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Susan Kaech and colleagues report that in chronic viral infection, prostaglandin E2 and PD-1 signaling suppressed the function and survival of cytotoxic T cells. More than 10% of the world's population is chronically infected with HIV, hepatitis C virus (HCV) or hepatitis B virus (HBV), all of which can cause severe disease and death. These viruses persist in part because continuous antigenic stimulation causes the deterioration of virus-specific cytotoxic T lymphocyte (CTL) function and survival. Additionally, antiviral CTLs autonomously suppress their responses to limit immunopathology by upregulating inhibitory receptors such as programmed cell death 1 (PD-1). Identification and blockade of the pathways that induce CTL dysfunction may facilitate the clearance of chronic viral infections. We found that the prostaglandin E2 (PGE 2 ) receptors EP2 and EP4 were upregulated on virus-specific CTLs during chronic lymphocytic choriomeningitis virus (LCMV) infection and suppressed CTL survival and function. We show that the combined blockade of PGE 2 and PD-1 signaling was therapeutic in terms of improving viral control and augmenting the numbers of functional virus-specific CTLs. Thus, PGE 2 inhibition is both an independent candidate therapeutic target and a promising adjunct therapy to PD-1 blockade for the treatment of HIV and other chronic viral infections.

Breast cancer progression is influenced by tumor subtype, metabolic environment, and patient factors, including menopausal status and BMI. In this study, we utilized publicly available data to investigate the prognostic relevance of PPARγ gene's expression across different subgroups. We also examined adipose tissue proliferation in patients with various tumor subtypes and phenotypic cohorts. We analyzed RNA‐seq data from primary breast cancer patients in the TCGA‐BRCA cohort, stratifying patients by PPARγ expression, menopausal status, and tumor receptor subtype. Kaplan–Meier analyses revealed that high PPARγ expression was associated with improved overall survival, particularly in premenopausal patients. Complementing this, we analyzed PET‐CT scans from breast cancer patients in the ACRIN‐6888 trial, focusing on standardized uptake value (SUV) metrics of a cell cycle tracer, 3′‐deoxy‐3′‐[18F]‐fluorothymidine (18F‐FLT) in visceral and subcutaneous adipose tissue. Postmenopausal patients had lower visceral adipose tissue SUVmean, and patients with ER+ or non‐TNBC tumors showed lower SUVpeak and SUVmax of both adipose tissue types, indicating metabolic/proliferative reprogramming of adipose tissue based on tumor subtype. We hypothesize that PPARγ expression and adipose proliferation differentially affect survival across subtypes and menopausal status, providing deeper insight into PPARγ as a therapeutic target in breast cancer and the potential implications for precision medicine treatments.

Prospective and between trial comparisons indicate that first-line treatment with immune checkpoint inhibitors improves survival outcomes compared to first-line therapy with combined BRAF and MEK inhibitors in metastatic melanoma containing BRAFV600E/K mutations. Long-term outcomes for BRAF/MEK inhibition after progression on immunotherapy have not been reported. Moreover, clinical variables associated with outcome from treatment with combined BRAF/MEK inhibition were previously identified in the first-line setting but have not been investigated when targeted therapies are administered after progression on immune therapy. We performed a retrospective single institution analysis of 40 metastatic melanoma patients receiving combined BRAF/MEK inhibitors after progression on an anti-PD-1 or ipilimumab plus nivolumab to assess response rate by RECIST 1.1, progression-free and overall survival (PFS and OS). Pretreatment clinical variables were analyzed for association with OS. Ipilimumab/nivolumab was the first-line immunotherapy regimen in 39 patients (97.5%), and BRAFV600E/K mutations were present in 33 (83%) and 7 (17%) patients, respectively. The median OS from start of BRAF/MEK inhibitors was 20.3 months (1.73-106.4+, 95% CI of median 13.3-30.7). Clinical characteristics associated with worse survival prior to starting BRAF/MEK inhibitors included age > 60 years (median OS 14 vs. 28 months; HR 2.5; 95% CI 0.91-6.87, P = .023), ECOG-PS > 2 (median OS 7 vs. 33 months; HR 2.89; 95% CI 0.78-10.76, P = .018), and presence of bone metastases (median OS 9 vs. 52 months; HR 3.17; 95% CI 1.33-7.54, P = .002). These associations with shorter survival maintained their significance on multivariate analysis. If confirmed in larger cohorts, the identified prognostic variables can be used for stratification of patients in future randomized trials. Long-term clinical outcomes for patients receiving BRAF/MEKi after progression on immune therapy have not been fully described, and clinical predictors of long-term response and survival are not known. This article reports an analysis of patients receiving BRAF/MEKi after progression on one or more lines of immune checkpoint inhibitor therapy to assess response and clinical variables associated with survival.

