Costimulatory domains direct distinct fates of CAR-driven T-cell dysfunction.

T cells engineered to express chimeric antigen receptors (CARs) targeting CD19 have demonstrated impressive activity against relapsed or refractory B-cell cancers yet fail to induce durable remissions for nearly half of all patients treated. Enhancing the efficacy of this therapy requires detailed understanding of the molecular circuitry that restrains CAR-driven antitumor T-cell function. We developed and validated an in vitro model that drives T-cell dysfunction through chronic CAR activation and interrogated how CAR costimulatory domains, central components of CAR structure and function, contribute to T-cell failure. We found that chronic activation of CD28-based CARs results in activation of classical T-cell exhaustion programs and development of dysfunctional cells that bear the hallmarks of exhaustion. In contrast, 41BB-based CARs activate a divergent molecular program and direct differentiation of T cells into a novel cell state. Interrogation using CAR T cells from a patient with progressive lymphoma confirmed the activation of this novel program in a failing clinical product. Furthermore, we demonstrate that 41BB-dependent activation of the transcription factor FOXO3 is directly responsible for impairing CAR T-cell function. These findings identify that costimulatory domains are critical regulators of CAR-driven T-cell failure and that targeted interventions are required to overcome costimulation-dependent dysfunctional programs.

Contemporary Public Health Finance: Varied Definitions, Patterns, and Implications.

The financing of public health systems and services relies on a complex and fragmented web of partners and funding priorities. Both underfunding and "dys-funding" contribute to preventable mortality, increases in disease frequency and severity, and hindered social and economic growth. These issues were both illuminated and magnified by the COVID-19 pandemic and associated responses. Further complicating issues is the difficulty in constructing adequate estimates of current public health resources and necessary resources. Each of these challenges inhibits the delivery of necessary services, leads to inequitable access and resourcing, contributes to resource volatility, and presents other deleterious outcomes. However, actions may be taken to defragment complex funding paradigms toward more flexible spending, to modernize and standardize data systems, and to assure equitable and sustainable public health investments.

Seasonal trends in lysogeny in an Appalachian oak-hickory forest soil.

Since 1989, investigations into viral ecology have revealed how bacteriophages can influence microbial dynamics within ecosystems at global scales. Most of the information we know about temperate phages, which can integrate themselves into the host genome and remain dormant via a process called lysogeny, has come from research in aquatic ecosystems. Soil environments remain under-studied, and more research is necessary to fully understand the range of impacts phage infections have on the soil bacteria they infect. The aims of this study were to compare the efficacy of different prophage-inducing agents and to elucidate potential temporal trends in lysogeny within a soil bacterial community. In addition to mitomycin C and acyl-homoserine lactones, our results indicated that halosulfuron methyl herbicides may also be potent inducing agents. In optimizing chemical induction assays, we determined that taking steps to reduce background virus particles and starve cells was critical in obtaining consistent results. A clear seasonal trend in inducible lysogeny was observed in an Appalachian oak-hickory forest soil. The average monthly air temperature was negatively correlated with inducible fraction and burst size, supporting the idea that lysogeny provides a mechanism for phage persistence when temperatures are low and host metabolism is slower. Furthermore, the inducible fraction was negatively correlated with both soil bacterial and soil viral abundance, supporting the idea that lysogeny provides a mechanism for temperate phage persistence when host density is lower. The present study is the first of its kind to reveal clear seasonal trends in inducible lysogeny in any soil.IMPORTANCELysogeny is a relationship in which certain viruses that infect bacteria (phages) may exist within their bacterial host cell as a segment of nucleic acid. In this state, the phage genome is protected from environmental damage and retains the potential to generate progeny particles in the future. It is thought that lysogeny provides a mechanism for long-term persistence for phages when host density is low or hosts are starved-two conditions likely to be found in soils. In the present study, we provide the first known evidence for a seasonal trend in lysogeny in a forest soil. Based on clear relationships observed between lysogeny, temperature, and soil microbial abundance, we find support for previous hypotheses regarding the factors governing lysogeny.

Technology, data, people, and partnerships in addressing unmet social needs within Medicaid Managed Care.

