Immunotherapy with IL12 and PD1/CTLA4 inhibition is effective in advanced ovarian cancer and associates with reversal of myeloid cell-induced immunosuppression.

The tumor microenvironment (TME) in ovarian cancer (OC) is characterized by immune suppression, due to an abundance of suppressive immune cells populations. To effectively enhance the activity of immune checkpoint inhibition (ICI), there is a need to identify agents that target these immunosuppressive networks while promoting the recruitment of effector T cells into the TME. To this end, we sought to investigate the effect of the immunomodulatory cytokine IL12 alone or in combination with dual-ICI (anti-PD1 + anti-CTLA4) on anti-tumor activity and survival, using the immunocompetent ID8-VEGF murine OC model. Detailed immunophenotyping of peripheral blood, ascites, and tumors revealed that durable treatment responses were associated with reversal of myeloid cell-induced immune suppression, which resulted in enhanced anti-tumor activity by T cells. Single cell transcriptomic analysis further demonstrated striking differences in the phenotype of myeloid cells from mice treated with IL12 in combination with dual-ICI. We also identified marked differences in treated mice that were in remission compared to those whose tumors progressed, further confirming a pivotal role for the modulation of myeloid cell function to allow for response to immunotherapy. These findings provide the scientific basis for the combination of IL12 and ICI to improve clinical response in OC.

Immunobiology of Melanoma.

Despite the ability of immune-based interventions to dramatically increase the survival of patients with melanoma, a significant subset fail to benefit from this treatment, underscoring the need for accurate means to identify the patient population likely to respond to immunotherapy. Understanding how melanoma evades natural or manipulated immune responses could provide the information needed to identify such resistant individuals. Efforts to address this challenge are hampered by the vast immune diversity characterizing tumor microenvironments that remain largely understudied. It is thus important to more clearly elucidate the complex interactions that take place between the tumor microenvironment and host immune system.

Effect of 6% Hydroxyethyl Starch 130/0.4 on Inflammatory Response and Pulmonary Function in Patients Having Cardiac Surgery: A Randomized Clinical Trial.

BACKGROUND: Cardiac surgery with cardiopulmonary bypass induces a profound inflammatory response that, when severe, can lead to multiorgan system dysfunction. Preliminary data suggest that administration of hydroxyethyl starch (HES) solutions may mitigate an inflammatory response and improve pulmonary function. Our goal was to examine the effect of 6% HES 130/0.4 versus 5% human albumin given for intravascular plasma volume replacement on the perioperative inflammatory response and pulmonary function in patients undergoing cardiac surgery. METHODS: This was a subinvestigation of a blinded, parallel-group, randomized clinical trial of patients undergoing elective aortic valve replacement surgery at the Cleveland Clinic main campus, titled "Effect of 6% Hydroxyethyl Starch 130/0.4 on Kidney and Haemostatic Function in Cardiac Surgical Patients." Of 141 patients who were randomized to receive either 6% HES 130/0.4 or 5% human albumin for intraoperative plasma volume replacement, 135 patients were included in the data analysis (HES n = 66, albumin n = 69). We assessed the cardiopulmonary bypass-induced inflammatory response end points by comparing the 2 groups' serum concentrations of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and macrophage migration inhibitory factor (MIF), measured at baseline and at 1 and 24 hours after surgery. We also compared the 2 groups' postoperative pulmonary function end points, including the ratio of partial pressure of arterial oxygen to fraction of inspired oxygen (Pao2:Fio2 ratio), dynamic lung compliance, oxygenation index (OI), and ventilation index (VI) at baseline, within 1 hour of arrival to the intensive care unit, and before tracheal extubation. The differences in the postoperative levels of inflammatory response and pulmonary function between the HES and albumin groups were assessed individually in linear mixed models. RESULTS: Serum concentrations of the inflammatory markers (TNF-α, IL-6, MIF) were not significantly different (P ≥ .05) between patients who received 6% HES 130/0.4 or 5% albumin, and there was no significant heterogeneity of the estimated treatment effect over time (P ≥ .15). The results of pulmonary function parameters (Pao2:Fio2 ratio, dynamic compliance, OI, VI) were not significantly different (P ≥ .05) between groups, and there was no significant heterogeneity of the estimated treatment effect over time (P ≥ .15). CONCLUSIONS: Our investigation found no significant difference in the concentrations of inflammatory markers and measures of pulmonary function between cardiac surgical patients who received 6% HES 130/0.4 versus 5% albumin.

