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CD8+T cells with stem cell-like properties (TSCM) sustain adaptive immunity to intracellular pathogens and tumors. However, the developmental origins and chromatin regulatory factors (CRFs) that establish their differentiation are unclear. Using an RNA interference screen of all CRFs we discovered the histone methylase Mll1 was required during T cell receptor (TCR) stimulation for development of a TSCMprecursor state and mature memory (TMEM) cells, but not short-lived or transitory effector cell-like states, in response to viral infections and tumors. Mll1 was essential for widespreadde novodeposition of histone H3 lysine 4 trimethylation (H3K4me3) upon TCR stimulation, which accounted for 70% of all activation-induced sites in mature TMEMcells. Mll1 promoted both H3K4me3 deposition and reduced TCR-induced Pol II pausing at genes whose single-cell transcriptional dynamics explained trajectories into nascent TSCMprecursor states during viral infection. Our results suggest Mll1-dependent control of Pol II elongation and H3K4me3 establishes and maintains differentiation of CD8+TSCMcell states.

HIV gene expression is modulated by the combinatorial activity of the HIV transcriptional activator, Tat, host transcription factors, and chromatin remodeling complexes. To identify host factors regulating HIV transcription, we used specific single-guide RNAs and endonuclease-deficient Cas9 to perform chromatin affinity purification of the integrated HIV promoter followed by mass spectrometry. The scaffold protein, p32, also called ASF/SF2 splicing factor-associated protein, was identified among the top enriched factors present in actively transcribing HIV promoters but absent in silenced ones. Chromatin immunoprecipitation analysis confirmed the presence of p32 on active HIV promoters and its enhanced recruitment by Tat. HIV uses Tat to efficiently recruit positive transcription elongation factor b (p-TEFb) (CDK9/CCNT1) to TAR, an RNA secondary structure that forms from the first 59 bp of HIV transcripts, to enhance RNAPII transcriptional elongation. The RNA interference of p32 significantly reduced HIV transcription in primary CD4 + T cells and in HIV chronically infected cells, independently of either HIV splicing or p32 anti-splicing activity. Conversely, overexpression of p32 specifically increased Tat-dependent HIV transcription. p32 was found to directly interact with Tat’s basic domain enhancing Tat stability and half-life. Conversely, p32 associates with Tat via N- and C-terminal domains. Likely due its scaffold properties, p32 also promoted Tat association with TAR, p-TEFb, and RNAPII enhancing Tat-dependent HIV transcription. In sum, we identified p32 as a host factor that interacts with and stabilizes Tat protein, promotes Tat-dependent transcriptional regulation, and may be explored for HIV-targeted transcriptional inhibition.

The 18S rRNA sequence is highly conserved, particularly at its 3’-end. In contrast, the sequence around the 3’-end is degenerate with similar sites nearby. How RNA is correctly processed by the endonuclease Nob1 is not known, especially because in vitro experiments have shown it to be error-prone. Here we used yeast genetics, biochemistry, and next generation sequencing to investigate a role for Rio1 in monitoring the 3’-end of 18S rRNA. We demonstrate that Nob1 can miscleave its rRNA substrate and that miscleaved rRNA accumulates upon bypassing the Rio1-mediated quality control step, but not in healthy cells with intact quality control mechanisms. Mechanistically, we show that Rio1 binding to miscleaved rRNA is weakened. Accordingly, excess Pno1 results in accumulation of miscleaved rRNA. Ribosomes containing miscleaved rRNA enter the polysomes and produce dominant negative growth defects, suggesting that they cause defects during translation. Our data strongly suggest that ribosome collisions identify these miscleaved 18S rRNA-containing ribosomes as partially functional and target them for degradation. Altogether, the data support a model in which Rio1 inspects the 3’-end of the nascent 18S rRNA, only removing Nob1 and Pno1 from the ribosomes with precisely cleaved 18S rRNA to prevent miscleaved 18S rRNA-containing ribosomes from erroneously engaging in translation, where they induce ribosome collisions. The data also demonstrate how ribosome collisions “purify” the cells of altered ribosomes with different functionalities, with important implications for the concept of ribosome heterogeneity.