BACKGROUND: Individuals with unmet social needs experience adverse health outcomes and are subject to greater inequities in health and social outcomes. Given the high prevalence of unmet needs among Medicaid enrollees, many Medicaid managed care organizations (MCOs) are now screening enrollees for unmet social needs and connecting them to community-based organizations (CBOs) with knowledge and resources to address identified needs. The use of screening and referral technology and data sharing are often considered key components in programs integrating health and social services. Despite this emphasis on technology and data collection, research suggests substantial barriers exist in operationalizing effective systems. METHODS: We used qualitative methods to examine cross-sector perspectives on the use of data and technology to facilitate MCO and CBO partnerships in Kentucky, a state with high Medicaid enrollment, to address enrollee social needs. We recruited participants through targeted sampling, and conducted 46 in-depth interviews with 26 representatives from all six Kentucky MCOs and 20 CBO leaders. Qualitative descriptive analysis, an inductive approach, was used to identify salient themes. RESULTS: We found that MCOs and CBOs have differing levels of need for data, varying incentives for collecting and sharing data, and differing valuations of what data can or should do. Four themes emerged from interviewees' descriptions of how they use data, including 1) to screen for patient needs, 2) to case manage, 3) to evaluate the effectiveness of programs, and 4) to partner with each other. Underlying these data use themes were areas of alignment between MCOs/CBOs, areas of incongruence, and areas of tension (both practical and ideological). The inability to interface with community partners for data privacy and ownership concerns contributes to division. Our findings suggest a disconnect between MCOs and CBOs regarding terms of their technology interfacing despite their shared mission of meeting the unmet social needs of enrollees. CONCLUSIONS: While data and technology can be used to identify enrollee needs and determine the most critical need, it is not sufficient in resolving challenges. People and relationships across sectors are vital in connecting enrollees with the community resources to resolve unmet needs.

Identifying Value-Added Population Health Capabilities to Strengthen Public Health Infrastructure.

Policy Points While the coronavirus pandemic has underscored the important role of public health systems in protecting community health, it has also exposed weaknesses in the public health infrastructure that stem from chronic underfunding and fragmentation in delivery systems. The results of our study suggest that the public health system structure can be strengthened through the targeted implementation of high-value population health capabilities. Prioritizing the delivery of value-added population health capabilities can help communities efficiently use limited time and resources and identify the most effective pathways for building a stronger public health system and improving health outcomes over time. CONTEXT: While the novel coronavirus pandemic has underscored the important role of public health systems in protecting community health, it has also exposed weaknesses in the public health infrastructure that stem from chronic underfunding and fragmentation in public health delivery systems. Information about the relative value in the implementation of recommended population health capabilities can help communities prioritize their use of limited time and resources and identify the most effective pathways for building a stronger public health system. METHODS: We used a longitudinal cohort design with data from the National Longitudinal Survey of Public Health Systems to examine longitudinal and geographic trends in the delivery of population health capabilities and their impact on system strength across communities in the United States. We used linear probability models to ascertain whether the delivery of certain capabilities added value to public health system strength. FINDINGS: Those communities with the strongest classification of public health system structure in both urban and rural areas implemented the largest set of population health capabilities. Results from the linear probability model indicate that a set of population health capabilities are associated with increased public health system strength. Key activities include allocating resources based on a community health plan, surveying the community for behavioral risk factors, analyzing the data on preventive services use, and engaging community stakeholders in health improvement planning (p < 0.01). CONCLUSIONS: The results of this study suggest that public health systems can be strengthened through the targeted implementation of high-value population health capabilities. Prioritizing the delivery of value-added population health capabilities may help communities increase their public health system's capacity and improve health outcomes.

Assessment of Kentucky's Local Health Department Cross-Jurisdictional Sharing: Strategy for Maximizing Efficiency.

