Myocardial infarction (MI) results in aberrant cardiac metabolism, but no therapeutics have been designed to target cardiac metabolism to enhance heart repair. We engineer a humanized monoclonal antibody against the ectonucleotidase ENPP1 (hENPP1mAb) that targets metabolic crosstalk in the infarcted heart. In mice expressing human ENPP1, systemic administration of hENPP1mAb metabolically reprograms myocytes and non-myocytes and leads to a significant rescue of post-MI heart dysfunction. Using metabolomics, single-nuclear transcriptomics, and cellular respiration studies, we show that the administration of the hENPP1mAb induces organ-wide metabolic and transcriptional reprogramming of the heart that enhances myocyte cellular respiration and decreases cell death and fibrosis in the infarcted heart. Biodistribution and safety studies showed specific organ-wide distribution with the antibody being well tolerated. In humanized animals, with drug clearance kinetics similar to humans, we demonstrate that a single "shot" of the hENPP1mAb after MI is sufficient to rescue cardiac dysfunction.
Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.

Chronic stimulation can cause T cell dysfunction and limit the efficacy of cellular immunotherapies. Improved methods are required to compare large numbers of synthetic knockin (KI) sequences to reprogram cell functions. Here, we developed modular pooled KI screening (ModPoKI), an adaptable platform for modular construction of DNA KI libraries using barcoded multicistronic adaptors. We built two ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors (SRs). Over 30 ModPoKI screens across human TCR- and CAR-T cells in diverse conditions identified a transcription factor AP4 (TFAP4) construct that enhanced fitness of chronically stimulated CAR-T cells and anti-cancer function in vitro and in vivo. ModPoKI's modularity allowed us to generate an ∼10,000-member library of TF combinations. Non-viral KI of a combined BATF-TFAP4 polycistronic construct enhanced fitness. Overexpressed BATF and TFAP4 co-occupy and regulate key gene targets to reprogram T cell function. ModPoKI facilitates the discovery of complex gene constructs to program cellular functions.
Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is characterized by a range of symptoms in which host immune response have been associated with disease progression. However, the putative role of regulatory T cells (Tregs) in determining COVID-19 outcomes has not been thoroughly investigated. Here, we compared peripheral Tregs between volunteers not previously infected with SARS-CoV-2 (healthy control [HC]) and volunteers who recovered from mild (Mild Recovered) and severe (Severe Recovered) COVID-19. Peripheral blood mononuclear cells (PBMC) were stimulated with SARS-CoV-2 synthetic peptides (Pool Spike CoV-2 and Pool CoV-2) or staphylococcal enterotoxin B (SEB). Results of a multicolor flow cytometric assay showed higher Treg frequency and expression of IL-10, IL-17, perforin, granzyme B, PD-1, and CD39/CD73 co-expression in Treg among the PBMC from the Mild Recovered group than in the Severe Recovered or HC groups for certain SARS-CoV-2 related stimulus. Moreover, Mild Recovered unstimulated samples presented a higher Tregs frequency and expression of IL-10 and granzyme B than did that of HC. Compared with Pool CoV-2 stimuli, Pool Spike CoV-2 reduced IL-10 expression and improved PD-1 expression in Tregs from volunteers in the Mild Recovered group. Interestingly, Pool Spike CoV-2 elicited a decrease in Treg IL-17+ frequency in the Severe Recovered group. In HC, the expression of latency-associated peptide (LAP) and cytotoxic granule co-expression by Tregs was higher in Pool CoV-2 stimulated samples. While Pool Spike CoV-2 stimulation reduced the frequency of IL-10+ and CTLA-4+ Tregs in PBMC from volunteers in the Mild Recovered group who had not experienced certain symptoms, higher levels of perforin and perforin+granzyme B+ co-expression by Tregs were found in the Mild Recovered group in volunteers who had experienced dyspnea. Finally, we found differential expression of CD39 and CD73 among volunteers in the Mild Recovered group between those who had and had not experienced musculoskeletal pain. Collectively, our study suggests that changes in the immunosuppressive repertoire of Tregs can influence the development of a distinct COVID-19 clinical profile, revealing that a possible modulation of Tregs exists among volunteers of the Mild Recovered group between those who did and did not develop certain symptoms, leading to mild disease.
Copyright © 2023 de Sousa Palmeira, Peixoto, Csordas, de Medeiros, de Azevedo, Veras, Janebro, Amaral and Keesen.

