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Targeting Cbx3/HP1γ Induces LEF-1 and IL-21R to Promote Tumor-Infiltrating CD8 T-Cell Persistence

Immune checkpoint blockade (ICB) relieves CD8+ T-cell exhaustion in most mutated tumors, and TCF-1 is implicated in converting progenitor exhausted cells to functional effector cells. However, identifying mechanisms that can prevent functional senescence and potentiate CD8+ T-cell persistence for ICB non-responsive and resistant tumors remains elusive. We demonstrate that targeting Cbx3/HP1γ in CD8+ T cells augments transcription initiation and chromatin remodeling leading to increased transcriptional activity at Lef1 and Il21r. LEF-1 and IL-21R are necessary for Cbx3/HP1γ-deficient CD8+ effector T cells to persist and control ovarian cancer, melanoma, and neuroblastoma in preclinical models. The enhanced persistence of Cbx3/HP1γ-deficient CD8+ T cells facilitates remodeling of the tumor chemokine/receptor landscape ensuring their optimal invasion at the expense of CD4+ Tregs. Thus, CD8+ T cells heightened effector function consequent to Cbx3/HP1γ deficiency may be distinct from functional reactivation by ICB, implicating Cbx3/HP1γ as a viable cancer T-cell-based therapy target for ICB resistant, non-responsive solid tumors.


INTRODUCTION
Under persistent antigen exposure as in cancer or chronic infections, CD8 + effector T cells enter an altered differentiation program known as T-cell exhaustion (T EX ) (1). In the tumor microenvironment (TME), CD8 + progenitor T EX cells express unique transcription factors as well as those shared with naïve and memory cells including the high mobility group (HMG) transcription factor, T-cell factor 1 (TCF-1, encoded by Tcf7). They have minimal effector capacity but can be reinvigorated by ICB to proliferate and differentiate into terminally exhausted cells having reduced proliferative capacity but efficient tumor killing ability mediated by perforin 1 (PRF1), granzyme B (GrB) and interferon k (INF-k) (2)(3)(4). TCF-1 is implicated in the conversion of CD8 + progenitor T EX to terminally differentiated T EX (5)(6)(7)(8)(9). However, given that only~20% of all solid tumors and primarily those harboring high mutation loads respond to ICB, in addition to the emergence of ICB resistance (10), there is a need to identify novel targets. In humans and mice, TCF-1, and the related lymphoid enhancer-binding factor 1 (LEF-1) are functionally linked during T-cell development in the thymus and the generation of precursor as well as functional memory CD8 + and CD4 + T cells (11)(12)(13)(14)(15)(16)(17)(18)(19). Nevertheless, a role for LEF-1 in tumor response has not been documented despite Lef1 expression being detected in human and mouse stem-like tumor infiltrating lymphocyte (TIL) subsets that also express Tcf7 (7,9,(20)(21)(22).
IL-21R is widely expressed on various innate and adaptive immune cell-lineages including activated CD8 + T, CD4 + T FH and NK cells. During a chronic viral infection or under IL-2-deprived conditions IL-21R signaling is critical for preventing CD8 + T-cell exhaustion (23,24). In acute viral infections, IL-21R signaling is essential for the proliferation and survival of activated CD8 + T cells as well as the generation of long-lived memory cells (25)(26)(27). However, the function of IL-21R signaling in cancer is controversial and not completely understood (28)(29)(30)(31)(32).
Here, we demonstrate that targeting Cbx3/HP1g promotes increased/sustained expression of LEF-1 and IL-21R in CD8 + effector T cells. Cbx3/HP1g deficiency leads to augmented transcription initiation and chromatin remodeling that results in increased transcriptional activity at Lef1 and Il21r. Genetic ablation of Lef1 and Il21r in Cbx3/HP1g-deficient mice causes a loss of tumor CD8 + effector T cells accompanied by the reduction of Ifng, Gzmb and Prf1 expression, which results in uncontrolled ovarian, melanoma and neuroblastoma growth. Our data establish that LEF-1 and IL-21R are necessary for Cbx3/HP1gdeficient CD8 + T cells to maintain their effector capacity and persist in tumors. Our findings together with those of others (22,47,48) illustrate the complex mechanisms governing CD8 + Tcell effector differentiation and function within a given TME. They underscore the need for continuing exploration of novel targets that can reverse CD8 + T-cell dysfunction in ICB resistant and unresponsive solid tumors.

