Type 1 Innate Lymphoid Cells Limit the Antitumoral Immune Response.

Natural killer (NK) cells are known to be able to kill established tumor cell lines, but important caveats remain regarding their roles in the detection and elimination of developing primary tumors. Using a genetic model of selective ILC1 and NK cell deficiency, we showed that these cells were dispensable for tumor immunosurveillance and immunoediting in the MCA-induced carcinogenesis model. However, we were able to generate primary cell lines derived from MCA-induced tumors with graded sensitivity to NK1.1+ cells (including NK cells and ILC1). This differential sensitivity was associated neither with a modulation of intratumoral NK cell frequency, nor the capacity of tumor cells to activate NK cells. Instead, ILC1 infiltration into the tumor was found to be a critical determinant of NK1.1+ cell-dependent tumor growth. Finally, bulk tumor RNAseq analysis identified a gene expression signature associated with tumor sensitivity to NK1.1+ cells. ILC1 therefore appear to play an active role in inhibiting the antitumoral immune response, prompting to evaluate the differential tumor infiltration of ILC1 and NK cells in patients to optimize the harnessing of immunity in cancer therapies.

NKTR-214 immunotherapy synergizes with radiotherapy to stimulate systemic CD8+ T cell responses capable of curing multi-focal cancer.

BACKGROUND: High-dose radiotherapy (RT) is known to be immunogenic, but is rarely capable of driving clinically relevant abscopal antitumor immunity as monotherapy. RT is known to increase antigen presentation, type I/II interferon responses, and immune cell trafficking to irradiated tumors. Bempegaldesleukin (NKTR-214) is a CD122-preferential interleukin 2 (IL-2) pathway agonist that has been shown to increase tumor-infiltrating lymphocytes, T cell clonality, and increase PD-1 expression. NKTR-214 has increased drug half-life, decreased toxicity, and increased CD8+ T cell and natural killer cell stimulation compared with IL-2. METHODS: Animals bearing bilateral subcutaneous MCA-205 fibrosarcoma or CT26 colorectal tumors were treated with NKTR-214, RT, or combination therapy, and tumor growth of irradiated and abscopal lesions was assessed. Focal RT was delivered using a small animal radiation research platform. Peripheral and tumor-infiltrating immune phenotype and functional analyses were performed by flow cytometry. RNA expression profiling from both irradiated and abscopal lesions was performed using microarray. RESULTS: We demonstrate synergy between RT of a single tumor and NKTR-214 systemic therapy resulting in dramatically increased cure rates of mice bearing bilateral tumors compared with RT or NKTR-214 therapy alone. Combination therapy resulted in increased magnitude and effector function of tumor-specific CD8+ T cell responses and increased trafficking of these T cells to both irradiated and distant, unirradiated, tumors. CONCLUSIONS: Given the increasing role of hypofractionated and stereotactic body RT as standard of care treatments in the management of locally advanced and metastatic cancer, these data have important implications for future clinical trial development. The combination of RT and NKTR-214 therapy potently stimulates systemic antitumor immunity and should be evaluated for the treatment of patients with locally advanced and metastatic solid tumors.

Vaccine efficacy against primary and metastatic cancer with in vitro-generated CD103+ conventional dendritic cells.

