Targeting NKT cells and PD-L1 pathway results in augmented anti-tumor responses in a melanoma model

Invariant or Type 1 NKT cells (iNKT cells) are a unique population of lymphocytes that share characteristics of T cells and natural killer (NK) cells. Various studies have shown that positive costimulatory pathways such as the CD28 and CD40 pathways can influence the expansion and cytokine production by iNKT cells. However, little is understood about the regulation of iNKT cells by negative costimulatory pathways. Here, we show that in vivo activation with α-GalCer results in increased cytokine production and expansion of iNKT cells in the absence of programmed cell death ligand-1 (PD-L1, B7-H1, and CD274). To study whether PD-L1 deficiency on NKT cells would enhance antigen-specific T-cell responses, we utilized CD8⁺ OT-1 OVA transgenic T cells. α-GalCer enhanced the expansion and cytokine production of OT-1 CD8⁺ cells after adoptive transfer into wild-type recipients. However, this expansion was significantly enhanced when OT-1 CD8⁺ T cells were adoptively transferred into PD-L1^−/− recipients. To extend these results to a tumor model, we used the B16 melanoma system. PD-L1^−/− mice given dendritic cells loaded with antigen and α-GalCer had a significant reduction in tumor growth and this was associated with increased trafficking of antigen-presenting cells and CD8⁺ T cells to the tumors. These data demonstrate that abrogating PDL1:PD-1 interactions during the activation of iNKT cells amplifies an anti-tumor response when coupled with DC vaccination.     

Targeting autophagy blocks melanoma growth by bringing natural killer cells to the tumor battlefield

Solid tumors are able to establish and sustain an immune suppressive microenvironment, which prevents the infiltration of cytotoxic effector immune cells into the tumor bed. We showed that genetic targeting of the macroautophagy/autophagy gene Becn1/Beclin1 in B16-F10 tumors inhibits their growth by inducing a massive infiltration of functional natural killer (NK) cells into the tumor bed. Such infiltration is primarily due to the ability of BECN1-defective tumor cells to overexpress and release CCL5 cytokine in the tumor microenvironment by a mechanism involving the activation of the MAPK8/JNK-JUN/c-Jun signaling pathway. Clinically, we reported a strong positive correlation between the expression of NK cell marker and CCL5 in human melanoma tumors and more importantly, a significant increased survival is found in melanoma patients expressing a high level of CCL5. Overall, these findings highlight the impact of targeting autophagy in breaking the immunosuppressive tumor microenvironment barrier, thus allowing the trafficking of cytotoxic NK cells into the tumor bed. This study underscore the importance of autophagy inhibition in tumors as a novel therapeutic strategy to fully exploit NK cells antitumor properties in clinical settings.     

Diverse manifestations of tumorigenicity and immunogenicity displayed by the poorly immunogenic B16-BL6 melanoma transduced with cytokine genes

 We evaluated the in vivo response to the poorly immunogenic B16-BL6 (BL6) murine melanoma genetically altered to secrete interleukin-2 (IL-2), IL-4, interferon γ (IFNγ) and granulocyte/macrophage-colony-stimulating factor (GM-CSF). Three parameters were evaluated: (1) tumorigenicity, (2) vaccination of naive animals, and (3) assessment of antitumor reactivity of T cells derived from tumor-draining lymph nodes (TDLN). Secretion of IL-2 abrogated the tumorigenicity of BL6, while IFNγ and IL-4 partially reduced tumorigenicity, and GM-CSF had no effect. Protective immunity to wild-type tumor challenge could not be achieved by vaccination with irradiated cytokine-secreting tumors, although IL-2 and IL-4 secretion appeared to retard the growth of the challenge inoculum significantly. An alternative method to evaluate the immunogenicity of the cytokine-secreting tumors was to measure the ability of T cells obtained from TDLN to mediate regression of wild-type tumor in adoptive immunotherapy. Neither IL-2 nor IFNγ secretion resulted in the induction of immune T cells. By contrast, GM-CSF and IL-4 secretion were found to induce immune T cells in the TDLN with GM-CSF being superior to IL-4. The combined secretion of GM-CSF and IL-4 did not lead to enhanced induction of immune T cells. GM-CSF secretion was found to up-regulate B7-1 expression in TDLN, consistent with an increase in the population of antigen-presenting cells. These studies demonstrated that reduced tumorigenicity by cytokine secretion did not correlate with increased immunogenicity. With the cytokines examined, there was limited capability of developing protective immunity against the BL6 tumor. Nevertheless, GM-CSF and IL-4 secretion significantly enhanced T cell immune reactivity to the poorly immunogenic BL6 tumor. Received: 30 January 1996 / Accepted: 22 March 1996     