Recent advancements in single-cell technologies allow characterization of experimental perturbations at single-cell resolution. While methods have been developed to analyze such experiments, the application of a strict causal framework has not yet been explored for the inference of treatment effects at the single-cell level. Here we present a causal-inference-based approach to single-cell perturbation analysis, termed CINEMA-OT (causal independent effect module attribution + optimal transport). CINEMA-OT separates confounding sources of variation from perturbation effects to obtain an optimal transport matching that reflects counterfactual cell pairs. These cell pairs represent causal perturbation responses permitting a number of novel analyses, such as individual treatment-effect analysis, response clustering, attribution analysis, and synergy analysis. We benchmark CINEMA-OT on an array of treatment-effect estimation tasks for several simulated and real datasets and show that it outperforms other single-cell perturbation analysis methods. Finally, we perform CINEMA-OT analysis of two newly generated datasets: (1) rhinovirus and cigarette-smoke-exposed airway organoids, and (2) combinatorial cytokine stimulation of immune cells. In these experiments, CINEMA-OT reveals potential mechanisms by which cigarette-smoke exposure dulls the airway antiviral response, as well as the logic that governs chemokine secretion and peripheral immune cell recruitment. CINEMA-OT is a framework that enables accurate causal inference of the effects of perturbation experiments at the single-cell level.

Prospective and between trial comparisons indicate that first-line treatment with immune checkpoint inhibitors improves survival outcomes compared to first-line therapy with combined BRAF and MEK inhibitors in metastatic melanoma containing [BRAF.sup.V600]E,K mutations. Long-term outcomes for BRAF/MEK inhibition after progression on immunotherapy have not been reported. Moreover, clinical variables associated with outcome from treatment with combined BRAF/MEK inhibition were previously identified in the first-line setting but have not been investigated when targeted therapies are administered after progression on immune therapy. We performed a retrospective single institution analysis of 40 metastatic melanoma patients receiving combined BRAF/MEK inhibitors after progression on an anti-PD-1 or ipilimumab plus nivolumab to assess response rate by RECIST 1.1, progression-free and overall survival (PFS and OS). Pretreatment clinical variables were analyzed for association with OS. Ipilimumab/nivolumab was the first-line immunotherapy regimen in 39 patients (975%), and [BRAF.sup.V600E/K] mutations were present in 33 (83%) and 7 (17%) patients, respectively. The median OS from start of BRAF/MEK inhibitors was 20.3 months (1.73-106.4+, 95% CI of median 13.3-30.7). Clinical characteristics associated with worse survival prior to starting BRAF/MEK inhibitors included age > 60 years (median OS 14 vs. 28 months; HR 2.5; 95% CI 0.91-6.87 P =.023), ECOG-PS > 2 (median OS 7 vs. 33 months; HR 2.89; 95% CI 0.78-10.76, P =.018), and presence of bone metastases (median OS 9 vs. 52 months; HR 3.17; 95% CI 1.33-754, P =.002). These associations with shorter survival maintained their significance on multivariate analysis. If confirmed in larger cohorts, the identified prognostic variables can be used for stratification of patients in future randomized trials. Key words: immunotherapy; targeted therapy; melanoma; survival.

