The Lupus Susceptibility Gene Pbx1 Regulates the Balance Between

The Lupus Susceptibility Gene Pbx1 Regulates the Balance between Follicular Helper T Cell and Regulatory T Cell Differentiation This information is current as of September 25, 2021. Seung-Chul Choi, Tarun E. Hutchinson, Anton A. Titov, Howard R. Seay, Shiwu Li, Todd M. Brusko, Byron P. Croker, Shahram Salek-Ardakani and Laurence Morel J Immunol 2016; 197:458-469; Prepublished online 13 June 2016; Downloaded from doi: 10.4049/jimmunol.1502283 http://www.jimmunol.org/content/197/2/458 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2016/06/11/jimmunol.150228 Material 3.DCSupplemental References This article cites 56 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/197/2/458.full#ref-list-1

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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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Lupus Susceptibility Gene Pbx1 Regulates the Balance between Follicular Helper T Cell and Regulatory T Cell Differentiation

Seung-Chul Choi, Tarun E. Hutchinson, Anton A. Titov, Howard R. Seay, Shiwu Li, Todd M. Brusko, Byron P. Croker, Shahram Salek-Ardakani, and Laurence Morel

Pbx1 controls chromatin accessibility to a large number of genes and is entirely conserved between mice and humans. The Pbx1-d dominant-negative isoform is more frequent in CD4+ T cells from lupus patients than from healthy controls. Pbx1-d is associated with the production of autoreactive T cells in mice carrying the Sle1a1 lupus-susceptibility locus. Transgenic (Tg) expression of Pbx1-d in CD4+ T cells reproduced the phenotypes of Sle1a1 mice, with increased inﬂammatory functions of CD4+ T cells and Downloaded from impaired Foxp3+ regulatory T cell (Treg) homeostasis. Pbx1-d–Tg expression also expanded the number of follicular helper T cells

(TFHs) in a cell-intrinsic and Ag-speciﬁc manner, which was enhanced in recall responses and resulted in Th1-biased Abs. More- over, Pbx1-d–Tg CD4+ T cells upregulated the expression of miR-10a, miR-21, and miR-155, which were implicated in Treg and follicular helper T cell homeostasis. Our results suggest that Pbx1-d impacts lupus development by regulating effector T cell + differentiation and promoting TFHs at the expense of Tregs. In addition, our results identify Pbx1 as a novel regulator of CD4 T cell effector function. The Journal of Immunology, 2016, 197: 458–469. http://www.jimmunol.org/

