Gfi1-Foxo1 Axis Controls the Fidelity of Effector Gene Expression And

Gfi1-Foxo1 axis controls the fidelity of effector gene PNAS PLUS expression and developmental maturation of thymocytes

Lewis Zhichang Shia,1,2, Jordy Saraviaa,1, Hu Zenga, Nishan S. Kalupahanaa,3, Clifford S. Guya, Geoffrey Nealeb, and Hongbo Chia,4

aDepartment of Immunology, St. Jude Children’s Research Hospital, Memphis, TN 38105; and bHartwell Center for Bioinformatics and Biotechnology, St. Jude Children’s Research Hospital, Memphis, TN 38105

Edited by Stephen M. Hedrick, University of California, San Diego, La Jolla, CA, and accepted by Editorial Board Member Arthur Weiss November 28, 2016 (received for review October 26, 2016) Double-positive (DP) thymocytes respond to intrathymic T-cell re- immature thymocytes while repressing the expression of effector ceptor (TCR) signals by undergoing positive selection and lineage molecules unique to mature T cells (4). However, given the multi- differentiation into single-positive (SP) mature cells. Concomitant faceted functions of E-proteins in thymocyte selection and survival with these well-characterized events is the acquisition of a mature T- (5), how E-proteins propagate signals in DP cells is unclear (1). cell gene expression program characterized by the induction of the Therefore, despite our increasing knowledge of the cellular events in α effector molecules IL-7R , S1P1, and CCR7, but the underlying mech- thymocyte selection and lineage differentiation, the underlying anism remains elusive. We report here that transcription repressor mechanisms and transcriptional circuitry for the maturation of DP Growth factor independent 1 (Gfi1) orchestrates the fidelity of the thymocytes remain to be determined. DP gene expression program and developmental maturation into SP Growth factor independent 1 (Gfi1), a transcriptional repressor, cells. Loss of Gfi1 resulted in premature induction of effector genes has emerged as an important regulator of hematopoietic and im- and the transcription factors forkhead box protein O1 (Foxo1) and Klf2 in DP thymocytes and the accumulation of postselection inter- mune system cells. Gfi1 is required for the normal development and mediate populations and accelerated transition into SP cells. Strik- homeostasis of hematopoietic stem cells and both myeloid and ingly, partial loss of Foxo1 function, but not restored survival fitness, lymphoid progenitors (6). Specifically, loss of Gfi1 impairs the de- rectified the dysregulated gene expression and thymocyte matura- velopment of neutrophils and B cells while expanding the monocyte tion in Gfi1-deficient mice. Our results establish the Gfi1-Foxo1 axis and myeloid populations (7). During T-cell development, Gfi1 ex- and the transcriptional circuitry that actively maintain DP identity pression is dynamically regulated (8), and its germ-line deficiency and shape the proper generation of mature T cells. impairs DN cell generation (9). Recently, we have established a role

