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.

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.

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.

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.

TGF-ß1 programmed myeloid-derived suppressor cells (MDSC) acquire immune-stimulating and tumor killing activity capable of rejecting established tumors in combination with radiotherapy.

Cancer-induced myeloid-derived suppressor cells (MDSC) play an important role in tumor immune evasion. MDSC programming or polarization has been proposed as a strategy for leveraging the developmental plasticity of myeloid cells to reverse MDSC immune suppressive functions, or cause them to acquire anti-tumor activity. While MDSC derived ex vivo from murine bone marrow precursor cells with tumor-conditioned medium efficiently suppressed T cell proliferation, MDSC derived from conditioned medium in presence of TGF-ß1 (TGFß-MDSC) acquired a novel immune-stimulatory phenotype, losing the ability to inhibit T cell proliferation and acquiring enhanced antigen-presenting capability. Altered immune function was associated with SMAD-2 dependent upregulation of maturation and costimulatory molecules, and downregulation of inducible nitric oxide synthase (iNOS), an effector mechanism of immunosuppression. TGFß-MDSC also upregulated FAS-ligand expression, leading to FAS-dependent killing of murine human papillomavirus (HPV)-associated head and neck cancer cells and tumor spheroids in vitro and anti-tumor activity in vivo. Radiation upregulated FAS expression on tumor cells, and the combination of radiotherapy and intratumoral injection of TGFß-MDSC strongly enhanced class I expression on tumor cells and induction of HPV E7 tetramer-positive CD8 + T cells, leading to clearance of established tumors and long-term survival. TGFß-MDSC derived from human PBMC with tumor conditioned medium also lost immunosuppressive function and acquired tumor-killing activity. Thus, TGFß1 mediated programming of nascent MDSC leads to a potent anti-tumor phenotype potentially suitable for adoptive immunotherapy.

Myeloid cell-derived inducible nitric oxide synthase suppresses M1 macrophage polarization.

Here we show that iNOS-deficient mice display enhanced classically activated M1 macrophage polarization without major effects on alternatively activated M2 macrophages. eNOS and nNOS mutant mice show comparable M1 macrophage polarization compared with wild-type control mice. Addition of N6-(1-iminoethyl)-L-lysine dihydrochloride, an iNOS inhibitor, significantly enhances M1 macrophage polarization while S-nitroso-N-acetylpenicillamine, a NO donor, suppresses M1 macrophage polarization. NO derived from iNOS mediates nitration of tyrosine residues in IRF5 protein, leading to the suppression of IRF5-targeted M1 macrophage signature gene activation. Computational analyses corroborate a circuit that fine-tunes the expression of IL-12 by iNOS in macrophages, potentially enabling versatile responses based on changing microenvironments. Finally, studies of an experimental model of endotoxin shock show that iNOS deficiency results in more severe inflammation with an enhanced M1 macrophage activation phenotype. These results suggest that NO derived from iNOS in activated macrophages suppresses M1 macrophage polarization.

Requirement for innate immunity and CD90⁺ NK1.1⁻ lymphocytes to treat established melanoma with chemo-immunotherapy.

We sought to define cellular immune mechanisms of synergy between tumor-antigen-targeted monoclonal antibodies and chemotherapy. Established B16 melanoma in mice was treated with cytotoxic doses of cyclophosphamide in combination with an antibody targeting tyrosinase-related protein 1 (αTRP1), a native melanoma differentiation antigen. We find that Fcγ receptors are required for efficacy, showing that antitumor activity of combination therapy is immune mediated. Rag1(-/-) mice deficient in adaptive immunity are able to clear tumors, and thus innate immunity is sufficient for efficacy. Furthermore, previously treated wild-type mice are not significantly protected against tumor reinduction, as compared with mice inoculated with irradiated B16 alone, consistent with a primarily innate immune mechanism of action of chemo-immunotherapy. In contrast, mice deficient in both classical natural killer (NK) lymphocytes and nonclassical innate lymphocytes (ILC) due to deletion of the IL2 receptor common gamma chain IL2γc(-/-)) are refractory to chemo-immunotherapy. Classical NK lymphocytes are not critical for treatment, as depletion of NK1.1⁺ cells does not impair antitumor effect. Depletion of CD90⁺NK1.1⁻ lymphocytes, however, both diminishes therapeutic benefit and decreases accumulation of macrophages within the tumor. Tumor clearance during combination chemo-immunotherapy with monoclonal antibodies against native antigen is mediated by the innate immune system. We highlight a novel potential role for CD90⁺NK1.1⁻ ILCs in chemo-immunotherapy.

iNOS expression in CD4+ T cells limits Treg induction by repressing TGFß1: combined iNOS inhibition and Treg depletion unmask endogenous antitumor immunity.

