Chromatin remodeling by the NuRD complex regulatesdevelopment of follicular helper and regulatory T cellsErxia Shena,b,c,1, Qin Wanga,d,1, Hardis Rabea,e, Wenquan Liua,f, Harvey Cantora,c,2, and Jianmei W. Leavenwortha,c,g,h,2

aDepartment of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA 02115; bDepartment of Pathogenic Biology and Immunology,Sino-French Hoffmann Institute, School of Basic Sciences, Guangzhou Medical University, 511436 Guangzhou, China; cDepartment of Microbiology &Immunobiology, Division of Immunology, Harvard Medical School, Boston, MA 02115; dDepartment of Immunology, Medical College of Soochow University,Suzhou, 215123 Jiangsu, China; eDepartment of Infectious Diseases, Institute of Biomedicine, The Sahlgrenska Academy, University of Gothenburg, 405 30Göteborg, Sweden; fDepartment of Parasitology, Wenzhou Medical University, Wenzhou, 325035 Zhejiang, China; gDepartment of Neurosurgery,University of Alabama at Birmingham, Birmingham, AL 35233; and hDepartment of Microbiology, University of Alabama at Birmingham, Birmingham, AL35233

Contributed by Harvey Cantor, May 11, 2018 (sent for review March 26, 2018; reviewed by Mari L. Shinohara and George C. Tsokos)

Lineage commitment and differentiation into CD4+ T cell subsetsreflect an interplay between chromatin regulators and transcriptionfactors (TF). Follicular T cell development is regulated by the Bcl6 TF,which helps determine the phenotype and follicular localization ofboth CD4+ follicular helper T cells (TFH) and follicular regulatoryT cells (TFR). Here we show that Bcl6-dependent control of follicularT cells is mediated by a complex formed between Bcl6 and the Mi-2β-nucleosome-remodeling deacetylase complex (Mi-2β-NuRD). For-mation of this complex reflects the contribution of the intracellularisoform of osteopontin (OPN-i), which acts as a scaffold to stabilizebinding between Bcl6 and the NuRD complex that together regulatethe genetic program of both TFH and TFR cells. Defective assemblyof the Bcl6–NuRD complex distorts follicular T cell differentiation,resulting in impaired TFR development and skewing of the TFH line-age toward a TH1-like program that includes expression of Blimp1,Tbet, granzyme B, and IFNγ. These findings define a core Bcl6-directedtranscriptional complex that enables CD4+ follicular T cells to regulatethe germinal center response.

follicular helper T cells | follicular regulatory T cells |germinal center response | osteopontin | Bcl6 transcription factor

The germinal center (GC) response is a highly dynamic pro-cess in tissues where high levels of dying cells provide a

battery of self-antigens that can activate autoreactive antibodyresponses (1). Generation of high-affinity antibodies and avoid-ance of autoimmune responses after microbial infection or vacci-nation requires precise control of the GC reaction, depending, to alarge degree, on the combined activities of CD4+ follicular helperT (TFH) and follicular regulatory T (TFR) cells (2–6). TFH cells thatarise from naive CD4+ T cells induce GC formation and help Bcells to produce protective antibody responses to invading patho-gens through generation of memory B cells and long-lived plasmacells (2, 3, 7). TFR cells that originate from FoxP3+ Treg precursorsdampen TFH-driven GC responses and can prevent the emergence ofautoreactive B cells and consequent autoantibody production (4–6).While TFH and TFR cells have opposing functions, shared expressionof the Bcl6 TF serves to repress alternative differentiation pathways(5, 6). Although engagement of the T cell antigen receptor (TCR)and costimulatory receptor inducible T cell costimulator (ICOS) hasbeen implicated in this process (4–6, 8), the conserved genetic andepigenetic mechanisms that ensure Bcl6-directed differentiation ofthis critical pair of follicular T cells remain largely unknown.Differentiation of CD4+ T cells following engagement of the

TCR and costimulatory receptors is determined by changes ingene expression, which in part reflect chromatin modificationsthat shape transcription, differentiation, and cellular replica-tion. Regulation of gene expression during differentiation ofTFH and TFR cells depends on Bcl6-dependent recruitment ofcorepressor complexes that help shape the chromatin landscapesurrounding Bcl6 target loci, including Prdm1 (encodingBlimp1) and other genes that may promote alternative T-helper(TH)-cell fates (9, 10).

