The Unfolded Protein Response Mediator PERK Governs Myeloid Cell-Driven Immunosuppression in Tumors through Inhibition of STING Signaling.

The primary mechanisms supporting immunoregulatory polarization of myeloid cells upon infiltration into tumors remain largely unexplored. Elucidation of these signals could enable better strategies to restore protective anti-tumor immunity. Here, we investigated the role of the intrinsic activation of the PKR-like endoplasmic reticulum (ER) kinase (PERK) in the immunoinhibitory actions of tumor-associated myeloid-derived suppressor cells (tumor-MDSCs). PERK signaling increased in tumor-MDSCs, and its deletion transformed MDSCs into myeloid cells that activated CD8+ T cell-mediated immunity against cancer. Tumor-MDSCs lacking PERK exhibited disrupted NRF2-driven antioxidant capacity and impaired mitochondrial respiratory homeostasis. Moreover, reduced NRF2 signaling in PERK-deficient MDSCs elicited cytosolic mitochondrial DNA elevation and, consequently, STING-dependent expression of anti-tumor type I interferon. Reactivation of NRF2 signaling, conditional deletion of STING, or blockade of type I interferon receptor I restored the immunoinhibitory potential of PERK-ablated MDSCs. Our findings demonstrate the pivotal role of PERK in tumor-MDSC functionality and unveil strategies to reprogram immunosuppressive myelopoiesis in tumors to boost cancer immunotherapy.

Accelerating the development of genetically engineered cellular therapies: a framework for extrapolating data across related products.

BACKGROUND: Significant advancements have been made in the field of cellular therapy as anti-cancer treatments, with the approval of chimeric antigen receptor (CAR)-T cell therapies and the development of other genetically engineered cellular therapies. CAR-T cell therapies have demonstrated remarkable clinical outcomes in various hematological malignancies, establishing their potential to change the current cancer treatment paradigm. Due to the increasing importance of genetically engineered cellular therapies in the oncology treatment landscape, implementing strategies to expedite development and evidence generation for the next generation of cellular therapy products can have a positive impact on patients. METHODS: We outline a risk-based methodology and assessment aid for the data extrapolation approach across related genetically engineered cellular therapy products. This systematic data extrapolation approach has applicability beyond CAR-T cells and can influence clinical development strategies for a variety of immune therapies such as T cell receptor (TCR) or genetically engineered and other cell-based therapies (e.g., tumor infiltrating lymphocytes, natural killer cells and macrophages). RESULTS: By analyzing commonalities in manufacturing processes, clinical trial designs, and regulatory considerations, key learnings were identified. These insights support optimization of the development and regulatory approval of novel cellular therapies. CONCLUSIONS: The field of cellular therapy holds immense promise in safely and effectively treating cancer. The ability to extrapolate data across related products presents opportunities to streamline the development process and accelerate the delivery of novel therapies to patients.

Epigenetic reprogramming of tumor cell-intrinsic STING function sculpts antigenicity and T cell recognition of melanoma.

Lack or loss of tumor antigenicity represents one of the key mechanisms of immune escape and resistance to T cell-based immunotherapies. Evidence suggests that activation of stimulator of interferon genes (STING) signaling in tumor cells can augment their antigenicity by triggering a type I IFN-mediated sequence of autocrine and paracrine events. Although suppression of this pathway in melanoma and other tumor types has been consistently reported, the mechanistic basis remains unclear. In this study, we asked whether this suppression is, in part, epigenetically regulated and whether it is indeed a driver of melanoma resistance to T cell-based immunotherapies. Using genome-wide DNA methylation profiling, we show that promoter hypermethylation of cGAS and STING genes mediates their coordinated transcriptional silencing and contributes to the widespread impairment of the STING signaling function in clinically-relevant human melanomas and melanoma cell lines. This suppression is reversible through pharmacologic inhibition of DNA methylation, which can reinstate functional STING signaling in at least half of the examined cell lines. Using a series of T cell recognition assays with HLA-matched human melanoma tumor-infiltrating lymphocytes (TIL), we further show that demethylation-mediated restoration of STING signaling in STING-defective melanoma cell lines can improve their antigenicity through the up-regulation of MHC class I molecules and thereby enhance their recognition and killing by cytotoxic T cells. These findings not only elucidate the contribution of epigenetic processes and specifically DNA methylation in melanoma-intrinsic STING signaling impairment but also highlight their functional significance in mediating tumor-immune evasion and resistance to T cell-based immunotherapies.