he NZM2410 murine strain was derived from the classical the development of autoreactive B cells and B cell hyperac- (NZB 3 NZW) F1 model of systemic lupus erythematosus tivity (3). T (SLE), which is an autoimmune disease characterized by Sle1 on telomeric chromosome 1 was the locus with the the production of autoantibodies to nuclear Ags, immune activa- strongest linkage to lupus nephritis, and its expression is required tion, and immune complex–mediated inflammation in various tis- for the development of systemic autoimmunity and pathogenesis sues. We mapped three major NZM2410 lupus-susceptibility loci in the NZM2410 model (4, 5). Sle1 expression results in the and generated three congenic strains, B6.NZMSle1, B6.NZMSle2, production of autoantibodies specific for chromatin (6) through by guest on September 25, 2021 and B6.NZMSle3, each carrying the corresponding NZM2410- intrinsic defects in B and CD4+ T cells (7). Three Sle1-indepen- derived genomic interval on the C57BL/6J (B6) genome (1, 2). dent subloci, Sle1a, Sle1b, and Sle1c, contribute to autoimmune Analysis of immunological properties of each congenic strain in phenotypes (8). Sle1a and Sle1c induce the production of activated comparison with B6 showed that Sle1 and Sle3 increase T cell autoreactive T cells and decrease the number and function of activation and effector functions and that Sle1 and Sle2 promote Foxp3+ regulatory T cells (Tregs) (9–11). Sle1b is associated with extensive polymorphisms between two divergent haplotypes of the SLAM family, and it regulates B cell (12) and T cell (13) toler- Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, Gainesville, FL 32610 ance. Sle1a was mapped to two interacting loci: Sle1a1 and Sle1a2 (14). Sle1a1 contains only one functional known gene, Pbx1, and ORCIDs: 0000-0001-6230-9087 (S.-C.C.); 0000-0002-9175-7222 (T.E.H.); 0000- 0003-2751-2340 (A.A.T.); 0000-0001-7002-0187 (H.R.S.); 0000-0003-2878-9296 the lupus-associated allele corresponds to the increased expression (T.M.B.); 0000-0002-2336-8452 (S.S.-A.). of a novel splice isoform, Pbx1-d, in CD4+ T cells (15). Pbx1,a Received for publication October 26, 2015. Accepted for publication May 11, 2016. member of the three-amino-acid-loop-extension class of homeo- This work was supported by National Institutes of Health Grants R01 AI045050 (to domain proteins, is a transcriptional factor that regulates chro- L.M.) and R01 AI087734 (to S.S.-A.). matin access of multimeric complexes that include Hox factors, S.-C.C., T.M.B., S.S.-A., and L.M. designed experiments; S.-C.C., T.E.H., A.A.T., as well as Meis and Prep-1, two other three-amino-acid-loop- H.R.S., S.L., and B.P.C. performed experiments; S.-C.C., T.E.H., A.A.T., and L.M. analyzed data; and S.-C.C., S.S.-A., and L.M. wrote the manuscript. extension proteins that regulate chromatin remodeling and coac- Address correspondence and reprint requests to Dr. Laurence Morel, Department of tivator access (16). The interactions of Pbx-Meis/Prep-1 complexes Pathology, Immunology, and Laboratory Medicine, University of Florida, JHMHC with Hox and non-Hox factors ultimately contribute to gene acti- 275, Box 100275, Gainesville, FL 32610-0275. E-mail address: morel@ufl.edu. vation or repression (17). Pbx1-d lacks exon 6 and exon 7 corre- The online version of this article contains supplemental material. sponding to the DNA-binding domain and the Hox-binding domain, egfp Abbreviations used in this article: ANA, anti-nuclear Ab; B6, C57BL/6J; B6.Foxp , respectively, which confers this splice isoform a dominant-negative B6.Foxp3-enhanced GFP; B6.Ly5a, B6.SJL-PtprcaPep3b/BoyJ; BM, bone marrow; B6.OT-II, B6.Cg-Tg(TcraTcrb)425Cbn/J; B6.Rag-12/2, B6.129S7-Rag1tm1Mom/J; function (18). B6.Sle1a1,B6.Sle1a.1NZW/NZW; GC, germinal center; iTreg, induced Treg; miRNA, Sle1a1 expands the number of activated and autoreactive CD4+ microRNA; mLN, mesenteric lymph node; pTreg, peripheral Treg; RA, retinoic acid; NZM2410/Aeg NZM2410/Aeg T cells and reduces the number of peripheral Tregs (pTregs) in a SLE, systemic lupus erythematosus; TC, B6.NZM-Sle1 Sle2 NZM2410/Aeg + Sle3 /LmoJ; TD, T dependent; TEff, effector T cell; TEM, effector memory CD4 T cell–intrinsic manner (15). However, these T cell phe- T cell; TFH, follicular helper T cell; TFR, follicular regulatory T cell; Tg, transgene/ notypes were not sufficient to induce a robust production of transgenic; T , naive T cell; Treg, Foxp3+ regulatory T cell; tTreg, thymic Treg. N autoantibodies in B6.Sle1a.1NZW/NZW (B6.Sle1a1) mice, which re- Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 quires the expression of Sle1 in B cells (14). In addition, Pbx1-d www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502283 The Journal of Immunology 459 expression is associated with abnormal responses to TGF-b and (104), CD62L (MEL-14), CD69 (H1.2F3), CD95 (Jo2), CD90.1 (HIS51), retinoic acid (RA) in murine and human T cells (19). Pbx1 ex- CD90.2 (53-2.1), Foxp3 (FJK-16s), Ly-77 (GL7), Neuropilin-1 (761705), pression is necessary for B cell development (20), but its function in PD-1 (RMP1-30), IFN-g (XMG1.2), IL-2 (JES6-5H4), IL-17a (eBio17B7), and IL-21 (FFA21). Follicular T cells were stained as previously described T cells is unknown. The goal of this study was to directly address (23) in a three-step process using purified CXCR5 (2G8), followed by the role of Pbx1-d overexpression in CD4+ T cells. We showed that biotinylated anti-rat IgG (Jackson ImmunoResearch) and then PerCP- transgenic (Tg) B6 mice that overexpress Pbx1-d in their CD4+ Cy5.5–labeled streptavidin in FACS staining buffer on ice. Dead cells Tcells(Pbx1-d–Tg mice) reproduce the phenotypes of B6.Sle1a1 were excluded with fixable viability dye (eFluor 780; eBioscience). Data + were collected on an LSRFortessa (BD Biosciences) and analyzed with mice, with increased inflammatory functions of CD4 T cells and FlowJo software (TreeStar). impaired Treg homeostasis. In addition, Pbx1-d–Tg mice possessed Mixed bone marrow chimera and adoptive cell transfers a follicular helper T cell (TFH) population that expanded in an Ag- specific and T cell–intrinsic manner, with an enhanced capacity to Chimeras were prepared as previously described (9), with the following locate in B cell follicles and to promote affinity maturation of Th1- modifications. Eight- to ten-week-old (B6 3 B6.Ly5a)F1 recipient mice associated Ab isotypes. These results suggest that Pbx1 regulates were lethally irradiated with two doses of 452 rad (4 h apart) using an the balance between pTreg and T maintenance or differentiation X-RAD 320 irradiator (Precision X-Ray). Donor bone marrow (BM) cells FH were depleted of mature T cells using CD5 MicroBeads (Miltenyi Biotec). and that Pbx1-d contributes to autoimmunity by tilting the balance BM cells (1 3 107 cells) mixed 1:1 between the two donors were given to in favor of TFHs over Tregs. the recipient mice by tail vein injection. Tregs were analyzed in chimeric mice 8 wk later. For T-dependent (TD) immune responses, chimeric mice Materials and Methods were immunized with 100 mgNP23-KLH (Biosearch Technologies) in alum 8 wk after BM cell transplantation, and TFHs were analyzed 1 wk Mice after immunization. CD4+ T cells from Pbx1-d–Tg.OT-II, B6.Sle1a1.OT- Downloaded from II, and B6.OT-II mice were purified by negative selection using MACS Pbx1-d–Tg mice were generated at the University of Florida transgenic MicroBeads, and 0.5 3 106 cells were injected into the tail vein of B6.Ly5a core using