VL is a life-threatening protozoal infection chiefly impinging the rural and poor population in the tropical and sub-tropical countries. The deadly affliction is rapidly expanding after its association with AIDS, swiftly defying its status of a neglected disease. Despite successful formulation of vaccine against canine leishmaniasis, no licensed vaccine is yet available for human VL, chemotherapy is in appalling state, and the development of new candidate drugs has been painfully slow. In face of lack of proper incentives, immunostimulatory plant preparations owing antileishmanial efficacy bear potential to rejuvenate awful antileishmanial chemotherapy. We have earlier reported profound leishmanicidal activity of P. nigrum seeds hexane (PNH) and P. nigrum ethanolic (PNE) fractions derived from P. nigrum seeds against Leishmania donovani promastigotes and amastigotes. In the present study, we illustrate that the remarkable anti-promastigote activity exhibited by PNH and PNE is mediated via apoptosis as evidenced by phosphatidylserine externalization, DNA fragmentation, arrest in sub G0/G1 phase, loss of mitochondrial membrane potential and generation of reactive oxygen species. Further, P. nigrum bioactive fractions rendered significant protection to L. donovani infected BALB/c mice in comparison to piperine, a known compound present in Piper species. The substantial therapeutic potential of PNH and PNE was accompanied by elicitation of cell-mediated immune response. The bioactive fractions elevated the secretion of Th1 (INF-γ, TNF-α and IL-2) cytokines and declined IL-4 and IL-10. PNH and PNE enhanced the production of IgG2a, upregulated the expression of co-stimulatory molecules CD80 and CD86, augmented splenic CD4+ and CD8+ T cell population, induced strong lymphoproliferative and DTH responses and partially stimulated NO production. PNH and PNE were devoid of any hepatic or renal toxicity. These encouraging findings merit further exploration of P. nigrum bioactive fractions as a source of potent and non-toxic antileishmanials.
Background Immunotherapy in prostate cancer (PCa) lags behind the progresses obtained in other cancer types partially because of its limited immune infiltration. Tumor-resident immune cells have been detected in the prostate, but the regulatory mechanisms that govern tumor infiltration are still poorly understood. To address this gap, we investigated the role of Wolf-Hirschhorn syndrome candidate 1 (WHSC1), a histone methyltransferase enzyme that targets dimethyl and trimethyl H3K36. WHSC1 is known to promote malignant growth and progression in multiple tumors, but its role in the interface between PCa and immune system is unknown. Methods RNA Sequencing (RNASeq) data from patients with PCa from The Cancer Genome Atlas (TCGA) were collected and divided into top/bottom 30% based on the expression of WHSC1 and disease-free survival was calculated. Publicly available chromatin immunoprecipitation (ChIPSeq) data were obtained from Cistrome and integrated with the available RNASeq data. RNASeq, ATACSeq and methylomic were analyzed using R Bioconductor packages comparing C42 cells with or without stable knockdown on WHSC1. Flow cytometry was used to measure Major Histocompatibility complex (MHC) levels, MHC-bound ovalbumin and tumor infiltration. C57B6 and NOD scid gamma (NSG) mice were subcutaneously grafted with TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) C2 cells and treated with MCTP39 (10 mg/kg); tumor size was monitored over time and curves were compared using permutation analyses. All analyses used a significance threshold of 0.05. Results Leveraging TCGA data, we demonstrated that elevated WHSC1 levels positively correlate with the presence of an immunosuppressive microenvironment. We validated those results in vitro, demonstrating that genetic and pharmacological inhibition of WHSC1 restores antigen presentation. This occurs via an elegant epigenetic regulation of gene expression at the chromatin and DNA methylation levels. In vivo studies in immunocompetent mice also show an increased frequency of CD8 + T cells in tumors from mice treated with WHSC1 inhibitor, supporting the hypothesis that the antitumor effect following WHSC1 inhibition requires a fully functional immune system. Conclusions This study demonstrates a novel role for WHSC1 in defining immune infiltration in PCa, with significant future implications for the use of immunotherapies in prostate malignancies.