Gfi1 | thymocyte maturation | transcription repressor | Foxo1 | apoptosis Significance INFLAMMATION IMMUNOLOGY AND ormal development of T cells in the thymus is essential for the A fundamental question in immunology is the mechanism of Ngeneration of a functional peripheral T-cell pool that mediates thymocyte development, but how differentiating CD4+CD8+ + + efficient immune responses against infections and tumors while double-positive thymocytes progress into CD4 or CD8 single- avoiding uncontrolled self-reactive responses. Generation of T cells positive cells remains poorly defined. We have now determined starts from the thymic settling of early thymic progenitors (ETPs), that the transcription repressor Growth factor independent 1 − − which progress from the CD4 CD8 double-negative (DN) stage (Gfi1) plays a central role in controlling double-positive cell fate + + + through the CD4 CD8 double-positive (DP) stage to mature CD4 and thymocyte maturation. Deletion of Gfi1 in double-positive + or CD8 single-positive (SP, namely CD4SP and CD8SP) thymo- thymocytes induces premature induction of single-positive– cytes. Whereas the majority of thymocytes are eliminated due to specific effector genes and transcription factors Foxo1 and Klf2 either lack of T-cell receptor (TCR) signals (death by neglect) or and accelerated transition into single-positive cells. These de- high-affinity interaction with self-peptide MHC ligands (negative fects are largely rectified upon partial loss of Foxo1 functions, selection), DP cells with low affinity are rescued from death and indicating the critical contribution of aberrant Foxo1 induction to undergo positive selection and develop into SP cells. Recent studies disrupted thymocyte maturation. Our study establishes a mo- have identified a number of transcriptional regulators, such as RAR- lecular mechanism that actively maintains double-positive cell relatedorphanreceptorgamma(t)(RORγt), runt-related tran- identity and shapes the proper generation of mature T cells. scription factor 3 (Runx3), and Th-inducing POZ-kruppel factor (ThPOK), that control positive selection and lineage decision be- Author contributions: L.Z.S., J.S., H.Z., and H.C. designed research; L.Z.S., J.S., H.Z., N.S.K., tween CD4SP and CD8SP T cells (1–3). and C.S.G. performed research; L.Z.S., J.S., H.Z., N.S.K., C.S.G., G.N., and H.C. analyzed In contrast, we have a very limited understanding of the mecha- data; and L.Z.S., J.S., and H.C. wrote the paper. nisms underlying the progression of DP into SP cells, a related but The authors declare no conflict of interest. rather distinct process from positive selection and CD4/CD8 lineage This article is a PNAS Direct Submission. S.M.H. is a Guest Editor invited by the Editorial Board. differentiation (1). Such developmental maturation is characterized by the acquisition of a mature T-cell gene expression program, Data deposition: The data reported in this paper have been deposited in the Gene Ex- α pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE89898). notably the induction of effector molecules IL-7 receptor chain (IL- 1 L.Z.S. and J.S. contributed equally to this work. 7Rα), C-C motif chemokine receptor 7 (CCR7), and sphingosine 2Present address: Department of Genitourinary Medical Oncology, MD Anderson Cancer 1-phosphate receptor 1 (S1P1) in SP cells that are essential for their Center, Houston, TX 77030. proper survival and trafficking (1). Among the few pathways identi- 3Present address: Department of Physiology, Faculty of Medicine, University of Peradeniya, fied to function in DP-to-SP transition, E-proteins play a pivotal role Sri Lanka. by serving as upstream “sensor” transcription factors in differentiating 4To whom correspondence should be addressed. Email: [email protected]. DP thymocytes (1). E-protein activity in DP thymocytes promotes This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. TCRα gene rearrangement and expression of genes characteristic of 1073/pnas.1617669114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1617669114 PNAS | Published online December 19, 2016 | E67–E74 Downloaded by guest on September 29, 2021 CD4 of Gfi1 in the differentiation of thymus-derived T cells (Treg) via (Gfi1 ) that resulted in effective deletion of Gfi1 starting at shaping the production of IL-2 in the thymic microenvironment (10, the DP stage (10). Loss of Gfi1 resulted in profound up-regulation 11). These results indicate a cell context-dependent function for of mature T-cell genes including Foxo1, Klf2, S1pr1, Ccr7, and Gfi1 in the immune system. Il7r in DP cells. In contrast, Gfi1 deletion did not affect ex- To ascertain the functions of Gfi1 in thymocyte maturation, we pression of DP-specific immature genes including Cxcr4 and developed several complementary genetic systems to modulate Gfi1 Rorc (Fig. 1C), nor did it affect DN-to-DP transition as evi- functions. We determined that Gfi1 orchestrates a unique gene denced by comparable percentages of DN and immature single- expression program. Loss of Gfi1 in DP thymocytes resulted in positive cells (Fig. S1D). Notably, the elevated expression of aberrant up-regulation of multiple SP-specific genes in a cell-in- mature T-cell genes in Gfi1CD4 mice was not due to an expanded + + trinsic manner; these include effector molecules for T-cell survival CD69 TCR subset within DP cells because the up-regulation of − − and trafficking such as IL-7Rα, CCR7, and S1P1 and the associated Foxo1, Klf2, S1pr1, Ccr7,andIl7r was evident even in CD69 TCR transcription factors forkhead box protein O1 (Foxo1) and Kruppel- preselection DP cells lacking Gfi1 (Fig. 1D). In addition, the absence like factor 2 (Klf2). Under physiological conditions of DP-to-SP of Gfi1 in DP cells led to increases in both total cellular and nuclear transition, Gfi1 expression was gradually down-regulated, thus Foxo1 protein levels (Fig. 1E), indicating an increased expression of allowing expression of mature T-cell genes when preselection DP both Foxo1 mRNA and protein. Therefore, Gfi1 specifically re- cells progress to postselection DP and SP stages. Therefore, Gfi1 presses induction of mature T-cell genes in DP thymocytes without enforces the identity