PURPOSE: Expression of inducible nitric oxide synthase (iNOS) in different cellular compartments may have divergent effects on immune function. We used a syngeneic tumor model to functionally characterize the role of iNOS in regulation of CD4(+)FOXP3(+) regulatory T cells (Treg), and optimize the beneficial effects of iNOS inhibition on antitumor immunity. EXPERIMENTAL DESIGN: Wild-type (WT) or iNOS knockout mice bearing established MT-RET-1 melanoma were treated with the small-molecule iNOS inhibitor L-NIL and/or cyclophosphamide alone or in combination. The effect of iNOS inhibition or knockout on induction of Treg from mouse and human CD4(+) T cells in ex vivo culture was determined in parallel in the presence or absence of TGFß1-depleting antibodies, and TGFß1 levels were assessed by ELISA. RESULTS: Whereas intratumoral myeloid-derived suppressor cells (MDSC) were suppressed by iNOS inhibition or knockout, systemic and intratumoral FOXP3(+) Treg levels increased in tumor-bearing mice. iNOS inhibition or knockout similarly enhanced induction of Treg from activated cultured mouse splenocytes or purified human or mouse CD4(+) T cells in a TGFß1-dependent manner. Although either iNOS inhibition or Treg depletion with low-dose cyclophosphamide alone had little effect on growth of established MT-RET1 melanoma, combination treatment potently inhibited MDSC and Treg, boosted tumor-infiltrating CD8(+) T-cell levels, and arrested tumor growth in an immune-dependent fashion. CONCLUSIONS: iNOS expression in CD4(+) T cells suppresses Treg induction by inhibiting TGFß1 production. Our data suggest that iNOS expression has divergent effects on induction of myeloid and lymphoid-derived regulatory populations, and strongly support development of combinatorial treatment approaches that target these populations simultaneously.

Inducible nitric oxide synthase drives mTOR pathway activation and proliferation of human melanoma by reversible nitrosylation of TSC2.

Melanoma is one of the cancers of fastest-rising incidence in the world. Inducible nitric oxide synthase (iNOS) is overexpressed in melanoma and other cancers, and previous data suggest that iNOS and nitric oxide (NO) drive survival and proliferation of human melanoma cells. However, specific mechanisms through which this occurs are poorly defined. One candidate is the PI3K-AKT-mTOR pathway, which plays a major role in proliferation, angiogenesis, and metastasis of melanoma and other cancers. We used the chick embryo chorioallantoic membrane (CAM) assay to test the hypothesis that melanoma growth is regulated by iNOS-dependent mTOR pathway activation. Both pharmacologic inhibition and siRNA-mediated gene silencing of iNOS suppressed melanoma proliferation and in vivo growth on the CAM in human melanoma models. This was associated with strong downregulation of mTOR pathway activation by Western blot analysis of p-mTOR, p70 ribosomal S6 kinase (p-P70S6K), p-S6RP, and p-4EBP1. iNOS expression and NO were associated with reversible nitrosylation of tuberous sclerosis complex (TSC) 2, and inhibited dimerization of TSC2 with its inhibitory partner TSC1, enhancing GTPase activity of its target Ras homolog enriched in brain (Rheb), a critical activator of mTOR signaling. Immunohistochemical analysis of tumor specimens from stage III melanoma patients showed a significant correlation between iNOS expression levels and expression of the mTOR pathway members. Exogenously supplied NO was also sufficient to reverse the mTOR pathway inhibition by the B-Raf inhibitor vemurafenib. In summary, covalent modification of TSC2 by iNOS-derived NO is associated with impaired TSC2/TSC1 dimerization, mTOR pathway activation, and proliferation of human melanoma. This model is consistent with the known association of iNOS overexpression and poor prognosis in melanoma and other cancers.

T cell­derived 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 T(H)17 cell differentiation but without major effects on either T(H)1 or T(H)2 cell lineages, whereas endothelial NOS (eNOS) or neuronal NOS (nNOS) mutant mice showed comparable T(H)17 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, dosedependently reduced the percentage of IL-17­producing 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 T(H)17 phenotype. These results suggest that NO derived from iNOS in activated T cells plays a negative role in the regulation of T(H)17 cell differentiation and highlight the importance of intrinsic programs for the control of T(H)17 immune responses.

Tumor-expressed inducible nitric oxide synthase controls induction of functional myeloid-derived suppressor cells through modulation of vascular endothelial growth factor release.

