RNA polymerase II pausing factor NELF in CD8+ T cells promotes antitumor immunity

Wu, Bogang; Zhang, Xiaowen; Chiang, Huai-Chin; Pan, Haihui; Yuan, Bin; Mitra, Payal; Qi, Leilei; Simonyan, Hayk; Young, Colin N.; Yvon, Eric; Hu, Yanfen; Zhang, Nu; Li, Rong

doi:10.1038/s41467-022-29869-2

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Published: 20 April 2022

RNA polymerase II pausing factor NELF in CD8⁺ T cells promotes antitumor immunity

Bogang Wu ORCID: orcid.org/0000-0003-1654-198X¹^na1,
Xiaowen Zhang¹^na1,
Huai-Chin Chiang¹,
Haihui Pan¹,
Bin Yuan¹,
Payal Mitra²,
Leilei Qi²,
Hayk Simonyan³,
Colin N. Young³,
Eric Yvon⁴,
Yanfen Hu²,
Nu Zhang ORCID: orcid.org/0000-0001-9695-210X⁵ &
…
Rong Li ORCID: orcid.org/0000-0002-6471-6580¹

Nature Communications volume 13, Article number: 2155 (2022) Cite this article

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Abstract

T cell factor 1 (TCF1) is required for memory and stem-like CD8⁺ T cell functions. How TCF1 partners with other transcription factors to regulate transcription remains unclear. Here we show that negative elongation factor (NELF), an RNA polymerase II (Pol II) pausing factor, cooperates with TCF1 in T cell responses to cancer. Deletion of mouse Nelfb, which encodes the NELFB subunit, in mature T lymphocytes impairs immune responses to both primary tumor challenge and tumor antigen-mediated vaccination. Nelfb deletion causes more exhausted and reduced memory T cell populations, whereas its ectopic expression boosts antitumor immunity and efficacy of chimeric antigen receptor T-cell immunotherapy. Mechanistically, NELF is associated with TCF1 and recruited preferentially to the enhancers and promoters of TCF1 target genes. Nelfb ablation reduces Pol II pausing and chromatin accessibility at these TCF1-associated loci. Our findings thus suggest an important and rate-limiting function of NELF in anti-tumor immunity.

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Introduction

T cells undergo rapid proliferation and acquire effector function after encountering antigens, co-stimulation signals, and inflammatory cytokines^1,2,3. Short-lived effector cells often undergo apoptosis after the clearance of foreign antigens. A small subset of effector cells develops into long-lived memory T cells². Both effector and memory T cells play critical roles in controlling virus infection and tumor outgrowth. However, unlike acute virus infection, tumor antigens often persist in an excessive amount and thus induce an exhaustion phenotype of tumor-reactive T cells⁴. Exhausted T cells, which exhibit upregulation of multiple inhibitory receptors and loss of polyfunctionality, have been the major target for reinvigorating T cell function in anticancer immunotherapy³. Despite clinical success in adoptive cell therapy and immune checkpoint blockade-based therapeutics^5,6, most patients still cannot benefit from these immunotherapies, partly due to the lack of persistent tumor-reactive memory T cells.

Recent studies reported a number of key transcription factors that control memory T cell differentiation, including TCF1⁷, FOXO1⁸, MYB⁹, BACH2¹⁰, and BATF3¹¹. In particular, TCF1 protein, encoded by the gene TCF7, plays a critical role in the regulation of T cell development¹², differentiation¹³, and effector function preservation during exhaustion^14,15. TCF1 promotes chromatin accessibility that favors memory T cell differentiation¹⁶. Meanwhile, TCF1 depletion exacerbates, and enforced TCF1 expression ameliorates T cell exhaustion^15,16. TCF1 also promotes long-term T cell survival by promoting the anti-apoptotic factor BCL2¹⁵ while suppressing the pro-apoptotic factor BIM¹⁶. A TCF1⁺ T cell population is critical in response to immune checkpoint blockade immunotherapy¹⁷. Although the role of TCF1 in the determination of T cell fate is well established, little is known about the partners that facilitate its function in T cell differentiation-related transcriptional programming.

Pol II pausing plays a pivotal role in regulating metazoan gene expression^18,19,20. At the molecular level, promoter-proximal Pol II pausing prevents nucleosome assembly and thus maintains a permissive chromatin structure for future rounds of transcriptional activation in response to environmental stimuli²¹. Recent studies also showed that Pol II pausing and release play a role in transcriptional enhancers²². NELFB is one of the four subunits of the NELF complex that controls Pol II pausing²³. Mouse genetic studies demonstrate that NELFB is indispensable for early embryogenesis²⁴ and mammary gland development²⁵. In addition, NELF is involved in various aspects of adult tissue homeostasis including energy metabolism in the myocardium²⁶, inflammatory responses in macrophages²⁷, myofiber repair after injury²⁸, and maintenance of junctional integrity²⁹. However, the functional significance of NELF-dependent Pol II pausing in the context of T cell function remains unclear.

Here, we define a CD8⁺ T cell-intrinsic role for NELF during antitumor immune response. Using tissue-specific mouse genetic models, we demonstrate that NELFB in CD8⁺ T lymphocytes is important for antitumor immunity. Mechanistically, NELF is associated with TCF1 and facilitates chromatin accessibility at TCF1-bound transcriptional enhancers and promoters. We further establish that ectopic NELFB expression boosts host antitumor immunity in mouse models and the efficacy of CAR-T immunotherapy.

Results

Nelfb is required for antitumor CD8⁺ T cell function

To investigate the role of NELF in mature T cell function, we deleted Nelfb in a mature T cell-specific manner by crossing Nelfb^f/f and distal Lck-Cre (dLck-Cre) mouse strains. Unlike the proximal Lck-Cre system, dLck-Cre is only activated after thymocyte positive selection and therefore has minimal impact on thymocyte development^30,31. NELFB protein levels were significantly reduced in primary CD8⁺ T cells, but not in non-CD8⁺ cells, from Nelfb^f/f,dLck-Cre knockout (hereafter KO) mice versus their Nelfb^f/f counterparts (Supplementary Fig. 1a, b). Levels of the other three NELF subunits were also decreased in KO mice versus the Nelfb^f/f control (Supplementary Fig. 1b), consistent with the previous finding that protein stability of the four NELF subunits is mutually dependent³². KO mice had a slightly lower percentage of CD8⁺ and increased CD4⁺ T cells than Nelfb^f/f controls (Supplementary Fig. 1c). Cell numbers of CD4⁺ subpopulations, including naïve, central memory, effector memory, and regulatory T (Treg) cells, were comparable between Nelfb^f/f and KO (Supplementary Fig. 1d, e). Among CD8⁺ T cells, the number of naïve T cells was comparable between Nelfb^f/f and KO mice (Supplementary Fig. 1f). However, central memory and effector memory T cell populations were significantly reduced in KO mice compared to those in Nelfb^f/f mice (Supplementary Fig. 1f), which could be indicative of a defective response of these mice to environmental antigens.

To investigate the role of NELF in T cell response to tumor growth, we challenged Nelfb^f/f and KO mice with syngeneic mouse mammary tumor cells (E0771 and AT-3). Tumors grew more robustly in KO versus Nelfb^f/f hosts (Fig. 1a–d). Tumor-infiltrating lymphocytes (TILs) contained substantially fewer total leukocytes (Fig. 1e) and CD8⁺ T cells (Fig. 1f) of KO versus Nelfb^f/f tumor-bearing mice. In addition, KO mice had smaller effector memory (Fig. 1g) and proliferative CD8⁺ T cell populations (Fig. 1h). To ascertain intrinsic defects of CD8⁺ T cells from KO mice, we adoptively transferred CD8⁺ T cells from Nelfb^f/f or KO mice to immunodeficient Rag1^−/− recipient mice and subsequently challenged the chimeric mice with E0771 tumors. As expected, mice that received Nelfb^f/f CD8⁺ T cells exhibited a strong antitumor response compared to those that received vehicles (Fig. 1i, j). In contrast, adoptively transferred KO CD8⁺ T cells did not confer any tumor-inhibiting activity versus controls (Fig. 1i, j). Tumors harvested from chimeric mice with KO CD8⁺ T cells had significantly smaller total and effector memory CD8⁺ populations versus those with Nelfb^f/f CD8⁺ T cells (Fig. 1k, l). These findings demonstrate a CD8⁺ T cell-intrinsic function of NELF in antitumor immunity.