OBJECTIVE: The purpose of this study was to examine patterns of cross-jurisdictional sharing across the 61 local public health jurisdictions (LHJs) in Kentucky. The opportunities to reduce the cost-of-service delivery for Kentucky's LHJs via cross-jurisdictional sharing present a mechanism to address financial instability across the state by achieving economies of scale, especially among smaller jurisdictions. DESIGN: A cross-sectional study design was used to examine patterns of cross-jurisdictional sharing across the 61 LHJs in Kentucky. The survey tool utilized was designed by the Center for Sharing Public Health Services, an initiative managed by the Kansas Health Institute with support from the Robert Wood Johnson Foundation. RESULTS: Seventy-two percent of the 61 LHJs in Kentucky responded to the survey. The majority of responding jurisdictions sharing services were rural, single-county jurisdictions, utilizing service-related informal sharing arrangements. The majority of health departments, when asked to identify which programmatic areas shared service arrangements were focused in, listed those services requiring intensive staff training such as Health Access Nurturing Development Services (HANDS) and epidemiology. Of particular interest were the services most infrequently shared such as communicable disease screening and treatment. CONCLUSIONS: This study suggests that, pre-COVID-19, a core group of primarily rural, single-county Kentucky local health departments has experience with cross-jurisdictional sharing. Among this group, engagement in informal arrangements was the form of cross-jurisdictional sharing predominantly used, with few jurisdictions reporting shared functions with joint oversight. When considering the potential benefits and efficiencies that cross-jurisdictional sharing can provide to public health departments and their communities, for some, COVID-19 may have been a catalyst to engage in sharing across health department jurisdictional lines.

Physical inactivity among American single-female caregivers: An analysis of the 2020 behavioral risk factor surveillance system.

BACKGROUND: Many adults in the United States do not reach the recommended levels of activity needed for health benefits. Single-female caregivers present a unique and vulnerable population that is often less active than their partnered peers or single-male caregivers. OBJECTIVE: The primary objective of this cross-sectional investigation was to identify the prevalence of physical inactivity in single-family, female-led households and determine differences in personal factors and social characteristics between physically active and not physically active single-female caregivers. A secondary objective was to examine associations among the social characteristic variables and physical inactivity in single-female caregiver households. Finally, we examined the odds single-female caregivers who are physically inactive reported chronic health conditions. DESIGN: Cross-sectional survey design. METHODS: We used the 2020 Behavioral Risk Factor Surveillance System (BRFSS) data. Participants were selected based on pre-determined criteria for inclusion. RESULTS: There was a 39.3% prevalence of physical inactivity among single-female caregivers in the sample. Single-female caregivers who were physically inactive had greater odds of having a history of multiple chronic health conditions. CONCLUSION: Healthcare providers and other community stakeholders should explore existing physical activity promotion strategies to increase physical activity in single-female caregivers. Future research should employ more rigorous, prospective research designs to determine if these chronic conditions and various social characteristics are caused by physical inactivity.

Longitudinal Trends in Insurer Participation in Multisector Population Health Activities.

Healthcare organizations increasingly engage in activities to identify and address social determinants of health (SDOH) among their patients to improve health outcomes and reduce costs. While several studies to date have focused on the evolving role of hospitals and physicians in these types of population health activities, much less is known about the role health insurers may play. We used data from the National Longitudinal Survey of Public Health Systems for the period 2006 to 2018 to examine trends in health insurer participation in population health activities and in the multi-sector collaborative networks that support these activities. We also used a difference-in-differences approach to examine the impact of Medicaid expansion on insurer participation in population health networks. Insurer participation increased in our study period both in the delivery of population health activities and in the integration into collaborative networks that support these activities. Insurers were most likely to participate in activities focusing on community health assessment and policy development. Results from our adjusted difference-in-differences models showed variation in association between insurer participation in population health networks and Medicaid expansion (Table 2). Population health networks in expansion states experienced significant increases insurer participation in assessment (4.48 percentage points, P < .05) and policy and planning (7.66 percentage points, P < .05) activities. Encouraging insurance coverage gains through policy mechanisms like Medicaid expansion may not only improve access to healthcare services but can also act as a driver of insurer integration into population health networks.

