Activated CD8 T cells differentiate into effector memory (TEM) cells that recirculate in the vasculature and tissue resident memory (TRM) cells that do not, but the early transcriptional control that drives their specification is unclear. Here, we provide evidence that transcription factors (TFs) ETS1 and RUNX3 have distinct functions that polarize formation of TEM and TRM cells. Ets1-deficient effector (TEFF) cells inefficiently formed TEM cells, redistributed into the small intestine (SI) epithelium, exhibited increased TGFβ responsiveness and more strongly differentiated into CD69hi CD103hi cells, whereas Runx3-deficiency caused the alternative phenotype. ETS1 promoted chromatin accessibility of cis-regulatory regions encoding ETS and composite ETS:RUNX motifs, distinct from those that required RUNX3, and activated expression of both TEM cell genes and the TGFβ-inhibitory factor encoded by Smad7. Enforced Smad7 expression restored wildtype TGFβ responsiveness and CD103 expression in Ets1-deficient cells. Thus, ETS1 activates TEM cell development and represses TRM cell formation, whereas RUNX3 promotes TRM cell formation. Supported by P01AI145815; U01AI163063; and Frenchman Creek’s Women for Scripps Research. Lymphocyte Differentiation and Peripheral Maintenance (LYM)

The 18S rRNA sequence is highly conserved, particularly at its 3′-end, which is formed by the endonuclease Nob1. How Nob1 identifies its target sequence is not known, and in vitro experiments have shown Nob1 to be error-prone. Moreover, the sequence around the 3′-end is degenerate with similar sites nearby. Here, we used yeast genetics, biochemistry, and next-generation sequencing to investigate a role for the ATPase Rio1 in monitoring the accuracy of the 18S rRNA 3′-end. We demonstrate that Nob1 can miscleave its rRNA substrate and that miscleaved rRNA accumulates upon bypassing the Rio1-mediated quality control (QC) step, but not in healthy cells with intact QC mechanisms. Mechanistically, we show that Rio1 binding to miscleaved rRNA is weaker than its binding to accurately processed 18S rRNA. Accordingly, excess Rio1 results in accumulation of miscleaved rRNA. Ribosomes containing miscleaved rRNA can translate, albeit more slowly, thereby inviting collisions with trailing ribosomes. These collisions result in degradation of the defective ribosomes utilizing parts of the machinery for mRNA QC. Altogether, the data support a model in which Rio1 inspects the 3′-end of the nascent 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 cells of altered ribosomes with different functionalities, with important implications for the concept of ribosome heterogeneity.

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 TSCM precursor 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 widespread de novo deposition of histone H3 lysine 4 trimethylation (H3K4me3) upon TCR stimulation, which accounted for 70% of all activation-induced sites in mature TMEM cells. Mll1 promoted both H3K4me3 deposition and reduced TCR-induced Pol II pausing at genes whose single-cell transcriptional dynamics explained trajectories into nascent TSCM precursor states during viral infection. Our results suggest Mll1-dependent control of Pol II elongation and H3K4me3 establishes and maintains differentiation of CD8+ TSCM cell states.

T follicular helper (Tfh) cells were discovered as CD4 +T cells in the B cell follicles that express CXCR5 and help germinal center (GC) B cell responses. However, it is not clear which PD-1 +CXCR5 +Bcl6 +CD4 +T cells will differentiate into PD-1 hiCXCR5 hiBcl6 hiGC-Tfh cells while the others have a different fate, nor is it clear how GC-Tfh cell differentiation is regulated. Here we report that the sustained Tigit expression in PD-1 +CXCR5 +CD4 +T cells marked the precursor Tfh (pre-Tfh) to GC-Tfh transition, whereas Tigit −PD-1 +CXCR5 +CD4 +T cells upregulated IL-7Ra to become CXCR5 +CD4 +T memory precursor/memory cells—with or without a CCR7 +(or CCR7 +CD62L +) central memory phenotype. Our study further shows that pre-Tfh cells undergo substantial further differentiation at the transcriptome and chromatin accessibility levels to become GC-Tfh cells. Mechanistically, we find that c-Maf exerted an important function in the pre- to GC-Tfh transition, and we have identified c-Maf downstream factor Plekho1 as a novel regulator that played a stage-specific role in GC-Tfh cell differentiation.