BACKGROUND: Type 1 conventional dendritic cells (cDC1s) possess efficient antigen presentation and cross-presentation activity, as well as potent T cell priming ability. Tissue-resident cDC1s (CD103+ cDC1s in mice, CD141+ cDC1s in humans) are linked with improved tumor control, yet the efficacy of immunotherapy using this population is understudied. METHODS: We generated murine CD103+ cDC1s in vitro and examined their expression of cDC1-related factors, antigen cross-presentation activity, and accumulation in tumor-draining lymph nodes (TdLNs). The antitumor efficacy of the in vitro-generated CD103+ cDC1s was studied in murine melanoma and osteosarcoma models. We evaluated tumor responses on vaccination with CD103+ cDC1s, compared these to vaccination with monocyte-derived DCs (MoDCs), tested CD103+ cDC1 vaccination with checkpoint blockade, and examined the antimetastatic activity of CD103+ cDC1s. RESULTS: In vitro-generated CD103+ cDC1s produced cDC1-associated factors such as interleukin-12p70 and CXCL10, and demonstrated antigen cross-presentation activity on stimulation with the toll-like receptor 3 agonist polyinosinic:polycytidylic acid (poly I:C). In vitro-generated CD103+ cDC1s also migrated to TdLNs following poly I:C treatment and intratumoral delivery. Vaccination with poly I:C-activated and tumor antigen-loaded CD103+ cDC1s enhanced tumor infiltration of tumor antigen-specific and interferon-γ+ CD8+ T cells, and suppressed melanoma and osteosarcoma growth. CD103+ cDC1s showed superior antitumor efficacy compared with MoDC vaccination, and led to complete regression of 100% of osteosarcoma tumors in combination with CTLA-4 antibody-mediated checkpoint blockade. In vitro-generated CD103+ cDC1s effectively protected mice from pulmonary melanoma and osteosarcoma metastases. CONCLUSIONS: Our data indicate an in vitro-generated CD103+ cDC1 vaccine elicits systemic and long-lasting tumor-specific T cell-mediated cytotoxicity, which restrains primary and metastatic tumor growth. The CD103+ cDC1 vaccine was superior to MoDCs and enhanced response to immune checkpoint blockade. These results indicate the potential for new immunotherapies based on use of cDC1s alone or in combination with checkpoint blockade.

Multifaceted effects of soluble human CD6 in experimental cancer models.

BACKGROUND: CD6 is a lymphocyte surface co-receptor physically associated with the T-cell receptor (TCR)/CD3 complex at the center of the immunological synapse. There, CD6 assists in cell-to-cell contact stabilization and modulation of activation/differentiation events through interaction with CD166/ALCAM (activated leukocyte cell adhesion molecule), its main reported ligand. While accumulating evidence is attracting new interest on targeting CD6 for therapeutic purposes in autoimmune disorders, little is known on its potential in cancer. In an attempt to elucidate the in vivo relevance of blocking CD6-mediated interactions in health and disease, we explored the consequences of expressing high circulating levels of a soluble form CD6 (sCD6) as a decoy receptor. METHODS: High sCD6 serum levels were achieved by using transgenic C57BL/6 mice expressing human sCD6 under the control of lymphoid-specific transcriptional elements (shCD6LckEµTg) or wild type either transduced with hepatotropic adeno-associated virus coding for mouse sCD6 or undergoing repeated infusions of recombinant human sCD6 protein. Characterization of sCD6-induced changes was performed by ex vivo flow cytometry and functional analyses of mouse lymphoid organ cells. The in vivo relevance of those changes was explored by challenging mice with subcutaneous or metastatic tumors induced by syngeneic cancer cells of different lineage origins. RESULTS: Through a combination of in vitro and in vivo studies, we show that circulating sCD6 expression induces defective regulatory T cell (Treg) generation and function, decreased CD166/ALCAM-mediated tumor cell proliferation/migration and impaired galectin-induced T-cell apoptosis, supporting the fact that sCD6 modulates antitumor lymphocyte effector function and tumorigenesis. Accordingly, sCD6 expression in vivo resulted in delayed subcutaneous tumor growth and/or reduced metastasis on challenge of mice with syngeneic cancer cells. CONCLUSIONS: Evidence is provided for the disruption of CD6 receptor-ligand interactions as a feasible immunomodulatory approach in cancer.

Nanotechnology-mediated immunochemotherapy with Ingenol-3-Mebutate for Systematic Anti-tumor Effects.