C-C receptor 7 (CCR7) is important to allow T cells and dendritic cells to migrate toward CCL19- and CCL21-producing cells in the T-cell zone of the spleen and lymph nodes. The role of this chemokine receptor in regulating the homeostasis of effector and memory T cells during acute viral infection is poorly defined, however. In this study, we show that CCR7 expression alters memory CD8 T-cell homeostasis following lymphocytic choriomeningitis virus infection. Greater numbers of CCR7-deficient memory T cells were formed and maintained compared with CCR7-sufficient memory T cells, especially in the lung and bone marrow. The CCR7-deficient memory T cells also displayed enhanced rates of homeostatic turnover, which may stem from increased exposure to IL-15 as a consequence of reduced exposure to IL-7, because removal of IL-15, but not of IL-7, normalized the numbers of CCR7-sufficient and CCR7-deficient memory CD8 T cells. This result suggests that IL-15 is the predominant cytokine supporting augmentation of the CCR7−/− memory CD8 T-cell pool. Taken together, these data suggest that CCR7 biases memory CD8 T cells toward IL-7–dependent niches over IL-15–dependent niches, which provides insight into the homeostatic regulation of different memory T-cell subsets.

Memory T cells respond rapidly to repeated antigen exposure and can maintain their population for extended periods through self-renewal. These characteristics of memory T cells have mainly been studied during viral infections, whereas their existence and functions in allergic diseases have been studied incompletely. Since allergic patients can suffer repeated relapses caused by intermittent allergen exposure, we hypothesized that allergen- specific memory Th2 cells are present and the factors necessary for the maintenance of these cells are provided by the lung and airways. Using a murine model of airway inflammation, we found that allergen-specific CD4 T cells survived longer than 70 days in the lung and airways in an IL-7 dependent fashion. These T cells showing homeostatic proliferation were largely found in the mediastinal lymph node (mLN), rather than the airways; however, cells residing in the lung and airways developed recall responses successfully. We also found that CD4 T cells exhibited differential phenotypes in the mLN and in the lung. Altogether, we believe that allergen-specific memory T cells reside and function in the lung and airways, while their numbers are replenished through homeostatic turnover in the mLNs. Furthermore, we determined that IL-7 signaling is important for the homeostasis of these cells.

Background/Objectives: Immune checkpoint inhibitors (ICIs) are frontline treatment for advanced Merkel Cell Carcinoma (MCC), regardless of viral status. Frontline ICIs provide durable benefit to only half of patients, highlighting a need for alternative therapies. In this study, the objective is to leverage whole exome sequencing (WES) and transcriptome sequencing (WTS) to distinguish genomic alterations associated with ICI response. Investigate differential genomic alterations between virus-positive (VP) and virus-negative (VN)-MCC to identify novel therapeutic targets. Methods: A total of 95 MCC cases underwent WES and WTS. Utilizing computational pipelines applied to WES, we identified viral status and tumor mutational burden (TMB). RNA-seq data was used to characterize the immune microenvironment. Results: Of 95 MCC cases, 57 (60%) were VP-MCC and 38 (40%) were VN-MCC. Median TMB was higher in VN-MCC (27.5 vs. 1 Muts/Mb). Mutations in TP53, RB1, NOTCH1, KMTD2, KMT2C, and PIK3CA were primarily found in VN-MCC. MAPK Pathway Activity Score, NK cell infiltration, and the immune checkpoint gene CD276 in VN-MCC tumors were upregulated. No overall survival (OS) difference was identified between VP and VN-MCC, even after ICIs. Conclusions: MCC oncogenesis and treatment response transcend viral status. While mutational analysis confirms previous findings, assessment of the transcriptome and tumor microenvironment suggests alternate therapeutic targets.