a bicistronic Pbx1-d/GFP cDNA controlled by the CD4 pro- mice, followed by s.c. immunization with 50 mgNP -OVA in alum 4 h moter (Supplemental Fig. 1A) on a B6 background. Four Tg lines were 16 after cell transfer. For mixed adoptive transfer, CD4+ T cells from Pbx1-d– obtained, with a Tg copy number of 8–13 in the A886, C855, and A872 Tg.OT-II or B6.Sle1a1.OT-II and B6.Thy1a.OT-II mice were mixed at a 1:1 lines, and a Tg copy number of 30 in the C861 line (Supplemental Fig. ratio, and 1 3 106 cells were injected into (B6 3 B6.Thy1a)F1 recipient 1B). GFP expression was not achieved in any of the lines. The following mice prior to immunization with NP16-OVA. http://www.jimmunol.org/ primers were used to detect Pbx1-d Tg: forward (spanning exons 5–8): 5 To assess the function of effector T cells (TEffs) in vivo, 4 3 10 purified 59-ATCACAGTCTCCCAGGTGGA-39, and reverse (in exon 9): 59- + 2 CD4 CD25 TEffs from 2-mo-old Pbx1-d–Tg and B6 mice were injected ATCCTGCCAACCTCCATTAG-39. Pbx1-d expression was restricted to 2 2 into the tail vein of B6.Rag-1 / mice. The function of Tregs was eval- CD4+ T cells (Supplemental Fig. 1B, 1C). Primer and TaqMan probe se- uated in coinjections of 1 3 105 CD4+CD25+ Tregs from Pbx1-d–Tg or B6 quences used to measure Pbx1-d message expression were described (21). 2 mice along with 4 3 105 CD4+CD25 B6 T . Recipient mice were Mice from all four lines were healthy at least up to 1 y of age. The results Effs monitored for clinical signs of colitis for up to 8 wk, and body weight was reported in this article were obtained using mice from the first three lines, monitored weekly. The recipient mice that lost .15% of body weight or without any difference observed among them. C861 mice were not in- showed overt clinical signs of disease were sacrificed. Colon histology was cluded because of poor breeding performance. Initial characterization was ranked blindly in a semiquantitative fashion, as previously described (9). performed with Tg-negative littermates, which presented phenotypes identical to those of B6 controls. No difference was observed between Immunohistochemistry by guest on September 25, 2021 hemizygous and homozygous lines, indicating that, within the observed range, the Tg copy number was not critical for the phenotypes. The results To stain splenic germinal centers (GCs) in B6.Ly5a mice that received reported in this article were obtained with homozygous mice. B6, B6.SJL- Pbx1-d–Tg.OT-II or B6.OT-II CD4+ T cells, 7-mm-thick OCT-embedded PtprcaPep3b/BoyJ (B6.Ly5a), B6.Cg-Tg(TcraTcrb)425Cbn/J (B6.OT-II), cryosections were prepared on Superfrost slides, washed with PBS, fixed 2 2 and B6.129S7-Rag1tm1Mom/J (B6.Rag-1 / ) mice were originally obtained with 4% paraformaldehyde for 15 min at 4˚C, and subsequently per- from The Jackson Laboratory. B6.Thy1a.OT-II mice were graciously pro- meabilized with 0.1% Triton X-100 for 5 min at 4˚C. The sections were vided by Dr. Stephen Schoenberger (La Jolla Institute for Allergy and washed with cold PBS and incubated overnight in the dark at 4˚C with Immunology). The B6.Sle1a1 and B6.NZM-Sle1NZM2410/AegSle2NZM2410/Aeg anti-CD45.2 conjugated to PE, anti-GL7 conjugated to Alexa Fluor 488, Sle3NZM2410/Aeg/LmoJ (TC) congenic mice were described previously (5, 14). and anti-IgD conjugated to allophycocyanin (all from eBioscience). Sec- B6.Foxp3-enhanced GFP (B6.Foxp egfp) mice (22) were kindly provided by tions were washed three times with cold PBS, mounted with Cytoseal, and Dr. Vijay Kuchroo (Harvard Medical School). Pbx1-d–Tg.Foxp3 egfp mice covered with a glass coverslip. The stained sections were analyzed using were bred from the A886 line, and Pbx1-d–Tg.OT-II mice were bred from an EVOS FL digital inverted fluorescence microscope (Fisher Scientific, the C855 line. All mice were bred and maintained at the University of Waltham, MA); images were captured using 103,203, and 403 objec- Florida in specific pathogen–free conditions. With the exception of the tives, keeping all microscope conditions and software settings identical for colitis experiment, only female mice were used in this study under a pro- all treatments and controls. tocol approved by the Institutional Animal Care and Use Committee of the University of Florida. Immunization and Ab measurements T cell polarization For TD responses, 8- to 10-wk-old mice were immunized i.p. with 100 mg NP23-KLH in alum and boosted at weeks 2 and 6. Serum samples were In vitro–induced Tregs (iTregs) were differentiated as previously de- collected 1, 3, 5, and 7 wk after the first immunization. NP-specific Abs + 2 scribed (15). Briefly, CD4 CD25 cells were negatively selected from were measured by ELISA using plates coated with NP4-BSA or NP25-BSA B6 or Pbx1-d–Tg splenocytes using the CD4+CD25+ Regulatory T Cell (Biosearch Technologies), followed by incubation with 1:1000 diluted Isolation Kit (Miltenyi Biotec); 5 3 105 cells were stimulated with mouse T- serum samples and developed with alkaline phosphatase–conjugated goat activator CD3/CD28 beads (Life Technologies) at a concentration of 1 3 106 anti-mouse IgG1, IgG2a, IgG2c, IgG3, or IgM (Southern Biotech). All beads/ml in the presence of 100 U IL-2, 20 ng/ml TGF-b, (PeproTech), and samples were run in duplicate. Anti-dsDNA and anti-chromatin IgG were 0.5 nM RA for 5 d. Th1 and Th17 polarization was performed as previously measured, as previously described (6), in sera diluted 1:100, and relative described (23). units were standardized using serial dilutions of a positive serum from TC mice, setting the 1:100 dilution reactivity to 100 U. Serum anti-nuclear Flow cytometry Abs (ANAs) were measured by applying 1:40 diluted sera to fixed Hep-2 cells (Inova) and revealed with anti-mouse IgG-FITC (Invitrogen). Single-cell suspensions were prepared using standard procedures from spleen, thymus, and mesenteric lymph node (mLN). After RBC lysis, cells MicroRNA analysis were blocked with anti-CD16/32 Ab (2.4G2) and stained in FACS staining buffer (2.5% FBS, 0.05% sodium azide in PBS). Fluorochrome-conjugated Splenic CD4+ T cells from Pbx1-d–Tg.Foxp3egfp,B6.Sle1a1.Foxp3egfp,and Abs, purchased from BD Biosciences, eBioscience, BioLegend, and R&D B6.Foxp3egfp mice were enriched by negative selection using MACS MicroBe- Systems, were to B220 (RA3-6B2), BCL6 (K112-91), CD4 (RAM4-5), ads and then GFP+ and GFP2 cells were sorted with a FACSAria cytometer with CD8 (53-6.7), CD25 (PC61.5), CD44 (IM7), CD45.1 (A20), CD45.2 a purity $ 95%. MicroRNAs (miRNAs) were isolated and reverse transcribed 460 Pbx1 REGULATES TFH VERSUS Treg DIFFERENTIATION using Life Technologies reagents. Quantification of miRNA expression was frequency of CD4+Foxp3+ Tregs was reduced in Pbx1-d–Tg mice, performed using TaqMan MicroRNA assays (Life Technologies) and the Applied with the largest difference in the mLN (Fig. 2C). In addition, Biosystems StepOne real-time PCR machine. U6 small nuclear RNA was used as 2DDCt in vitro iTreg polarization by TGF-b and RA was reduced in Pbx1- internal control. Comparisons were made using the 2 method, and values + 2 were normalized to the B6 average values. d–Tg CD4 CD25 T cells compared with B6 cells (Fig. 2D). These results indicate that, as found in B6.Sle1a1 mice, Pbx1-d Luciferase assay overexpression in CD4+ T cells impairs iTreg differentiation by Putative human PBX1 binding sites in the ∼2 kb flanking miR-10a, miR-21, interfering with the response to RA in the presence of TGF-b. and miR-155 precursors were identified using Jaspar3 (set for 75% accu- To further evaluate the impact of Pbx1-d on Treg differentiation, racy). We generated luciferase reporter constructs containing the 798-bp, we reconstituted lethally irradiated (B6 3 B6.Ly5a)F1 mice with 1071-bp, and 850-bp regions upstream of miR-10a, miR-21, and miR-155, a respectively (Fig. 8A), cloned into the pGL4.25 Luciferase vector (Promega). T cell–depleted BM cells mixed from Pbx1-d–Tg and B6.Ly5 PBX1-b or PBX1-d expression plasmids were generated by inserting cDNAs mice (Fig. 2E). Pbx1-d–Tg BM yielded a decreased percentage of 2 (15) into the pHAGE-CMV-MCS-IzsGreen plasmid. The luciferase reporter CD4+Foxp3+Nrp-1 pTregs compared with B6.Ly5a BM in the constructs and the pGL3-Basic plasmid were each cotransfected with PBX1-b thymus of the recipient mice, whereas the proportion of CD4+ or PBX1-d expression plasmid into HEK293T cells. Cells were harvested Foxp3+Nrp-1+ thymic Tregs (tTregs) was similar between the two after 48 h. After lysis with Passive Lysis Buffer (Promega), luciferase activity was measured using a dual-luciferase reporter assay system (Prom- genotypes (Fig. 2F–H). However, in these conditions, the pro- ega), according to the manufacturer’s instructions, with a Lumat LB 9507 portions of Tregs in the peripheral organs of recipient mice were luminometer (Berthold Technologies). similar for BM-derived cells of either genotype (data not shown), Statistical analysis possibly as a result of the relatively short time between BM transfer and phenotype readout. Together, these data indicate that Downloaded from Differences between groups were evaluated by two-tailed statistics: unpaired Pbx1-d Tg overexpression in CD4+ T cells impairs the induction t tests or Mann–Whitney U tests depending on whether the data were nor- mally distributed, paired t tests for mixed BM chimeras, x2 tests to compare or maintenance of pTregs in a cell-intrinsic manner. distributions, and two-way ANOVA tests for time-course experiments. Un- + less specified, graphs show mean and standard deviations of the mean. Pbx1-d Tg overexpression in CD4 T cells expanded the number of TFHs + Results Because Pbx1-d–overexpressing CD4 T cells showed an acti- http://www.jimmunol.org/ Pbx1-d–Tg mice replicated the phenotypes of B6.Sle1a1 mice vated phenotype (Fig. 1D), we next examined their cytokine + To address whether the overexpression of Pbx1-d in CD4+ T cells production. Ex vivo Pbx1-d–Tg CD4 T cells showed a signifi- was sufficient to induce their activation and loss of tolerance, we cantly increased IL-21 expression (Supplemental Fig. 2A, 2B) generated Pbx1-d–Tg mice that express Pbx1-d driven by the Cd4 compared with B6 mice, but there was no difference in the pro- promoter (Supplemental Fig. 1). Pbx1-d Tg overexpression in duction of IL-2, IL-17A, or IFN-g (Supplemental Fig. 2A, 2C–E) + + + CD4+ T cells resulted in the production of serum ANAs with the or the frequency of IL-17A or IFN-g CD4 T cells (data not characteristic homogenous nuclear staining pattern (Fig. 1A), as shown). Under in vitro Th1-polarization conditions, a small, but + well as anti-dsDNA and anti-chromatin IgG in 7–12-mo-old mice significant, difference was observed, with Pbx1-d–Tg CD4 T (Fig. 1B, 1C). A similar nuclear staining pattern was observed in cells producing more IFN-g than B6 cells, whereas there was no by guest on September 25, 2021 the serum of B6.Sle1a1 mice, although it had a lower intensity, difference for IL-17A (Supplemental Fig. 2F, 2G). These results which is consistent with a low level of anti-dsDNA and anti- prompted us to investigate TFH differentiation in Pbx1-d–Tg mice. chromatin IgG (Fig. 1B, 1C), as we reported previously for this TFHs secrete high levels of IL-21, and IL-21 signaling upregulates strain (14, 15). This suggested that Pbx1-d overexpression results Bcl6 expression, which is required for TFH development and GC in the production of autoreactive CD4+ T cells that are sufficient to formation (24–27). At steady-state, young (6–8 wk) Pbx1-d–Tg induce humoral autoimmunity against nuclear Ags. B6.Sle1a1 and B6.Sle1a1 mice showed an increased cellularity in the mLN, mice were characterized by the expansion of CD44+CD62L2 ef- where the numbers and percentages of GC B cells were also in- 2 + creased compared with B6 mice (Fig. 3A–C). The percentages and fector memory T cells (TEMs) relative to CD44 CD62L naive absolute numbers of CD4+PD1+CXCR5+Bcl6+Foxp32 T were T cells (TNs) (15). Aged Pbx1-d–Tg mice also presented a skewed FHs significantly higher in the mLN, but not in the spleen, from Pbx1- TN/TEM ratio (Fig. 1D). We examined the consequence of Pbx1-d + 2 d–Tgmice(Fig.3D–F).Asimilartrendwasobservedfor overexpression in CD4 CD25 TEffs in vivo with the experimental 2/2 + + + colitis model that we used with B6.Sle1a1 T cells (9). B6.Rag1 B6.Sle1a1 TFHs. In contrast, the number of CD4 PD1 CXCR5 Bcl6+Foxp3+ follicular regulatory T cells (T ) was significantly mice that received Pbx1-d–Tg TEffs showed greater body weight FRs reduced in the spleen of Pbx1-d–Tg mice (Fig. 3F). We next tested loss and more severe colitis than the mice that received B6 TEffs (Fig. 1E, 1F). These results suggest that Pbx1-d expression is the differentiation of TFHs and TFRs 7 d after administering a TD sufficient to induce an intrinsic activation in CD4+ T cells. Ag to chimeric mice reconstituted with a 1:1 mix of Pbx1-d–Tg However, we observed a similar ability of Pbx1-d–Tg or B6 CD4+ and B6 BM (Fig. 3G). The number and percentage of Pbx1-d + Tg-derived T and T were increased significantly compared CD25 T cells to suppress B6 TEffs in the same colitis model (data FHs FRs not shown), suggesting that the in vivo functions of Pbx1-d–Tg with B6-derived cells (Fig. 3H, 3I). The discrepancy between the Tregs were less affected than those of B6.Sle1a1 Tregs (14). unchanged frequency or fewer Pbx1-d–Tg TFRs in unmanipulated mice (Fig. 3E, 3F) on one hand and the increased number and Pbx1-d overexpression impaired Treg homeostasis frequency of Pbx1-d Tg-derived TFRs in chimeric mice (Fig. 3H, To assess the role of Pbx1-d in Tregs compared with conventional 3I) on the other hand is likely due to homeostatic expansion that CD4+ Tcells,webredtheEGFP-Foxp3 reporter construct to favors all Bcl6-expressing Pbx1-d Tg-derived T cells. The num- B6.Sle1a1 and Pbx1-d–Tg mice. Pbx1-d expression in Pbx1-d–Tg bers of B6 and Pbx1-d Tg-derived total CD4+ T cells were similar CD4+ T cells was ∼10,000-fold higher than in B6 T cells and was before immunization, indicating an Ag-driven enhancement of + 2 similar in Foxp3 and Foxp3 cells (Fig. 2A, 2B). Pbx1-d ex- TFH differentiation by Pbx1-d overexpression. These steady-state pression in Pbx1-d–Tg T cells was also much higher (∼100-fold) and immunization results are consistent with Pbx1-d over- + 2 + than in B6.Sle1a1 mice in Foxp3 and Foxp3 T cells (Fig. 2A, expression in CD4 T cells increasing TFH differentiation in a cell- 2B). As previously reported for B6.Sle1a1 mice (14, 15), the intrinsic manner. The Journal of Immunology 461 Downloaded from FIGURE 1. Pbx1-d overexpression in CD4+ T cells reproduced the phenotypes of B6.Sle1a1 mice. (A) Representative serum ANA staining patterns in B6, Pbx1-d–Tg2 (Tg2), B6.Sle1, B6.Sle1a1, Pbx1-d–Tg (Tg+), and TC mice as positive control (original magnification 320). The pattern distribution (nuclear, cytoplasmic, or negative) was significantly different between Pbx1-d–Tg+ and B6.Sle1a1 and (B6 + Tg2) combined mice (n = 10–18 per strain).