of DP thymocytes by preventing the premature impacting expression of DP-specific immature genes. expression of SP genes. Mechanistically, this process is contingent upon the ability of Gfi1 to actively suppress Foxo1 expression in DP Gfi1 Intrinsically Represses a Mature Gene Expression Program in DP thymocytes, as heterozygous loss of Foxo1 rescued defective gene Cells. We next investigated whether the alteration of thymic gene expression, thymocyte maturation, and generation of mature T-cell expression due to Gfi1 deficiency was a cell-autonomous defect. subsets in Gfi1-deficient mice. In contrast, although Gfi1 deficiency To this end, we generated mixed bone marrow (BM) chimeras by − − increased thymocyte apoptosis, a process intimately involved in all reconstituting alymphoid Rag1 / mice with a 1:1 mixture of WT + + TCR selection events, Gfi1 impinged upon thymocyte maturation or Gfi1CD4 (CD45.2 ; “donor”) BM cells and WT (CD45.1 ; “spike”) independently of its prosurvival effect. These data point to a control BM cells. Following reconstitution, we purified DP thymocytes mechanism mediated by the Gfi1-Foxo1 axis that actively maintains from the respective BM-derived cells in the mixed chimeras and DP identity to direct proper DP-to-SP transition and development found that the up-regulation of SP-associated genes was retained of mature T-cell subsets. in Gfi1CD4-derived donor cells (relative to WT donors or the spike cells) (Fig. 2A). Therefore, altered gene expression is intrinsic to Results the loss of Gfi1 function. Gfi1 Is Highly Expressed in DP Thymocytes to Prevent Premature To verify our aforementioned results and to ensure optimal Induction of SP Genes. To explore the role of Gfi1 in thymocyte Gfi1 deletion, we developed an independent genetic system by maturation, we first analyzed the dynamics of Gfi1 expression in the using hCD2-iCre mice to delete the floxed Gfi1 allele in early thymus, relative to the levels of the prototypical effector molecules thymocytes with high efficiency (Gfi1hCD2 mice) (10). Similar and transcription factors characteristic of immature and mature to Gfi1CD4 mice, marked up-regulation of SP-associated genes thymocytes (1). These included the DP-specific molecules C-X-C was observed in DP thymocytes from Gfi1hCD2 mice (Fig. S1E). To motif chemokine receptor 4 (CXCR4) and RORγt (encoded by gain a global view on the gene expression program regulated by Rorc) and the SP-specific molecules CCR7, IL-7Rα (encoded by Gfi1, we next used a functional genomics approach to analyze Il7r), S1P1 (encoded by S1pr1), Foxo1, and Klf2. Among thymo- the Gfi1-dependent gene signature. We purified DP cells from cyte subsets, Gfi1 was highly expressed in DP thymocytes but was Gfi1hCD2 mice and used Affymetrix oligonucleotide arrays to markedly down-regulated in SP cells (Fig. 1A). This pattern par- compare their gene expression profiles with those from WT DP alleled the expression of Cxcr4 and Rorc (Fig. S1A) but was re- cells. A total of 566 probes (corresponding to 403 individual ciprocal to that of SP-specific genes (Fig. 1A). We next performed genes) showed equal or greater than twofold change [false dis- a more detailed analysis of Gfi1 expression in thymocytes at var- covery rate (FDR) <0.05] between WT and Gfi1-deficient DP ious stages of DP-to-SP transition. Upon productive interactions cells, including 443 up-regulated and 123 down-regulated probes of TCRs with the self-peptide–MHC complex presented by cor- upon Gfi1 deletion. The up-regulated genes included cytokines tical thymic epithelia, DP cells up-regulate their surface expression and effector molecules (Lta, Gzma, Tnfrsf25, and Tnfsf11), of CD69 and TCR (12–14). These postselection DP thymocytes transcription factors (Foxo1, Klf2, Runx3, and Id2), and traf- + then down-regulate CD8 expression levels to become CD4 CD8lo ficking receptors (Ccr7, Ccr10, and S1pr1), all of which are as- cells, an obligatory precursor stage for the development of sociated with the homeostasis or function of mature T cells both CD4SP and CD8SP cells (1, 2). We therefore purified instead of DP thymocytes (Fig. 2B). Gfi1 deficiency also en- andanalyzeddifferentsubsetsofthymocytes,includingTCR- hanced expression of additional transcriptional regulators (Relb − − inexperienced preselection CD69 TCR DP, postselection and Hdac4), kinases (Sgk1 and Map3k5), and apoptotic and + + + + + CD69 TCR DP, CD4 CD8lo,CD69 CD4SP, and CD69 cell cycle regulators (Gadd45g, Nod1, and Ccnd2). Real-time CD8SP cells (the latter two populations were not fully mature SP PCR analysis verified the alteredexpressionofselectedgenesin cells) (see gating strategy described in Fig. S1B). Gfi1 down-reg- Gfi1hCD2 mice,aswellasinGfi1CD4 mice (Fig. S1F). Therefore, the ulation was gradual during this process (Fig. 1B), associated with gene-profiling analysis of DP thymocytes reveals the aberrant up- more drastic down-regulation of Cxcr4 and Rorc (Fig. S1C)and regulation of the mature T-cell geneprogramintheabsenceofGfi1. with steady up-regulation of S1pr1, Ccr7, Il7r, Foxo1,andKlf2 Finally, we used a gain-of-function approach by transducing + (although the up-regulation of Foxo1 and Klf2 from CD69 T cells with a retrovirus expressing Gfi1 or an empty vector. Ec- + + TCR DP to CD4 CD8lo cells was not obvious) (Fig. 1B). topic expression of Gfi1 in T cells inhibited the mRNA levels of Collectively, these results indicate that Gfi1 expression in the Foxo1, Klf2,andIl7r (Fig. 2C). Altogether, Gfi1 function is nec- thymus is actively down-regulated during DP-to-SP transition. essary and sufficient to repress expression of mature T-cell genes. Given the dynamic regulation of Gfi1 during thymocyte maturation, we next determined the functional effects of Gfi1 de- Gfi1 Deficiency Facilitates Developmental Maturation During DP-to- ficiency on the gene expression program in this process. To this SP Transition. A key inhibitory role of Gfi1 in mature T-cell gene end, we generated T-cell–specific Gfi1-deficient mice by crossing expression prompted us to examine whether loss of Gfi1 accelera- loxP-flanked Gfi1 alleles (Gfi1fl/fl) with CD4-Cre transgenic mice ted developmental progression of thymocytes, using several