Tumor Cell Lines
The mouse syngeneic ID8 ovarian tumor line was obtained from Dr. Katherine F. Roby under an MTA executed by the University of Kansas Medical Center (56). Dr. Eva M. Schmelz provided the mouse syngeneic MOSE-L TICv ovarian tumor line (57,58). The NB9464 neuroblastoma cell line was a kind gift from Dr. Crystal L. MacKall (Stanford University) (59). The B16-F10 melanoma cell line was purchased from ATCC and tested negative for mycoplasma.
Melanoma. Mice were implanted subcutaneously with B16 tumor cells (1 x 10 5 /mouse in 100µL PBS). Using a digital caliber, B16 tumor size was measured and calculated on day 14 and at 2-day or 3-day intervals until day 20 depending on the size of the tumor, at which time mice were euthanized for analysis.
Neuroblastoma. Mice were implanted subcutaneously with NB-9464 tumor cells (1 x 10 6 /mouse in 100µL PBS). Using a digital caliber, NB-9464 tumor volume was measured and calculated (W x L x 0.4) starting on day 22 and at 2-day intervals until day 30 depending on the size of the tumor, at which time mice were euthanized for analysis.

Co-Culturing Effector Cells With B16 Tumor Cells
Target B16 tumor cells were plated in 12-well plates at a density of 5 x 10 5 cells per well in 750ml of medium (high glucose DMEM, 10% FBS, penicillin/streptomycin, non-essential amino acids, HEPES, L-glutamate and sodium pyruvate). CD8 + or CD4 + effector T cells were added. For ratio of 1:1 (effector: target), 5 x 10 5 CD8 + or CD4 + T cells: 5 x 10 5 B16 cells were cocultured in the same well; ratio 5:1, 2.5 x 10 6 CD8 + or CD4 + T cells: 5 x 10 5 B16 cells. Plates were incubated at 37°CC in 5% CO 2 for 24 hours. Wells were washed to remove non-adherent T cells. Adherent B16 cells were collected and washed with 1 ml cold PBS. Pellets were resuspended in Radio-Immunoprecipitation Assay (RIPA) lysis buffer containing a protease inhibitor cocktail (Roche) and used immediately or stored at -80°CC for further analysis.

Western Blots
Cells (1 x 10 6 ) were lysed with RIPA buffer (Boston BioProducts) containing a protease inhibitor cocktail on ice for 30 minutes. Lysates were centrifuged at 13,000 rpm for 10 minutes at 4°CC. Protein extracts were denatured at 95°CC for 10 minutes, separated by SDS-PAGE, and transferred to PVDF membranes (EMD Millipore). Membranes were probes with primary antibodies. Proteins of interest were detected with HRPconjugated secondary antibodies and the Pierce ™ ECL Western Blotting Substrate (Thermo Fisher Scientific). Membranes were exposed with HyBlot CL Autoradiography films (Denville Scientific, Inc.), and developed with the Kodax X-OMAT 2000 Processor.

Chromatin Immunoprecipitation Followed by qPCR (ChIP-qPCR) and ChIP Followed by Deep Sequencing (ChIP-Seq)
ChIP was performed using the SimpleChIP Kit #9003 according to manufacturer's protocol (Cell Signaling). Briefly, 2 x 10 7 CD8 + T cells were used for each ChIP. Cells were fixed in 1% formaldehyde to cross-link proteins to DNA, then lysed with 500mL of lysis buffer containing 1X ChIP buffer and protease inhibitor cocktail. Chromatin was sheared using a Sonic Dismembrator (Fisher Scientific Model 120) at 120W-20kHz power, at 15 seconds per cycle, 45 seconds break in between, for 3 cycles total. Chromatin was subjected to immunoprecipitation using specific antibodies at 4°C overnight with rotation. Following incubation, ChIP grade protein G magnetic beads were added to each ChIP and incubated for 2 hours at 4°C with rotation. Samples were placed in a magnetic separation rack for 2 minutes each time and washed 3X with high and low salt buffers. Chromatin was eluted from antibody/protein G magnetic beads using 1X ChIP elution buffer at 65°C for 30 minutes. Eluted chromatin was collected and subjected to cross-link reversal using 5M NaCl and 20 mg/ml Proteinase K, incubated for 2 hours at 65°C. After reversal of protein-DNA cross-link, the DNA was purified using DNA purification spin columns and eluted with DNA elution buffer provided in the kit. Purified ChIP DNA was immediately used for qPCR. BioRad hard-shell PCR Plates (BioRad) were used. In each well 2mL of purified ChIP DNA was added in triplicates along with 18ml primer mixture, which consisted of 1mL forward primer (5mM), 1mL reverse primer (5mM), 6mL nuclease-free water, and 10mL of 2X QuaniNova SYBR Green PCR Master Mix (Qiagen) or SimpleChIP Universal qPCR Master Mix (Cell Signaling). The plate was centrifuged at 300 RCF for 1 minute and read in the BioRad CFX384 Real-Time System (BioRad). The following qPCR settings were used: initial denaturation 95°C for 3:00 minutes, denatured at 95°C for 0:15 minutes, annealing and extension at 60°C for 1:00 minute, GOTO step denature and extension for a total of 40 cycles. Quantitative PCR result was analyzed, and the IP efficiency was calculated per formula provided in Simple ChIP Kit #9003 (Cell Signaling). Primers are listed in Table S2.
ChIP-Seq detailed experiments and analyses have been described previously (60).

Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR)
Total RNA was extracted from tumors or activated/differentiated CD8 + T cells using TRIzol (Thermo Fisher Scientific). After DNAse I treatment and RNA cleanup with the RNeasy kit (Qiagen), 1 mg of total RNA was reversed transcribed into cDNA using the Invitrogen SuperScript II reverse transcriptase kit according to manufacturer's recommendations (Thermo Fisher Scientific). The BioRad hard-shell PCR Plates (BioRad) were used. Typically, 5-10 ng of cDNA was added to each well in triplicates along with 7.5mL of the primer mixture, which consisted of 0.5mL forward primer (10mM), 0.5mL reverse primer (10mM), 1.7mL PCR grade water, and 5mL of Roches LightCycler 480 SYBR Green I Master (Roche). The plate was centrifuged at 300 RCF for 1 minute and read in the BioRad CFX384 Real-Time System (BioRad). The qPCR settings: 95°C for 10:00 minutes, 95°C for 0:10 minutes, 62°C for 0:20 minutes, 72°C for 0:20 minutes (Read Plate), GOTO step 2 for 39 times, 92°C for 0:05 minutes, melt curve 65°C to 97°C, at increment of 0.5°C for 1:00 minute (Read Plate), 40°C for 0:10 minutes. Primers are listed in Table S3.

Immunohistochemistry (IHC)
Tumor section processing and TUNEL staining were performed by HistoWiz (Brooklyn, NY). Briefly, all IHC staining is automated using BOND Rx. Slides were treated with Dewax, a solvent-based solution (Leica Biosystems). Tissue sections were fixed by adding 10% neutral buffered formalin for 15 minutes. Slides were treated with Proteinase K at 1:500 dilution. Tissue sections were re-fixed using 10% neutral buffered formalin. Tissue sections were then treated with the equilibration buffer and incubated for 12 minutes at room temperature (RT). Subsequently the TdT reaction mix was added and incubated for 60 minutes at 37°C. The TUNEL reaction was stopped by adding 2X SSC. Tissue slides were treated with Peroxide Block (3-4% Hydrogen Peroxide) followed by wash buffer. Streptavidin HRP was added and incubated for 30 minutes at RT. DAB was added onto the slides and incubated for 10 minutes. Counterstaining was done by adding hematoxylin. Slides were covered using the Sakura Tissue Tek strainer and cover slips then scanned at 40x using the Leica AT2 scanner.

Statistical Analysis
Statistical analysis of tumor growth was determined by the Graphpad Two-Way Anova, for survival the Log-rank (Mantel-Cox) test and for all others the Graphpad unpaired student t test (comparison between 2 groups) or One-Way Anova (multiple comparisons of group means) were performed. The number of mice used were determined using the GraphPad StatMate ® , no animals were excluded from the analysis, and no randomization or blinding was applied.

