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Human regulatory T (Treg) cells are essential for immune homeostasis. The transcription factor FOXP3 maintains Tregcell identity, yet the complete set of key transcription factors that control Tregcell gene expression remains unknown. Here, we used pooled and arrayed Cas9 ribonucleoprotein screens to identify transcription factors that regulate critical proteins in primary human Tregcells under basal and proinflammatory conditions. We then generated 54,424 single-cell transcriptomes from Tregcells subjected to genetic perturbations and cytokine stimulation, which revealed distinct gene networks individually regulated by FOXP3 and PRDM1, in addition to a network coregulated by FOXO1 and IRF4. We also discovered that HIVEP2, to our knowledge not previously implicated in Tregcell function, coregulates another gene network with SATB1 and is important for Tregcell–mediated immunosuppression. By integrating CRISPR screens and single-cell RNA-sequencing profiling, we have uncovered transcriptional regulators and downstream gene networks in human Tregcells that could be targeted for immunotherapies.

Regulatory T (Treg) cells are required to control immune responses and maintain homeostasis, but are a significant barrier to antitumour immunity1. Conversely, Treginstability, characterized by loss of the master transcription factor Foxp3 and acquisition of proinflammatory properties2, can promote autoimmunity and/or facilitate more effective tumour immunity3,4. A comprehensive understanding of the pathways that regulate Foxp3 could lead to more effective Tregtherapies for autoimmune disease and cancer. The availability of new functional genetic tools has enabled the possibility of systematic dissection of the gene regulatory programs that modulate Foxp3 expression. Here we developed a CRISPR-based pooled screening platform for phenotypes in primary mouse Tregcells and applied this technology to perform a targeted loss-of-function screen of around 500 nuclear factors to identify gene regulatory programs that promote or disrupt Foxp3 expression. We identified several modulators of Foxp3 expression, including ubiquitin-specific peptidase 22 (Usp22) and ring finger protein 20 (Rnf20). Usp22, a member of the deubiquitination module of the SAGA chromatin-modifying complex, was revealed to be a positive regulator that stabilized Foxp3 expression; whereas the screen suggested that Rnf20, an E3 ubiquitin ligase, can serve as a negative regulator of Foxp3. Treg-specific ablation of Usp22 in mice reduced Foxp3 protein levels and caused defects in their suppressive function that led to spontaneous autoimmunity but protected against tumour growth in multiple cancer models. Foxp3 destabilization in Usp22-deficient Tregcells could be rescued by ablation of Rnf20, revealing a reciprocal ubiquitin switch in Tregcells. These results reveal previously unknown modulators of Foxp3 and demonstrate a screening method that can be broadly applied to discover new targets for Tregimmunotherapies for cancer and autoimmune disease.

The majority of genetic variants associated with common human diseases map to enhancers, non-coding elements that shape cell-type-specific transcriptional programs and responses to extracellular cues. Systematic mapping of functional enhancers and their biological contexts is required to understand the mechanisms by which variation in non-coding genetic sequences contributes to disease. Functional enhancers can be mapped by genomic sequence disruption, but this approach is limited to the subset of enhancers that are necessary in the particular cellular context being studied. We hypothesized that recruitment of a strong transcriptional activator to an enhancer would be sufficient to drive target gene expression, even if that enhancer was not currently active in the assayed cells. Here we describe a discovery platform that can identify stimulus-responsive enhancers for a target gene independent of stimulus exposure. We used tiled CRISPR activation (CRISPRa) to synthetically recruit a transcriptional activator to sites across large genomic regions (more than 100 kilobases) surrounding two key autoimmunity risk loci, CD69 and IL2RA. We identified several CRISPRa-responsive elements with chromatin features of stimulus-responsive enhancers, including an IL2RA enhancer that harbours an autoimmunity risk variant. Using engineered mouse models, we found that sequence perturbation of the disease-associated Il2ra enhancer did not entirely block Il2ra expression, but rather delayed the timing of gene activation in response to specific extracellular signals. Enhancer deletion skewed polarization of naive T cells towards a pro-inflammatory T helper (TH17) cell state and away from a regulatory T cell state. This integrated approach identifies functional enhancers and reveals how non-coding variation associated with human immune dysfunction alters context-specific gene programs.