**Fig. 1: *Nelfb* is required for CD8⁺ T cell-intrinsic antitumor function.**

Nelfb deletion impairs memory T cell recall response

Memory T cell response is critical for mediating the protective role of vaccination^33,34. To determine the impact of Nelfb KO on memory function during tumor antigen-initiated vaccination, we chose two aggressive, ovalbumin (OVA)-expressing tumor models: lymphoma E.G7-OVA and melanoma B16-OVA (Fig. 2a and Supplementary Fig. 2a). In both tumor models, non-vaccinated (non-vac), tumor-bearing KO mice tended to have worse survival than their Nelfb^f/f counterparts, though the difference was not statistically significant (Fig. 2b and Supplementary Fig. 2b). This is likely because the robust tumor growth outstripped host antitumor immunity. When mice were vaccinated with OVA protein and then challenged with tumor cells, vaccination significantly lengthened survival in both Nelfb^f/f and KO hosts. However, vaccination exhibited substantially greater protection against tumors in Nelfb^f/f than KO mice (p = 0.002 in Fig. 2b, p = 0.01 in Supplementary Fig. 2b). In addition, vaccination markedly increased CD8⁺ T cell abundance in Nelfb^f/f, but not KO, hosts (Fig. 2c), suggesting that Nelfb KO impairs memory T cell response.

**Fig. 2: *Nelfb* deletion in T cells impairs memory response to tumor antigen vaccination.**

To ascertain the role of NELF in memory T cell function, we next used heat-inactivated B16 tumor cells as the vaccine based on an established protocol³⁵. Consistent with the findings from OVA-mediated vaccination, B16 tumor growth was comparable in non-vaccinated Nelfb^f/f and KO mice (Fig. 2d). In contrast, vaccination provided robust survival benefits in Nelfb^f/f, but not in KO, mice (Fig. 2d), further corroborating the importance of NELF in memory T cell response. Following vaccination, tumor-bearing Nelfb^f/f hosts, but not KO ones, displayed significant increases in CD8⁺ T cell abundance in the spleen, and central memory T cells in both spleen and lymph nodes (Supplementary Fig. 2c–e). Taken together, our studies from multiple models of tumor antigen-initiated vaccination demonstrate that NELF plays a pivotal role in promoting tumor antigen-initiated T cell recall response.

Nelfb KO causes functional defects in CD8⁺ T cells

To further characterize CD8⁺ T cell defects in Nelfb KO mice, we sought to define cellular functions affected by Nelfb loss during T cell receptor (TCR) activation. Carboxyfluorescein succinimidyl ester (CFSE) labeling showed that KO CD8⁺ T cells had relatively normal proliferation rates shortly after in vitro TCR activation (days 1–4), but exhibited significant proliferation defects on day 5 (Fig. 3a and Supplementary Fig. 3a). This indicates that Nelfb deletion unlikely directly impaired TCR-activated cell cycle entry. On the other hand, significantly more KO CD8⁺ T cells underwent apoptosis compared to their Nelfb^f/f counterparts as early as 24 hr after TCR activation (Fig. 3b and Supplementary Fig. 3b).

**Fig. 3: *Nelfb* deletion promotes apoptosis and exhaustion of CD8⁺ T cells.**

Interleukin 2 (IL2) is critical for the expansion of activated T cells but also promotes an exhaustion phenotype³⁶. As expected, under prolonged in vitro incubation with IL2, both Nelfb^f/f and KO CD8⁺ T cells gradually increased expression of the immune inhibitory receptors PD1 and TIM3 (Fig. 3c–f). Notably, the magnitude of increase in either PD1/TIM3 single or double-positive exhausted populations was significantly higher in KO versus Nelfb^f/f cells (Fig. 3c–f). Loss of polyfunctionality is a hallmark of reduced memory stem cell population and increased T cell exhaustion³⁷. Extended in vitro proliferation also resulted in a substantially lower abundance of interferon γ (IFNγ) and tumor necrosis factor α (TNFα) double-positive, polyfunctional KO T cells versus Nelfb^f/f (Fig. 3g, h and Supplementary Fig. 3c, d). In aggregate, our data strongly indicate that Nelfb ablation exacerbates CD8⁺ T cell exhaustion during ex vivo expansion.

To confirm the impact of Nelfb deletion on CD8⁺ T cell functionality, we performed single-cell RNA-sequencing (scRNA-seq) using total CD8⁺ T cells isolated from Nelfb^f/f and KO mice. Based on canonical marker gene expression patterns, we identified four major subsets—naïve, memory, exhausted, and senescent populations (Fig. 4a and Supplementary Fig. 4a). Compared to Nelfb^f/f CD8⁺ T cells, KO samples had smaller naïve and memory, but larger exhausted and senescent, cell populations (Fig. 4b and Supplementary Fig. 4b–d). Using an established algorithm for trajectory analysis³⁸, we found two differentiation trajectories initiated from the naïve stage; one leads to the exhausted stage and the other to the senescent stage (Fig. 4c). Pseudotime is a parameter that describes the positioning of individual cells from the differentiation starting point along the specific trajectory, therefore representing the degree of the differentiation from the naïve to terminally differentiated stage³⁹. Pseudotime analysis of the differentiation trajectory clearly showed that for both exhausted and senescent lineages, KO cells had significantly higher pseudotimes versus Nelfb^f/f (Fig. 4d). This indicates that KO cells are further downstream from their differentiation origin and therefore are more terminally differentiated than Nelfb^f/f cells. Together, our data support the notion that NELF prevents precocious terminal differentiation of CD8⁺ T cells.

**Fig. 4: *Nelfb* deletion promotes terminal differentiation of CD8⁺ T cells.**

Preferential NELF-dependent Pol II pausing at TCF1 targets

To elucidate the molecular mechanism by which NELF regulates T cell functionality, we performed NELFB ChIP-seq in primary mouse CD8⁺ T cells. An unbiased analysis of transcription factor binding motif enrichment showed that NELFB chromatin binding overlapped most significantly with that of TCF1 (Fig. 5a)⁴⁰. Of note, NELFB ChIP-seq signals were substantially higher at both enhancers and transcription start sites (TSS) of TCF1 targets^13,41 versus non-TCF1 targets (Fig. 5b). Next, we conducted Pol II ChIP-seq in primary Nelfb^f/f and KO CD8⁺ T cells. We used Pol II pausing index to assess the degree of promoter-proximal enrichment of Pol II, and value D to denote the maximum difference in vertical distance between two cumulative distributions^42,43 (Fig. 5c). In Nelfb^f/f CD8⁺ T cells, TCF1 targets had a significantly higher Pol II pausing index versus non-TCF1 targets (Fig. 5c, D = 0.45736, p < 2.2e-16). Nelfb KO decreased the Pol II pausing index to a greater extent at TCF1 targets (D = 0.3172, p < 2.2e-16) than at non-TCF1 targets (D = 0.13387, p < 2.2e-16, Fig. 5c, Supplementary Fig. 5a). When enhancers and TSS were analyzed separately, the reduction in Pol II binding upon Nelfb KO was more pronounced at TCF1 targets than at TCF1 non-bound targets (Fig. 5d and Supplementary Fig. 5b). Together, our data indicate that NELF-dependent Pol II binding in primary CD8⁺ T cells is preferentially associated with TCF1-bound regulatory regions.

**Fig. 5: NELF-mediated Pol II pausing at *cis*-regulatory elements of TCF1 targets genes.**

NELF regulates chromatin accessibility of TCF1 targets

Pol II accumulation at promoters and enhancers have been implicated in chromatin accessibility^21,44. We, therefore, used an assay for transposase-accessible chromatin sequencing (ATAC-seq) to assess global chromatin openness in primary naive Nelfb^f/f and KO CD8⁺ T cells. Principal component analysis showed that Nelfb^f/f and KO had distinct chromatin opening states (Supplementary Fig. 5c). Three-quarters of the differentially accessible regions exhibited reduced chromatin openness upon Nelfb deletion (Supplementary Fig. 5d), suggesting a role of NELF in promoting chromatin accessibility. Reminiscent of its enrichment in NELFB chromatin binding regions (Fig. 5a), TCF1 binding was the most significantly enriched motif in the chromatin regions with KO-impaired accessibility (Fig. 6a). Nelfb KO-triggered reduction in chromatin openness occurred more at TCF1 targets than non-TCF1 targets, and furthermore, the KO effect was more pronounced at enhancers than TSS of TCF1 targets (Fig. 6b and Supplementary Figs. 5e, 6a, b). These data support the notion that NELF in naïve CD8⁺ T cells preferentially facilitates chromatin accessibility at TSS and to a greater extent, enhancers of TCF1 targets.

