1 apr. 2026
Chen et al. (BioRxiv)
Keywords
● T Cell Dysfunction
● Tumor Immunotherapy
● CRISPR Screening
Main Findings
Aging is associated with a progressive decline in immune function and surveillance, which mirrors a global disproportionate rise in cancer incidence among the elderly population. Functional CD8+ T cell immunity decreases with age, as cancer risk increases, which limits tumor control and efficacy of immune checkpoint blockade (ICB) therapies. The intrinsic and extrinsic factors which constrain the expansion, persistence, and sustained effector function of CD8+ T cells within the tumor microenvironment (TME) of aged individuals remains poorly understood. In this study Chen et al. sought to identify genetic regulators that limit CD8+ T cell function within the aged TME and whether targeting these genes in vivo could restore T cell function while improving responses to ICB therapies.
To evaluate their research question, the authors utilized an in vivo single-cell CRISPR-Cas9 loss-of-function screening platform within a B16-OVA tumor model. A lentiviral CRISPR library was designed to target 60 transcriptional regulators and/or nuclear proteins. Genes of interest were selected based on both literature review and differential gene expression analyses between young and aged endogenous CD8+ T cells as well as OT-1 CD8+ T cells isolated from a B16-OVA tumor model in a study by Chen et al. (2024). The design of a robust CRISPR library was facilitated by the inclusion of 6 single guide RNAs (sgRNAs) per gene with two additional positive controls (Pdcd1 and Ptpn2), 20 non-targeting control (NTC) sgRNAs, and 20 intergenic control (OTC) sgRNAs. Cas9+OT-1 CD45.1+ CD8+ T cells transduced with the designed CRISPR library were transferred intravenously into young (10-weeks) and aged (68-weeks) CD45.2+ wild-type C57BL/6 mice one day prior to B16-OVA tumor implantation. Following 15 days of tumor growth, OT-1 T cells were sorted from tumors and tumor-draining lymph nodes (TDLNs) by mAmetrine expression. Perturb-seq on these recovered OT-1 T cells was performed to integrate single sgRNA CRISPR/Cas9-mediated gene perturbations at single-cell resolution. A set of candidate gene regulators, including Dusp5 and Zfp219, which influence T cell persistence and expansion during tumor progression in aged environments were identified by comparing sgRNA enrichment in both tumor-resident and TDLN-derived OT-1 cells.
Through scRNA-seq analysis, the authors observed that aged tumors display increases in T Effector-Like (TEff-Like) and Tumor-Infiltrating Age-Associated Dysfunctional (TTAD) populations. However, it was found that loss of Dusp5 and Zfp219 reprogrammed CD8+ T cells towards a TEff-Like phenotype, suggesting that loss of these regulators not only increases T cell numbers, but actively reprograms the functional states of CD8+ T cells.
Dusp5 is a nuclear phosphatase which is known to act as a negative feedback regulator of ERK1/2 signalling, where it dephosphorylates ERK and limits downstream signalling and cellular proliferation. When compared to controls, gene expression and pathway analyses demonstrated that Dusp5 knockout T cells were enriched for the expression of genes associated with the cell cycle, hinting at an increased proliferative capacity. Accordingly, the authors observed that Dusp5knockout led to increased ERK phosphorylation and elevated BrdU incorporation within CD8+ T cells, in both young and aged mice. Accumulation at higher frequencies within tumors, elevated IL-2 production, and improved tumor control was also observed, suggesting that modulation of ERK signalling enhances T cell expansion and persistence within the TME.
Zfp219, in contrast, functions as a DNA-binding transcriptional repressor which is a previously unrecognized regulator of CD8+ T cell activity. Zfp219-deficient CD8+T cells exhibited increased expression of cytotoxic effector genes and were enriched for granzyme-mediated cytolysis pathways. ATAC-seq revealed that Zfp219loss increased chromatin accessibility at the Gzma locus in CD8+T cells exclusively in aged mice. Correspondingly, elevated expression of both Gzmaand Gzmb was observed in an age-dependent context within Zfp219 knockout CD8+ TEff-Like cells. The authors demonstrated that Zfp219 loss improved survival in multiple in vivo tumor models, however, this anti-tumor benefit was restricted to aged hosts. In adoptive transfer experiments, young T cells placed into aged hosts upregulated Zfp219expression, but this increase was not observed when aged T cells were transferred into young hosts, suggesting that the aged TME actively imposes dysfunctional programs onto T cells.