Normalization of Tumor Microenvironment by Neem Leaf Glycoprotein Potentiates Effector T Cell Functions and Therapeutically Intervenes in the Growth of Mouse Sarcoma

We have observed restriction of the murine sarcoma growth by therapeutic intervention of neem leaf glycoprotein (NLGP). In order to evaluate the mechanism of tumor growth restriction, here, we have analyzed tumor microenvironment (TME) from sarcoma bearing mice with NLGP therapy (NLGP-TME, in comparison to PBS-TME). Analysis of cytokine milieu within TME revealed IL-10, TGFβ, IL-6 rich type 2 characters was switched to type 1 microenvironment with dominance of IFNγ secretion within NLGP-TME. Proportion of CD8⁺ T cells was increased within NLGP-TME and these T cells were protected from TME-induced anergy by NLGP, as indicated by higher expression of pNFAT and inhibit related downstream signaling. Moreover, low expression of FasR⁺ cells within CD8⁺ T cell population denotes prevention from activation induced cell death. Using CFSE as a probe, better migration of T cells was noted within TME from NLGP treated mice than PBS cohort. CD8⁺ T cells isolated from NLGP-TME exhibited greater cytotoxicity to sarcoma cells in vitro and these cells show higher expression of cytotoxicity related molecules, perforin and granzyme B. Adoptive transfer of NLGP-TME exposed T cells, but not PBS-TME exposed cells in mice, is able to significantly inhibit the growth of sarcoma in vivo. Such tumor growth inhibition by NLGP-TME exposed T cells was not observed when mice were depleted for CD8⁺ T cells. Accumulated evidences strongly suggest NLGP mediated normalization of TME allows T cells to perform optimally to inhibit the tumor growth.     

Enhanced anti-tumor activity of interferon-alpha in SOCS1-deficient mice is mediated by CD4<Superscript>+</Superscript> and CD8<Superscript>+</Superscript> T cells

Interferon-alpha (IFN-α) is an immunomodulatory cytokine that is used clinically for the treatment of melanoma in the adjuvant setting. The cellular actions of IFN-α are regulated by the suppressors of cytokine signaling (SOCS) family of proteins. We hypothesized that the anti-tumor activity of exogenous IFN-α would be enhanced in SOCS1-deficient mice. SOCS1-deficient (SOCS1^−/−) or control (SOCS1^+/+) mice on an IFN-γ^−/− C57BL/6 background bearing intraperitoneal (i.p.) JB/MS murine melanoma cells were treated for 30 days with i.p. injections of IFN-A/D or PBS (vehicle). Log-rank Kaplan-Meier survival curves were used to evaluate survival. Tumor-bearing control SOCS1^+/+ mice receiving IFN-A/D had significantly enhanced survival versus PBS–treated mice (P = 0.0048). The anti-tumor effects of IFN-A/D therapy were significantly enhanced in tumor-bearing SOCS1^−/− mice; 75% of these mice survived tumor challenge, whereas PBS-treated SOCS1^−/− mice all died at 13-16 days (P = 0.00038). Antibody (Ab) depletion of CD8⁺ T cells abrogated the anti-tumor effects of IFN-A/D in SOCS1^−/− mice as compared with mice receiving a control antibody (P = 0.0021). CD4⁺ T-cell depletion from SOCS1^−/− mice also inhibited the effects of IFN-A/D (P = 0.0003). IFN-A/D did not alter expression of CD80 or CD86 on splenocytes of SOCS1^+/+ or SOCS1^−/− mice, or the proportion of T regulatory cells or myeloid-derived suppressor cells in SOCS1^+/+ or SOCS1^−/− mice. An analysis of T-cell function did reveal increased proliferation of SOCS1-deficient splenocytes at baseline and in response to mitogenic stimuli. These data suggest that modulation of SOCS1 function in T-cell subsets could enhance the anti-tumor effects of IFN-α in the setting of melanoma.     

