CD8+ T cells differentiate into diverse states that shape immune outcomes in cancer and chronic infection1-4. To define systematically the transcription factors (TFs) driving these states, we built a comprehensive atlas integrating transcriptional and epigenetic data across nine CD8+ T cell states and inferred TF activity profiles. Our analysis catalogued TF activity fingerprints, uncovering regulatory mechanisms governing selective cell state differentiation. Leveraging this platform, we focused on two transcriptionally similar but functionally opposing states that are critical in tumour and viral contexts: terminally exhausted T (TEXterm) cells, which are dysfunctional5-8, and tissue-resident memory T (TRM) cells, which are protective9-13. Global TF community analysis revealed distinct biological pathways and TF-driven networks underlying protective versus dysfunctional states. Through in vivo CRISPR screening integrated with single-cell RNA sequencing (in vivo Perturb-seq) we delineated several TFs that selectively govern TEXterm cell differentiation. We also identified HIC1 and GFI1 as shared regulators of TEXterm and TRM cell differentiation and KLF6 as a unique regulator of TRM cells. We discovered new TEXterm-selective TFs, including ZSCAN20 and JDP2, with no previous known function in T cells. Targeted deletion of these TFs enhanced tumour control and synergized with immune checkpoint blockade but did not interfere with TRM cell formation. Consistently, their depletion in human T cells reduces the expression of inhibitory receptors and improves effector function. By decoupling exhaustion TEX-selective from protective TRM cell programmes, our platform enables more precise engineering of T cell states, accelerating the rational design of more effective cellular immunotherapies.

Exhausted T cells (TEX) in cancer and chronic viral infections undergo metabolic and epigenetic remodeling, impairing their protective capabilities. However, the impact of nutrient metabolism on epigenetic modifications that control TEX differentiation remains unclear. We showed that TEX cells shifted from acetate to citrate metabolism by downregulating acetyl-CoA synthetase 2 (ACSS2) while maintaining ATP-citrate lyase (ACLY) activity. This metabolic switch increased citrate-dependent histone acetylation, mediated by histone acetyltransferase KAT2A-ACLY interactions, at TEX signature-genes while reducing acetate-dependent histone acetylation, dependent on p300-ACSS2 complexes, at effector and memory T cell genes. Nuclear ACSS2 overexpression or ACLY inhibition prevented TEX differentiation and enhanced tumor-specific T cell responses. These findings unveiled a nutrient-instructed histone code governing CD8 + T cell differentiation, with implications for metabolic- and epigenetic-based T cell therapies.

Persistent antigen signaling is known to drive CD8+ T cell exhaustion (TEX) in cancer and chronic infection, but the role of downstream kinase cascades remains unclear. We found that activation of protein kinase C (PKC) triggers degradation of PKC theta while sparing PKC eta, leading to terminal TEX cells. In chronic infection, PKC theta supports progenitor exhausted (TEX-PROG) cells and maintains the antigen-specific T cell response, whereas PKC eta drives terminal exhaustion (TEX-TERM) both in vitro and in vivo. These kinases activate distinct phospho-cascades: PKC theta promotes MAPK and CDK pathways, whereas targets downstream of PKC eta include casein kinase I G2 (CK1G2). An engineered PKC theta variant resistant to degradation, or deletion of CK1G2, enhances CD8+ T cell function and tumor control. These findings reveal that TCR signaling engages distinct phospho-proteomes to regulate effector or exhausted states, opening new therapeutic avenues for T cell engineering and immunotherapy. Supported by NIH/NCI T32CA009370; NIH/NIAID 5R01AI066232; NIH/NCI 5R01CA216101; Cancer Research Institute CRI4859; Damon Runyon Cancer Research Foundation DRG2358-19; Salary support via sponsored research with Arvinas. Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Chronic antigen signaling drives CD8+ T cell exhaustion (TEX) in cancer and chronic infection. However, how the kinase cascades downstream of the T cell receptor drive exhaustion is not understood. We found that continuous agonism of protein kinase C (PKC) causes degradation of PKC theta, but not PKC eta, and induces terminal TEX cells. During chronic infection, PKC theta is necessary to maintain the progenitor exhausted (TEX-PROG) cells, and thus the antigen-specific T cell response, while agonism of PKC eta promotes terminal exhaustion (TEX-TERM) in vitro and in vivo . The cascades downstream of these kinases are distinct, with PKC theta promoting activity of canonical PKC targets in the MAPK and CDK families, while eta promotes activity of other targets, including casein kinase I G2 (CK1G2). Expression of an engineered, degradation-resistant PKC theta, or deletion of the gene encoding CK1G2, improves T cell function and tumor control. Our illustration of multiple therapeutic avenues arising from targeting PKC highlights its centrality in TEX differentiation and its clinical potential in cancer immunotherapy. ### Competing Interest Statement T.H.M. and S.M.K. are coinventors on a provisional patent related to use of degradation-resistant PKC theta or perturbations of CK1G2 activity in adoptive cell transfer therapy. S.M.K. is a scientific advisory board member for Pfizer, EvolveImmune Therapeutics, Arvinas and Affini-T, and an Academic Editor at the Journal of Experimental Medicine . T.H.M., J.F., V.T., M.L., and S.M.K have conducted sponsored research in partnership with Arvinas.

