Loss of T Cell Progenitor Checkpoint Control Underlies Leukemia Initiation in Rag1 -Deficient Nonobese Diabetic Mice

This information is current as Mary A. Yui, Ni Feng, Jingli A. Zhang, Chen Yee Liaw, of September 25, 2021. Ellen V. Rothenberg and Jeffrey A. Longmate J Immunol published online 25 February 2013 http://www.jimmunol.org/content/early/2013/02/24/jimmun ol.1202970 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 © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 25, 2013, doi:10.4049/jimmunol.1202970 The Journal of Immunology

Loss of T Cell Progenitor Checkpoint Control Underlies Leukemia Initiation in Rag1-Deficient Nonobese Diabetic Mice

Mary A. Yui,* Ni Feng,* Jingli A. Zhang,* Chen Yee Liaw,* Ellen V. Rothenberg,* and Jeffrey A. Longmate†

NOD mice exhibit major defects in the earliest stages of T cell development in the thymus. Genome-wide genetic and transcriptome analyses were used to investigate the origins and consequences of an early T cell developmental checkpoint breakthrough in Rag1- deficient NOD mice. Quantitative trait analysis mapped the presence of checkpoint breakthrough cells to several known NOD diabetes susceptibility regions, particularly insulin-dependent diabetes susceptibility (Idd)9/11 on 4,

suggesting common genetic origins for T cell defects affecting this trait and autoimmunity. Genome-wide RNA deep-se- Downloaded from quencing of NOD and B6 Rag1-deficient thymocytes revealed the effects of genetic background prior to breakthrough, as well as the cellular consequences of the breakthrough. Transcriptome comparison between the two strains showed enrichment in differentially expressed signal transduction genes, prominently tyrosine and actin-binding genes, in accord with their divergent sensitivities to activating signals. Emerging NOD breakthrough cells aberrantly expressed both stem cell–associated proto-oncogenes, such as Lmo2, Hhex, Lyl1, and Kit, which are normally repressed at the commitment checkpoint, and post–b-

selection checkpoint genes, including Cd2 and Cd5. Coexpression of genes characteristic of multipotent progenitors and more http://www.jimmunol.org/ mature T cells persists in the expanding population of thymocytes and in the thymic leukemias that emerge with age in these mice. These results show that Rag1-deficient NOD thymocytes have T cell defects that can collapse regulatory boundaries at two early T cell checkpoints, which may predispose them to both leukemia and autoimmunity. The Journal of Immunology, 2013, 190: 000–000.

ll T cells arise from a small pool of multipotent pro- by decreased Kit expression from the multipotent DN2a stage to genitors, which undergo proliferation and tightly con- the committed DN2b stage (5) and dependent on Bcl11b (6, 7). trolled developmental programming induced and sus- DN2a cells, similar to more immature DN1/ETP cells, undergo

A by guest on September 25, 2021 tained by interactions with the thymic environment rich in Notch proliferative expansion and express legacy stem/progenitor genes ligands and cytokines (1, 2). Thymic precursors are CD4 and such as Kit, Lyl1, Tal1, Gfi1b,andSfpi1 (PU.1). These features CD8 double-negative (DN), and they are defined by sequential decline sharply in the DN2b/DN3 stages when T cell specification changes in surface Kit (CD117), CD25, and CD44 into stages: genes are strongly activated, proliferation slows, and efficient DN1 (or early T cell progenitor, ETP; KithiCD44hiCD252), DN2 TCR rearrangement begins (5, 8). Thus, the T cell commit- (KithiCD44hiCD25+), DN3 (KitloCD44loCD25+), and DN4 (Kitlo ment checkpoint divides the TCR-negative stages of development CD44loCD25lo) (3, 4). A TCR-independent commitment check- into two phases: phase I, wherein cells proliferate and retain al- point has recently been identified within the DN2 stage, marked ternative lineage potential, and phase II, which prepares com- mitted DN3 cells for the first TCR-dependent checkpoint, b-se- lection (9). Normally, only cells that successfully rearrange a *Division of Biology, California Institute of Technology, Pasadena, CA 91125; and TCRb and assemble a signaling pre-TCR complex are permitted †Division of Biostatistics, City of Hope, Duarte, CA 91010 to pass through the b-selection checkpoint to DN4 and proliferate. Received for publication October 31, 2012. Accepted for publication January 21, + + 2013. These cells then become CD4 CD8 double-positive (DP), ex- press TCRab, and undergo positive and negative selection (10, This work was supported by National Institutes of Health Grant AI64590 (to M.A.Y.); California Institute of Technology Summer Undergraduate Research Fellowships (to 11). Rag-deficient T cells are blocked at b-selection and do not C.Y.L.); the Albert Billings Ruddock Professorship (to E.V.R.); the Louis A. Garfinkle generate DP cells. Memorial Laboratory Fund; and the Al Sherman Foundation. The NOD mouse is a model of T cell–mediated autoimmune The sequences presented in this article have been submitted to the type 1 diabetes, with .20 genetic regions associated with diabetes Omnibus (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= GSE40688) under accession number GSE40688. susceptibility, many of which specifically affect T cell activities Address correspondence and reprint requests to Dr. Mary A. Yui, California Institute (12–15). Most T lineages have been implicated in autoimmune of Technology, 1200 E. California Boulevard, Pasadena, CA 91125. E-mail address: susceptibility or resistance in NOD mice, including CD4, CD8, [email protected] NKT, regulatory T, and gdT cells (16), raising the possibility that The online version of this article contains supplemental material. all NOD T cells may share fundamental abnormalities that con- Abbreviations used in this article: chr, chromosome; DL, delta-like; DN, double- tribute to loss of self-tolerance and might be traceable to their negative; DP, double-positive; ETP, early T cell progenitor; GeoMean, geometric mean; Idd, insulin-dependent diabetes susceptibility gene; LOD, logarithm of the common progenitors (8). We previously reported defects in the odds; MFI, mean fluorescence intensity; QTL, quantitative trait locus; RPKM, read earliest stages of T cell development in both wild-type (WT) and per kilobase per million reads; SNP, single nucleotide polymorphism; T-ALL, T cell TCR-deficient NOD mice. Precursor cells from WT NOD mice acute lymphoblastic leukemia; WT, wild-type. exhibit impaired abT cell development and enhanced gdT cell Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 generation apparently arising from the ETP-DN2 stages (17).

