Cannabinoid exposure during pregnancy and its impact on immune function.

Cannabinoids are the most commonly abused illicit drugs worldwide. While cannabis can be beneficial for certain heath conditions, abuse of potent synthetic cannabinoids has been on the rise. Exposure to cannabinoids is also prevalent in women of child-bearing age and pregnant women. These compounds can cross the placental barrier and directly affect the fetus. They mediate their effects primarily through G-protein coupled cannabinoid receptors, CB1 and CB2. In addition to significant neurological effects, cannabinoids can trigger robust immunomodulation by altering cytokine levels, causing apoptosis of lymphoid cells and inducing suppressor cells of the immune system. Profound effects of cannabinoids on the immune system as discussed in this review, suggest that maternal exposure during pregnancy could lead to dysregulation of innate and adaptive immune system of developing fetus and offspring potentially leading to weakening of immune defenses against infections and cancer later in life. Emerging evidence also indicates the underlying role of epigenetic mechanisms causing long-lasting impact following cannabinoid exposure in utero.

MDSCs drive the process of endometriosis by enhancing angiogenesis and are a new potential therapeutic target.

Endometriosis affects women of reproductive age via unclear immunological mechanism(s). Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of myeloid cells with potent immunosuppressive and angiogenic properties. Here, we found MDSCs significantly increased in the peripheral blood of patients with endometriosis and in the peritoneal cavity of a mouse model of surgically induced endometriosis. Majority of MDSCs were granulocytic, produced ROS, and arginase, and suppressed T-cell proliferation. Depletion of MDSCs by antiGr-1 antibody dramatically suppressed development of endometrial lesions in mice. The chemokines CXCL1, 2, and 5 were expressed at sites of lesion while MDSCs expressed CXCR-2. These CXC-chemokines promoted MDSC migration toward endometriotic implants both in vitro and in vivo. Also, CXCR2-deficient mice show significantly decreased MDSC induction, endometrial lesions, and angiogenesis. Importantly, adoptive transfer of MDSCs into CXCR2-KO mice restored endometriotic growth and angiogenesis. Together, this study demonstrates that MDSCs play a role in the pathogenesis of endometriosis and identifies a novel CXC-chemokine and receptor for the recruitment of MDSCs, thereby providing a potential target for endometriosis treatment.

Critical Role of Mast Cells and Peroxisome Proliferator-Activated Receptor γ in the Induction of Myeloid-Derived Suppressor Cells by Marijuana Cannabidiol In Vivo.

Cannabidiol (CBD) is a natural nonpsychotropic cannabinoid from marijuana (Cannabis sativa) with anti-epileptic and anti-inflammatory properties. Effect of CBD on naive immune system is not precisely understood. In this study, we observed that administering CBD into naive mice triggers robust induction of CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSC) in the peritoneum, which expressed functional arginase 1, and potently suppressed T cell proliferation ex vivo. Furthermore, CBD-MDSC suppressed LPS-induced acute inflammatory response upon adoptive transfer in vivo. CBD-induced suppressor cells were comprised of CD11b(+)Ly6-G(+)Ly6-C(+) granulocytic and CD11b(+)Ly6-G(-)Ly6-C(+) monocytic subtypes, with monocytic MDSC exhibiting higher T cell-suppressive function. Induction of MDSC by CBD was markedly attenuated in Kit-mutant (Kit(W/W-v)) mast cell-deficient mice. MDSC response was reconstituted upon transfer of wild-type bone marrow-derived mast cells in Kit(W/W-v) mice, suggesting the key role of cKit (CD117) as well as mast cells. Moreover, mast cell activator compound 48/80 induced significant levels of MDSC in vivo. CBD administration in mice induced G-CSF, CXCL1, and M-CSF, but not GM-CSF. G-CSF was found to play a key role in MDSC mobilization inasmuch as neutralizing G-CSF caused a significant decrease in MDSC. Lastly, CBD enhanced the transcriptional activity of peroxisome proliferator-activated receptor γ in luciferase reporter assay, and PPAR-γ selective antagonist completely inhibited MDSC induction in vivo, suggesting its critical role. Together, the results suggest that CBD may induce activation of PPAR-γ in mast cells leading to secretion of G-CSF and consequent MDSC mobilization. CBD being a major component of Cannabis, our study indicates that marijuana may modulate or dysregulate the immune system by mobilizing MDSC.

