validated in an independent cohort of active disease patients. Suppressive function of Tregs was assessed in allogeneic mixed lymphocyte reactions. RESULTS: Tregs from non-relapse patients had increased expression of proliferation associated genes. An increase in the proportion of circulating Tregs and pSTAT3 expression and a reduction in Treg-suppressive capacity were observed in non-relapsing, but not relapsing patient samples 13 weeks after starting treatment. In vitro blockade of PD-1 increased Treg percentages and pSTAT3 expression, and reduced Treg-suppressive function. PD-1 blockade also led to IL10 production by T cells, resulting in higher Treg proliferation. The addition of a STAT3 inhibitor ameliorated the increase in Tregs, enhanced suppressive function, and decreased T-cell IL10 production in vitro. CONCLUSIONS: These results demonstrate that induction of pSTAT3, reduced suppressive function, and a paradoxical increase in Treg proliferation are novel correlates of patient benefit from PD-1 blockade.

The role of mannose-6-phosphate receptor and autophagy in influencing the outcome of combination therapy.

Combining two different treatment modalities for targeting malignancies is gaining importance, with preclinical/clinical results indicating higher success rates in eradicating tumors or having longer remission periods. A better understanding of the synergy between the treatments helps in optimizing the dose and time of administration. We found that chemotherapy enhanced the levels of insulin-like growth factor 2 receptor/cation-independent mannose-6-phosphate receptor (IGF2R) on the surface of tumor cells, which leads to better tumor targeting by cytotoxic T cells (CTLs). Early evidence indicates that upregulation of IGF2R involves the autophagy pathway.

Autophagy induced by conventional chemotherapy mediates tumor cell sensitivity to immunotherapy.

Autophagy attenuates the efficacy of conventional chemotherapy but its effects on immunotherapy have been little studied. Here, we report that chemotherapy renders tumor cells more susceptible to lysis by CTL in vivo. Moreover, bystander tumor cells that did not express antigen were killed by CTL. This effect was mediated by transient but dramatic upregulation of the mannose-6-phosphate receptor (MPR) on the tumor cell surface. Antitumor effects of combined treatment related to the kinetics of MPR upregulation and abrogation of this event abolished the combined effect of immunotherapy and chemotherapy. MPR accumulation on the tumor cell surface during chemotherapy was observed in different mouse tumor models and in patients with multiple myeloma. Notably, this effect was the result of redistribution of the receptor caused by chemotherapy-inducible autophagy. Together, our findings reveal one molecular mechanism through which the antitumor effects of conventional cancer chemotherapy and immunotherapy are realized.

Combination of external beam radiotherapy (EBRT) with intratumoral injection of dendritic cells as neo-adjuvant treatment of high-risk soft tissue sarcoma patients.

PURPOSE: The goal of this study was to determine the effect of combination of intratumoral administration of dendritic cells (DC) and fractionated external beam radiation (EBRT) on tumor-specific immune responses in patients with soft-tissue sarcoma (STS). METHODS AND MATERIAL: Seventeen patients with large (>5 cm) high-grade STS were enrolled in the study. They were treated in the neoadjuvant setting with 5,040 cGy of EBRT, split into 28 fractions and delivered 5 days per week, combined with intratumoral injection of 10(7) DCs followed by complete resection. DCs were injected on the second, third, and fourth Friday of the treatment cycle. Clinical evaluation and immunological assessments were performed. RESULTS: The treatment was well tolerated. No patient had tumor-specific immune responses before combined EBRT/DC therapy; 9 patients (52.9%) developed tumor-specific immune responses, which lasted from 11 to 42 weeks. Twelve of 17 patients (70.6%) were progression free after 1 year. Treatment caused a dramatic accumulation of T cells in the tumor. The presence of CD4(+) T cells in the tumor positively correlated with tumor-specific immune responses that developed following combined therapy. Accumulation of myeloid-derived suppressor cells but not regulatory T cells negatively correlated with the development of tumor-specific immune responses. Experiments with (111)In labeled DCs demonstrated that these antigen presenting cells need at least 48 h to start migrating from tumor site. CONCLUSIONS: Combination of intratumoral DC administration with EBRT was safe and resulted in induction of antitumor immune responses. This suggests that this therapy is promising and needs further testing in clinical trials design to assess clinical efficacy.

Tumor-infiltrating myeloid cells induce tumor cell resistance to cytotoxic T cells in mice.

Cancer immunotherapeutic approaches induce tumor-specific immune responses, in particular CTL responses, in many patients treated. However, such approaches are clinically beneficial to only a few patients. We set out to investigate one possible explanation for the failure of CTLs to eliminate tumors, specifically, the concept that this failure is not dependent on inhibition of T cell function. In a previous study, we found that in mice, myeloid-derived suppressor cells (MDSCs) are a source of the free radical peroxynitrite (PNT). Here, we show that pre-treatment of mouse and human tumor cells with PNT or with MDSCs inhibits binding of processed peptides to tumor cell-associated MHC, and as a result, tumor cells become resistant to antigen-specific CTLs. This effect was abrogated in MDSCs treated with a PNT inhibitor. In a mouse model of tumor-associated inflammation in which the antitumor effects of antigen-specific CTLs are eradicated by expression of IL-1ß in the tumor cells, we determined that therapeutic failure was not caused by more profound suppression of CTLs by IL-1ß-expressing tumors than tumors not expressing this proinflammatory cytokine. Rather, therapeutic failure was a result of the presence of PNT. Clinical relevance for these data was suggested by the observation that myeloid cells were the predominant source of PNT in human lung, pancreatic, and breast cancer samples. Our data therefore suggest what we believe to be a novel mechanism of MDSC-mediated tumor cell resistance to CTLs.