Cancer-Immunotherapy was the most exciting topic. However, either insensitivity due to singleness of therapeutic target or immune evasion leads to the failure of the treatment. Ingenol-3-mebutate (I3A) can inhibit cancer through synergy between immunotherapy and chemotherapy, however, the speculation and accurate mechanism haven't been confirmed in vivo limited by its hydrophobicity and pH-instability, which also hindered its clinical translation. Herein we developed a polymeric micelle with 'acidic core' provided by single alcoholic hydroxyl (-CH(CH3)-OH) encapsulating I3A (I3A-PM), which successfully overcome the aforementioned problems and reduce the toxicity in vivo. To test the synergy, S180 tumor-bearing mice were subjected to I3A-PM through intravenous and intratumoral administration, we found I3A-PM presented significant antitumor effect, and promoted Th1 polarization by upregulating the level of Th1 cytokines (IL-12, IL-2, IFN-γ and TNF-α), and accelerated the expansion of CD4+ and CD8+ T cells, meanwhile, I3A-PM depleted regulatory T cells, Th2 cytokine IL-6 through inhibiting TGF-ß signaling pathway. Furthermore, we appealed to virtual screening of tumor target, and found a new pathway of I3A as a TGF-ß receptor type I inhibitor to improve immunostimulatory effects. These results demonstrated I3A-PM as a promising nanoagent for cancer immunotherapy strategy. The synergistic therapeutic effects are encouraged to further evaluate in different cancer model compared with commercial products to facilitate research finding (I3A-PM) entering the clinic.

Targeted Delivery of miRNA 155 to Tumor Associated Macrophages for Tumor Immunotherapy.

Tumor associated macrophages (TAMs) are important components residing in the tumor microenvironment. They are immunosuppressive and promote tumor progression. Targeting TAMs and reprogramming their phenotype may be a promising strategy that can restore antitumor immune responses. In this study, we developed a microRNA delivery system based on lipid-coated calcium phosphonate nanoparticles (CaP/miR@pMNPs) containing conjugated mannose and sterically shielded with a pH-responsive material. The nanocarrier could respond to the low pH in the tumor microenvironment and expose mannose to promote cellular internalization in TAMs. The carrier could reactivate TAMs and reprogram their functions, reverse the immunosuppressive tumor microenvironment, and inhibit tumor growth in a tumor-bearing mouse model. In summary, redirecting the polarization of TAMs is a potential therapeutic strategy for tumor immunotherapy.

The anticancer peptide RT53 induces immunogenic cell death.

In recent years, immunogenic cell death (ICD) has emerged as a revolutionary concept in the development of novel anticancer therapies. This particular form of cell death is able, through the spatiotemporally defined emission of danger signals by the dying cell, to induce an effective antitumor immune response, allowing the immune system to recognize and eradicate malignant cells. To date, only a restricted number of chemotherapeutics can trigger ICD of cancer cells. We previously reported that a peptide, called RT53, spanning the heptad leucine repeat region of the survival protein AAC-11 fused to a penetrating sequence, selectively induces cancer cell death in vitro and in vivo. Interestingly, B16F10 melanoma cells treated by RT53 were able to mediate anticancer effects in a tumor vaccination model. Stimulated by this observation, we investigated whether RT53 might mediate ICD of cancer cells. Here, we report that RT53 treatment induces all the hallmarks of immunogenic cell death, as defined by the plasma membrane exposure of calreticulin, release of ATP and the exodus of high-mobility group box 1 protein (HMGB1) from dying cancer cells, through a non-regulated, membranolytic mode of action. In a prophylactic mouse model, vaccination with RT53-treated fibrosarcomas prevented tumor growth at the challenge site. Finally, local intratumoral injection of RT53 into established cancers led to tumor regression together with T-cell infiltration and the mounting of an inflammatory response in the treated animals. Collectively, our results strongly suggest that RT53 can induce bona fide ICD of cancer cells and illustrate its potential use as a novel antitumor and immunotherapeutic strategy.

The dual effects of a novel peptibody on angiogenesis inhibition and M2 macrophage polarization on sarcoma.