New genetic tools are needed to understand the functional interactions between HIV and human host factors in primary cells. We recently developed a method to edit the genome of primary CD4+T cells by electroporation of CRISPR/Cas9 ribonucleoproteins (RNPs). Here, we adapted this methodology to a high-throughput platform for the efficient, arrayed editing of candidate host factors. CXCR4 or CCR5 knockout cells generated with this method are resistant to HIV infection in a tropism-dependent manner, whereas knockout of LEDGF or TNPO3 results in a tropism-independent reduction in infection. CRISPR/Cas9 RNPs can furthermore edit multiple genes simultaneously, enabling studies of interactions among multiple host and viral factors. Finally, in an arrayed screen of 45 genes associated with HIV integrase, we identified several candidate dependency/restriction factors, demonstrating the power of this approach as a discovery platform. This technology should accelerate target validation for pharmaceutical and cell-based therapies to cure HIV infection.

Tenofovir disoproxil fumarate (TDF) is a widely used antiretroviral agent with favorable efficacy, safety, and tolerability profiles. However, renal adverse events, including the rare Fanconi syndrome (FS), may occur in a small subset of patients treated for HIV infections.

This study investigated 2 hypotheses about genotype‐phenotype relationships for the efflux transporter, P‐glycoprotein: (1) the presence of a synonymous C3435T variant in exon 26 of the MDR1 gene correlates to higher plasma concentrations of a P‐glycoprotein substrate, dicloxacillin, and (2) the effects of genotypic differences decrease under conditions of P‐glycoprotein induction by rifampin. Eighteen healthy volunteers received two 1‐g doses of dicloxacillin, one on the 1st study day and the other on the 11th day of rifampin dosing (600 mg daily). Dicloxacillin and its 5‐hydroxymethyl metabolite were analyzed using liquid chromatography/tandem mass spectrometry. Mean dicloxacillin Cmaxmeasurements were 30.5 ± 13.5, 33.3 ± 4.7, and 31.1 ± 12.8 μg/mL in individuals with the CC, CT, and TT genotype at position 3435 in exon 26 of the MDR1 gene. Following rifampin dosing, the mean dicloxacillin Cmaxacross genotypes decreased from 31.4 ± 10.8 to 22.9 ± 7.0 μg/mL (P < .05), whereas the mean oral clearance increased from 235 ± 82 to 297 ± 71 mL/min (P < .001), and the mean absorption time increased from 0.71 ± 0.55 to 1.34 ± 0.77 h (P 7< .05). Rifampin treatment increased the formation clearance, Cmax, and AUC of the 5‐hydroxymethyl metabolite by 135%, 119%, and 59%, respectively. The C3435T variant had no effect on dicloxacillin pharmacokinetics. The data suggested that rifampin induced intestinal P‐glycoprotein and increased dicloxacillin metabolism.

We have investigated the variability of the binary X-ray pulsar SMC X-1 in data from several X-ray observatories. We confirm the ~60 day cyclic variation of the X-ray flux in the long-term monitoring data from the RXTE and CGRO observatories. X-ray light curves and spectra from the ROSAT, Ginga, and ASCA observatories show that the uneclipsed flux varies by as much as a factor of 20 between a high-flux state when 0.71 s pulses are present and a low-flux state when pulses are absent. In contrast, during eclipses when the X-rays consist of radiation scattered from circumsource matter, the fluxes and spectra in the high and low states are approximately the same. These observations prove that the low state of SMC X-1 is not caused by a reduction in the intrinsic luminosity of the source or a spectral redistribution thereof, but rather by a quasi-periodic blockage of the line of sight, most likely by a precessing tilted accretion disk. In each of two observations in the midst of low states a brief increase in the X-ray flux and reappearance of 0.71 s pulses occurred near orbital phase 0.2. A similar brief flux increase near orbital phase 0.2 was observed during a low-state observation that did not have sufficient time resolution to detect 0.71 s pulses. These brief increases result from an opening of the line of sight to the pulsar that may be caused by wobble in the precessing accretion disk. The records of spin-up of the neutron star and decay of the binary orbit are extended during 1991-1996 by pulse-timing analysis of ROSAT, ASCA, and RXTE PCA data. The pulse profiles in various energy ranges from 0.1 to greater than 21 keV are well represented as a combination of a pencil beam and a fan beam. Finally, there is a marked difference between the power spectra of random fluctuations in the high-state data from the RXTE PCA below and above 3.4 keV. In the higher energy range the spectrum has a sharp break at 3.3 Hz, with fitted power-law indices of 0.45 and 1.76 below and above the break. No break is evident in the power spectrum below 3.4 keV, and the fitted power-law index is 0.51. In both spectra there is a positive deviation from the fitted power law around 0.06 Hz that may be quasi-periodic oscillation.