The Mi-2β-nucleosome-remodeling deacetylase complex (Mi-2β-NuRD) couples a histone deacetylase and a nucleosome-stimulated ATPase to several corepressors, including a familyof metastasis-associated (MTA) proteins (11, 12), which canrepress transcription following interactions with site-specificDNA binding proteins (11). Previous studies have indicatedthat B cell development may reflect recruitment of Mi-2β-NuRDto Bcl6 target loci by MTA3, a cell-type-specific subunit of theMi-2β-NuRD complex (12). Recent analysis of the Bcl6 sec-ondary repression domain (Bcl6-RD2) has also suggested thatMTA3 may interact with Bcl6 in CD4+ TFH cells (13). However,whether Bcl6, MTA3, and Mi-2β-NuRD form a complex in TFHand TFR cells and the impact of a putative Bcl6–MTA3–Mi-2β-NuRD complex on follicular T cell differentiation during animmune response is unknown.Our recent analysis of CD4+ T-helper responses has revealed

that expression of the intracellular isoform of osteopontin (OPN-i) is essential for the differentiation of both follicular T cellsubsets –TFH and TFR cells (4). For example, analysis of TFHcells indicates that engagement of ICOS on TFH and TFR cellspromotes nuclear translocation of OPN-i, binding to Bcl6 via theRD2 domain and protection of the Bcl6–OPN-i complex fromproteasomal degradation to allow sustained TFH/TFR responsesfollowing initial lineage commitment (4).

Significance

Production of high-affinity antibody responses after infectionor vaccination requires precise control of germinal center B cellsby follicular helper T cells and follicular regulatory T cells. Al-though the Bcl6 transcription factor plays a central role in fol-licular T cell differentiation, the molecular basis of Bcl6 controlhas been clouded in uncertainty. Here we report that Bcl6-dependent control reflects the formation of a macromolecularcomplex between Bcl6 and the Mi-2β-nucleosome remodelingdeacetylase complex (Mi-2β-NuRD). The repressive activity ofthis intranuclear complex potentiates the follicular T cell phe-notype and inhibits alternative T cell fates. Identification of thisintracellular complex may facilitate new targeted approachesto the treatment of autoimmune disorders.

Author contributions: E.S., Q.W., H.C., and J.W.L. designed research; E.S., Q.W., H.R., W.L.,and J.W.L. performed research; E.S., Q.W., H.R., W.L., H.C., and J.W.L. analyzed data; andE.S., H.C., and J.W.L. wrote the paper.

Reviewers: M.L.S., Duke University School of Medicine; and G.C.T., Beth Israel DeaconessMedical Center and Harvard Medical School.

The authors declare no conflict of interest.

Published under the PNAS license.1E.S. and Q.W. contributed equally to this work.2To whom correspondence may be addressed. Email: Harvey_Cantor@dfci.harvard.edu orjleavenworth@uabmc.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1805239115/-/DCSupplemental.

Published online June 11, 2018.

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Here we analyze the transcriptional events that confer com-mitment to the two major follicular T cell lineages. We noted asurprising and profound defect in early TFH/TFR lineage com-mitment by OPN-i–deficient cells despite intact Bcl6 protein levels.Analyses of the complex formed by OPN-i, Bcl6, and Mi-2β-NuRDrevealed that the OPN-i protein acts as a scaffold that supportsthe formation of a complex between Bcl6 and MTA3 that me-diates the genetic programming of TFH and TFR cells (SI Ap-pendix, Fig. S1). Additional interrogation of the biologic activityof this complex revealed that OPN-i–dependent recruitment ofthe Bcl6–Mi-2β-NuRD complex to Bcl6 target loci is a criticalstep in the transcriptional repression of the Prdm1 locus andcommitment to the TFH and TFR cell genetic program.

ResultsOPN-i Deficiency Impairs TFH and TFR Early Commitment. To definethe impact of OPN-i deficiency on early commitment of TFH andTFR cells, we used Spp1flstop mice bearing a mutated Spp1 allelethat allows expression of the OPN-i isoform after Cre-mediatedrecombination. These Spp1flstopCre+ mice are termed OPN-i-knock-in (OPN-i-KI) mice, while Spp1flstopCre– mice are OPN-knockout (OPN-KO) mice (4). We then isolated CD4+ T cellsfrom OPN-i-KI or OPN-KO mice that coexpress the OT-II[ovalbumin (OVA)-specific] TCR transgene. Since TFH com-mitment occurs as early as 72 h in vivo (8), we analyzed TH celldifferentiation at 2.5 d after transfer of these CD4+ T cells alongwith B cells into Rag2

−/−Prf1−/− mice followed by immunizationwith NP13-OVA in Complete Freunds’ Adjuvant (CFA) (Fig. 1).Bcl6 protein levels were not affected by OPN-i deficiency at thisearly time point (Fig. 1A) (4). However, OPN-KO CD4+ T cellsdisplayed a marked impairment in TFH commitment, as reflectedby reduced proportions of CD4+ T cells expressing CXCR5compared with OPN-i-KI cells (Fig. 1B). A bifurcation betweenTFH and other effector TH cells, particularly TH1 cells, occursduring early TH cell fate determination (8). Analysis of the TH1-cell-associated phenotype of these differentiating cells revealedthat a substantially increased proportion of OPN-KO CD4+

