T cell–derived inducible nitric oxide synthase switches off TH17 cell differentiation

RORγt is necessary for the generation of TH17 cells but the molecular mechanisms for the regulation of TH17 cells are still not fully understood. We show that activation of CD4+ T cells results in the expression of inducible nitric oxide synthase (iNOS). iNOS-deficient mice displayed enhanced TH17 cell differentiation but without major effects on either TH1 or TH2 cell lineages, whereas endothelial NOS (eNOS) or neuronal NOS (nNOS) mutant mice showed comparable TH17 cell differentiation compared with wild-type control mice. The addition of N6-(1-iminoethyl)-l-lysine dihydrochloride (L-NIL), the iNOS inhibitor, significantly enhanced TH17 cell differentiation, and S-nitroso-N-acetylpenicillamine (SNAP), the NO donor, dose-dependently reduced the percentage of IL-17–producing CD4+ T cells. NO mediates nitration of tyrosine residues in RORγt, leading to the suppression of RORγt-induced IL-17 promoter activation, indicating that NO regulates IL-17 expression at the transcriptional level. Finally, studies of an experimental model of colitis showed that iNOS deficiency results in more severe inflammation with an enhanced TH17 phenotype. These results suggest that NO derived from iNOS in activated T cells plays a negative role in the regulation of TH17 cell differentiation and highlight the importance of intrinsic programs for the control of TH17 immune responses

Tumor microenvironment modulation enhances immunologic benefit of chemoradiotherapy

Background Chemoradiotherapy (CRT) remains one of the most common cancer treatment modalities, and recent data suggest that CRT is maximally effective when there is generation of an anti-tumoral immune response. However, CRT has also been shown to promote immunosuppressive mechanisms which must be blocked or reversed to maximize its immune stimulating effects. Methods Therefore, using a preclinical model of human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC), we developed a clinically relevant therapy combining CRT and two existing immunomodulatory drugs: cyclophosphamide (CTX) and the small molecule inducible nitric oxide synthase (iNOS) inhibitor L-n6-(1-iminoethyl)-lysine (L-NIL). In this model, we treated the syngeneic HPV-HNSCC mEER tumor-bearing mice with fractionated (10 fractions of 3 Gy) tumor-directed radiation and weekly cisplatin administration. We compared the immune responses induced by CRT and those induced by combinatory treatment (CRT + CTX/L-NIL) with flow cytometry, quantitative multiplex immunofluorescence and by profiling immune-related gene expression changes. Results We show that combination treatment favorably remodels the tumor myeloid immune microenvironment including an increase in anti-tumor immune cell types (inflammatory monocytes and M1-like macrophages) and a decrease in immunosuppressive granulocytic myeloid-derived suppressor cells (MDSCs). Intratumoral T cell infiltration and tumor antigen specificity of T cells were also improved, including a 31.8-fold increase in the CD8+ T cell/ regulatory T cell ratio and a significant increase in tumor antigen-specific CD8+ T cells compared to CRT alone. CTX/LNIL immunomodulation was also shown to significantly improve CRT efficacy, leading to rejection of 21% established tumors in a CD8-dependent manner. Conclusions Overall, these data show that modulation of the tumor immune microenvironment with CTX/L-NIL enhances susceptibility of treatment-refractory tumors to CRT. The combination of tumor immune microenvironment modulation with CRT constitutes a translationally relevant approach to enhance CRT efficacy through enhanced immune activation

Induction of SARS-CoV-2 Protein S-Specific CD8+ T Cells in the Lungs of gp96-Ig-S Vaccinated Mice

Given the aggressive spread of COVID-19-related deaths, there is an urgent public health need to support the development of vaccine candidates to rapidly improve the available control measures against SARS-CoV-2. To meet this need, we are leveraging our existing vaccine platform to target SARS-CoV-2. Here, we generated cellular heat shock chaperone protein, glycoprotein 96 (gp96), to deliver SARS-CoV-2 protein S (spike) to the immune system and to induce cell-mediated immune responses. We showed that our vaccine platform effectively stimulates a robust cellular immune response against protein S. Moreover, we confirmed that gp96-Ig, secreted from allogeneic cells expressing full-length protein S, generates powerful, protein S polyepitope-specific CD4+ and CD8+ T cell responses in both lung interstitium and airways. These findings were further strengthened by the observation that protein-S -specific CD8+ T cells were induced in human leukocyte antigen HLA-A2.1 transgenic mice thus providing encouraging translational data that the vaccine is likely to work in humans, in the context of SARS-CoV-2 antigen presentation

Induction of SARS-CoV-2 protein S-specific CD8+ T cells in the lungs of gp96-Ig-S vaccinated mice

Given the aggressive spread of COVID-19-related deaths, there is an urgent public health need to support the development of vaccine candidates to rapidly improve the available control measures against SARS-CoV-2. To meet this need, we are leveraging our existing vaccine platform to target SARS-CoV-2. Here, we generated cellular heat shock chaperone protein, glycoprotein 96 (gp96), to deliver SARS-CoV-2 protein S (spike) to the immune system and to induce cell-mediated immune responses. We showed that our vaccine platform effectively stimulates a robust cellular immune response against protein S. Moreover, we confirmed that gp96-Ig, secreted from allogeneic cells expressing full-length protein S, generates powerful, protein S polyepitope-specific CD4+ and CD8+ T cell responses in both lung interstitium and airways. These findings were further strengthened by the observation that protein-S -specific CD8+ T cells were induced in human leukocyte antigen (HLA)-A2-02-01 transgenic mice thus providing encouraging translational data that the vaccine is likely to work in humans, in the context of SARS-CoV-2 antigen presentation. Competing Interest Statement NS is inventor on the patent application No 62/983,783 entitled Immune-mediated coronavirus treatments; NS is a member of Heat Biologics COVID-19 Advisory Board. MMS is the Executive Director of Special Projects. PJ is the Associate Director of Business Development, both are employed by Heat Biologics, Inc. RJ is the CEO of Pelican Therapeutics, a subsidiary of Heat Biologics, Inc. MSS, PJ, RJ, and KP hold stock options in Heat Biologics, Inc