Overexpression of NTPDase2 in gliomas promotes systemic inflammation and pulmonary injury

Gliomas are the most common and devastating type of primary brain tumor. Many non-neoplastic cells, including immune cells, comprise the tumor microenvironment where they create a milieu that appears to dictate cancer development. ATP and the phosphohydrolytic products ADP and adenosine by activating P2 and P1 receptors may participate in these interactions among malignant and immune cells. Purinergic receptor-mediated cell communication is closely regulated by ectonucleotidases, such as by members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family, which hydrolyze extracellular nucleotides. We have shown that gliomas, unlike astrocytes, exhibit low NTPDase activity. Furthermore, ATP induces glioma cell proliferation and the co-administration of apyrase decreases progression of injected cells in vivo. We have previously shown that NTPDase2 reconstitution dramatically increases tumor growth in vivo. Here we evaluated whether NTPDase2 reconstitution to gliomas modulates systemic inflammatory responses. We observed that NTPDase2 overexpression modulated pro-inflammatory cytokine production and platelet reactivity. Additionally, pathological alterations in the lungs were observed in rats bearing these tumors. Our results suggest that disruption of purinergic signaling via ADP accumulation creates an inflammatory state that may promote tumor spread and dictate clinical progression.     

Leaf anatomy during leaf development of photoautotrophically <Emphasis Type="Italic">in vitro</Emphasis>-grown tobacco plants as affected by growth irradiance

Tobacco (Nicotiana tabacum L.) plants were cultured in vitro photoautotrophically at three levels of irradiance (PAR 400–700 nm): low (LI, 60 μmol m⁻² s⁻¹), middle (MI, 180 μmol m⁻² s⁻¹) and high (HI, 270 μmol m⁻² s⁻¹). Anatomy of the fourth leaf from bottom was followed during leaf development. In HI and MI plants, leaf area expansion started earlier as compared to LI plants, and both HI and MI plants developed some adaptations of sun species: leaves were thicker with higher proportion of palisade parenchyma to spongy parenchyma tissue. Furthermore, in HI and MI plants palisade and spongy parenchyma cells were larger and relative abundance of chloroplasts in parenchyma cells measured as chloroplasts cross-sectional area in the cell was lower than in LI plants. During leaf growth, chloroplasts crosssectional area in both palisade and spongy parenchyma cells in all treatments considerably decreased and finally it occupied only about 5 to 8 % of the cell cross-sectional area. Thus, leaf anatomy of photoautotrophically in vitro cultured plants showed a similar response to growth irradiance as in vivo grown plants, however, the formation of chloroplasts and therefore of photosynthetic apparatus was strongly impaired.     

Impaired melanoma growth in VASP deficient mice

Progression of tumors depends on interactions of cancer cells with the host environment. Expression of the cytoskeleton protein VASP is upregulated in various cancer entities. We analyzed the role of VASP for melanoma growth in murine allograft models. Growth of VASP expressing melanomas was retarded in VASP^?/? versus wild-type animals. Over time tumor size was <50% in VASP^?/? versus wild-type animals and independent of expression levels of Ena/VASP protein family members. Histological analyses showed smaller cells with impaired nutrition status and less vascularization in melanomas derived from VASP^?/? versus counterparts from wild-type mice. Cumulatively, the data reveal a critical role of VASP in non-tumor cells in the tumor environment for melanoma growth in vivo.     

Activation of anti-tumor immune response and reduction of regulatory T cells with Mycobacterium indicus pranii (MIP) therapy in tumor bearing mice

Background

Role of immune system in protecting the host from cancer is well established. Growing cancer however subverts immune response towards Th2 type and escape from antitumor mechanism of the host. Activation of both innate and Th1 type response is crucial for host antitumor activity. In our previous study it was found, that Mycobacterium indicus pranii (MIP) also known as M. w induces Th1 type response and activates macrophages in animal model of tuberculosis. Hence, we studied the immunotherapeutic potential of MIP in mouse tumor model and the underlying mechanisms for its antitumor activity.

Methodology and Principal Findings

Tumors were implanted by injecting B16F10 melanoma cells subcutaneously into C57BL/6 mice. Using the optimized dose and treatment regimes, anti-tumor efficacy of heat killed MIP was evaluated. In MIP treated group, tumor appeared in only 50–60% of mice, tumor growth was delayed and tumor volume was less as compared to control. MIP mediated immune activation was analysed in the tumor microenvironment, tumor draining lymph node and spleen. Induction of Th1 response and higher infiltration of immune cells in the tumor microenvironment was observed in MIP treated mice. A large fraction of these immune cells were in activated state as confirmed by phenotypic and functional analysis. Interestingly, percentage of Treg cells in the tumor milieu of treated mice was less. We also evaluated efficacy of MIP along with chemotherapy and found a better response as compared to chemotherapy alone.