Cbx3/HP1g Deficiency Modulates LEF-1 and IL-21R Expression in CD8 + Effector T Cells
Previously we showed that germline deletion of Cbx3/HP1g impairs lymphoid-tissue germinal center (GC) reaction and high-affinity antibody response against a thymus (T)dependent antigen (Ag) in a CD8 + T-cell-intrinsic manner (61). Cbx3/HP1g deficiency releases the effector capacity of CD8 + T cells to control neuroblastoma (NBL) growth (60). However, it is not understood why Cbx3/HP1g-deficient CD8 + effector T cells are not subjected to functional senescence and persist in NBL or if they can control tumors with varied mutation loads. To ensure Cbx3/HP1g deletion was restricted to CD8 + T cells, all experiments were performed using the CD8a-Cre strain (52) crossed with our Cbx3/HP1g-floxed mice (resulting animals were Cbx3/HP1g fl/+ and Cbx3/HP1g fl/fl ; collectively designated as Cbx3/HP1g-deficient). Cre recombinase from this CD8a-Cre strain was active in CD8 + CD4 -T cells not in CD4 + CD8or CD11b + cells ( Figure S1A). Ablation of Cbx3/HP1g in CD8 + T cells resulted in a near or complete loss of protein expression and phosphorylation ( Figure S1B). Expression of Bcl6 and Tbx21 (Tbet) (62) mRNAs was increased in Cbx3/HP1g-deficient CD8 + effector T cells over control cells ( Figure S1C). By contrast Eomes transcript and protein levels were reduced ( Figures S1C, D). We observed an upregulation of Prf1, Gzmb and Ifng mRNA expression in Cbx3/HP1g-deficient CD8 + effector T cells compared to control cells ( Figure S1E). More cleaved caspase 3 (CC3) was detected in B16 melanoma tumor cells co-cultured with Cbx3/HP1g-deficient CD8 + effector T cells compared to control co-cultures ( Figure S1F). Cbx3/HP1g deficiency in CD4 + T cells did not enhance tumor cells killing. Thus, after activation, Cbx3/HP1g-deficient CD8 + T cells differentiate into effector-like cells armed with a heightened effector/killing capacity to induce tumor-cell apoptosis in vitro. To determine mechanisms conferring persistence on Cbx3/HP1g-deficient CD8 + effector T cells, ChIP-Seq data was analyzed (60). In wild-type day 5 activated/ differentiated CD8 + T cells, Cbx3/HP1g was bound to the 5' untranslated region (UTR) surrounding transcriptional start sites (TSSs) of Lef1 ( Figures 1A, B) and Table S1). Western immunoblots showed an induction of LEF-1 expression in activated/differentiated control CD8 + T cells; however, its expression was further increased when Cbx3/HP1g was ablated ( Figure 1C). LEF-1 levels in control and Cbx3/HP1g-deficient thymocytes were similar ( Figure S1G). The effects of IL-21 on IL-21R expression are controversial. In humans, IL-21 was shown to induce IL-21R expression on naïve CD8 + T cells (63). In mice, IL-21 exposure led to the downregulation of its receptor expression on all T-cell subsets (64). To mitigate IL-21 confounding effects, all experiments were done without added IL-21. In the absence of exogenous IL-21, activated/differentiated Cbx3/HP1g-deficient CD8 + T cells expressed more IL-21R protein (measured as mean fluorescence intensity or MFI) and transcript, compared to control cells ( Figures 1F-H). There was a steady expansion of Cbx3/HP1gdeficient CD8 + IL-21R + T cells after activation/differentiation while the growth of control cells remained low throughout the activation/ differentiation period ( Figures 1F, I). IL-21R was not detected on thymocytes from control or Cbx3/HP1g-deficient mice ( Figure  S1H). We establish that Cbx3/HP1g deficiency induces the sustained increase of LEF-1 and IL-21R in CD8 + effector T cells as well as enhancing their effector capacity. IL-21R elevated expression likely provides signals mediating Cbx3/HP1g-deficient CD8 + effector T-cell expansion in the absence of exogenous IL-21.

Cbx3/HP1g Regulates Lef1 and Il21r Transcription Initiation and Chromatin Remodeling
The rate of gene expression is governed in part by RNA Pol II initiation, elongation and/or chromatin remodeling. Pol II is phosphorylated at serine 5 (Pol II S5) during initiation of transcription while phosphorylation at serine 2 (Pol II S2) generally indicates chromatin remodeling concomitant with transcriptional elongation. To test whether alterations in transcriptional initiation, elongation and/or chromatin remodeling caused Lef1 and Il21r increased expression in Cbx3/HP1g-deficient CD8 + effector T cells, ChIP-qPCR experiments were performed using ChIP-tested antibodies specific for Pol II S2 or S5, and H3K9me3. These results revealed augmented levels of Pol II S5 in or around TSSs of Lef1 and Il21r loci in Cbx3/HP1g-deficient CD8 + effector T cells compared to control cells (Figures 2A, B). Similar levels of Pol II S2 density were observed in control and Cbx3/HP1g-deficient CD8 + T cells (Figures 2C, D). The levels of H3K9me3 deposition at Lef1 locus was unaltered ( Figure 2E). However, there was a general loss of H3K9me3 around the TSS of Il21r locus in Cbx3/ HP1g-deficient CD8 + effector T cells ( Figure 2F). Thus, genetic deletion of Cbx3/HP1g in CD8 + effector T cells results in enhanced transcription initiation at Lef1 and Il21r loci, with chromatin remodeling activity taking place in an extended region around the TSS of the Il21r locus. These changes likely underpinned the increased and sustained transcriptional activity at Lef1 and Il21r.