Histone modifications are associated with Δ9-tetrahydrocannabinol-mediated alterations in antigen-specific T cell responses.

Marijuana is one of the most abused drugs due to its psychotropic effects. Interestingly, it is also used for medicinal purposes. The main psychotropic component in marijuana, Δ(9)-tetrahydrocannabinol (THC), has also been shown to mediate potent anti-inflammatory properties. Whether the immunomodulatory activity of THC is mediated by epigenetic regulation has not been investigated previously. In this study, we employed ChIP-Seq technology to examine the in vivo effect of THC on global histone methylation in lymph node cells of mice immunized with a superantigen, staphylococcal enterotoxin B. We compared genome-wide histone H3 Lys-4, Lys-27, Lys-9, and Lys-36 trimethylation and histone H3 Lys-9 acetylation patterns in such cells exposed to THC or vehicle. Our results showed that THC treatment leads to the association of active histone modification signals to Th2 cytokine genes and suppressive modification signals to Th1 cytokine genes, indicating that such a mechanism may play a critical role in the THC-mediated switch from Th1 to Th2. At the global level, a significant portion of histone methylation and acetylation regions were altered by THC. However, the overall distribution of these histone methylation signals among the genomic features was not altered significantly by THC, suggesting that THC activates the expression of a subset of genes while suppressing the expression of another subset of genes through histone modification. Functional classification of these histone marker-associated genes showed that these differentially associated genes were involved in various cellular functions, from cell cycle regulation to metabolism, suggesting that THC had a pleiotropic effect on gene expression in immune cells. Altogether, the current study demonstrates for the first time that THC may modulate immune response through epigenetic regulation involving histone modifications.

Distinct microRNA expression profile and targeted biological pathways in functional myeloid-derived suppressor cells induced by Δ9-tetrahydrocannabinol in vivo: regulation of CCAAT/enhancer-binding protein α by microRNA-690.

Δ(9)-Tetrahydrocannabinol (THC), the major bioactive component of marijuana, has been shown to induce functional myeloid-derived suppressor cells (MDSCs) in vivo. Here, we studied the involvement of microRNA (miRNA) in this process. CD11b(+)Gr-1(+) MDSCs were purified from peritoneal exudates of mice administered with THC and used for genome-wide miRNA profiling. Expression of CD31 and Ki-67 confirmed that the THC-MDSCs were immature and proliferating. THC-induced MDSCs exhibited distinct miRNA expression signature relative to various myeloid cells and BM precursors. We identified 13 differentially expressed (>2-fold) miRNA in THC-MDSCs relative to control BM precursors. In silico target prediction for these miRNA and pathway analysis using multiple bioinformatics tools revealed significant overrepresentation of Gene Ontology clusters within hematopoiesis, myeloid cell differentiation, and regulation categories. Insulin-like growth factor 1 signaling involved in cell growth and proliferation, and myeloid differentiation pathways were among the most significantly enriched canonical pathways. Among the differentially expressed, miRNA-690 was highly overexpressed in THC-MDSCs (∼16-fold). Transcription factor CCAAT/enhancer-binding protein α (C/EBPα) was identified as a potential functional target of miR-690. Supporting this, C/EBPα expression was attenuated in THC-MDSCs as compared with BM precursors and exhibited an inverse relation with miR-690. miR-690 knockdown using peptide nucleic acid-antagomiR was able to unblock and significantly increase C/EBPα expression establishing the functional link. Further, CD11b(+)Ly6G(+)Ly6C(+) and CD11b(+)Ly6G(-)Ly6C(+) purified subtypes showed high levels of miR-690 with attenuated C/EBPα expression. Moreover, EL-4 tumor-elicited MDSCs showed increased miR-690 expression. In conclusion, miRNA are significantly altered during the generation of functional MDSC from BM. Select miRNA such as miR-690 targeting genes involved in myeloid expansion and differentiation likely play crucial roles in this process and therefore in cannabinoid-induced immunosuppression.

miR-155 deficiency protects mice from experimental colitis by reducing T helper type 1/type 17 responses.