SARS-CoV-2 Period Seroprevalence and Related Factors, Hillsborough County, Florida, USA, October 2020-March 2021.

Estimating the actual extent of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is challenging because virus test positivity data undercount the actual number and proportion of persons infected. SARS-CoV-2 seroprevalence is a marker of past SARS-CoV-2 infection regardless of presence or severity of symptoms and therefore is a robust biomarker of infection period prevalence. We estimated SARS-CoV-2 seroprevalence among residents of Hillsborough County, Florida, USA, to determine factors independently associated with SARS-CoV-2 antibody status overall and among asymptomatic antibody-positive persons. Among 867 participants, SARS-CoV-2 period prevalence (October 2020-March 2021) was 19.5% (asymptomatic seroprevalence was 8%). Seroprevalence was 2-fold higher than reported SARS-CoV-2 virus test positivity. Factors related to social distancing (e.g., essential worker status, not practicing social distancing, contact with a virus-positive person, and length of contact exposure time) were consistently associated with seroprevalence but did not differ by time since suspected or known infection (<6 months vs. >6 months).

Intratumoral Activation of 41BB Costimulatory Signals Enhances CD8 T Cell Expansion and Modulates Tumor-Infiltrating Myeloid Cells.

The activation of 41BB costimulatory signals by agonistic Abs enhances the expansion and function of tumor-infiltrating lymphocytes (TILs) for treating cancer patients with adoptive cell therapy. However, the impact of 41BB agonism is not limited to enhancing the activity of T cells, and the mechanism by which additional activation of this costimulatory axis in tumor-associated myeloid cells is poorly understood. In this study, we describe that the intratumoral administration of 41BB agonistic Abs led to increases in CD8 T cell infiltration followed by tumor regression in murine models. We found that granulocytes and monocytes rapidly replaced macrophages and dendritic cells in tumors following administration of anti-41BB Abs. Overall, myeloid cells from anti-41BB-treated tumors had an improved capacity to stimulate T cells in comparison with control-treated tumors. In human coculture systems, we demonstrated that the agonism of the 41BB-41BBL axis enhanced costimulatory signals and effector functions among APC and autologous TILs. Overall, these findings suggest that the effect of 41BB agonistic Abs are supported by additional costimulatory signals from tumor-associated myeloid cells,v leading to enhanced TIL expansion and function.

Diversity Outbred Mice Reveal the Quantitative Trait Locus and Regulatory Cells of HER2 Immunity.

The genetic basis and mechanisms of disparate antitumor immune response was investigated in Diversity Outbred (DO) F1 mice that express human HER2. DO mouse stock samples nearly the entire genetic repertoire of the species. We crossed DO mice with syngeneic HER2 transgenic mice to study the genetics of an anti-self HER2 response in a healthy outbred population. Anti-HER2 IgG was induced by Ad/E2TM or naked pE2TM, both encoding HER2 extracellular and transmembrane domains. The response of DO F1 HER2 transgenic mice was remarkably variable. Still, immune sera inhibited HER2+ SKBR3 cell survival in a dose-dependent fashion. Using DO quantitative trait locus (QTL) analysis, we mapped the QTL that influences both total IgG and IgG2(a/b/c) Ab response to either Ad/E2TM or pE2TM. QTL from these four datasets identified a region in chromosome 17 that was responsible for regulating the response. A/J and NOD segments of genes in this region drove elevated HER2 Ig levels. This region is rich in MHC-IB genes, several of which interact with inhibitory receptors of NK cells. (B6xA/J)F1 and (B6xNOD)F1 HER2 transgenic mice received Ad/E2TM after NK cell depletion, and they produced less HER2 IgG, demonstrating positive regulatory function of NK cells. Depletion of regulatory T cells enhanced response. Using DO QTL analysis, we show that MHC-IB reactive NK cells exert positive influence on the immunity, countering negative regulation by regulatory T cells. This new, to our knowledge, DO F1 platform is a powerful tool for revealing novel immune regulatory mechanisms and for testing new interventional strategies.