**Fig. 6: NELF is associated with TCF1 and facilitates chromatin accessibility and transcriptional program of TCF target genes.**

The propensity of NELF chromatin binding and its action for TCF1 targets prompted us to discern a physical relationship between these two transcription factors. TCF1 protein levels were comparable in naïve Nelfb^f/f and KO CD8⁺ T cells (Supplementary Fig. 6c), suggesting that NELF unlikely impacts TCF1 targets by regulating its expression. Using proximity labeling⁴⁵, we found that NELF was in close proximity with TCF1 (Fig. 6c). As a positive control, similar physical proximity was detected between NELFB and NELFE, another NELF subunit (Fig. 6c). This finding is consistent with the idea that NELF and TCF1 work at a common set of targets to regulate chromatin accessibility in naïve CD8⁺ T cells.

Chromatin accessibility at transcriptionally regulatory regions often precedes transcription of associated genes^46,47. We, therefore, conducted deep RNA-sequencing using Nelfb^f/f and KO CD8⁺ T cells before and after in vitro TCR activation by anti-CD3/CD28 plus IL2. While baseline transcriptomes were similar between Nelfb^f/f and KO cells, TCR-activated transcriptomes of WT and KO cells were quite distinct (Fig. 6d, Supplementary Fig. 6d, and Supplementary Data 1). Of note, gene set enrichment analysis (GSEA) showed that Nelfb deletion was associated with enriched gene signatures for TCF1 deficiency, T cell exhaustion, and aging signature (Fig. 6e). Furthermore, KO cells exhibited reduced gene signatures for memory T cells and fatty acid metabolism, a hallmark of memory T cells⁴⁸ (Fig. 6e). In keeping with our findings, GSEA analysis of published scRNA-seq data from human melanoma showed that high NELFB expression in tumor-infiltrating CD8⁺ cells significantly correlated with gene signatures for memory T cell and fatty acid metabolism⁴⁹ (Fig. 6f). Thus, our data strongly suggest that NELF facilitates TCR-triggered transcription that favors memory T cell fate and mitigates T cell exhaustion and aging.

NELFB overexpression boosts antitumor immunity

To determine whether NELFB overexpression could enhance adaptive immunity, we established a T cell-specific transgenic mouse model (referred to as Tg hereafter, Supplementary Fig. 7a). In addition to elevated NELFB levels (Fig. 7a), the expression of NELFA and NELFC, two other NELF subunits, were also increased in CD8⁺ T cells of Tg mice (Supplementary Fig. 7b), likely through stabilization of the entire NELF complex. In an in vivo competitive assay, KO or Tg CD45.2⁺CD8⁺ T cells were mixed at a 1:1 ratio with their corresponding control CD8⁺ T cells carrying a congenic marker CD45.1⁺, which were subsequently transferred into B16 tumor-bearing recipient mice. Tumor-infiltrating CD8⁺ cells were analyzed 2–3 weeks post-transfer (Fig. 7b). As expected, KO cells were significantly outcompeted by control cells (Fig. 7c, d). In contrast, Tg cells comprised most of the tumor-infiltrating CD8⁺ cell population (Fig. 7e, f), indicating that NELFB-overexpressing T cells are superior to their WT counterparts. When purified WT and Tg CD8⁺ cells were transferred separately into Rag1^−/− immunodeficient hosts followed by E0771 tumor challenge, Tg CD8⁺ T cells again exhibited more potent antitumor activity than their WT counterparts (Fig. 7g–i). Furthermore, mice receiving Tg CD8⁺ cells had significantly more total leukocytes (Fig. 7j) and CD8⁺ TILs (Fig. 7k). NELFB overexpression increased effector memory (Fig. 7l) and proliferative CD8⁺ T cell populations (Fig. 7m) while reducing the exhaustion marker PD1 expression (Fig. 7n). In addition, NELFB overexpression increased both single and double IFNγ⁺/TNFα⁺ CD8⁺ cells (Fig. 7o–q). Collectively, our data suggest that NELF is a rate-limiting factor in boosting CD8⁺ T cell antitumor activity, which likely occurs through reducing T cell exhaustion and increasing memory and polyfunctionality.

**Fig. 7: NELFB overexpression promotes the tumor-killing capacity of CD8⁺ T cells.**

T cell exhaustion and the lack of sustained persistence are the major barriers to successful CAR-T therapy⁵⁰. We, therefore, sought to determine whether human NELFB (hNELFB) can boost T cell functionality in a more clinically relevant setting. We engineered a bi-cistronic CD19-specific CAR vector based on an established CAR construct, anti-CD19-28z⁵¹ (anti-CD19-28z-P2A-hNELFB). Lentivirus-mediated transduction of primary human T cells resulted in overexpression of hNELFB (Supplementary Fig. 8a) without boosting CAR expression or altering the CD4:CD8 ratio during in vitro expansion (Supplementary Fig. 8b–d). In vitro expanded human T cells with hNELFB overexpression displayed an increased percentage of CD62L⁺CD45RA⁺, a reported hallmark shared by naïve and memory stem cells⁵², in both CD4⁺ and CD8⁺ populations (Fig. 8a, b). In a Raji lymphoma model using NSG immunodeficient mice, T cells carrying the parental anti-CD19-CAR-28z vector significantly prolonged the survival of tumor-bearing mice when compared with mice receiving PBS or mock-infected T cells (Fig. 8c). Of note, T cells with anti-CD19-CAR-28z-hNELFB conferred markedly superior host survival benefits over those with the parental CAR-T vector (Fig. 8c). In a solid tumor model, in which CD19 antigen was engineered in human breast cancer cell line MDA-231, host mice receiving hNELFB-expressing CAR-T cells exhibited smaller tumor growth than those with parental CAR-T cells (Supplementary Fig. 8e). Furthermore, compared to parental CAR-T, hNELFB-expressing CAR-T conferred more tumor infiltration of CD8⁺ and CD4⁺ T cells (Supplementary Fig. 8f, g), and higher memory marker CD127 expression and fewer cells with the exhaustion markers TIM3⁺CD39⁺ in both CD8⁺ and CD4⁺ populations (Supplementary Fig. 8h–k). Our data, therefore, provide the proof of principle that hNELFB overexpression could boost CAR-T anticancer immunotherapy.

**Fig. 8: NELFB overexpression boosts human CAR-T efficacy.**

Discussion

In the current study, we defined a CD8⁺ T cell-intrinsic function of the Pol II pausing factor NELF in regulating adaptive immunity. Upon genetic ablation of Nelfb, CD8⁺ T cells exhibit accelerated apoptosis, impaired proliferation, loss of polyfunctionality, and reduced memory cell populations, all of which likely contribute to defective antitumor immune responses. The effect of Nelfb KO on memory T cell response can be attributed to a direct role of NELF in the memory cell population or an indirect consequence of NELF functions in effector T cells, analogous to the findings from the genetic studies of other T cell transcription factors such as Batf and Irf4^53,54. Combining scRNA-seq and differentiation trajectory analyses, we found that T cell-specific deletion of Nelfb led to more terminally differentiated populations while diminishing naïve and memory progenitor pools. These findings are consistent with a recent study using a muscle stem cell (MuSC)-specific Nelfb KO model, in which Nelfb deletion impaired MuSC replenishment in response to repetitive muscle injury due to premature terminal differentiation of myogenic progenitors²⁸. We point out that NELFB protein in CD8⁺ T cells was incompletely depleted in our T cell-specific genetic model, similar to what was observed in other tissue-specific Nelfb KO mouse models^26,27,28. It is conceivable that both the protein amount of individual NELF subunits and the stability of the NELF complex can affect T cell lifespan and functionality. The robust phenotypes of our KO and transgenic mouse models provide compelling evidence for an important and rate-limiting role of NELF in sustaining CD8⁺ cell functions in antitumor immunity.