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1202970 2 T CELL PROGENITOR CHECKPOINT LOSS AND LEUKEMIA INITIATION

Additionally, the NOD genetic background severely com- isotype controls (Cell Signaling Technology, Danvers, MA) before wash- promises b-selection checkpoint control: despite the absence of ing and flow cytometric analysis. 2/2 TCR expression in NOD.SCID and NOD.Rag1 (NOD.Rag) Genome-wide transcriptome analysis mice, aberrant breakthrough DP thymocytes spontaneously appear + 2 in all young adult animals (18). These TCR-negative DP cells do T thymocytes were FACS sorted to obtain CD25 CD4 cells from NOD. Rag mice at 4 wk age (prebreakthrough) and 7 wk (at the time of first not fully mimic normal b-selected cells, as they still express breakthrough), and age-matched B6.Rag mice for RNA extraction. mRNA receptors characteristic of earlier DN stages, such as Kit, IL7Ra, purification and cDNA library building were performed as described (24). and CD25, which are normally turned off by the DP stage (18). Sequencing was done using Illumina high-throughput genome analyzer Furthermore, older NOD.SCID and NOD.Rag mice develop thy- IIx sequencers at the California Institute of Technology’s Jacobs Genetics and Genomics Laboratory, and data have been deposited in National Center mic tumors at high whereas mice of other strains with for Biotechnology Information’s Gene Expression Omnibus (25) and are these immunodeficiencies do not (18–21). This suggests a possible accessible through Gene Expression Omnibus series accession no. GSE40688 link between early T cell checkpoint control and tumor suppres- (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= GSE40688). Com- sion that may be jointly defective in the NOD genetic background. putational analysis was carried out using software developed at the Cal- We used genome-wide genetic and transcriptome analytical ifornia Institute of Technology (26, 27) as well as Mathematica and R. For analysis, 38-bp, single-read raw sequence reads were trimmed to 32 bp methods to investigate the source and consequences of the NOD.Rag and mapped onto the mouse genome build NCBI37/mm9 using Bowtie thymocyte checkpoint defect. First, we found quantitative trait loci (bowtie-0.12.1; http://bowtie-bio.sourceforge.net/index.shtml) with setting (QTLs) for this trait, all within several of known diabetes suscep- “-v 2 -k 11 -m 10 -t–best–strata”. The mappable data were then processed tibility regions mapped in WT NOD mice. A major QTL localized by the ERANGE v. 3.3 RNA-Seq analysis program to obtain the expres- sion level in reads per kilobase per million reads (RPKM) for each gene Idd 9/11 6 Downloaded from within the insulin-dependent diabetes susceptibility gene ( ) (26). We obtained 13–17 3 10 mappable reads per sample for analysis. region of chromosome (chr)4 was confirmed using congenic mice. Based on a pairwise comparison between samples, B4 values were divided Additionally, genome-wide transcriptome analyses revealed distinct by 1.27 to remove a scaling artifact. Genes were required to have at least differences in gene expression between thymocytes from NOD.Rag one sample with 32 mappable reads (based on a significance level of 0.05, and B6.Rag control mice. The genes differentially expressed be- with a Bonferonni adjustment for 12,000 comparisons) and have an an- notated transcript name, leaving 11,098 named genes for further analysis. tween the two strains were enriched for those encoding signaling For each gene, comparisons of expression in the four samples were cal- proteins, suggesting aberrant signal transduction as a possible pre- culated as differences on a log2 scale, after replacing any zeros by 0.01 as http://www.jimmunol.org/ condition for breakthrough. Furthermore, newly emergent NOD. an approximate limit of detection. The significance probabilities (p values) Rag breakthrough cells fail to terminate gene expression programs were based on the assumption of Poisson distributions of counts, under the null hypothesis of equal rates, allowing for the difference in the number of from earlier stages: they coexpress phase I stem/progenitor genes megabases sequenced (and mapped). Three-fold change is typically the along with T cell–specific genes characteristic of phase II and more stringent requirement and was used to determine the differentially post–b-selection stages. This mixed gene expression profile fore- expressed genes. The statistical significance requirement screens out some shadows the phenotype of thymic tumors found in older mice of this genes with low expression whose 3-fold differences might be attributable strain, which share characteristics with classes of human early type to sampling variation. The design does not permit assessing extra-Poisson variation, which would be more important at larger expression levels, acute T cell lymphoblastic leukemia (T-ALL), suggesting that pri- where the 3-fold criterion rather than the statistical comparison is the domi- mary defects in early T cell checkpoint control underlie some forms nant concern. by guest on September 25, 2021 of T-ALL. To profile the connection between different populations, hierarchical clustering was carried out for each selected subgroup of genes from B6.Rag and NOD.Rag samples or WT B6 ETP to DP populations (24). Individual Materials and Methods mRNA data for the selected genes were first normalized by the corre- Mice and crosses sponding geometric mean (GeoMean) of comparing populations, and then one-dimensional hierarchical clustering (along genes) was performed on B6.129S7-Rag1tm1Mom/J (B6.Rag) and NOD.Cg-Rag1tm1Mom Prf1tm1Sdz/ Idd9 the results after log2 transformation. Euclidean distance and Ward linkage SzJ (NOD.Rag) (The Jackson Laboratory) and NOD.B10 line 905 (14) were used (MATLAB 7.10.0). Clusters were visualized as heat maps. (Taconic Farms) mice were bred and maintained in the Caltech Laboratory Animal Facility using autoclaved cages, food, and water. All animal pro- Real-time quantitative PCR analysis tocols were reviewed and approved by the Animal Care and Use Com- mittee of the California Institute of Technology. Quantitative PCR was carried out based on selected RNA-Seq results using FACS-sorted thymocyte samples. RNA was extracted and reverse-transcribed Genetic crosses, QTL analysis, and congenic mice and quantitative PCRs were carried out using SYBR GreenER (Invitrogen) in a GeneAmp 7900HT sequence detection system (Applied Biosystems), as Rag Rag For the QTL analysis B6. and NOD. mice were crossed and previously described (17). Relative expression was calculated for each gene intercrossed for F2 or backcrossed to NOD.Rag for N2 progeny. Thymo- by the DCT (change in cycling threshold) method, normalized to b-actin cytes from 12 to 14 wk progeny were phenotyped by flow cytometric expression. Primer sequences were as previously published (6, 28), plus: analysis. DNA was extracted from tail tips of 150 N2 and 30 F2 cross mice Zap70,forward,59-TGTCCTCCTGAGATGTATGCAC-39,reverse,59-AT- and 150 polymorphic single nucleotide polymorphisms (SNPs) were AGTTCCGCATACGTTGTTCC-39; Ptcra, forward, 59-CTGGCTCCACC- genotyped by The Jackson Laboratory Genetic Services. QTL analysis was CATCACACT-39, reverse, 59-TGCCATTGCCAGCTGAGA-39; Notch1, p carried out using the R/qtl program (22) and values were obtained from forward, 59-CCACTGTGAACTGCCCTATGT-39, reverse, 59-TTGTTTC- genome-wide significance test using 5000 permutations (23). Congenic Idd9 CTGGACAGTCATCC-39; Heyl, forward, 59-AAGCTGGAGAAAGCTG- NOD.B10 Rag mice were created by crossing NOD.Rag and NOD. AGGTC-39,reverse,59-CCAATACTCCGGAAGTCAACA-39; Kit,forward, B10Idd9 mice and repeated backcrossing until the knockout Rag1 gene and Idd9 59-ACTTCGCCTGACCAGATTAAA-39, reverse, 59-CGTACGTCAGGA- the B10 region were homozygous, as determined by PCR analysis. TTTCTGGTT-39; Hhex, forward, 59-ACTACACGCACGCCCTACTC-39, Cell cultures and Ab staining reverse, 59-GTCGTTGGAGAACCTCACTTG-39; Epha2, forward, 59- CAGGAAGGCTACGAGAAGGTC-39, reverse, 59-CAGGGTATGCTCT- Freshly isolated thymocytes were either stained immediately for flow GGACACTC-39; Mllt4, forward, 59-GACTGGACAGTGACAGGGTGT- cytometetric analysis or cultured on OP9-delta-like (DL)1 or OP9-DL4 cells 39, reverse, 59-CAGTATCAGTTCAGGCCCAGT-39; Dapk1, forward, 59- with 5 ng/ml IL-7, as previously described (17). For cell stimulations, CATCACCCTGCATGAGGTCTA-39, reverse, 59-TGCCTCCTCTTCAG- thymocytes were cultured for 1 h in RPMI 1640 supplemented with 10% TCAGAGA-39; Vegfa, forward, 59-CTCCGAAACCATGAACTTTCT-39, FBS (Life Technologies) before treatment with PMA. Cells were imme- reverse, 59-ATGGGACTTCTGCTCTCCTTC-39; Tnfrsf9, forward, 59-G- diately fixed in 1.5% formaldehyde in PBS at 37˚C and permeabilized by CTGGCCCTGATCTTCATTA-39, reverse, 59-ATCGGCAGCTACAAGC- slow addition of ice-cold methanol to a final concentration of 90%. Cells ATCT -39. The Tcf7 primers (28) yielded lower values than previously were incubated on ice for 30 min, washed with PBS plus 0.5% BSA, and published (5); however, the relative efficiency was consistent between incubated with either phospho-p42/p44 (Erk1/2)-Alexa Fluor 647 Abs or samples in these experiments. The Journal of Immunology 3