BackgroundMCC and SCLC remain among the most treatment-refractory solid tumors despite first-line ICB, with durable responses in <25% of patients. Loss of HLA-I antigen presentation represents a common immune-escape route in MCC. MDK, a heparin-binding growth factor that is over-expressed in many cancers and dampens IFN signalling and DC maturation, is highly transcribed in MCC and SCLC tumors and lines, yet its contribution to neuroendocrine ICB failure is undefined.MethodsUsing CRISPR/Cas9 we generated isogenic MDK-KO human MCC (WaGa, MKL-1) and SCLC (NCI-H69, NCI-H82) models. Bulk RNA-seq, phospho-proteomics, flow cytometry, and monocyte-derived DC co-cultures quantified IFN responsiveness, antigen-presentation machinery, and DC activation. Public bulk- and single-cell RNA-seq datasets (102 MCC, 70 SCLC) were mined for MDK-immune correlations.ResultsMDK deletion restored type I/II IFN signalling, re-engaging STAT1/IRF1 networks and driving 4– to 9-fold up-regulation of HLA-A/B/C surface levels across all lines (P < 0.01). MDK-KO tumors re-sensitized to low-dose IFN-γ, yielding >300 differentially up-regulated inflammatory transcripts, including TAP1/2 and CXCL9/10. Co-culture with MDK-KO cells elicited a two-fold increase in CD86^hi HLA-DR^hi DCs versus wild-type (WT) controls, consistent with prior evidence that MDK restrains DC maturation Patient-omics analyses confirmed an inverse correlation between MDK expression and IFN-stimulated, antigen-processing, and cytotoxic-lymphocyte signatures (Spearman ρ = –0.61 to –0.44; FDR < 0.001). High-MDK tumors were enriched in transcriptionally defined ‘immune-cold’ neuroendocrine subtypes and associated with early ICB progressionConclusionsMDK functions as a nodal suppressor of IFN-driven anti-tumor immunity in MCC and SCLC, simultaneously curbing tumor-intrinsic antigen presentation and extrinsic DC activation. Genetic or pharmacologic MDK blockade may synergize with ICB to convert immune-cold neuroendocrine tumors into responsive disease.Ethics ApprovalWe obtain de-identified human surgical samples through the Yale Spore in Skin Cancer (IRB protocol # 0609001869). The Spore systematically informs and provides the opportunity for all patients treated for any skin cancer, including women and minorities (including special focus on translated consent documents/processes for the substantial international patient population in Connecticut). Patient/sample relevant clinical characteristics are provided upon request by the Spore, and we operate with only a de-identified sample ID, to avoid any risk to patient privacy. All testing including sequencing is approved via the IRB approved consent.

Metabolic homeostasis is one of the most exquisitely tuned systems in mammalian physiology. Metabolic homeostasis requires multiple redundant systems to cooperate to maintain blood glucose concentrations in a narrow range, despite a multitude of physiological and pathophysiological pressures. Cancer is one of the canonical pathophysiological settings in which metabolism plays a key role. In this study, we utilized REnal Gluconeogenesis Analytical Leads (REGAL), a liquid chromatography-mass spectrometry/mass spectrometry-based stable isotope tracer method that we developed to show that in conditions of metabolic stress, the fasting hepatokine fibroblast growth factor-21 (FGF-21) coordinates a liver-brain-kidney axis to promote renal gluconeogenesis. FGF-21 promotes renal gluconeogenesis by enhancing β2 adrenergic receptor (Adrb2)-driven, adipose triglyceride lipase (ATGL)-mediated intrarenal lipolysis. Further, we show that this liver-brain-kidney axis promotes gluconeogenesis in the renal parenchyma in mice and humans with renal cell carcinoma (RCC). This increased gluconeogenesis is, in turn, associated with accelerated RCC progression. We identify Adrb2 blockade as a new class of therapy for RCC in mice, with confirmatory data in human patients. In summary, these data reveal a new metabolic function of FGF-21 in driving renal gluconeogenesis, and demonstrate that inhibition of renal gluconeogenesis by FGF-21 antagonism deserves attention as a new therapeutic approach to RCC.