Inhibition of the VEGF/VEGF receptor (VEGFR) and angiopoietin-2 (Ang-2)/TEK receptor tyrosine kinase (Tie-2) pathway is a potential target for tumor angiogenesis. We previously showed that a peptide AS16 which dually inhibits VEGFR/Ang-2 could reduce the tumor growth and decrease the number of microvessels in tumor. However, its short circulating half-life in the serum limits its clinical applications. In this study, as an effort to prolong the short in vivo half-life of AS16, we designed a fusion protein containing peptide AS16 and an IgG Fc fragment. Pharmacokinetic study also revealed that AS16-Fc has a prolonged circulating half-life of about 231 min in rats. We examined the effects of treatment on the tumor vasculature and immune cell populations, tumor growth, in both the MCA-205 and S180 tumor models. We found that AS16-Fc dramatically reduced tumor volume, vascular density and tumor-associated macrophages. Macrophages were identified as potential novel targets following anti-angiogenic therapy, our findings imply a novel role for anti-angiogenic peptide AS16-Fc. These findings indicate that AS16-Fc could be more effective on inhibiting tumor growth angiogenesis and tumor immune microenvironment than that of peptide AS16.

Checkpoint blockade immunotherapy reshapes the high-dimensional phenotypic heterogeneity of murine intratumoural neoantigen-specific CD8+ T cells.

The analysis of neoantigen-specific CD8+ T cells in tumour-bearing individuals is challenging due to the small pool of tumour antigen-specific T cells. Here we show that mass cytometry with multiplex combinatorial tetramer staining can identify and characterize neoantigen-specific CD8+ T cells in mice bearing T3 methylcholanthrene-induced sarcomas that are susceptible to checkpoint blockade immunotherapy. Among 81 candidate antigens tested, we identify T cells restricted to two known neoantigens simultaneously in tumours, spleens and lymph nodes in tumour-bearing mice. High-dimensional phenotypic profiling reveals that antigen-specific, tumour-infiltrating T cells are highly heterogeneous. We further show that neoantigen-specific T cells display a different phenotypic profile in mice treated with anti-CTLA-4 or anti-PD-1 immunotherapy, whereas their peripheral counterparts are not affected by the treatments. Our results provide insights into the nature of neoantigen-specific T cells and the effects of checkpoint blockade immunotherapy.Immune checkpoint blockade (ICB) therapies can unleash anti-tumour T-cell responses. Here the authors show, by integrating MHC tetramer multiplexing, mass cytometry and high-dimensional analyses, that neoantigen-specific, tumour-infiltrating T cells are highly heterogeneous and are subjected to ICB modulations.

A Multifunctional Role for Adjuvant Anti-4-1BB Therapy in Augmenting Antitumor Response by Chimeric Antigen Receptor T Cells.

Adoptive immunotherapy utilizing chimeric antigen receptor (CAR) T cells has demonstrated high success rates in hematologic cancers, but results against solid malignancies have been limited to date, due in part to the immunosuppressive tumor microenvironment. Activation of the 4-1BB (CD137) pathway using an agonistic α-4-1BB antibody is known to provide strong costimulatory signals for augmenting and diversifying T-cell responses. We therefore hypothesized that a combination of α-4-1BB and CAR T-cell therapy would result in improved antitumor responses. Using a human-Her2 self-antigen mouse model, we report here that α-4-1BB significantly enhanced CAR T-cell efficacy directed against the Her2 antigen in two different established solid tumor settings. Treatment also increased the expression of IFNγ and the proliferation marker Ki67 in tumor-infiltrating CAR T cells when combined with α-4-1BB. Strikingly, α-4-1BB significantly reduced host immunosuppressive cells at the tumor site, including regulatory T cells and myeloid-derived suppressor cells, correlating with an increased therapeutic response. We conclude that α-4-1BB has a multifunctional role for enhancing CAR T-cell responses and that this combination therapy has high translational potential, given current phase I/II clinical trials with α-4-1BB against various types of cancer. Cancer Res; 77(6); 1296-309. ©2017 AACR.

Immune deficiency augments the prevalence of p53 loss of heterozygosity in spontaneous tumors but not bi-directional loss of heterozygosity in bone marrow progenitors.