Inflammatory bowel disease (IBD), a chronic intestinal inflammatory condition that affects millions of people worldwide, results in high morbidity and exorbitant health-care costs. The critical features of both innate and adaptive immunity are to control inflammation and dysfunction in this equilibrium is believed to be the reason for the development of IBD. miR-155, a microRNA, is up-regulated in various inflammatory disease states, including IBD, and is a positive regulator of T-cell responses. To date, no reports have defined a function for miR-155 with regard to cellular responses in IBD. Using an acute experimental colitis model, we found that miR-155(-/-) mice, as compared to wild-type control mice, have decreased clinical scores, a reversal of colitis-associated pathogenesis, and reduced systemic and mucosal inflammatory cytokines. The increased frequency of CD4+ lymphocytes in the spleen and lamina propria with dextran sodium sulphate induction was decreased in miR-155(-/-) mice. Similarly, miR-155 deficiency abrogated the increased numbers of interferon-γ expressing CD4+ T cells typically observed in wild-type mice in this model. The frequency of systemic and mucosal T helper type 17-, CCR9-expressing CD4+ T cells was also reduced in miR-155(-/-) mice compared with control mice. These findings strongly support a role for miR-155 in facilitating pro-inflammatory cellular responses in this model of IBD. Loss of miR-155 also results in decreases in T helper type 1/type 17, CD11b+) and CD11c+ cells, which correlated with reduced clinical scores and severity of disease. miR-155 may serve as a potential therapeutic target for the treatment of IBD.

TP-0184 inhibits FLT3/ACVR1 to overcome FLT3 inhibitor resistance and hinder AML growth synergistically with venetoclax.

We identified activin A receptor type I (ACVR1), a member of the TGF-ß superfamily, as a factor favoring acute myeloid leukemia (AML) growth and a new potential therapeutic target. ACVR1 is overexpressed in FLT3-mutated AML and inhibition of ACVR1 expression sensitized AML cells to FLT3 inhibitors. We developed a novel ACVR1 inhibitor, TP-0184, which selectively caused growth arrest in FLT3-mutated AML cell lines. Molecular docking and in vitro kinase assays revealed that TP-0184 binds to both ACVR1 and FLT3 with high affinity and inhibits FLT3/ACVR1 downstream signaling. Treatment with TP-0184 or in combination with BCL2 inhibitor, venetoclax dramatically inhibited leukemia growth in FLT3-mutated AML cell lines and patient-derived xenograft models in a dose-dependent manner. These findings suggest that ACVR1 is a novel biomarker and plays a role in AML resistance to FLT3 inhibitors and that FLT3/ACVR1 dual inhibitor TP-0184 is a novel potential therapeutic tool for AML with FLT3 mutations.

Characterization and gene cloning of an acidic thaumatin-like protein (TLP 1), an allergen from sapodilla fruit (Manilkara zapota).

BACKGROUND: Allergy to sapodilla (Manilkara zapota) fruit ingestion is rare. An independent study from our group has identified a basic thaumatin-like protein (TLP 2) as the major allergen. The present study was aimed at identifying and characterizing additional allergens from sapodilla. METHODS: Allergic subjects were identified by case history, skin prick test (SPT) and allergen-specific IgE. Sapodilla extract was fractionated using SP-Sepharose into 3 components (SP1, SP2 and SP3) which were analyzed by native/SDS-PAGE, IgE-immunoblot, isoelectric focusing (IEF) and N-terminal sequencing. The conserved regions of plant TLPs and the N-terminal sequence were used to design primers for PCR. RESULTS: SPT and ELISA confirmed a subject with oral allergy syndrome (OAS) to sapodilla and custard apple. Two proteins (26.9 and 24.5kDa; reducing conditions) were detected as allergens, of which the latter in SP2 has already been identified as basic TLP (TLP 2). The 26.9kDa protein present in SP1 was identified as an acidic TLP based on native PAGE, IEF and N-terminal sequencing. Presence of a basic ß-1,3-glucanase in SP3 was inferred by zymography. Sequence analysis of the genomic clone of the acidic TLP gene revealed that it is intronless and non-glycosylated. Evolutionary relatedness to olive, grape and kiwi fruit allergenic TLPs were inferred by phylogenetic analysis. CONCLUSIONS: An acidic TLP (TLP 1) was identified as a new allergen in sapodilla. TLP 1 is a single polypeptide (207 residues) belonging to the thaumatin family of the GH64-TLP-SF superfamily. Clinically, sapodilla should be considered in the list of fruits causing OAS.