T follicular helper (TFH) cells are a specialized subset of CD4 T cells that deliver critical help signals to B cells for the production of high-affinity Abs. Understanding the genetic program regulating TFH differentiation is critical if one wants to manipulate TFH cells during vaccination. A large number of transcription factor (TFs) involved in the regulation of TFH differentiation have been characterized. However, there are likely additional unknown TFs required for this process. To identify new TFs, we screened a large short hairpin RNA library targeting 353 TFs in mice using an in vivo RNA interference screen. Yin Yang 1 (YY-1) was identified as a novel positive regulator of TFH differentiation. Ablation of YY-1 severely impaired TFH differentiation following acute viral infection and protein immunization. We found that the zinc fingers of YY-1 are critical to support TFH differentiation. Thus, we discovered a novel TF involved in the regulation of TFH cells.

Individual naive CD8 T cells activated in lymphoid organs differentiate into functionally diverse and anatomically distributed T cell phylogenies in response to intracellular microbes. During infections that resolve rapidly, including live viral vaccines1, distinct effector (TEFF) and memory (TMEM) cell populations develop that ensure long term immunity2. During chronic infections, responding cells progressively become dysfunctional and “exhaust”3. A diverse taxonomy of TEFF, TMEM and exhausted (TEX) CD8 T cell populations is known, but the initial developmental basis of this phenotypic variation remains unclear4–10. Here, we defined single-cell trajectories and identified chromatin regulators that establish antiviral CD8 T cell heterogeneity using unsupervised analyses of single-cell RNA dynamics11–13 and an in vivo RNAi screen14. Activated naive cells differentiate linearly into uncommitted effector-memory progenitor (EMP) cells, which initially branch into an analogous manifold during either acute or chronic infection. Disparate RNA velocities in single EMP cells initiate divergence into stem, circulating, and tissue-resident memory lineages that generate diverse TMEM and TEX precursor states in specific developmental orders. Interleukin-2 receptor (IL-2R) signals are essential for formation and transcriptional heterogeneity of EMP cells, and promote trajectories toward TEFF rather than TEX states. Nucleosome remodelers Smarca4 and Chd7 differentially promote transcription that delineates divergent TMEM lineages before cooperatively driving terminal TEFF cell differentiation. Thus, the lineage architecture is established by specific chromatin regulators that stabilize diverging transcription in uncommitted progenitors.

BRD4 is a bromodomain-containing protein that binds acetylated histones to regulate transcription. In this issue of JEM, Milner et al. show that BRD4 plays a critical role in the effector function of CD8 T cells responding to infection and cancer., BRD4 is a bromodomain-containing protein that binds acetylated histones to regulate transcription. In this issue of JEM, Milner et al. (2021. J. Exp. Med. https://doi.org/10.1084/jem.20202512) show that BRD4 plays a critical role in the effector function of CD8 T cells responding to infection and cancer.

Tissue-resident memory CD8+ T (TRM) cells are found at common sites of pathogen exposure, where they elicit rapid and robust protective immune responses. However, the molecular signals that control TRM cell differentiation and homeostasis are not fully understood. Here we show that mouse TRM precursor cells represent a unique CD8+ T cell subset that is distinct from the precursors of circulating memory cell populations at the levels of gene expression and chromatin accessibility. Using computational and pooled in vivo RNA interference screens, we identify the transcription factor Runx3 as a key regulator of TRM cell differentiation and homeostasis. Runx3 was required to establish TRM cell populations in diverse tissue environments, and supported the expression of crucial tissue-residency genes while suppressing genes associated with tissue egress and recirculation. Furthermore, we show that human and mouse tumour-infiltrating lymphocytes share a core tissue-residency gene-expression signature with TRM cells that is associated with Runx3 activity. In a mouse model of adoptive T cell therapy for melanoma, Runx3-deficient CD8+ tumour-infiltrating lymphocytes failed to accumulate in tumours, resulting in greater rates of tumour growth and mortality. Conversely, overexpression of Runx3 enhanced tumour-specific CD8+ T cell abundance, delayed tumour growth, and prolonged survival. In addition to establishing Runx3 as a central regulator of TRM cell differentiation, these results provide insight into the signals that promote T cell residency in non-lymphoid sites, which could be used to enhance vaccine efficacy or adoptive cell therapy treatments that target cancer.