p53 loss of heterozygosity (LOH) is a frequent event in tumors of somatic and Li-Fraumeni syndrome patients harboring p53 mutation. Here, we focused on resolving a possible crosstalk between the immune-system and p53 LOH. Previously, we reported that p53 heterozygous bone-marrow mesenchymal progenitor cells undergo p53 LOH in-vivo. Surprisingly, the loss of either the wild-type p53 allele or mutant p53 allele was detected with a three-to-one ratio in favor of losing the mutant allele. In this study, we examined whether the immune-system can affect the LOH directionality in bone marrow progenitors. We found that mesenchymal progenitor cells derived from immune-deficient mice exhibited the same preference of losing the mutant p53 allele as immune-competent matched cells, nevertheless, these animals showed a significantly shorter tumor-free survival, indicating the possible involvement of immune surveillance in this model. Surprisingly, spontaneous tumors of p53 heterozygous immune-deficient mice exhibited a significantly higher incidence of p53 LOH compared to that observed in tumors derived of p53 heterozygous immune-competent mice. These findings indicate that the immune-system may affect the p53 LOH prevalence in spontaneous tumors. Thus suggesting that the immune-system may recognize and clear cells that underwent p53 LOH, whereas in immune-compromised mice, those cells will form tumors with shorter latency. In individuals with a competent immune-system, p53 LOH independent pathways may induce malignant transformation which requires a longer tumor latency. Moreover, this data may imply that the current immunotherapy treatment aimed at abrogating the inhibition of cellular immune checkpoints may be beneficial for LFS patients.

B lymphocytes as direct antigen-presenting cells for anti-tumor DNA vaccines.

In spite of remarkable preclinical efficacy, DNA vaccination has demonstrated low immunogenicity in humans. While efforts have focused on increasing cross-presentation of DNA-encoded antigens, efforts to increase DNA vaccine immunogenicity by targeting direct presentation have remained mostly unexplored. In these studies, we compared the ability of different APCs to present antigen to T cells after simple co-culture with plasmid DNA. We found that human primary peripheral B lymphocytes, and not monocytes or in vitro derived dendritic cells (DCs), were able to efficiently encode antigen mRNA and expand cognate tumor antigen-specific CD8 T cells ex vivo. Similarly, murine B lymphocytes co-cultured with plasmid DNA, and not DCs, were able to prime antigen-specific T cells in vivo. Moreover, B lymphocyte-mediated presentation of plasmid antigen led to greater Th1-biased immunity and was sufficient to elicit an anti-tumor effect in vivo. Surprisingly, increasing plasmid presentation by B cells, and not cross presentation of peptides by DCs, further augmented traditional plasmid vaccination. Together, these data suggest that targeting plasmid DNA to B lymphocytes, for example through transfer of ex vivo plasmidloaded B cells, may be novel means to achieve greater T cell immunity from DNA vaccines.

Eliminating roles for T-bet and IL-2 but revealing superior activation and proliferation as mechanisms underpinning dominance of regulatory T cells in tumors.

Foxp3(+) regulatory T cells (Tregs) are often highly enriched within the tumor-infiltrating T cell pool. Using a well-characterised model of carcinogen-induced fibrosarcomas we show that the enriched tumor-infiltrating Treg population comprises largely of CXCR3(+) T-bet(+) 'TH1-like' Tregs which are thymus-derived Helios(+) cells. Whilst IL-2 maintains homeostatic ratios of Tregs in lymphoid organs, we found that the perturbation in Treg frequencies in tumors is IL-2 independent. Moreover, we show that the TH1 phenotype of tumor-infiltrating Tregs is dispensable for their ability to influence tumor progression. We did however find that unlike Tconvs, the majority of intra-tumoral Tregs express the activation markers CD69, CD25, ICOS, CD103 and CTLA4 and are significantly more proliferative than Tconvs. Moreover, we have found that CD69(+) Tregs are more suppressive than their CD69- counterparts. Collectively, these data indicate superior activation of Tregs in the tumor microenvironment, promoting their suppressive ability and selective proliferation at this site.

CXCR2-mediated tumor-associated neutrophil recruitment is regulated by IFN-ß.

