Downregulation of E Activity Augments an ILC2 Differentiation Program in the Thymus

This information is current as Hong-Cheng Wang, Liangyue Qian, Ying Zhao, Joni of October 7, 2021. Mengarelli, Indra Adrianto, Courtney G. Montgomery, Joseph F. Urban, Jr., Kar-Ming Fung and Xiao-Hong Sun J Immunol published online 3 March 2017 http://www.jimmunol.org/content/early/2017/03/03/jimmun ol.1602009 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 3, 2017, doi:10.4049/jimmunol.1602009 The Journal of Immunology

Downregulation of E Protein Activity Augments an ILC2 Differentiation Program in the Thymus

Hong-Cheng Wang,*,1 Liangyue Qian,*,1 Ying Zhao,* Joni Mengarelli,* Indra Adrianto,* Courtney G. Montgomery,* Joseph F. Urban, Jr.,† Kar-Ming Fung,‡ and Xiao-Hong Sun*

Innate lymphoid cells (ILCs) are important regulators in various immune responses. The current paradigm states that all newly made ILCs originate from common lymphoid progenitors in the bone marrow. Id2, an inhibitor of E protein transcription factors, is indispensable for ILC differentiation. Unexpectedly, we found that ectopically expressing Id1 or deleting two E protein in the thymus drastically increased ILC2 counts in the thymus and other organs where ILC2 normally reside. Further evidence suggests a thymic origin of these mutant ILC2s. The mutant mice exhibit augmented spontaneous infiltration of eosinophils and heightened

responses to papain in the lung and increased ability to expulse the helminth parasite, Nippostrongylus brasiliensis. These results Downloaded from prompt the questions of whether the thymus naturally has the capacity to produce ILC2s and whether E restrain such a potential. The abundance of ILC2s in Id1 transgenic mice also offers a unique opportunity for testing the biological functions of ILC2s. The Journal of Immunology, 2017, 198: 000–000.

nnate lymphoid cells (ILCs) play pivotal roles in mounting entiate into adaptive lymphoid cells while retaining the ability to innate immunity against pathogens as well as in contributing generate all ILC subsets (2, 4–6). However, it is unclear whether http://www.jimmunol.org/ I to the pathogenesis of immunological illnesses (1, 2). ILC differentiation follows this single linear path or whether Therefore, understanding the regulation of their ontology will multipotent progenitors distinct from lymphoid-primed multi- provide insights into therapeutic strategies to modulate the func- potent progenitors or CLPs or those residing outside of the BM tion of these ILCs. Rapid progress has led to the notion that ILCs also possess the ability to produce ILCs. For example, progenitors originate from lymphoid-primed multipotent progenitors or com- thought to lie upstream of CLPs in the hierarchy of hematopoiesis mon lymphoid progenitors (CLPs), the same progenitors that give have been shown to travel to the thymus (7, 8), which then rise to adaptive lymphoid cells (1–3). Several populations of ILC propagate into the earliest T cell progenitors (9). Whether pro- progenitors (ILCPs) have been identified and are thought to de- genitors seeding the thymus can generate all ILC subsets has not

velop from CLPs in the bone marrow (BM). They cannot differ- been fully investigated, but thymus can be a fertile ground for by guest on October 7, 2021 ILC2s, because it provides ample IL-7 and Notch signals, essential for ILC2 differentiation (10–12). T cell progenitors also possess *Program in Arthritis and Clinical Immunology, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104; †Diet, Genomics, and Immunology Lab- transcription factors necessary for ILC2 differentiation, which oratory, Beltsville Human Nutrition Research Center, Agricultural Research Service, include Bcl11b, GATA3, and T cell–specific factor 1 (10, 13–16). ‡ United States Department of Agriculture, Beltsville, MD 20705; and Department of Although the thymus is not essential for ILC2 production, CD4 Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104 and CD8 double-negative (DN) thymocytes (DN1 and DN2) were 1H.-C.W. and L.Q. contributed equally to this work. shown to give rise to ILC2s when cultured extensively with IL-7 ORCIDs: 0000-0003-0265-1985 (L.Q.); 0000-0002-9973-3057 (I.A.); 0000-0002-9606- and IL-33 (11, 17). However, their capacity to produce ILC2 3772 (C.G.M.); 0000-0002-1590-8869 (J.F.U.); 0000-0002-4623-9469 (K.-M.F.); 0000- in vivo has not been demonstrated. 0002-7390-7506 (X.-H.S.). Id2 is a member of the family of helix–loop–helix Id proteins, Received for publication November 28, 2016. Accepted for publication February 10, consisting of Id1–4, which all inhibit the DNA-binding activity of 2017. a group of E protein transcription factors encoded by the E2A, This work was supported by National Institutes of Health Grants R01 AI126851-01 and R21 AI117895 and by funds from the Presbyterian Health Foundation (to X.-H.S.), as HEB, and E2-2 genes (18, 19). Id2-deficient mice lack not only well as by National Institutes of Health Centers of Biomedical Research Excellence NK cells but also all ILC subsets (20). Id2 is highly expressed in Grant P30GM110766 (to C.G.M.). X.-H.S. holds the Lew and Myra Chair in Biomed- both ILCs and ILCPs (6, 21, 22). In the thymus, E2A and HEB are ical Research. both expressed and play redundant roles in controlling T cell The RNA-sequencing data presented in this article have been submitted to the Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number differentiation (23–29). Ablation of both E2A and HEB blocks GSE94597. T cell development at the DN3 stage whereas loss of either of Address correspondence and reprint requests to Dr. Xiao-Hong Sun, Oklahoma Med- these genes leads to partial impairment (30). Id2 and Id3 are ical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104. E-mail known to be involved in later stages of T and NKT cell devel- address: [email protected] opment in the thymus, and the Id3 gene is known to be activated The online version of this article contains supplemental material. by pre-TCR and TCR signaling (31–35). Abbreviations used in this article: BALF, bronchoalveolar lavage fluid; BM, bone marrow; CLP, common lymphoid progenitor; DN, double-negative; ILC, innate lym- Although the Id1 gene is normally not expressed in lymphoid phoid cell; ILCP, ILC progenitor; Lin, lineage; MLN, mesenteric lymph node; PCA, cells, we have previously used it as a tool to inhibit all E proteins in principal component analysis; plck, proximal promoter of the lck gene; RNA-seq, thymocytes by ectopically expressing Id1 off the proximal pro- RNA sequencing; WT, wild-type. moter of the lck gene (plck). Homozygous Id1 transgenic mice Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 were found to have T cell development blocked at the DN stage in

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1602009 2 AUGMENTED THYMIC ILC2 PRODUCTION DUE TO E PROTEIN DEFICIT the thymus, lacking any T lineage–committed cells (36–38). Re- For protease challenge, 10 mg of papain was intranasally administrated examination of Id1 transgenic mice with ILC2 parameters into mice every 24 h on days 0, 1, and 2. Mice were sacrificed 12 h after revealed a large number of ILC2s in the thymus and in peripheral the last challenge. Bronchoalveolar lavage fluid (BALF) and lungs were analyzed. organs such as the lung, mesenteric lymph nodes (MLN), and For infections with N. brasiliensis, mice were s.c. inoculated with 500 spleen. Augmented ILC2 production in the thymus is found to be third-stage infective larvae and sacrificed 6 d later for analysis of ILC2 cell-autonomous and likely responsible for the accumulation of responses. BALF, lungs, mediastinal lymph nodes, and MLNs were ana- ILC2 in the periphery. Consistent with the role of Id proteins as lyzed, and adult worm burden was determined by enumerating worms present in the proximal half of the small intestine. inhibitors of E proteins, deletion of both E2A and HEB genes in the thymus (30) using the plck-Cre transgene also led to similar Isolation of lung hematopoietic cells phenotypes as those for Id1 transgenic mice. Id1 transgenic Perfused lungs were cut into small fragments and digested in HBSS ILC2s display overlapping but distinct transcriptional profiles containing 2.5 mg/ml collagenase (Sigma-Aldrich) and 160 U/ml DNase I compared with wild-type (WT) ILC2s. Furthermore, Id1 trans- (Sigma-Aldrich) for 30 min. Cells were filtered by using a 70-mm cell genic mice exhibit augmented recruitment of eosinophils to the strainer. lung before and after challenges with an allergen, papain. These Flow cytometry and cell sorting mice are more adept at expulsing the helminth parasite, Nip- All Abs used were purchased from BioLegend unless specified otherwise. postrongylus brasiliensis. Taken together, our findings suggest Abs in the lineage (Lin) mixture included anti-FcεR (MAR-1; eBioscience), that specific downregulation of E proteins in the thymus high- anti-B220 (RA3-6B2), anti-CD19 (ID3; BD Biosciences), anti–Mac-1 lights a previously unappreciated potential of the thymus as an (M1/70; BD Biosciences), anti–Gr-1 (R86-8C5; BD Biosciences), anti- CD11c (HL3; BD Biosciences), anti-NK1.1 (PK 136), anti–Ter-119 organ for ILC2 production.

(Ter-119), anti-CD3 (145-2C11), anti-CD8a (53-6.7; BD Biosciences), Downloaded from anti-CD5 (53-7.3), anti-TCRb (H57-597), and anti-gdTCR (GL-3; BD Materials and Methods Biosciences). Additional Abs used included anti-CD45.2 (104), anti- Mice and in vivo treatments CD45.1 (A20), anti-CD4 (RM4-5; BD Biosciences), anti–c-Kit (2B8), anti–Sca-1 (D7), anti-Thy1.2 (53-2.1), anti–IL-5 (TRFK5), anti–IL-13 2 2 C57BL/6 (CD45.2), TCRb / , and B6-CD45.1 mice were purchased from (eBio13A; eBioscience), anti–Siglec F (E50-2440; BD Biosciences), The Jackson Laboratory and Charles River Laboratories. pLck-Id1 trans- anti–IL-7Ra (A019D5), anti-a4b7 (DATK32; eBioscience), anti-CD25 genic mice and LckCre;E2Af/f;HEBf/f mice are as described (30, 37). All (PC61), anti-PLZF (Mags.21F7; eBioscience), and anti-ST2 (DIH9). animal experiments were performed according to protocols approved by Cell sorting was performed on a FACSAria II (BD Biosciences), and flow http://www.jimmunol.org/ the Institutional Animal Care and Use Committee at the Oklahoma cytometric analysis was performed on an LSR II (BD Biosciences). In- Medical Research Foundation. tracellular staining of transcription factors was done using a Foxp3 staining by guest on October 7, 2021

FIGURE 1. Downregulation of E protein function augments ILC2 production. (A) Analyses of Lin2 cells from C57BL/6J (WT) and Id1tg/tg mice. Cells from indicated tissues of mice of indicated genotypes were stained for Lin markers (FcεR, B220, CD19, Mac-1, Gr-1, CD11c, NK1.1, Ter-119, CD3, CD8a, TCRb, and gdTCR) together with Abs against Thy1 and ST2. The Lin2 gates and expression pro- files of Thy1 and ST2 on Lin2 cells are pre- sented. The percentage of ILC2s defined as Lin2Thy1+ST2+ is shown. Bar graphs show average cell numbers in each tissue (n = 4–9). (B) Additional analyses for the indicated markers on ILC2s defined as in (A). Shaded peaks represent the profiles of WT ILC2s in indicated tissues and the solid lines depict those of Id1tg/tg ILC2s. (C) Analyses of Lin2 cells from plck-Cre/E2Af/f;HEBf/f and littermate controls without plck-Cre as described for (A) (n = 6–8). Bar graphs show data pooled from five (A) or three (C) independent experiments. Error bars are SEM. A Student t test was used for statistical analyses. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 3