Intratumoral T-cell receptor repertoire composition predicts overall survival in patients with pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy that is refractory to immune checkpoint inhibitor therapy. However, intratumoral T-cell infiltration correlates with improved overall survival (OS). Herein, we characterized the diversity and antigen specificity of the PDAC T-cell receptor (TCR) repertoire to identify novel immune-relevant biomarkers. Demographic, clinical, and TCR-beta sequencing data were collated from 353 patients across three cohorts that underwent surgical resection for PDAC. TCR diversity was calculated using Shannon Wiener index, Inverse Simpson index, and "True entropy." Patients were clustered by shared repertoire specificity. TCRs predictive of OS were identified and their associated transcriptional states were characterized by single-cell RNAseq. In multivariate Cox regression models controlling for relevant covariates, high intratumoral TCR diversity predicted OS across multiple cohorts. Conversely, in peripheral blood, high abundance of T-cells, but not high diversity, predicted OS. Clustering patients based on TCR specificity revealed a subset of TCRs that predicts OS. Interestingly, these TCR sequences were more likely to encode CD8+ effector memory and CD4+ T-regulatory (Tregs) T-cells, all with the capacity to recognize beta islet-derived autoantigens. As opposed to T-cell abundance, intratumoral TCR diversity was predictive of OS in multiple PDAC cohorts, and a subset of TCRs enriched in high-diversity patients independently correlated with OS. These findings emphasize the importance of evaluating peripheral and intratumoral TCR repertoires as distinct and relevant biomarkers in PDAC.

Association Between Community Social Vulnerability and Preventable Hospitalizations.

Preventable hospitalizations are common and costly events that burden patients and our health care system. While research suggests that these events are strongly linked to ambulatory care access, emerging evidence suggests they may also be sensitive to a patient's social, environmental, and economic conditions. This study examines the association between variations in social vulnerability and preventable hospitalization rates. We conducted a cross-sectional analysis of county-level preventable hospitalization rates for 33 states linked with data from the 2020 Social Vulnerability Index (SVI). Preventable hospitalizations were 40% higher in the most vulnerable counties compared with the least vulnerable. Adjusted regression results confirm the strong relationship between social vulnerability and preventable hospitalizations. Our results suggest wide variation in community-level preventable hospitalization rates, with robust evidence that variation is strongly related to a community's social vulnerability. The human toll, societal cost, and preventability of these hospitalizations make understanding and mitigating these inequities a national priority.

Stromal reprogramming overcomes resistance to RAS-MAPK inhibition to improve pancreas cancer responses to cytotoxic and immune therapy.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy that is often resistant to therapy. An immune suppressive tumor microenvironment (TME) and oncogenic mutations in KRAS have both been implicated as drivers of resistance to therapy. Mitogen-activated protein kinase (MAPK) inhibition has not yet shown clinical efficacy, likely because of rapid acquisition of tumor-intrinsic resistance. However, the unique PDAC TME may also be a driver of resistance. We found that long-term focal adhesion kinase (FAK) inhibitor treatment led to hyperactivation of the RAS/MAPK pathway in PDAC cells in mouse models and tissues from patients with PDAC. Concomitant inhibition of both FAK (with VS-4718) and rapidly accelerated fibrosarcoma and MAPK kinase (RAF-MEK) (with avutometinib) induced tumor growth inhibition and increased survival across multiple PDAC mouse models. In the TME, cancer-associated fibroblasts (CAFs) impaired the down-regulation of MYC by RAF-MEK inhibition in PDAC cells, resulting in resistance. By contrast, FAK inhibition reprogramed CAFs to suppress the production of FGF1, which can drive resistance to RAF-MEK inhibition. The addition of chemotherapy to combined FAK and RAF-MEK inhibition led to tumor regression, a decrease in liver metastasis, and improved survival in KRAS-driven PDAC mouse models. Combination of FAK and RAF-MEK inhibition alone improved antitumor immunity and priming of T cell responses in response to chemotherapy. These findings provided the rationale for an ongoing clinical trial evaluating the efficacy of avutometinib and defactinib in combination with gemcitabine and nab-paclitaxel in patients with PDAC and may suggest further paths for combined stromal and tumor-targeting therapies.

The Appalachian Gap in Preventable Hospitalizations: Are We Seeing Any Progress?