Through analysis of human pan-cancer datasets, elevated expression of the Zfp219 human ortholog, ZNF219, in tumor-infiltrating CD8+ T cells was found to be associated with poorer overall and progression-free survival in pre-treatment patients and those receiving anti-PD-1 therapy. Applying this to an in vivo model, the authors found that Zfp219 loss in combination with PD-1 blockade resulted in improved tumor control in aged mice, with corresponding increases in T cell infiltration and frequencies of CD8+ TEff-Like cells. Interestingly, a subset of animals achieved complete tumor clearance and durable protection upon tumor rechallenge suggesting generation of long-term anti-tumor immunity.
Collectively, this preprint indicates that Dusp5 and Zfp219 are new regulators of CD8+ T cells that mediate improved effector differentiation and anti-tumor immunity, highlighting these genes as promising targets to enhance immunotherapy responses in the aged TME.
Limitations
● Although previous scRNA-seq experiments facilitated the identification of sgRNA targets to prioritize in the CRISPR library, the relatively small design constraint of the library limited novel and unbiased discovery of regulators of T cell function. Combinatorial gene perturbations were not assessed, despite T activation and function being governed by interacting and synergistic gene networks rather than single transcriptional regulators and nuclear proteins. Despite the known influence of CD4+ T cell gene perturbations in antitumor immunity and response to ICB therapies, these cells were not evaluated which limits the understanding of how Dusp5 and Zfp219influence broader immune networks.
● There existed several limitations of the model systems used in this study. Experiments relied on an artificial antigen OT-1 transgenic system which evaluates monoclonal high-affinity TCRs specific for the OVA peptide. This antigen system fails to recapitulate low-affinity tumor neoantigens and polyclonal TCR repertoires which are observed in humans, which may overestimate T cell expansion and efficacy to ICB therapies. Additionally, experiments were performed using subcutaneous tumor models of B16-OVA and Lewis Lung Carcinoma (LLC)-OVA which do not recapitulate highly immunosuppressive and complex TMEs, such as those observed in pancreatic cancer. For future experiments, the authors could evaluate whether these findings translate to in vivo orthotopic models of immunosuppressive cancers.
● Despite integration of human data with stratified ZNF219 expression in CD8+ T cells in the presence or absence of PD-1 blockade, the study did not assess other ICBs or combination therapies. Similarly, in vivo experiments solely utilized PD-1 blockade, therefore it remains unclear if targeting Znf219 in combination with other immunotherapeutic strategies would have similar effects. Lastly, the limited in vitro experiments the authors performed were using healthy PBMCs, however, it would be interesting to evaluate these cells derived from young and aged patients with cancer.
Significance/Novelty
This high-quality study combined in vivo CRISPR screening with scRNA-seq to identified key regulators that drive age-associated T cell dysfunction in the TME. Most previous T cell CRISPR screens are conducted in vitro or with in vivo pre-activated cells, which may not capture the physiological priming, activation, and differentiation of T cells. This study represents the first in vivo Perturb-seq experiment in naïve T cells which is highly novel and best recapitulates T cell immunology. Utilizing this technological advancement, the authors uncover Zfp219 as a previously unrecognized regulator of T cell function in the aged TME. Through demonstrating that targeting both Dusp5 and Znf219 can restore antitumor immunity in vivo, and connecting these findings to clinical data, this work provides evidence for the rational development of strategies to enhance immunotherapy responses in aged patients with cancer. Future work may evaluate the role of these key regulators in broader immune regulatory networks and in combination with other immunotherapeutic modalities.
Credit
Reviewed by Matthew Bianca as part of a cross-institutional journal club between the Icahn School of Medicine at Mount Sinai, the University of Oxford, the Karolinska Institute and the University of Toronto.
The author declares no conflict of interests in relation to their involvement in the review.