Th1 cytokine interferon gamma improves response in HER2 breast cancer by modulating the ubiquitin proteasomal pathway.

HER2 breast cancer (BC) remains a significant problem in patients with locally advanced or metastatic BC. We investigated the relationship between T helper 1 (Th1) immune response and the proteasomal degradation pathway (PDP), in HER2-sensitive and -resistant cells. HER2 overexpression is partially maintained because E3 ubiquitin ligase Cullin5 (CUL5), which degrades HER2, is frequently mutated or underexpressed, while the client-protective co-chaperones cell division cycle 37 (Cdc37) and heat shock protein 90 (Hsp90) are increased translating to diminished survival. The Th1 cytokine interferon (IFN)-γ caused increased CUL5 expression and marked dissociation of both Cdc37 and Hsp90 from HER2, causing significant surface loss of HER2, diminished growth, and induction of tumor senescence. In HER2-resistant mammary carcinoma, either IFN-γ or Th1-polarizing anti-HER2 vaccination, when administered with anti-HER2 antibodies, demonstrated increased intratumor CUL5 expression, decreased surface HER2, and tumor senescence with significant therapeutic activity. IFN-γ synergized with multiple HER2-targeted agents to decrease surface HER2 expression, resulting in decreased tumor growth. These data suggest a novel function of IFN-γ that regulates HER2 through the PDP pathway and provides an opportunity to impact HER2 responses through anti-tumor immunity.

Reactive myelopoiesis and the onset of myeloid-mediated immune suppression: Implications for adoptive cell therapy.

Adoptive T cell therapy (ACT) in combination with lymphodepleting chemotherapy is an effective strategy to induce the eradication of cancer, providing long-term regressions in patients. However, only a minority of patients that receive ACT with tumor-specific T cells exhibit durable benefit. Thus, there is an urgent need to characterize mechanisms of resistance and define strategies to alleviate immunosuppression in the context of ACT in cancer. This article reviews the importance of lymphodepleting regimens in promoting the optimal engraftment and expansion of T cells in hosts after adoptive transfer. In addition, we discuss the role of concomitant immunosuppression and the accumulation of myeloid derived suppressor cells (MDSCs) during immune recovery after lymphodepleting regimens and mobilization regimens.

Intralesional injection of rose bengal augments the efficacy of gemcitabine chemotherapy against pancreatic tumors.

BACKGROUND: Chemotherapy regimens that include the utilization of gemcitabine are the standard of care in pancreatic cancer patients. However, most patients with advanced pancreatic cancer die within the first 2 years after diagnosis, even when treated with standard of care chemotherapy. This study aims to explore combination therapies that could boost the efficacy of standard of care regimens in pancreatic cancer patients. METHODS: In this study, we used PV-10, a 10% solution of rose bengal, to induce the death of human pancreatic tumor cells in vitro. Murine in vivo studies were carried out to examine the effectiveness of the direct injection of PV-10 into syngeneic pancreatic tumors in causing lesion-specific ablation. Intralesional PV-10 treatment was combined with systemic gemcitabine treatment in tumor-bearing mice to investigate the control of growth among treated tumors and distal uninjected tumors. The involvement of the immune-mediated clearance of tumors was examined in immunogenic tumor models that express ovalbumin (OVA). RESULTS: In this study, we demonstrate that the injection of PV-10 into mouse pancreatic tumors caused lesion-specific ablation. We show that the combination of intralesional PV-10 with the systemic administration of gemcitabine caused lesion-specific ablation and delayed the growth of distal uninjected tumors. We observed that this treatment strategy was markedly more successful in immunogenic tumors that express the neoantigen OVA, suggesting that the combination therapy enhanced the immune clearance of tumors. Moreover, the regression of tumors in mice that received PV-10 in combination with gemcitabine was associated with the depletion of splenic CD11b+Gr-1+ cells and increases in damage associated molecular patterns HMGB1, S100A8, and IL-1α. CONCLUSIONS: These results demonstrate that intralesional therapy with PV-10 in combination with gemcitabine can enhance anti-tumor activity against pancreatic tumors and raises the potential for this strategy to be used for the treatment of patients with pancreatic cancer.