Serum anti-dsDNA (B) and anti-chromatin (C) IgG (n = 10–15). (D)TN/TEM ratio in the spleen and mLN from B6 and Pbx1-d–Tg mice (n = 3–4). Seven- to twelve–month-old mice are shown in (A)–(D). (E) Maximum weekly percentage of body weight loss by B6.Rag12/2 mice up to 8 wk after transfer of Pbx1- d–Tg or B6 TEffs.(F) Colitis pathology score 8 wk after transfer (left panel). Representative colon histology images (H&E stain, original magniﬁcation

340) (right panels). Each point in (B), (C), (E), and (F) represents an individual mouse. *p , 0.05, **p , 0.01. http://www.jimmunol.org/

+ Pbx1-d Tg overexpression enhanced Ag-specific TFH Pbx1-d–Tg.OT-II and B6.OT-II total CD4 T cells were expanded differentiation and follicular localization to a similar extent, and there was no difference for the recipient CD45.1+ T cells (Fig. 4A). However, CD45.2+ Pbx1-d–Tg.OT-II To track TFHs in an Ag-specific manner, Pbx1-d–Tg mice were + bred with B6.OT-II mice, which express an MHC class II–re- CD4 T cells contained a higher frequency and number of TFHs + stricted TCR specific for OVA (28). Pbx1-d–Tg.OT-II or B6.OT-II (Fig. 4B, 4C) than did the B6.OT-II CD4 T cells. Pbx1-d–Tg.OT- CD4+ T cells were transferred into B6.Ly5a mice that were sub- II or B6.OT-II transferred CD4+ T cells produced very low

sequently immunized with NP-OVA. By day 5 after immunization, numbers of TFRs (Fig.4A).HomingofprimedTFHs to B cell by guest on September 25, 2021

FIGURE 2. Pbx1-d overexpression impaired pTreg induction. Pbx1-d mRNA expression in Foxp3+ and Foxp32 CD4+ T cells puriﬁed from B6.Foxp3egfp, Pbx1-d–Tg.Foxp3egfp, and B6.Sle1a1.Foxp3egfp mice analyzed by conventional (A) and quantitative (B) RT-PCR, with the data presented relative to B6 (RQ; n = 3). (C) Frequency of Foxp3+ Tregs in the mLN of 10–12-mo-old B6, Pbx1-d–Tg, and B6.Sle1a1 mice. Each point represents an individual mouse. (D) Foxp3 induction in CD4+CD252 T cells in the presence of TGF-b, presented as the ratio of induction with or without RA. (E–H) Mixed BM chimera analysis of Tregs. (E) Experimental design. (F) Gates for Foxp3+Nrp-1+ tTregs and Foxp3+Nrp-12 pTregs within total CD4+CD82 CD45.1+ or CD45.2+ thymocytes. Percentage of pTregs (G) and tTregs (H) in the chimeras; each linked pair of points represents an individual mouse. *p , 0.05, **p , 0.01. 462 Pbx1 REGULATES TFH VERSUS Treg DIFFERENTIATION Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 3. Pbx1-d Tg expression in CD4+ T cells expanded GC lymphocytes. Absolute numbers of lymphocytes (A) and percentages (B) and absolute numbers (C) of B220+GL-7+FAS+ GC B cells in the spleen and mLN from 2 mo-old B6, Pbx1-d–Tg, and B6.Sle1a1 mice. (D) Representative FACS plots + + + + + + + + + 2 for CD4 CXCR5 PD-1 Bcl6 Foxp3 TFRs and CD4 CXCR5 PD-1 Bcl6 Foxp3 TFHs. Percentages (E) and absolute numbers (F)ofTFHs and TFRs (n =5– 10). (G–I) Follicular T cell expansion in response to NP-KLH immunization in mixed BM chimeras. (G) Experimental strategy. Absolute numbers (H) and percentages (I)ofTFHs and TFRs 7 d after immunization, gated according to their strain of origin (CD45.1, B6; CD45.2, Pbx1-d Tg). Each linked pair of points in (I) represents an individual mouse. *p , 0.05, **p , 0.01. follicles is a critical step to generate GCs. The number of trans- 507.20 6 23.21 versus 436.60 6 13.86, p , 0.05). At day 7 after ferred Pbx1-d–Tg.OT-II CD4+ T cells within the B cell zone was immunization, there was a similar frequency of Pbx1-d–Tg.OT-II + signiﬁcantly higher than the number of B6.OT-II CD4 T cells, and B6.OT-II TFHs (Supplemental Fig. 3A) and a similar number whereas there was no difference in the number of transferred of these cells in the spleen (Supplemental Fig. 3B), although the T cells in the T cell zone (Fig. 4D, 4E). In addition, CXCR5 and number of Pbx1-d–Tg.OT-II TFHs was still higher in the mLN Bcl6 expression was signiﬁcantly higher in the transferred Pbx1- (Supplemental Fig. 3B). Interestingly, the number of total Pbx1-d– d–Tg.OT-II T cells than in the transferred B6.OT-II CD4+ T cells Tg.OT-II T cells was increased in both spleen and mLN (CXCR5: 483 6 18.14 versus 430.20 6 10.14, p , 0.001; Bcl6: (Supplemental Fig. 3C). This expanded population was largely The Journal of Immunology 463 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

+ FIGURE 4. Pbx1-d Tg expression in CD4 T cells regulated Ag-specific TFH differentiation. TFHs were analyzed 5 d after NP-OVA immunization of CD45.1+ B6 mice transferred with CD45.2+ naive B6.OT-II or Pbx1-d–Tg.OT-II cells. (A) Representative flow cytometric plots showing mLN CD4+ + + + + CD45.1 (endogenous) and CD45.2 (transferred) gated CXCR5 PD-1 TFHs and TFRs.(B) Percentages of donor and recipient TFHs.(C) Absolute numbers + + of donor TFHs. Each individual point represents a recipient mouse. (D) Localization of donor OT-II CD4 T cells (CD45.2 , red) relative to B cell follicles (IgD+, F), GCs (GL7+, green), and periarteriolar lymphoid sheaths (PALS). The boxed areas (top panels) are shown at higher magnification (middle and bottom panels). For a better comparison of the number and location of CD45.2+ T cells, only the outline of IgG+ B cell follicles are shown (bottom panels). (E) Numbers of B6.OT-II or Pbx1-d–Tg.OT-II CD4+ T cells in B or T cell zones. Data are mean 6 SEM from three to seven GC regions per mouse from + + three mice per strain. (F) Percentages of donor and recipient TFHs 5 d after NP-OVA immunization of CD45.1 B6 mice transferred with CD45.2 naive B6.OT-II or Sle1a1.OT-II cells. (G) Representative spleen sections of B6.OT-II or Sle1a1.OT-II cell recipients, as in (D). (H) Numbers of B6.OT-II or Sle1a1.OT-II CD4+ T cells in B or T cell zones. Data are mean 6 SEM from three to seven GC regions per mouse from five mice per strain. *p , 0.05, **p , 0.01, ***p , 0.001. 464 Pbx1 REGULATES TFH VERSUS Treg DIFFERENTIATION