APEX2-mediated proximal labeling confers cross-linking of neighboring proteins in the radius of 1–10 nm⁵⁵. Therefore, our result strongly suggests physical proximity, but not necessarily direct protein–protein interaction, between NELFB and TCF1. Nevertheless, together with the genome-wide findings, the proximal labeling data are consistent with the notion that NELF and TCF1 are part of the same transcription complex co-occupied at a common set of transcriptional enhancers and promoters in CD8⁺ T cells. It is also worth pointing out that our ATAC-seq was conducted in primary naïve CD8⁺ T cells, whereas bulk RNA-seq was done in CD8⁺ T cells before and after TCR activation. Unlike baseline transcriptomes prior to TCR activation, which were similar between Nelfb^f/f and KO cells, TCR-activated transcriptomes of Nelfb^f/f and KO cells were substantially divergent. Therefore, our data favor the model in which NELF-dependent chromatin openness prior to TCR activation precedes and likely facilitates TCR-stimulated transcription of TCF1 target genes (Fig. 8d).

NELF-dependent Pol II pausing at promoter-proximal regions is known to facilitate transcription by maintaining an accessible chromatin architecture²¹ and stabilizing the transcription initiation complex²⁶. In contrast, relatively little is known about the functional consequence of Pol II accumulation at transcriptional enhancers. Our genome-wide survey of primary CD8⁺ T cells supports the notion that NELF-dependent Pol II accumulation has even stronger effects on chromatin accessibility in enhancers versus promoters. While the exact mechanism by which enhancer-associated Pol II regulates transcription remains to be determined, we envision at least two plausible scenarios: (1) enhancer-associated Pol II may facilitate enhancer–promoter looping/interaction (Fig. 8d); and (2) Pol II-transcribed enhancer RNA could serve as the anchor for additional transcription factors/cofactors^44,56. Histone-based epigenetic programming has been implicated in various stages of T cell development and differentiation⁵⁷. For example, terminally exhausted and stem-like memory T cells have distinctive histone modification and chromatin accessibility profiles⁵⁸. In addition, histone methyltransferase Suv39h1 has been shown to restrain the transcriptional program of memory T cell differentiation by enabling H3K9me3 deposition at memory-related genes⁵⁹. We propose that NELF-mediated Pol II pausing may be part of the epigenetic programming that dictates the differentiation and function of memory T cells.

Our work suggests a strategy to improve outcomes of CD8⁺ T cell-based adoptive cell therapy. NELFB overexpression in both human and mouse T cells significantly enhances antitumor immunity and prolongs the survival of tumor-bearing hosts. Consistent with our findings, TCF1 overexpression has been shown to boost antitumor immunity and thus proposed to enhance CAR-T efficacy⁶⁰. We propose that NELF belongs to an expanding list of transcription factors whose overexpression bolsters CAR-T efficiency^52,61. It will be of interest to determine whether these transcription factors regulate the same or distinct transcriptional programs in T cell-based immunotherapy. The anti-CD19-28z CAR construct we used is more exhaustion-prone than newer generations of anti-CD19 CAR constructs carrying the 4-1BB or ICOS signaling domain⁶². To strengthen the translational potential of NELFB overexpression in boosting adoptive cell therapy, future experiments are needed to test NELFB overexpression in other CAR constructs with tonic signaling and exhaustion-prone phenotypes, such as GD2.28z CAR-T⁶³. Because CD8⁺ T cell exhaustion was observed as a pan-cancer phenotype⁶⁴, NELF function in mitigating immune exhaustion could be applied to multiple cancer types.

In summary, our current study shows that NELF in CD8⁺ T cells works at both enhancers and promoters of TCF1 target genes to potentiate chromatin accessibility and transcriptional activation. We propose that the functional cooperation between NELF-dependent Pol II pausing and TCF1 is a mechanism that controls conversion between memory T cells and differentiated effector cells, ultimately dictating antitumor immunity and efficacy of cell-based immunotherapy against cancer.

Methods

Mice

All animal protocols were approved by the Institutional Animal Care and Use Committee at George Washington University. Nelfb^f/f and distal Lck-cre (dLck-cre) mice were created as previously described^24,65. Nelfb^Tg mice were generated by CRISPR-based gene editing (Cyagen Biosciences). Briefly, the gRNA to ROSA26, a donor vector containing CAG promoter-loxP-Stop-loxP-mouse Nelfb cDNA-polyA, and Cas9 mRNA were co-injected into fertilized mouse eggs to generate targeted knockin offspring. F0 founder animals, identified by PCR followed by sequence analysis, were bred to WT mice and assessed for germline transmission and F1 mouse generation. T cell-specific Nelfb KO or transgenic mice were generated by crossing dLck-cre with Nelfb^f/f or Nelfb^Tg, respectively. The Cre and floxed strain behaved the same as WT B6 mice; we, therefore, used the floxed mice as WT. Rag1^−/− (stock no. 002216), NSG (stock no. 005557), and C57BL/6 CD45.1 congenic mice (stock no. 002014) were purchased from The Jackson Laboratory. Mice that are 10-week-old were considered adults and were used for tumor studies.

In vivo tumor challenge and assessment

For direct tumor challenge, syngeneic mammary tumor E0771 cells (5 × 10⁵ cells) (CH3 Biosystems, 940001) or AT-3 (2 × 10⁵ cells) (a generous gift from Dr. Scott Abrams at Roswell Park Comprehensive Cancer Center)⁶⁶ were inoculated into the fourth mammary fat pad of female C57BL/6 mice.

For adoptive CD8⁺ T cell transfer experiments, total CD8⁺ cells were isolated from mouse spleen using an EasySep™ Mouse CD8⁺ T Cell Isolation Kit (STEMCELL Technologies, 19853). WT/KO (3.5 × 10⁶) or WT/Tg CD8⁺ cells (5 × 10⁵) were transferred by tail vein injection into Rag1^−/− recipients. E0771 (5 × 10⁵ cells) mammary tumor cells were inoculated into the fourth mammary fat pad one day after CD8⁺ adoptive transfer.

Heat-inactivated tumor cell vaccination experiments were done using an established protocol with some modifications³⁵. Briefly, 5 × 10⁶ heat-killed (boiled for 30 min) B16 mouse melanoma cells (ATCC, CRL-6475) were subcutaneously inoculated into the back flanks of mice. Two weeks after the initial vaccination, 5 × 10⁶ heat-killed B16 cells were subcutaneously inoculated into the back flanks of mice as a booster. Following another 2 weeks, 1 × 10⁵ live B16 cells were subcutaneously inoculated into the back flanks of mice and tumor growth was monitored.

OVA immunization was done following a previously published protocol⁶⁷. Briefly, OVA emulsified in CFA (Hooke Laboratories, EK-0301) was subcutaneously injected at two sites on the backs of mice (0.05 ml per site). Two weeks later, OVA emulsified in IFA (Hooke Laboratories, EK-0311) was subcutaneously injected at one site on the backs of mice (0.1 ml) as a booster. Control CFA emulsions (Hooke Laboratories, CK-0301) and IFA emulsions (Hooke Laboratories, CK-0311) were used as the corresponding controls. After at least 2 weeks of booster injection, 2 × 10⁶ E.G7-OVA mouse T cell lymphoma cells (ATCC, CRL-2113) or 2.4 × 10⁶ B16-OVA mouse melanoma cells (generated by Dr. Tyler Curiel’s lab at the University of Texas Health San Antonio) were subcutaneously injected into the backs of mice. All solid tumors were measured by digital calipers (tumor volume = 0.5 × length × width²). Survival analyses used animal death or tumor size >1000 mm³ as endpoints.

For CAR-T experiments, NSG mice were inoculated with Raji lymphoma cells (5 × 10⁵ in 100 μl PBS) through tail vein injections. One day after the tumor challenge, 1 × 10⁵ CAR-T cells in 100 μl PBS were administered through tail vein injections. Survival analyses used animal death or hind limb paralysis as endpoints. MDA-MB-231 human breast cancer cells were infected with lentiviruses expressing human CD19 (pLenti-GIII-EF1a, Abmgood, 154800610695). Cells were then selected by Puromycin, successful CD19 overexpression were confirmed by flow cytometry using an anti-CD19 antibody (Biolegend, 302239). About 4 × 10⁵ MDA-MB231-hCD19 tumor cells were orthotopically injected into NSG mice. After 10 days, tumor sizes were measured and mice were randomized for receiving either mock treatment (PBS), 1.8 × 10⁶ 28z, or 1.8 × 10⁶ 28z-hNELFB CAR-T cells through tail vein injection. Tumor sizes were then monitored.