Introduction: Previous studies have documented geographic variation in preventable hospitalizations between rural and urban areas, but much less is known about preventable hospitalization patterns between heterogeneous rural areas. Unique challenges related to access of care and poverty may put the rural Appalachian Region at risk for higher rates of preventable hospitalizations. Purpose: This study examines whether within-rural differences in Kentucky's preventable hospitalization rates exist and how these differences may be changing over time. Methods: Longitudinal and geographic trends in county-level preventable hospitalization rates were examined using Kentucky hospital discharge data from 2016 to 2019. Regression models were run to determine whether changes over time in preventable hospitalization rates led to an increasing or decreasing gap in outcomes between rural Appalachian counties and their urban and rural non- Appalachian counterparts. Results: Rural Appalachian counties consistently had significantly higher preventable hospitalizations rates compared to their rural non-Appalachian and urban counterparts ( p < 0.01). A downward trend in overall preventable hospitalizations was observed for rural Appalachia over time, but trends were relatively stable for rural non-Appalachian and urban counties. Regression results indicate that there was no significant reduction in the "Appalachian gap" over time. Implications: The analyses confirm that rural areas within Kentucky experienced highly heterogeneous rates of preventable hospitalizations. Despite Medicaid expansion, there is little evidence of any narrowing of the "Appalachian gap." Focus on improving access to care alone may be insufficient to improve outcomes. Alternative strategies that leverage population health approaches may improve capacity to address complex health and social needs in rural Appalachia.

Association Between Insurer Connectivity in Appalachian Population Health Networks and Preventable Hospitalizations: Evidence from Kentucky.

Introduction: Addressing complex health and social needs requires cross-sector collaboration to deliver medical, social, and population health services at the community level. Capacity in community health and social services networks may be constrained in regions like Appalachia due to the combined effects of rurality and persistently poor health and social outcomes. One way that cross-sector networks serving low-resource communities can expand their capacity is by engaging partners, like health insurers, who can leverage resources from outside the local area. Purpose: This study examines insurer connectivity in cross-sector networks across Kentucky's geographic regions and the association between connectivity and the probability of an individual experiencing a preventable hospitalization. Methods: A cross-sectional design was used that linked data from the National Longitudinal Survey of Public Health Systems (NALSYS) with 2018 patient-level Kentucky hospital discharge data to examine the association between insurer connectivity in community networks and preventable hospitalizations across urban, rural non-Appalachian, and Appalachian regions. Results: Analysis of the data shows substantial geographic variation in the association between insurer connectivity in community networks and preventable hospitalization. Insurer connectivity in rural Appalachian communities was associated with lower likelihood that an individual was admitted for a preventable hospitalization ( p < 0.01). Implications: Findings suggest insurer connectivity in cross-sector community health and social services networks has the potential to strengthen network capacity to address preventable hospitalizations and improve health outcomes and well-being for the people of Appalachia.

Impact of state Medicaid expansion on cross-sector health and social service networks: Evidence from a longitudinal cohort study.

OBJECTIVE: To examine the impact of state Medicaid expansion on the delivery of population health activities in cross-sector health and social services networks. Community networks are multisector, interorganizational networks that provide services ranging from the direct provision of individual social services to the implementation of population-level initiatives addressing community outcomes. DATA SOURCES: We used data measuring the composition of cross-sector population health networks 2006-2018 National Longitudinal Survey of Public Health Systems (NALSYS) linked with the Area Health Resource File. STUDY DESIGN: A difference-in-differences approach was used to examine the impact of expansion on organization engagement in population health activities and network structure. DATA COLLECTION/EXTRACTION METHODS: Stratified random sampling of local public health jurisdictions in the United States. We restricted our data to jurisdictions serving populations of 100,000 or more and states that had NALSYS observations across all time periods, resulting in a final sample size of 667. PRINCIPAL FINDINGS: Results from our adjusted difference-in-differences estimates indicated that Medicaid expansion was associated with a 2.3 percentage point increase in the density of population health networks (p < 0.10). Communities in states that expanded Medicaid experienced significant increases in the participation of local public health, local government, hospital, nonprofit, insurer, and K-12 schools. Of the organizations with significant increases in expansion communities, nonprofits (7.7 percentage points, p < 0.01), local public health agencies (6.5 percentage points, p < 0.01), hospitals (5.8 percentage points, p < 0.01), and local government agencies (6.0 percentage points, p < 0.05) had the largest gains. CONCLUSIONS: Our study found increases in cross-sector participation in population health networks in states that expanded Medicaid compared with nonexpansion states, suggesting that additional coverage gains are associated with positive changes in population health network structure.