Circulating neutrophils and tumor-associated myeloid cells function as a powerful biomarker for response to chemoradiation in locally advanced cervical cancer.

Purpose: The immune system's role in mediating the cytotoxic effects of chemoradiotherapy remains not completely understood. The integration of immunotherapies into treatment will require insight into features and timing of the immune microenvironment associated with treatment response. Here, we investigated the role of circulating neutrophils and tumor-associated myeloid cells (TSAMs) as potential agents and biomarkers for disease-related outcomes in locally advanced cervical cancer (LACC). Material and Methods: Hematologic parameters for two LACC patient cohorts, a retrospective clinical and a prospective translational cohort, were obtained at baseline, weekly during chemoradiotherapy for the retrospective cohort, biweekly during chemoradiotherapy for the prospective cohort, and at the first follow-up visit for both cohorts (mean 14.7 weeks, range 8.1-25.1 weeks for the prospective cohort and 5.3 weeks with a range of 2.7-9.0 weeks for the retrospective cohort). In both cohorts, baseline as well as mean and lowest on-treatment values for platelets, hemoglobin, absolute neutrophil count (ANC), and absolute lymphocyte count (ALC) were analyzed for correlations with disease-related outcomes. In the prospective cohort, circulating myeloid cells were isolated from peripheral blood mononuclear cells (PBMCs), and TSAMs were isolated from tumor tissue via a novel serial cytobrush sampling assay. The samples were analyzed by flow cytometry. Results: In both cohorts, the only hematologic parameter significantly associated with survival was elevated on-treatment mean ANC (mANC), which was associated with lower local failure-free and overall survival rates in the retrospective and prospective cohorts, respectively. mANC was not associated with a difference in distant metastases. CD11b+CD11c- TSAMs, which act as a surrogate marker for intratumoral neutrophils, steadily decreased during the course of chemoRT and nadier'd at week 5 of treatment. Conversely, circulating myeloid cells identified from PBMCs steadily increased through week 5 of treatment. Regression analysis confirmed an inverse relationship between circulating myeloid cells and TSAMs at this time point. Conclusions: These findings identify on-treatment mean neutrophil count as a predictor of disease-related outcomes, suggest that neutrophils contribute to chemoradiation treatment resistance, and demonstrate the importance of techniques to measure intratumoral immune activity.

CD44 receptor targeted nanoparticles augment immunity against tuberculosis in mice.

We describe a role of CD44-mediated signaling during host-defense against tuberculosis (TB) using a mouse model of TB and studies in M. tuberculosis (Mtb) infected human macrophage (MФ). Liposomes targeting CD44 using thioaptamers (CD44TA-LIP) were designed and tested as new vaccines to boost host immunity in TB. CD44TA-LIP enhanced killing of Mtb in human MФ, which correlated with an increased production of pro-inflammatory cytokines IL-1ß, TNF-α and IL-12. CD44TA-LIP activated MФ showed an enhanced MHC-II dependent antigen presentation to CD4 T-cells. Inhibition of cellular proliferation and cytoskeleton rearrangement pathways downstream of CD44 signaling abrogated CD44TA-LIP-induced antimicrobial effects. Blockade of inflammatory pathways also reduced antigen presentation by MФ and activation of CD4 T cells. Mtb infected MФ treated with CD44TA-LIP exhibited increased nitric oxide and HßD2 defensin peptide production. Among Mtb infected mice with increased lung and spleen loads of organisms, intranasal administration of CD44TA-LIP led to a ten-fold reduction of colony forming units of Mtb and elevated IFN-γ + CD4, effector, central and resident memory T cells. Biodistribution studies demonstrated that CD44TA-LIP preferentially accumulated in the lungs and were associated with CD11b + cells. CD44TA-LIP treated mice showed no weight loss or increased liver LDH levels. This study highlights the importance of CD44-mediated signaling in host-defense during TB and the therapeutic potential of CD44TA-LIP.