Flow cytometry

Tumor tissues were first minced and passed through 70 μm filters to obtain single-cell suspension. Cells were then stained with Viability Ghost Dye 510 (Tonbo Biosciences, 13-0870) for 10 min at 4 °C and blocked by anti-CD16/32 (Tonbo Bioscience, 70-0161) (1 to 100 dilution) at 4 °C for 10 min. Cells were further stained with anti-CD45 (Invitrogen, 11-0451-82), anti-CD3 (Tonbo Biosciences, 65-0031-U100), anti-CD8 (BD Pharmingen, 557654), anti-CD44 (BioLegend, 103041), anti-CD62L (BioLegend, 104424), anti-PD1 (BD Biosciences, 563059), anti-TIM3 (BioLegend, 119704), and anti-LAG3 (Invitrogen, 25223182) in staining buffer (2% FBS in PBS). For nuclear transcription factor staining, cells were permeabilized using a FoxP3/transcription factor staining kit (eBioscience, 00-5523-00) and then stained with anti-Ki67 (Invitrogen, 48-5698-82). For cytokine staining, cells were stimulated by anti-CD3/CD28 (Thermo Fisher, 11452D) at 37 °C overnight and then treated with BD GolgiPlug (BD Biosciences, 550583) at 37 °C for 5 h. After surface staining, cells were permeabilized using a BD Cytofix/Cytoperm kit (BD Biosciences, 554714) and stained with anti-IFNγ (BioLegend, 505826) and anti-TNFα (BioLegend, 506314). All antibodies were used at 1:150 dilution. Cells were then fixed with 1% paraformaldehyde and analyzed by BD FACSCelesta. Data were analyzed using BD FACSDiva and FlowJo software.

In vitro CD8⁺ T cell characterization

For apoptosis detection, naïve CD8⁺ T cells were isolated from mouse splenocytes using a negative selection protocol (STEMCELL Technologies, 19858). Cells were then activated by anti-CD3/CD28 (25 μL beads per million cells) (Thermo Fisher, 11452D) following the manufacturer’s instructions. After 24 h of activation, cells were stained using an Annexin V Apoptosis Detection Kit (eBioscience, 88-8007) and analyzed by flow cytometry.

For CFSE labeling experiments, isolated naïve CD8⁺ T cells were first labeled with a CFSE Cell Division Tracker Kit (BioLegend, 50-712-280) and activated by anti-CD3/CD28 (Thermo Fisher, 11452D) for 24 h. The activation beads were then removed by a magnet and recombinant mouse IL2 (R&D SYSTEMS, 402-ML-020) was added to the culture medium (10 ng/ml). Fresh IL2-containing medium was replaced every other day. Cell seeding density was maintained between 0.5–1 × 10⁶/ml. CFSE intensity was examined by flow cytometry.

To characterize cytokine production and exhaustion phenotypes, total CD8⁺ T cells were first isolated from mouse splenocytes (STEMCELL Technologies, 19853). Cells were then activated by anti-CD3/CD28 (Thermo Fisher, 11452D) and subsequently cultured in an IL2-containing (R&D Systems, 402-ML-020) medium. Freshly prepared IL2-containing medium was replaced every other day. Anti-PD1 and anti-TIM3 were stained and examined by flow cytometry at the indicated timepoints. For cytokine production, cells were treated with BD GolgiPlug (BD Biosciences, 550583) at 37 °C for 5 h and permeabilized using a BD Cytofix/Cytoperm kit (BD Biosciences, 554714) and subsequently stained with anti-IFNγ and anti-TNFα.

Bulk RNA-seq and single-cell RNA-seq analyses

For bulk RNA-seq, total CD8⁺ T cells directly isolated from 8-month-old mouse splenocytes were studied in the pre-activation stage. CD8⁺ T activated by anti-CD3/CD28 and subsequently expanded in IL2-containing medium for 8 days were used as the post-activation stages. Total RNA was extracted by an RNeasy Mini Kit (Qiagen, 74104) following the manufacturer’s instructions. RNA samples were processed further as follows. Briefly, about 500 ng total RNA was used for library preparation following the protocol for Illumina TruSeq stranded mRNA-seq. PolyA-containing mRNA were enriched and converted into first-strand cDNA by random primers and reverse transcriptase. Second-strand cDNA were then synthesized and final RNA-seq libraries were generated by PCR. An Illumina HiSeq 3000 platform was used to carry out 50 bp single-read sequencing.

For single-cell RNA-seq, total CD8⁺ T cells were isolated from 10-month-old mouse spleens using a negative selection protocol according to the manufacturer’s instructions (STEMCELL Technologies, 19853). Freshly isolated, >90% viable CD8⁺ T cells were immediately loaded onto a Chromium Next GEM Chip G (10x Genomics, PN-1000127) and processed with the Chromium Controller (10x Genomics), with a target of 3000 cells per sample. Single-cell RNA-seq libraries were built using Chromium Next GEM Single Cell 3′ GEM, Library & Gel Bead Kit v3.1 (10x Genomics, PN-1000128), and barcoded with unique Illumina sample indexes (10x Genomics, PN-120262). Libraries were sequenced on an Illumina Hiseq instrument with a 10x Genomics-compatible configuration, and 50,000 reads per cell were targeted.

Sequencing data were demultiplexed and processed by Cell Ranger (version 3.1.0). STAR⁶⁸ was used to map reads to the mouse reference genome (refdata-cell ranger-mm10-3.0.0). The outputs of individual samples were loaded into the Seurat R package⁶⁹ (version 3.1.5). High-quality cells were filtered based on the number of genes detected (between 1000 and 5000) and the percentage of unique molecular identifiers (UMIs) mapped to mitochondrial genes (<12%). Individual samples were integrated, and principal components were calculated. The first ten principal components were used for cell clustering and tSNE visualization. Data QC analysis for scRNA-seq were included in Supplementary Fig. 9a–d. Bimod test was used for marker gene expression comparison between different populations (Supplementary Fig. 4c, d).

ChIP-seq and ATAC-seq assays

For ChIP-seq assays, cells were first cross-linked using 1% formaldehyde for 10 min and subsequently terminated by 125 mM glycine at room temperature for 5 min. The cross-linking reagent was removed by spinning at 1000 g at 4 °C for 5 min. Cells were then washed with cold PBS three times. From this step until ChIP elution, all buffers were prepared with a freshly added cocktail of protease inhibitors (1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 μg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride). Cells were lysed on ice for 10 min using lysis buffer (5 mM HEPES, pH 7.9, 85 mM KCl, 0.5% Triton X-100). The supernatant was removed after spinning at 1600 × g at 4 °C for 5 min, and pellets were resuspended with nuclei lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS). Chromosomal DNA was sonicated using a probe sonicator on ice, then centrifuged at 14,000 × g for 15 min, and the supernatant saved for immunoprecipitation. 10% of the sonicated DNA was saved as input. Antibodies used for ChIP include anti-Pol II (BioLegend; 664906) and anti-NELFB (Cell Signaling Technology, 14894 S). Sonicated DNA was incubated with antibodies at 4 °C overnight. Dynabeads Protein A (Thermo Fisher Scientific, 10002D) was added the following day and incubated for 2 h. After incubation, Dynabeads was washed as previously described⁴². Samples were subsequently eluted and reverse-cross-linked at 65 °C overnight. Immunoprecipitated chromatin and input chromatin were ethanol-precipitated and used for library construction.

Sequencing and bioinformatic analysis were conducted at the UT Health San Antonio Genome Sequencing Facility. Briefly, the size distribution of ChIP-DNA was checked by Fragment Analyzer High Sensitivity DNA assays (Agilent Technologies). 0.1–20 ng ChIP-DNA (100–400 bps) was used for ChIP-seq library preparation using SwiftBiosciences Accel-NGS 2 S Plus DNA Library Kit (SwiftBiosciences, 21024). ChIP-seq libraries were quantified by Qubit and Bioanalyzer, and then pooled for cBot amplification and sequenced with 50 bp single-read sequencing using an Illumina HiSeq 3000 platform. After sequencing, fastq files were generated with Bcl2fastq2. ChIP-seq data quality was checked by MultiQC (v1.9).