COVID-19 and MCO-community partnerships to address enrollee social needs.

OBJECTIVES: Many Medicaid managed care organizations (MCOs) now screen enrollees and connect them to community-based organizations (CBOs) to address unmet social needs. COVID-19 has significantly disrupted health care delivery and overall economic activity in the United States. We examined how partnerships between Medicaid MCOs and CBOs to address social determinants of health have been affected by the pandemic. STUDY DESIGN: Guided by questions and recruitment strategies developed with our stakeholder advisory board, we conducted 26 interviews with representatives from all 6 of Kentucky's Medicaid MCOs. METHODS: In-depth, structured interviews for data collection and iterative content analyses to identify themes. RESULTS: Several themes emerged, including substantial increases in enrollees' unmet needs and the demand to find new ways to be responsive, changing funding patterns, disruptions to and evolving modes of communication, and shifting partner relationships. In virtually all areas of impact, COVID-19 has been associated with both negative and positive change. CONCLUSIONS: Unmet social needs associated with the pandemic placed tremendous strain on CBOs, limiting their capacity to sustain some programs and partnerships. Isolation associated with COVID-19 also had wide-ranging effects on service delivery, communication with enrollees and partners, and the ability to maintain relationships. Nonetheless, the pandemic also had some silver linings, including additional resources and flexibility for addressing unmet needs. Federal and state agencies, along with MCO leaders, should carefully evaluate what innovations have been particularly effective during the pandemic and craft new flexibilities into their policies, procedures, and regulations.

Tumor-associated fibrosis impairs immune surveillance and response to immune checkpoint blockade in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related deaths. Immune checkpoint blockade has improved survival for many patients with NSCLC, but most fail to obtain long-term benefit. Understanding the factors leading to reduced immune surveillance in NSCLC is critical in improving patient outcomes. Here, we show that human NSCLC harbors large amounts of fibrosis that correlates with reduced T cell infiltration. In murine NSCLC models, the induction of fibrosis led to increased lung cancer progression, impaired T cell immune surveillance, and failure of immune checkpoint blockade efficacy. Associated with these changes, we observed that fibrosis leads to numerically and functionally impaired dendritic cells and altered macrophage phenotypes that likely contribute to immunosuppression. Within cancer-associated fibroblasts, distinct changes within the Col13a1-expressing population suggest that these cells produce chemokines to recruit macrophages and regulatory T cells while limiting recruitment of dendritic cells and T cells. Targeting fibrosis through transforming growth factor-ß receptor signaling overcame the effects of fibrosis to enhance T cell responses and improved the efficacy of immune checkpoint blockade but only in the context of chemotherapy. Together, these data suggest that fibrosis in NSCLC leads to reduced immune surveillance and poor responsiveness to checkpoint blockade and highlight antifibrotic therapies as a candidate strategy to overcome immunotherapeutic resistance.

Systemic Alterations in Type-2 Conventional Dendritic Cells Lead to Impaired Tumor Immunity in Pancreatic Cancer.