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Fig. 1. Gfi1 deficiency results in aberrant induction of mature T-cell genes in DP thymocytes. (A) Expression profiles of Gfi1 and mature T-cell genes in thymic main populations (DN, DP, CD4SP, and CD8SP) in C57BL/6 mice, as assessed by real-time PCR. (B) Dynamic regulation of Gfi1 and mature T-cell genes in − − + + thymocytes at different developmental stages, as assessed by real-time PCR analysis of CD69 TCRβ preselection DP thymocytes and CD69 TCRβ postselection + + + + − − + thymocytes including CD4 CD8 , CD4 CD8lo, CD4 CD8 , and CD4 CD8 cells in C57BL/6 mice. (C) Expression of prototypical mature and immature T-cell genes − − in DP thymocytes from WT and Gfi1CD4 mice. (D) Expression of mature T-cell genes in CD69 TCRβ preselection DP thymocytes sorted from WT and Gfi1CD4 mice. (E) Total cellular and nuclear expression of Foxo1 protein (red) in preselection DP thymocytes sorted from WT and Gfi1CD4 mice. (Scale bar, 5 μm.) Data represent at least two independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant.

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Fig. 2. An intrinsic role of Gfi1 in repressing a mature gene expression program. (A) Mixed BM chimeras were generated by reconstituting sublethally ir- − − + + radiated Rag1 / mice with a 1:1 mixture of spike (CD45.1 ) and WT or Gfi1CD4 (CD45.2 ) BM cells and analyzed after reconstitution for the expression of mature T-cell genes in DP thymocytes. (B) Microarray analyses of gene expression profiles of WT and Gfi1hCD2 DP cells. Heat maps display replicate gene

expression differences relative to the WT on a log2 scale. (C) Naive CD4 T cells were activated with anti-CD3/CD28 and transduced with Thy1.1-tagged vector control retrovirus (Ctrl RV) or Gfi1-expressing retrovirus (Gfi1 RV). Thy1.1+ or Thy1.1– cells were sorted, and expression of Gfi1, Foxo1, Klf2, and Il7r was analyzed by real-time PCR. Data represent two independent experiments. **P < 0.01.

complementary approaches. We first analyzed induction of CD69 CD8SP cells (16). To synchronize the initiation of thymocyte devel- − + and TCR on DP cells, which are early events associated with pos- opment in vitro, we purified DN3 (CD44 CD25 ) thymocytes from itive selection (12–14). The frequency and number of TCR-signaled WT and Gfi1CD4 mice and cocultured them with OP9-DL1 cells. + + CD69 TCR cells among DP cells were significantly increased in Compared with WT cells, Gfi1-deficient thymocytes developed into Gfi1CD4 mice relative to WT mice (Fig. 3A). Analysis of additional CD8SP cells at a much faster rate (Fig. 3C). As a parallel system, we maturation markers including β7 integrin up-regulation and CD24 performed fetal thymus organ culture (FTOC), in which embryonic down-regulation also revealed altered maturation of Gfi1-deficient thymic lobes were isolated and cultured in vitro for T-cell maturation DP thymocytes (Fig. S2A). Thus, the increased expression of ma- (17).SimilartotheOP9-DL1results,weobservedanincreaseinthe ture T-cell genes was intrinsic to Gfi1 deficiency and not simply a ratio of CD8SP to CD4SP T cells (Fig. S2C). Finally, to ensure that consequence of the altered maturation. This conclusion was further the thymocyte developmental and maturational effects caused by supported by the altered maturation of DP thymocytes from Gfi1 deficiency are indeed cell-intrinsic (i.e., not the result of a trans Gfi1CD4 neonatal mice, which precluded the secondary effects of factor), we generated asymmetric BM chimeras with a 10:1 ratio (WT chronic loss of Gfi1 (Fig. S2B). or Gfi1CD4 CD45.2:WT CD45.1, respectively). Following re- Although the increased postselection DP thymocytes in Gfi1CD4 constitution, only Gfi1CD4 CD45.2 cells displayed an increased per- + + mice suggested an accelerated developmental maturation, an al- centage of CD69 TCR DP cells and CD8/CD4 ratio, whereas these ternative interpretation was a stalled progression to the ensuing SP parameters were unchanged in spike CD45.1 cells (Fig. S2D). Taken stage. Arguing against this possibility, Gfi1 deficiency did not cause together, these data indicate an intrinsic role of Gfi1 in restraining the a major reduction in SP thymocytes but led to increased CD8SP transition of DP-to-CD8SP cells. frequency (9, 10). To further address this issue and to investigate Another possible explanation for the increased postselection theimpactofTCRsignalingonGfi1-mediated gene expression, we DP thymocytes in Gfi1CD4 mice was due to an altered TCR reper- − − crossed Gfi1CD4 mice with TCRα-deficient (Tcra / )micethatlack toire under the polyclonal background. Thus, we assessed the dis- fully differentiated SP thymocytes but maintain DN and DP thy- tribution of TCRβ chains in CD4SP thymocytes and found mocytes (15). Importantly, the up-regulation of mature T-cell genes, comparable TCRβ chain usage between WT and Gfi1CD4 mice (Fig. including Foxo1, Klf2, Ccr7,andRunx3, was maintained in DP cells S2E). To more definitively exclude this possibility, we introduced a − − − − from Gfi1CD4 Tcra / mice, whereas Gfi1-competent Tcra / con- single class II-restricted αβTCR transgene, the ovalbumin peptide- trols exhibited similar gene expression as WT cells (Fig. 3B). In- specific OT-II system, into WT and Gfi1CD4 mice. This TCR- terestingly, S1pr1 and Il7r up-regulation due to Gfi1 deficiency was transgenic system allowed us to assess the effects of Gfi1 loss on T cells − − not retained in Gfi1CD4 Tcra / mice (Fig. 3B), suggesting the with defined specificity of TCR chains. In line with the observations + + involvement of TCR signaling on selective Gfi1 target genes. in the polyclonal background, the frequency of CD69 TCR cells To directly assess the dynamics of CD8SP generation, and to ex- among DP cells was elevated in the thymus from Gfi1CD4 OT-II mice clude the contribution of complex in vivo factors that could non- compared with WT OT-II mice (Fig. 3D). Interestingly, even in this specifically affect thymic development, we used the in vitro OP9-DL1 MHC class II-restricted background, mature CD8SP cells expressing + + system. In this system, OP9 stromal cells ectopically express the components of the OT-II transgene (Vα2 Vβ5 )wereselectedin + Notch ligand Delta-like 1, which acts in synergy with pre-TCR signals the absence of Gfi1 (Fig. 3D). Moreover, postselection CD4 CD8lo to drive thymocyte differentiation in vitro, including the generation of intermediate thymocytes, which can be unequivocally detected in the

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WT Gfi1CD4 17.3 49.9 20.3 35.3 3.24 69.0 1.96 56.5 5 3 * of DP