For ATAC-seq assays, freshly isolated naïve CD8⁺ T cells from mouse spleen (STEMCELL Technologies, 19858) were washed and centrifuged at 500 × g for 5 min. Cell pellets were then resuspended in a cryopreservation solution containing FBS and 10% DMSO. Approximately 100,000 frozen cells of each sample were used by the Active Motif to perform ATAC-seq and analyses. Briefly, cells were thawed at 37 °C. Cell nuclei were first isolated to perform Tn5 tagmentation and make libraries of open chromatin, as previously described^70,71. Tagmented DNA was then purified using a MinElute PCR purification kit (Qiagen, 28004), amplified with ten cycles of PCR, and purified using Agencourt AMPure SPRI beads (Beckman Coulter). Afterward, paired-end (PE42) sequencing with a depth of 30 million reads (a total of 60 million reads) was performed using NextSeq 500 (Illumina). FRIP scores and peak counting were used as quality controls. Reads were aligned to the mouse genome (mm9) using the BWA algorithm. Duplicate reads were removed, and only uniquely mapped reads (mapping quality ≥1) and reads mapping as matched pairs were used for further analysis. Peaks were identified using the MACS 2.1.0 algorithm. Peak read numbers correlation were included in Supplementary Fig. 9e. For genomic analyses, active enhancers were defined by H3K27ac binding peaks at non-TSS regions in CD8⁺ T cells, and active promoters/TSS were defined as H3K4me3 peaks that overlap with TSS in CD8⁺ T cells⁷². TCF1 targets were defined using previously published CD8⁺ TCF1 ChIP-seq data⁴¹.

Co-transfer experiment

Splenic CD8⁺ T cells were isolated from congenic marked WT C57BL/6 (carrying CD45.1) or dLck-cre;Nelfb^f/f mice (carrying CD45.2), mixed at a ratio of 50:50, and intravenously transferred into B16 melanoma tumor-bearing Rag1^−/− immunodeficient mice. When comparing WT versus Nelfb-overexpressing cells, WT (CD45.1) or dLck-cre;Nelfb^Tg CD8⁺ (CD45.2) were co-transferred. A total of 2 × 10⁴ cells were transferred in 100 μl PBS injected via the tail vein. Percentages of CD45.1 (BioLegend, 110706) and CD45.2 (BioLegend, 109814) in tumor-infiltrating CD8⁺ cells were assessed by flow cytometry after 2–3 weeks post-transfer.

Proximity labeling and western blotting

APEX2-mediated proximity biotinylation was done as previously described⁷³. Briefly, APEX2-encoding DNA sequences were fused to the mouse Nelfb gene using standard molecular cloning techniques, and the overexpression plasmid was packaged with helper plasmids in 293 T cells to generate lentivirus stocks. Afterward, Jurkat cell lines (ATCC, TIB-152) were infected with the lentivirus and a stable cell line was selected using neomycin. Biotin-phenol labeling was conducted with 30 min incubation in 500 μM biotin-phenol. Cells were then exposed to 1 mM H₂O₂ at room temperature for 1 min. The reactions were then stopped with ice-cold Dulbecco’s phosphate-buffered saline with quenchers (10 mM sodium azide, 10 mM sodium ascorbate, and 5 mM Trolox). Cells were then pelleted by centrifugation and lysed by RIPA lysis buffer containing 1 mM PMSF, 5 mM Trolox, 10 mM sodium ascorbate, and 10 mM sodium azide. A slurry of streptavidin magnetic beads (NEB, S1420S) was incubated with cell lysate and rotated at room temperature for 1 h. The beads were subsequently washed and boiled to elute biotinylated proteins. Cells without H₂O₂ exposure served as negative controls. The resultant protein lysates were analyzed using standard Western blotting techniques. Primary antibodies included anti-TCF1 (CST, 2203 S), anti-NELFE (Proteintech, 10705-1-AP), and anti-GAPDH (Bio-Rad, 12004167). Other antibodies used for Western blotting included anti-NELFB (Cell Signaling Technology, 14894 S), anti-NELFA (Proteintech, 10456-1-AP), and anti-NELFC (Cell Signaling Technology, 12265 S). All antibodies were used at 1:1000 dilution.

CAR-T generation and characterization

A lentiviral vector pELPS-CAR19-28z was a generous gift from Dr. Carl H. June’s lab at the University of Pennsylvania⁵¹. The human NELFB gene coding sequence was inserted downstream of the CAR19-28z-encoding sequence, with the P2A cleavage sequence in between (Gene Universal, Inc.). Ultra-purified high-titer viruses for both unmodified and modified lentiviral vectors were packaged and generated by VectorBuilder Inc.

Freshly isolated (STEMCELL Technologies, 200-0046) or frozen (STEMCELL Technologies, 70024) human peripheral blood pan-T cells were stimulated by Dynabeads™ Human T-Activator CD3/CD28 (Gibco, 11131D) following the manufacturer’s instructions. After 24 h of activation, high-titer CAR virus, recombinant human IL2 protein (Gibco, PHC0027), and polybrene (MilliporeSigma, TR1003G) were added to the wells and spinoculated at 800 × g at 32 °C for 90 min. Infected T cells were expanded in vitro for 10 days in 10 ng/ml human recombinant IL2 (Gibco, PHC0027) containing RPMI (Corning, 45000-412) media before being injected into NSG mice via the tail vein. Subsets of cells were expanded in vitro, stained by anti-CD4 (BioLegend, 317418), anti-CD8 (BioLegend, 344710), anti-CD45RA (BioLegend, 304112), and anti-CD62L (BioLegend, 304806), and analyzed by flow cytometry using BD FACSCelesta. In vitro expanded cells were stained by FITC-labeled human CD19 (ACROBiosystems, CD9HF2H225UG) and subsequently purified by Anti-FITC MicroBeads (Miltenyi Biotec, 130-048-701) before the detection of NELFB overexpression by western blot or CAR expression by FITC-labeled human CD19. All antibodies were used at 1:100 dilution.

Statistics

Mean differences between the two groups were tested using Student’s t-test. Mean differences between three or more groups were tested using one-way ANOVA. Tumor curves were compared using two-way ANOVA followed by multiple comparisons. Two-sided tests were used. Survival analyses were done by Log-rank (Mantel–Cox) and Gehan–Breslow–Wilcoxon tests. GSEA was done using GSEA software⁷⁴. To analyze human melanoma tumor-infiltrating lymphocytes (TIL), we downloaded the single-cell RNA-seq dataset from GSE72056⁴⁹. Activated CD8⁺ TILs (CD8a ≥5 and CD44 ≥2) were used for GSEA as previously described⁷⁵. Population distributions in scRNA-seq data analyses were examined using Chi-square tests. Wilcoxon rank-sum test with continuity correction was used for genomic analysis. Statistics were performed using GraphPad Prism software. p < 0.05 was considered significant.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

The sequencing data that support the findings of this study have been deposited to NCBI Gene Expression Omnibus under accession codes GSE182862. Human melanoma tumor-infiltrating lymphocytes (TIL) data were downloaded from GSE72056⁴⁹. The remaining data were available within the Article, Supplementary Information, or Source Data file. Source data are provided with this paper.