Human immunodeficiency virus (HIV) transcription requires assembly of cellular transcription factors at the human immunodeficiency virus type 1 (HIV-1) promoter. The TFIIH general transcription factor facilitates transcription initiation by opening the DNA strands around the transcription start site and phosphorylating the C-terminal domain of RNA polymerase II (RNAPII) for activation. Spironolactone (SP), an FDA approved aldosterone antagonist, triggers the proteasomal degradation of the XPB subunit of TFIIH and concurrently suppresses acute HIV infection in vitro. Here, we investigated SP as a possible block-and-lock agent for a functional cure, aimed at the transcriptional silencing of the viral reservoir. The long-term activity of SP was investigated in primary and cell line models of HIV-1 latency and reactivation. We show that SP rapidly inhibits HIV-1 transcription by reducing RNAPII recruitment to the HIV-1 genome. Short hairpin RNA (shRNA) knockdown of XPB confirmed XPB loss as the mechanism of action of HIV inhibition. Unfortunately, long-term pretreatment with SP does not result in long-term epigenetic suppression of HIV upon SP treatment interruption, since the virus rapidly rebounds when XPB reemerges; however, SP alone without antiretroviral therapy (ART) maintains transcriptional silencing of HIV. Importantly, SP inhibits HIV reactivation from latency in both cell line models and resting CD4(+) T cells isolated from aviremic individuals living with HIV upon cell stimulation with latency-reversing agents. Furthermore, long-term treatment with concentrations of SP that potently degrade XPB does not lead to global dysregulation of cellular mRNA expression. Overall, these results suggest that XPB plays a key role in HIV transcriptional regulation, and XPB degradation by SP strengthens the potential of HIV transcriptional inhibitors in block-and-lock cure approaches. IMPORTANCE Antiretroviral therapy (ART) effectively reduces an individual’s HIV loads to below the detection limit; nevertheless, rapid viral rebound immediately ensues upon treatment interruption. Furthermore, virally suppressed individuals experience chronic immune activation from ongoing low-level viral expression. Thus, the importance of identifying novel therapeutics to explore in block-and-lock HIV functional cure approaches, aimed at the transcriptional and epigenetic silencing of the viral reservoir to block reactivation from latency, is apparent. We investigated the potential of repurposing the FDA-approved spironolactone (SP) as one such drug. SP treatment rapidly degrades a host transcription factor subunit, XPB, inhibiting HIV transcription and blocking reactivation from latency. Long-term SP treatment does not affect cellular viability, cell cycle progression, or global cellular transcription. SP alone blocks HIV transcription in the absence of ART but does not delay rebound upon drug removal, as XPB rapidly reemerges. This study highlights XPB as a novel drug target in block-and-lock therapeutic approaches.

MDR1 is considered a dedicated drug efflux pump in tumor cells. Chen et al. show that endogenous MDR1 expression in CD8+ T cells is required for immunity to pathogens, revealing new concepts in cell-mediated immunity and informing use of MDR1 inhibitors in human cancer trials., Multidrug resistance-1 (MDR1) acts as a chemotherapeutic drug efflux pump in tumor cells, although its physiological functions remain enigmatic. Using a recently developed MDR1-knockin reporter allele (Abcb1aAME), we found that constitutive MDR1 expression among hematopoietic cells was observed in cytolytic lymphocytes—including CD8+ cytotoxic T lymphocytes (CTLs) and natural killer cells—and regulated by Runt-related (Runx) transcription factors. Whereas MDR1 was dispensable for naive CD8+ T cell development, it was required for both the normal accumulation of effector CTLs following acute viral infection and the protective function of memory CTLs following challenge with an intracellular bacterium. MDR1 acted early after naive CD8+ T cell activation to suppress oxidative stress, enforce survival, and safeguard mitochondrial function in nascent CTLs. These data highlight an important endogenous function of MDR1 in cell-mediated immune responses and suggest that ongoing efforts to intentionally inhibit MDR1 in cancer patients could be counterproductive., Graphical Abstract

In response to infection and malignancy, antigen-specific CD8 T cells differentiate into functionally diverse populations that are critical for resolving disease and providing durable immunity. Here, we utilized an in vivo RNAi screening strategy to identify transcriptional and epigenetic regulators controlling CD8 T cell fate. Through small-molecule inhibition, RNAi studies, and induced genetic deletion, we revealed an essential role for the chromatin modifier and BET family member BRD4 in supporting T cell differentiation during infection and cancer. BRD4 was found to bind diverse regulatory regions critical to effector T cell differentiation and was essential for transcriptional activity of terminal effector-specific super-enhancers in vivo. Consequentially, induced deletion or chemical inhibition of BRD4 resulted in impaired maintenance of a terminal effector phenotype. In preclinical models of cancer, BRD4 was also required for formation of a terminally differentiated CD8 T cell state in the tumor microenvironment, which we show holds implications for immunotherapies. Tailored inhibition of BRD4 enhanced immune checkpoint blockade efficacy; however, complete ablation of BRD4 activity blunted T cell effector function and adoptive cell therapy efficacy. Taken together, these data reveal an unappreciated requirement for BRD4 in coordinating activity of cis-regulatory elements to control CD8 T cell fate and stability of lineage identity, and provide insight for understanding the efficacy and full therapeutic potential of BET inhibition and T cell immunotherapies.