Intratumoral T-cell dysfunction is a hallmark of pancreatic tumors, and efforts to improve dendritic cell (DC)-mediated T-cell activation may be critical in treating these immune therapy unresponsive tumors. Recent evidence indicates that mechanisms that induce dysfunction of type 1 conventional DCs (cDC1) in pancreatic adenocarcinomas (PDAC) are drivers of the lack of responsiveness to checkpoint immunotherapy. However, the impact of PDAC on systemic type 2 cDC2 development and function has not been well studied. Herein, we report the analysis of 3 cohorts, totaling 106 samples, of human blood and bone marrow (BM) from patients with PDAC for changes in cDCs. We found that circulating cDC2s and their progenitors were significantly decreased in the blood of patients with PDAC, and repressed numbers of cDC2s were associated with poor prognosis. Serum cytokine analyses identified IL6 as significantly elevated in patients with PDAC and negatively correlated with cDC numbers. In vitro, IL6 impaired the differentiation of cDC1s and cDC2s from BM progenitors. Single-cell RNA sequencing analysis of human cDC progenitors in the BM and blood of patients with PDAC showed an upregulation of the IL6/STAT3 pathway and a corresponding impairment of antigen processing and presentation. These results suggested that cDC2s were systemically suppressed by inflammatory cytokines, which was linked to impaired antitumor immunity.

Costimulatory domains direct distinct fates of CAR-driven T cell dysfunction.

Chimeric antigen receptor (CAR) engineered T cells often fail to enact effector functions after infusion into patients. Understanding the biological pathways that lead CAR T cells to failure is of critical importance in the design of more effective therapies. We developed and validated an in vitro model that drives T cell dysfunction through chronic CAR activation and interrogated how CAR costimulatory domains contribute to T cell failure. We found that dysfunctional CD28-based CARs targeting CD19 bear hallmarks of classical T cell exhaustion while dysfunctional 41BB-based CARs are phenotypically, transcriptionally and epigenetically distinct. We confirmed activation of this unique transcriptional program in CAR T cells that failed to control clinical disease. Further, we demonstrate that 41BB-dependent activation of the transcription factor FOXO3 is a significant contributor to this dysfunction and disruption of FOXO3 improves CAR T cell function. These findings identify that chronic activation of 41BB leads to novel state of T cell dysfunction that can be alleviated by genetic modification of FOXO3. Summary: Chronic stimulation of CARs containing the 41BB costimulatory domain leads to a novel state of T cell dysfunction that is distinct from T cell exhaustion.

Stromal and therapy-induced macrophage proliferation promotes PDAC progression and susceptibility to innate immunotherapy.

Tumor-associated macrophages (TAMs) are abundant in pancreatic ductal adenocarcinomas (PDACs). While TAMs are known to proliferate in cancer tissues, the impact of this on macrophage phenotype and disease progression is poorly understood. We showed that in PDAC, proliferation of TAMs could be driven by colony stimulating factor-1 (CSF1) produced by cancer-associated fibroblasts. CSF1 induced high levels of p21 in macrophages, which regulated both TAM proliferation and phenotype. TAMs in human and mouse PDACs with high levels of p21 had more inflammatory and immunosuppressive phenotypes. p21 expression in TAMs was induced by both stromal interaction and/or chemotherapy treatment. Finally, by modeling p21 expression levels in TAMs, we found that p21-driven macrophage immunosuppression in vivo drove tumor progression. Serendipitously, the same p21-driven pathways that drive tumor progression also drove response to CD40 agonist. These data suggest that stromal or therapy-induced regulation of cell cycle machinery can regulate both macrophage-mediated immune suppression and susceptibility to innate immunotherapy.

Context-dependent activation of STING-interferon signaling by CD11b agonists enhances anti-tumor immunity.

Chronic activation of inflammatory pathways and suppressed interferon are hallmarks of immunosuppressive tumors. Previous studies have shown that CD11b integrin agonists could enhance anti-tumor immunity through myeloid reprograming, but the underlying mechanisms remain unclear. Herein we find that CD11b agonists alter tumor-associated macrophage (TAM) phenotypes by repressing NF-κB signaling and activating interferon gene expression simultaneously. Repression of NF-κB signaling involves degradation of p65 protein and is context independent. In contrast, CD11b agonism induces STING/STAT1 pathway-mediated interferon gene expression through FAK-mediated mitochondrial dysfunction, with the magnitude of induction dependent on the tumor microenvironment and amplified by cytotoxic therapies. Using tissues from phase I clinical studies, we demonstrate that GB1275 treatment activates STING and STAT1 signaling in TAMs in human tumors. These findings suggest potential mechanism-based therapeutic strategies for CD11b agonists and identify patient populations more likely to benefit.