FIGURE 2. Augmented ILC2 production in Id1 transgenic mice is cell intrinsic. (A) Mixed BM chimera. Lin2Thy12 BM cells of C57BL/6 (CD45.2+)or Id1 transgenic (CD45.2+) mice were mixed with the same cells from CD45.1+ B6 mice at a 1:1 ratio and transplanted into lethally irradiated CD45.1+ B6 mice (five per cohort). Total CD45+ and ILC2s in indicated organs were analyzed 7 wk after transplantation. Chimerism was defined as percentage of CD45.2+ fractions of CD45.1+ plus CD45.2+ cells. The numbers of CD45.2+ILC2s are shown on the right. Error bars are SEM. Data shown are repre- sentative of three independent experiments. (B) Comparison of ILC2 counts in WT and TCRb2/2 mice. ILC2s were defined as described in the legend for Fig. 1 except that anti-CD5 was also included in the lineage mixture. A Student t test was used for statistical analyses. ***p , 0.001. Downloaded from buffer (eBioscience). Staining of cytokines was carried out using a Statistical analysis Cytofix/Cytoperm kit (BD Biosciences). Pairwise comparison was performed using a Student t test or one-way BM transplantation ANOVA. To generate mixed BM chimeras, 105 sorted Lin2Thy12 BM cells from CD45.2+ C57BL/6 or Id1 transgenic mice were mixed with 105 WT Results 2 2 CD45.1+Lin Thy1 cells and i.v. transferred into lethally irradiated WT Downregulation of E proteins promotes ILC2 production http://www.jimmunol.org/ hosts (CD45.1). Recipients were analyzed 7 wk after transplantation. To study the collective role of E proteins in T cell development, we RNA sequencing analysis previously created Id1 transgenic mice by using the proximal promoter of the lck gene, known to direct spe- Total RNA was purified from ILC2s of Id1 transgenic thymus and MLN as tg/tg well as from MLN ILC2s cultured as described (16). Approximately 300,000 cifically in the thymus at an early stage. In homozygotes (Id1 ), cells were used in the preparation of each sample. RNA sequencing T cell differentiation is completely blocked (36–38). Within the (RNA-seq) was performed in duplicate by the Genomics Core Facility at the CD4 and CD8 DN compartment depleted of B, myeloid, or NK Oklahoma Medical Research Foundation using Illumina TruSeq stranded cells, we detected subsets of cells with atypical CD44 and CD25 mRNA library preparation kits followed by sequencing on an Illumina HiSeq 2500 in rapid mode with 50-bp, paired-end reads (Illumina, San Diego, CA). profiles (Supplemental Fig. 1A). Interestingly, these cells could by guest on October 7, 2021 Additionally, transcriptomes of WT ILC2s from small intestine (16) and WT produce IL-5 and IL-13 but very little IFN-g and IL-17 upon DN3 thymocytes (39) were included in our analyses. Sequencing reads were stimulation with PMA and ionomycin (Supplemental Fig. 1B). aligned to the Mus musculus genome reference (mm9) using STAR v2.4.0h. Further analyses of the Lin2 fraction for the expression of ILC2 Gene-level read counts were determined using HTSeq v0.5.3p9 with the surface markers such as Thy1 and ST2 revealed an ∼60-fold in- Ensembl M. musculus NCBIM37 (release 67) annotation. Non- and low- expressed genes were filtered out and 20,471 genes remained in the analy- crease in ILC2 counts when comparing Id1 transgenic to WT sis. The read counts were normalized using DESeq v1.18.0. Principal thymuses (Fig. 1A). These cells were also found to be able to component analysis (PCA) was performed using the Fluidigm SINGuLAR produce IL-5 and IL-13 (Supplemental Fig. 1C). Moreover, the analysis toolset 2.0 in R (40). The top-ranked PCA genes were determined ILC2 levels in other organs such as the lung, spleen, BM, and based on the maximum absolute value of each gene loading score in the first three principal components. Hierarchical clustering using complete linkage MLN were also 10- to 100-fold higher (Fig. 1A). We also detected and Euclidean distance as the similarity metric and heat map analyses were increased numbers of ILC2s in the white adipose tissue, blood, performed using Cluster 3.0 and Java TreeView, respectively (41, 42). and small intestine of the transgenic mice (data not shown). In

FIGURE 3. The frequency of ILCPs was not affected by thymus-specific expression of the Id1 transgene. (A) The Id1 transgene was specifically expressed in transgenic thymocytes. Total Id1 mRNA levels in thymocytes and BM cells of WT and Id1tg mice were determined using quantitative RT-PCR and normalized against the levels of b-actin. BM cells of WT and Id1tg/tg mice were fractionated by first enriching for Lin2Sca-1lo/2ST22 cells and then dividing the resulting population into c-Kit+IL-7Ra+, c-Kit+IL-7Ra2, and c-Kit2 subsets. The c-Kit+ fractions include both c-Kithi and c-Kitint cells. The 2 + + 2 2 + + normalized Id1 levels relative to the WT fractions are shown. (B) Similar numbers of BM ILCPs (Lin Thy1 IL-7Ra ST2 Sca-1 a4b7 PLZF ) were found in WT and Id1tg/tg mice. Gating strategy is shown on the left and the average ILCP number is shown on the right (n = 3). Data shown are repre- sentative of at least two independent experiments. A Student t test was used for statistical analyses. ****p , 0.0001. ns, not significant. 4 AUGMENTED THYMIC ILC2 PRODUCTION DUE TO E PROTEIN DEFICIT Downloaded from

FIGURE 4. Lineage tracing data suggest the thymic origin of Id1 transgenic ILC2s. (A) The Cre transgene is driven by the proximal promoter of the lck gene in plck-Cre mice, which were crossed with ROSA26-STOP-tdTomato reporter mice. Percentages of tdTomato+ cells in subsets of thymocytes and B220+ or Mac-1+ BM cells as well as ILCPs are shown. CD4, CD8, CD19, B220, Mac-1, Gr1, FcgR, Ter119, NK1.1, and TCRgd-negative thymocytes were further defined by c-Kit and CD25 expression in DN populations: DN1 (c-Kit+CD252), DN2 (c-Kit+CD25+), DN3 (c-Kit2CD25+), and DN4 (c-Kit2CD252). (B) Analyses of tdTomato expression in ILC2s in mice of indicated genotypes. To ensure the elimination of any potential contamination of T lineage cells, ILC2s were defined as in Fig. 2B except that the Lin2 population was further gated for TCRb2TCRgd2 based on staining with Abs against these TCRs http://www.jimmunol.org/ conjugated with different fluorophores from that used for lineage staining, and analyzed for Thy1 and ST2. Note that the fluorescence intensity in WT thymic ILC2s was reproducibly lower than that in their Id1tg counterparts. Bar graph shows the average percentage of tdTomato+ cells in the ILC2 population of pooled data from several experiments (n = 3–10). One-way ANOVAwas used to determine the statistical significance of the variations of tdTomato levels in different tissues in each strain. WAT, white adipose tissue. heterozygous transgenic mice (Id1tg), the numbers of ILC2 in Id1tg/tg mice were mixed with equal numbers of CD45.1+Lin2Thy1.12 different organs were also dramatically elevated even though WT cells and injected into lethally irradiated CD45.1+C57BL/6 T cell development was not completely blocked (data not shown). hosts. Seven weeks later, the recipients were analyzed for chi-

In contrast, the numbers of ILC1 in the liver and ILC3 in the small merism and ILC2 production (Fig. 2A). BM reconstitution with by guest on October 7, 2021 intestine of Id1 transgenic mice were not dramatically altered CD45.2+ WT and Id1tg/tg cells achieved 44 and 37% chimerism, (data not shown). respectively. Likewise, CD45.2+ cells also represented substantial 2 Further characterization of ILC2s (Lin Thy1+ST2+) was carried fractions of hematopoietic cells in the lung. Whereas WT CD45.2+ out with additional markers associated with ILC2 phenotypes, which thymocytes were abundant, Id1 transgenic cells in the thymus include IL-7Ra, CD25, ICOS, and KLRG1. Although the expression were barely detectable due to a complete block in T cell devel- of these markers on WT ILC2s in different organs is variable, Id1 opment. In contrast, ILC2s of Id1tg/tg origin in the BM, lung, and transgenic ILC2s in the thymus, BM, and lung displayed a similar thymus vastly outnumbered CD45.1+ ILC2s derived from the 2 + + + + int +/2 pattern, namely Lin Thy1 ST2 IL-7Ra ICOS CD25 KLRG1 CD45.1+ donors or hosts even though the chimerism of WT (Fig. 1B). This result suggests that the putative ILC2s found in CD45.2+ ILC2s was only ∼20% in each of these locations Id1 transgenic mice carry multiple characteristics of well- (Fig. 2A). These results suggest that the promotion of ILC2 recognized ILC2s. Interestingly, the fact that Id1 transgenic production by the Id1 transgene is likely through cell-autonomous ILC2s from different organs exhibit a similar phenotype raised mechanisms rather than alterations in secreted factors, which the possibility that these cells come from the same source, such would have stimulated ILC2 differentiation from both transgenic as the thymus. and CD45.1+ progenitors. Admittedly, the number of ILC2s pro- To test whether Id1 acted by inhibiting E protein transcription duced from Id1 donors in the thymus of these recipients (Fig. 2A) factors (encoded by the E2A, HEB, and E2-2 genes), we generated tg/tg was not as high as that found in Id1 mice themselves (Fig. 1A). plck-Cre/E2Af/f/HEBf/f mice to specifically delete both the E2A ILC2 differentiation of Id1tg/tg progenitors was probably hindered and HEB genes with the plck-Cre transgene, whose expression is by robust T cell development from CD45.1+ progenitors, which known to be restricted to the thymus. These mice were reported to competes for space and cytokines. However, the massive pro- have impaired T cell development (30), but we found them to duction of ILC2s in Id1tg/tg mice is not caused by a blockage of possess large numbers of ILC2s in the thymus and other organs 2 2 T cell development itself because TCRb / mice, despite the (Fig. 1C), suggesting that E protein ablation in the thymus en- block in T cell development and lack of regulatory T cells, do not hances the production of ILC2s, a phenotype similar to that found have dramatically augmented ILC2 counts (Fig. 2B). We have also in Id1 transgenic mice. ruled out the possibility that Id1 expression stimulates the prolif- Augmented ILC2 differentiation in the thymus is eration of ILC2s because no increase in cycling cells was detected cell-autonomous in various tissues of Id1tg/tg mice as measured using Ki67 and DAPI To determine whether the Id1 transgene promoted ILC2 differ- staining (Supplemental Fig. 2A). In fact, WT thymic ILC2s had tg/tg entiation in a cell-autonomous manner, we created mixed BM a larger fraction of cells in S–G2 phases than did Id1 coun- chimeras where Lin2Thy1.22 BM cells from CD45.2+ WT or terparts, possibly due to more robust homeostatic proliferation. The Journal of Immunology 5