1.1 2.23 + 4 * DP cells DP ) WT WT + 6 2 3 10 TCR ( + 2 13.4 16.1 21.8 35.9 TCR 1 1 + 7.51 36.6 4.66 16.6 0.68 32.5 0.49 17.5 CD69 0 0

TCR CD4 CD69 CD4 CD4 % CD69 WT Gfi1 WT Gfi1 Gfi1

CD4 26.2 32.3 53.2 73.6 CD8 B / WT Gfi1CD4 Tcra / Gfi1CD4 Tcra 8080 WT 400400 ) Gfi1CD4 )

Foxo1 Klf2 S1pr1 Ccr7 Runx3 hi Il7r *** hi ** 55 2020 44 88 33 88 6060 300300 * * ** ** ** ** * ** 44 ** 4040 *** 200200 1515 33 66 66 ** *** 22 33 1010 22 44 44 2020 ** 100100 CD8/CD4 (TCR 22 (TCR % CD8SP 11 00 00 55 11 22 22 11 (Day) 9 15 21 27 9 15 21 27

Relative mRNA expression Relative mRNA 00 00 00 00 00 00

D Thymus TCR (V 2V 5)+ DP E CD4SP CD8SP F CD4SP CD8SP

23.4 5.68 57 93.2 3.4 1.5 1.84 21.6 52.8 WT WT WT WT

WT WT 3.51 95.3 2.03 91.8 OT-II OT-II

0.44 Rag2-GFP 1.57 INFLAMMATION 10.7 12.6 CD4

16.4 4.27 IMMUNOLOGY AND CD4 49.6 72.5 3.44 65.2

CD4 38.7 OT-II Gfi1 15 81.3 5.75 86.2 Gfi1 Rag2-GFP Rag2-GFP Gfi1

7.29 Cells CD8 7.42 CD4 CD4 CD4 CD69 Qa-2 CD8 CD8 TCR GFP

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8 (%) * (%) + (%) 6 lo 2V lo 10 6 4 CD8 TCR CD8

4 + + + 5 2 2 CD4 CD4 among (V 0 0 CD69 0 WT Gfi1CD4 WT Gfi1CD4 WT Gfi1CD4

+ + Fig. 3. Gfi1 deficiency facilitates thymic maturation during DP-to-SP transition. (A) Proportion and absolute number of CD69 TCRβ cells in DP thymocytes. − − (B) Expression of prototypical mature T-cell genes in preselection (CD69 TCRβ ) DP thymocytes of Gfi1CD4 or Gfi1CD4 mice with additional TCRα deficiency (Gfi1CD4 Tcra−/−) compared with WT and Tcra−/− controls. (C) Expression of CD4 and CD8, frequencies of TCRhi CD8SP cells, and TCRhi CD8/CD4 ratios of in vitro differentiated T cells (7-AAD− live cells) in OP9-DL1 cells cultured with DN3 cells from WT and Gfi1CD4 mice. (D) Frequencies of DP, CD8SP, CD4SP, and CD4+ + + + CD8lo populations among total thymocytes (Left) or antigen-specific TCR (Vα2Vβ5) thymocytes (Middle), and proportions of CD69 TCRβ cells in DP cells + + (Right) from OT-II TCR-transgenic mice. (Lower panels) Proportions of CD4 CD8lo intermediate cells among total thymocytes (Left), among TCR (Vα2Vβ5) + + thymocytes (Middle), and CD69 TCRβ cells in gated DP cells (Right). (E) GFP expression of CD4SP or CD8SP thymocytes from WT Rag2-GFP and Gfi1CD4 Rag2- GFP mice. (F) Qa-2 expression on CD4SP or CD8SP cells from WT and Gfi1CD4 mice. Data represent three to five independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

TCR-transgenic system, showed a higher frequency in Gfi1CD4 OT-II timer” to follow thymocyte development (18). To this end, we mice compared with OT-II mice (Fig. 3D). Altogether, the increased bred Gfi1CD4 mice to Rag2-GFP mice and found that deletion of + + + − frequencies of CD69 TCR DP and CD4 CD8lo populations in the Gfi1 resulted in a marked expansion of Rag2-GFP SP thymo- TCR-transgenic system further illustrate the “expedited” maturation cytes (Fig. 3E). Notably, this expansion was not attributable to CD4 of thymocytes in the absence of Gfi1. increased Treg cells as previously seen in Gfi1 mice (10) be- + To more directly examine DP-to-SP transition in vivo, we took cause exclusion of such a population (CD25 ) (18) did not affect advantage of several well-established systems. First, the decline the conclusion (Fig. S2F). In fact, the accelerated down-regula- of GFP expression in the Rag2-GFP system acts as a “molecular tion of GFP expression levels was already initiated in DP cells