Conventional antiviral memory CD4 T cells typically arise during the first two weeks of acute infection. Unlike most viruses, cytomegalovirus (CMV) exhibits an extended persistent replication phase followed by lifelong latency accompanied with some gene expression. We show that during mouse CMV (MCMV) infection, CD4 T cells recognizing an epitope derived from the viral M09 protein only develop after conventional memory T cells have already peaked and contracted. Ablating these CD4 T cells by mutating the M09 genomic epitope in the MCMV Smith strain, or inducing them by introducing the epitope into the K181 strain, resulted in delayed or enhanced control of viral persistence, respectively. These cells were shown to be unique compared to their conventional memory counterparts; producing higher IFNγ and IL-2 and lower IL-10 levels. RNAseq analyses revealed them to express distinct subsets of effector genes as compared to classical CD4 T cells. Additionally, when M09 cells were induced by epitope vaccination they significantly enhanced protection when compared to conventional CD4 T cells alone. These data show that late-rising CD4 T cells are a unique memory subset with excellent protective capacities that display a development program strongly differing from the majority of memory T cells.

Figure S5 shows human splenic and skin expressions of CCR8

Figure S4 shows donor immune cell reconstitution in the blood post Ccr8-/- donor bone marrow transplant

The same types of cells can assume diverse states with varying functionalities. Effective cell therapy can be achieved by specifically driving a desirable cell state, which requires the elucidation of key transcription factors (TFs). Here, we integrated epigenomic and transcriptomic data at the systems level to identify TFs that define different CD8+ T cell states in an unbiased manner. These TF profiles can be used for cell state programming that aims to maximize the therapeutic potential of T cells. For example, T cells can be programmed to avoid a terminal exhaustion state (TexTerm), a dysfunctional T cell state that is often found in tumors or chronic infections. However, TexTerm exhibits high similarity with the beneficial tissue-resident memory T states (TRM) in terms of their locations and transcription profiles. Our bioinformatic analysis predicted Zscan20 , a novel TF, to be uniquely active in TexTerm. Consistently, Zscan20 knock-out thwarted the differentiation of TexTerm in vivo , but not that of TRM. Furthermore, perturbation of Zscan20 programs T cells into an effector-like state that confers superior tumor and virus control and synergizes with immune checkpoint therapy. We also identified Jdp2 and Nfil3 as powerful TexTerm drivers. In short, our multiomics-based approach discovered novel TFs that enhance anti-tumor immunity, and enable highly effective cell state programming.One sentence summary Multiomics atlas enables the systematic identification of cell-state specifying transcription factors for therapeutic cell state programming.### Competing Interest StatementThe authors have declared no competing interest.

Figure S6 shows lower FC receptor transcription in normal skin versus tumor tissues

The limited efficacy of immunotherapies against glioblastoma underscores the urgency of better understanding immunity in the central nervous system. We found that treatment with degrees cCTLA-4, but not degrees cPD-1, prolonged survival in a mouse model of mesenchymal-like glioblastoma. This effect was lost upon the depletion of CD4+ T cells but not CD8+ T cells. degrees cCTLA-4 treatment increased frequencies of intratumoral IFNy-producing CD4+ T cells, and IFNy blockade negated the therapeutic impact of degrees cCTLA-4. The anti-tumor activity of CD4+ T cells did not require tumor-intrinsic MHC-II expression but rather required conventional dendritic cells as well as MHC-II expression on microglia. CD4+ T cells interacted directly with microglia, promoting IFNy-dependent microglia activation and phagocytosis via the AXL/MER tyrosine kinase receptors, which were necessary for tumor suppression. Thus, degrees cCTLA-4 blockade in mesenchymal-like glioblastoma promotes a CD4+ T cell-microglia circuit wherein IFNy triggers microglia activation and phagocytosis and microglia in turn act as antigen-presenting cells fueling the CD4+ T cell response.