Likewise, we obtained no evidence of increased survival of Id1 transgenic ILC2s in the thymus or periphery. Instead, Id1 trans- genic ILC2s appeared to exhibit increased apoptosis in the BM (Supplemental Fig. 2B). Data from the transplantation studies also showed that ILC2s generated from donor BM readily repopulated adult lymphoid and nonlymphoid organs in irradiated recipients (Fig. 2A). Although the representation of WT CD45.2+ ILC2s was somewhat lower than the overall chimerism, possibly due to the radio resistance of host ILC2s, Id1 transgenic donors supplied a large number of ILC2s to all of the organs examined. These results suggest that transplanted BM progenitors are capable of giving rise to ILC2s either in the BM or thymus, and these ILC2s can home to pe- ripheral tissues such as the lung of irradiated hosts. These results are in contrast to the observations made with parabiotic mice (43), which showed little exchange of lung ILC2s between the two jointed mice under physiological conditions. ILC2 differentiation in the BM is not perturbed in Id1

transgenic mice Downloaded from To verify whether increased production of ILC2s is due to the specific expression of the Id1 transgene in the thymus, we analyzed Id1 mRNA in total thymocytes of Id1tg mice and different fractions of Lin2 BM of Id1tg/tg mice. Because ILC2s generated in the thymus can migrate to the BM and may express the transgene, ILC2s were excluded from the BM preparations (Fig. 3A). The http://www.jimmunol.org/ c-Kit+/loIL-7R+ fraction likely contains common lymphoid and ILCPs, whereas the c-Kit+/loIL-7R2 fraction includes myeloid progenitors. The c-Kit2 fraction consists of the remainder of the Lin2Sca-1lo/2ST22 BM. Whereas Id1 levels were 760-fold higher in Id1tg thymus than in WT controls, the levels in the different populations of BM were comparable, suggesting that the trans- gene was not expressed in the BM, although the possibility of the transgene expression in rare unknown subsets has not been ruled by guest on October 7, 2021 out (Fig. 3A). To further verify whether any undetectable levels of Id1 ex- pression in rare progenitors promoted ILC2 differentiation in the BM, we determined the numbers of PLZF+ ILCPs in the BM (6). We found that WT and Id1tg/tg mice have similar numbers, suggesting that early stages of ILC differentiation in the BM was not altered (Fig. 3B). The large number of Lin2Thy1+ST2+ cells in the trans- genic BM, which can also be characterized as Lin2Sca-1+CD25int (data not shown), could be attributed to increased thymus output. Lineage tracing data support the thymic origin of excess ILC2s in Id1 transgenic mice In an attempt to mark ILC2s generated in the thymus, we took advantage of the thymus-specific expression driven by plck. We created plck-Cre/ROSA26-Stop-tdTomato mice with or without the Id1 transgene and first confirmed that Cre expression labeled most cells in the thymus, but not non–T cells, including ILCPs in the BM (Fig. 4A). Within the CD4 and CD8 DN compartment of thymocytes, an appreciable proportion of DN3 cells were tdTo- FIGURE 5. Id1 transgenic ILC2s have overlapping and distinct tran- mato+, but DN1 and DN2 cells had very low levels of reporter criptomes from WT ILC2s. (A) Representative sorting data for ILC2s from expression (Fig. 4A). In the absence of the Id1 transgene, a tg/tg the thymus and MLN of Id1 mice. For each sort, single-cell preparations significant fraction of ILC2s in the thymus were tdTomato+ but from three mice were depleted with Abs against CD3, B220, Mac1, Ter119, BM ILC2s were not (Fig. 4B). Interestingly, a remarkable per- and NK1.1. The unbound cells were stained and gated as indicated. Ap- centage of lung ILC2s expressed tdTomato, but other peripheral proximately 300,000 ILC2s were obtained from each sort. (B)PCAs.RNA- tg/tg organs such as white adipose tissue had much lower fractions of seq analyses of Id1 ILC2s from the thymus (T) and MLNs (M) compared + with WT ILC2 from small intestine (SI) and CD42CD82CD442CD25+ tdTomato ILC2s (Fig. 4B). This result suggests that thymus- thymocytes (DN3). Data for MLN ILC2s cultured in vitro for 7 d were also derived ILC2s preferentially home to the lung under natural included for both WT and Td1tg/tg mice. (C) Hierarchical clustering and heat conditions. Whether thymus-derived ILC2s play an important map of top 500 PCA genes. The log2 of gene-level read counts was centered role in lung physiology or pathophysiology, however, remains to on the mean and normalized. be investigated. 6 AUGMENTED THYMIC ILC2 PRODUCTION DUE TO E PROTEIN DEFICIT

FIGURE 6. Id1 transgenic mice ex- hibit spontaneous inflammation and hyperresponse to allergen. (A)H&E staining of lung sections of 2-mo-old WT and Id1tg/tg lungs. Areas with higher magnification are indicated by arrows. Representative graphs from two mice of each genotype are shown. (B) Eosinophil infiltration in mice of indicated geno- types following intranasal exposure to papain or in untreated controls. Eosino- phil gates in total BALF are shown on the left, percentages of eosinophils in total CD45+ cells of BALF and lungs of un- treated and treated mice are shown on the right (n = 3–6). (C) Lung ILC2s in the same mice as (B). CD45+Lin2 cells were analyzed for Thy1 and ST2 expression, and ILC2s are gated as shown. Percent- ages of lung ILC2s and IL-5+IL-13+ ILC2s of total CD45+ cells are shown Downloaded from on the right. Error bars are SEM. Data shown are representative of two to three experiments. A Student t test was used for statistical analyses. *p , 0.05, ***p , 0.001. http://www.jimmunol.org/

Id1tg/plck-Cre/ROSA26-Stop-tdTomato mice had ∼12% of DN3 or ILC2s (Fig. 5C). Id1tg/tg ILC2s share a group of .100 tdTomato+ ILC2s in the thymus. This percentage was lower than ILC2 signature genes with WT ILC2s, which include Rora, Rarg, that found in mice on the WT background, probably because Id1 Icos, Il1rl1, Klrg1, Arg1, IL2rb, IL13, and IL6 (Supplemental expression beginning at the DN1 or DN2 stages (38) diverted the Table I). Noticeably, there are also small clusters of genes that cells at these stages to ILC2s before tdTomato was turned on. are different among WT ILC2s from the small intestine, as well as Nevertheless, the percentages of tdTomato+ ILC2s were found to Id1tg/tg ILC2s from the MLN and thymus (clusters 123, Fig. 5C, be ∼10% in all organs of Id1 transgenic mice (Fig. 4B). These Supplemental Table I). These differences may be due to the in-

results are in contrast to the observations in WT mice, where fluences of tissue environments, Id1 overexpression, or the ma- by guest on October 7, 2021 ILC2s in the BM, MLN, spleen, and white adipose tissue showed turity of the ILC2s, even though the possibility of technical little tdTomato expression (Fig. 4B). One-way ANONA analyses variations among data generated in different laboratories has not showed that the difference in tdTomato percentages in different been ruled out. Interestingly, ILC2s from the thymus also tran- organs on the WT background was highly significant (p , 0.0001) scribe some genes found in DN3 developing T lineage cells whereas that on the Id1 transgenic background was not (p = 0.24). (cluster 4). This is unlikely due to T cell contamination in the Therefore, these findings, combined with data shown in Figs. 1–3, preparation, as Id1tg/tg mice do not have T cells. Although a few of suggest that ILC2s overproduced in the Id1 transgenic thymus the genes in cluster 4 are related to cell cycle control, the simi- supply these cells to all peripheral organs, leading to the dramatic larities also raise a question whether both DN3 cells and ILC2s increases of ILC2s with similar characteristics of the thymic in the thymus are derived from common progenitors. Taken to- ILC2s in these organs. gether, the transcriptome analyses provide further evidence to suggest that the phenotypic ILC2s found in Id1 transgenic mice Id1 transgenic ILC2s display ILC2 signature gene expression exhibit characteristics of WT ILC2s. As a means of further phenotyping Id1 transgenic ILC2s, we Incidentally, comparison of the transcript levels of E and Id 2 + + performed RNA-seq analyses of ILC2s (Lin Thy1 ST2 ) freshly proteins between Id1tg/tg thymic ILC2s and WT DN3 cells tg/tg isolated from Id1 thymus and MLN (Fig. 5A) and compared revealed a slight decrease (∼2.5-fold) in E2A and HEB levels in data reported by Yagi et al. (16) for WT ILC2s from the small ILC2s. In contrast, Id1 expression was 725-fold higher as a result tg/tg intestine. We also cultured Id1 ILC2s from MLN in the pres- of transgene expression. Id2 levels in Id1tg/tg cells were 59-fold ence of IL-7, IL-25, and IL-33 in the same manner as Yagi et al. higher, which is consistent with their commitment to the ILC2 (16). Additionally, we included gene expression profiles of WT lineage. However, Id3 expression in transgenic ILC2s decreased tg/tg DN3 thymocytes in our analyses (39). PCA showed that Id1 by 55-fold, probably due to a lack of pre-TCR signaling, which ILC2s from the thymus and MLN closely resemble each other and drives Id3 expression (33). The levels of E2A, HEB, Id2, and Id3 share substantial similarities with WT ILC2s from the small in- were similar in all ILC2 preparations analyzed. Therefore, these testine (Fig. 5B). In contrast, these ILC2s are distinct from DN3 results reflect the expression patterns of E and Id proteins in their tg/tg thymocytes. After culturing for a week, WT and Id1 ILC2s respective cell types. remain closely related, although the cultured cells have very dif- ferent transcriptomes from freshly isolated cells. These results Id1 transgenic mice exhibit spontaneous inflammation and help validate the identity of the Id1 transgenic ILC2s. hypersensitivity to allergen stimulation Indeed, hierarchical clustering and heat map analyses of the top To test the functionality of Id1 transgenic ILC2s, we examined the 500 PCA genes in different preparations of ILC2s and DN3 thy- type 2 immune responses in the lung. First, histological exami- mocytes revealed large blocks of genes specifically expressed in nation revealed that naive Id1 transgenic lungs showed more The Journal of Immunology 7

burden in the small intestine 6 d later (Fig. 7A). At this time point, WT mice retained ∼200 adult worms, whereas T cell–deficient TCRb2/2 mice had significantly greater numbers of worms. In contrast, Id1tg/tg mice, despite their T cell defect, exhibited en- hanced worm clearance compared with both WT and TCRb2/2 mice, suggesting enhanced worm expulsion in the presence of excessive ILC2s (Fig 7B). Consistent with the data on worm burden, Id1tg/tg mice harbored many more ILC2s capable of pro- ducing IL-5 and IL-13 (Fig. 7C) and recruited significantly larger numbers of eosinophils in the BALF and lung (Fig. 7D). These results suggest that the large numbers of ILC2s in Id1 transgenic mice lead to augmented type 2 immune responses.