Cbx3/HP1g-Deficient CD8 + Effector T Cells Persist and Cause Tumor Rejection
The increased/sustained expression of LEF-1 and IL-21R together with the enhanced effector capacity exhibited by Cbx3/HP1g-deficient CD8 + effector T cells suggest they can persist to control tumor development. Thus, the ability of Cbx3/HP1g-deficient CD8 + T cells to eradicate solid tumors was evaluated using the mouse ID8 or MOSE-L TICv ovarian, B16 melanoma and NB-9464 NBL tumor models. Ovarian and NBL tumors have low mutation rates, no clear defining tumorassociated antigens (TAAs) and are minimally responsive to ICB (65)(66)(67). Accordingly, syngeneic ID8 or MOSE-L TICv ovarian tumor cells were injected intraperitoneally (IP) into control and Cbx3/HP1g-deficient mice. Mice were monitored until abdominal distension was visible, which indicated increased ascites and mirrored metastasis observed in humans. Tumor growth and production of ascites were inhibited in Cbx3/HP1gdeficient mice compared to controls or mice ectopically expressing Cbx3/HP1g driven by the human T-cell-restricted Cd2 promoter (Cbx3/HP1g Tg ) ( Figures 3A, B and S2A, B). As a result, Cbx3/HP1g-deficient mice lived longer than controls ( Figure 3C). Cbx3/HP1g-deficient mice were equally effective in reducing B16 melanoma ( Figure 3D) and NB-9464 NBL tumor burden ( Figure 3F), leading to their increased survival compared to controls ( Figures 3E, G). On day 120 after ID8 injection, there was an enrichment of CD8 + NKG2D + effector T cells and a decrease in CD4 + CD25 + FOXP3 + regulatory T cells (Tregs) in ascites from Cbx3/HP1g-deficient mice compared to control or Cbx3/HP1g Tg animals ( Figures 3H, I and S2C, D). Similarly, enrichment of CD8 + NKG2D + effector T cells and decrease in CD4 + CD25 + FOXP3 + Tregs were observed in B16 melanoma as well as NBL tumors ( Figures 3J-M and S2E-H). Comparable frequencies of NK1.1 + NKG2D + or CD4 + NKG2D + T cells was recovered from tumors of control, Cbx3/HP1g Tg and Cbx3/HP1g-deficient mice ( Figure S3). Expression of Ifng, Gzmb and Prf1 was elevated in B16 melanoma and NBL tumors excised from Cbx3/HP1g-deficient mice compared to controls or Cbx3/ HP1g Tg animals ( Figures S4A-F). TUNEL staining revealed more apoptotic tumor cells (brown) in melanoma and NBL tumors from Cbx3/HP1g-deficient mice compared to controls ( Figures 3N, O). Genetic ablation of Prf1 in Cbx3/HP1gdeficient animals led to uncontrolled B16 melanoma growth and decreased TUNEL positivity indicative of reduced tumorcell apoptosis ( Figures 4SG, H) while NBL tumor burden was incompletely inhibited and tumor-cell death was readily detected (Figures S4I, J). By contrast, Ifng deletion resulted in uncontrolled NBL tumor growth in Cbx3/HP1g-deficient mice ( Figure S4K). Thus, PRF1 and INF-g likely mediate the killing of tumors in Cbx3/HP1g-deficient mice. To show that Cbx3/HP1gdeficient CD8 + effector T cells cause tumor rejection in vivo,    thereafter once per week. NKG2D blockade resulted in uncontrolled tumor growth and decreased survival of treated animals ( Figures S6A-D), accompanied by the reduction of Cbx3/HP1g-deficient CD8 + NKG2D + effector T cells in tumors, to a level similar to PBS-treated control mice ( Figures S6E, F). NKG2D blockade did not affect CD4 + NKG2D + and NK1.1 + NKG2D + T-cell frequencies ( Figures S6G, H). Together, our results demonstrate that Cbx3/HP1g-deficient CD8 + effector T cells expressing NKG2D can persist and cause tumor rejection, irrespective of tumor mutation status. The engagement of NKG2D with its ligands expressed on tumor cells likely facilitates tumor killing by Cbx3/HP1g-deficient CD8 + effector T cells through the enhanced production of PRF1, GrB and INF-g. Additionally, B16 and NBL tumors have differential sensitivity to killing by PRF1 and IFN-g, and surveying for their presence in the TME alone may not reveal the precise mechanism whereby tumor cells are eradicated. Furthermore, deletion of even one Cbx3/HP1g allele is sufficient to control solid tumor growth suggesting that Cbx3/ HP1g is haploinsufficient.
In short, the presence of Cbx3/HP1g-deficient CD8 + effector T cells in the TME induces remodeling of the chemokine/receptor landscape that favors their optimal trafficking into tumors at the expense of CD4 + Tregs.