We present an astrophysical extended X-ray absorption fine-structure (EXAFS) analysis (AEA) tool. The AEA tool is designed to generate a numerical model of the modification to the X-ray absorption coefficient due to the EXAFS phenomenon. We have constructed a complete database (elements up to the atomic number 92) of EXAFS parameters: central atom phase shift (2?1), backscattering phase shift (b), and backscattering amplitude (F). Using the EXAFS parameter data base, the AEA tool can generate a numerical model of any compound when the atomic numbers of neighboring atoms and their distances to the central X-ray-absorbing atom are given.

The X-ray binary pulsar Cen X-3 was observed with the ASCA satellite over an entire binary eclipse. With unprecedented energy resolution at the iron K-energy band (FWHM ∼ 2%), three iron emission lines at 6.4 keV, 6.7 keV, and 6.97 keV were clearly resolved. At lower energies, lines from hydrogenic ions of Ne, Mg, Si, and S were also observed. Spectral variations over the eclipse were studied, in particular variations of the iron emission lines. Although the intensity of the 6.4 keV line, which is due to fluorescence from low ionized iron, decreased about one order of magnitude during the eclipse, the intensity drop of the 6.7 and 6.97 keV lines, which are respectively from Fe XXV and Fe XXVI, was at most a factor of three. This, confirming the conjecture by Nagase et al. (1992, AAA56.117.175), indicates that the 6.4 keV fluorescent line is emitted from the region close to the neutron star while the highly photoionized plasma which emits 6.7 and 6.97 keV lines is more extended than the size of the companion star (∼ 12 R⊙). The 6.4 keV line equivalent-width did not show a large variation over the eclipse, and was 75–145 eV. The dense Alfvén shell or an optically thick accretion disk is a likely candidate for reprocessing the fluorescent iron line. From the intensity ratio of the 6.7 and 6.97 keV lines during the eclipse, the ionization parameter of the photoionized plasma is estimated as ξ ∼ 103.4. The size and the density of the plasma are estimated as ∼ 16 R⊙ and ∼ 1.3 × 1011cm−3, respectively. It is suggested that a lower ionization state is dominated in the pre-eclipse phase, since the 6.7 and 6.97 keV lines were not clearly seen and other K-lines from lighter elements were strong.

We perform precise measurements of the x-ray transmission of the thin films comprising CCD gate structure, namely, phosphorus doped polysilicon, silicon dioxide, and silicon nitride. The x-ray transmission of these films shows large oscillations with small changes in energy in the vicinity of the following absorption edges: nitrogen K(400 eV), oxygen K(536 eV), silicon Land K(100 and 1840 eV, respectively). As a result, quantum efficiency of a CCD in the soft x-ray range deviates significantly from simple model predictions based on Henke (1993) mass absorption coefficients. The measurements covered the range of energies from 60 to 3000 eV, using synchrotron beamlines at the Advanced Light Source (ALS; Berkeley), Physikalisch-Technische Bundesanstalt BESSY (Berlin), the Synchrotron Radiation Center (SRC; University of Wisconsin-Madison). Our model of the CCD response includes near edge x-ray absorption structure and predicts a very complicated shape of the energy dependence of the quantum efficiency around silicon and oxygen absorption edges. Experimental measurements of CCD quantum efficiency relative to a calibrated detector at the BESSY synchrotron confirmed our model predictions for both frontside and backside illuminated CCDs. © 1998 Society of Photo-Optical Instrumentation Engineers.