Results developmental stages). As shown in Fig. 1A, B6.Rag and (B6 3 The NOD.Rag breakthrough trait maps to the Idd9/11 region of NOD)F1.Rag mice at 12 wk age exhibited very few CD4+ or Kit+ chr4 cells, whereas most thymocytes from NOD.Rag mice were CD4+ + We previously reported that aberrant Kit+ DP breakthrough cells and Kit . Results for individual progeny differed; for example, no. 87 exhibited a non-breakthrough phenotype similar to B6.Rag, spontaneously appear in the thymuses of all male and female + + NOD.Rag mice by 8 wk age, whereas B6.Rag and (B6 3 NOD) and no. 86 showed Kit CD4 breakthrough cells like NOD.Rag F1.Rag thymocytes arrest normally in DN3 at the b-selection mice. The F2 intercross yielded a low percentage of progeny with + + , checkpoint (18). To map the genetic basis of the T cell break- elevated percentages of CD4 and/or Kit cells ( 10%), whereas ∼ through, we tested progeny from a (B6 3 NOD)F2.Rag intercross 35% of N2 backcross progeny exhibited T cell breakthrough. andanN2 backcross with NOD.Rag as the recurrent parent. Despite indicating differing developmental defects, the traits are Thymocytes from individual parental and cross progeny were not independent, as shown in the strong correlation (r2 = 0.82) phenotyped by flow cytometry at 12–14 wk age for two traits: 1) between the log-transformed percentages of CD4+ and Kit+ cells the percentage of CD4+ (including DP) cells, which is indicative for individual progeny (Fig. 1B). of illegitimate progression past the b-selection checkpoint, and 2) For the QTL analysis, 150 N2 and 30 F2 phenotyped progeny the percentage of Kit+ cells, indicative of a failure to downregulate were genotyped for 150 polymorphic SNPs distributed over the a key phase I stem/progenitor gene (see Fig. 1E for a model of 19 autosomal . Genome-wide one-dimensional QTL Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 1. Genome-wide QTL analysis of the NOD.Rag early T cell breakthrough trait shows mapping to known diabetes susceptibility regions. (A) Flow cytometric data showing representative percentage CD4+ versus percentage CD8+ and percentage Kit+ versus percentage CD25+ phenotyping plots for thymocytes from control parental B6.Rag, NOD.Rag, and F1.Rag mice as well as two individual (NOD 3 B6)F2.Rag cross mice, at 14 wk age. (B) Scatterplot showing the correlation between the two measured thymocyte phenotypic traits: percentage CD4+ and percentage Kit+ cells. (C) Genome-wide + + + QTL scan for percentage CD4 and percentage Kit thymocytes, using the combined N2 plus F2 crosses. LOD scores are plotted for percentage CD4 cells (black) and percentage Kit+ cells (gray) for all 19 autosomal chromosomes. Black line indicates a LOD score of 3. (D) Expanded plots of LOD scores for the chromosomes with the highest peaks in the combined cross, chr4 and chr17, for the two phenotypes, percentage CD4+ and percentage Kit+ thymocytes. Approximate regions of known diabetes susceptibility Idd loci are also noted. (E) Diagram of key early T cell developmental stages (based on Ref. 9) and possible pathways (dashed and solid arrows) leading to pro– T cell checkpoint breakthrough and T-ALL in NOD.Rag mice. 4 T CELL PROGENITOR CHECKPOINT LOSS AND LEUKEMIA INITIATION

scans were performed using R/qtl on data from the N2 and F2 gion contains at least three distinct diabetes resistance alleles from crosses alone and combined (22). Peak QTL logarithm of the odds B10 mice, Idd9.1, Idd9.2, and Idd9.3 (Fig. 2A), as well as a pos- Idd9 (LOD) scores for the two phenotypes and the N2 and F2 crosses, sible Idd11 locus (30). The NOD.B10 Rag mice showed sig- individually and combined, are shown in Table I, and genome- nificantly lower percentages of CD4+ and DP breakthrough cells wide QTL scans for the combined cross data are shown in Fig. 1C. than did NOD.Rag mice (Fig. 2B, 2C), demonstrating that the chr4 A highly significant QTL (LOD . 12) was detected on distal chr4 congenic region includes at least one gene involved in suscepti- + + for both percentage CD4 and percentage Kit in both the N2 and bility to thymocyte breakthrough. Other genetic regions also con- the combined cross (Fig. 1C, 1D). Weaker QTLs were detected on tribute, as NOD.B10Idd9Rag congenic thymocytes exhibited more chr17 for both phenotypes and N2 and combined crosses (LOD . breakthrough cells than did B6.Rag (Fig. 2B, 2C), in accord with + 3 for percentage Kit ) (Fig. 1C, 1D), and on chr13, in the N2 cross the QTL analysis. only (LOD . 2.5). A scan using the F cross alone revealed only 2 Differentially expressed genes between Rag-deficient NOD and a suggestive locus on chr11. Remarkably, all four of these QTLs B6 CD25+DN cells are enriched for actin-binding and signal map to known NOD Idd QTL regions. The chr4 QTL lies within transduction genes the Idd9/11 region (14, 29, 30), the chr17 QTL encompasses the region containing Idd1, Idd16, Idd23, and Idd24 (31–33) (Fig. Insights into the breakthrough program should emerge from both 1D), the chr13 QTL region overlaps with Idd14 (34), and the determining underlying strain-specific gene expression differences chr11 QTL region overlaps with Idd4 (29). These results allow that precede the loss of checkpoint control, as well as investigating the possibility that the same genes could be involved in the loss of the earliest postbreakthrough changes. To first evaluate differences checkpoint control and susceptibility to autoimmune diabetes in between NOD.Rag and B6.Rag thymocytes that restrict check- Downloaded from NOD mice. point control loss to the NOD background, we carried out a ge- Control of percentage CD4+ and percentage Kit+ mapped to nome-wide RNA deep sequencing analysis (RNA-Seq) (26), 2 + the same genetic regions, and therefore they arise from linked comparing sorted CD4 CD25 DN thymocytes from NOD.Rag or identical genetic sources and are distinct from the Cd4 (chr6) or mice at 4 (N4, before breakthrough) and 7 wk (N7, early break- Kit (chr5) genes themselves. The aberrant upregulation of CD4 through), with corresponding non-breakthrough cells from B6.Rag

