Relative expression of exhaustion and activation markers on T cells across stimulations of the exhaustion protocol under normoxic or hypoxic conditions, gating strategy for T cell phenotype annotation, and gating strategy for the analysis of CD39+ and TCF7+ T cells.

Relative proteasome activity in iTEX treated with bortezomib, relative T-cell survival following bortezomib exposure, correlation between T-cell viability and proteasome activity under different doses of bortezomib, and the cytotoxic capacity of hCD19-CAR iTEX in the presence of bortezomib against SK-MEL-28 cells engineered to overexpress hCD19.

Experimental design for the induction of an exhausted phenotype in murine OT-I CD8+ T cells, relative expression of co-receptors, exhaustion markers, and TCF7 in murine CD8+ T cells at different time points of the exhaustion protocol, relative RNA expression of Ccl3, Cxcl13, and Entpd1 genes after one or three stimulations under hypoxia, relative proinflammatory cytokine production and secretion in singly-stimulated or continuously-stimulated T cells under hypoxia, relative expression of PRF1 and GZMB in murine CD8+ T cells after one or three stimulations upon hypoxic exposure, and killing capacity of challenged OT-I cells against B16-OVA melanoma cells.

T cell therapies have shown limited success in solid tumors, mainly due to the difficulty of T cells to penetrate the tumor tissue. Here, we develop injectable and viscoelastic click alginate hydrogels for local and sustained delivery of T cells, with the goal to improve T cell administration, viability, proliferation, and persistence in vivo. Oxidized alginate (Alg), functionalized with norbornene and tetrazine for inverse electron demand Diels-Alder covalent click crosslinking, at varying low (1% Alg) and high (2% Alg) alginate concentration were used. 1% Alg hydrogels showed better injectability in a fully crosslinked state, characterized by lower stiffness, larger mesh size, viscoelastic behavior, lower injection forces and higher cell viability upon injection. In vitro experiments demonstrated that 1% Alg supported T cell viability and proliferation, and promoted sustained release for 10 days. Using an in vivo chick chorioallantoic membrane (CAM) model, hydrogel-based T cell administration exhibited better local delivery, proliferation and persistence over time compared to bolus injection, with 1% Alg showing enhanced T cell release compared to 2% Alg. Further, in a murine model with a local injection in the mammary gland, 1% Alg showed enhanced T cell persistence within the mammary gland and high tissue integration. In conclusion, we engineered injectable, viscoelastic click alginate hydrogels that support T cell administration, local injection, viability, proliferation and persistence in vivo, opening future opportunities for spatio-temporal control of T cell immunotherapies.

Puromycin incorporation in iTEX and non-iTEX, and relative RNA expression of target genes belonging to two different branches of the UPR pathway.

Contribution of each study and cancer type to the total pool of CD8+ T cells analyzed in the single-cell dataset.

Maximal respiration, spare respiratory capacity, and ATP-production coupled respiration of T cells across time points and oxygen conditions of the exhaustion protocol, glucose dependence and FAO and AAO capacity in T cells, compensatory glycolysis of T cells, and MitoTracker Deep Red to Green ratio in chronically stimulated T cells under normoxic or hypoxic conditions.

Row-normalized UCell-based scores for pathways across different T cell subtypes and correlations among gene signatures related to exhaustion, hypoxia, stress responses, and the proteasome in CD8+ T cells based on single-cell transcriptomic data.

Normalized RNA expression levels of hypoxia markers and proteasome-related genes in T cells exposed to acute hypoxia or normoxic conditions, proteasome activity in non-activated, singly-stimulated or continuously stimulated T cells under hypoxia, expression of proteasome subunit-encoding genes in murine CD8+ TILs from B16-F10 bearing mice with different expression levels of PD-1 and TIM-3 exhaustion markers, and relative RNA expression of proteasome-specific chaperones in murine TILs classified according to the expression levels of PD-1 and TIM-3 exhaustion markers.

Fluorochrome-conjugated antibodies and fluorescent probes used in flow cytometry, primers used for mRNA quantification by quantitative PCR, antibodies used for immunoblotting, primers used for mouse genotyping, and differentially expressed oxidized proteins in T cells treated with TWS119 or DMSO control.