LEF-1 and IL-21R Are Indispensable for Halting Tumor Growth
To establish that LEF-1 and IL-21R contribute to the control of tumor growth by Cbx3/HP1g-deficient CD8 + effector T cells, compound mutant mice were created ( Figure S7A). In the Cbx3-Lef1-deficient mouse, Lef1 and Cbx3/HP1g deletion was restricted to CD8 + T cells; in the Cbx3-Il21r-deficient mouse, Cbx3/HP1g ablation was restricted to CD8 + T cells while Il21r was deleted in all tissues. LEF-1 and IL-21R expression was abolished in deficient CD8 + T cells compared to control cells ( Figures 5A, B). Loss of one Lef1 or Il21r allele showed a decrease in protein levels that were almost completely abrogated upon deletion of both alleles. Progenitor T-cell development proceeded normally in the thymus of compound mutant animals ( Figures S7B, C). Mesenteric lymph nodes (mLNs) of compound mutant mice displayed normal frequencies of mature CD8 + and CD4 + T cells ( Figures S7D, E). Normal ratios of naïve (CD44 -CD62L + ), effector (CD44 + CD62L -) and memory (CD44 + CD62L + ) T cells were observed in the mLNs of compound mutant animals ( Figures S7F, G). In the mLNs, IL-21R expression was restricted to the CD8 + effector (CD44 + CD62L -) T-cell population and was also dependent on gene dosage ( Figure  S7H). IL-21R was not detected on any CD4 + T-cell populations ( Figure S7I). To determine LEF-1 contribution to tumor control, ID8 (ovarian), B16 (melanoma) or NB-9464 (NBL) tumor cells were injected into Lef1-deficient, Cbx3-Lef1-deficient and control mice. CD8 + T-cell-restricted Lef1 ablation resulted in uncontrolled ovarian, melanoma and NBL tumor growth ( Figures 5C, D, F).
Tumor burden in Lef1-and Cbx3-Lef1-deficient animals was higher than that observed for Cbx3/HP1g-deficient and control mice. Because Il21r was deleted in all tissues, adoptive transfer of activated/differentiated CD8 + T cells from Il21r -/-, Cbx3-Il21rdeficient and control animals was performed. Melanoma and NBL growth proceeded unchecked in tumor-bearing B6.SJL congenic mice treated with control, Il21r -/or Cbx3-Il21r-deficient CD8 + effector T cells compared to animals receiving Cbx3/HP1gdeficient CD8 + effector T cells (Figures 5E, G). Our data establish that LEF-1 and IL-21R are necessary for the optimal control of ovarian, melanoma and NBL tumor growth. Moreover, LEF-1 expression in control CD8 + effector T cells is induced, yet they are less capable of persisting in tumors, suggesting there may be a threshold of expression level required for LEF-1 to function effectively. IL-21R expression is confined to the CD8 + T-cell effector population and its loss following deletion suggest that IL-21R function is essential to this population, which is critical for halting tumor development.

LEF-1 and IL-21R Are Required for CD8 + Effector T Cells to Persist in Tumors
The inability of Lef1-, Il21r-, Cbx3-Lef1-and Cbx3-Il21r-deficient CD8 + T cells to halt tumor development suggests that LEF-1 and  IL-21R are necessary for their persistence in tumors. Analyses of tumors from Cbx3-Lef1-deficient mice revealed that the frequencies of CD8 + NKG2D + T cells were reduced to control levels in all three tumors ( Figures 6A, B, D). CD8 + NKG2D + T cells were also decreased to control levels in B16 tumors from Lef1deficient mice ( Figure 6B). There was no enrichment of CD8 + NKG2D + T cells in B16 or NBL tumors from animals treated with Cbx3-Il21r-deficient CD8 + effector T cells ( Figures 6C, E). Likewise, CD8 + NKG2D + T cells were not augmented in B16 tumors receiving Il21r -/-CD8 + effector T cells ( Figure 6C). CD8 + TIM3 + CXCR5 + (progenitor exhausted) or CD8 + TIM3 + CXCR5 -(terminally exhausted) T-cell populations were not perturbed ( Figure S8). The frequency of endogenous NK1.1 + NKG2D + , CD4 + NKG2D + T and myeloid cells in all tumor types was similar to that of controls ( Figure S9). These results demonstrate that Lef1 and/or Il21r genetic ablation results in the exclusive loss of CD8 + effector T cells in all three tumor types. Our data underscore the fact that LEF-1 and IL-21R are requisite for the persistence of Cbx3/HP1g-deficient CD8 + effector T cells in tumors with varied mutation loads that are ICB responsive (B16 melanoma) or non-responsive (ovarian and NBL).