occurs in cells that already express high levels of Kit (18), and miceat4(B4)and7(B7)wks.Ineachsample,thesortedcells http://www.jimmunol.org/ results in Fig. 1B support this: no progeny were high for %CD4+ were predominantly pre–b-selection DN3 cells and they were and low for %Kit+ (right bottom quadrant), whereas a few prog- phenotypically similar (Fig. 3A). eny were high for %Kit+ and low for %CD4+ (left top quadrant). Of .11,000 expressed genes, 412 genes were differentially Also, QTL LOD scores for %Kit+ were typically higher than for % expressed by at least 3-fold between the two strains (excluding 10 CD4 (Fig. 1C, 1D, Table I), indicating that Kit may be a more genes more prominently differing between N7 and N4, see below) sensitive measure of breakthrough. These cells express CD25 (Fig. 3B, 3C, Supplemental Table I). These genes are normally and IL7Ra, as well as Kit (18), and their abnormal coexpression expressed in at least one stage of T cell development, from ETP to with CD4 (and CD8) suggests that the breakthrough arises from DP, based on our data from WT B6 mice (24), as shown in heat- specific genes on the NOD genetic background that cause bypass maps in Fig. 3D. Genes with strain-specific differences in ex- by guest on September 25, 2021 of both the T cell commitment and b-selection checkpoints, as pression, at higher and lower levels in NOD cells, exhibit highly shown in Fig. 1E (and discussed further below). varied patterns of expression across these stages of T cell de- Idd9 velopment, in contrast to what we find with genes specific to N7 The congenic B10 region of chr4 attenuates the NOD.Rag postbreakthrough cells (see below). Overall, 34% of the strain- thymocyte breakthrough dependent differentially expressed genes have peak expression in To confirm the presence of a gene or genes in the NOD Idd9/11 phase I (23% ETP, 11% DN2a), 40% in phase II (19% DN2b, 21% chr4 QTL region controlling the breakthrough trait, NOD.B10Idd9 DN3), and 26% in post–b-selection DP cells. congenic mice (line 905) (14) were bred to NOD.Rag to generate analysis of the differentially expressed genes a NOD.B10Idd9Rag congenic mouse line. The congenic chr4 re- using DAVID bioinformatics resources 6.7 (35) showed three types

Table I. Peak regions from a genome-wide QTL scan of N2, F2, and combined N2 plus F2 crosses for percentage CD4+ and percentage Kit+ thymocytes

Cross Trait Chr Position (cM)a Peak (Mb)b LODc p Valued N2 %CD4+ 4 91.6 142.8 12.49 ,,0.01 13 40.3 76.2 2.85 ,0.05 17 19.2 40.2 2.69 ,0.1 %Kit+ 4 91.6 142.8 18.97 ,,0.01 13 40.3 76.2 2.59 ,0.1 17 19.2 40.2 3.2 ,0.05 F2 %CD4+ 11 42.3 70.3 2.85 ,0.5 %Kit+ 11 31 60 3.46 ,0.2 Combined %CD4+ 4 91.6 142.8 13.67 ,,0.01 N2 plus F2 17 19.2 40.2 2.6 ,0.025 %Kit+ 4 91.6 142.8 21.13 ,,0.01 17 19.2 40.2 3.87 ,0.01 aGenetic map position in cM, calculated from cross genotypes. bPeak locations in NCBI37/mm9 mouse assembly (Mb) obtained from peak SNP location. c Log10 likelihood ratio (LOD) calculated with R/qtl using a simple model for the N2 and F2 crosses and adding cross to the model for the combined N2 plus F2 cross data; only LOD scores .2.5 are shown. dThe p values are based on genome-wide significance tests for LOD thresholds (5000 permutations) for each cross and phenotype. The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/