Mucosal Vaccination With Recombinant Tm-WAP49 Protein Induces Protective Humoral and Cellular Immunity Against Experimental Trichuriasis in AKR Mice.

Trichuriasis is one of the most common neglected tropical diseases of the world's poorest people. A recombinant vaccine composed of Tm-WAP49, an immunodominant antigen secreted by adult Trichuris stichocytes into the mucosa of the cecum to which the parasite attaches, is under development. The prototype is being evaluated in a mouse model of Trichuris muris infection, with the ultimate goal of producing a mucosal vaccine through intranasal delivery. Intranasal immunization of mice with Tm-WAP49 formulated with the adjuvant OCH, a truncated analog of alpha-GalCer with adjuvanticity to stimulate natural killer T cells (NKT) and mucosal immunity, induced significantly high levels of IgG and its subclasses (IgG1 and IgG2a) in immunized mice. This also resulted in a significant reduction of worm burden after challenge with T. muris-infective eggs. The addition of QS-21 adjuvant to this vaccine formulation further reduced worm counts. The improved protection from the dual-adjuvanted vaccine correlated with higher serum antibody responses (IgG, IgG1, IgG2a, IgA) as well as with the induction of antigen-specific IgA in the nasal mucosa. It was also associated with the robust cellular responses including functional subsets of CD4 T cells producing IL-4, and cytotoxic CD8 T cells expressing granzyme B. The worm reduction achieved by mucosal immunization was higher than that induced by subcutaneous immunization. Intranasal immunization also induced a significantly higher nasal mucosa-secreted antigen-specific IgA response, as well as higher functional cellular responses including CD4+IL4+ (Th1) and CD8+GnzB+ (Th2) T cells, and antigen-specific INFγ-producing T cells in both spleen and MLNs and antibody-producing B cells (CD19+B220+/B220+GL7+). Mucosal immunization further induced long-term T lymphocyte memory with increased central (CD62L+CD44+) and effector (CD62L-CD44+) memory subsets of both CD4 and CD8 T cells at 60 days after the last immunization. In summary, intranasal immunization with recombinant Tm-WAP49 protein induced strong protection versus murine trichuriasis. It represents a promising vaccination approach against intestinal nematodes.

Cannabinoid receptor activation leads to massive mobilization of myeloid-derived suppressor cells with potent immunosuppressive properties.

Cannabinoid receptor activation by agents such as Δ(9)-tetrahydrocannabinol (THC) is known to trigger immune suppression. Here, we show that administration of THC in mice leads to rapid and massive expansion of CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSC) expressing functional arginase and exhibiting potent immunosuppressive properties both in vitro and in vivo. The induction of MDSC by THC was associated with a significant increase in granulocyte CSF. Moreover, administration of anti-granulocyte CSF Ab inhibited the induction of MDSC by THC. THC was able to induce MDSC in TLR4 mutant C3H and C57BL10/ScN mice and hence acted independently of TLR4. Accumulation of MDSC in the periphery with a corresponding decrease in the proportion of CD11b(+)Gr-1(+) cells in the bone marrow, as well as in vivo BrdU labeling and cell-cycle analysis, showed that THC induced mobilization of these cells from bone marrow and their expansion in the periphery. Use of selective antagonists SR141716A and SR144528 against cannabinoid receptors 1 and 2, respectively, as well as receptor-deficient mice showed that induction of MDSC was mediated through activation of both cannabinoid receptors 1 and 2. These studies demonstrate that cannabinoid receptor signaling may play a crucial role in immune regulation via the induction of MDSC.

Targeting cannabinoid receptors as a novel approach in the treatment of graft-versus-host disease: evidence from an experimental murine model.