LEF-1 and IL-21R Are Required to Maintain Effector Activity in Tumors
To show that uncontrolled tumor growth resulted primarily from the loss of CD8 + T-cell effector capacity, RT-qPCR assays were done using tumor RNA samples from compound mutant and control mice. Prf1 and Gzmb levels in melanoma tumors from control and compound mutant mice as well as those treated with Cbx3-Il21r-or Il21r-deficient CD8 + effector T cells were low compared to Cbx3/HP1g-deficient mice (Figures 7A, B). Prf1, Gzmb and Ifng expression in NBL tumors from Cbx3-Lef1deficient mice and those treated with Cbx3-Il21r-deficient CD8 + effector T cells was diminished compared to Cbx3/HP1gdeficient animals ( Figures 7C-E). Next, we asked whether the loss of effector capacity is inherent to Lef1 and/or Il21r deficiency or is caused by extrinsic factors in the TME. RT-qPCR analyses showed that Prf1, Gzmb and Ifng levels were reduced in in vitrogenerated CD8 + effector T cells from compound mutant mice compared to those from Cbx3/HP1g-deficient cells (Figures 7F-H). Therefore, our findings demonstrate that LEF-1 and IL-21R are indispensable for maintaining Prf1, Gzmb and Ifng in tumors. The loss of CD8 + effector T cells likely contributes to the observed blunted effector activity observed in tumors from Lef1-and Cbx3-Lef1-deficient mice as well as those treated with Il21r-or Cbx3-Il21r-deficient CD8 + T cells.