Discussion The unexpected findings in our Id1 transgenic mice taught us several important lessons: 1) multipotent progenitors that normally seed the thymus or T cell precursors are capable of generating functional ILC2s; 2) E proteins suppress the innate lymphoid fate while driving T cell differentiation; and 3) elevated ILC2 counts lead to heightened, spontaneous, and induced type 2 immune re- Downloaded from sponses. These findings also prompt the question of whether downregulation of E protein activities simply highlights a natural capacity of the thymus as a second site for ILC2 production. In- deed, previous studies have shown that DN thymocytes are able to give rise to ILC2s in vitro (11, 17). The phenotypes of Id1 transgenic mice are, to our knowledge, the first examples that il- http://www.jimmunol.org/ lustrate the extraordinary capacity of the thymus to generate ILC2s in vivo. This is likely due to the ideal environment the thymus provides and the intrinsic properties of thymic T cell FIGURE 7. Augmented helminth expulsion by Id1 transgenic mice. Mice progenitors poised for ILC2 differentiation, namely the combi- of indicated genotypes were inoculated s.c. with 500 third-stage N. brasiliensis nation of Notch and IL-7 signaling and the presence of critical larvae. Six days later mice were analyzed. (A) Worm counts in the small in- transcription factors such as T cell–specific factor 1, GATA3, and testine (n =5–9).(B). ILC2s in the lung. (C) ILC2s in mediastinal lymph node Bcl11b (10, 13, 15, 16). It would be extremely interesting to de- (mLN) and MLN. (D) Percentages of eosinophils in the CD45+ fractioninthe termine how E proteins restrain such a potential. by guest on October 7, 2021 BALF and lung. Error bars are SEM. Data shown are representative of at least It would also be informative to determine the developmental three experiments. A Student t test was used for statistical analyses. *p , 0.05, stages at which T cell progenitors can be hijacked to become ILC2s , , **p 0.01, ***p 0.001. by downregulating E protein function. Reporter expression induced by plck-Cre is helpful in addressing this question. Although ILCs pronounced infiltration of lymphoid-looking cells around small have been shown to express some levels of the lck gene (44), bronchioles, a location where ILC2s are thought to congregate patterns of gene expression driven by the proximal promoter of the (Fig. 6A). This is consistent with the dramatic increases in the lck gene in transgenic constructs are unlikely the same as those of numbers of ILC2s but not B or T cells in the lung in Id1 transgenic the native lck gene. Indeed, we have shown that plck-Cre only mice. Moreover, marked elevations in steady-state eosinophil marks T lineage cells and ILC2s in the thymus but not ILC2s in counts were observed in the BALF and lung of untreated Id1tg/tg the BM, MLN, spleen, white adipose tissue, and skin (Fig. 4B and + mice, which suggests increased ILC2 function (Fig. 6B). Becaise S. Bajana and X.-H. Sun, data not shown). The tdTomato fraction Id1tg/tg mice lack any T cells, we used TCRb2/2 mice to control in thymic ILC2s is generally higher than that in DN3 thymocytes for the lack of Th2 and regulatory T cell functions and they did not (Fig. 4), which would mean that ILC2s arise after the DN3 stage, show elevated eosinophil recruitment. or that tdTomato is turned on independently in ILC2s in the thy- When the mice were treated with papain, an allergen delivered mus. Although the Id1 transgene is also driven by the proximal intranasally, substantial increases in eosinophil recruitment were promoter of the lck gene, its expression is known to occur at the seen in mice of all three genotypes. Additionally, marked increases DN1 to DN2 stage (38). As a result, a significant portion of ILC2s of IL-5– and IL-13–producing ILC2s were detected (Fig. 6C). were probably derived from T cell progenitors before plck-Cre is + These results suggest that Id1 transgenic ILC2s can mount type 2 expressed, leading to a lower percentage of tdTomato ILC2s in immune responses, and the elevated ILC2 counts in the lung of the Id1 transgenic thymus. Id1tg/tg mice lead to greater allergic reactions in steady-state and That ∼10% of lung ILC2s were labeled with tdTomato suggests upon allergen exposure. that thymus-derived ILC2s preferentially home to the lung, al- though the possibility that tdTomato is expressed independently in Id1 transgenic mice exhibit enhanced ability to expulse the lung remains to be ruled out. Considering that only 30% of the helminth parasites thymic ILC2s were tdTomato+, the actual fraction of lung ILC2s One of the major functions of ILC2s is to help the hosts clear that come from the thymus could be as high as 30%. If thymic parasite infection. Infection with helminth parasite such as ILC2s indeed contribute to the pool of lung ILC2s, it would be N. brasiliensis has been widely used as a test for ILC2 functions. interesting to determine whether these cells have distinct properties We inoculated WT, TCRb2/2, and Id1tg/tg transgenic mice with and serve different functions, which could be of great significance 500 third-stage infective larvae s.c. and measured adult worm in understanding certain illnesses such as childhood asthma. 8 AUGMENTED THYMIC ILC2 PRODUCTION DUE TO E PROTEIN DEFICIT