Ovarian cancer (OC) is the fifth leading cause of cancer death in the US, presenting a low mutational burden and a diverse degree of infiltrating T cells. Neoantigens derived from somatic mutations represent an attractive immunotherapeutic target, however, mouse models for the development of personalized immunotherapies are still poor and do not fully recapitulate the individualized nature of OC in patients. To address this hurdle, our study established a patient-derived xenograft (PDX) of an OC patient as segue to studying neoantigen-driven autologous T cell response. We first evaluated tumor mutations in the primary tumor (Pr) and two successive passages (P0, P1) via WES. As tumors from P0 and P1 have a higher mutational count than Pr, we hypothesize that this difference is caused by the loss of immune-surveillance in immunocompromised mice, which allows for the outgrowth of previously repressed clones. By using the variant allele frequency (VAF) to cluster 123 shared mutations, we identified three clusters with unique VAF dynamics (Fig 1A-D). Tumors from Pr, P0 and P1 had a conserved functional with a strong conservation of antigen presenting pathways (Fig 2A-E). In PDX P0 we identified 184 non-synonymous mutations, leading to 30 potential neoantigens with high affinity for the patient's HLAs (Fig 3A-B). Neoantigens were ranked based on differential predicted HLA affinity of the WT versus mutated peptide (Fig 3C). T cell activation by neoantigens was tested in vitro via ELISA and flow cytometry. Interferon-γ production and upregulation of CD137 identified a core set of six neoantigens selectively recognized by patient's autologous CD8+ T cells (Fig 4A-B). Of those, 3/6 neoantigens were common between PDX and primary tumor, corroborating the role of the patient's own immune system in repressing the expansion of selected tumor clones (Fig 5). In vivo ACT studies showed that mice injected with neoantigen-stimulated autologous PBMCs (ACT_MUT) have reduced tumor growth when compared to mice injected with unstimulated cells (ACT_NP) (Fig 6A). ACT_MUT mice have higher levels of circulating T cells 15 days post-ACT and higher intratumoral T cells at end point than ACT_NP (Fig 6 B-C). We then sought to identify the TCR moieties that determine T cell response. Single cell TCRSeq analyses on the two strongest neoantigens identified multiple TCR activated by a single cancer neoantigen (Fig 7A-B), suggesting oligoclonal T cell activation. We tested this hypothesis by generating a motif with the most expanded clones and comparing it with the motif from clones that did not expend. Results indicate a significant difference in the frequency of amino acid in multiple CDR3 locations, suggesting the presence of an oligoclonal response to neoantigenic T cell stimulation. In conclusion, we have successfully established PDX models of OC that can be used to study and predict autologous T cell response to neoantigens.Citation Format: Muzamil Y. Want, Anna Konstorum, Ruea-Yea Huang, Richard Koya, Sebastiano Battaglia. Neoantigens retention by patient derived xenograft model mediate autologous T cells activation in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1076.
There is a pressing need for drug discovery against visceral leishmaniasis, a life-threatening protozoal infection, as the available chemotherapy is antiquated and not bereft of side effects. Plants as alternate drug resources has rewarded mankind in the past and aimed in this direction, we investigated the antileishmanial potential of Cinnamomum cassia.Dichloromethane, ethanolic and aqueous fractions of C. cassia bark, prepared by sequential extraction, were appraised for their anti-promastigote activity along with apoptosis-inducing potential. The most potent, C. cassia dichloromethane fraction (CBD) was evaluated for anti-amastigote efficacy in infected macrophages and nitric oxide (NO) production studied. The in vivo antileishmanial efficacy was assessed in L. donovani infected BALB/c mice and hamsters and various correlates of host protective immunity ascertained. Toxicity profile of CBD was investigated in vitro against peritoneal macrophages and in vivo via alterations in liver and kidney functions. The plant secondary metabolites present in CBD were identified by gas chromatography-mass spectroscopy (GC-MS).CBD displayed significant anti-promastigote activity with 50% inhibitory concentration (IC50) of 33.6 μg ml-1 that was mediated via apoptosis. This was evidenced by mitochondrial membrane depolarization, increased proportion of cells in sub-G0-G1 phase, ROS production, PS externalization and DNA fragmentation (TUNEL assay). CBD also inhibited intracellular amastigote proliferation (IC50 14.06 μg ml-1) independent of NO production. The in vivo protection achieved was 80.91% (liver) and 82.92% (spleen) in mice and 75.61% (liver) and 78.93% (spleen) in hamsters indicating its profound therapeutic efficacy. CBD exhibited direct antileishmanial activity, as it did not specifically induce a T helper type (Th)-1-polarized mileu in cured hosts. This was evidenced by insignificant modulation of NO production, lymphoproliferation, DTH (delayed type hypersensitivity), serum IgG2a and IgG1 levels and production of Th2 cytokines (IL-4 and IL-10) along with restoration of pro-inflammatory Th1 cytokines (INF-γ, IL-12p70) to the normal range. CBD was devoid of any toxicity in vitro as well as in vivo. The chemical constituents, cinnamaldehyde and its derivatives present in CBD may have imparted the observed antileishmanial effect.Our study highlights the profound antileishmanial efficacy of C. cassia bark DCM fraction and merits its further exploration as a source of safe and effective antieishmanial compounds.
