Gene Screens in Aged Mice Reveal Two Distinct Routes to Restore Tumor-Fighting T Cells

Two genetic targets that can rejuvenate exhausted tumor-fighting T cells have been identified in aged mice, and strikingly, only one of them works specifically in older hosts. Through genetic perturbation screens performed directly in aged animals, Chen and colleagues pinpointed Dusp5 and Zfp219 as age-associated regulators of anti-tumor immunity. Deleting Dusp5 enhances ERK1/2 phosphorylation and T cell proliferation, while knocking out Zfp219 increases epigenetic accessibility of cytotoxic gene loci. In murine melanoma models, Dusp5 deletion improves tumor elimination in both young and aged hosts, but Zfp219 disruption restores anti-tumor immunity only in aged tumor-bearing mice — a finding that suggests aging may demand distinct therapeutic considerations when designing next-generation immunotherapies.

The work, published in Cell and highlighted in Cell Research by Chris J. Frangieh and Benjamin Izar, tackles a central challenge in cancer immunology: distinguishing what T cells can do from what their macroenvironment and microenvironment actually allow them to do. Aging influences both axes. As immune cells age, they increase expression of genes associated with immune dysfunction and of immune checkpoints that impede effective anti-tumor immunity. At the same time, the tumor immune microenvironment, or TME, is significantly remodeled with age, which may impede the function of tumor-infiltrating T cells. Dissecting the respective contributions of intrinsic T cell aging and the aged environment has potential implications both for understanding the biology of anti-tumor immunity and for improving immunotherapies.

To attack the problem, the researchers combined functional genetic screening with transfer experiments that separately interrogate donor T cell age and recipient host age. They first designed a library of 60 perturbations targeting T cell genes known to be differentially expressed between young and old T cells, with the goal of measuring each gene’s effect on functional restoration of old T cells. The in vivo efficacy evaluation relied on the ovalbumin-expressing B16 melanoma (B16-OVA) tumor model in mice. In this model, the abundance of OT-1 T cells engineered to target B16-OVA should correlate with the ability of each perturbed T cell to expand and persist within the TME and the tumor-draining lymph node.

The first set of impactful genetic knockouts was identified by measuring sgRNA abundance in intratumoral T cells harvested from B16-OVA tumors and in lymph node-resident T cells. This analysis yielded an initial list of targets whose disruption enhances tumor infiltration specifically in aged mice. A subset of these perturbations was enriched in both aged tumors and tumor-draining lymph nodes — Dusp5, Ikzf2, Prdm1, Dusp4 and Bcl6 — while others, including Irf2bp2, Ets1 and Zfp219, were enriched only within aged tumors. These patterns nominate regulators of cellular accumulation, but they do not distinguish proliferation from survival, trafficking and retention. Crucially, increased T cell abundance does not necessarily indicate more effective tumor killing, so the team coupled the screen to single-cell transcriptomic profiling to determine which perturbations favor functionally relevant states.

The single-cell analysis revealed the phenotypic effects of each perturbation, focusing on the ability to induce distinct age-associated states in tumor-infiltrating T cells. It separated dividing, effector-like and several exhaustion-associated states from the tumor-infiltrating age-associated dysfunctional state, termed TTAD. Earlier work had identified TTAD cells as functionally, transcriptionally and epigenetically distinct from conventional exhausted T cells. Disruption of Dusp5, Ets1, Irf2bp2 and Zfp219 favored effector-like programs, helping distinguish promising interventions from perturbations that simply increase proliferation. The divergent effects of the two related phosphatases were particularly informative: Dusp4 loss induces a stress response that is counterproductive for T cell activation, whereas Dusp5 loss enhances cycling and cytolytic programs. Arrayed validation identified perturbation of Dusp5 and Zfp219 as the strongest candidates for improved tumor control, and the findings extended to a second model, the OVA-expressing Lewis lung carcinoma (LLC-OVA).

Reciprocal-transfer experiments then helped separate donor age from the influence of the recipient environment on Zfp219 expression. Young donor T cells showed higher Zfp219 expression in young hosts, whereas aged donor T cells transferred into young hosts expressed levels comparable to endogenous T cells in young recipients. These findings support environmental regulation of Zfp219 expression and argue against donor age as its sole determinant. However, as Frangieh and Izar caution, these expression measurements alone do not establish whether persistent effects of donor aging influence the functional response to Zfp219 deletion.

The applicability of these findings to human cancers is key to clinical translation. ZNF219, the human ortholog of Zfp219, is more highly expressed in intratumoral CD8+ T cells from older patients, and elevated expression is associated with poorer outcomes. In experimental systems, ZNF219 overexpression reduced human T cell persistence in an immunodeficient mouse model, while Zfp219 overexpression impaired tumor control even in young mice. These experiments support the ability of the regulator to constrain T cell function, but they do not establish a causal link showing that inhibiting endogenous ZNF219 will restore human anti-tumor immunity. Moreover, patient age encompasses T cell history, systemic aging and age-associated tumor biology, making simple extrapolation from mice hazardous.

Several limitations constrain how far the conclusions can extend. The focused library leaves many potential regulators unexplored, including genes whose activity changes without altered transcript abundance. Although B16-OVA and LLC-OVA provide distinct tumor contexts, both retain the same foreign antigen and the high-affinity OT-1 receptor, limiting inference about endogenous antigens and polyclonal responses. Short tumor-growth experiments cannot fully capture chronic disease, prior treatment or recurrent cancer, even when rechallenge experiments demonstrate protection. In addition, healthy donor T cells studied under favorable culture conditions and in immunodeficient recipients do not reproduce the combined cellular and environmental constraints encountered in older patients.

The age-specificity of Zfp219 knockout carries a broader lesson for the field. Many current therapeutics development efforts focus on cells isolated from young mice, which tend to be more viable and therefore easier to work with than aged cells. The fact that the benefit of Zfp219 knockout manifests only in old hosts suggests that target discovery could benefit from models specifically designed to mimic the aged tumor microenvironment. Human loss-of-function studies remain a critical step toward establishing therapeutic utility, and a key open question is whether this and similar studies will significantly influence the selection of patients to receive immunotherapies.

Notably, existing clinical evidence indicates that elderly patients with advanced melanoma or kidney cancer have no worse clinical outcomes, and no differences in the rate of major toxicities, compared with younger individuals. Nonetheless, because the study has defined both aging-dependent and aging-independent approaches toward improving immunotherapies, the translational impact may extend beyond a defined patient age group. Future directions include screening a more comprehensive, possibly genome-wide, library to interrogate a much larger set of possible targets, and incorporating restrictive tumor-like growth conditions — hypoxia, low amino acid availability, low glucose and acidosis — into in vitro screening to improve clinical translatability. Identifying patients likely to resist immune checkpoint blockade remains a key challenge, and this work introduces aging as a variable that, in the correct context of aged individuals, may contribute to patient selection.

Subject of Research: Age-specific genetic regulators of anti-tumor T cell immunity identified by in vivo perturbation screening in aged mice

Article Title: Age-specific targets for T cell rejuvenation

Article References: Frangieh, C. J., & Izar, B. (2026). Age-specific targets for T cell rejuvenation. Cell Research. https://doi.org/10.1038/s41422-026-01313-w

Image Credits: AI Generated

DOI: 10.1038/s41422-026-01313-w

Keywords: T cells, aging, cancer immunology, CRISPR screens, Dusp5, Zfp219, tumor microenvironment, melanoma, single-cell transcriptomics, immunotherapy, epigenetics, mouse models