ADA/CD26 axis increases intra-tumor PD-1+CD28+CD8+ T-cell fitness and affects NSCLC prognosis and response to ICB.

Improving cancer immunotherapy efficacy hinges on identifying key T-cell populations critical for tumor control and response to Immune Checkpoint Blockade (ICB). We have recently reported that while the co-expression of PD-1 and CD28 is associated with impaired functionality in peripheral blood, it significantly enhances T-cell fitness in the tumor site of non-small cell lung cancer (NSCLC) patients. To uncover the underlying mechanisms, we explored the role of CD26, a key player in T-cell activation through its interaction with adenosine deaminase (ADA), a crucial intra/extracellular enzyme able to neutralize local adenosine (ADO). We found that an autocrine ADA/CD26 axis enhances CD8+PD-1+CD28+ T-cell function, particularly within an immunosuppressive environment marked by CD39 expression. Then, we interrogated the TCGA and OAK datasets to gain insight into the prognostic/predictive potential of our findings. We identified a signature predicting overall survival (OS) in LUAD patients and response to atezolizumab in advanced LUAD cases. These findings suggest promising avenues for therapeutic intervention targeting the ADA/CD26 axis.

Tumoral and stromal hMENA isoforms impact tertiary lymphoid structure localization in lung cancer and predict immune checkpoint blockade response in patients with cancer.

BACKGROUND: Tertiary Lymphoid Structures (TLS) correlate with positive outcomes in patients with NSCLC and the efficacy of immune checkpoint blockade (ICB) in cancer. The actin regulatory protein hMENA undergoes tissue-specific splicing, producing the epithelial hMENA11a linked to favorable prognosis in early NSCLC, and the mesenchymal hMENAΔv6 found in invasive cancer cells and pro-tumoral cancer-associated fibroblasts (CAFs). This study investigates how hMENA isoforms in tumor cells and CAFs relate to TLS presence, localization and impact on patient outcomes and ICB response. METHODS: Methods involved RNA-SEQ on NSCLC cells with depleted hMENA isoforms. A retrospective observational study assessed tissues from surgically treated N0 patients with NSCLC, using immunohistochemistry for tumoral and stromal hMENA isoforms, fibronectin, and TLS presence. ICB-treated patient tumors were analyzed using Nanostring nCounter and GeoMx spatial transcriptomics. Multiparametric flow cytometry characterized B cells and tissue-resident memory T cells (TRM). Survival and ICB response were estimated in the cohort and validated using bioinformatics pipelines in different datasets. FINDINGS: Findings indicate that hMENA11a in NSCLC cells upregulates the TLS regulator LTßR, decreases fibronectin, and favors CXCL13 production by TRM. Conversely, hMENAΔv6 in CAFs inhibits LTßR-related NF-kB pathway, reduces CXCL13 secretion, and promotes fibronectin production. These patterns are validated in N0 NSCLC tumors, where hMENA11ahigh expression, CAF hMENAΔv6low, and stromal fibronectinlow are associated with intratumoral TLS, linked to memory B cells and predictive of longer survival. The hMENA isoform pattern, fibronectin, and LTßR expression broadly predict ICB response in tumors where TLS indicates an anti-tumor immune response. INTERPRETATION: This study uncovers hMENA alternative splicing as an unexplored contributor to TLS-related Tumor Immune Microenvironment (TIME) and a promising biomarker for clinical outcomes and likely ICB responsiveness in N0 patients with NSCLC. FUNDING: This work is supported by AIRC (IG 19822), ACC (RCR-2019-23669120), CAL.HUB.RIA Ministero Salute PNRR-POS T4, "Ricerca Corrente" granted by the Italian Ministry of Health.

CD28/PD1 co-expression: dual impact on CD8+ T cells in peripheral blood and tumor tissue, and its significance in NSCLC patients' survival and ICB response.