The posttranslational modification of proteins critically influences many biological processes and is a key mechanism that regulates the function of the RNA-binding protein Hu antigen R (HuR), a hub in liver cancer. Here, we show that HuR is SUMOylated in the tumor sections of patients with hepatocellular carcinoma in contrast to the surrounding tissue, as well as in human cell line and mouse models of the disease. SUMOylation of HuR promotes major cancer hallmarks, namely proliferation and invasion, whereas the absence of HuR SUMOylation results in a senescent phenotype with dysfunctional mitochondria and endoplasmic reticulum. Mechanistically, SUMOylation induces a structural rearrangement of the RNA recognition motifs that modulates HuR binding affinity to its target RNAs, further modifying the transcriptomic profile toward hepatic tumor progression. Overall, SUMOylation constitutes a mechanism of HuR regulation that could be potentially exploited as a therapeutic strategy for liver cancer.

Relative RNA expression of proteasome subunits and assembly-associated proteins in human memory CD8+ T cells treated with TWS119 or DMSO and differentially expressed E3 ubiquitin ligases and deubiquitinases in TWS119-treated versus DMSO control T cells.

Relative RNA expression of CCL3 and CRTAM across stimulations of the exhaustion protocol under hypoxic conditions, dysregulated GSEA pathways related to cell cycle progression and apoptosis between singly-stimulated and continuously stimulated T cells under hypoxia, and DEGs in chronically stimulated T cells under low glucose levels in hypoxic versus normoxic conditions.

Immunoblots of NRF1-, POMP-, and β1-overexpresing T cells, relative proteasome activity of NRF1-OE and POMP-OE T cells compared to controls, killing capacity of NRF1-OE and POMP-OE hCD19-CAR T cells against hCD19-overexpressing SK-MEL-28 melanoma cells, relative frequency of PD-1+TIM-3+LAG-3+CD39+ T cells in β1-OE or control T cells, and relative expression of TOX in β1-OE T cells or mock controls.

Immunoblot analysis of β-catenin in TWS119-treated or DMSO control T cells, NFAT activity assessed by eGFP fluorescence in non-activated or activated TPR cells treated with TWS119 or DMSO control, relative proteasome activity of T cells treated with 20SPA1 or DMSO control, relative protein aggregates accumulation in T cells treated with 20SPA1 or DMSO control, and relative expression of exhaustion markers in 20SPA1-treated or DMSO control T cells.

Chronic T-cell receptor (TCR) stimulation combined with adverse conditions in the tumor microenvironment (TME), such as hypoxia and nutrient deprivation, frequently results in T-cell exhaustion. Exhausted CD8+ T cells (TEX) experience oxidative stress, which causes an accumulation of oxidized proteins within the cells. We hypothesized that oxidized protein formation might exceed proteasomal degradation capacity, leading to their accumulation and impairing T-cell fitness. Single-cell transcriptomics analysis across 16 tumor types revealed increased expression of proteasome genes in TEX compared with non-TEX. In a robust in vitro model for the generation of human TEX, the cells exhibited hallmarks of exhaustion, with higher levels of reactive oxygen species-induced protein oxidation and increased expression of proteasome genes. Pharmacologic and genetic enhancement of proteasome activity delayed the onset of T-cell exhaustion, improved T-cell fitness, and translated into superior antitumor immunity and tumor control. These findings identify proteasome modulation as a promising strategy to counteract TME-driven T-cell dysfunction, potentially overcoming a major obstacle to the efficacy of cell-based immunotherapies in solid tumors. SIGNIFICANCE:Increased proteasome activity serves as a compensatory mechanism to manage oxidative stress, alleviate proteotoxicity, and prevent exhaustion in T cells, offering opportunities for cancer immunotherapy.

Mean percentage of positive cells for each panel and marker.

Proinflammatory cytokine production in T cells at different stages of the exhaustion protocol under normoxia or hypoxia, secretion of proinflammatory cytokines after one or four stimulations under hypoxia with standard or reduced levels of glucose, and in vitro cytotoxicity assay comparing the killing capacity of hCD19-CAR T cells after one or four stimulations under normoxia or hypoxia against Ramos cell line at different effector to target ratios.

PDF file - 537K, This file includes multipanelled figures with their corresponsding legend showing data regarding CD137 expression on other leukocyte types, hypoxia in mouse tumors,functional data on CD137 induction and immune chages uupon anti-CD137 therapy

Experimental workflow for sorting and treating distinct T cell phenotypes with TWS119 or DMSO.