Abstract Ovarian cancer (OC) is the fifth leading cause of cancer death in the United States with approximately 20,000 women being diagnosed every year. Since OC development is mainly asymptomatic, patients are often diagnosed at late stage and present local and distal metastases. This offers a clinical challenge, as roughly 70% of the patients develop chemoresistant disease. With the advent of cancer immunogenomics it is now possible to identify tumor specific mutations, or neoantigens/neoepitopes, that can be exploited for the development of personalized T cell therapies via DC vaccines or adoptive cell therapy (ACT). We therefore hypothesize that tumor cells isolated from malignant ascites reflect the genomic landscape of the primary tumor, offering a unique opportunity to identify clinically actionable tumor neoantigens. We utilized primary tumor, tumor cells isolated from malignant ascites and metastatic samples to create patient-derived xenografts (PDX) mice and to interrogate the tumor mutational landscape. Somatic mutations were identified by whole exome sequencing (WES) and RNASeq analysis in tumor samples, using PBMCs as germline control. In parallel 20 PDX of ovarian cancer were established in NSG mice by injecting 2×106 cells/mouse subcutaneously (SQ, n=10) and intraperitoneally (IP, n=10). Tumor growth was monitored weekly via caliper measurement for mice injected SQ and via abdominal circumference for mice injected IP. Mutational analysis and prediction of high affinity peptides was done using bioinformatics approach. We have utilized different algorithms that are based on Artificial Neural Networks or published motifs for predicting binding of 8-11 mer peptides to number of Human MHC alleles (HLA) and out of 168 mutated peptides identified, 30 were found to have high affinity for different HLAs compared to wild type peptide. Mutated and wild type peptides were pulsed into dendritic cells and co-cultured with CD8+ T cells for 20 days. Validation of stimulated CD8+ T cells by these mutated peptides using ELISPOT is underway. TCR specific for neoantigen from CD8+T cells will be identified and cloned using retroviral vectors. Transfected CD8+ T cells with neoantigen specific TCR will be confirmed by co-culturing them with dendritic cells pulsed with wild type and mutated peptides. Further engineered CD8+ T cells specific for neoantigens will be tested for adoptive cell therapy in the established PDX models of ovarian cancer. Citation Format: Muzamil Y. Want, Takemasa Tsuji, Richard Koya, Sebastiano Battaglia. Cancer immunogenomics for the development of personalized ovarian cancer vaccine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5674. doi:10.1158/1538-7445.AM2017-5674
Major progress in the analysis of human immune responses to cancer has been made through the molecular characterization of human tumour antigens. The development of therapeutic strategies for eliciting immune-mediated rejection of tumours has accelerated due to the elucidation of the molecular basis for tumour cell recognition and destruction by immune cells. Of the various human tumour antigens defined to date in ovarian cancer, the cancer-testis (CT) family of antigens have been studied extensively preclinically and clinically because of their testis-restricted expression in normal tissues and ability to elicit robust immune responses. Recent developments in cancer sequencing technologies offer a unique opportunity to identify tumour mutations with the highest likelihood of being expressed and recognized by the immune system. Such mutations, or neoantigens, could potentially serve as specific immune targets for T-cell-mediated destruction of cancer cells. This review will highlight current work in selecting tumour rejection antigens in ovarian cancer for improving the efficacy of immunotherapy.