During infections, naive CD8 T cells differentiate into terminal effector cells (TE) that are relatively short-lived, and memory precursor (MP) cells that give rise to long-lived memory CD8 T cells, but the transcriptional control of this process is still unclear. In naïve CD8 T cells, cis-regulatory regions that become accessible in chromatin during the first 24 hours of TCR stimulation and that remain accessible in mature memory T cell subsets are highly enriched with motifs encoding binding sites for the ETS- and bZIP-families of transcription factors (TFs), many of which also overlap Runx-TF binding sites. The ETS and bZIP TF families are encoded by 88 genes, and are differentially expressed between naïve, early effector, and memory CD8 T cell subsets. To interrogate their requirements functionally, we applied an in vivo pooled RNA interference screen using short hairpin RNAs in microRNA contexts (shRNAmirs) to suppress individually all TFs from both families in CD8 T cells responding to lymphocytic choriomeningitis virus (LCMV) infection. In addition, we focused on Ets1, the most highly expressed ETS TF in naïve, effector and memory cell subsets. Ets1 suppression with shRNAmirs impaired overall effector CD8 T cell numbers in vivo, and increased the frequency of TE-like cells at early times after LCMV infection, which resulted in reduced long-lived effector cells and an increased fraction of altered central memory-like cells at later time points. Enforced Runx3 expression in Ets1-suppressed cells partially restored normal MP cell formation at early times. These results suggest that Ets1 and Runx3 are each necessary for the normal differentiation of MP cells, and could function cooperatively to program their differentiation.

Transcription factors controlling T follicular helper (Tfh) cell differentiation have been extensively studied in recent years. However, there is limited knowledge of the different chromatin regulators involved in the control of Tfh differentiation. Most transcription factors control gene expression with the help of chromatin regulators, which modify chromatin structure to regulate gene expression. We hypothesize that chromatin modifying enzymes are involved in the control of Tfh differentiation. To uncover novel chromatin modifying enzymes essential for Tfh differentiation, we screened a shRNA library targeting all known chromatin regulators using our in vivo RNAi screen. Utilizing this screen, we identified the histone methyltransferase MLL1 as a positive regulator of Tfh differentiation after viral infection. We found MLL1 to be functionally important for Tfh differentiation. Knockdown of MLL1 utilizing multiple shRNAs impaired Tfh differentiation during LCMV infection. The requirement for MLL1 for proper Tfh differentiation after LCMV infection was further confirmed using a CRISPR/Cas9 approach. Additionally, ablation of MLL1 expression selectively impaired production of IL-21 by Tfh cells. To further understand the role of MLL1 in regulating the Tfh differentiation network, we performed RNA-Seq of MLL1-deficient Tfh as well as MLL1-sufficient Tfh. Analysis revealed that the expression of several genes critical for the regulation of Tfh differentiation is dysregulated in the absence of MLL1. Specific examples will be discussed.

Tissue-resident memory CD8

Summary T cell receptor (TCR) stimulation of naive CD8+ T cells initiates reprogramming of cis-regulatory landscapes that specify effector and memory cytotoxic T lymphocyte (CTL) differentiation. We mapped regions of hyper-accessible chromatin in naive cells during TCR stimulation and discovered that the transcription factor (TF) Runx3 promoted accessibility to memory CTL-specific cis-regulatory regions before the first cell division and was essential for memory CTL differentiation. Runx3 was specifically required for accessibility to regions highly enriched with IRF, bZIP and PRDM1-like TF motifs, upregulation of TFs IRF4 and Blimp1, and activation of fundamental CTL attributes in early effector and memory precursor cells. Runx3 ensured that nascent CTLs differentiated into memory CTLs by preventing high expression of the TF T-bet, slowing effector cell proliferation, and repressing terminal CTL differentiation. Runx3 overexpression enhanced memory CTL differentiation during iterative infections. Thus, Runx3 governs chromatin accessibility during TCR stimulation and enforces the memory CTL developmental program.

Nature 552, 253–257 (2017); doi:10.1038/nature24993 In this Letter, owing to errors introduced during the proofreading process, the words ‘infection with’ were missing from the sentence “Furthermore, Runx3 RNAi also impaired TRM cell differentiation in the context of a localized infection with enteric Listeria monocytogenes expressing GP33–41 (LM–GP33–41) (Fig. 2b).”