DISCUSSION
We establish that genetic ablation of Cbx3/HP1g induces the elevated, sustained expression of factors conferring both persistence and heightened effector/killing capacity on CD8 + T cells, which in turn enables them to control the growth of diverse tumor types. Once in the TME, Cbx3/HP1g-deficient CD8 + T cells can remodel the chemokine/receptor landscape to ensure their optimal trafficking into tumors at the expense of CD4 + Tregs. Mechanistically, Cbx3/HP1g deficiency allows for a higher rate of transcriptional initiation and chromatin remodeling at Lef1 and Il21r loci. Consequently, Cbx3/HP1g-deficient CD8 + T cells express elevated levels of LEF-1 and IL-21R that can mitigate functional senescence and enable their persistence in tumors without the assistance of ICB.
To date, no essential functions for LEF-1 are identified in CD8 + effector T cells despite Lef1 expression being detected in human and mouse stem-like tumor infiltrating lymphocyte (TIL) subsets that also express Tcf7. Here, we provide compelling evidence that LEF-1 has a critical and non-redundant function in tumor rejection. The exclusive increase of Cbx3/HP1gdeficient CD8 + effector T cells in tumors and their subsequent disappearance when Lef1 is ablated suggest that LEF-1 is requisite for their persistence in varied tumor types irrespective of mutational status. Lef1-and Cbx3-Lef1-deficient mice, in which Tcf7 is not deleted, fail to control tumor growth suggesting that Lef1 function is not redundant. Nevertheless, it is possible that in our models LEF-1 + CD8 + and TCF-1 + CD8 + T cells represent two separate populations with defined functions, and the former subset is endowed with an enhanced proliferative potential, or TCF-1 expression is downregulated in LEF-1 + CD8 + T cells. Our data are distinct to those found with ICB where TCF-1 activity is implicated in the conversion of CD8 + T EX progenitor cells into terminally differentiated effector cells that can control the growth of tumors with high mutation rates. Instead, our findings are consistent with those recently published by Weber and colleagues (78). These authors demonstrate that CAR-T cells, when allowed to rest after receptor signaling is shut off, can recover from exhaustion, and regain their anti-tumor activity, in the absence of ICB or other pharmacological interventions. B-cell proliferation and survival through Wnt signaling (80). In the latter two models, the authors posit that LEF-1 exhibits a more dominant role while TCF-1 function is not sufficient for the differentiation and expansion of the immune-cell populations examined. Our observation that IL-21R + CD8 + effector T cells increase in number after activation and are lost upon Il21r deletion suggest that IL-21R provides survival and expansion signals to these cells. This is in keeping with works showing that during a chronic viral infection or under IL-2-deprived conditions, IL-21R signaling is critical for preventing CD8 + T-cell exhaustion (23,24). In acute viral infections, IL-21R signaling is essential for the proliferation and survival of activated CD8 + T cells as well as the generation of long-lived memory cells (25)(26)(27). In these models, IL-21R can activate the STAT1/STAT3 signaling pathways, which subsequently upregulate pro-survival factors BCL-2 and BCL-X L and downregulate TRAIL (26,27). Furthermore, several lines of evidence indicate that IL-21R is involved in tumor immunity. First, high expression of IL-21R on CD8 + T cells in tumors correlates positively with overall survival and lack of tumor recurrence in hepatocellular carcinoma (HCC) patients (28). Second, in mice, IL-21R signaling reduces accumulation of myeloid derived suppressor cells (MDSCs) in the TME to control rapid HCC growth and maintains an immunological memory response to tumor re-challenge (28). Third, newly diagnosed HER2 + breast cancer patients with higher Il21r expression may have a reduced risk of distant relapse when treated with trastuzumab (anti-HER2/ErB2 mAb) in combination with chemotherapy; IL-21R expression on CD8 + effector T cells, not NK cells, is required for optimal anti-ErB2 mAb efficacy (29). Moreover, similar to HER2 + breast tumors, Il21 is not readily detected in B16 and NBL tumors from control or Cbx3/HP1gdeficient mice. By contrast, IL-21 is visible in subcutaneous tumors (28). Differences in IL-21 expression are also observed in viral infection models. IL-21 is produced during both the acute and chronic phases; however, its levels decrease during the chronic phase (81,82). This reduction is attributed to a contraction in the number of virus specific CD4 + T cells, which are the primary source of IL-21 production. Though reduced, IL-21 produced during the chronic phase can provide help to CD8 + T cells to resolve the infection. Thus, local endogenous IL-21 production in tumors may also be temporal. Because IL-21R expression is induced and sustained in Cbx3/ HP1g-deficient CD8 + effector T cells without exogenous IL-21, we posit that whether IL-21 production is high or low during tumor development, it can still positively affect Cbx3/HP1gdeficient CD8 + T-cell effector functions.
Within tumors, activated tumor-associated-MØs and/or -DCs set up a milieu to be either activating or suppressing through the production of chemokines that attract CD8 + T cells or CD4 + Tregs (77). Our data demonstrate that Cbx3/ HP1g-deficient CD8 + effector T cells can remodel the chemokine landscape favoring their accumulation while preventing CD4 + Tregs from trafficking into tumors. It is tempting to speculate that the imbalance of chemokine production is caused by functional alterations and/or selective expansion of tumor-associated myeloid cells imposed by Cbx3/HP1g-deficient CD8 + effector T cells.
Our results suggest a model whereby Cbx3/HP1g normally restrains the effector and persistence potential of CD8 + T cells, which eventually succumb to functional inactivation and apoptosis thus incapable of controlling tumor development. Removal of Cbx3/HP1g restraint allows CD8 + T cells to become effector-like cells armed with an intrinsic heightened killing and persistence capacity to control tumor growth; LEF-1 function and IL-21R signaling are necessary. Alternatively, Cbx3/ HP1g-deficient CD8 + effector T cells are preferentially recruited to tumors compared to control cells. Moreover, our model does not preclude the possibility that LEF-1 and IL-21R may also be implicated in CD8 + T-cell responses downstream of ICB. Since mouse and human Cbx3/HP1g peptides are 100% identical, we suspect that human Cbx3/HP1g will behave identically to the mouse ortholog: Cbx3/HP1g deficiency would have the same effects on human CD8 + T cells. Ovarian and neuroblastoma are tumors with low mutation rates that respond minimally to ICB, yet their growth is effectively controlled by Cbx3/HP1g-deficient CD8 + effector T cells. Thus, our findings provide a rationale for targeting Cbx3/HP1g in human T cells to treat tumors harboring low mutation loads and do not respond to ICB.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi. nlm.nih.gov/geo/GSE183238.

ETHICS STATEMENT
The animal study was reviewed and approved by BIDMC Institutional Animal Care and Use Committee.