References

Philip, M. & Schietinger, A. CD8+ T cell differentiation and dysfunction in cancer. Nat. Rev. Immunol. 22, 209–223 (2021).
Kaech, S. M. & Cui, W. Transcriptional control of effector and memory CD8+ T cell differentiation. Nat. Rev. Immunol. 12, 749–761 (2012).
Article CAS PubMed PubMed Central Google Scholar
Waldman, A. D., Fritz, J. M. & Lenardo, M. J. A guide to cancer immunotherapy: from T cell basic science to clinical practice. Nat. Rev. Immunol. 20, 651–668 (2020).
Article CAS PubMed PubMed Central Google Scholar
Ando, M., Ito, M., Srirat, T., Kondo, T. & Yoshimura, A. Memory T cell, exhaustion, and tumor immunity. Immunol. Med. 43, 1–9 (2020).
Article PubMed Google Scholar
Morotti, M. et al. Promises and challenges of adoptive T-cell therapies for solid tumours. Br. J. Cancer 124, 1759–1776 (2021).
Article PubMed PubMed Central Google Scholar
Principe, N. et al. Tumor infiltrating effector memory Antigen-Specific CD8+ T Cells predict response to immune checkpoint therapy. Front. Immunol. 11, 2907 (2020).
Article CAS Google Scholar
Zhou, X. et al. Differentiation and persistence of memory CD8+ T cells depend on T cell factor 1. Immunity 33, 229–240 (2010).
Article CAS PubMed PubMed Central Google Scholar
Michelini, R. H., Doedens, A. L., Goldrath, A. W. & Hedrick, S. M. Differentiation of CD8 memory T cells depends on Foxo1. J. Exp. Med. 210, 1189–1200 (2013).
Article CAS PubMed Central Google Scholar
Gautam, S. et al. The transcription factor c-Myb regulates CD8+ T cell stemness and antitumor immunity. Nat. Immunol. 20, 337–349 (2019).
Article CAS PubMed PubMed Central Google Scholar
Roychoudhuri, R. et al. BACH2 regulates CD8+ T cell differentiation by controlling access of AP-1 factors to enhancers. Nat. Immunol. 17, 851–860 (2016).
Article CAS PubMed PubMed Central Google Scholar
Ataide, M. A. et al. BATF3 programs CD8+ T cell memory. Nat. Immunol. 21, 1397–1407 (2020).
Article CAS PubMed Google Scholar
Wang, F. et al. Exploring the stage-specific roles of Tcf-1 in T cell development and malignancy at single-cell resolution. Cell. Mol. Immunol. 18, 644–659 (2021).
Article CAS PubMed Google Scholar
Kim, C., Jin, J., Weyand, C. M. & Goronzy, J. J. The transcription factor TCF1 in T cell differentiation and aging. Int. J. Mol. Sci. 21, 6497 (2020).
Article CAS PubMed Central Google Scholar
Chen, Z. et al. TCF-1-centered transcriptional network drives an effector versus exhausted CD8 T cell-fate decision. Immunity 51, 840–855. e845 (2019).
Article CAS PubMed PubMed Central Google Scholar
Wang, Y. et al. The transcription factor TCF1 preserves the effector function of exhausted CD8 T cells during chronic viral infection. Front. Immunol. 10, 169 (2019).
Article CAS PubMed PubMed Central Google Scholar
Shan, Q. et al. Ectopic Tcf1 expression instills a stem-like program in exhausted CD8+ T cells to enhance viral and tumor immunity. Cell. Mol. Immunol. 18, 1262–1277 (2021).
Article CAS PubMed Google Scholar
Siddiqui, I. et al. Intratumoral Tcf1+ PD-1+ CD8+ T cells with stem-like properties promote tumor control in response to vaccination and checkpoint blockade immunotherapy. Immunity 50, 195–211. e110 (2019).
Article CAS PubMed Google Scholar
Core, L. & Adelman, K. Promoter-proximal pausing of RNA polymerase II: a nexus of gene regulation. Genes Dev. 33, 960–982 (2019).
Article CAS PubMed PubMed Central Google Scholar
Chen, F. X., Smith, E. R. & Shilatifard, A. Born to run: control of transcription elongation by RNA polymerase II. Nat. Rev. Mol. Cell Biol. 19, 464–478 (2018).
Article CAS PubMed Google Scholar
Wissink, E. M., Vihervaara, A., Tippens, N. D. & Lis, J. T. Nascent RNA analyses: tracking transcription and its regulation. Nat. Rev. Genet. 20, 705–723 (2019).
Article CAS PubMed PubMed Central Google Scholar
Gilchrist, D. A. et al. NELF-mediated stalling of Pol II can enhance gene expression by blocking promoter-proximal nucleosome assembly. Genes Dev. 22, 1921–1933 (2008).
Article CAS PubMed PubMed Central Google Scholar
Henriques, T. et al. Widespread transcriptional pausing and elongation control at enhancers. Genes Dev. 32, 26–41 (2018).
Article MathSciNet CAS PubMed PubMed Central Google Scholar
Yamaguchi, Y., Shibata, H. & Handa, H. Transcription elongation factors DSIF and NELF: promoter-proximal pausing and beyond. Biochim. Biophys. Acta 1829, 98–104 (2013).
Article CAS PubMed Google Scholar
Amleh, A. et al. Mouse cofactor of BRCA1 (Cobra1) is required for early embryogenesis. PLoS ONE 4, e5034 (2009).
Article ADS CAS PubMed PubMed Central Google Scholar
Nair, S. J. et al. Genetic suppression reveals DNA repair-independent antagonism between BRCA1 and COBRA1 in mammary gland development. Nat. Commun. 7, 1–9 (2016).
Article CAS Google Scholar
Pan, H. et al. Negative elongation factor controls energy homeostasis in cardiomyocytes. Cell Rep. 7, 79–85 (2014).
Article CAS PubMed PubMed Central Google Scholar
Yu, L. et al. Negative elongation factor complex enables macrophage inflammatory responses by controlling anti-inflammatory gene expression. Nat. Commun. 11, 1–13 (2020).
ADS CAS Google Scholar
Robinson, D. C. et al. Negative elongation factor regulates muscle progenitor expansion for efficient myofiber repair and stem cell pool repopulation. Dev. Cell 56, 1014–1029. e1017 (2021).
Article CAS PubMed PubMed Central Google Scholar
Ou, J. et al. Epithelial NELF guards intestinal barrier function to ameliorate colitis by maintaining junctional integrity. Mucosal Immunol. 15, 279–288 (2021).
Zhang, D. J. et al. Selective expression of the Cre recombinase in late-stage thymocytes using the distal promoter of the Lck gene. J. Immunol. 174, 6725–6731 (2005).
Article CAS PubMed Google Scholar
Zhang, N. & Bevan, M. J. TGF-beta signaling to T cells inhibits autoimmunity during lymphopenia-driven proliferation. Nat. Immunol. 13, 667–673 (2012).
Article CAS PubMed PubMed Central Google Scholar
Liu, X. et al. Dynamic change of transcription pausing through modulating NELF protein stability regulates granulocytic differentiation. Blood Adv. 1, 1358–1367 (2017).
Article CAS PubMed PubMed Central Google Scholar
Butler, N. S., Nolz, J. C. & Harty, J. T. Immunologic considerations for generating memory CD8 T cells through vaccination. Cell. Microbiol. 13, 925–933 (2011).
Article CAS PubMed PubMed Central Google Scholar
van Duikeren, S. et al. Vaccine-induced effector-memory CD8+ T cell responses predict therapeutic efficacy against tumors. J. Immun. 189, 3397–3403 (2012).
Article CAS PubMed Google Scholar
Yoon, T. J. et al. Antitumor immunostimulatory effect of heat-killed tumor cells. Exp. Mol. Med. 40, 130–144 (2008).
Article CAS PubMed PubMed Central Google Scholar
Liu, Y. et al. IL-2 regulates tumor-reactive CD8+ T cell exhaustion by activating the aryl hydrocarbon receptor. Nat. Immunol. 22, 358–369 (2021).
Article CAS PubMed Google Scholar
Yi, J. S., Cox, M. A. & Zajac, A. J. T‐cell exhaustion: characteristics, causes and conversion. Immunology 129, 474–481 (2010).
Article CAS PubMed PubMed Central Google Scholar
Saelens, W., Cannoodt, R., Todorov, H. & Saeys, Y. A comparison of single-cell trajectory inference methods. Nat. Biotechnol. 37, 547–554 (2019).
Article CAS PubMed Google Scholar
Iturri, L. et al. Megakaryocyte production is sustained by direct differentiation from erythromyeloid progenitors in the yolk sac until midgestation. Immunity 54, 1433–1446.e5 (2021).
Chen, E. Y. et al. Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinforma. 14, 1–14 (2013).
Article Google Scholar
Xing, S. et al. Tcf1 and Lef1 transcription factors establish CD8+ T cell identity through intrinsic HDAC activity. Nat. Immunol. 17, 695–703 (2016).
Article CAS PubMed PubMed Central Google Scholar
Zhang, X. et al. Attenuation of RNA polymerase II pausing mitigates BRCA1-associated R-loop accumulation and tumorigenesis. Nat. Commun. 8, 1–12 (2017).
ADS CAS Google Scholar
Chen, F. X. et al. PAF1, a molecular regulator of promoter-proximal pausing by RNA polymerase II. Cell 162, 1003–1015 (2015).
Article CAS PubMed PubMed Central Google Scholar
Mousavi, K. et al. eRNAs promote transcription by establishing chromatin accessibility at defined genomic loci. Mol. Cell 51, 606–617 (2013).
Article CAS PubMed PubMed Central Google Scholar
Ummethum, H. & Hamperl, S. Proximity labeling techniques to study chromatin. Front. Genet. 11, 450 (2020).
Article CAS PubMed PubMed Central Google Scholar
Pálfy, M., Schulze, G., Valen, E. & Vastenhouw, N. L. Chromatin accessibility established by Pou5f3, Sox19b and Nanog primes genes for activity during zebrafish genome activation. PLoS Genet. 16, e1008546 (2020).
Article CAS PubMed PubMed Central Google Scholar
Gennert, D. G. et al. Dynamic chromatin regulatory landscape of human CAR T cell exhaustion. Proc. Natl Acad. Sci. USA 118, e2104758118 (2021).
Article CAS PubMed PubMed Central Google Scholar
Lochner, M., Berod, L. & Sparwasser, T. Fatty acid metabolism in the regulation of T cell function. Trends Immunol. 36, 81–91 (2015).
Article CAS PubMed Google Scholar
Tirosh, I. et al. Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq. Science 352, 189–196 (2016).
Article ADS CAS PubMed PubMed Central Google Scholar
Rafiq, S., Hackett, C. S. & Brentjens, R. J. Engineering strategies to overcome the current roadblocks in CAR T cell therapy. Nat. Rev. Clin. Oncol. 17, 147–167 (2020).
Article PubMed Google Scholar
Milone, M. C. et al. Chimeric receptors containing CD137 signal transduction domains mediate enhanced survival of T cells and increased antileukemic efficacy in vivo. Mol. Ther. 17, 1453–1464 (2009).
Article CAS PubMed PubMed Central Google Scholar
Lynn, R. C. et al. c-Jun overexpression in CAR T cells induces exhaustion resistance. Nature 576, 293–300 (2019).
Article ADS CAS PubMed PubMed Central Google Scholar
Kurachi, M. et al. The transcription factor BATF operates as an essential differentiation checkpoint in early effector CD8+ T cells. Nat. Immunol. 15, 373–383 (2014).
Article CAS PubMed PubMed Central Google Scholar
Yao, S. et al. Interferon regulatory factor 4 sustains CD8+ T cell expansion and effector differentiation. Immunity 39, 833–845 (2013).
Article CAS PubMed Google Scholar
Qin, W., Cho, K. F., Cavanagh, P. E. & Ting, A. Y. Deciphering molecular interactions by proximity labeling. Nat. Methods 18, 133–143 (2021).
Article CAS PubMed Google Scholar
De Santa, F. et al. A large fraction of extragenic RNA pol II transcription sites overlap enhancers. PLoS Biol. 8, e1000384 (2010).
Article CAS PubMed PubMed Central Google Scholar
Chen, Y., Zander, R., Khatun, A., Schauder, D. M. & Cui, W. Transcriptional and epigenetic regulation of effector and memory CD8 T cell differentiation. Front. Immunol. 9, 2826 (2018).
Yao, C. et al. BACH2 enforces the transcriptional and epigenetic programs of stem-like CD8+ T cells. Nat. Immunol. 22, 370–380 (2021).
Article CAS PubMed PubMed Central Google Scholar
Pace, L. et al. The epigenetic control of stemness in CD8+ T cell fate commitment. Science 359, 177–186 (2018).
Article ADS CAS PubMed Google Scholar
Chan, J. D. et al. Cellular networks controlling T cell persistence in adoptive cell therapy. Nat. Rev. Immunol. 21, 769–784 (2021).
Seo, H. et al. BATF and IRF4 cooperate to counter exhaustion in tumor-infiltrating CAR T cells. Nat. Immunol. 22, 983–995 (2021).
Guedan, S. et al. Enhancing CAR T cell persistence through ICOS and 4-1BB costimulation. JCI Insight 3, e96976 (2018).
Long, A. H. et al. 4-1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors. Nat. Med. 21, 581–590 (2015).
Article CAS PubMed PubMed Central Google Scholar
Zheng, L. et al. Pan-cancer single-cell landscape of tumor-infiltrating T cells. Science 374, abe6474 (2021).
Article CAS PubMed Google Scholar
Zhang, N. & Bevan, M. J. TGF-β signaling to T cells inhibits autoimmunity during lymphopenia-driven proliferation. Nat. Immunol. 13, 667–673 (2012).
Article CAS PubMed PubMed Central Google Scholar
Stewart, T. J. & Abrams, S. I. Altered immune function during long-term host-tumor interactions can be modulated to retard autochthonous neoplastic growth. J. Immun. 179, 2851–2859 (2007).
Article CAS PubMed Google Scholar
Jiang, S., Yan, W., Wang, S. E. & Baltimore, D. Let-7 suppresses B cell activation through restricting the availability of necessary nutrients. Cell Metab. 27, 393–403. e394 (2018).
Article CAS PubMed Google Scholar
Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
Article CAS PubMed Google Scholar
Butler, A., Hoffman, P., Smibert, P., Papalexi, E. & Satija, R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat. Biotechnol. 36, 411–420 (2018).
Article CAS PubMed PubMed Central Google Scholar
Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y. & Greenleaf, W. J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat. Methods 10, 1213 (2013).
Article CAS PubMed PubMed Central Google Scholar
Corces, M. R. et al. An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nat. Methods 14, 959–962 (2017).
Article CAS PubMed PubMed Central Google Scholar
He, B. et al. CD8+ T cells utilize highly dynamic enhancer repertoires and regulatory circuitry in response to infections. Immunity 45, 1341–1354 (2016).
Article CAS PubMed PubMed Central Google Scholar
Lam, S. S. et al. Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat. Methods 12, 51–54 (2015).
Article ADS CAS PubMed Google Scholar
Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. USA 102, 15545–15550 (2005).
Article ADS CAS PubMed PubMed Central Google Scholar
Milner, J. J. et al. Runx3 programs CD8+ T cell residency in non-lymphoid tissues and tumours. Nature 552, 253–257 (2017).
Article ADS CAS PubMed PubMed Central Google Scholar