BACKGROUND: Immune checkpoint blockade (ICB) has significantly prolonged survival of non-small cell lung cancer (NSCLC) patients, although most patients develop mechanisms of resistance. Recently single-cell RNA-sequencing (scRNA-Seq) revealed a huge T-cell phenotypic and (dys)functional state variability. Accordingly, T-cell exhaustion is recognized as a functional adaptation, with a dynamic progression from a long-lived "pre-exhausted stem-like progenitor" to a "terminally exhausted" state. In this scenario it is crucial to understand the complex interplay between co-stimulatory and inhibitory molecules in CD8+ T-cell functionality. METHODS: To gain a baseline landscape of the composition, functional states, and transcriptomic signatures predictive of prognosis, we analyzed CD8+ T-cell subsets characterized by the presence/absence of PD1 and CD28 from periphery, adjacent non-tumor tissue and tumor site of a cohort of treatment-naïve NSCLC patients, by integrated multiparametric flow cytometry, targeted multi-omic scRNA-seq analyses, and computational pipelines. RESULTS: Despite the increased PD1 levels, an improved PD1+CD28+ T-cell polyfunctionality was observed with the transition from periphery to tumor site, associated with lack of TIGIT, TIM-3 and LAG-3, but not with Ag-experienced-marker CD11a. Differently from CD28+ T cells, the increased PD1 levels in the tumor were associated with reduced functionality in PD1+CD28- T cells. CD11ahigh, although expressed only in a small fraction of this subset, still sustained its functionality. Absence of TIGIT, TIM-3 and CTLA-4, alone or combined, was beneficial to CD28- T cells. Notably, we observed distinct TRM phenotypes in the different districts, with CD28+ T cells more capable of producing TGFß in the periphery, potentially contributing to elevated CD103 levels. In contrast CD28- TRM mainly produced CXCL13 within the tumor. ScRNA-seq revealed 5 different clusters for each of the two subsets, with distinctive transcriptional profiles in the three districts. By interrogating the TCGA dataset of patients with lung adenocarcinoma (LUAD) and metastatic NSCLC treated with atezolizumab, we found signatures of heterogeneous TRM and "pre-exhausted" long-lived effector memory CD8+ T cells associated with improved response to ICB only in the presence of CD28. CONCLUSIONS: Our findings identify signatures able to stratify survival of LUAD patients and predict ICB response in advanced NSCLC. CD28 is advocated as a key determinant in the signatures identified, in both periphery and tumor site, thus likely providing feasible biomarkers of ICB response.

A cytofluorimetric assay to evaluate T cell polyfunctionality.

The magnitude and the quality of T-cell response are critical endpoints in the immune-monitoring of cancer patients. The release of cytokines and lytic factors by T cells operate in each phase of the cancer immunity cycle. The simultaneous cytokine production, defined as T-cell polyfunctionality, is a dynamic state of different T-cell subsets and may represent a surrogate biomarker of response to immune-mediated therapies. To quantify the T-cell immune mediators, different methodologies are available. Herein we describe two flow cytometry-based protocols to detect the simultaneous intracellular cytokine production, by using different methods of T-cell activation. Among the different procedures, multicolor flow cytometry is considered a powerful, quick, and semiquantitative technique, able to achieve the analysis of both surface and intracellular proteins, expressed by specific T-cell subsets. We report the capability of this technique to study simultaneous cytokine production within a heterogeneous population. As the field of novel single-cell high-throughput methodological approaches advance in this exciting era of Immuno-Oncology, we expect to gather even more information on the immunodynamics of polyfunctional T cells and their role in improving disease control.

Survival and prognostic factors in cats with restrictive cardiomyopathy: a review of 90 cases.

OBJECTIVES: Large studies focusing on restrictive cardiomyopathy (RCM) in the cat are scarce. The aims of this retrospective study were to describe epidemiological characteristics and to analyse prognostic factors affecting survival in cats with RCM. METHODS: The clinical archives of the Gran Sasso Veterinary Clinic (Milan, Italy) and of the cardiology unit of the Department of Veterinary Medicine (University of Milan, Italy) from 1997-2015 were reviewed for all cats diagnosed with RCM based on an echocardiographic examination (left atrial/biatrial enlargement, normal left ventricle wall thickness, normal or mildly decreased systolic function and restrictive left ventricle filling pattern with pulsed Doppler echocardiography). RESULTS: The study population comprised 90 cats (53 male, 37 female) with an echocardiographic diagnosis of RCM. Most were domestic shorthairs (n = 60) with a mean ± SD age of 10.0 ± 4.3 years and a median weight of 3.8 kg (interquartile range 3.2-5 kg). Most cats were symptomatic (n = 87). The most common clinical sign was respiratory distress (n = 75). Follow-up was available for 60 cats and the median survival time (MST) was 69 days (95% confidence interval [CI] 0-175 days). Cardiac-related death occurred in 50 cats. In the multivariate Cox analysis only respiratory distress showed a statistically significant effect on survival. The cats without respiratory distress showed an MST of 466 days (95% CI 0-1208); cats with respiratory distress showing an MST of 64 days (95% CI 8-120; P = 0.011). CONCLUSIONS AND RELEVANCE: RCM can be considered an end-stage condition associated with a poor prognosis, with few cats not showing clinical signs and surviving >1 year. Most cats died of cardiac disease within a very short time.