The discovery of immune checkpoints and the development of their specific inhibitors was acclaimed as a major breakthrough in cancer therapy. However, only a limited patient cohort shows sufficient response to therapy. Hence, there is a need for identifying new checkpoints and predictive biomarkers with the objective of overcoming immune escape and resistance to treatment. Having been associated with both, treatment response and failure, LDL seems to be a double-edged sword in anti-PD1 immunotherapy. Being embedded into complex metabolic conditions, the impact of LDL on distinct immune cells has not been sufficiently addressed. Revealing the effects of LDL on T cell performance in tumor immunity may enable individual treatment adjustments in order to enhance the response to routinely administered immunotherapies in different patient populations. The object of this work was to investigate the effect of LDL on T cell activation and tumor immunity in-vitro.
Experiments were performed with different LDL dosages (LDLlow = 50 μg/ml and LDLhigh = 200 μg/ml) referring to medium control. T cell phenotype, cytokines and metabolism were analyzed. The functional relevance of our findings was studied in a HCT116 spheroid model in the context of anti-PD-1 blockade.
The key points of our findings showed that LDLhigh skewed the CD4+ T cell subset into a central memory-like phenotype, enhanced the expression of the co-stimulatory marker CD154 (CD40L) and significantly reduced secretion of IL-10. The exhaustion markers PD-1 and LAG-3 were downregulated on both T cell subsets and phenotypical changes were associated with a balanced T cell metabolism, in particular with a significant decrease of reactive oxygen species (ROS). T cell transfer into a HCT116 spheroid model resulted in a significant reduction of the spheroid viability in presence of an anti-PD-1 antibody combined with LDLhigh.
Further research needs to be conducted to fully understand the impact of LDL on T cells in tumor immunity and moreover, to also unravel LDL effects on other lymphocytes and myeloid cells for improving anti-PD-1 immunotherapy. The reason for improved response might be a resilient, less exhausted phenotype with balanced ROS levels.
Copyright © 2023 Babl, Hofbauer, Matos, Voll, Menevse, Rechenmacher, Mair, Beckhove, Herr, Siska, Renner, Kreutz and Schnell.

Synthesis of extracellular adenosine by the ectonucleotidases CD39 and CD73 represents an important pathway of immune suppression in the tumor microenvironment. Using two mouse models (RET transgenic melanoma and Panc02 orthotopic pancreatic adenocarcinoma), we identified an elevated frequency of ectonucleotidase-expressing T cells in tumors and spleens. Importantly, these ectonucleotidase-positive T cells also showed a pronounced expression of PD-1. Conversely, the PD-1+ T cell subsets in tumors contained substantially larger proportions of ectonucleotidase-expressing cells compared to their counterparts lacking PD-1 expression. Our in vitro experiments showed that the activation of normal T cells resulted in an increase in the CD39 expression. CD39+ and CD73+ T cells displayed effector or memory phenotypes and produced IFN-γ, thereby linking ectonucleotidase expression to T cell effector functions. An accumulation of conventional and regulatory T cells expressing CD39 and/or CD73 was also detected in the peripheral blood of patients with melanoma and pancreatic cancer. Moreover, we demonstrated a significant association between low frequencies of circulating CD73+CD8+ T cells and CD73+CD4+ regulatory T cells and better overall survival of melanoma patients. Tumor-derived soluble factors (in particular, TGF-β) significantly enhanced the frequencies of ectonucleotidase-expressing cells in mice. Our findings suggest that the upregulation of ectonucleotidase expression in T cells promotes extracellular adenosine accumulation and represents an important mechanism of homeostatic immune auto-regulation, which could be hijacked by tumors to evade anti-cancer immunity. Targeting CD39 and CD73 can open new avenues for cancer immunotherapy.
© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.