Shi et al. PNAS | Published online December 19, 2016 | E71 Downloaded by guest on September 29, 2021 lacking Gfi1 (Fig. S2G). Second, Gfi1-deficient SP thymocytes, es- cell population among DP thymocytes (Fig. S3B). Development of pecially CD4SP cells, contained an increased frequency of cells various thymic subsets including CD4SP, CD8SP, and Treg cells expressing the maturation marker Qa-2 (Fig. 3F). Third, we per- and the induction of Qa-2 among CD4SP thymocytes also formed a BrdU pulse-chase by injecting BrdU into WT or Gfi1CD4 appeared normal (Fig. S3 C–E). However, under the Gfi1- + mice, followed by flow cytometry analysis of BrdU cells 2 d later deficient background, the aberrant induction of Klf2, S1pr1,andIl7r (18). Gfi1-deficient CD4SP cells contained an increased pro- in DP thymocytes was markedly suppressed upon the heterozy- portion labeled with BrdU (Fig. S2H), indicative of faster gener- gous loss of Foxo1, associated with the largely restored expression ation of CD4SP cells. These results collectively indicate that of Foxo1 itself (Fig. 4A). There was also a trend for the rescue of deficiency of Gfi1 accelerates developmental progression of thy- Ccr7 expression, although this did not reach statistical signifi- mocytes in vivo. cance (Fig. 4A). Moreover, heterozygous deletion of Foxo1 considerably rescued the phenotypes of Gfi1CD4 mice in thymo- + + The Gfi-Foxo1 Axis Controls DP Thymocyte Maturation and Development cyte maturation, including the increased frequencies of CD69 TCR + of SP Cells. We next investigated the molecular mechanism by which and Qa-2 populations among DP and CD4SP thymocytes, re- Gfi1 regulates thymocyte maturation. Among the plethora of gene spectively (Fig. 4 B and C). These results indicate that aberrant targets altered in the absence of Gfi1, we chose Foxo1 for further induction of Foxo1 in the absence of Gfi1 plays a central role in analysis because Gfi1 deficiency substantially enhanced Foxo1 ex- disrupting thymocyte maturation. pression in DP thymocytes (Fig. 1C)toalevelcomparabletothat We further investigated the effect of Foxo1 dysregulation on of SP thymocytes (Fig. 1A). Furthermore, Foxo1 has been shown the altered thymic development of mature T-cell subsets in CD4 to regulate Klf2, S1P1, CCR7, and IL-7Rα expression in peripheral Gfi1 mice. The altered CD8/CD4 ratio in Gfi1-deficient mice Tcells(19–21), although its function in thymic development is (9, 10) was considerably rescued upon heterozygous deletion of much less understood. To examine the role of Foxo1 in mediating Foxo1 (Fig. 4 D and E). We recently reported that Gfi1 deletion induction of mature T-cell genes in Gfi1-deficient DP thymocytes, results in an expanded thymic Treg population due to the over- + + − we crossed Gfi1CD4 mice with CD4-Cre;Foxo1 /fl (Foxo1 / )mice production of IL-2 from conventional CD4SP thymocytes (10, 11). + + − to derive CD4-Cre;Gfi1fl/fl;Foxo1 /fl (Gfi1CD4Foxo1 / ) mice. Under Importantly, these two defects were substantially, albeit incom- the Gfi1-sufficient background, the partial loss of Foxo1 neither pletely, rescued by the heterozygous deletion of Foxo1 (Fig. 4 F exerted major effects on Klf2, S1pr1, Ccr7,andIl7r expression (Fig. and G). Altogether, Gfi1-mediated suppression of Foxo1 expres- + + S3A) nor affected the frequency of the TCR-signaled CD69 TCR sion links multiple processes in the thymus, including thymocyte

A WT Gfi1CD4 Gfi1CD4Foxo1 Foxo1 Klf2 S1pr1 Ccr7 Il7r 5 10 15 NS 15 5 ** NS NS 44 8 4 *** *** *** *** 10 10 *** 33 6 *** *** 3 *** NS *** ** 22 4 2 5 5 11 2 1

Relative mRNA expression Relative mRNA 00 0 0 0 0

B Gfi1CD4 Gfi1CD4Foxo1 WT 3 3 * * * * of DP

0.55 + 0.55 2.192.19 2

0.93 ) 0.93 2 cells DP 6 + 10 TCR + ( 1

1 CD69 +

0 CD69

% CD69 0 TCR TCR

C WT Gfi1CD4 Gfi1CD4Foxo1 D WT Gfi1CD4 Gfi1CD4Foxo1 16.6 4.18 32.6 6.83 22.4 6 7.99 85.1 5.80 85.2 6.14 85.1

6.78 53.8 CD4 1.89 3.65 2.46 Qa-2 8.74 8.14 CD69 CD8

1.0 Il2 WT Gfi1CD4 Gfi1CD4Foxo1 E F G 5 0.8 * WT 3.5 11 7.41 4 0.6 Gfi1CD4 0.4 3 ** * Gfi1CD4 CD8/CD4 Foxo1 0.2 2

0 1 WT Gfi1CD4 Gfi1CD4 Foxp3 CD25 Foxo1 0 Relative mRNA expression Relative mRNA

Fig. 4. Heterozygous loss of Foxo1 largely restores the altered gene expression and maturation of DP thymocytes and development of SP subsets in Gfi1CD4 mice. (A) Prototypical mature T-cell gene expression in sorted DP thymocytes from WT, Gfi1CD4,andGfi1CD4Foxo1+/− mice, as assessed by real-time PCR. + + + − (B) Flow cytometry analysis, percentage, and absolute number of CD69 TCRβ cells in DP thymocytes from WT, Gfi1CD4, and Gfi1CD4Foxo1 / mice. (C) Qa-2 + − and CD69 surface expression on CD4SP thymocytes from WT, Gfi1CD4, and Gfi1CD4Foxo1 / mice. (D) CD4 and CD8 surface expression among total thymocytes CD4 CD4 +/− CD4 CD4 +/− + from WT, Gfi1 , and Gfi1 Foxo1 mice. (E) Ratio of CD8/CD4 SP thymocytes from WT, Gfi1 , and Gfi1 Foxo1 mice. (F) Frequency of Foxp3 Treg + − cells in CD4SP thymocytes in WT, Gfi1CD4, and Gfi1CD4Foxo1 / mice. (G) Il2 mRNA expression in sorted CD4SP thymocytes from WT, Gfi1CD4,andGfi1CD4 Foxo1+/− mice, as assessed by real-time PCR. Data represent at least three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; NS: not significant.