absent from the GC (Supplemental Fig. 3D), suggesting that they We then examined the effect of Pbx1-d overexpressiononTFH may correspond to extrafollicular Th cells, a population that is differentiation in a competitive setting by cotransferring B6.Thy1a. expanded in lupus-prone mice (29). OT-II and Pbx1-d–Tg.OT-II (1:1) CD4+ T cells into (B6 3 We performed the same experiment with B6.Sle1a1.OT-II B6.Thy1a)F1 recipient mice. Five days after primary NP-OVA CD4+ T cells, which generated a similar frequency and num- immunization (Fig. 5A), the percentage of total B6.Thy1a.OT-II + + + berofTFHs, as well as a similar number of CD4 T cells in the CD4 T cells was higher than that of Pbx1-d.OT-II–Tg CD4 B cell follicles than B6.OT-II CD4+ T cells 5 d after immu- T cells in the spleen and mLN, and the same trend was observed nization (Fig. 4F, 4G). At day 7 after immunization, the for Sle1a1.OT-II CD4+ T cells (Fig. 5B). A higher percentage of number and frequency of TFHs were also similar between cells of TFHs was only found with Pbx1-d Tg origin in the mLN (Fig. 5C). Sle1a1 or B6 origin (Supplemental Fig. 3E, 3F). However, the total We next asked whether Pbx1-d overexpression influenced the number of Sle1a1.OT-II CD4+ T cells was higher than the total recall Ag response in the same model (Fig. 5A). In contrast to number of B6.OT-II T cells in the spleen, and there was a similar the primary response, the percentages of Pbx1-d.OT-II–Tg and + trend in the mLN (Supplemental Fig. 3G). Therefore, these results Sle1a1.OT-II total CD4 T cells (Fig. 5D) and TFHs (Fig. 5E) were suggest that a high level of Pbx1-d expression in CD4+ T cells higher than those of corresponding cells of B6 origin in the spleen increased Ag-specific TFH differentiation, as well as early entry and mLN at 3 d after secondary immunization. Moreover, the in B cell follicles, whereas the more modest increased expression relative expansion of the TFH subset was significantly greater in of Pbx1-d in Sle1a1 CD4+ T cells increased their Ag-specific cells from Pbx1-d Tg or Sle1a1 origin compared with cells of B6 expansion. origin in the recall compared with the primary response (Fig. 5F). Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 5. Pbx1-d intrinsically regulated TFH differentiation and ampliﬁed recall responses. (A) Experimental design for primary and recall response after mixed adoptive transfer of CD90.1+ B6.OT-II and CD90.2+ Pbx1-d–Tg.OT-II or Sle1a1.OT-II CD4+ T cells into (B6 3 B6.Thy1a)F1 mice. Per- + + + centages of CD90.1 B6.OT-II and CD90.2 Pbx1-d–Tg.OT-II or Sle1a1.OT-II CD4 T cells (B and D) and TFHs (C and E) from each genotype in the + + primary (B and C) and recall (D and E) responses. (F) Fold increase in TFHs in recall versus primary response, calculated as the ratio of CD90.2 /CD90.1 + + + TFHs. Representative ﬂow cytometric plots and corresponding percentages of CD4 CD69 activated T cells (G and H) and TEM/TN CD4 T cell ratios (I and J) in CD90.1+ B6.OT-II and CD90.2+ Pbx1-d.OT-II–Tg donor cells in the recall response. Each linked pair of points in (H) and (J) represents a recipient mouse. n = 5–6. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 465

Finally, the percentage of Pbx1-d–Tg–activated T cells (Fig. 5G, B6.Sle1a1.Foxp3egfp, and B6.Foxp3egfp mice. miR-10a, miR-21, 5H) and CD44hi memory CD4+ T cells (Fig. 5I, 5J) was enhanced, and miR-155 expression was 2–3-fold higher in Pbx1-d–Tg or which we did not observe during the primary response. Taken B6.Sle1a1 Tregs than in B6.Foxp3-GFP+ Tregs (Fig. 7A). The together, these results indicate that Pbx1-d overexpression con- same result was obtained for Pbx1-d–Tg Foxp3-GFP2 conven- ferred a cell-intrinsic competitive advantage during Ag-specific tional CD4+ T cells, whereas expression of miR-21 and miR-155 2 + TFH differentiation that was enhanced during recall responses. was more variable in B6.Sle1a1 Foxp3-GFP CD4 T cells, although with differences going in the same direction (Fig. 7B). The Pbx1-d–Tg mice responded to TD Ags with increased expression of miRNAs from the miR-17–92 and miR-181 families Th1-related Ig isotypes was significantly increased in Foxp3-GFP+ Tregs from B6.Sle1a1 To assess the effect of Pbx1-d overexpression on affinity matu- mice but not in Tregs from Pbx1-d–Tg mice, and there was no ration and class switching, B6 and Pbx1-d–Tg mice were boosted consistent difference in Foxp3-GFP2 conventional T cells be- 2 and 6 wk after primary immunization with NP-KLH. Pbx1-d–Tg tween strains (Supplemental Table I). These findings indicate that mice produced significantly more high- and low-affinity anti–NP- Pbx1-d affects the expression of specific miRNAs that are asso- + IgG3 and high-affinity anti-NP IgG2a and IgG2c than did B6 mice ciated with CD4 Treg and TFH differentiation and function, either (Fig. 6A, 6B). Moreover, the affinity of anti-NP IgG2a and IgG2c, directly or indirectly. as measured by the NP4/NP25 ratio, was significantly increased To address whether the increased expression of miR-10a, miR-21, in Pbx1-d–Tg mice after boosting (Fig. 6C), suggesting that and miR-155 in Pbx1-d–Tg and B6.Sle1a1 CD4+ T cells was Pbx1-d overexpression increases affinity maturation of Th1- maintained in mice with clinical lupus phenotypes, we analyzed TC associated Abs. mice, which carry the Sle1a1 allele of Pbx1 (5). TC mice present Downloaded from spontaneously expanded subsets of TFHs (Fig. 7C, 7D). miR-10a, Pbx1-d overexpression increased the expression of miR-10, + + miR-21, and miR-155 were expressed at higher levels in TC CD4 miR-21, and miR-155 in CD4 T cells T cells starting at 2 mo of age, before the mice produce autoanti- Numerous studies implicated miRNAs in Treg and TFH differen- bodies and show overt signs of autoimmune activation (Fig. 7E). tiation and function (30). We analyzed the effect of Pbx1-d over- These results suggest that the upregulation of these three miRNAs

expression on candidate miRNAs selected for their role in this driven by Pbx1-d overexpression in Tg mice contributes to the http://www.jimmunol.org/ process (miR-10a, miR-17, miR-19a, miR-19b, miR-20a, miR-21, autoimmune phenotype in lupus mice that express the Pbx1-d allele. miR-92, miR-155, miR-181a, and miR-181b) in Foxp3-GFP+ and miR-21 expression is regulated by the PBX1/HOX9 complex in Foxp3-GFP2 CD4+ T cells from young Pbx1-d–Tg.Foxp3egfp, leukemia (31), suggesting that Pbx1 could directly regulate the by guest on September 25, 2021

FIGURE 6. Pbx1-d-Tg expression in CD4+ T cells increased afﬁnity maturation of Th1-related isotypes. B6 and Pbx1-d–Tg mice were immunized with NP-KLH and boosted 2 and 6 wk later (arrows on x-axes). Serum levels of NP-speciﬁc Abs 1 wk before and after boosting measured in plates coated with