Inducible NO synthase (iNOS) is a hallmark of chronic inflammation that is also overexpressed in melanoma and other cancers. Whereas iNOS is a known effector of myeloid-derived suppressor cell (MDSC)-mediated immunosuppression, its pivotal position at the interface of inflammation and cancer also makes it an attractive candidate regulator of MDSC recruitment. We hypothesized that tumor-expressed iNOS controls MDSC accumulation and acquisition of suppressive activity in melanoma. CD11b(+)GR1(+) MDSC derived from mouse bone marrow cells cultured in the presence of MT-RET-1 mouse melanoma cells or conditioned supernatants expressed STAT3 and reactive oxygen species (ROS) and efficiently suppressed T cell proliferation. Inhibition of tumor-expressed iNOS with the small molecule inhibitor L-NIL blocked accumulation of STAT3/ROS-expressing MDSC, and abolished their suppressive function. Experiments with vascular endothelial growth factor (VEGF)-depleting Ab and recombinant VEGF identified a key role for VEGF in the iNOS-dependent induction of MDSC. These findings were further validated in mice bearing transplantable MT-RET-1 melanoma, in which L-NIL normalized elevated serum VEGF levels; downregulated activated STAT3 and ROS production in MDSC; and reversed tumor-mediated immunosuppression. These beneficial effects were not observed in iNOS knockout mice, suggesting L-NIL acts primarily on tumor- rather than host-expressed iNOS to regulate MDSC function. A significant decrease in tumor growth and a trend toward increased tumor-infiltrating CD8(+) T cells were also observed in MT-RET transgenic mice bearing spontaneous tumors. These data suggest a critical role for tumor-expressed iNOS in the recruitment and induction of functional MDSC by modulation of tumor VEGF secretion and upregulation of STAT3 and ROS in MDSC.

Targeted inhibition of inducible nitric oxide synthase inhibits growth of human melanoma in vivo and synergizes with chemotherapy.

PURPOSE: Aberrant expression of inflammatory molecules, such as inducible nitric oxide (NO) synthase (iNOS), has been linked to cancer, suggesting that their inhibition is a rational therapeutic approach. Whereas iNOS expression in melanoma and other cancers is associated with poor clinical prognosis, in vitro and in vivo studies suggest that iNOS and NO can have both protumor and antitumor effects. We tested the hypothesis that targeted iNOS inhibition would interfere with human melanoma growth and survival in vivo in a preclinical model. EXPERIMENTAL DESIGN: We used an immunodeficient non-obese diabetic/severe combined immunodeficient xenograft model to test the susceptibility of two different human melanoma lines to the orally-given iNOS-selective small molecule antagonist N(6)-(1-iminoethyl)-l-lysine-dihydrochloride (L-nil) with and without cytotoxic cisplatin chemotherapy. RESULTS: L-nil significantly inhibited melanoma growth and extended the survival of tumor-bearing mice. L-nil treatment decreased the density of CD31+ microvessels and increased the number of apoptotic cells in tumor xenografts. Proteomic analysis of melanoma xenografts with reverse-phase protein array identified alterations in the expression of multiple cell signaling and survival genes after L-nil treatment. The canonical antiapoptotic protein Bcl-2 was downregulated in vivo and in vitro after L-nil treatment, which was associated with increased susceptibility to cisplatin-mediated tumor death. Consistent with this observation, combination therapy with L-nil plus cisplatin in vivo was more effective than either drug alone, without increased toxicity. CONCLUSIONS: These data support the hypothesis that iNOS and iNOS-derived NO support tumor growth in vivo and provide convincing preclinical validation of targeted iNOS inhibition as therapy for solid tumors.

Unrestrained glycogen synthase kinase-3 beta activity leads to activated T cell death and can be inhibited by natural adjuvant.

Activated T cell death (ATCD) after peak clonal expansion is required for effective homeostasis of the immune system. Using a mouse model of T cell clonal expansion and contraction, we found that regulation of the proapoptotic kinase glycogen synthase kinase (GSK)-3beta plays a decisive role in determining the extent to which T cells are eliminated after activation. Involvement of GSK-3beta in ATCD was tested by measuring T cell survival after GSK-3beta inhibition, either ex vivo with chemical and pharmacological inhibitors or in vivo by retroviral expression of a dominant-negative form of GSK-3. We also measured amounts of inactivating phosphorylation of GSK-3beta (Ser9) in T cells primed in the presence or absence of LPS. Our results show that GSK-3beta activity is required for ATCD and that its inhibition promoted T cell survival. Adjuvant treatment in vivo maintained GSK-3beta (Ser9) phosphorylation in activated T cells, whereas with adjuvant-free stimulation it peaked and then decayed as the cells became susceptible to ATCD. We conclude that the duration of GSK-3beta inactivation determines activated T cell survival and that natural adjuvant stimulation decreases the severity of clonal contraction in part by keeping a critical proapoptotic regulatory factor, GSK-3beta, inactivated.