Follicular helper T (Tfh) cells are essential for germinal center (GC) B cell responses. However, it is not clear which PD-1 + CXCR5 + Bcl6 + CD4 + T cells will differentiate into PD-1 hi CXCR5 hi Bcl6 hi GC-Tfh cells and how GC-Tfh cell differentiation is regulated. Here, we report that the sustained Tigit expression in PD-1 + CXCR5 + CD4 + T cells marks the precursor Tfh (pre-Tfh) to GC-Tfh transition, whereas Tigit – PD-1 + CXCR5 + CD4 + T cells upregulate IL-7Rα to become CXCR5 + CD4 + T memory cells with or without CCR7. We demonstrate that pre-Tfh cells undergo substantial further differentiation at the transcriptome and chromatin accessibility levels to become GC-Tfh cells. The transcription factor c-Maf appears critical in governing the pre-Tfh to GC-Tfh transition, and we identify Plekho1 as a stage-specific downstream factor regulating the GC-Tfh competitive fitness. In summary, our work identifies an important marker and regulatory mechanism of PD-1 + CXCR5 + CD4 + T cells during their developmental choice between memory T cell fate and GC-Tfh cell differentiation.

T follicular helper (TFH) cells are essential for developing protective Ab responses following vaccination. Greater understanding of the genetic program leading to TFH differentiation is needed. Chromatin modifications are central in the control of gene expression. However, detailed knowledge of how chromatin regulators (CRs) regulate differentiation of TFH cells is limited. We screened a large short hairpin RNA library targeting all known CRs in mice and identified the histone methyltransferase mixed lineage leukemia 1 (Mll1) as a positive regulator of TFH differentiation. Loss of Mll1 expression reduced formation of TFH cells following acute viral infection or protein immunization. In addition, expression of the TFH lineage-defining transcription factor Bcl6 was reduced in the absence of Mll1. Transcriptomics analysis identified Lef1 and Tcf7 as genes dependent on Mll1 for their expression, which provides one mechanism for the regulation of TFH differentiation by Mll1. Taken together, CRs such as Mll1 substantially influence TFH differentiation.

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.

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.

HIV transcription requires assembly of cellular transcription factors at the HIV-1promoter. The TFIIH general transcription factor facilitates transcription initiation by opening the DNA strands around the transcription start site and phosphorylating the C-terminal domain for 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. shRNA knockdown of XPB confirmed XPB degradation as the mechanism of action. Unfortunately, long-term pre-treatment with SP does not result in epigenetic suppression of HIV upon SP treatment interruption, since virus rapidly rebounds when XPB reemerges; however, SP alone without ART maintains the transcriptional suppression. Importantly, SP inhibits HIV reactivation from latency in both cell line models and resting CD4+T cells isolated from aviremic infected individuals 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 HIV 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 virus 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. 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.

During infections, naive CD8 T cells differentiate into terminal effector cells (TE) and long-lived memory CD8 T cells that recirculate through lymphoid organs using the vasculature (TCIRC), and tissue resident memory (TRM) cells that do not recirculate. The transcriptional control that delineates differentiation of TE cells from memory cell subsets is still unclear. DNA motifs recognized by ETS-family transcription factors (TFs) are highly frequent in cis-regulatory regions that become chromatin accessible in naïve CD8 T cells 24 hours after TCR stimulation, and in mature memory T cell subsets. The ETS-family is encoded by 27 genes, and expression analyses revealed Ets1 is the most highly expressed member. Ets1 is transiently downregulated during activation and is re-expressed in TCIRC cells, but remains lower in intestinal TRM cells. P14 CD8 T cells responding to acute LCMV infection that were depleted of Ets1 using retrovirally-delivered shRNAmirs preferentially became KLRG1hi CD127lo TE-like cells, and their numbers in the circulation rapidly declined, resulting in an absence of long-lived effector cells and residual numbers of central memory-like cells. Concomitantly, Ets1-depleted cells re-distributed into the gut TRM compartment and had increased fractions of CD69hi CD103hi cells. Early after infection in the spleen, a larger fraction of Ets1-depleted P14 cells express CCR9 and α4β7. Depletion or overexpression of Ets1 in P14 cells both indicate it negatively regulates CD25 and Prdm1 expression, and promotes Tcf7 and Bcl6 expression. These results suggest that Ets1 functions early during CD8 T cell activation to establish the normal pattern of TCIRC and TRM cell differentiation during acute viral infection.