NP4 (A)orNP25 (B) BSA. (C) Affinity measured as the NP4/NP25 ratio for each isotype. n =5. 466 Pbx1 REGULATES TFH VERSUS Treg DIFFERENTIATION transcription of miR-10a, miR-21, and miR-155. An in silico the challenge, two possibilities that we will explore in future analysis identified several putative PBX1 binding sites for each of experiments. the three miRNAs that are overexpressed in Pbx1-d–Tg T cells. Interestingly, we found that the imbalance between Tregs and We cotransfected HEK293T cells with one of three luciferase TFHs was enhanced in the mLN. We previously associated Pbx1-d constructs containing the upstream regulatory region for miR-10a, expression with an impaired response to RA [(15) and this study], miR-21, or miR-155 that contained the most proximal putative and the major effect of RA in vivo occurs in GALT. Therefore, it is PBX1 binding sites (Fig. 8A) and plasmids expressing either the possible that the anatomy of TFH expansion in Pbx1-d–Tg mice normal allele PBX1-b or the lupus allele PBX1-d cDNA. As reflects the relative response to RA and commensal microbiota, an shown in Fig. 8B, PBX1-b and PBX1-d increased the transcription issue that will be explored in future studies. Pbx1-d led to TFH of each of the three miRNAs, with higher levels obtained for expansion despite a relatively expanded TFR subset, which is PBX1-d than for PBX1-b for miR-10a and miR-155. These data reminiscent of a recent study of mice with a specific deletion of + strongly support transcriptional regulation of miR-10a, miR-21, PTEN in Foxp3 T cells, in which TFH expansion occurred as a and miR-155 by PBX1. result of impaired Treg functions but in the presence of unchanged or expanded TFR numbers (41). In addition to Pbx1-d expression Discussion level, other factors may contribute to the differences between This study examined the role of Pbx1-d expression in CD4+ T B6.Sle1a1 and Pbx1-d–Tg mice. B6.Sle1a1 mice express Pbx1-d cells relative to the phenotypes induced by the Sle1a1 lupus- in other cell types, such as mesenchymal stem cells, leading to susceptibility locus. Sle1a1 increases CD4+ T cell effector func- an increased production of proinflammatory cytokines (21). Pbx1 tions and reduces the number and function of Tregs, resulting in also regulates the production of inflammatory cytokines in mac- Downloaded from the production of autoreactive CD4+ T cells (9, 11). Pbx1- rophages in response to apoptotic cells (42). Although we showed d overexpression in the CD4+ T cells of B6 mice resulted in au- that the Sle1a1 T cell phenotypes are cell intrinsic (15), we cannot toantibody production and T cell activation, as well as fewer rule out that Pbx1-d expression in other cell types may have ad- pTregs and reduced iTreg differentiation in vitro. These results are ditional modulatory effects on CD4+ T cell differentiation. fully consistent with Pbx1-d overexpression being responsible for MicroRNAs are essential mediators of Th cell plasticity, in- + Sle1a1 CD4 T cell activation and impaired iTreg differentiation cluding the balance between Tregs and TFHs (30). Numerous http://www.jimmunol.org/ or maintenance (9). However, the most striking phenotype of studies documented different miRNA profiles in human and mu- Pbx1-d–Tg mice was the expansion of TFHs and associated TD rine SLE (43–45), and causal relationships between over- immune responses. BM chimera and adoptive transfer studies expression of either miR-21 or miR-155 and lupus were showed that Pbx1-d overexpression resulted in a cell-intrinsic Ag- established (46, 47). We showed that increased expression of miR- + specific expansion of TFHs. This phenotype was accentuated in 10a, miR-21, and miR-155 occurs in CD4 T cells before TC mice recall responses, which represent the chronic stimulation by exhibit autoimmune manifestations. Importantly, increased Pbx1- autoantigens better than primary responses. This suggests that d expression in the B6.Sle1a1 congenic and Pbx1-d–Tg CD4+ Pbx1-d expression contributes to lupus by promoting TFHs at the T cells showed the same pattern of miRNA expression, impli- expense of iTreg differentiation, resulting in the production of cating Pbx1-d as a primary driver regulating the intrinsic ex- by guest on September 25, 2021 class-switched affinity matured autoantibodies that are the hall- pression of these three miRNAs in T cells, either directly or mark of the Sle1 susceptibility locus and lupus pathogenesis. indirectly. The existence of PBX1 binding sites in the distal pro- SLE patients present high levels of circulating TFH-like cells that moter of each of these three miRNAs that are sufficient to increase correlate with disease activity (32, 33), and functional TFHs were transcription in the presence of Pbx1 supports this hypothesis. If found in the kidneys of patients with lupus nephritis (34). TFHs are Pbx1-d functions as a dominant-negative transcription factor, it the limiting factor for GC size and function, and there is abundant suggests that Pbx1-b is a negative regulator of these three miR- evidence from several mouse models that unrestricted TFH ex- NAs, especially miR-10a and miR-155. However, Pbx1 can bind pansion leads to systemic autoimmunity (35, 36). Multiple studies to DNA indirectly through numerous cofactors (48, 49). Detailed also found an association between decreased numbers or impaired studies will be necessary to unravel the molecular mechanisms by function of Tregs in lupus patients and mouse models (37). The which Pbx1 isoforms regulate the expression of miR-10a, miR-21, differentiation of effector CD4+ T cell subsets is a dynamic and and miR-155. plastic process, and cross-talk between the TFH and Treg pathways The exact role of each of these miRNAs in T cells overexpress- was described (30, 38). Although rapid progress has been made in ing Pbx1-d could provide important cues on the mechanisms by which recent years in mapping out the molecular pathways leading to Pbx1 regulates T cell differentiation. miR-10a is overexpressed in Treg (39) and TFH (40) differentiation, many questions still re- B cells from lupus patients (44), and miR-10a stabilizes Tregs (50) main, including the mechanisms by which the balance between and prevents the conversion of pTregs into TFHs by targeting Bcl6 these two subsets is maintained. We propose that PBX1 is in- and its corepressor Ncor2 in a TGF-b and RA–dependent manner volved in this process through its ability to regulate gene ex- (51). Therefore, it was unexpected to find consistently high levels pression in a cell-specific manner through complex interactions of miR-10a expression in the CD4+ T cells, including Foxp3+ with its cofactors. The overexpression of dominant-negative T cells, of the lupus congenic mice characterized in this study. Pbx1-d leads to an imbalance in T cell homeostasis; a modest Moreover, the level of miR-10a was similar between Pbx1-d–Tg Pbx1-d overexpression limits the spontaneous expansion or main- and TC CD4+ T cells, arguing that Pbx1-d overexpression is the tenance of the Treg subset in B6.Sle1a1 mice, whereas a high main determinant of this dysregulation. miR-21 is elevated in overexpression of Pbx1-d expands TFHs in Pbx1-d–Tg mice. lupus (46), and it regulates aberrant T cell responses by blocking Homeostatic expansion in the context of BM chimera revealed a PDCD4 expression (45). miR-21 expression has not been directly role for Pbx1-d–Tg overexpression in Treg maintenance. Simi- linked to either Tregs or TFHs, but it is activated by STAT3 in larly, a recall response in a competitive setting revealed a much T cells (52), and it is possible that its increased expression in + stronger response of TFHs expressing Pbx1-d either as a Tg or Pbx1-d–Tg CD4 T cells is secondary to their increased IL-21 endogenously. This could be due to a better survival of memory expression. miR-155 is a central modulator of T cell functions TFHs after the primary response or to a better ability to respond to (53). Receptor activation increases miR-155 expression in T cells, The Journal of Immunology 467