The current therapeutic armory for visceral leishmaniasis (VL) caused by Leishmania donovani complex is inadequate, coupled with serious limitations. Combination therapy has proved ineffective due to mounting resistance; however, the search for safe and effective drugs is desirable, in the absence of any vaccine. There is a growing interest in the application of nanoparticles for the therapeutic effectiveness of leishmaniasis. Aimed in this direction, we assessed the antileishmanial effect of gold nanoparticles (GNP) against L. donovani in vitro.GNP were synthesized and characterized for particle size by dynamic light scattering (DLS) and atomic force microscopy (AFM) and for optical properties by UV-visible spectroscopy. Cytotoxicity of GNP was measured by the MTT proliferation assay. The antileishmanial activity of the nanoparticles was evaluated against L. donovani promastigotes and macrophage-infected amastigotes in vitro.GNP showed a strong SPR peak at 520 nm and mean particle size, polydispersity index (PDI), and zeta potential of 56.0 ± 10 nm, 0.3 ± 0.1 and -27.0 ± 3 mV, respectively. The GNPs were smooth and spherical with a mean particle diameter of 20 ± 5 nm. Nanoparticles [1.2-100 µM] did not reveal any cytotoxicity on RAW 264.7 murine macrophage cell line, but exerted significant activity against both promastigotes and amastigote stages of L. donovani with 50% inhibitory concentrations (IC50) of 18.4 ± 0.4 µM and 5.0 ± 0.3 µM, respectively. GNP showed significant antileishmanial activity with deformed morphology of parasites and the least number of surviving promastigotes after growth reversibility analysis.GNP may provide a platform to conjugate antileishmanial drugs onto the surface of nanoparticles to enhance their therapeutic effectiveness against VL. Further work is warranted, involving more in-depth mechanistic studies and in vivo investigations.
Abstract Ovarian cancer (OC) is the fifth leading cause of cancer death in the US, presenting a low mutational burden and a diverse degree of infiltrating T cells. Neoantigens derived from somatic mutations represent an attractive immunotherapeutic target, however, mouse models for the development of personalized immunotherapies are still poor and do not fully recapitulate the individualized nature of OC in patients. To address this hurdle, our study established a patient-derived xenograft (PDX) of an OC patient as segue to studying neoantigen-driven autologous T cell response. We first evaluated tumor mutations in the primary tumor (Pr) and two successive passages (P0, P1) via WES. As tumors from P0 and P1 have a higher mutational count than Pr, we hypothesize that this difference is caused by the loss of immune-surveillance in immunocompromised mice, which allows for the outgrowth of previously repressed clones. By using the variant allele frequency (VAF) to cluster 123 shared mutations, we identified three clusters with unique VAF dynamics (Fig 1A-D). Tumors from Pr, P0 and P1 had a conserved functional with a strong conservation of antigen presenting pathways (Fig 2A-E). In PDX P0 we identified 184 non-synonymous mutations, leading to 30 potential neoantigens with high affinity for the patient’s HLAs (Fig 3A-B). Neoantigens were ranked based on differential predicted HLA affinity of the WT versus mutated peptide (Fig 3C). T cell activation by neoantigens was tested in vitro via ELISA and flow cytometry. Interferon-γ production and upregulation of CD137 identified a core set of six neoantigens selectively recognized by patient’s autologous CD8+ T cells (Fig 4A-B). Of those, 3/6 neoantigens were common between PDX and primary tumor, corroborating the role of the patient’s own immune system in repressing the expansion of selected tumor clones (Fig 5). In vivo ACT studies showed that mice injected with neoantigen-stimulated autologous PBMCs (ACT_MUT) have reduced tumor growth when compared to mice injected with unstimulated cells (ACT_NP) (Fig 6A). ACT_MUT mice have higher levels of circulating T cells 15 days post-ACT and higher intratumoral T cells at end point than ACT_NP (Fig 6 B-C). We then sought to identify the TCR moieties that determine T cell response. Single cell TCRSeq analyses on the two strongest neoantigens identified multiple TCR activated by a single cancer neoantigen (Fig 7A-B), suggesting oligoclonal T cell activation. We tested this hypothesis by generating a motif with the most expanded clones and comparing it with the motif from clones that did not expend. Results indicate a significant difference in the frequency of amino acid in multiple CDR3 locations, suggesting the presence of an oligoclonal response to neoantigenic T cell stimulation. In conclusion, we have successfully established PDX models of OC that can be used to study and predict autologous T cell response to neoantigens. Citation Format: Muzamil Y. Want, Anna Konstorum, Ruea-Yea Huang, Richard Koya, Sebastiano Battaglia. Neoantigens retention by patient derived xenograft model mediate autologous T cells activation in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1076.
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