Mediators of Inflammation-Driven Expansion, Trafficking, and Function of Tumor-Infiltrating MDSCs.

Myeloid-derived suppressor cells (MDSC) are induced by and accumulate within many histologically distinct solid tumors, where they promote disease by secreting angiogenic and immunosuppressive molecules. Although IL1ß can drive the generation, accumulation, and functional capacity of MDSCs, the specific IL1ß-induced inflammatory mediators contributing to these activities remain incompletely defined. Here, we identified IL1ß-induced molecules that expand, mobilize, and modulate the accumulation and angiogenic and immunosuppressive potencies of polymorphonuclear (PMN)-MDSCs. Unlike parental CT26 tumors, which recruited primarily monocytic (M)-MDSCs by constitutively expressing GM-CSF- and CCR2-directed chemokines, IL1ß-transfected CT26 produced higher G-CSF, multiple CXC chemokines, and vascular adhesion molecules required for mediating infiltration of PMN-MDSCs with increased angiogenic and immunosuppressive properties. Conversely, CT26 tumors transfected with IL1ß-inducible molecules could mobilize PMN-MDSCs, but because they lacked the ability to upregulate IL1ß-inducible CXCR2-directed chemokines or vascular adhesion molecules, additional PMN-MDSCs could not infiltrate tumors. IL1ß-expressing CT26 increased angiogenic and immunosuppressive factors of tumor-infiltrating MDSCs, as did CT26 tumors individually transfected with G-CSF, Bv8, CXCL1, or CXCL5, demonstrating that mediators downstream of IL1ß could also modulate MDSC functional activity. Translational relevance was indicated by the finding that the same growth factors, cytokines, chemokines, and adhesion molecules responsible for the mobilization and recruitment of PMN-MDSCs into inflammatory CT26 murine tumors were also coordinately upregulated with increasing IL1ß expression in human renal cell carcinoma tumors. These studies demonstrated that IL1ß stimulated the components of a multifaceted inflammatory program that produces, mobilizes, chemoattracts, activates, and mediates the infiltration of PMN-MDSCs into inflammatory tumors to promote tumor progression.

Ex vivo conditioning with IL-12 protects tumor-infiltrating CD8+ T cells from negative regulation by local IFN-γ.

Optimal ex vivo expansion protocols for adoptive cell therapy (ACT) must yield T cells able to effectively home to tumors and survive the inhospitable conditions of the tumor microenvironment (TME), while simultaneously exerting persistent anti-tumor effector functions. Our previous work has shown that ex vivo activation in the presence of IL-12 can induce optimal expansion of murine CD8+ T cells, thus resulting in significant tumor regression after ACT mostly via sustained secretion of IFN-γ. In this report, we further elucidate the mechanism of this potency, showing that IL-12 additionally counteracts the negative regulatory effects of autocrine IFN-γ. IL-12 not only downregulates PD-1 expression by T cells, thus minimizing the effects of IFN-γ-induced PD-L1 upregulation by tumor stromal cells, but also inhibits IFNγR2 expression, thereby protecting T cells from IFN-γ-induced cell death. Thus, the enhanced anti-tumor activity of CD8+ T cells expanded ex vivo in the presence of IL-12 is due not only to the ability of IL-12-stimulated cells to secrete sustained levels of IFN-γ, but also to the additional capacity of IL-12 to counter the negative regulatory effects of autocrine IFN-γ.

Talimogene Laherparepvec combined with anti-PD-1 based immunotherapy for unresectable stage III-IV melanoma: a case series.