FIGURE 2. NOD.B10Idd9Rag congenic mouse strain analysis of the chr4 Idd9 QTL peak region. (A) Diagram of the chr4 congenic region from NOD. B10Idd9 (L. Wicker, Cambridge, U.K.), which was crossed onto NOD.Rag congenic mice. Indicated are the genomic regions derived from B10Idd9 (black) and NOD (white) and the recombination region (gray), as well as the Mappair markers used in the congenic cross and the QTL peak (arrow). Approximate Idd9 locations for Idd9.1, 9.2, and 9.3 are shown (from T1Dbase.org). (B) Flow cytometric analysis of B6.Rag, NOD.Rag, and four NOD.B10 Rag congenic by guest on September 25, 2021 mice at 12 wk age. (C) Summary of percentage CD4, percentage DP, and percentage Kit+ cells for individual NOD.B10Idd9Rag (NOD.chr4Rag) congenic mice in comparison with parental B6.Rag, NOD.Rag, and F1.Rag mice, all at 12–16 wk old. A p value , 0.0001 is from t tests comparing data from NOD. Rag and NOD.B10Idd9Rag mice. of genes were overrepresented: MHC (H2), signaling, and actin- are differentially expressed between the two strains, including at binding protein genes (Table II). Enrichment of H2 genes is not least three in a region near the chr4 peak that is predicted to en- unexpected owing to their expression diversity between mouse code multiple KRAB-zinc finger proteins (unpublished observa- strains, although no role is known for them in early T cell de- tion). Differentially expressed genes of interest were also found velopment. genes, including tyrosine and serine/ within other QTL regions, including Vegfa; Notch4; Mllt4 (Afadin), threonine , were enriched, as were adherens junction pro- a Ras-associated adherens junction gene; and Ddr1, discoidin tein genes, which include actin-binding, tyrosine kinase, and domain tyrosine kinase receptor, found in the chr17 QTL region, -binding protein genes. All of these protein classes plus a tumor suppressor gene, death-associated protein kinase, play critical roles in modulating signal transduction activity in Dapk1, located within the chr13 QTL region and expressed in B6. T cells (36). Notably, all 11 of the differentially expressed tyrosine Rag but not in NOD.Rag cells (Fig. 3F). kinases were higher in NOD.Rag cells, suggesting that the two strains likely exhibit differences in signaling activity. This may not Signaling and developmental differences between NOD.Rag only contribute to checkpoint failure, but also promote the thymic and B6.Rag thymocytes lymphomas that develop in these mice, as tyrosine kinases are The enrichment of differentially expressed signal transduction common oncogenes and therapeutic targets in leukemia and other pathway genes suggests that inappropriate signaling might underlie malignancies (37). the NOD.Rag checkpoint breakthrough. Thymocytes from sex- Several genes differentially expressed between NOD.Rag and matched 4- to 5-wk-old prebreakthrough NOD.Rag and B6.Rag B6.Rag are located within and near the chr4 QTL peak region mice were assayed for differences in responses to the protein ki- (Supplemental Table I). RNA-Seq tracks for several of these genes nase C activator PMA, in short- and long-term assays. Thymocytes are shown in Fig. 3E, including Epha2, an ephrin tyrosine kinase from both strains used for these experiments were phenotypically receptor strongly expressed in both NOD samples; Padi3, a pep- similar and .90% were DN3 stage cells (18). Phosphorylation tidylarginine deiminase; Tnfrsf9 (CD137, 4-1BB, an Idd9.3 can- of Erk1/2, mediators of the Ras/MAPK pathway, was measured by didate gene) (14); Ctnnbip1 (ICAT), a Wnt pathway inhibitor; and p-Erk1/2 intracellular staining of thymocytes from mice stimu- Hdac1, a histone deacetylase, which is differentially spliced. lated with PMA (Fig. 4A). NOD.Rag populations exhibited cells These genes contrast with the stably expressed chr4 region gene, with consistently lower levels of pErk than B6.Rag in response to Lck. Furthermore, a number of poorly defined transcription units PMA stimulation, over all PMA concentrations tested, as shown 6 T CELL PROGENITOR CHECKPOINT LOSS AND LEUKEMIA INITIATION Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 3. RNA-Seq transcriptome analysis comparing CD25+ DN thymocytes from B6.Rag and NOD.Rag mice. (A) Gates used for sorting pheno- typically similar CD42CD25+ cells for RNA purification: B6.Rag at 4 and 7 wk (B4, B7) and NOD.Rag at 4 and 7 wk (N4, N7). (B) Difference versus mean of log2 expression of N4/N7 and B4/B7, including 11,098 genes expressed at .32 reads in at least one sample. Horizontal gray lines show 3- and 4-fold differences. Genes differing by .3-fold are shown in dark circles, ,3-fold by gray circles. (C) Heatmap for hierarchical clustering of 412 genes differing by .3-fold between N(4 and 7) versus B(4 and 7). Data are shown as log2(RPKM/GeoMean). (D) Hierarchical cluster heatmaps showing the normal (WT B6) developmentally regulated expression (log2(RPKM/GeoMean)) of differentially expressed genes that are higher (top) and lower (bottom) in N(4 and 7) compared with B(4 and 7) using our previously published data (24). (E and F) RNA-Seq tracks showing sequence read histograms for expressed genes of interest in QTL regions from B6.Rag and NOD.Rag CD25+ DN cells at 4 and 7 wk age. Gene tracks are shown using the University of California at Santa Cruz browser, and the data are mapped onto the mouse genome build NCBI37/mm9. (E) Genes located in the chr4 major QTL (Figure legend continues) The Journal of Immunology 7 by plotting mean fluorescence intensity (MFI) ratios between pairs key phase I genes, Lmo2, Hhex, Lyl1, Kit,andMef2c (9), as well of matched cells (NOD:B6), over multiple experiments, which are as a post–b-selection gene, Cd2, are shown in Fig. 5D. An addi- almost uniformly below an expected equal value of 1 (red line) tional phase I gene, Dlk1 (delta-like 1; Pref1), was expressed only (Fig. 4B). These results are in agreement with a recent report showing in N7, as a previously unreported truncated splice form (arrow). differences in Erk1/2 phosphorylation between B6 and NOD Dlk1 supports proliferation in hematopoietic progenitors, main- TCR-transgenic T cells (38). tains cells in an undifferentiated state, and is overexpressed in PMA with calcium ionophore can mimic b-selection–promoting some leukemias (40). signals in vitro to drive Rag-deficient thymocytes to generate DP The prominent expression of phase I stem/progenitor genes cells in long-term cultures. Therefore, NOD.Rag and B6.Rag suggests that the N7 emergent population either failed to silence or thymocytes were also tested for developmental responses to these re-established the early T cell program, which normally occurs at signals in coculture with OP9 stromal cells expressing Notch li- the DN2 commitment checkpoint. At the same time, the emerging gand, DL1 (OP9-DL1). OP9-DL1 coculture supports T cell de- cells also appear to bypass the b-selection checkpoint, as shown by velopment through the b-selection checkpoint in thymic expression of Cd2 and Cd5, which are normally turned on in DN4 progenitors from WT B6 and NOD mice (17, 39), but not from (http://www.immgen.org). Whereas the assayed cells were se- Rag-deficient mice unless an artificial TCR signal is delivered. lected to be negative for surface CD4, Cd4 gene expression was Cells were treated with graded doses of PMA and calcium iono- detectably upregulated (1.9-fold higher in N7 than in N4 cells). phore and analyzed after 7 d. NOD.Rag cells (dashed lines) were These results show that the emerging NOD.Rag breakthrough more sensitive to PMA than B6.Rag cells (solid lines) in this as- cells have greatly disordered developmental programming and say, as seen by the shift in dose-response curves for both total cell fail to passage or arrest properly at the early T cell checkpoints Downloaded from numbers (Fig. 4C, left), and the percentage of cells that had pro- (Fig. 1E). gressed through the b-selection checkpoint, as measured by per- centage CD4+ cells (including DP) (Fig. 4C, right). These results NOD.Rag thymocyte numbers expand with age and in vitro demonstrate consistent differences between the two strains in the These features of the transcriptome analysis suggest a possible responses of their thymocytes to a specific stimulus. linkage between T cell breakthrough and the high incidence of