Reactive Myelopoiesis Triggered by Lymphodepleting Chemotherapy Limits the Efficacy of Adoptive T Cell Therapy.

Adoptive T cell therapy (ACT) in combination with lymphodepleting chemotherapy is an effective strategy to induce the eradication of tumors, providing long-term regression in cancer patients. Despite that lymphodepleting regimens condition the host for optimal engraftment and expansion of adoptively transferred T cells, lymphodepletion concomitantly promotes immunosuppression during the course of endogenous immune recovery. In this study, we have identified that lymphodepleting chemotherapy initiates the mobilization of hematopoietic progenitor cells that differentiate to immunosuppressive myeloid cells, leading to a dramatic increase of peripheral myeloid-derived suppressor cells (MDSCs). In melanoma and lung cancer patients, MDSCs rapidly expanded in the periphery within 1 week after completion of a lymphodepleting regimen and infusion of autologous tumor-infiltrating lymphocytes (TILs). This expansion was associated with disease progression, poor survival, and reduced TIL persistence in melanoma patients. We demonstrated that the interleukin 6 (IL-6)-driven differentiation of mobilized hematopoietic progenitor cells promoted the survival and immunosuppressive capacity of post-lymphodepletion MDSCs. Furthermore, the genetic abrogation or therapeutic inhibition of IL-6 in mouse models enhanced host survival and reduced tumor growth in mice that received ACT. Thus, the expansion of MDSCs in response to lymphodepleting chemotherapy may contribute to ACT failure, and targeting myeloid-mediated immunosuppression may support anti-tumor immune responses.

Correction: Acidity promotes tumour progression by altering macrophage phenotype in prostate cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Anti-tumor efficacy of plasmid encoding emm55 in a murine melanoma model.

Emm55 is a bacterial gene derived from Streptococcus pyogenes (S. pyogenes) that was cloned into a plasmid DNA vaccine (pAc/emm55). In this study, we investigated the anti-tumor efficacy of pAc/emm55 in a B16 murine melanoma model. Intralesional (IL) injections of pAc/emm55 significantly delayed tumor growth compared to the pAc/Empty group. There was a significant increase in the CD8+ T cells infiltrating into the tumors after pAc/emm55 treatment compared to the control group. In addition, we observed that IL injection of pAc/emm55 increased antigen-specific T cell infiltration into tumors. Depletion of CD4+ or CD8+ T cells abrogated the anti-tumor effect of pAc/emm55. Combination treatment of IL injection of pAc/emm55 with anti-PD-1 antibody significantly delayed tumor growth compared to either monotherapy. pAc/emm55 treatment combined with PD-1 blockade enhanced anti-tumor immune response and improved systemic anti-tumor immunity. Together, these strategies may lead to improvements in the treatment of patients with melanoma.

Acidity promotes tumour progression by altering macrophage phenotype in prostate cancer.

BACKGROUND: Tumours rapidly ferment glucose to lactic acid even in the presence of oxygen, and coupling high glycolysis with poor perfusion leads to extracellular acidification. We hypothesise that acidity, independent from lactate, can augment the pro-tumour phenotype of macrophages. METHODS: We analysed publicly available data of human prostate cancer for linear correlation between macrophage markers and glycolysis genes. We used zwitterionic buffers to adjust the pH in series of in vitro experiments. We then utilised subcutaneous and transgenic tumour models developed in C57BL/6 mice as well as computer simulations to correlate tumour progression with macrophage infiltration and to delineate role of acidity. RESULTS: Activating macrophages at pH 6.8 in vitro enhanced an IL-4-driven phenotype as measured by gene expression, cytokine profiling, and functional assays. These results were recapitulated in vivo wherein neutralising intratumoural acidity reduced the pro-tumour phenotype of macrophages, while also decreasing tumour incidence and invasion in the TRAMP model of prostate cancer. These results were recapitulated using an in silico mathematical model that simulate macrophage responses to environmental signals. By turning off acid-induced cellular responses, our in silico mathematical modelling shows that acid-resistant macrophages can limit tumour progression. CONCLUSIONS: This study suggests that tumour acidity contributes to prostate carcinogenesis by altering the state of macrophage activation.