The chemokine receptor CXCR2 and its ligands CXCL1, CXCL2 and CXCL5 play an important role in homing of tumor-associated neutrophils (TANs) into developing tumors. TANs are known to support the development of blood vessels in growing solid tumors, hence contributing to tumor growth. Here, we show that the migration of neutrophils is influenced by endogenous interferon-beta (IFN-ß) via regulation of such chemokines and their receptor. We could demonstrate that CXCL1 and CXCL2 gradients are formed in tumor-bearing mice, i.e., low chemokine level in bone marrow (BM) and high level in the tumor. This supports migration of neutrophils into the tumor. Moreover, expression of CXCR2 was highest on neutrophils from BM and lowest in TANs. Importantly, although IFN-ß appears to have only a minor influence on the expression of CXCR2, it strongly regulates the CXCR2 ligands. In the absence of endogenous IFN-ß, they were expressed significantly higher in tumor-infiltrating neutrophils. Treatment of such neutrophils from tumor-bearing Ifnb1(-/-) mice with recombinant IFN-ß downregulated CXCR2 ligand expression to wild-type levels. This explains the reduced migration of neutrophils into tumors and the diminished tumor angiogenesis in IFN-ß-sufficient mice. Our results add a novel functional aspect of the type I IFN system as effector molecules of natural cancer surveillance and open interesting possibilities for antineutrophil therapies against cancer.

Signs of cell-cell interactions in sarcoma 45 tissue under conditions of antitumor effect caused by injection of magnetite nanoparticles.

Changes in transplanted sarcoma 45 tissue in outbred albino rats with tumor regression under the effect of magnetite nanoparticles (magnetic fluid) were studied by light and electron microscopy. The ultrastructure and cell death types in regressing tumors and signs of cell-cell interactions with participation of macrophages, lymphocytes, neutrophils, and degranulating mast cells were described. Some possible mechanisms of a pronounced antitumor activity of magnetite nanoparticles were discussed.

Immune microenvironment profiles of tumor immune equilibrium and immune escape states of mouse sarcoma.

Cancer immunoediting consists of three distinct phases: elimination, equilibrium and escape. Here, for the first time, we investigated the immune microenvironment profiles of tumor immune equilibrium and immune escape states in 3'-methylcholanthrene-induced murine sarcoma model. Our study indicates the relative balance of monocytic MDSCs and antitumor immunity cells (especially CTLs, NK cells and γδT cells) may involve in maintaining tumor cells in a state of immune-mediated dormancy. In addition, high percentages of Treg cells and PMN-MDSCs are associated with the tumor immune escape state - mice with progressing sarcomas. In summary, the relative balance of immune effector cells and suppressive populations in the tumor microenvironment may involve in determining the fate of tumors.

Modulation of antitumour immune responses by intratumoural Stat1 expression.

Signal transducer and activator of transcription 1 (Stat1) mediates anti-viral responses and cytokine-driven anti-proliferative, apoptotic and immunomodulatory activities. As de-regulated Stat1 function can affect tumour progression we sought to elucidate the effects of tumour cell-intrinsic Stat1 expression on immunosurveillance. Knockout of Stat1 enhanced the development of sarcomas induced by the chemical carcinogen 3-methylcholanthrene (MCA). Growth of transplanted MCA-induced Stat1⁻/⁻ sarcomas was suppressed in wild-type mice compared to growth in Stat1⁻/⁻ and immunocompromised recipients. Co-depletion of NK and CD8⁺ T cells from wild-type mice facilitated Stat1-deficient tumour growth whereas depletion of CD4⁺ T cells and CD8⁺ T cells did not. In vitro and in vivo analysis of the tumours implicated a role for NK cell-mediated, perforin-dependent killing of Stat1-deficient tumours. Interestingly, restoration of Stat1 expression in Stat1⁻/⁻ tumours resulted in diminished involvement of NK cells and increased contribution of CD8⁺ T cells in anti-tumour responses. Therefore, Stat1 expression within tumour cells modulated anti-tumour immune responses by altering the dominant immune effector cell involvement from NK cells to CD8⁺ T cells in the absence or presence of Stat1 respectively.

Extra-hepatic cancer represses hepatic drug metabolism via interleukin (IL)-6 signalling.