thymic lymphomas that appear in NOD.Rag mice (18, 20, 21). http://www.jimmunol.org/ Breakthrough NOD.Rag DN cells upregulate phase I NOD.Rag thymic cell numbers increase dramatically with age stem/progenitor cell genes whereas B6.Rag thymocyte cell numbers change very little or NOD.Rag DP breakthrough cells only partially resemble post–b- decline over time (Fig. 6A). Beginning at 20 wk, NOD.Rag mice selection cells. They express surface CD4, CD8, and CD2 and exhibiting thymic lymphomas appeared, as previously reported a initiate TCR-C and Bcl-xL transcription, like normal or induced (18, 21). Additionally, when DN thymocytes from young NOD. DP cells, while also retaining characteristics of pre–b-selection Rag and B6.Rag mice were cultured with Notch ligands and IL-7, DN cells such as surface CD25, KIT, and IL-7Ra, and Spib and both expanded but only NOD.Rag DN cells violated the b-se- Hes1 transcription (18). To determine whether these were isolated lection checkpoint and upregulated CD4 (Fig. 6B). Furthermore, cases or indicators of more general program derangement, the NOD.Rag cells isolated at 9–12 wk age, after breakthrough but by guest on September 25, 2021 transcriptome data were analyzed for the earliest breakthrough- before lymphoma formation, expanded continuously in cultures in specific gene changes by comparing NOD.Rag cells (N7) with the presence of Notch ligand and IL-7. Whereas sorted NOD.Rag prebreakthrough NOD.Rag (N4) and normal B6.Rag (B4, B7) DP cells from three individuals expanded 6.7 6 3.2-fold (SEM) cells. Sorted N7 cells excluded breakthrough cells expressing during 5 d, and only generated more DP cells, sorted CD25+ DN surface CD4 but included their precursors (Fig. 3A). cells from the same mice expanded 3.1 6 1.0 and generated both Very few genes exhibited a .3-fold difference between N7 and DP cells and a small population of DN cells (Fig. 6C), suggesting N4, and almost all were higher in N7 (Fig. 5A). This finding is that cells within the DN population may be a source of illegitimate consistent with the emergence of a subpopulation of abnormal differentiation. These thymocytes failed to survive when cultured cells, expressing novel genes; any downregulated genes are likely in the absence of Notch ligands or IL-7 (data not shown), indi- to be masked by the dominant presence of conventional DN3 cells. cating that they are committed to the T lineage. Fifty-two breakthrough-specific differentially expressed genes were identified, including only those genes that distinguished N7 Transcriptional regulatory relationship between NOD.Rag from all other samples, N4, B4, and B7, by at least 3-fold (Fig. 5B, breakthrough thymocytes and thymic lymphoma cells Supplemental Table II). To determine whether the emergent If gene expression abnormalities in NOD.Rag breakthrough cells population more closely resembles pre– or post–b-selected cells, favored their differentiation into lymphoma cells, then at least we used our RNA-Seq data for WT B6 DN1 to DP cells (24) as some of their aberrant features should be preserved in the lym- a reference for the developmentally regulated expression of the phomas that develop a few months after the breakthrough. To test N7-specific genes, and their patterns of expression are shown in a this prediction, we used real-time quantitative PCR on populations heatmap in Fig. 5C. Strikingly, although the breakthrough phe- of NOD.Rag cells to track changes in gene expression related to notype was originally identified by expression of post–b-selection age and lymphoma status. We tested the persistence of represen- markers CD2, CD4, and CD8, most of the genes preferentially tative genes from various developmental stages, as well as a few expressedbyN7arenormalphase I genes. Of the differentially genes that showed differential expression between NOD.Rag and expressed genes, 71% exhibited peak expression in phase I (48% B6.Rag DN cells (Fig. 6D–G), using RNA from the following in DN1/ETP and 23% in DN2a), 21% in phase II (15% in DN2b sorted thymocyte populations: CD25+ DN cells from 4- to 10-wk- and 6% in DN3), and only 8% in DP. RNA-Seq tracks for some old B6.Rag (arrested at the b-selection checkpoint, brown); CD25+

region, including differentially expressed genes, Epha2, Padi3, Tnfrsf9 (4-1BB), Ctnnbip1 (Icat), a differentially spliced gene, Hdac1 (differentially spliced exons indicated with arrows), and Lck, a nondifferentially expressed gene for reference. (F) Differentially expressed genes in the chr17 QTL region, in- cluding Vegfa, Notch4, Mllt4 (Afadin), and Ddr1, and in the chr13 QTL region, Dapk1. 8 T CELL PROGENITOR CHECKPOINT LOSS AND LEUKEMIA INITIATION

Table II. Gene ontology analysis on .3-fold differentially expressed genes between NOD.Rag and B6.Rag CD25+ DN cells showing major enriched categories of genes

Fold GOTERM Categorya Genesb Enrichment p Value Benjamini MHC protein complex H2-K1, H2-Q2, H2-M6-PS, H2-Q10, C920025E04rik, H2-OA, 9.8 6.5 3 1027 1.6 3 1024 H2-BL, H2-Q1, H2-T10, H2-AB1, H2-Q7, H2-T3 (all chr17) Actin binding Lima1, Ccdc88a, Mybpc2, Tln2, Inppl1, Myo7a, Evl, Spire2, 3.5 1.8 3 1025 7.9 3 1023 Tpm3, Coro2b, Fmn2, Syne2, Mtap1a, Capg, Tmod4, Eps8l1, Mylk, Myh10 (chr11) Protein tyrosine Tyro3, Ddr1 (chr17), Fgfr1, Ptk2, Ltk, Ptpn3, Ptk2b, Hck, Txk, 3.7 7.7 3 1024 1.1 3 1021 kinase activity Ephb4, Epha2 (chr4) Adherens junction Ptk2, Lima1, Itga6, Ptk2b, Tln2, Lmo7, Evl, Mllt4 (chr17), Arhgap26 4.3 1.1 3 1023 6.8 3 1022 Protein kinase activity Tyro3, Fgfr1, Alpk1, Ltk, Ptpn3, Hck, Ulk4, Ephb4, Epha2 (chr4), 2.0 4.3 3 1023 1.9 3 1021 Dapk1 (chr13), Rps6kl1, Ddr1 (chr17), Ptk2, Ptk2b, Camk1, Stk39, Camk2B, Txk, Grk5, Camk2a, Mylk Calmodulin binding Adcy1, Myo7a, Camk1, Camk2b, Camk2a, Mylk, Myh10 (chr11), 3.9 4.5 3 1023 1.8 3 1021 Dapk1 (chr13) aGOTERM: Gene Ontology Term. bGenes expressed at higher amounts in NOD.Rag cells are indicated in bold type. Genes located within a mapped QTL region in this study are noted (chr). Downloaded from DN cells from 4- to 6-wk-old NOD.Rag mice (arrested at the NOD.Rag mice (light green); unsorted thymic lymphoma cells from b-selection checkpoint, before overt breakthrough, dark green); .4-mo-old NOD.Rag mice (pink); and ETP, DN2a, DN2b, DN3a, DN, CD4+, and DP cells from postbreakthrough 10- to 16-wk-old and DP cells from WT NOD thymuses for comparison (blue). http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. NOD.Rag and B6.Rag thymocytes exhibit differential responses to PMA stimulation. (A) Representative flow cytometric histogram showing intracellular Erk1/2 phosphorylation (pErk1/2) in freshly isolated thymocytes (.90% DN3 cells) from prebreakthrough NOD.Rag and B6.Rag mice treated for 2 min with PMA. (B) Summary plot of the NOD/B6 ratios of p-Erk1/2 (using geometric MFI from intracellular staining) for thymocytes from age- and sex-matched NOD.Rag and B6.Rag mice treated with 1–25 ng/l PMA. The MFI was consistently lower for NOD.Rag cells than for B6.Rag cells as shown by ratios ,1 (equal expression, indicated with a red line). Data included are combined from five independent experiments. (C) Responses of thymocytes from 6-wk-old NOD.Rag (dashed lines) and B6.Rag (solid lines), placed in coculture with OP9-DL1 cells and stimulated with graded doses of PMA, and calcium ionophore A23187 (CI; doses of 0, 22, 43, and 87 nM as indicated) and assayed at day 7 for cell number (left graph) and percentage CD4+ cells (right graph). Results are representative of three independent experiments. The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 5. RNA-Seq transcriptome analysis of NOD.Rag breakthrough cells. (A) Difference versus mean of log2 expression of NOD.Rag 7wk(N7,at breakthrough) versus 4 wk (N4, prebreakthrough). Horizontal gray lines show 3- and 4-fold differences. Genes differing by . 3-fold are shown in dark circles, ,3- fold by gray circles. (B) Heatmap for hierarchical clustering of 52 genes differing by .3-fold between N7 versus all other samples. Data are shown as log2(RPKM/ GeoMean). (C) Hierarchical cluster heatmap showing the WT B6 expression profile (log2[RPKM/GeoMean]) of 52 genes differentially expressed between N7 and other samples using data from ETP to DP developmental stages (24). (D) RNA-Seq gene tracks from B6.Rag and NOD.Rag CD25+ DNcellsat4and7wkage showing a selection of genes of interest expressed .3-fold higher in N7 in comparison with N4, B4, and B7, including phase I genes, Lmo2, Hhex, Lyl1 Kit,and Mef2c, post–b-selection gene, Cd2, and a differentially expressed and spliced Notch-related gene, Dlk1 (lack of expression of 39 exons is indicated with an arrow).