BACKGROUND: Talimogene Laherparepvec (T-VEC) is an oncolytic virus approved as an intratumoral therapy for treating unresectable stage IIIB-IV metastatic melanoma. The mechanisms of action for T-VEC and checkpoint inhibitor are highly complementary. Recent studies have shown that combining checkpoint inhibitor therapy with T-VEC injection can lead to improved response rates for stage IIIB-IV melanoma patients. METHODS: We reviewed 10 consecutive cases of stage IIIC to stage IVM1b melanoma patients that received T-VEC plus checkpoint inhibitor(s) therapy (pembrolizumab, ipilimumab/nivolumab, or nivolumab) treated between June 2016 and August 2017 at the Cleveland Clinic with a median follow-up of 7 months (range: 4 to 13 months). Responses of injected (on-target) and uninjected (off-target) lesions were evaluated according to RECIST 2.0. RESULTS: The overall response rate for on-target lesions was 90%, with 6 patients experiencing a complete response in injected lesions. Two patients had off-target lesions, which were completely resolved after treatment. Blood samples were tested for 3 complete responders and 2 partial responders. CD4:CD8 ratio and frequencies of circulating PD1+ CD4 and CD8 T cells were elevated in complete responders but not partial responders. One patient died due to causes unrelated to melanoma and one patient died of progression of the disease. CONCLUSION: Our data suggest that combining checkpoint inhibitor(s) with T-VEC injection may provide a synergistic efficacy for patients with unresectable melanoma. We observed a better overall response rate and complete response rate compared to published studies on similar therapeutic regimens.

Myeloid-Derived Suppressor Cell Subset Accumulation in Renal Cell Carcinoma Parenchyma Is Associated with Intratumoral Expression of IL1ß, IL8, CXCL5, and Mip-1α.

Purpose: Little is known about the association between myeloid-derived suppressor cell (MDSC) subsets and various chemokines in patients with renal cell carcinoma (RCC) or the factors that draw MDSC into tumor parenchyma.Experimental Design: We analyzed polymorphonuclear MDSC (PMN-MDSC), monocytic MDSC (M-MDSC), and immature MDSC (I-MDSC) from the parenchyma and peripheral blood of 48 patients with RCC, isolated at nephrectomy. We analyzed levels of IL1ß, IL8, CXCL5, Mip-1α, MCP-1, and Rantes. Furthermore, we performed experiments in a Renca murine model to assess therapeutic synergy between CXCR2 and anti-PD1 and to elucidate the impact of IL1ß blockade on MDSC.Results: Parenchymal PMN-MDSC have a positive correlation with IL1ß, IL8, CXCL5, and Mip-1α, and I-MDSC correlate with IL8 and CXCL5. Furthermore, peripheral PMN-MDSC correlate with tumor grade. Given that PMN-MDSC express CXCR2 and parenchymal PMN-MDSC correlated with IL8 and CXCL5, we assessed the response of CXCR2 blockade with or without anti-PD1. Combination therapy reduced tumor weight and enhanced CD4+ and CD8+ T-cell infiltration. In addition, anti-IL1ß decreased PMN-MDSC and M-MDSC in the periphery, PMN-MDSC in the tumor, and peripheral CXCL5 and KC. Anti-IL1ß also delayed tumor growth.Conclusions: Parenchymal PMN-MDSC have a positive correlation with IL1ß, IL8, CXCL5, and Mip-1α, suggesting they may attract PMN-MDSC into the tumor. Peripheral PMN-MDSC correlate with tumor grade, suggesting prognostic significance. Anti-CXCR2 and anti-PD1 synergized to reduce tumor weight and enhanced CD4+ and CD8+ T-cell infiltration in a Renca murine model, suggesting that CXCR2+ PMN-MDSC are important in reducing activity of anti-PD1 antibody. Finally, anti-IL1ß decreases MDSC and delayed tumor growth, suggesting a potential target for MDSC inhibition. Clin Cancer Res; 23(9); 2346-55. ©2016 AACR.

Structure-Function Analysis of the Mcl-1 Protein Identifies a Novel Senescence-regulating Domain.