Conclusion

MIP therapy is effective in protecting mice from tumor. It activates the immune cells, increases their infiltration in tumor, and abrogates tumor mediated immune suppression.     

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.  Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune “escape,” including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.     

Immunological tumor destruction in a murine melanoma model by targeted LTα independent of secondary lymphoid tissue

Background We previously demonstrated that targeting lymphotoxin α (LTα) to the tumor evokes its immunological destruction in a syngeneic B16 melanoma model. Since treatment was associated with the induction of peritumoral tertiary lymphoid tissue, we speculated that the induced immune response was initiated at the tumor site. Methods and results In order to directly test this notion, we analyzed the efficacy of tumor targeted LTα in LTα knock-out (LTα^−/−) mice which lack peripheral lymph nodes. To this end, we demonstrate that tumor-targeted LTα mediates the induction of specific T-cell responses even in the absence of secondary lymphoid organs. In addition, this effect is accompanied by the initiation of tertiary lymphoid tissue at the tumor site in which B and T lymphocytes are compartmentalized in defined areas and which harbor expanded numbers of tumor specific T cells as demonstrated by in situ TRP-2/K^b tetramer staining. Mechanistically, targeted LTα therapy seems to induce changes at the tumor site which allows a coordinated interaction of immune competent cells triggering the induction of tertiary lymphoid tissue. Conclusion Thus, our data demonstrate that targeted LTα promotes an accelerated immune response by enabling the priming of T cells at the tumor site.     

Tumor-derived death receptor 6 modulates dendritic cell development

Studies in murine models of cancer as well as in cancer patients have demonstrated that the immune response to cancer is often compromised. This paradigm is viewed as one of the major mechanisms of tumor escape. Many therapies focus on employing the professional antigen presenting dendritic cells (DC) as a strategy to overcome immune inhibition in cancer patients. Death receptor 6 (DR6) is an orphan member of the tumor necrosis factor receptor superfamily (TNFRSF21). It is overexpressed on many tumor cells and DR6^−/− mice display altered immunity. We investigated whether DR6 plays a role in tumorigenesis by negatively affecting the generation of anti-tumor activity. We show that DR6 is uniquely cleaved from the cell surface of tumor cell lines by the membrane-associated matrix metalloproteinase (MMP)-14, which is often overexpressed on tumor cells and is associated with malignancy. We also demonstrate that >50% of monocytes differentiating into DC die when the extracellular domain of DR6 is present. In addition, DR6 affects the cell surface phenotype of the resulting immature DC and changes their cytokine production upon stimulation with LPS/IFN-γ. The effects of DR6 are mostly amended when these immature DC are matured with IL-1β/TNF-α, as measured by cell surface phenotype and their ability to present antigen. These results implicate MMP-14 and DR6 as a mechanism tumor cells can employ to actively escape detection by the immune system by affecting the generation of antigen presenting cells.     

Lack of apoE causes alteration of cytokines expression in young mice liver

To investigate the effect of apolipoprotein E (apoE) on cytokine expression profile of the liver of young mice, quantitative RT-PCR (qRT-PCR) assay and cytokine antibody array for multiplex analysis of 62 cytokines have been used to analyze characteristics of expression of cytokines in the liver of 6-week-old apoE-null (apoE^−/−) mice. The levels of plasma cytokines were also analyzed. The mRNA level of IL-1β, IL-2, IL-6, ICAM-1, VCAM-1, MCP-1, NF-κB (p65), IFN-γ and IκB-α were increased significantly in apoE^−/− mice comparative to wild-type (WT) mice. IL-4, IL-10 and GM-CSF, however, were slightly decreased. Compared with WT, levels of 21 cytokines altered twofold or more in apoE^−/− mice, including 10 cytokines increased and 11 decreased. Expression patterns of IL-1β, IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-γ and VCAM-1 showed identical trend between cytokine antibody array and qRT-PCR analysis. Moreover, levels of IL-1β, IFN-γ and IL-6 in the plasma were elevated, while IL-4 was lightly decreased in apoE^−/− mice compared to those in WT mice. These results implied that promotion of type I immune response in the liver of young apoE^−/− mice due to alteration of these cytokines, and the phenotypes may be caused by the regulation of NF-κB. The inflammation and lipid metabolism dysfunction in the liver cooperated in dysfunction of the liver in young apoE^−/− mice.     