NOD.Rag thymocytes obtained from all ages, including CD4+ Notch signaling is required for early but not later stages of and DP breakthrough cells and thymic lymphomas, expressed T cell development (44), and constitutive Notch activation is one critical T cell genes, including Bcl11b, Tcf7, CD3e,andZap70 of the most potent and common oncogenic factors in T-ALL (Fig. 6D). Bcl11b expression in all NOD.Rag samples is particu- (45). Expression of Notch1, and target genes Dtx1, Hes1, Heyl, larly significant, as it is critical for phase I gene repression and and Ptcra, was sustained in all stages, including in lymphoma T cell differentiation past the DN2a stage (6, 7). Bcl11b was re- cells (Fig. 6E), implying sustained Notch signaling. ported to be a haploinsufficient tumor suppressor in human T- Among phase I genes (Fig. 6F), Kit expression was sustained at ALL (41, 42), and expression of Bcl11b,aswellasCD3e,was high levels in NOD.Rag cells at all ages, as well as in lymphoma indeed lower by 2- to 3-fold in breakthrough and lymphoma cells cells. Lmo2 was upregulated .100-fold between 4 and 6 wk in in comparison with B6.Rag and NOD WT DN3a/DP controls. A NOD.Rag cells, declined in breakthrough CD4+ and DP cells, but key TCR signaling gene, Zap70, was consistently expressed at was still expressed in all lymphoma cells. Hhex was also elevated higher levels in NOD.Rag cells than in B6.Rag cells, and Il7ra, in most NOD.Rag cells, including all lymphomas. Lyl1 and Sfpi1 a critical early T cell cytokine receptor, was sustained at high levels levels varied between different NOD.Rag cell populations, but, in almost all populations. Continuing expression of Il7ra in all similar to Lmo2 and Hhex, both genes were still expressed in all breakthrough cell stages and in thymic lymphoma cells supports thymic lymphoma cells at levels higher than in WT NOD DP cells. the requirement for IL-7 for cell proliferation in early T cells, and These phase I genes are known proto-oncogenes and are likely to be this pathway has been reported to be critical in human early T- major contributors to the thymocyte expansion and lymphomas ALL (43). found in NOD.Rag mice. 10 T CELL PROGENITOR CHECKPOINT LOSS AND LEUKEMIA INITIATION Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 6. Changes in thymocyte cell number, in vitro potential, and gene expression with age and lymphoma. (A) Plot of thymocyte cell numbers with age showing the increase in cell numbers with age in NOD.Rag but not B6.Rag mice. (B) DN cells from 6–wk-old prebreakthrough NOD.Rag mice cultured with OP9-DL1 stroma plus IL-7 exhibit upregulation of CD4 whereas B6.Rag cells do not express CD4. (C) Sorted CD25+ DN and DP cells from a 12-wk- old NOD.Rag mouse proliferate and differentiate when cultured on OP9-DL4 stroma supplemented with 5 ng/ml IL-7 for 3 and 16 d before FACS analysis for expression of CD4 and CD8. Data are representative of two experiments. (D–G) Real-time quantitative PCR expression of some developmentally regulated genes in sorted B6.Rag CD25+ DN cells, from 4, 6, and 10 wk age (brown bars), sorted NOD.Rag prelymphoma CD25+ DN, CD4+, and DP cells, from 4, 6, 10, 13, and 16 wk age (green bars), unsorted cells from five independent NOD.Rag lymphomas (pink bars), and representative WT NOD DN1/ ETP, DN2a, DN2b, DN3a, and DP cells for reference (blue bars). Relative gene expression levels are shown for representatives of (D) key T cell genes, (E) Notch and target genes, (F) phase I (ETP/DN2a) genes, and (G) differentially expressed genes from the transcriptome analysis. The Journal of Immunology 11