Unlike other antiapoptotic Bcl-2 family members, Mcl-1 also mediates resistance to cancer therapy by uniquely inhibiting chemotherapy-induced senescence (CIS). In general, Bcl-2 family members regulate apoptosis at the level of the mitochondria through a common prosurvival binding groove. Through mutagenesis, we determined that Mcl-1 can inhibit CIS even in the absence of its apoptotically important mitochondrion-localizing domains. This finding prompted us to generate a series of Mcl-1 deletion mutants from both the N and C termini of the protein, including one that contained a deletion of all of the Bcl-2 homology domains, none of which impacted anti-CIS capabilities. Through subsequent structure-function analyses of Mcl-1, we identified a previously uncharacterized loop domain responsible for the anti-CIS activity of Mcl-1. The importance of the loop domain was confirmed in multiple tumor types, two in vivo models of senescence, and by demonstrating that a peptide mimetic of the loop domain can effectively inhibit the anti-CIS function of Mcl-1. The results from our studies appear to be highly translatable because we discerned an inverse relationship between the expression of Mcl-1 and of various senescence markers in cancerous human tissues. In summary, our findings regarding the unique structural properties of Mcl-1 provide new approaches for targeted cancer therapy.

GBM Derived Gangliosides Induce T Cell Apoptosis through Activation of the Caspase Cascade Involving Both the Extrinsic and the Intrinsic Pathway.

Previously we demonstrated that human glioblastoma cell lines induce apoptosis in peripheral blood T cells through partial involvement of secreted gangliosides. Here we show that GBM-derived gangliosides induce apoptosis through involvement of the TNF receptor and activation of the caspase cascade. Culturing T lymphocytes with GBM cell line derived gangliosides (10-20 µg/ml) demonstrated increased ROS production as early as 18 hrs as indicated by increased uptake of the dye H2DCFDA while western blotting demonstrated mitochondrial damage as evident by cleavage of Bid to t-Bid and by the release of cytochrome-c into the cytosol. Within 48-72 hrs apoptosis was evident by nuclear blebbing, trypan blue positivity and annexinV/7AAD staining. GBM-ganglioside induced activation of the effector caspase-3 along with both initiator caspases (-9 and -8) in T cells while both the caspase-8 and -9 inhibitors were equally effective in blocking apoptosis (60% protection) confirming the role of caspases in the apoptotic process. Ganglioside-induced T cell apoptosis did not involve production of TNF-α since anti-human TNFα antibody was unable to protect T cells from nuclear blebbing and subsequent cell death. However, confocal microscopy demonstrated co-localization of GM2 ganglioside with the TNF receptor and co-immunoprecipitation experiments showed recruitment of death domains FADD and TRADD with the TNF receptor post ganglioside treatment, suggesting direct interaction of gangliosides with the TNF receptor. Further confirmation of the interaction between GM2 and TNFR1 was obtained from confocal microscopy data with wild type and TNFR1 KO (TALEN mediated) Jurkat cells, which clearly demonstrated co-localization of GM2 and TNFR1 in the wild type cells but not in the TNFR1 KO clones. Thus, GBM-ganglioside can mediate T cell apoptosis by interacting with the TNF receptor followed by activation of both the extrinsic and the intrinsic pathway of caspases.

Immunological response to renal cryoablation in an in vivo orthotopic renal cell carcinoma murine model.

PURPOSE: The immunological consequences of cryoablation for renal cell carcinoma are largely unknown. Cryoablation is an attractive therapeutic option for tumors due to its minimally invasive nature. Cryoablation is also potentially immunogenic. We describe the development of an animal model to deliver in vivo renal cryotherapy to orthotopically implanted renal cell carcinoma and the results of multiple immunological interrogations after cryoablation. MATERIALS AND METHODS: Four to 6-week-old female Balb/c mice (Jackson Laboratories, Bar Harbor, Maine) underwent renal subcapsular implantation of the syngeneic murine renal cell carcinoma Renca. Two weeks later contact cryoablation was done in tumor bearing kidneys. Another group of animals underwent cryoablation of normal kidneys. Animals were sacrificed 2 weeks after tumor injection or 1 and 2 weeks after cryoablation, respectively. Kidneys, spleens and draining lymph nodes were harvested. Evaluation consisted of immunohistochemistry, immunofluorescence and gene expression profiling using reverse-transcriptase polymerase chain reaction. RESULTS: Subcapsular tumor implantation was successful in all cases and confirmed histologically. No significant lymphocytic infiltrate was seen in tumor only animals but those treated with cryoablation (tumor and nontumor bearing) had a significant inflammatory response primarily in sublethal tissue injury and perivascular areas. After cryoablation most infiltrating cells were neutrophils, macrophages and T cells. Polymerase chain reaction showed increased interferon-gamma production in kidneys after cryoablation. CONCLUSIONS: This study shows the potential feasibility of this animal model for studying cryo-immunology. We confirm the absence of any significant immune cell infiltration in tumor bearing kidneys and report a significant inflammatory infiltrate after cryoablation, consisting primarily of neutrophils, macrophages, and CD4+ and CD8+ T cells with an increase in the T helper type 1/2 ratio. This orthotopic murine model can form the basis of future studies of additional immunological aspects of renal cryoablation.