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Nat. lymphoid cell subsets possess tissue-type based heterogeneity in phenotype and Immunol. 6: 1079–1086. frequency. Immunity 46: 148–161. Supplemental Figure 1. Id1 transgenic thymocytes are capable of producing type 2 cytokines. (A) The developmental profiles were determined by staining for CD4, CD8, CD44 and CD25 in thymocytes negative for B220, Mac-1, Gr-1, TCRγδ and NK1.1. The CD4 and CD8 double negative subset was further analyzed for CD44 and CD25 expression. (B) Total thymocytes from WT and Id1tg/tg mice were stimulated with 500 ng/ml of ionomycin, 50 ng/ml PMA and monensin for 4 h. The CD4- CD8-B220-DX5- fraction was analyzed for the expression of indicated cytokines after intracellular staining with the Cytofix/Cytoperm Kit (BD Biosciences). (C) IL-5 and IL-13 production by thymic ILC2s (Lin- Thy1+ST2+) after stimulated as in (B). Data are representative of two independent experiments. Supplemental Figure 2. Augmented ILC2 counts in Id1 transgenic ILC2s are not due to increased proliferation or survival (A) ILC2s in indicated tissues of WT and Id1tg/tg mice were stained intracellularly for Ki67 and DAPI along with antibodies described for Fig. 2B. Lin-Thy1+ST2+ cells were gated to analyze Ki67 and DAPI fluorescence. Analyses of WT DN3 thymocytes and Lin- bone marrow cells served as controls for cycling cells. Numbers show the percentage in each quadrant. Ki67+DAPI+ cells are considered to be in S-G2 phases of the cell cycle whereas Ki67+DAPI- mark cells in the G1 phase. Average percentages are shown in the bar graphs (n=3). Data are representative of two independent experiments. (B) ILC2s in indicated tissues of WT and Id1tg mice were stained with Annexin V and 7AAD along with antibodies described for Fig. 2B. Annexin-V+7AAD- cells are considered apoptotic. Average percentages are shown in the bar graphs (n=3 or 4). Data are representative of two independent experiments. * < 0.05. Supplemental Table 1 Lists of genes shown in Fig. 3B WT-ILC2-SIId1_M1 Id1_M2 Id1_T1 Id1_T2 ThyDN3_1ThyDN3_2Gene -0.3056 -0.3782 -0.5911 0.2702 0.2795 0.3561 0.3690 Pif1 -0.2213 -0.4805 -0.5614 0.2685 0.3293 0.2943 0.3712 Ska1 -0.1881 -0.5136 -0.5501 0.2769 0.2783 0.3213 0.3753 Troap -0.1922 -0.5008 -0.5597 0.2914 0.2741 0.3061 0.3811 Top2a -0.1998 -0.4994 -0.5585 0.2916 0.2824 0.3349 0.3488 Spag5 -0.2062 -0.4600 -0.5823 0.2607 0.2340 0.3785 0.3752 Clspn -0.2341 -0.4727 -0.5515 0.2639 0.2362 0.3307 0.4276 Pbk -0.2313 -0.4934 -0.5340 0.2479 0.2538 0.3257 0.4312 Ccna2 -0.2439 -0.5128 -0.5114 0.2631 0.2593 0.3454 0.4003 Shcbp1 -0.2129 -0.5258 -0.5212 0.2941 0.2450 0.3493 0.3715 Fignl1 -0.2754 -0.5243 -0.4853 0.3099 0.3284 0.3032 0.3436 Neil3 -0.2699 -0.5448 -0.4654 0.3334 0.3180 0.2703 0.3584 Prr11 -0.3132 -0.5142 -0.4639 0.3296 0.3137 0.2724 0.3756 Rrm2 -0.2893 -0.4859 -0.5050 0.2881 0.2884 0.2767 0.4270 2810417H13Rik -0.2891 -0.4471 -0.5443 0.2949 0.2728 0.3075 0.4053 Ncapg -0.3162 -0.4733 -0.4954 0.2654 0.2523 0.3497 0.4175 Espl1 -0.3098 -0.4610 -0.5123 0.2606 0.2570 0.3507 0.4149 Ncaph -0.2808 -0.4879 -0.5129 0.2766 0.2791 0.3299 0.3960 Kif4 -0.2781 -0.4628 -0.5362 0.2665 0.2584 0.3610 0.3911 Cdc6 -0.3102 -0.5282 -0.4369 0.2339 0.2667 0.3240 0.4507 Cdca3 -0.3550 -0.4569 -0.4798 0.2721 0.3054 0.2773 0.4369 Nusap1 -0.4015 -0.3673 -0.5210 0.2521 0.2647 0.3810 0.3920 Matn2 -0.4072 -0.3774 -0.5131 0.2827 0.2977 0.3217 0.3956 Kif18b -0.3821 -0.4084 -0.4988 0.2437 0.2611 0.3528 0.4317 Cdca5 -0.4204 -0.3982 -0.4755 0.2560 0.2646 0.3428 0.4307 Cdk1 -0.4484 -0.4268 -0.4301 0.3024 0.3005 0.3108 0.3917 Ttk -0.4105 -0.4413 -0.4534 0.2988 0.2846 0.3456 0.3761 Bub1 -0.4348 -0.4348 -0.4348 0.2787 0.2927 0.3449 0.3881 Hnf4a -0.4434 -0.4345 -0.4172 0.2491 0.2601 0.3615 0.4244 Aurkb -0.4460 -0.4314 -0.4185 0.2466 0.2642 0.3705 0.4146 Nek2 -0.4222 -0.4350 -0.4437 0.2700 0.2687 0.3755 0.3867 Cenpf -0.3983 -0.4229 -0.4771 0.2790 0.2552 0.3808 0.3833 Mcoln3 -0.3756 -0.4789 -0.4444 0.2815 0.2748 0.3438 0.3989 Ckap2l -0.3962 -0.4455 -0.4538 0.2442 0.2916 0.3275 0.4321 F630043A04Rik -0.4088 -0.4634 -0.4341 0.3230 0.3456 0.2831 0.3546 Gm11223 -0.4381 -0.4381 -0.4308 0.3150 0.3706 0.2911 0.3304 Tmem40 -0.4020 -0.4410 -0.4643 0.3447 0.3399 0.3076 0.3151 E2f7 -0.4360 -0.4360 -0.4360 0.3521 0.3479 0.3067 0.3011 2010110K18Rik -0.4361 -0.4361 -0.4361 0.3502 0.3315 0.2936 0.3329 Gbx2 -0.4649 -0.3619 -0.4649 0.3257 0.3974 0.2102 0.3586 Gm11832 -0.1676 -0.5985 -0.4642 0.2356 0.2513 0.3321 0.4114 Fam64a -0.1376 -0.5519 -0.5408 0.2702 0.2987 0.2918 0.3696 Birc5 -0.1482 -0.5752 -0.5094 0.3141 0.2840 0.2593 0.3756 Hmmr -0.1681 -0.5609 -0.5150 0.3028 0.2725 0.2984 0.3702 Casc5 -0.1315 -0.5322 -0.5656 0.3044 0.2919 0.2904 0.3426 Esco2 -0.1448 -0.5215 -0.5683 0.3180 0.2988 0.2710 0.3468 Cep55 -0.0971 -0.5368 -0.5780 0.3117 0.2783 0.3233 0.2986 Mki67 -0.0735 -0.5683 -0.5571 0.2969 0.2752 0.2916 0.3352 Cenpe -0.0876 -0.4803 -0.6226 0.2431 0.2546 0.2757 0.4172 Ube2c -0.0741 -0.5304 -0.5900 0.2648 0.2580 0.2987 0.3730 Kif11 -0.0771 -0.5115 -0.6016 0.2390 0.2286 0.3583 0.3643 Aspm -0.1474 -0.5131 -0.5733 0.2868 0.2592 0.3307 0.3571 Stil -0.1323 -0.4847 -0.6068 0.2846 0.2606 0.3266 0.3519 Sgol1 -0.1253 -0.5096 -0.5818 0.2555 0.2387 0.3241 0.3985 Kif15 -0.1523 -0.5065 -0.5686 0.2458 0.2446 0.3178 0.4192 Spc25 -0.0520 -0.5490 -0.5663 0.1955 0.2062 0.3562 0.4094 E2f2 -0.0823 -0.5393 -0.5568 0.2099 0.1697 0.3851 0.4137 Mcm10 -0.0735 -0.5758 -0.5363 0.2283 0.2152 0.3436 0.3987 Rad51 -0.0324 -0.5103 -0.6084 0.2112 0.1753 0.3640 0.4007 5730590G19Rik -0.1001 -0.6459 -0.4369 0.2340 0.2489 0.4505 0.2496 Spns2 0.0303 -0.6237 -0.5361 0.2858 0.2586 0.2538 0.3313 E2f8 0.0076 -0.6112 -0.5409 0.3219 0.2242 0.2770 0.3213 Muc13 0.0064 -0.6704 -0.4729 0.2576 0.2507 0.3061 0.3224 Mastl 0.0187 -0.5805 -0.5699 0.3047 0.3167 0.1696 0.3406 Cenpk 0.0961 -0.5543 -0.6168 0.2145 0.2142 0.2893 0.3571 BC030867 0.0865 -0.5716 -0.5995 0.2341 0.2117 0.2833 0.3555 Kif14 0.0412 -0.5131 -0.6356 0.2269 0.1889 0.2982 0.3936 Chek1 0.1273 -0.5229 -0.6453 0.2167 0.1581 0.3213 0.3447 Kntc1 0.0903 -0.5473 -0.6099 0.1945 0.1541 0.3389 0.3794 Dtl 0.1357 -0.5893 -0.5890 0.1970 0.2210 0.2645 0.3601 Ccnb2 0.1502 -0.5901 -0.5836 0.1639 0.2330 0.2389 0.3878 Cks1b -0.1212 -0.3561 -0.7056 0.2982 0.2539 0.3797 0.2512 Gm17356 -0.1692 -0.5141 -0.5560 0.3206 0.3399 0.3674 0.2113 2810442I21Rik -0.2273 -0.4942 -0.4898 0.1660 0.1291 0.4440 0.4721 Blk -0.1382 -0.5490 -0.4717 0.1161 0.1158 0.4491 0.4779 Syt13 -0.1577 -0.5085 -0.5085 0.1297 0.1420 0.5326 0.3705 Gm11579 -0.1279 -0.6024 -0.3610 0.0756 0.0328 0.4741 0.5088 Tspan2 0.0603 -0.5042 -0.5769 0.0707 0.0546 0.4633 0.4321 Gria3 0.0218 -0.4642 -0.5804 0.0658 0.0163 0.4610 0.4796 Angptl2 -0.1075 -0.4121 -0.6132 0.0789 0.1453 0.4881 0.4206 Ddc -0.5000 -0.3690 -0.3823 0.1867 0.1634 0.4489 0.4523 Myb -0.4945 -0.3576 -0.3537 0.1350 0.0981 0.4593 0.5135 Tmem121 -0.4497 -0.3240 -0.4467 0.1563 0.1111 0.5098 0.4432 Rab44 -0.4465 -0.3309 -0.4465 0.1810 0.0945 0.4865 0.4619 Ybx2 -0.5344 -0.2535 -0.3969 0.1128 0.1034 0.4944 0.4741 Endou -0.4078 -0.4078 -0.4078 0.1386 0.1179 0.4668 0.5001 BC048355 -0.4148 -0.4148 -0.4148 0.1752 0.1369 0.4688 0.4634 Fads2 -0.4151 -0.4141 -0.4276 0.1885 0.1616 0.4356 0.4712 Gm5648 -0.3557 -0.4248 -0.4762 0.1918 0.1697 0.4386 0.4565 Uhrf1 -0.3978 -0.3967 -0.4592 0.1831 0.1662 0.4118 0.4927 Clec12a -0.4224 -0.4224 -0.4224 0.2356 0.1561 0.4680 0.4073 Prtg -0.5003 -0.5003 -0.2836 0.3049 0.2649 0.3395 0.3749 Pcyt1b -0.4674 -0.4674 -0.3456 0.2366 0.2206 0.4135 0.4098 Dmwd -0.4741 -0.4741 -0.2877 0.1942 0.1343 0.4627 0.4447 Gm10384 -0.4791 -0.3316 -0.4735 0.2399 0.2900 0.4484 0.3059 S1pr3 -0.4617 -0.4617 -0.3629 0.2563 0.2931 0.4657 0.2711 Hist1h2bj -0.5674 -0.3239 -0.3847 0.2465 0.2712 0.3387 0.4196 2010317E24Rik -0.5860 -0.3110 -0.3796 0.3025 0.2732 0.3052 0.3957 Lrr1 -0.5081 -0.3729 -0.4204 0.3053 0.3008 0.3306 0.3648 Nuf2 -0.5062 -0.2702 -0.5006 0.2597 0.2887 0.3200 