Chemotherapy enhances tumor cell susceptibility to CTL-mediated killing during cancer immunotherapy in mice.

Cancer immunotherapy faces a serious challenge because of low clinical efficacy. Recently, a number of clinical studies have reported the serendipitous finding of high rates of objective clinical response when cancer vaccines are combined with chemotherapy in patients with different types of cancers. However, the mechanism of this phenomenon remains unclear. Here, we tested in mice several cancer vaccines and an adoptive T cell transfer approach to cancer immunotherapy in combination with several widely used chemotherapeutic drugs. We found that chemotherapy made tumor cells more susceptible to the cytotoxic effect of CTLs through a dramatic perforin-independent increase in permeability to GrzB released by the CTLs. This effect was mediated via upregulation of mannose-6-phosphate receptors on the surface of tumor cells and was observed in mouse and human cells. When combined with chemotherapy, CTLs raised against specific antigens were able to induce apoptosis in neighboring tumor cells that did not express those antigens. These data suggest that small numbers of CTLs could mediate a potent antitumor effect when combined with chemotherapy. In addition, these results provide a strong rationale for combining these modalities for the treatment of patients with advanced cancers.

Molecular events in the activation of B cells and macrophages by a non-microbial TLR4 agonist, G1-4A from Tinospora cordifolia.

G1-4A, a polysaccharide from an Indian medicinal plant Tinospora cordifolia, was recently shown to protect mice against septic shock by modulating the proinflammatory cytokines. G1-4A also activated B cells polyclonally. The present report describes in detail the molecular events associated with G1-4A-induced immunomodulation in vitro and in vivo. G1-4A treatment led to an increase in the CD69 expression in lymphocytes. G1-4A-induced proliferation of B cells was completely inhibited by PI3K inhibitor Ly294002, mTOR inhibitor rapamycin and NF-kappaB inhibitor plumbagin. Akt, ERK and JNK were activated by G1-4A which finally resulted in the activation of IKK, degradation of IkappaB-alpha and translocation of NF-kappaB to the nucleus. Administration of G1-4A to mice led to splenomegaly and an increase in the numbers of T cells, B cells and macrophages. This increase in spleen cellularity was due to in vivo proliferation of lymphocytes and upregulation of anti-apoptotic genes. Anti-TLR4-MD2 complex antibody inhibited G1-4A-induced B cell proliferation and degradation of IkappaB-alpha suggesting that TLR-4 was a receptor for G1-4A on B cells. Activation of RAW 264.7 macrophages by G1-4A was found to be dependent on ERK and NF-kappaB-mediated signals. The phagocytosis index in peritoneal exudate cells (PEC) isolated from G1-4A treated mice was significantly higher as compared to that in PEC from control mice. G1-4A administration also increased the number of CD11b(+) cells in the PEC without an increase in the total number of PEC. Thus the present understanding of the molecular mechanism of action of G1-4A, a novel non-microbial TLR4 agonist, will pave the way for its application as an immunomodulator and adjuvant.

Combined modality immunotherapy and chemotherapy: a new perspective.

The results of recent clinical trials have demonstrated that cancer vaccines continue to struggle to achieve tangible clinical benefits as monotherapy. Tumor-induced abnormalities in the immune system hamper anti-tumor T cell responses limiting the effectiveness of cancer immunotherapy. Recently, evidence has been mounting to suggest that immunotherapy has the possibility of achieving better success when used in combination with conventional chemotherapy. In clinical trials, immune responses elicited by cancer vaccines appear to augment the effectiveness of subsequent conventional cancer therapies.

Role of CD4(+)CD25(+) T regulatory cells in IL-2-induced vascular leak.

T regulatory cells (CD4(+)CD25(+)) play an important role in the regulation of the immune response. However, little is known about the ability of T regulatory cells to regulate endothelial cell (EC) damage following activation of lymphocytes with IL-2. Therefore, in the current study, we examined the role of T regulatory cells and the subsequent T(h)1/T(h)2 bias in IL-2-mediated EC injury using the well-characterized C57BL/6 (T(h)1-biased) and BALB/c (T(h)2-biased) models. Following IL-2 treatment, BALB/c mice were less susceptible to IL-2-induced vascular leak syndrome (VLS) compared with C57BL/6 mice. Splenocytes from BALB/c mice displayed less cytotoxicity against ECs compared with those from C57BL/6 mice. Interestingly, BALB/c mice had significantly higher numbers of CD4(+)CD25(+) T regulatory cells, which proliferated more profoundly following IL-2 treatment, compared with CD4(+)CD25(+) T regulatory cells from C57BL/6 mice. In addition, T regulatory cells from naive BALB/c mice were more potent suppressors of anti-CD3 mAb-stimulated proliferation of T cells than similar cells from C57BL/6 mice. Depletion of T regulatory cells in both BALB/c and C57BL/6 mice led to a significant increase in IL-2-induced VLS. Together, the results from this study suggest that CD4(+)CD25(+) T regulatory cells play an important role in the regulation of IL-2-induced EC injury.