Elevated levels of select gangliosides in T cells from renal cell carcinoma patients is associated with T cell dysfunction.

Increased expression of gangliosides by different tumor types including renal cell carcinoma (RCC) is thought to contribute to the immune suppression observed in cancer patients. In this study, we report an increase in apoptotic T cells from RCC patients compared with T cells from normal donors that coincided with the detection of T cells staining positive for GM2 and that the apoptosis was predominantly observed in the GM2(+) but not the GM2(-) T cell population. Ganglioside shedding from tumor rather than endogenous production accounts for GM2(+) T cells since there was no detectable level of mRNA for GM2 synthase in RCC patient T cells and in T cells from normal healthy donors after incubation with either purified GM2 or supernatant from RCC cell lines despite their staining positive for GM2. Moreover, reactive oxygen species as well as activated caspase 3, 8, and 9 were predominantly elevated in GM2(+) but not GM2(-) T cells. Similarly, increased staining for GD2 and GD3 but not GD1a was detected with patient T cells with elevated levels of apoptosis in the GD2(+) and GD3(+) cells. These findings suggest that GM2, GD2, and GD3 play a significant role in immune dysfunction observed in RCC patient T cells.

GD3, an overexpressed tumor-derived ganglioside, mediates the apoptosis of activated but not resting T cells.

We previously elucidated an important role for gangliosides in renal cell carcinoma-mediated T lymphocyte apoptosis, although the mechanism by which they mediated lymphocyte death remained unclear. Here, we show that when added in purified form, GD3 is internalized by activated T cells, initiating a series of proapoptotic events, including the induction of reactive oxygen species (ROS), an enhancement of p53 and Bax accumulation, an increase in mitochondrial permeability, cytochrome c release, and the activation of caspase-9. GD3-induced apoptosis of activated T cells was dose dependent and inhibitable by pretreating the lymphocytes with N-acetylcysteine, cyclosporin A, or bongkrekic acid, emphasizing the essential role of ROS and mitochondrial permeability to the process. Ganglioside-induced T-cell killing was associated with the caspase-dependent degradation of nuclear factor-kappaB-inducible, antiapoptotic proteins, including RelA; this suggests that their loss is initiated only after the cascade is activated and that their disappearance amplifies but not triggers GD3 susceptibility. Resting T cells did not internalize appreciable levels of GD3 and did not undergo any of the proapoptotic changes that characterize activated T lymphocytes exposed to the ganglioside. RelA overexpression endows Jurkat cells with resistance to GD3-mediated apoptosis, verifying the role of the intact transcription factor in mediating protection from the ganglioside.

Renal cell carcinoma tumors induce T cell apoptosis through receptor-dependent and receptor-independent pathways.

Tumors can promote their own progressive growth by inducing T cell apoptosis. Though previous studies suggested that tumor-mediated T cell killing is receptor dependent, we recently showed that tumor gangliosides also participate, a notion consistent with reports indicating that, in some cell types, gangliosides can activate the intrinsic apoptotic pathway by stimulating reactive oxygen species production, cytochrome c release, and caspase-9 activation. In this study, we used normal peripheral blood T cells, as well as caspase-8-, caspase-9-, and Fas-associated death domain protein-deficient Jurkat cells, to assess whether the death ligands and gangliosides overexpressed by the renal cell carcinoma (RCC) cell line SK-RC-45 can independently stimulate T cell apoptosis as a mechanism of immune escape. Anti-FasL Abs and the glycosylceramide synthase inhibitor 1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (PPPP) each partially inhibited the ability of SK-RC-45 to kill cocultured activated T cells; together, as purified molecules, RCC gangliosides and rFasL induced a more extensive mitochondrial permeability transition and greater levels of apoptosis than either agent alone, equivalent to that induced by the FasL- and ganglioside-expressing RCC line itself. rFasL-mediated apoptosis was completely inhibited in caspase-8- and Fas-associated death domain protein-negative Jurkat cells, though apoptosis induced by purified gangliosides remained intact, findings that correlate with the observed partial inhibition of SK-RC-45-induced apoptosis in the Jurkat lines with defective death receptor signaling. Western blot analysis performed on lysates made from wild-type and mutant Jurkat cells cocultured with SK-RC-45 revealed caspase activation patterns and other biochemical correlates which additionally supported the concept that tumor-associated gangliosides and FasL independently activate the caspase cascade in T cells through the intrinsic and extrinsic pathways, respectively.