PURPOSE: In many cancer patients, the malignancy causes reduced hepatic drug clearance leading to potentially serious complications from the use of anticancer drugs. The mechanisms underlying this phenomenon are poorly understood. We aimed to identify tumor-associated inflammatory pathways that alter drug response and enhance chemotherapy-associated toxicity. METHODS: We studied inflammatory pathways involved in extra-hepatic tumor mediated repression of CYP3A, a major hepatic drug metabolizing cytochrome P450 subfamily, using a murine Engelbreth-Holm-Swarm sarcoma model. Studies in IL-6 knockout mice determined the source of elevated IL-6 in tumor-bearing animals and monoclonal antibodies against IL-6 were used to intervene in this inflammatory pathway. RESULTS: Our studies confirm elevated plasma IL-6 levels and reveal activation of Jak/Stat and Mapk signalling pathways and acute phase proteins in livers of tumor-bearing mice. Circulating IL-6 was predominantly produced by the tumor xenograft, rather than being host derived. Anti IL-6 antibody intervention partially reversed tumor-mediated inflammation and Cyp3a gene repression. CONCLUSIONS: IL-6 is an important player in cancer-related repression of CYP3A-mediated drug metabolism and activation of the acute phase response. Targeting IL-6 in cancer patients may prove an effective approach to alleviating cancer-related phenomena, such as adverse drug-related outcomes commonly associated with cancer chemotherapy.

Antitumor and immunomodulatory activity of a polysaccharide from fungus Coprinus comatus (Mull.:Fr.) Gray.

In our study, a water-soluble polysaccharide (CCPa-1) was successfully purified from the fruiting bodies of Coprinus comatus by DEAE-cellulose and Sepharose CL-6B column chromatography. The molecular weight was evaluated to be 53.6 kDa as determined by high performance size exclusion chromatography (HPSEC). Sugar composition analysis revealed that CCPa-1 consisted primarily of galactose, glucose and arabinose in a molar ratio of 6.6:1.2:2.2. CCPa-1 could not only inhibit the growth of sarcoma 180 (S180) tumor transplanted in mice, but also increase the relative spleen/thymus indexes and body weight of tumor bearing mice. Moreover, Con A- or LPS-induced splenocyte proliferation was also enhanced after CCPa-1 administration in tumor-bearing mice. Furthermore, CCPa-1 significantly enhanced the Con A- or LPS-induced splenocyte proliferation and increased the production of TNF-α and IL-2. All the data demonstrated that CCPa-1 had a potential application as natural antitumor agent with immunomodulatory activity.

Neem leaf glycoprotein activates CD8(+) T cells to promote therapeutic anti-tumor immunity inhibiting the growth of mouse sarcoma.

In spite of sufficient data on Neem Leaf Glycoprotein (NLGP) as a prophylactic vaccine, little knowledge currently exists to support the use of NLGP as a therapeutic vaccine. Treatment of mice bearing established sarcomas with NLGP (25 µg/mice/week subcutaneously for 4 weeks) resulted in tumor regression or dormancy (Tumor free/Regressor, 13/24 (NLGP), 4/24 (PBS)). Evaluation of CD8(+) T cell status in blood, spleen, TDLN, VDLN and tumor revealed increase in cellular number. Elevated expression of CD69, CD44 and Ki67 on CD8(+) T cells revealed their state of activation and proliferation by NLGP. Depletion of CD8(+) T cells in mice at the time of NLGP treatment resulted in partial termination of tumor regression. An expansion of CXCR3(+) and CCR5(+) T cells was observed in the TDLN and tumor, along with their corresponding ligands. NLGP treatment enhances type 1 polarized T-bet expressing T cells with downregulation of GATA3. Treg cell population was almost unchanged. However, T∶Treg ratios significantly increased with NLGP. Enhanced secretion/expression of IFNγ was noted after NLGP therapy. In vitro culture of T cells with IL-2 and sarcoma antigen resulted in significant enhancement in cytotoxic efficacy. Consistently higher expression of CD107a was also observed in CD8(+) T cells from tumors. Reinoculation of sarcoma cells in tumor regressed NLGP-treated mice maintained tumor free status in majority. This is correlated with the increment of CD44(hi)CD62L(hi) central memory T cells. Collectively, these findings support a paradigm in which NLGP dynamically orchestrates the activation, expansion, and recruitment of CD8(+) T cells into established tumors to operate significant tumor cell lysis.