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 vaccines 1 , distinct effector (T EFF ) and memory (T MEM ) cell populations develop that ensure long term immunity 2 . During chronic infections, responding cells progressively become dysfunctional and “exhaust” 3 . A diverse taxonomy of T EFF , T MEM and exhausted (T EX ) CD8 T cell populations is known, but the initial developmental basis of this phenotypic variation remains unclear 4–10 . Here, we defined single-cell trajectories and identified chromatin regulators that establish antiviral CD8 T cell heterogeneity using unsupervised analyses of single-cell RNA dynamics 11–13 and an in vivo RNAi screen 14 . 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 T MEM and T EX 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 T EFF rather than T EX states. Nucleosome remodelers Smarca4 and Chd7 differentially promote transcription that delineates divergent T MEM lineages before cooperatively driving terminal T EFF cell differentiation. Thus, the lineage architecture is established by specific chromatin regulators that stabilize diverging transcription in uncommitted progenitors.

In response to infection, pathogen-specific CD8 T cells differentiate into functionally diverse effector and memory T cell populations critical for resolving disease and providing durable immunity. Through small-molecule inhibition, RNAi studies, and induced genetic deletion, we reveal an essential role for the chromatin modifier and BET family member BRD4 in supporting the differentiation and maintenance of terminally fated effector CD8 T cells during infection. BRD4 bound diverse regulatory regions critical to effector T cell differentiation and controlled transcriptional activity of terminal effector-specific super-enhancers in vivo. Consequentially, induced deletion of Brd4 or small molecule-mediated BET inhibition impaired maintenance of a terminal effector T cell phenotype. BRD4 was also required for terminal differentiation of CD8 T cells in the tumor microenvironment in murine models, which we show has implications for immunotherapies. Taken together, these data reveal an unappreciated requirement for BRD4 in coordinating activity of cis regulatory elements to control CD8 T cell fate and lineage stability.

CD8+ T cells with stem cell-like qualities (TSTEM) provide central memory following resolved intracellular infections, and serve as progenitor cells that sustain effector-like cell development during chronic infections and tumors, both naturally and in response to immunotherapeutic PD-1 blockade. However, the chromatin regulatory factors (CRFs) and transcriptional programs that establish TSTEM cell differentiation are unclear. Using an RNA interference screen of all CRFs we discovered that Mixed Lineage Leukemia 1 (MLL1) was required in activated CD8 T cells to promote Id3-GFP reporter expression and to coordinately restrain PD-1, LAG3 and TIM3 expression. Single cell RNA-seq analysis demonstrated Kmt2a (MLL1) was preferentially expressed in P14 CD8 T cells enriched with TSTEM gene expression during lymphocytic choriomeningitis virus (LCMV) infection. P14 CD8 T cells depleted of MLL1 failed to establish TSTEM-like cells, terminally differentiated prematurely, failed to sustain responses to chronic LCMV infection and tumors, and failed to develop central memory subsets during acute LCMV infection. MLL1 preferentially bound transcriptional start sites (TSSs) in CD8 T cells, and was essential for global accumulation of H3K4me3, H4K16ac, and RNA Polymerase II CTD-phosphorylation during TCR stimulation. These results suggest that MLL1-COMPASS organizes TSS activity to establish transcriptional programs that delineate TSTEM and terminal 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.

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.

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.

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).”