Allogeneic hematopoietic cell transplantation (HCT) is widely used to treat patients with life-threatening malignant and nonmalignant hematological diseases. However, allogeneic HCT often is accompanied by severe and lethal complications from graft-versus-host disease (GVHD), in which activated donor T cells recognize histocompatibility antigenic mismatches and cause significant toxicity in the recipient. In the current study, we tested the hypothesis that activation of cannabinoid receptors on donor-derived T cells may prevent GVHD. We tested the effect of Δ(9)-tetrahydrocannabinol (THC) in an acute model of GVHD that was induced by transferring parental C57BL/6 (B6) spleen cells into (C57BL/6 × DBA/2) F(1)(BDF1) mice. Transfer of B6 cells into BDF1 mice produced severe acute GVHD in the recipient, characterized by lymphoid hyperplasia, weight loss, T helper l cytokine production and mortality. THC administration led to early recovery from body weight loss, reduced tissue injury in the liver and intestine, as well as complete survival. THC treatment reduced the expansion of donor-derived effector T cells and blocked the killing of host-derived immune cells while promoting Foxp3(+) regulatory T cells. Impaired hematopoiesis seen during GVHD was rescued by treatment with THC. The ability of THC to reduce the clinical GVHD was reversed, at least in part, by administration of cannabinoid receptor (CB) 1 and CB2 antagonists, thereby demonstrating that THC-mediated amelioration of GVHD was cannabinoid receptor-dependent. Our results demonstrate for the first time that targeting cannabinoid receptors may constitute a novel treatment modality against acute GVHD.

Perinatal exposure to Δ9-tetrahydrocannabinol triggers profound defects in T cell differentiation and function in fetal and postnatal stages of life, including decreased responsiveness to HIV antigens.

Marijuana abuse is very prominent among pregnant women. Although marijuana cannabinoids have been shown to exert immunosuppression in adults, virtually nothing is known about the effects of marijuana use during pregnancy on the developing immune system of the fetus and during postnatal life. We noted that murine fetal thymus expressed high levels of the cannabinoid receptors CB1 and CB2. Moreover, perinatal exposure to Δ(9)-tetrahydrocannabinol (THC) had a profound effect on the fetus as evidenced by a decrease in thymic cellularity on gestational days 16, 17, and 18 and postgestational day 1 and marked alterations in T cell subpopulations. These outcomes were reversed by CB1/CB2 antagonists, suggesting that THC-mediated these effects through cannabinoid receptors. Thymic atrophy induced in the fetus correlated with caspase-dependent apoptosis in thymocytes. Thymic atrophy was the result of direct action of THC and not based on maternal factors inasmuch as THC was able to induce T cell apoptosis in vitro in fetal thymic organ cultures. It is noteworthy that perinatal exposure to THC also had a profound effect on the immune response during postnatal life. Peripheral T cells from such mice showed decreased proliferative response to T cell mitogen as well as both T cell and antibody response to HIV-1 p17/p24/gp120 antigens. Together, our data demonstrate for the first time that perinatal exposure to THC triggers profound T cell dysfunction, thereby suggesting that the offspring of marijuana abusers who have been exposed to THC in utero may be at a higher risk of exhibiting immune dysfunction and contracting infectious diseases including HIV.

Gut microbiome diversity is an independent predictor of survival in cervical cancer patients receiving chemoradiation.

Diversity of the gut microbiome is associated with higher response rates for cancer patients receiving immunotherapy but has not been investigated in patients receiving radiation therapy. Additionally, current studies investigating the gut microbiome and outcomes in cancer patients may not have adjusted for established risk factors. Here, we sought to determine if diversity and composition of the gut microbiome was independently associated with survival in cervical cancer patients receiving chemoradiation. Our study demonstrates that the diversity of gut microbiota is associated with a favorable response to chemoradiation. Additionally, compositional variation among patients correlated with short term and long-term survival. Short term survivor fecal samples were significantly enriched in Porphyromonas, Porphyromonadaceae, and Dialister, whereas long term survivor samples were significantly enriched in Escherichia Shigella, Enterobacteriaceae, and Enterobacteriales. Moreover, analysis of immune cells from cervical tumor brush samples by flow cytometry revealed that patients with a high microbiome diversity had increased tumor infiltration of CD4+ lymphocytes as well as activated subsets of CD4 cells expressing ki67+ and CD69+ over the course of radiation therapy. Modulation of the gut microbiota before chemoradiation might provide an alternative way to enhance treatment efficacy and improve treatment outcomes in cervical cancer patients.

Intranasal Therapeutic Peptide Vaccine Promotes Efficient Induction and Trafficking of Cytotoxic T Cell Response for the Clearance of HPV Vaginal Tumors.