E72 | www.pnas.org/cgi/doi/10.1073/pnas.1617669114 Shi et al. Downloaded by guest on September 29, 2021 gene expression, DP-to-SP maturation, and development of both Gfi1 in restraining DP-to-SP transition that can be ascribed to its PNAS PLUS conventional and Treg cells, thereby pointing to a key role of the ability to repress the expression of genes characteristic of mature Gfi1-Foxo1 axis in thymic development. thymocytes in DP cells, in line with the dynamic regulation of Gfi1 expression by intrathymic developmental signals. Moreover, the Survival-Independent Functions of Gfi1 in Thymocyte Maturation. An- aberrant up-regulation of Foxo1 in Gfi1-deficient DP thymocytes other process intimately involved in thymocyte development is largely accounts for the expedited maturational program and dys- apoptotic cell death. Indeed, all TCR selection events, including regulated development of SP thymocytes (including CD8SP and positive selection, negative selection, and death by neglect, are Treg cells), whereas the prosurvival effect of Gfi1 is dispensable in intricately linked to the decision of cell death or survival (1). Gfi1 these processes. We therefore propose that Gfi1-dependent tran- mutation is frequently associated with elevated apoptosis of mul- scriptional repression of Foxo1 and Foxo1-mediated mature T-cell tiple cell types (6). In hematopoietic stem cells deficient in Gfi1, genes maintains DP developmental integrity and that this serves as the survival defect is a central mechanism for impaired stem cell a central mechanism to orchestrate thymocyte maturation and function, as ectopic Bcl-2 expression allows Gfi1-deficient stem development of mature T cells (Fig. S4). cells to self-renew and initiate multilineage differentiation (22). Identifying the mechanisms that orchestrate the differentia- Notably, Gfi1-deficient BM donor cells were less efficient in tion of DP thymocytes into mature T cells has been a challenging populating the thymic DP compartments in a competitive envi- issue to unravel, partly because of our incomplete understanding ronment introduced by the mixed chimeras (Fig. 5A). Further- of the multiplicity of events in DP thymocytes experiencing TCR more, Gfi1CD4 DP thymocytes were impaired in ability to survive selection signals. In addition to undergoing positive selection when cultured in vitro (Fig. 5B). Thus, Gfi1 is required for optimal (defined by rescue from cell death) and lineage differentiation into + + survival of DP thymocytes. CD4 or CD8 cells, DP-to-SP transition requires proper acqui- To address the effect of the prosurvival effect of Gfi1 on DP sition of effector functions (1). These include the following: (i)the maturation, we crossed Gfi1CD4 mice with lymphocyte-specific expression of CCR7, which promotes the migration of thymocytes transgenic mice for Bcl-2 that potently inhibits thymocyte death from the cortex to the medulla; (ii) the initiation of S1P1 expression (10). As expected, the introduction of the Bcl-2 transgene rectified for thymic egress; and (iii) the acquisition of IL-7Rα expression for the survival defects of Gfi1-deficient DP thymocytes in vitro (Fig. survival fitness (1). Remarkably, all of these genes are expressed in + + 5B). However, the increased CD69 TCR frequency was retained a reciprocal manner to Gfi1, and they are profoundly up-regulated in DP thymocytes from Gfi1CD4 Bcl2-TG mice (Fig. 5C). More- in Gfi1-deficient DP thymocytes. Associated with this up-regula- over, these mutant DP cells showed marked up-regulation of tion is the induction of the transcription factors Foxo1 and Klf2. SP-associated genes compared with the control Bcl2-TG cells Our genetic rescue approaches have established that Gfi1 restrains (Fig. 5D). Therefore, Gfi1 regulates thymic maturation largely Foxo1 expression that in turn affects Klf2 and the effector mole- independently of cell survival. cules. In further support of this model, although the levels of S1pr1, Ccr7,andIl7r in Gfi1-deficient DP thymocytes did not reach those Discussion of WT SP cells, Gfi1-deficient DP thymocytes expressed Foxo1 at a Although Gfi1 has been implicated in the generation or function similar level as WT SP thymocytes. Although Foxo1 has been of various hematopoietic cells including T cells, its roles in the shown to affect the expression of these molecules in peripheral INFLAMMATION

development of DP thymocytes have not been addressed. In this T cells (19–21), whether this regulation is operational in thymocyte IMMUNOLOGY AND study, we have identified previously unrecognized functions of maturation is unclear, especially considering that Foxo1 is normally

A B WT Bcl2-TG C 1.63 48.2 19.9 75.6 3.85

23.8 Bcl2-TG WT

47.3 2.86 74.9 Gfi1CD4 Gfi1CD4 Bcl2-TG 27.6 1.36 88.7 21.9 18 Gfi1CD4 71.6 Gfi1CD4 Bcl2-TG CD69 CD45.2 8.75 1.2 73.5

CD45.1 7-AAD TCR Annexin V

D WT Gfi1CD4 Bcl2-TG Gfi1CD4 Bcl2-TG Foxo1 Klf2 S1pr1 Ccr7 Il7r 15 4 15 15 ** 15 20 * ** ** ** * 3 15 ** 10 10 10 10 2 10 ** ** ** 5 55 5 1 5

Relative mRNA expression Relative mRNA 0 0 00 0 0

Fig. 5. Survival defect of Gfi1-deficient thymocytes does not contribute to DP maturation. (A) Competitive fitness of DP cells in vivo. Mixed BM chimeras were + + set up as described in Fig. 2A, and the contributions of WT or Gfi1CD4 donor (CD45.2 ) and spike (CD45.1 ) cells were analyzed. (B) DP thymocytes from WT, + + Gfi1CD4, Bcl2-TG, or Gfi1CD4 Bcl2-TG mice were cultured for 2 d, followed by Annexin V and 7-AAD staining. (C) Proportion of the postselection CD69 TCRβ population in DP cells from Bcl2-TG or Gfi1CD4 Bcl2-TG mice. (D) Expression of mature T-cell genes in DP thymocytes from WT, Gfi1CD4, Bcl2-TG, or Gfi1CD4 Bcl2-TG mice. Data represent five (A–C) and three (D) independent experiments. *P < 0.05; **P < 0.01.