GM1 and tumor necrosis factor-alpha, overexpressed in renal cell carcinoma, synergize to induce T-cell apoptosis.

The ability to induce T-cell apoptosis is one mechanism by which tumors evade the immune system, although the molecules involved remain controversial. We found that renal cell carcinoma (RCC)-induced T-cell apoptosis was inhibited by >50% when cocultures were performed with ganglioside-depleted tumor cells, caspase-8-negative lymphocytes, or anti-tumor necrosis factor-alpha (TNFalpha) antibodies, suggesting that tumor gangliosides synergize with signals delivered through TNFalpha death receptors to mediate T-cell killing. The synergy between tumor-derived TNFalpha and the RCC-overexpressed ganglioside GM1 for killing resting T cells is corroborated by studies using purified GM1 and rTNF alpha, which indicate that a 48-hour pretreatment with the ganglioside optimally sensitizes the lymphocytes to a TNFalpha-induced apoptotic death. However, activated T cells, which synthesize TNFalpha themselves, can be killed by exogenous GM1 alone. RelA-overexpressing lymphocytes are protected from GM1 plus TNFalpha-mediated apoptosis, a finding consistent with our previous studies indicating that gangliosides inhibit nuclear factor-kappaB activation. These results are clinically relevant because, similar to T-cells cocultured with GM1-overexpressing RCC lines, T cells isolated from the peripheral blood of patients with metastatic RCC are also heavily coated with that tumor-shed ganglioside. This population of patient cells, unlike T cells isolated from normal donors, is highly susceptible to apoptosis induced by rTNF alpha or by metastatic patient sera, which contain elevated levels of the cytokine. This report thus extends our previous studies by demonstrating that tumor-derived TNFalpha enhances RCC apoptogenicity not only by inducing ganglioside synthesis but also by initiating receptor-dependent apoptosis in T cells in which the nuclear factor-kappaB activation pathway has been inhibited by GM1.

TNF-alpha induction of GM2 expression on renal cell carcinomas promotes T cell dysfunction.

Previous studies from our laboratory demonstrated the role of tumor-derived gangliosides as important mediators of T cell apoptosis, and hence, as one mechanism by which tumors evade immune destruction. In this study, we report that TNF-alpha secreted by infiltrating inflammatory cells and/or genetically modified tumors augments tumor-associated GM2 levels, which leads to T cell death and immune dysfunction. The conversion of weakly apoptogenic renal cell carcinoma (RCC) clones to lines that can induce T cell death requires 3-5 days of TNF-alpha pretreatment, a time frame paralleling that needed for TNF-alpha to stimulate GM2 accumulation by SK-RC-45, SK-RC-54, and SK-RC-13. RCC tumor cell lines permanently transfected with the TNF-alpha transgene are similarly toxic for T lymphocytes, which correlates with their constitutively elevated levels of GM2. TNF-alpha increases GM2 ganglioside expression by enhancing the mRNA levels encoding its synthetic enzyme, GM2 synthase, as demonstrated by both RT-PCR and Southern analysis. The contribution of GM2 gangliosides to tumor-induced T cell death was supported by the finding that anti-GM2 Abs significantly blocked T cell apoptosis mediated by TNF-alpha-treated tumor cells, and by the observation that small interfering RNA directed against TNF-alpha abrogated GM2 synthase expression by TNF-transfected SK-RC-45, diminished its GM2 accumulation, and inhibited its apoptogenicity for T lymphocytes. Our results indicate that TNF-alpha signaling promotes RCC-induced killing of T cells by stimulating the acquisition of a distinct ganglioside assembly in RCC tumor cells.