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Acknowledgements

We thank Drs. Tyler Curiel, Carl June, and Scott Abrams for the B16-OVA cell line, anti-CD19 CAR plasmids, and AT-3 cells, respectively. We also thank Dr. Xiujie Sun and Ms. Madeleine Prevost for technical assistance with tail vein injection and mouse genotyping, respectively. The model demonstration was created with BioRender.com. The work was supported by grants to R.L. from NIH (CA246707 and CA220578) and the Keck Foundation, to Y.H. from NIH (CA212674) and the Congressionally Directed Medical Research Program (W81XWH-17-1-0008), to C.N.Y. from NIH (R01HL141393 and R01DK117007). The Genome Sequencing Facility at UT Health San Antonio is supported by NIH-NCI P30 CA054174 (Mays Cancer Center at UT Health San Antonio), an NIH Shared Instrument grant 1S10OD021805-01 (S10 grant), and a CPRIT Core Facility Award (RP160732).

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These authors contributed equally: Bogang Wu, Xiaowen Zhang.

Authors and Affiliations

Department of Biochemistry & Molecular Medicine, The George Washington University, Washington, DC, 20037, USA
Bogang Wu, Xiaowen Zhang, Huai-Chin Chiang, Haihui Pan, Bin Yuan & Rong Li
Department of Anatomy & Cell Biology, The George Washington University, Washington, DC, 20037, USA
Payal Mitra, Leilei Qi & Yanfen Hu
Department of Pharmacology & Physiology, The George Washington University, Washington, DC, 20037, USA
Hayk Simonyan & Colin N. Young
Department of Medicine, The George Washington University Cancer Center School of Medicine & Health Sciences, The George Washington University, Washington, DC, 20037, USA
Eric Yvon
Department of Microbiology, Immunology & Molecular Genetics, University of Texas Health San Antonio, San Antonio, TX, 78229, USA
Nu Zhang

Authors

Bogang Wu
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Xiaowen Zhang
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Huai-Chin Chiang
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Haihui Pan
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Bin Yuan
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Payal Mitra
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Leilei Qi
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Hayk Simonyan
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Colin N. Young
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Eric Yvon
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Yanfen Hu
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Nu Zhang
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Rong Li
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Contributions

R.L. managed and oversaw the overall project. R.L. and B.W. designed the experiments and wrote the manuscript. B.W., X.Z., H-C.C., H.P., B.Y., P.M., L.Q., and H.S. carried out the experiments. B.W., X.Z., C.N.Y., E.Y., Y.H., N.Z., and R.L. analyzed the data.

Corresponding author

Correspondence to Rong Li.

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Competing interests

R.L. and B.W. are co-inventors of a pending patent (application number 62/982,514) filed by The George Washington University on the therapeutic utility of NELFB in CAR-T therapy. The remaining authors declare no competing interests.

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Wu, B., Zhang, X., Chiang, HC. et al. RNA polymerase II pausing factor NELF in CD8⁺ T cells promotes antitumor immunity. Nat Commun 13, 2155 (2022). https://doi.org/10.1038/s41467-022-29869-2

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Received: 20 January 2022
Accepted: 31 March 2022
Published: 20 April 2022
DOI: https://doi.org/10.1038/s41467-022-29869-2

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