Formulation, characterization and evaluation of gelatin-syringic acid/zinc oxide nanocomposite for its effective anticancer, antioxidant and anti-inflammatory activities

Background: Hepatocellular carcinoma (HCC) is the most typical form of liver cancer. Apart from modern therapies, various natural chemical constituents have been tested for the treatment of HCC. However, the usage of the latter has declined due to their low bioavailability and stability. Objectives: Considering the above drawback’s, in this study, we describe a gelatin-syringic acid/ zinc oxide (ZnO) nanocomposite for liver cancer treatment, which showed antioxidant, anti-inflammatory and anticancer activities. Methods: The hydrothermal method was used to synthesize ZnO nanoparticle and it was encapsulated with gelatin-syringic acid by coacervation technique. The nanocomposites were characterized by UV spec, FTIR, SEM, XRD, EDX, and DLS. The drug release profile of nanocomposite was studied by dialysis bag method, which exhibits a sustained drug release. Results: The antioxidant ability of nano-composite was studied by performing an ABTS and DPPH assay and the IC50 was recorded as 76.5 and 47.63 µg/mL, respectively. The anti-inflammatory potential was studied by assessing the ability of nanocomposite on denaturation of protein and the results exhibited a dose-dependent inhibition. The acute toxicity of nanocomposite tested on zebrafish liver and heart showed that it is non-toxic. Moreover, the nanocomposite inhibits the Hep G2 cell viability with an increasing concentration and it increases oxidative stress caused by the mitochondrial damage, which leads to cell death. Conclusion: Based on the findings of the present study, gelatin-syringic acid/ZnO nanocomposite has been shown to possess antioxidant, and anti-inflammatory properties, and thus can be used against HCC

Secreted heat shock protein gp96-Ig and OX40L-Fc combination vaccine enhances SARS-CoV-2 Spike (S) protein-specific B and T cell immune responses

A cell-based vaccine co-secreting gp96-Ig, protein S1 and OX40L-Fc leads to activation of dendritic cells (DC) and cross-presentation of gp96-chaperoned protein S peptides to CD8+ T cells. Co-secretion of OX40L-Fc further augments CD8+T cell activity through OX40/OX40L axis. Secreted protein S1 is endocytosed by activated DC and S1 peptides are presented within the MHC class II molecules to TFH cells that also receive co-stimulatory signals through OX40L-Fc. In addition, B cells recognize secreted protein S1 through BCR and present S1 peptides within the MHC class II molecules to TFH cells. TFH cells through OX40/OX40L axis provide essential signals to promote B cell differentiation into memory B cells and long-lived plasma cells, and secretion of high-affinity specific antibodies. Local modulation of the gp96-Ig vaccine microenvironment by OX40L-Fc has the advantage to enhance immunological protection through providing additional support to CD8+ T cell and immunoglobulin antiviral response in the lungs and simplifying the clinical translation of such combination immunotherapies into humans. [Display omitted] •gp96-Ig-S-OX40L-Fc vaccine enhances S-specific IgG responses.•gp96-Ig-S-OX40L-Fc vaccine enhances TFH cell responses.•gp96-Ig-S-OX40L-Fc vaccine enhances lungs S-specific CD8 + T cell responses. Encouraging protection results from current mRNA-based SARS-CoV-2 vaccine platforms are primarily due to the induction of SARS- CoV-2- specific B cell antibody and CD4 + T cell. Even though, current mRNA vaccine platforms are adept in inducing SARS-CoV2-specific CD8 + T cell, much less is known about CD8 T cells contribution to the overall vaccine protection. Our allogeneic cellular vaccine, based on a secreted form of the heat-shock protein gp96-Ig, achieves high frequencies of polyclonal CD8 + T cell responses to tumor and infectious antigens through antigen cross-priming in vivo. We and others have shown that gp96-Ig, in addition to antigen-specific CD8 + T cell anti-tumor and anti-pathogen immunity, primes antibody responses as well. Here, we generated a cell-based vaccine that expresses SARS-Cov-2 Spike (S) protein and simultaneously secretes gp96-Ig and OX40L-Fc fusion proteins. We show that co-secretion of gp96-Ig-S peptide complexes and the OX40L-Fc costimulatory fusion protein in allogeneic cell lines results in enhanced activation of S protein-specific IgG antibody responses. These findings were further strengthened by the observation that this vaccine platform induces T follicular helper cells (TFH) and protein-S -specific CD8 + T cells. Thus, a cell-based gp96-Ig vaccine/OX40-L fusion protein regimen provides encouraging translational data that this vaccine platform induces pathogen-specific CD8+, CD4 + T and B cell responses, and may cohesively work as a booster for FDA-approved vaccines. Our vaccine platform can be rapidly engineered and customized based on other current and future pathogen sequences