0.4086 Gm6091 -0.5798 -0.3402 -0.3628 0.3366 0.3532 0.2604 0.3326 Depdc1b -0.5753 -0.2940 -0.4131 0.3058 0.3222 0.3093 0.3449 Uchl1 -0.5160 -0.3209 -0.4390 0.2676 0.2009 0.3905 0.4167 I830077J02Rik -0.5482 -0.2728 -0.4317 0.2361 0.1788 0.4113 0.4264 Bcl11a -0.5194 -0.3658 -0.3982 0.2443 0.2402 0.3732 0.4257 2610002D18Rik -0.5415 -0.3696 -0.3592 0.2210 0.2266 0.3756 0.4471 Spc24 -0.6024 -0.3054 -0.3281 0.2116 0.1749 0.4259 0.4235 Ephb2 -0.6327 -0.2640 -0.3338 0.1966 0.2320 0.3530 0.4488 Cenpm -0.6372 -0.3824 -0.2225 0.2569 0.2568 0.3431 0.3853 Iqgap3 -0.6288 -0.3266 -0.3096 0.3102 0.2889 0.3139 0.3520 Neurl1b -0.6217 -0.3388 -0.3027 0.2660 0.2903 0.3203 0.3866 Mxd3 -0.6207 -0.3159 -0.3274 0.2773 0.2798 0.3168 0.3901 Cdc25c -0.6207 -0.3692 -0.2713 0.2948 0.3022 0.2777 0.3865 Ccnb1 -0.6620 -0.3406 -0.2403 0.3389 0.3066 0.2801 0.3173 Slc45a3 -0.5957 -0.3771 -0.2898 0.3335 0.2002 0.3959 0.3330 Olfr524 -0.6899 -0.2253 -0.3057 0.2598 0.2810 0.3081 0.3721 Kif2c -0.6640 -0.2321 -0.3418 0.2717 0.2821 0.3647 0.3194 Ankle1 -0.7441 -0.2249 -0.2056 0.2465 0.2543 0.3130 0.3608 Ccnf -0.7375 -0.1942 -0.2514 0.2755 0.2490 0.3149 0.3437 Rad51ap1 -0.5645 -0.3398 -0.2892 0.1295 0.1123 0.4724 0.4793 Gpr56 -0.5917 -0.3222 -0.2729 0.1307 0.1170 0.4741 0.4649 Epb4.1l4b -0.5700 -0.3373 -0.2381 0.0947 0.0584 0.4963 0.4960 Evpl -0.6988 -0.2059 -0.1603 0.0856 0.0478 0.4715 0.4601 Actn1 -0.3153 -0.1014 -0.1169 -0.3153 -0.3153 0.5891 0.5751 Scn4b -0.3752 -0.0373 -0.0015 -0.3174 -0.3752 0.5979 0.5088 Lipg -0.1259 -0.0928 -0.0966 -0.3810 -0.4307 0.5778 0.5492 Spib -0.0991 -0.2678 -0.2331 -0.3214 -0.2564 0.6144 0.5635 Xist -0.0852 -0.2732 -0.2732 -0.2732 -0.2732 0.6042 0.5737 Fam163b -0.2373 -0.2373 -0.2373 -0.2373 -0.2373 0.6782 0.5083 Gm14236 -0.2637 -0.2637 -0.2637 -0.1265 -0.2637 0.6533 0.5282 Vwce -0.2548 -0.1727 -0.2548 -0.2548 -0.2548 0.5796 0.6121 Hmgcs2 -0.2390 -0.2390 -0.2390 -0.2390 -0.2390 0.5910 0.6041 Spats2 -0.2390 -0.2390 -0.2390 -0.2390 -0.2390 0.6118 0.5832 Camkv -0.2390 -0.2390 -0.2390 -0.2390 -0.2390 0.6113 0.5837 Fbln2 -0.2390 -0.2390 -0.2390 -0.2390 -0.2390 0.6106 0.5844 A930002I21Rik -0.2396 -0.2396 -0.2368 -0.2396 -0.2396 0.5983 0.5969 Ptcra -0.2390 -0.2390 -0.2390 -0.2390 -0.2390 0.6053 0.5898 Gm525 -0.2396 -0.2396 -0.2396 -0.2396 -0.2367 0.6030 0.5922 Myzap -0.2088 -0.2443 -0.2141 -0.2410 -0.2844 0.6133 0.5795 Notch3 -0.2387 -0.2387 -0.2387 -0.2387 -0.2387 0.6337 0.5599 Slc5a9 -0.2385 -0.2385 -0.2385 -0.2385 -0.2385 0.6427 0.5500 Adamtsl2 -0.2389 -0.2389 -0.2389 -0.2389 -0.2389 0.6222 0.5723 Odz1 -0.2389 -0.2389 -0.2389 -0.2389 -0.2389 0.6240 0.5704 Rtkn -0.2388 -0.2388 -0.2388 -0.2388 -0.2388 0.6269 0.5673 Kcnh3 -0.2388 -0.2388 -0.2388 -0.2388 -0.2388 0.6261 0.5682 Cldn4 -0.2888 -0.1540 -0.2888 -0.1624 -0.2888 0.5985 0.5843 Tdrd5 -0.2630 -0.2630 -0.1796 -0.2231 -0.2630 0.6097 0.5820 Uaca -0.2567 -0.2567 -0.1671 -0.2567 -0.2541 0.5868 0.6045 2010001M09Rik -0.3069 -0.2515 -0.0951 -0.2195 -0.3041 0.5969 0.5801 Mst1r -0.3840 -0.1931 -0.2408 -0.1534 -0.2048 0.5964 0.5797 Bst1 -0.4229 -0.2042 -0.1157 -0.1831 -0.2371 0.5742 0.5887 Tmem151b -0.3094 -0.3094 -0.3094 -0.0778 -0.1603 0.6025 0.5639 Rag1 -0.3078 -0.3078 -0.3078 -0.0501 -0.1894 0.6087 0.5540 H19 -0.2926 -0.2926 -0.2926 -0.0836 -0.2134 0.6029 0.5719 Mpzl2 -0.2947 -0.2947 -0.2910 -0.0534 -0.2353 0.5853 0.5838 Lhfpl2 -0.3058 -0.3058 -0.3058 -0.0474 -0.1992 0.5699 0.5940 Trbv5 -0.3401 -0.3401 -0.2775 -0.0784 -0.1220 0.5722 0.5858 Dntt -0.3522 -0.3522 -0.2640 -0.0407 -0.1407 0.5774 0.5723 Slc16a5 -0.3290 -0.3290 -0.3290 -0.0389 -0.1219 0.5754 0.5725 Elfn2 -0.3241 -0.3241 -0.3241 -0.0387 -0.1407 0.5874 0.5644 1810059H22Rik -0.3216 -0.3216 -0.3216 -0.0322 -0.1558 0.5774 0.5754 C1qtnf1 -0.4104 -0.2156 -0.3280 -0.0962 -0.0977 0.5787 0.5691 Gfra1 -0.3235 -0.2539 -0.3235 -0.1131 -0.1585 0.5661 0.6065 Ly6d -0.3092 -0.3092 -0.3092 -0.1265 -0.1152 0.5920 0.5774 Gm5111 -0.3008 -0.3008 -0.2968 -0.1452 -0.1329 0.5853 0.5913 Cpne5 -0.3110 -0.2520 -0.3071 -0.1356 -0.1730 0.6253 0.5533 Col15a1 -0.2695 -0.2695 -0.2664 -0.2695 -0.1048 0.6356 0.5442 Gm12503 -0.2675 -0.2675 -0.2640 -0.2209 -0.1707 0.6098 0.5809 Pgpep1l -0.3986 -0.2856 -0.1840 -0.1741 -0.1236 0.5988 0.5671 A930013B10Rik -0.3444 -0.3444 -0.2051 -0.2180 -0.0436 0.6215 0.5340 Chrna9 -0.5428 -0.2115 -0.1507 -0.1166 -0.0877 0.5807 0.5286 Pld4 -0.4982 -0.2104 -0.1387 -0.1146 -0.1729 0.5923 0.5426 Adamts17 -0.3869 -0.2919 -0.3419 -0.0421 -0.0697 0.5784 0.5541 Ptprf -0.3454 -0.3454 -0.3454 -0.0383 -0.0544 0.5762 0.5528 Rag2 -0.3527 -0.3527 -0.3527 -0.0008 -0.0578 0.5566 0.5601 Ptk7 -0.3580 -0.3580 -0.3543 0.0152 -0.0533 0.5335 0.5748 Tubb3 -0.4397 -0.2966 -0.3164 -0.0052 -0.0519 0.5561 0.5537 Sox13 -0.3539 -0.3539 -0.3501 -0.0696 0.0168 0.5052 0.6055 Id3 -0.3907 -0.3907 -0.3907 0.0886 0.0524 0.5182 0.5127 Prss57 -0.3922 -0.3922 -0.3888 0.0878 0.0553 0.5140 0.5160 Slc43a1 -0.3747 -0.3747 -0.3747 0.0209 0.0283 0.5397 0.5351 Sstr2 0.5291 -0.4272 -0.1444 0.3699 0.3601 -0.2604 -0.4272 Stc2 0.5843 -0.3636 -0.3636 0.2930 0.3836 -0.1702 -0.3636 Igfbp7 0.5443 -0.3337 -0.1910 0.3400 0.3931 -0.4126 -0.3400 Ccr6 0.6636 -0.2962 -0.1486 0.2887 0.2617 -0.4169 -0.3523 Sox8 0.6106 -0.2929 -0.1487 0.2863 0.3437 -0.3995 -0.3995 2810468N07Rik 0.7032 -0.4078 -0.3203 0.2444 0.2440 -0.1607 -0.3027 Il1r1 0.7206 -0.3818 -0.3818 0.2452 0.2090 -0.2269 -0.1844 Mapk10 0.7577 -0.3470 -0.3419 0.1918 0.2021 -0.1867 -0.2760 Scrt1 0.7394 -0.3305 -0.3900 0.2153 0.2125 -0.2028 -0.2438 Abi3bp 0.7398 -0.2968 -0.2968 0.2094 0.2378 -0.2968 -0.2968 1700006J14Rik 0.6935 -0.3081 -0.3017 0.2941 0.2384 -0.3081 -0.3081 Tdrd9 0.7914 -0.1657 -0.2684 0.1662 0.1472 -0.3354 -0.3354 Fzd4 0.7945 -0.1855 -0.3042 0.1882 0.1248 -0.3089 -0.3089 Irg1 0.7980 -0.3179 -0.2098 0.1568 0.1533 -0.3179 -0.2625 Kcnk1 0.8159 -0.2733 -0.2679 0.1080 0.1639 -0.2733 -0.2733 Smarca1 0.7796 -0.2703 -0.1801 0.1675 0.1774 -0.3371 -0.3371 Il23r 0.7952 -0.2132 -0.1423 0.1343 0.1476 -0.3907 -0.3309 Plekhg1 0.7334 -0.2451 -0.1614 0.2167 0.2109 -0.3772 -0.3772 Upp1 0.7708 -0.4411 -0.3643 0.0905 0.0243 -0.2224 0.1423 Hspa1a 0.7373 -0.4203 -0.4963 0.1664 0.0393 0.0308 -0.0573 Lingo4 0.7428 -0.4871 -0.2879 0.1845 0.1890 -0.1644 -0.1769 Mc5r 0.6922 -0.4070 -0.4683 0.2789 0.1588 -0.1444 -0.1101 Sdc1 0.6346 -0.5840 -0.4039 0.2346 0.1864 -0.0567 -0.0110 Tox2 0.8992 -0.2280 -0.1693 -0.2280 -0.2251 0.0347 -0.0836 Gm17056 0.9062 -0.2188 -0.2219 -0.1986 -0.1934 -0.0040 -0.0694 Fosb 0.9074 -0.2091 -0.2043 -0.2091 -0.2091 -0.0586 -0.0173 4930532G15Rik 0.9186 -0.1866 -0.1823 -0.1866 -0.1866 -0.0503 -0.1263 9630028B13Rik 0.9188 -0.1756 -0.1756 -0.1756 -0.1756 -0.0411 -0.1756 Hs6st2 0.9079 -0.2479 -0.0420 -0.2479 -0.1793 -0.1138 -0.0770 Slc24a1 0.8668 -0.3352 -0.2789 -0.1856 -0.1445 0.0285 0.0488 mt-Rnr2 0.8854 -0.2661 -0.2621 -0.0360 -0.0475 -0.0035 -0.2702 Ptgs2 0.9042 -0.2840 -0.1250 -0.1775 -0.0692 -0.0266 -0.2218 Snord118 0.8702 -0.0280 -0.1182 -0.2709 -0.2709 0.0888 -0.2709 SNORA17 0.9197 -0.0485 -0.1742 -0.1742 -0.1742 -0.1742 -0.1742 Cxcl2 0.9233 -0.1674 -0.1674 -0.0862 -0.1674 -0.1674 -0.1674 Il22 0.9258 -0.1543 -0.1543 -0.1543 -0.1543 -0.1543 -0.1543 Cxcl1 0.9258 -0.1543 -0.1543 -0.1543 -0.1543 -0.1543 -0.1543 Gm12473 0.9258 -0.1549 -0.1549 -0.1549 -0.1515 -0.1549 -0.1549 Zmat4 0.8875 -0.0428 -0.0760 -0.0585 -0.0880 -0.3111 -0.3111 Atf3 0.9009 -0.1349 -0.0976 -0.1104 -0.0389 -0.3375 -0.1817 Gjb2 0.3797 -0.4122 -0.6332 -0.0105 -0.0713 0.3582 0.3893 Ctla4 0.1692 -0.4067 -0.7403 0.1984 0.1950 0.2224 0.3619 Cdkn3 0.1983 -0.3574 -0.7457 0.2280 0.0072 0.3161 0.3535 Pou2af1 0.3883 -0.6554 -0.5042 0.1939 0.1216 0.1596 0.2961 Gm5465 0.4797 -0.6083 -0.5234 0.1264 0.1183 0.1233 0.2841 Gas2l3 0.4328 -0.5301 -0.6259 0.2576 0.1858 0.1428 0.1370 Auts2 0.3358 -0.5227 -0.6562 0.2499 0.1894 0.2439 0.1599 Tacstd2 0.5866 -0.6068 -0.4216 -0.0256 -0.0001 0.2375 0.2300 Dock1 0.5246 -0.6072 -0.4898 0.0515 0.1915 0.2711 0.0583 Rnf150 0.5745 -0.5802 -0.4791 0.0619 0.0509 0.3044 0.0677 Gls2 