T cells undergo extensive chromatin remodeling over several days following stimulation through the T cell receptor. However, the kinetics and gene loci targeted by early remodeling events within the first 24 hours of T cell priming to orchestrate effector differentiation have not been well described. We identified that chromatin accessibility is rapidly and extensively remodeled within 1 hour of stimulation of naïve CD8+ T cells, leading to increased global chromatin accessibility at many effector T cell-associated genes that are enriched for AP-1, early growth response (EGR), and nuclear factor of activated T cells (NFAT) binding sites, but this short duration of stimulation is insufficient for commitment to clonal expansion in vivo. Sustained 24-hour stimulation led to further chromatin remodeling and was sufficient to enable clonal expansion. These data suggest that the duration of antigen receptor signaling is intimately coupled to chromatin remodeling and activation of genes involved in effector cell differentiation and highlight a potential mechanism that helps CD8+ T cells discriminate between foreign- and self-antigens.

Binding, blocking and Fc engaging characteristics of anti-mouse CCR8 antibodies

Figure S8 shows binding and CD16 cross-linking characteristics of anti-human CCR8 antibodies

During cancer and chronic viral infections, the persistence of antigen progressively causes CD8+ T cells to differentiate into a dysfunctional PD1+ “exhausted” state, with reduced production of inflammatory cytokines relative to effector cells that form during acute infections. Antigen and costimulation signals activate kinase cascades to induce distinct T cell transcription programs, but how T cells distinguish acute and chronic signals to program the exhausted or effector transcriptional states remains poorly understood. We found that members of the protein kinase C (PKC) family function together as a “molecular clock,” sensing acute or chronic agonism to drive distinct transcriptional programs. Continuous stimulation of PKC induces many features of T cell exhaustion, including a loss of production of the cytokines IFNγ and TNF, upregulation of inhibitory receptors and TOX, and altered expression of the proteins in the AP-1 family. Mechanistically, CD8+ T cells express several different PKC proteins, and chronic agonism of PKC leads to degradation of multiple family members and selective maintenance of only one PKC protein, PKC-η. This “PKC switch” alters downstream signaling to support the transcriptional reprogramming of T cells into a terminally exhausted state. In summary, continuous signaling through PKCs causes changes in the output from these kinases initially at the protein level, driving transcriptional changes downstream of PKC targets in the AP-1 transcription factor family and thus allowing further widespread transcriptional and functional changes that characterize T cell exhaustion. Supported by a Damon Runyon Cancer Research Fellowship (DRG-2358-19) and an NIH training grant, T32-CA009370.

Figure S7 shows 4T1 mouse tumor reductions resulting from anti-mCCR8 or anti-mCCR8/anti-mPD1 treatments

Supplemental materials and methods

Figure S2 shows single cell RNAseq analysis of CCR8+ and CCR8- subpopulations of tumor CD4+FOXP3+ T cells

CD8+ T cells provide host protection against pathogens by differentiating into distinct effector and memory cell subsets, but how chromatin is site-specifically remodeled during their differentiation is unclear. Due to its critical role in regulating chromatin and enhancer accessibility through its nucleosome remodeling activities, we investigated the role of the canonical BAF (cBAF) chromatin remodeling complex in antiviral CD8+ T cells during infection. ARID1A, a subunit of cBAF, was recruited early after activation and established de novo open chromatin regions (OCRs) at enhancers. Arid1a deficiency impaired the opening of thousands of activation-induced enhancers, leading to loss of TF binding, dysregulated proliferation and gene expression, and failure to undergo terminal effector differentiation. Although Arid1a was dispensable for circulating memory cell formation, tissue-resident memory (Trm) formation was strongly impaired. Thus, cBAF governs the enhancer landscape of activated CD8+ T cells that orchestrates TF recruitment and activity and the acquisition of specific effector and memory differentiation states.