We present a theoretical model and database designed to provide analysis of astrophysical X-ray absorption fine structure (XAFS). The model includes spherical wave corrections and multiple-scattering contributions to the modification of the X-ray absorption coefficient. The model and database provide the basis for the astrophysical XAFS analysis (AXA) tool, which may be used to analyze properties of interstellar grains and molecules from high-resolution X-ray spectra expected to be observed by future satellite missions (e.g., the Advanced X-ray Astrophysics Facility).

We present a theoretical model and database designed to provide analysis of astrophysical X-ray absorption fine structure (XAFS). The model includes spherical wave corrections and multiple-scattering contributions to the modification of the X-ray absorption coefficient. The model and database provide the basis for the astrophysical XAFS analysis (AXA) tool, which may be used to analyze properties of interstellar grains and molecules from high-resolution X-ray spectra expected to be observed by future satellite missions (e.g., the Advanced X-ray Astrophysics Facility).

We have investigated the variability of the binary X-ray pulsar SMC X-1 in data from several X-ray observatories. We confirm the ~60 day cyclic variation of the X-ray flux in the long-term monitoring data from the RXTEand CGROobservatories. X-ray light curves and spectra from the ROSAT, Ginga, and ASCAobservatories show that the uneclipsed flux varies by as much as a factor of 20 between a high-flux state when 0.71 s pulses are present and a low-flux state when pulses are absent. In contrast, during eclipses when the X-rays consist of radiation scattered from circumsource matter, the fluxes and spectra in the high and low states are approximately the same. These observations prove that the low state of SMC X-1 is not caused by a reduction in the intrinsic luminosity of the source or a spectral redistribution thereof, but rather by a quasi-periodic blockage of the line of sight, most likely by a precessing tilted accretion disk. In each of two observations in the midst of low states a brief increase in the X-ray flux and reappearance of 0.71 s pulses occurred near orbital phase 0.2. A similar brief flux increase near orbital phase 0.2 was observed during a low-state observation that did not have sufficient time resolution to detect 0.71 s pulses. These brief increases result from an opening of the line of sight to the pulsar that may be caused by wobble in the precessing accretion disk. The records of spin-up of the neutron star and decay of the binary orbit are extended during 1991-1996 by pulse-timing analysis of ROSAT, ASCA, and RXTEPCA data. The pulse profiles in various energy ranges from 0.1 to greater than 21 keV are well represented as a combination of a pencil beam and a fan beam. Finally, there is a marked difference between the power spectra of random fluctuations in the high-state data from the RXTEPCA below and above 3.4 keV. In the higher energy range the spectrum has a sharp break at 3.3 Hz, with fitted power-law indices of 0.45 and 1.76 below and above the break. No break is evident in the power spectrum below 3.4 keV, and the fitted power-law index is 0.51. In both spectra there is a positive deviation from the fitted power law around 0.06 Hz that may be quasi-periodic oscillation.

In this Letter, analysis of steady-state regulatory T (Treg) cell percentages from Il2ra enhancer deletion (EDEL) and wild-type (WT) mice revealed no differences between them (Extended Data Fig. 9d). This analysis included two mice whose genotypes were incorrectly assigned. Even after correction of the genotypes, no significant differences in Treg cell percentages were seen when data across experimental cohorts were averaged (as was done in Extended Data Fig. 9d). However, if we normalize the corrected data to account for variation among experimental cohorts, a subtle decrease in EDEL Treg cell percentages is revealed and, using the corrected and normalized data, we have redrawn Extended Data Fig. 9d in Supplementary Fig. 1. The Supplementary Information to this Amendment contains the corrected and reanalysed Extended Data Fig. 9d. The sentence “This enhancer deletion (EDEL) strain also had no obvious T cell phenotypes at steady state (Extended Data Fig. 9).” should read: “This enhancer deletion (EDEL) strain had a small decrease in the percentage of Treg cells (Extended Data Fig. 9).”. This error does not affect any of the main figures in the Letter or the data from mice with the human autoimmune-associated single nucleotide polymorphism (SNP) knocked in or with a 12-base-pair deletion at the site (12DEL). In addition, we stated in the Methods that we observed consistent immunophenotypes of EDEL mice across three founders, but in fact, we observed consistent phenotypes in mice from two founders. This does not change any of our conclusions and the original Letter has not been corrected.

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