0.4332 -0.3232 -0.3232 -0.3232 -0.3232 0.5418 0.3177 Gm12913 0.4375 -0.3265 -0.3265 -0.3265 -0.3265 0.4855 0.3829 mt-Tl1 0.3568 -0.3308 -0.2693 -0.4427 -0.2308 0.5614 0.3554 Mir5115 0.4883 -0.2533 -0.2486 -0.4109 -0.3547 0.5264 0.2528 Mir5109 0.5765 -0.3228 -0.3228 -0.3228 -0.3194 0.3587 0.3527 Gm5263 0.7845 -0.2834 -0.2834 -0.2834 -0.2800 0.1205 0.2253 Gm7618 0.7407 -0.2829 -0.2294 -0.4038 -0.2528 0.2092 0.2191 Gm12420 0.0044 -0.4524 -0.4524 0.5456 0.5202 -0.0164 -0.1490 Svep1 0.3101 -0.4581 -0.4581 0.4447 0.3541 0.1705 -0.3632 Cacna1e 0.4055 -0.5457 -0.5457 0.3261 0.3539 -0.0617 0.0677 Grb10 0.1249 -0.4878 -0.6175 0.4901 0.3068 0.0082 0.1754 Lrrc66 0.3621 -0.2443 -0.7250 0.4612 0.2500 -0.0134 -0.0907 Sprn -0.1141 -0.6483 -0.3389 0.4344 0.4362 -0.0367 0.2675 Depdc1a 0.0325 -0.4609 -0.3282 0.4806 0.5318 -0.3851 0.1293 Hhex -0.3659 -0.3659 -0.3659 0.6079 0.4333 -0.1121 0.1687 Gm16194 -0.4179 -0.2851 -0.4179 0.5290 0.3839 -0.1416 0.3495 42257 -0.3176 -0.3176 -0.3176 0.4964 0.5460 -0.3176 0.2279 Olfr523 -0.3358 -0.3358 -0.2540 0.6018 0.5593 -0.1778 -0.0576 Clec4e -0.3170 -0.3170 -0.3170 0.6237 0.5353 -0.1297 -0.0784 Cldn2 -0.4244 -0.2093 -0.2944 0.5854 0.5669 -0.1216 -0.1025 Gm8113 -0.2395 -0.2395 -0.2327 0.5336 0.6570 -0.2395 -0.2395 Syt6 -0.2607 -0.2607 -0.2607 0.5147 0.6664 -0.2607 -0.1383 Siglec5 -0.2626 -0.2626 -0.2626 0.5026 0.6748 -0.2626 -0.1269 Prss16 -0.2864 -0.1069 -0.2864 0.6078 0.5722 -0.2864 -0.2138 Cd34 -0.2750 -0.0824 -0.2676 0.6353 0.5396 -0.2750 -0.2750 Gcm2 -0.2388 -0.2388 -0.2388 0.6292 0.5648 -0.2388 -0.2388 Fgf3 -0.2390 -0.2390 -0.2390 0.6055 0.5897 -0.2390 -0.2390 Ripply1 -0.2390 -0.2390 -0.2390 0.6109 0.5841 -0.2390 -0.2390 Sema3c -0.2405 -0.2405 -0.2327 0.6154 0.5794 -0.2405 -0.2405 Gm15947 -0.2390 -0.2390 -0.2390 0.6170 0.5778 -0.2390 -0.2390 Aox1 -0.2383 -0.2383 -0.2383 0.6512 0.5404 -0.2383 -0.2383 Ahsg -0.2378 -0.2378 -0.2378 0.6669 0.5221 -0.2378 -0.2378 Ctsg -0.2374 -0.2374 -0.2374 0.6769 0.5100 -0.2374 -0.2374 Caly -0.2370 -0.2370 -0.2370 0.6856 0.4993 -0.2370 -0.2370 Trav14n-3 -0.4078 -0.2114 -0.4078 0.5766 0.5277 0.0277 -0.1048 Slc35d3 -0.3127 -0.3127 -0.3127 0.7026 0.4252 -0.0106 -0.1790 Gm11692 -0.2947 0.0418 -0.2947 0.5194 0.6174 -0.2947 -0.2947 Adamts2 -0.3427 -0.0422 -0.3340 0.6328 0.4968 -0.0680 -0.3427 Stbd1 -0.1874 0.0497 0.0613 0.5145 0.5020 -0.4657 -0.4745 Lgals3 -0.1692 0.0488 0.1016 0.4936 0.4949 -0.4849 -0.4849 Cx3cr1 -0.4426 0.1036 -0.0047 0.5512 0.4822 -0.4426 -0.2470 Parm1 -0.3073 -0.1526 -0.0954 0.5933 0.5765 -0.3073 -0.3073 Fam179a -0.3610 -0.0549 -0.0382 0.5586 0.5653 -0.4033 -0.2665 Igfbp4 -0.3546 -0.1209 0.0893 0.5521 0.5434 -0.3546 -0.3546 Mmp27 -0.3696 -0.0382 0.0665 0.5516 0.5291 -0.3696 -0.3696 Lama3 -0.3843 0.0233 0.0813 0.5095 0.5387 -0.3843 -0.3843 Abhd12b -0.4266 -0.4266 -0.4196 0.4613 0.3860 0.2748 0.1507 Morc4 -0.5206 -0.3192 -0.4411 0.4082 0.3801 0.2528 0.2398 Gm15428 -0.4316 -0.4316 -0.4316 0.4083 0.3834 0.2720 0.2312 Il31ra -0.4410 -0.4082 -0.4517 0.3726 0.3807 0.2978 0.2499 Cd163l1 -0.4478 -0.4107 -0.4324 0.3815 0.4195 0.2122 0.2778 Lgals1 -0.4333 -0.4333 -0.4333 0.3660 0.3908 0.2371 0.3061 Gm2788 -0.6235 -0.3308 -0.2922 0.3943 0.3676 0.3074 0.1771 Kif5a -0.4849 -0.3835 -0.3756 0.3990 0.4748 0.0767 0.2935 Gm12856 -0.5218 -0.4174 -0.2762 0.5154 0.3470 0.0564 0.2966 Mdga1 -0.4592 -0.4132 -0.3421 0.4643 0.4916 0.0563 0.2023 Stmn1 -0.4455 -0.4455 -0.3009 0.5377 0.4457 0.0660 0.1426 Odz4 -0.4621 -0.4621 -0.1797 0.5237 0.5068 0.0950 -0.0216 E030002O03Rik -0.4422 -0.3288 -0.2869 0.5780 0.5253 0.0156 -0.0609 Slc15a1 -0.4824 -0.3692 -0.2162 0.5454 0.5274 0.0631 -0.0680 Slc17a8 -0.6279 -0.3401 -0.0579 0.5022 0.4799 0.0628 -0.0189 Sox5 -0.6532 -0.2605 -0.2083 0.4730 0.4701 0.1142 0.0647 Gm5593 -0.7603 -0.1472 -0.1546 0.4273 0.4106 0.1090 0.1152 Tgfb3 -0.6027 -0.2326 -0.3502 0.4366 0.4586 0.2372 0.0531 Lyl1 -0.6737 -0.1559 -0.2654 0.4761 0.4329 0.1930 -0.0069 Tas1r1 -0.8596 0.0915 0.1164 0.3349 0.3412 -0.0839 0.0595 Cpa3 -0.7068 0.0370 0.0970 0.4630 0.4557 -0.2355 -0.1104 Emilin2 -0.9115 0.0552 0.0739 0.1644 0.1555 0.2671 0.1953 Adcy6 -0.8876 -0.0173 -0.0131 0.2333 0.2504 0.2067 0.2276 5830411N06Rik -0.9076 0.0272 0.0601 0.2281 0.2336 0.1572 0.2014 Sell -0.8855 -0.0028 0.0287 0.3039 0.2962 0.1034 0.1559 Slc22a3 0.6935 -0.1365 -0.0843 0.2182 0.2068 -0.4514 -0.4463 Fosl2 0.6026 -0.1774 -0.1227 0.3154 0.2847 -0.4158 -0.4868 Hey1 0.6050 -0.1310 -0.0770 0.3040 0.2526 -0.4768 -0.4768 Tns4 0.5129 -0.1126 -0.0500 0.3185 0.3376 -0.5032 -0.5032 Rhod 0.5697 -0.0196 0.0439 0.2261 0.2417 -0.5309 -0.5309 Calca 0.5213 -0.0771 -0.0068 0.2913 0.2940 -0.6296 -0.3932 Plaur 0.4660 0.0929 -0.0749 0.3214 0.2786 -0.5420 -0.5420 Sv2b 0.1855 -0.0551 0.0152 0.4492 0.4547 -0.4852 -0.5642 Ifitm1 0.1877 -0.0549 -0.0649 0.4934 0.4501 -0.5057 -0.5057 Edil3 0.1896 0.0612 0.0589 0.3992 0.3990 -0.6644 -0.4437 Fam20a 0.1232 -0.0750 -0.1854 0.5454 0.4920 -0.4556 -0.4446 Podxl 0.2691 -0.2535 -0.0686 0.4666 0.4856 -0.4686 -0.4307 Btnl9 0.3834 -0.1700 -0.1277 0.3530 0.4869 -0.5570 -0.3685 Nqo1 0.3280 -0.2113 -0.3586 0.5132 0.4457 -0.3586 -0.3586 Fam81a 0.4094 -0.2448 -0.3698 0.4645 0.4255 -0.2963 -0.3886 Dsp 0.2838 -0.3221 -0.3221 0.5077 0.4967 -0.3221 -0.3221 Eml1 0.2780 -0.3072 -0.3072 0.2593 0.6914 -0.3072 -0.3072 Clec4d 0.4409 0.2308 0.2028 0.1497 0.1297 -0.6334 -0.5205 Hs3st1 0.4776 0.2250 0.2065 0.1133 0.1191 -0.5708 -0.5708 Klrg1 0.4367 0.2149 0.2107 0.1520 0.1467 -0.5805 -0.5805 Ccr8 0.4201 0.2090 0.2137 0.1600 0.1647 -0.5837 -0.5837 Il4 0.4367 0.2327 0.2170 0.1497 0.1221 -0.5791 -0.5791 Fbxl21 0.4242 0.2319 0.2245 0.1497 0.1324 -0.5814 -0.5814 Naip6 0.3976 0.1997 0.2105 0.1748 0.1840 -0.6631 -0.5036 Fam46a 0.4018 0.1894 0.2043 0.1831 0.1923 -0.6359 -0.5349 Neurl3 0.4078 0.2028 0.1787 0.2077 0.1694 -0.6477 -0.5188 Abcb1a 0.4143 0.2073 0.1957 0.1816 0.1657 -0.6470 -0.5177 Myo1e 0.4159 0.1863 0.1519 0.2022 0.2023 -0.6698 -0.4888 Il2rb 0.4306 0.1868 0.2082 0.1664 0.1525 -0.6958 -0.4487 Lilrb4 0.4818 0.1201 0.1464 0.2059 0.1860 -0.5209 -0.6194 Gp49a 0.4427 0.1764 0.1637 0.2056 0.1725 -0.5705 -0.5904 Rora 0.4687 0.1745 0.1203 0.1770 0.2089 -0.5747 -0.5747 1700113H08Rik 0.6257 0.1705 0.1634 0.0392 0.0488 -0.5238 -0.5238 Csf2 0.5689 0.1764 0.1793 0.1055 0.0632 -0.5468 -0.5468 Clnk 0.6369 0.0918 0.0639 0.1138 0.1414 -0.5239 -0.5239 Il13 0.6970 0.0781 0.0609 0.0707 0.0845 -0.5182 -0.4730 Tgm2 0.6953 0.0402 0.0443 0.0926 0.1181 -0.4952 -0.4952 Klrb1b 0.4613 0.3035 0.3075 -0.0244 0.0393 -0.6042 -0.4831 Cxcl10 0.6073 0.2295 0.2460 -0.0535 -0.0150 -0.5227 -0.4916 Ccrl2 0.2632 0.3181 0.3217 0.1238 0.1254 -0.4511 -0.7011 Gm4956 0.2702 0.3468 0.3214 0.1219 0.1039 -0.5821 -0.5821 Art2b 0.2762 0.3369 0.3539 0.0831 0.1038 -0.5495 -0.6045 Ffar2 0.2490 0.3628 0.3691 0.0724 0.0925 -0.5729 -0.5729 Hpn 0.3144 0.2988 0.3328 0.1122 0.1058 -0.5820 -0.5820 Pard3 0.3131 0.3406 0.3160 0.0948 0.0920 -0.5782 -0.5782 Asb2 0.3359 0.3245 0.2981 0.1001 0.1013 -0.5800 -0.5800 Serpina3f 0.3312 0.3002 0.2883 0.1215 0.1297 -0.5854 -0.5854 Csf1 0.3829 0.2873 0.2778 0.1081 0.1027 -0.5794 -0.5794 Ret 0.3897 0.3051 0.2789 0.1108 0.0652 -0.5748 -0.5748 Art2a-ps 0.4132 0.3016 0.2857 0.0579 0.0792 -0.5688 -0.5688 Sdc4 0.4175 0.2933 0.2576 0.0752 0.1028 -0.5732 -0.5732 Il6 0.4006 0.2876 0.2716 0.0888 0.0977 -0.5073 -0.6390 Gimap4 0.3762 0.2303 0.2058 0.1786 0.1503 -0.4011 -0.7401 Adam19 0.4332 0.2026 0.1969 0.1503 0.1596 -0.4454 -0.6972 Dkk3 0.4832 0.2619 0.2242 0.0791 0.0483 -0.4113 -0.6855 Irak3 0.2573 0.3125 0.2975 0.1502 0.1641 -0.5908 -0.5908 Serpina3g 0.2538 0.2850 0.2927 0.1657 0.1903 -0.5937 -0.5937 1810011H11Rik 0.2483 0.2929 0.3046 0.1613 0.1778 -0.5925 -0.5925 Cpne7 0.2516 0.3074 0.3179 0.1507 0.1496 -0.6284 -0.5488 H2-Q6 0.2023 0.2990 0.3007 0.1822 0.2027 -0.6119 -0.5750 Gimap3 0.2286 0.2921 0.3172 0.1645 0.1819 -0.6062 -0.5781 Fam55d 0.2421 0.2746 0.2708 0.2003 0.2037 -0.6187 -0.5729 Naip2 0.2376 0.2587 0.2778 0.2115 0.2066 -0.6223 -0.5699 Mmp25 0.1580 0.2931 0.3101 0.2019 0.2178 -0.6516 -0.5293 Acsbg1 0.1768 0.3332 0.3398 0.1660 0.1575 -0.6156 -0.5576 H2-Q8 0.2292 0.2571 0.2524 0.2304 0.2195 -0.5237 -0.6649 Eif2s3y 0.2056 0.2519 0.2564 0.2261 0.2512 -0.5461 -0.6450 Gm14303 0.2043 0.2736 0.2696 0.2106 0.2335 -0.5648 -0.6268 Zbtb7b 0.2495 0.2358 0.2440 0.2209 0.2447 -0.5975 -0.5975 Krt80 0.2272 0.2323 0.2369 0.2401 0.2581 -0.6119 -0.5827 Ptgir 0.1491 0.2871 0.2755 0.2249 0.2513 -0.5940 -0.5940 Alox5 0.1783 0.2707 0.2834 0.2273 0.2314 -0.5863 -0.6047 Samhd1 0.1916 0.2805 0.2733 0.2211 0.2254 -0.5959 -0.5959 Fam19a3 0.1895 0.3046 0.2770 0.2039 0.2141 -0.5946 -0.5946 Metrnl 0.1879 0.2719 0.2659 0.2322 0.2338 -0.6210 -0.5707 Tspan32 0.1875 0.2876 0.2816 0.2131 0.2200 -0.6199 -0.5698 Gm5127 0.3410 0.2425 0.2225 0.1904 0.1895 -0.5930 -0.5930 1300014I06Rik 0.3577 0.2668 0.2370 0.1670 0.1493 -0.6088 -0.5691 Nmur1 0.3674 0.2172 0.2506 0.1734 0.1702 -0.5636 -0.6152 Pde7b 0.3564 0.1985 0.2594 0.1691 0.1984 -0.5909 -0.5909 Klrk1 0.2858 0.2652 0.2422 0.1977 0.2007 -0.5958 -0.5958 Rnf128 0.2981 0.2335 0.2355 0.2144 0.2107 -0.5961 -0.5961 Il18rap 0.2912 0.2709 0.2206 0.2131 0.1942 -0.5635 -0.6265 Cntn1 0.3069 0.2529 0.2398 0.1902 0.1996 -0.5728 -0.6166 Cxcr6 0.3084 0.2294 0.2659 0.1782 0.2066 -0.5736 -0.6149 Slc7a8 0.3336 0.2156 0.2279 0.2038 0.2060 -0.5621 -0.6248 Ddx3y 0.3715 0.2380 0.2223 0.1700 0.1667 -0.4937 -0.6749 Epas1 0.3440 0.2562 0.2310 0.1803 0.1556 -0.4722 -0.6950 Lpcat2 0.3886 0.2395 0.2242 0.1450 0.1720 -0.5254 -0.6440 Arg1 0.3142 0.2621 0.2681 0.1693 0.1691 -0.5471 -0.6357 Ttc39b 0.3299 0.2919 0.2381 0.1871 0.1281 -0.5260 -0.6490 Pcsk1 0.2736 0.2613 0.2703 0.1759 0.1929 -0.4722 -0.7019 Naip5 0.2300 0.2413 0.2454 0.2237 0.2300 -0.4425 -0.7280 Nt5e 0.2556 0.2172 0.1977 0.2602 0.2509 -0.4918 -0.6898 Gpr114 0.2510 0.2196 0.2305 0.2613 0.2271 -0.5301 -0.6594 Nav2 0.2743 0.2274 0.2133 0.2436 0.2275 -0.5123 -0.6738 Cd200r1 0.3068 0.2070 0.1779 0.2541 0.2353 -0.5073 -0.6738 Jag1 0.3497 0.1908 0.1622 0.2490 0.2231 -0.5066 -0.6683 Ahnak 0.2269 0.1275 0.1452 0.3365 0.3344 -0.6057 -0.5650 Il18r1 0.2381 0.1380 0.1185 0.3489 0.3244 -0.6097 -0.5582 Gzmb 0.3299 0.1222 0.1097 0.3004 0.3049 -0.5835 -0.5835 Stab2 0.3542 0.1205 0.1418 0.2785 0.2757 -0.6065 -0.5643 Ramp3 0.2814 0.1941 0.1756 0.2759 0.2624 -0.5907 -0.5987 Hlf 0.3255 0.1886 0.1843 0.2489 0.2401 -0.5937 -0.5937 Itga2 0.3582 0.1697 0.1671 0.2394 0.2460 -0.6067 -0.5737 Icos 0.2457 0.2582 0.2421 0.2227 0.2199 -0.6645 -0.5243 Fgl2 0.2833 0.2401 0.2378 0.2157 0.2057 -0.6846 -0.4980 Kdm5d 0.3258 0.2184 0.2306 0.2087 0.1938 -0.6858 -0.4915 Uty 0.3266 0.2165 0.2004 0.2244 0.2071 -0.6945 -0.4804 Cd226 0.0699 0.2841 0.2742 0.2608 0.2821 -0.6401 -0.5309 Rpsa-ps10 0.1362 0.2769 0.2687 0.2408 0.2616 -0.6427 -0.5414 Gm15500 0.1161 0.2858 0.2820 0.2525 0.2407 -0.6618 -0.5154 Fam124b 0.0757 0.2225 0.1871 0.3300 0.3496 -0.5825 -0.5825 S100a4 0.1409 0.2272 0.2364 0.2945 0.2869 -0.6073 -0.5786 Rab27b 0.1999 0.2023 0.2341 0.2714 0.2840 -0.5958 -0.5958 9430031J16Rik 0.1528 0.1214 0.2584 0.3160 0.3256 -0.5871 -0.5871 Zbtb16 -0.0809 0.2491 0.2616 0.3422 0.3467 -0.5593 -0.5593 S1pr1 0.0167 0.2713 0.2670 0.2934 0.3109 -0.5797 -0.5797 Rpl28-ps1 -0.0401 0.2785 0.2860 0.2980 0.3117 -0.6380 -0.4961 Rarg 0.0638 0.2847 0.2832 0.2503 0.2713 -0.4472 -0.7060 Gm13654 0.0194 0.3092 0.3334 0.2260 0.2589 -0.4807 -0.6661 Ltb4r1 0.1126 0.2798 0.2767 0.2572 0.2570 -0.5917 -0.5917 Il1rl1 0.0776 0.2784 0.2820 0.2623 0.2759 -0.5765 -0.5997 Tnfrsf25 0.0992 0.3084 0.2686 0.2577 0.2429 -0.5371 -0.6396 Tlr1 0.0833 0.2723 0.2744 0.2618 0.2826 -0.5352 -0.6393 Rps25-ps1 0.1435 0.3244 0.3335 0.1885 0.1857 -0.5563 -0.6193 Gvin1 0.1064 0.3470 0.3412 0.1866 0.1865 -0.5714 -0.5964 Grin2d 0.1247 0.3345 0.3406 0.1824 0.1817 -0.5019 -0.6621 Gm4070 -0.6358 0.3335 0.3195 0.2932 0.3144 -0.3479 -0.2769 Gm11361 -0.6136 0.3311 0.3274 0.2997 0.3150 -0.3260 -0.3337 Gm5045 -0.5881 0.3207 0.3282 0.3162 0.3180 -0.3241 -0.3708 Id1 -0.6026 0.3250 0.3301 0.2916 0.3288 -0.3001 -0.3729 Gm10709 -0.6861 0.2604 0.2726 0.3528 0.3411 -0.2741 -0.2668 Elovl7 -0.6634 0.3284 0.3113 0.2914 0.3158 -0.2818 -0.3018 Gm6274 -0.6931 0.3264 0.3338 0.2701 0.2937 -0.2384 -0.2926 Gm5148 -0.4650 0.3698 0.3512 0.3003 0.2808 -0.3721 -0.4650 St8sia6 -0.4657 0.3970 0.4032 0.2440 0.2438 -0.3565 -0.4657 Apol9b -0.4986 0.3356 0.3321 0.3062 0.3261 -0.3410 -0.4605 Gm7536 -0.4677 0.3472 0.3390 0.2952 0.3225 -0.3686 -0.4677 Gm12191 -0.4731 0.3368 0.3390 0.2962 0.3335 -0.3822 -0.4503 Gm10119 -0.4736 0.3144 0.3205 0.3146 0.3494 -0.3338 -0.4915 Gm10736 -0.5052 0.3370 0.3441 0.2781 0.3248 -0.2735 -0.5052 Gm13611 -0.5383 0.3311 0.3261 0.3082 0.3314 -0.3507 -0.4078 Gm5428 -0.5313 0.3412 0.3360 0.2957 0.3237 -0.4236 -0.3417 Gm13268 -0.4579 0.3381 0.3293 0.3110 0.3300 -0.4134 -0.4371 Gm13436 -0.4361 0.3396 0.3471 0.2981 0.3236 -0.4361 -0.4361 Gm12918 -0.4364 0.3268 0.3174 0.3362 0.3287 -0.4364 -0.4364 5430421N21Rik -0.4361 0.3065 0.3150 0.3303 0.3565 -0.4361 -0.4361 Gm10116 -0.4359 0.3064 0.2977 0.3479 0.3557 -0.4359 -0.4359 Gm10241 -0.4251 0.2126 0.2007 0.4129 0.4492 -0.4251 -0.4251 Ggt1 -0.4342 0.2949 0.2589 0.3630 0.3856 -0.4342 -0.4342 AI646023 -0.4314 0.2537 0.2445 0.3780 0.4180 -0.4314 -0.4314 Spon2 -0.3677 0.3239 0.3290 0.3064 0.3293 -0.5731 -0.3477 Gm8430 -0.2017 0.3278 0.3188 0.2971 0.3175 -0.5297 -0.5297 Rps27a-ps2 -0.2919 0.3442 0.3346 0.3015 0.3093 -0.4989 -0.4989 Rpl9-ps4 -0.4870 0.5179 0.5514 -0.0701 -0.0001 -0.0909 -0.4212 Fscn1 -0.4088 0.5673 0.5526 -0.0354 -0.0433 -0.2804 -0.3520 Gm4841 -0.3979 0.5830 0.5352 -0.0084 -0.0952 -0.2187 -0.3979 Gm5970 -0.6080 0.4849 0.4918 0.0274 0.1247 -0.2873 -0.2334 Ifit3 -0.5554 0.5243 0.5084 0.0562 0.0145 -0.3224 -0.2256 Gm4951 -0.5893 0.5039 0.5245 -0.0102 0.0551 -0.2830 -0.2011 I830012O16Rik -0.5960 0.5161 0.5240 -0.0127 0.0191 -0.2544 -0.1961 Ifi44 -0.4696 0.4955 0.4825 0.0872 0.1172 -0.4696 -0.2434 Ms4a4c -0.4982 0.4911 0.5059 0.0896 0.0956 -0.3850 -0.2989 Slfn1 -0.6663 0.3964 0.4193 0.1788 0.2122 -0.2643 -0.2762 Pydc3 -0.6989 0.4368 0.4621 0.0787 0.1293 -0.1806 -0.2273 Tlr7 -0.7469 0.4577 0.4664 -0.0697 0.0281 -0.0798 -0.0558 Gm14446 -0.8407 0.3459 0.3395 0.1384 0.1621 -0.0379 -0.1073 Ly6c1 -0.1229 0.5597 0.5354 -0.0444 -0.0687 -0.3614 -0.4977 F830016B08Rik 0.1025 0.5190 0.5192 -0.1364 -0.0872 -0.4153 -0.5018 Gbp10 0.1623 0.5275 0.5550 -0.3112 -0.3112 -0.3112 -0.3112 Apol7c 0.0369 0.5753 0.5492 -0.3394 -0.3394 -0.1433 -0.3394 Aldh1a2 -0.0176 0.5639 0.5713 -0.0996 -0.3393 -0.3393 -0.3393 Vmn2r100 -0.2675 0.5409 0.6396 -0.1105 -0.2675 -0.2675 -0.2675 Clu -0.2781 0.5749 0.6124 -0.1873 -0.1657 -0.2781 -0.2781 Klra14 -0.2386 0.6410 0.5519 -0.2386 -0.2386 -0.2386 -0.2386 Anxa3 -0.2389 0.5687 0.6255 -0.2389 -0.2389 -0.2389 -0.2389 Gm8221 -0.2388 0.5684 0.6259 -0.2388 -0.2388 -0.2388 -0.2388 Zfp366 -0.2390 0.5928 0.6024 -0.2390 -0.2390 -0.2390 -0.2390 Lyz1 -0.2331 0.4253 0.7400 -0.2331 -0.2331 -0.2331 -0.2331 Cd209b 0.5345 0.3458 0.4050 -0.3873 -0.2656 -0.2452 -0.3873 Ccl22 0.5224 0.3849 0.3906 -0.3181 -0.3470 -0.3637 -0.2692 Dgat2 0.5150 0.3705 0.3614 -0.1966 -0.1491 -0.4506 -0.4506 Cybb 0.3815 0.5408 0.3444 -0.2676 -0.1784 -0.4497 -0.3711 Cyp26b1 0.7509 0.2120 0.2010 -0.3391 -0.3391 -0.2058 -0.2801 Ahrr 0.7005 0.2774 0.2316 -0.2386 -0.2975 -0.2797 -0.3938 Atp8a2 0.8038 0.1909 0.0781 -0.2860 -0.3536 -0.2894 -0.1439 Ppfibp2 0.4294 0.4628 0.2500 -0.1762 -0.6633 -0.0462 -0.2566 Ceacam15 -0.1808 0.4432 0.3482 -0.4698 -0.5663 0.3067 0.1189 Gbp11 -0.4263 0.3477 0.3104 -0.4263 -0.4195 0.4726 0.1414 Dnahc2 -0.1121 0.3904 0.1626 -0.2991 -0.6375 0.5559 -0.0602 Gm12194 -0.4548 0.2581 0.1787 -0.3542 -0.4548 0.3129 0.5142 Akap2 -0.4585 0.4588 0.4745 -0.2721 -0.4585 -0.0081 0.2639 Lad1 -0.4696 0.4803 0.4063 -0.2673 -0.4630 0.3129 0.0004 Lama4 -0.4469 0.4339 0.5917 -0.2097 -0.4469 0.1261 -0.0483 Gm16340 -0.4462 0.4168 0.6050 -0.2090 -0.4462 0.1271 -0.0474 Ttc39a 0.0087 0.2248 0.2459 -0.5908 -0.5605 0.3300 0.3419 Pou6f1 0.2755 0.1192 0.1961 -0.6592 -0.5115 0.3500 0.2299 Tlr9