CXC chemokine ligand 9/monokine induced by IFN-gamma production by tumor cells is critical for T cell-mediated suppression of cutaneous tumors.

The role of tumor-produced chemokines in the growth of malignancies remains poorly understood. We retrieved an in vivo growing MCA205 fibrosarcoma and isolated tumor cell clones that produce both CXCL9/monokine induced by IFN-gamma (Mig) and CXCL10/IFN-gamma-inducible protein 10 following stimulation with IFN-gamma and clones that produce IFN-gamma-inducible protein 10 but not Mig. The Mig-deficient variants grew more aggressively as cutaneous tumors in wild-type mice than the Mig-producing tumor cells. The growth of Mig-expressing, but not Mig-deficient, tumor cells was suppressed by NK and T cell activity. Transduction of Mig-negative variants to generate constitutive tumor cell production of Mig resulted in T cell-dependent rejection of the tumors and in induction of protective tumor-specific CD8(+) T cell responses to Mig-deficient tumors. The results indicate a critical role for tumor-derived Mig in T cell-mediated responses to cutaneous fibrosarcomas and suggest the loss of Mig expression as a mechanism used by tumor cells to evade these responses.

Tumor-induced oxidative stress perturbs nuclear factor-kappaB activity-augmenting tumor necrosis factor-alpha-mediated T-cell death: protection by curcumin.

Cancer patients often exhibit loss of proper cell-mediated immunity and reduced effector T-cell population in the circulation. Thymus is a major site of T-cell maturation, and tumors induce thymic atrophy to evade cellular immune response. Here, we report severe thymic hypocellularity along with decreased thymic integrity in tumor bearer. In an effort to delineate the mechanisms behind such thymic atrophy, we observed that tumor-induced oxidative stress played a critical role, as it perturbed nuclear factor-kappaB (NF-kappaB) activity. Tumor-induced oxidative stress increased cytosolic IkappaBalpha retention and inhibited NF-kappaB nuclear translocation in thymic T cells. These NF-kappaB-perturbed cells became vulnerable to tumor-secreted tumor necrosis factor (TNF)-alpha (TNF-alpha)-mediated apoptosis through the activation of TNF receptor-associated protein death domain-associated Fas-associated protein death domain and caspase-8. Interestingly, TNF-alpha-depleted tumor supernatants, either by antibody neutralization or by TNF-alpha-small interfering RNA transfection of tumor cells, were unable to kill T cell effectively. When T cells were overexpressed with NF-kappaB, the cells became resistant to tumor-induced apoptosis. In contrast, when degradation-defective IkappaBalpha (IkappaBalpha super-repressor) was introduced into T cells, the cells became more vulnerable, indicating that inhibition of NF-kappaB is the reason behind such tumor/TNF-alpha-mediated apoptosis. Curcumin could prevent tumor-induced thymic atrophy by restoring the activity of NF-kappaB. Further investigations suggest that neutralization of tumor-induced oxidative stress and restoration of NF-kappaB activity along with the reeducation of the TNF-alpha signaling pathway can be the mechanism behind curcumin-mediated thymic protection. Thus, our results suggest that unlike many other anticancer agents, curcumin is not only devoid of immunosuppressive effects but also acts as immunorestorer in tumor-bearing host.

GM2 expression in renal cell carcinoma: potential role in tumor-induced T-cell dysfunction.

Multiple mechanisms have been proposed to account for immune escape by tumors. Although gangliosides have long been known to suppress T-cell immunity, few studies have examined the effect of human tumor-derived gangliosides on immune responses. Here, we show that gangliosides isolated from renal cell carcinoma (RCC) cell lines and clear cell tumor tissue can induce apoptosis in peripheral blood T cells. The RCC tissue-derived gangliosides also suppressed IFN-gamma and, in many cases, interleukin-4 production by CD4+ T cells at concentrations (1 ng/mL-100 pg/mL) well below those that induce any detectable T-cell death (4-20 microg/mL). Additional findings show that GM2 expressed by RCC plays a significant role in promoting T-cell dysfunction. This is supported by the demonstration that all RCC cell lines examined (n = 5) expressed GM2 as did the majority of tumors (15 of 18) derived from patients with clear cell RCC. Furthermore, an antibody specific for GM2 (DMF10.167.4) partially blocked (50-60%) T-cell apoptosis induced by coculturing lymphocytes with RCC cell lines or with RCC tissue-derived gangliosides. DMF10.167.4 also partially blocked the suppression of IFN-gamma production induced by RCC tissue-derived gangliosides, suggesting that GM2 plays a role in down-regulating cytokine production by CD4+ T cells.