Human papillomavirus (HPV)-induced cancers continue to affect millions of women around the world, and the five year survival rate under the current standard of care for these cancers is less than 60% in some demographics. Therefore there is still an unmet need to develop an effective therapy that can be easily administered to treat established HPV cervical cancer lesions. We sought to investigate the potential of an intranasal HPV peptide therapeutic vaccine incorporating the combination of α-Galactosylceramide (α-GalCer) and CpG-ODN adjuvants (TVAC) against established HPV genital tumors in a syngeneic C57BL/6J mouse model. We obtained evidence to show that TVAC, delivered by the mucosal intranasal route, induced high frequencies of antigen-specific CD8 T cells concurrent with significant reduction in the immunosuppressive regulatory T cells and myeloid derived suppressor cells in the tumor microenvironment (TME), correlating with sustained elimination of established HPV genital tumors in over 85% of mice. Inclusion of both the adjuvants in the vaccine was necessary for significant increase of antigen-specific CD8 T cells to the tumor and antitumor efficacy because vaccination incorporating either adjuvant alone was inefficient. These results strongly support the utility of the TVAC administered by needle-free intranasal route as a safe and effective strategy for the treatment of established genital HPV tumors.

Pervasive Genomic Damage in Experimental Intracerebral Hemorrhage: Therapeutic Potential of a Mechanistic-Based Carbon Nanoparticle.

Therapy for intracerebral hemorrhage (ICH) remains elusive, in part dependent on the severity of the hemorrhage itself as well as multiple deleterious effects of blood and its breakdown products such as hemin and free iron. While oxidative injury and genomic damage have been seen following ICH, the details of this injury and implications remain unclear. Here, we discovered that, while free iron produced mostly reactive oxygen species (ROS)-related single-strand DNA breaks, hemin unexpectedly induced rapid and persistent nuclear and mitochondrial double-strand breaks (DSBs) in neuronal and endothelial cell genomes and in mouse brains following experimental ICH comparable to that seen with γ radiation and DNA-complexing chemotherapies. Potentially as a result of persistent DSBs and the DNA damage response, hemin also resulted in senescence phenotype in cultured neurons and endothelial cells. Subsequent resistance to ferroptosis reported in other senescent cell types was also observed here in neurons. While antioxidant therapy prevented senescence, cells became sensitized to ferroptosis. To address both senescence and resistance to ferroptosis, we synthesized a modified, catalytic, and rapidly internalized carbon nanomaterial, poly(ethylene glycol)-conjugated hydrophilic carbon clusters (PEG-HCC) by covalently bonding the iron chelator, deferoxamine (DEF). This multifunctional nanoparticle, DEF-HCC-PEG, protected cells from both senescence and ferroptosis and restored nuclear and mitochondrial genome integrity in vitro and in vivo. We thus describe a potential molecular mechanism of hemin/iron-induced toxicity in ICH that involves a rapid induction of DSBs, senescence, and the consequent resistance to ferroptosis and provide a mechanistic-based combinatorial therapeutic strategy.

CD44 mobilization in allogeneic dendritic cell-T cell immunological synapse plays a key role in T cell activation.

CD44 is involved in several biological processes owing to its dual role as a cell adhesion and signaling molecule. In an allogeneic dendritic cell (DC)-T cell interaction model, we show here that CD44 gets clustered at the contact between T cells with mature but not immature DCs. Also, CD44 colocalized with lipid rafts at the immunological synapse (IS). Using DCs or T cells derived from CD44-deficient mice, we observed that the presence of CD44 on DCs and T cells is important for the formation of DC-T cell tight conjugates. However, deficiency of CD44 on DCs but not T cells affected the functional IS, as indicated by decreased phosphotyrosine and protein kinase C-theta enrichment at the synapse. Also, CD44-deficient DCs induced significantly decreased proliferation as well as IL-2 and IFN-gamma production from allogeneic T cells. The polarization of CD44 at the synapse was also noted in an antigen (OVA)-specific, syngeneic DC-T cell interaction using OVA-specific T cells derived from OT-II mice. It was believed that large molecules such as CD44 were excluded from the IS. Results presented here show for the first time that CD44 is recruited to the IS during allogeneic DC and T cell interactions and plays an important role in subsequent T cell activation.

Targeting interferon signaling and CTLA-4 enhance the therapeutic efficacy of anti-PD-1 immunotherapy in preclinical model of HPV+ oral cancer.