The lymphomas did not passively maintain gene expression is suggested by the transcriptome data showing a marked over- patterns from DN and breakthrough cells, as shown in Fig. 6G for representation of signaling genes, especially actin-binding pro- representative differentially expressed genes, with each showing teins and tyrosine kinases, among differentially expressed genes distinctive expression patterns. Of particular interest, the NOD- between NOD.Rag and B6.Rag thymocytes. Actin/cytoskeletal specific high-level expression of Epha2 (in the chr4 QTL peak) and signal transduction pathways are interrelated in TCR signal- began before breakthrough and was maintained in all stages, in- ing (36), and alterations in expression of key genes may trigger cluding all lymphomas. Quantitative PCR analysis also showed inappropriate signaling at the checkpoints. Evidence that activated Epha2 to be differentially expressed between WT NOD and B6 NOD CD4+ cells exhibit a higher overall level of phospho- cells, at all DN stages, and in DP and gdT cells (Supplemental Fig. tyrosine than B6 cells (46) supports a potential role for at least 1A). Higher NOD.Rag cell expression of Mllt4 (Afadin, in chr17 some of the 11 differentially overexpressed tyrosine kinases in QTL peak) was also confirmed, but expression levels declined NOD.Rag cells, and we show in vitro evidence for subtle but with age. Expression of the tumor suppressor kinase, Dapk1 consistent strain-specific differences in responses to PMA between (chr13 QTL peak), was lower in NOD.Rag cells, although some cells from the two strains. Furthermore, consistent differences in expression persisted in the lymphomas. WT NOD cells also Erk1/2 phosphorylation have also been reported for immature and showed a lower expression of Dapk1 in all stages of development mature NOD and B6 TCR-transgenic T cells (38). in comparison with B6 cells, especially in the DN2a and DP stages The NOD.Rag mixed gene expression signature of early break- where it is undetectable (Supplemental Fig. 1B). Expression of through T cells and thymic lymphoma cells that emerge at high Vegfa and Tnfrsf9 declined with age, but Vegfa was consistently frequency after 4–5 mo is shared with certain human acute T- expressed in the lymphomas whereas Tnfrsf9 was variable. ALL, especially of the early LYL1-LMO2 T-ALL subgroup (47), Downloaded from Overall, the lymphoma cells maintained many of the unique gene as well as the recently described ETP-ALL, whose gene signatures expression patterns found in NOD.Rag N7 cells, especially ex- share features with normal ETP and myeloid cells (48). ETP-ALL pression of phase I proto-oncogenes, suggesting that origins of the also shares similarities with both T cell and myeloid stem cells, transformed cells may lie in the early breakthrough cells which and mutations in cytokine (Flt3 and IL-7R) and Ras signaling fail to shut off the genes that sustain the pleuripotency and pro- pathways are prominent (43). In common with ETP-ALL, NOD.

liferation of progenitor T cells (Fig. 1E). Rag breakthrough cells express genes indicative of their early http://www.jimmunol.org/ T cell programming, for example, Bcl11b, Ptcra, and Il7r, in ad- Discussion dition to stem/progenitor genes such as Lmo2, Hhex, Lyl1, Kit, and Thymic lymphomas generated in mice with an NOD genetic Sfpi1, which also likely contribute to the myeloid potential of WT background preserve many gene expression features of the early NOD and B6 ETP/DN2a cells (5). The prominent overexpression breakthrough cells, which themselves emerge as the result of an of Lmo2 in the NOD.Rag breakthrough cells is noteworthy, as DN earlier dysfunction. From the transcriptome analysis, the dominant cells overexpressing transgenic Lmo2 develop into self-renewing feature of breakthrough cells emerging spontaneously in the NOD. thymocytes, with striking gene expression similarities to NOD. Rag thymus is the collapse of two regulatory boundaries: one that Rag breakthrough cells, followed by T-ALL 8 mo later (49). The normally distinguishes precommitment ETP and DN2a stage cells NOD.Rag breakthrough and thymic lymphoma development may by guest on September 25, 2021 from committed DN3 cells, with the other operating at the b-se- be only partially attributable to the enhanced expression of Lmo2, lection checkpoint that requires a TCR signal for DN3 to DP as thymic tumors appear much more rapidly in NOD.Rag mice, progression (Fig. 1E). The breakthrough cells and the later-arising beginning 2 mo after breakthrough cells first appear, possibly due lymphoma cells share a highly abnormal coexpression of legacy to the elevated expression of tyrosine kinases, which are com- progenitor cell genes, including proto-oncogenes, normally re- monly oncogenic (37). stricted to uncommitted ETP and DN2a cells, along with T cell Genes within the identified QTL regions control propensity for genes characteristic of later stage cells, before and after TCR- breakthrough in the NOD genetic background and they mapped to dependent b-selection. Unlike normal Rag-deficient cells, break- known autoimmune diabetes susceptibility regions. The strong through thymocytes undergo long-term expansion and aberrant chr4 QTL peak in the Idd9/11 region (14) clearly includes at least differentiation in vitro, and thymic cell numbers increase with age one recessive NOD allele needed to promote efficient break- in vivo, even before thymic lymphomas appear. Thus, the break- through, as shown by the reduced breakthrough in congenic NOD. through cells may include a population of preleukemic cells, which B10Idd9Rag mice. Possible breakthrough-enhancing QTL regions can provide cellular targets of oncogenic transformation. were also detected in the Idd16/23/1/24 region on chr17, Idd14 on One striking feature of the T cell commitment checkpoint in the chr13, and Idd4 on chr11. Although all of the QTL regions remain DN2 stage is the brief period of overlap in phase I and T cell too large to determine whether any of the same genes control both identity genes during the transition from dominant expression to autoimmunity and the early T cell breakthrough, the close map- silencing of legacy stem cell genes after the onset of expression of ping of the two different T cell–related phenotypes is highly T cell–specific genes (5, 9). The most notable feature of our suggestive and requires further analysis to pinpoint source genes. current transcriptome and quantitative PCR data are the apparent The B10 Idd9 congenic region, which protects against the break- failure to terminate the phase I ETP-DN2 progenitor/stem cell through trait, is reported to protect NOD mice against type 1 di- program in early breakthrough cells despite upregulation of abetes after lymphocytic infiltration in the pancreas, suggesting T cell–specific genes including Bcl11b, which is involved in their control over the balance between pathogenic and protective T cell silencing (6), making the breakthrough cells appear to be retained responses (14). If the differentially expressed genes found within in a DN2-like state. Expression of genes such as Cd2, Cd5, and the QTL regions are also used in establishing self-tolerance, then Cd4 indicate a bypass of the later b-selection checkpoint as well, the autoimmunity promoting T cell defects may be traceable to the possibly due to an aberrant pre–TCR-like signal. It is also possible earliest common T cell progenitors, even before TCR rearrange- that failure of the first checkpoint might prevent the normal es- ment occurs. tablishment of the second checkpoint, making the DN2 stage the To date, animal models of T-ALL have typically depended on critical point of breakthough vulnerability. Aberrant signal trans- artificial gene expression alterations such as ablation of tumor duction at one or both checkpoints as a promoter of breakthrough suppressor genes or transgenic overexpression of oncogenes (50). 12 T CELL PROGENITOR CHECKPOINT LOSS AND LEUKEMIA INITIATION

The results reported in this study suggest that the NOD.Rag mouse genetic interval influences the pathogenicity of insulitis and contains molecular variants of Cd30, Tnfr2, and Cd137. Immunity 13: 107–115. may be a useful unmanipulated model for studying oncogenic 15. Serreze, D. V., and E. H. Leiter. 2001. Genes and cellular requirements for au- progression from a preleukemic state, which arises spontaneously toimmune diabetes susceptibility in nonobese diabetic mice. Curr. Dir. Auto- and predictably from defects in early T cell checkpoint control. immun. 4: 31–67. 16. Todd, J. A., and L. S. Wicker. 2001. Genetic protection from the inflammatory Moreover, our genetic analysis suggests a potential linkage to disease type 1 diabetes in humans and animal models. Immunity 15: 387–395. autoimmune diabetes in WT NOD mice, possibly related to an 17. Feng, N., P. Vegh, E. V. Rothenberg, and M. A. Yui. 2011. 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