Shi et al. PNAS | Published online December 19, 2016 | E73 Downloaded by guest on September 29, 2021 expressed at very low levels in DP thymocytes. Our studies have aberrant induction of Runx3, a key factor specifying the CD8SP therefore identified a Gfi1-Foxo1 axis in DP thymocytes that acts T-cell lineage (23), as well as diminished E-protein activity due to Id2 to prevent the premature induction of a gene expression program up-regulation (Fig. S1F). Indeed, deletion of Gfi1 and loss of characteristic of mature T cells. E-protein activity result in overlapping phenotypes, including altered Functionally, the dysregulated gene expression program in Gfi1- CD8/CD4 ratios and up-regulation of mature T-cell genes in im- deficient thymocytes is likely to be a driving force to facilitate DP- mature DP thymocytes (24, 25), and this is consistent with a positive to-SP transition, as evidenced by the following: (i) up-regulation of regulatory loop composed of E-proteins, Gfi1, and Id2 to amplify and mature T-cell genes even in the preselection DP thymocytes, in- −/− perpetuate E-protein activity in DP cells (Fig. S4). However, it should cluding many of them in the Tcra background; (ii) the increases be noted that deficiency of Gfi1 does not impinge upon expression of in the proportions of DP thymocytes positive for TCR, CD69, and DP-specific immature genes (Rorc and Cxcr4)orTCRα expression, β + lo 7 integrin but negative for CD24, as well as CD4 CD8 transi- unlike the pleiotropic effects induced by the loss of E-proteins tional cells; (iii) faster and biased differentiation of DP to CD8SP (E2A and HEB) (4), suggesting that Gfi1 exerts a selective cells in vivo including asymmetric BM chimeras and in the in vitro effect at repressing the mature T-cell gene expression program. OP9-DL1 coculture and FTOC systems; and (iv)enhancedmat- In summary, we have identified complex and unique roles of uration of SP cells indicated by the down-regulation of GFP levels the transcriptional repressor Gfi1 in coupling the thymic gene in the Rag2-GFP system, up-regulation of the maturation marker expression program to thymocyte maturation and development Qa-2, and faster generation of CD4SP cells in the BrdU pulse- chase experiment. These results point to a control mechanism that of CD8SP and Treg cells. Gfi1, mainly by preventing the pre- actively maintains DP identity to prevent the premature induction mature acquisition of a SP-specific gene expression program in of SP-specific genes for a normal thymic development. DP cells, restrains DP-to-SP transition. In the absence of Gfi1, Maturation of DP thymocytes involves the critical decision to aberrant induction of Foxo1 disrupts the gene expression pro- enter the CD4SP or CD8SP lineage, which requires distinct tran- gram and leads to expedited maturation and development of scription factors and extracellular inputs, especially cytokine sig- thymocytes. Importantly, Gfi1 expression is tightly regulated by naling (1, 2). Previous work using the germ-line deletion system intrathymic developmental signals, and the transcriptional cir- revealed that Gfi1 inhibits CD8SP but promotes CD4SP T-cell cuitry composed of Gfi1, Foxo1, and its downstream targets lineage choices, but the mechanisms involved are not well un- shapes the identity of DP thymocytes and generation of SP cells. derstood (9). Although not the focus of our present study, we found that CD8SP T cells were increased in Gfi1CD4 mice, con- Materials and Methods − − − − sistent with a previous report using the class I-restricted HY TCR C57BL/6, CD45.1, Tcra / , and Rag1 / mice were purchased from the Jackson – system (9). However, it is remarkable that developing thymocytes Laboratory. CD4-Cre, hCD2-iCre, Rag2-GFP, Bcl-2-TG, Gfi1fl/fl,andFoxo1fl/ + that would normally be directed to the CD4 lineage, as in the mice were as described (10, 18, 26–28). Animal protocols were approved by OT-II TCR-transgenic system, could instead become CD8SP T the Institutional Animal Care and Use Committee of St. Jude Children’s Re- cells in the absence of Gfi1. Our results are best explained by search Hospital. Additional information about the experimental methods a “kinetic signaling” model (2), in that the accelerated DP transi- may be found in SI Materials and Methods. tion and therefore the shortened duration of TCR signaling in ACKNOWLEDGMENTS. We thank Hanno Hock and Stuart Orkin for Gfi1-targeted Gfi1-deficient DP thymocytes would favor the CD8SP lineage mice, J. C. Zuniga-Pflucker for OP9-DL1 cells, and Maureen McGargill for critical commitment. Mechanistically, the biased development of CD8SP scientific inputs. This work was supported by NIH Grants CA176624, AI101407, vs. CD4SP T cells in Gfi1-deficient thymocytes could result from AI105887, and NS064599 and by the American Asthma Foundation (H.C.).

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