Tumor-infiltrating lymphocytes: Streamlining a complex manufacturing process.

Adoptive cell therapy of tumor-infiltrating lymphocytes has shown promise for treatment of refractory melanoma and other solid malignancies; however, challenges to manufacturing have limited its widespread use. Traditional manufacturing efforts were lengthy, cumbersome and used open culture systems. We describe changes in testing and manufacturing that decreased the process cycle time, enhanced the robustness of critical quality attribute testing and facilitated a functionally closed system. These changes have enabled export of the manufacturing process to support multi-center clinical trials.

Cost-effectiveness of measles control during elimination in Ontario, Canada, 2015.

BackgroundGiven that measles is eliminated in Canada and measles immunisation coverage in Ontario is high, it has been questioned whether Ontario's measles outbreak response is worthwhile.AimOur objective was to determine cost-effectiveness of measles containment protocols in Ontario from the healthcare payer perspective.MethodsWe developed a decision-analysis model comparing Ontario's measles containment strategy (based on actual 2015 outbreak data) with a hypothetical 'modified response'. The modified scenario assumed 10% response costs with reduced case and contact tracing and no outbreak-associated vaccinations; it was based on local and provincial administrative and laboratory data and parameters from peer-reviewed literature. Short- and long-term health outcomes, quality-adjusted life years (QALYs) and costs discounted at 1.5%, were estimated. We conducted one- and two-way sensitivity analyses.ResultsThe 2015 outbreak in Ontario comprised 16 measles cases and an estimated 3,369 contacts. Predictive modelling suggested that the outbreak response prevented 16 outbreak-associated cases at a cost of CAD 1,213,491 (EUR 861,579). The incremental cost-effectiveness ratio was CAD 739,063 (EUR 524,735) per QALY gained for the outbreak response vs modified response. To meet the commonly accepted cost-effectiveness threshold of CAD 50,000 (EUR 35,500) per QALY gained, the outbreak response would have to prevent 94 measles cases. In sensitivity analyses, the findings were robust.ConclusionsOntario's measles outbreak response exceeds generally accepted cost-effectiveness thresholds and may not be the most efficient use of public health resources from a healthcare payer perspective. These findings should be balanced against benefits of increased vaccine coverage and maintaining elimination status.

Hypoxia and acidosis: immune suppressors and therapeutic targets.

Due to imbalances between vascularity and cellular growth patterns, the tumour microenvironment harbours multiple metabolic stressors including hypoxia and acidosis, which have significant influences on remodelling both tumour and peritumoral tissues. These stressors are also immunosuppressive and can contribute to escape from immune surveillance. Understanding these effects and characterizing the pathways involved can identify new targets for therapy and may redefine our understanding of traditional anti-tumour therapies. In this review, the effects of hypoxia and acidosis on tumour immunity will be summarized, and how modulating these parameters and their sequelae can be a useful tool for future therapeutic interventions is discussed.

Neurofibromatosis type 1: A neuro-psycho-cutaneous syndrome?

Neurofibromatosis type 1 (NF1) is an inherited disorder often associated with optic nerve gliomas, low-grade brain tumors, and readily visible signs. Though these features are frequently emphasized, the psychosocial and emotional morbidities are often overlooked. We present a patient with depressive disorder resulting in suicide in a patient with NF1.

Measles Outbreak with Unique Virus Genotyping, Ontario, Canada, 2015.