BACKGROUND: The US is experiencing an epidemic of HPV+ oropharyngeal cancers (OPC), the rates and burden of which now exceed that for cervical cancer. Immunotherapy targeting programmed death 1 (PD-1) on tumor-infiltrating lymphocytes and/or its ligand PD-L1 on tumor cells, which was effective in several cancers has however, showed efficacy in only less than 15% of patients. METHODS: We used a preclinical HPV+ oral tumor model, mEER, consisting of mouse tonsil derived epithelial cells expressing HPV-16 E6 and E7 genes, along with the H-ras oncogene to test strategies for enhancing the efficacy of anti-PD-1 therapy. RESULTS: Monotherapy with PD-1 blocking antibody was ineffective against flank-implanted tumors, but induced regression in 54% of mice bearing orthotopic tongue tumors that correlated with higher CD8 T cell responses. Since the CD8+ T cells derived from tongue tumors also showed high levels of the immune checkpoint inhibitory receptor CTLA-4, we tested combination immunotherapy targeting both CTLA-4 and PD-1 together and observed 93.3% survival of mice bearing tumors in the tongue for the duration of our 100-day study. Protective immunity correlated with a significant decrease in immunosuppressive lymphoid and myeloid populations within the tumor microenvironment. Consistent with the reported capacity of interferon-driven PD-L1/PD-1 pathway induction to serve as a biomarker of response to PD-1 blockade, we observed elevated interferon signaling and significantly higher levels of PD-1/PD-L1 in tongue-implanted mEER tumors compared to those growing on the flank correlating with their preferential responsiveness to PD-1 blockade. More importantly, in a pseudometastasic mouse model bearing both flank and tongue tumors to represent metastatic disease, delivery of Stimulator of Interferon Induced Genes (STING) agonist into the flank tumors combined with systemic treatment with α-PD-1 and α-CTLA-4 antibodies resulted in sustained tumor regression in 71% of mice. In this case, productive abscopal anti-tumor immunity was associated with robust increases in the ratios of cytotoxic CD8+ T cells (CTL) versus regulatory T cells (Treg) and versus functional myeloid-derived suppressor cells (MDSC). CONCLUSIONS: These results support combining α-PD-1 therapy with induction of IFN-α/ß signaling via provision of STING agonist and/or through CTLA-4 blockade as potential treatment option for HNSCC patients, especially, those not responding to α-PD-1 monotherapy.

Attenuation of experimental autoimmune hepatitis by exogenous and endogenous cannabinoids: involvement of regulatory T cells.

Immune-mediated liver diseases including autoimmune and viral hepatitis are a major health problem worldwide. Natural cannabinoids such as Delta(9)-tetrahydrocannabinol (THC) effectively modulate immune cell function, and they have shown therapeutic potential in treating inflammatory diseases. We investigated the effects of THC in a murine model of concanavalin A (ConA)-induced hepatitis. Intraperitoneal administration of THC after ConA challenge inhibited hepatitis as shown by significant decrease in liver enzymes and reduced liver tissue injury. Furthermore, THC treatment resulted in significant suppression of crucial inflammatory cytokines in ConA-induced hepatitis. It is noteworthy that THC treatment in ConA-injected mice led to significant increase in absolute number of Forkhead helix transcription factor p3+ T regulatory cells in liver. We were surprised to find that select cannabinoid receptor (CB1 or CB2) agonists were not able to block hepatitis either independently or in combination. However, CB1/CB2 mixed agonists were able to efficiently attenuate hepatitis similar to THC. The modulatory effect of THC in ConA-induced hepatitis was reversed by both CB1 and CB2 antagonists. We also observed that endogenous cannabinoid anandamide was able to reduce hepatitis by suppressing cytokine levels. In addition, deficiency or inhibition of endocannabinoid hydrolyzing enzyme fatty acid amide hydrolase (FAAH), which leads to increased levels of endogenous cannabinoids, resulted in decreased liver injury upon ConA challenge. Our data demonstrate that targeting cannabinoid receptors using exogenous or endogenous cannabinoids and use of FAAH inhibitors may constitute novel therapeutic modalities to treat immune-mediated liver inflammation.