FIGURE 7. Pbx1-d-Tg in CD4+ T cells increased the expression of miR-10a, miR-21, and miR-155. miRNA expression was analyzed by quantitative PCR in FOXP3+-GFP+ (A) and FOXP3-GFP2 (B) CD4+ T cells from Pbx1-d–Tg.Foxp3egfp, B6.Sle1a1. Foxp3egfp, and B6.Foxp3egfp spleens. Representative FACS plots for follicular T cells in spleens from 4-mo-old B6 and TC mice (C) and corresponding percentages and absolute numbers of TFHs (D). (E) + miRNA expression in CD4 T cells from B6 and TC Downloaded from mice at 2, 4, and 10–12 mo of age (n = 3–4). *p , 0.05, **p , 0.01. http://www.jimmunol.org/

which regulates effector subset differentiation and maintenance. expressing T cells showing high miR-155 expression, skewed Th1 miR-155 deficiency was associated with decreased Th1 responses, humoral responses, and a reduced Treg population. Specific de- by guest on September 25, 2021 as well as low Treg numbers, which fits with Pbx1-d Tg– letion of miR-155 in T cells showed an intrinsic requirement of

FIGURE 8. Human PBX1 binds to the promoter of miR-10a, miR-21, and miR-155. (A) miRNA luciferase constructs containing predicted PBX1 binding sites were selected using Jaspar3. The regions used in the luciferase assays are indicated for each locus. (B) Dual-luciferase analyses of miR-10a, miR-21, and miR-155 expression in the presence of PBX1-b or PBX1-d, showing fold change relative to the absence of PBX1 expression plasmid. All data are mean 6 SD from at least three independent experiments. *p , 0.05, **p , 0.01. 468 Pbx1 REGULATES TFH VERSUS Treg DIFFERENTIATION

miR-155 for TFH differentiation (54). miR-155 deficiency nor- 11. Chen, Y., C. Cuda, and L. Morel. 2005. Genetic determination of T cell help in loss of tolerance to nuclear antigens. J. Immunol. 174: 7692–7702. malized B cell functions and autoantibody production in FAS- 12. Wandstrat, A. E., C. Nguyen, N. Limaye, A. Y. Chan, S. Subramanian, deficient mice (47); however, the role of miR-155 expression in X. H. Tian, Y. S. Yim, A. Pertsemlidis, H. R. Garner, Jr., L. Morel, and T cells was not addressed in this model. Our results demonstrate E. K. Wakeland. 2004. Association of extensive polymorphisms in the SLAM/ + CD2 gene cluster with murine lupus. Immunity 21: 769–780. consistent high levels of miR-155 expression in the CD4 T cells 13. Keszei, M., C. Detre, S. T. Rietdijk, P. Munõz, X. Romero, S. B. Berger, of the lupus congenic mice examined in this study, including S. Calpe, G. Liao, W. Castro, A. Julien, et al. 2011. A novel isoform of the Ly108 Foxp3+ and Foxp32 Pbx1-d–Tg CD4+ T cells. This is consistent gene ameliorates murine lupus. J. Exp. Med. 208: 811–822. 14. Cuda, C. M., L. Zeumer, E. S. Sobel, B. P. Croker, and L. Morel. 2010. Murine with a model in which miR-155 favors TFH differentiation over lupus susceptibility locus Sle1a requires the expression of two sub-loci to induce Treg differentiation and demonstrates that Pbx1-d overexpression inflammatory T cells. Genes Immun. 11: 542–553. is sufficient to drive this process. Interestingly, we found over- 15. Cuda, C. M., S. Li, S. Liang, Y. Yin, H. H. Potula, Z. Xu, M. Sengupta, Y. Chen, E. Butfiloski, H. Baker, et al. 2012. Pre-B cell leukemia homeobox 1 is asso- expression of the miR-17–92 cluster in Sle1a1 Tregs but no ciated with lupus susceptibility in mice and humans. J. Immunol. 188: 604–614. difference in Pbx1-d–Tg Tregs or in non-Tregs in either strain. 16. Sagerstro¨m, C. G. 2004. PbX marks the spot. Dev. Cell 6: 737–738. 17. Laurent, A., R. Bihan, F. Omilli, S. Deschamps, and I. Pellerin. 2008. PBX miR-17–92 prevents pTreg differentiation (55), which fits with proteins: much more than Hox cofactors. Int. J. Dev. Biol. 52: 9–20. our findings that Tregs are more affected in Sle1a1 T cells than 18. Sengupta, M., S. Liang, H. H. S. Potula, L. J. Chang, and L. Morel. 2012. The in Pbx1-d–Tg T cells. Moreover, miR-17–92 overexpression SLE-associated Pbx1-d isoform acts as a dominant-negative transcriptional regulator. Genes Immun. 13: 653–657. in lymphocytes leads to autoimmune phenotypes in mice (56), 19. Sobel, E. S., T. M. Brusko, E. J. Butfiloski, W. Hou, S. Li, C. M. Cuda, suggesting that it may be a mechanism by which Pbx1-d over- A. N. Abid, W. H. Reeves, and L. Morel. 2011. Defective response of CD4(+) expression contributes to autoimmunity. T cells to retinoic acid and TGFb in systemic lupus erythematosus. Arthritis Res. Ther. 13: R106.

+ Downloaded from In conclusion, the phenotypes and CD4 T cell differentiation in 20. Sanyal, M., J. W. Tung, H. Karsunky, H. Zeng, L. Selleri, I. L. Weissman, Pbx1-d–Tg mice are similar to those of B6.Sle1a1 mice, which L. A. Herzenberg, and M. L. Cleary. 2007. B-cell development fails in the ab- establishes Pbx1 as the gene responsible for the Sle1a1 phenotype. sence of the Pbx1 proto-oncogene. Blood 109: 4191–4199. 21. Lu, S., L. Zeumer, H. Sorensen, H. Yang, Y. Ng, F. Yu, A. Riva, B. Croker, It also highlights critical roles for lupus development in regulating S. Wallet, and L. Morel. 2015. The murine Pbx1-d lupus susceptibility allele Treg and TFH differentiation and developing humoral autoimmu- accelerates mesenchymal stem cell differentiation and impairs their immuno- Pbx1-d suppressive function. J. Immunol. 194: 43–55. nity. –Tg mice provide a novel model to investigate T cell– 22. Bettelli, E., Y. Carrier, W. Gao, T. Korn, T. B. Strom, M. Oukka, H. L. 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