The province of Ontario continues to experience measles virus transmissions despite the elimination of measles in Canada. We describe an unusual outbreak of measles in Ontario, Canada, in early 2015 that involved cases with a unique strain of virus and no known association among primary case-patients. A total of 18 cases of measles were reported from 4 public health units during the outbreak period (January 25-March 23, 2015); none of these cases occurred in persons who had recently traveled. Despite enhancements to case-patient interview methods and epidemiologic analyses, a source patient was not identified. However, the molecular epidemiologic analysis, which included extended sequencing, strongly suggested that all cases derived from a single importation of measles virus genotype D4. The use of timely genotype sequencing, rigorous epidemiologic investigation, and a better understanding of the gaps in surveillance are needed to maintain Ontario's measles elimination status.

Metals exposure and risk of small-for-gestational age birth in a Canadian birth cohort: The MIREC study.

BACKGROUND: Lead, mercury, cadmium and arsenic are some of the most common toxic metals to which Canadians are exposed. The effect of exposure to current low levels of toxic metals on fetal growth restriction is unknown. OBJECTIVE: The aim of this study was to examine relationships between exposure to lead, mercury, cadmium and arsenic during pregnancy, and risk of small for gestational age (SGA) birth. METHODS: Lead, mercury, cadmium and arsenic levels were measured in blood samples from the first and third trimesters in 1835 pregnant women from across Canada. Arsenic species in first trimester urine were also assessed. Relative risks and 95% confidence intervals were estimated using log binomial multivariate regression. Important covariates including maternal age, parity, pre-pregnancy BMI, and smoking, were considered in the analysis. An exploratory analysis was performed to examine potential effect modification of these relationships by single nucleotide polymorphisms (SNPs) in GSTP1 and GSTO1 genes. RESULTS: No association was found between blood lead, cadmium or arsenic and risk for SGA. We observed an increased risk for SGA for the highest compared to the lowest tertile of exposure for mercury (>1.6 µg/L, RR=1.56.; 95% CI=1.04-2.58) and arsenobetaine (>2.25 µg/L, RR=1.65; 95% CI=1.10-2.47) after adjustment for the effects of parity and smoking. A statistically significant interaction was observed in the relationship between dimethylarsinic acid (DMA) levels in urinary arsenic and SGA between strata of GSTO1 A104A (p for interaction=0.02). A marginally significant interaction was observed in the relationship between blood lead and SGA between strata of GSTP1 A114V (p for interaction=0.06). CONCLUSIONS: These results suggest a small increase in risk for SGA in infants born to women exposed to mercury and arsenic. Given the conflicting evidence in the literature this warrants further investigation in other pregnant populations.

Blockade of myeloid-derived suppressor cells after induction of lymphopenia improves adoptive T cell therapy in a murine model of melanoma.

Administration of nonmyeloablative chemotherapeutic agents or total body irradiation (TBI) prior to adoptive transfer of tumor-specific T cells may reduce or eliminate immunosuppressive populations such as T regulatory cells (Tregs) and myeloid-derived suppressor cells (MDSC). Little is known about these populations during immune reconstitution. This study was designed to understand the reconstitution rate and function of these populations post TBI in melanoma tumor-bearing mice. Reconstitution rate and suppressive activity of CD4(+)CD25(+)Foxp3(+) Tregs and CD11b(+)Gr1(+) MDSC following TBI-induced lymphopenia was measured in B16 melanoma tumor-bearing mice. To ablate the rapid reconstitution of suppressive populations, we treated mice with docetaxel, a known chemotherapeutic agent that targets MDSC, in combination with adoptive T cell transfer and dendritic cell immunotherapy. Both Treg and MDSC populations exhibited rapid reconstitution after TBI-induced lymphopenia. Although reconstituted Tregs were just as suppressive as Tregs from untreated mice, MDSC demonstrated enhanced suppressive activity of CD8(+) T cell proliferation compared with endogenous MDSC from tumor-bearing mice. TBI-induced lymphopenia followed by docetaxel treatment improved the efficacy of adoptive T cell transfer and dendritic cell immunotherapy in melanoma-bearing mice, inducing a significant reduction in tumor growth and enhancing survival. Tumor regression correlated with increased CTL activity and persistence of adoptively transferred T cells. Overall, these findings suggest that TBI-induced MDSC are highly immunosuppressive and blocking their rapid reconstitution may improve the efficacy of vaccination strategies and adoptive immunotherapy.