Glioblastomas induce T-lymphocyte death by two distinct pathways involving gangliosides and CD70.

Here we report that glioblastoma multiforme (GBM) mediates immunosuppression by promoting T-cell death via tumor-associated CD70 and gangliosides that act through receptor-dependent and receptor-independent pathways, respectively. GBM lines cocultured with T cells induced lymphocyte death. The GBM lines were characterized for their expression of CD70, Fas ligand (FasL), and tumor necrosis factor-alpha (TNF-alpha), and the possible participation of those molecules in T-cell killing was assessed by doing GBM/T cell cocultures in the presence of anti-CD70 antibodies, Fas fusion proteins, or anti-TNF-alpha antibodies. CD70 but not TNF-alpha or FasL is responsible for initiating T-cell death via the receptor-dependent pathway. Of the four GBM cell lines that induced T-cell death, three highly expressed CD70. Two nonapoptogenic GBM lines (CCF3 and U138), on the other hand, had only minimally detectable CD70 expression. Blocking experiments with the anti-CD70 antibody confirmed that elevated CD70 levels were involved in the apoptogenicity of the three GBM lines expressing that molecule. Gangliosides were found to participate in the induction of T-cell apoptosis, because the glucosylceramide synthase inhibitor (PPPP) significantly reduced the abilities of all four apoptogenic lines to kill the lymphocytes. High-performance liquid chromatography (HPLC) and mass spectroscopy revealed that GM2, GM2-like gangliosides, and GD1a were synthesized in abundance by all four apoptogenic GBM lines but not by the two GBMs lacking activity. Furthermore, gangliosides isolated from GBM lines as well as HPLC fractions containing GM2 and GD1a were directly apoptogenic for T cells. Our results indicate that CD70 and gangliosides are both products synthesized by GBMs that may be key mediators of T-cell apoptosis and likely contribute to the T-cell dysfunction observed within the tumor microenvironment.

Effect of renal cell carcinomas on the development of type 1 T-cell responses.

PURPOSE: We reported that in renal cell carcinoma patients with active disease, T-cell reactions to the tumor-associated antigens MAGE-6 and EphA2 are highly skewed toward TH2-type cytokine responses [interleukin (IL) 5]. Herein, we determined whether tumor-derived products, including gangliosides isolated from renal cell carcinoma patients, participate in the down-regulation of type 1 T-cell responses. EXPERIMENTAL DESIGN: T cells from healthy volunteers or renal cell carcinoma patients were cultured in the presence and absence of supernatants derived from renal cell carcinoma explants or with gangliosides isolated from those tumor supernatants. T cells were stimulated or not with either autologous dendritic cells pulsed with superantigen (Staphylococcus enterotoxin B) or with phorbol 12-myristate 13-acetate and ionomycin and then were assessed for type 1 or type 2 responses (cytokine production and gene expression) and apoptosis. RESULTS: Tumor supernatants efficiently inhibited the TH1-type responses [interferon (IFN) gamma] of T cells stimulated with either S. enterotoxin B or phorbol 12-myristate 13-acetate and ionomycin but had no inhibitory effect on activated T-cell production of type 2 cytokines (IL-4, IL-5, and IL-10). Likewise, IFN-gamma mRNA and protein production were inhibited when T cells were cocultured with either renal cell carcinoma supernatant-derived gangliosides or a commercial source of purified GD1a. It was also determined that gangliosides impair type 1 responses by inducing apoptosis of activated T cells. CONCLUSIONS: We propose that renal cell carcinoma-derived tumor products such as gangliosides can induce a type 2 bias in antitumor immunity by initiating apoptosis in the IFN-gamma-producing type 1 effector cells. This represents a relevant mechanism by which renal cell carcinoma can inhibit protective antitumor immunity.