WSX1 expression in tumors induces immune tolerance via suppression of effector immune cells

Crosstalk between tumor cells and the cognate microenvironment plays a crucial role in tumor initiation and progression. However, only a few genes are known to affect such a crosstalk. This study reveals that WSX1 plays such a role when highly expressed in tumor cells. The expression of WSX1 in Lewis Lung Carcinoma (LLC) and the melanoma cell line AGS induces the death of T cells and inhibits the production of the effector cytokine IFNγ from NK and T cells, resulting in the promotion of tumor growth. These pro-tumorigenic properties of WSX1 are independent of IL27. This key observation reveals a new pathway of tumor-host interaction, which will ultimately lead to better strategies in immune therapy to reverse tumor tolerance.     

Anchoring cytokines to cancer cells using diphtheria toxin: better than immunotherapy by gene transfer?

Many cytokines are able to stimulate the antitumor immune response. However, in order to avoid the toxic effects due to systemic injection, it is necessary to concentrate the cytokines in the tumor microenvironment. Current methods, based on the transfer of cytokine genes into tumor cells, still suffer drawbacks. We describe an alternative approach using recombinant cytokines genetically conjugated to a membrane anchor derived from diphtheria toxin. Interleukin-2 anchored to lymphoma and melanoma cells remained displayed on their surface and were not internalized. Injection of these cell preparations to mice led to an immune response able to prevent or slow tumor growth following tumor challenge.     

Overcoming immunosuppression in the melanoma microenvironment induced by chronic inflammation

Malignant melanoma is known by its rapid progression and poor response to currently applied treatments. Despite the well-documented melanoma immunogenicity, the results of immunotherapeutic clinical trials are not satisfactory. This poor antitumor reactivity is due to the development of chronic inflammation in the tumor microenvironment characterized by infiltrating leukocytes and soluble mediators, which lead to an immunosuppression associated with cancer progression. Using the ret transgenic mouse melanoma model that closely resembles human melanoma, we demonstrated increased levels of chronic inflammatory factors in skin tumors and metastatic lymph nodes, which correlated with tumor progression. Furthermore, Gr1⁺CD11b⁺ myeloid-derived suppressor cells (MDSC), known to block tumor-reactive T cells, were enriched in melanoma lesions and showed an enhanced immunosuppressive capacity. This MDSC accumulation was associated with a strong TCR ζ-chain downregulation in T cells suggesting that the tumor inflammatory microenvironment supports MDSC recruitment and immunosuppressive activity. Indeed, upon administration of phosphodiesterase-5 inhibitor sildenafil or paclitaxel in non-cytotoxic doses, we observed reduced levels of chronic inflammatory mediators in association with decreased MDSC amounts and immunosuppressive function. This led to a partial restoration of ζ-chain expression in T cells and to a significantly increased survival of tumor-bearing mice. CD8 T-cell depletion resulted in an abrogation of beneficial outcome of both drugs, suggesting the involvement of MDSC and CD8 T cells in the observed therapeutic effects. Our data imply that inhibition of chronic inflammation in the tumor microenvironment should be applied in conjunction with melanoma immunotherapies to increase their efficacy.     

Monocytes secrete CXCL7 to promote breast cancer progression

Certain immune cells and inflammatory cytokines are essential components in the tumor microenvironment to promote breast cancer progression. To identify key immune players in the tumor microenvironment, we applied highly invasive MDA-MB-231 breast cancer cell lines to co-culture with human monocyte THP-1 cells and identified CXCL7 by cytokine array as one of the increasingly secreted cytokines by THP-1 cells. Further investigations indicated that upon co-culturing, breast cancer cells secreted CSF1 to induce expression and release of CXCL7 from monocytes, which in turn acted on cancer cells to promote FAK activation, MMP13 expression, migration, and invasion. In a xenograft mouse model, administration of CXCL7 antibodies significantly reduced abundance of M2 macrophages in tumor microenvironment, as well as decreased tumor growth and distant metastasis. Clinical investigation further suggested that high CXCL7 expression is correlated with breast cancer progression and poor overall survival of patients. Overall, our study unveils an important immune cytokine, CXCL7, which is secreted by tumor infiltrating monocytes, to stimulate cancer cell migration, invasion, and metastasis, contributing to the promotion of breast cancer progression.Subject terms: Breast cancer, Cancer microenvironment, Target identification, Chemokines