T cells expressed the Tbet, Ly6C, and granzyme B triad, whichcharacterize a TH1-like phenotype (14). As a consequence, OPN-KO CD4+ T cells displayed an increased ratio of triad+ CD4+T cells to CXCR5+ CD4+ T cells compared with their OPN-i-KIcounterparts (Fig. 1B), suggesting that OPN-i deficiency mightimpair early TFH commitment and precede the reducedBcl6 protein levels that occur later in the response (4).Bcl6-dependent differentiation of TFH cells includes repression

of an alternative Blimp1-associated non-TFH program (Fig. 1) (9,15). We therefore asked whether OPN-i deficiency altered theBcl6−Blimp1 balance during early CD4+ TH cell differentiation. Weused Blimp1-YFP reporter mice to generate Blimp1-YFP×OPN-KOmice and Blimp1-YFP×OPN-i-KI mice. Analysis of TFH differenti-ation at day 2.5 postimmunization revealed that the proportions ofBlimp1+ CD4 effector T cells (FoxP3−) were considerably higher inOPN-KO mice than OPN-i-KI mice, despite unimpaired Bcl6 pro-tein expression (SI Appendix, Fig. S2). Moreover, higher frequen-cies of Blimp1+ FoxP3+ CD4+ Treg were also noted in OPN-KOmice compared with OPN-i-KI mice (SI Appendix, Fig. S2), openingthe possibility that OPN-i deficiency might impair Bcl6-dependentrepression of Blimp1 transcription in both TFH and TFR cells.We then asked whether early TFR differentiation was also affected

by OPN-i deficiency using the approach described above (Fig. 1 Aand B). We transferred CD25hi Treg cells from WT, OPN-KO, orOPN-i-KI mice along with naive CD4+ T cells from CD45.1 con-genic mice into Tcra−/− mice followed by immunization with NP13-OVA in CFA. After 2.5 d, OPN-KO but not OPNWT or OPN-i-KITreg displayed elevated expression of Blimp1 and Tbet but reducedexpression of CXCR5 by FoxP3+ T cells (Fig. 1 C andD), suggestingthat OPN-i deficiency skewed Treg away from the conventionalfollicular phenotype. Taken together, these results suggested thatOPN-i might regulate early TFH and TFR commitment, in partthrough enhanced Bcl6-dependent repression of alternative geneticprograms that might depend on Blimp1 expression.

OPN-i Interacts with MTA3 to Promote the Formation of a Bcl6–MTA3–NuRD Complex. An interaction between Bcl6 and the Mi-2β-NuRDcomplex via the MTA3 corepressor contributes to Bcl6 transcrip-tional repressive activity and B cell fate (12). Previous studies havealso indicated that, in response to TCR and ICOS signals, apool of OPN-i translocates into the nucleus to interact withBcl6 via the Bcl6-RD2 domain (4). The above findings that acomplex formed by OPN-i and Bcl6 might regulate early TFHand TFR commitment led us to ask whether OPN-i–dependentformation of a Bcl6–MTA3–Mi-2β-NuRD complex might me-diate Bcl6-dependent TFH and TFR differentiation. We first ana-lyzed 293T cells transfected with vectors expressing HA-taggedMTA3, OPN-i, and Flag-tagged Bcl6 followed by immunopre-cipitation with anti-Flag antibody. Consistent with previous re-ports (4, 12), immunoblot analysis revealed that Bcl6 interactedwith both MTA3 and OPN-i, either directly or indirectly. Wealso noted that the Bcl6–MTA3 association was enhanced indirect proportion to increasing concentrations of OPN-i (Fig. 2A,Left). Deletion of the N-terminal portion of the Bcl6-RD2 region(Δ120–300), which disrupts the Bcl6–OPN-i interaction (4),prevented OPN-i–dependent enhancement of the Bcl6–MTA3association (Fig. 2A, Right). Moreover, enhanced binding ofBcl6 to MTA3, a component of the Mi-2β-NuRD complex, wasassociated with increased binding of Bcl6 to Mi-2β (Fig. 2A), acentral component of the Mi-2β-NuRD complex (11, 12).We then asked whether OPN-i–mediated enhancement of

Bcl6–MTA3–Mi-2β-NuRD complex formation noted above mightbe apparent in primary CD4+ T cells. Analysis of Bcl6-associated

Fig. 1. OPN-i deficiency impairs TFH and TFR early commitment. (A and B)FACS analysis of TH cell differentiation at day 2.5 after transfer of OT-II CD4+

T cells and B cells into Rag2−/−Prf1−/− mice followed by immunization with

NP13-OVA in CFA. (A) Histogram overlays of intracellular protein expression(gated on FoxP3– CD4+ T cells). (B) Plots of non-TFH and TFH phenotype(gated on FoxP3– CD4+ T cells) and mean ratios of non-TFH to TFH cells areshown for OPN-i-KI and OPN-KO mice (n = 3–4 for each group). GzmB,granzyme B. (C and D) Treg from CD45.2+ WT, OPN-i-KI, or OPN-KO micewere transferred along with naive CD45.1+ CD4+ T cells into Tcra−/− micefollowed by immunization with NP13-OVA in CFA. Analysis of CD45.2+

Treg cells (gated on FoxP3+) 3 d postimmunization. Histogram overlays (C )and quantitation of mean fluorescence intensity (MFI) (D) of each protein(n = 3 for each group). Data shown are representative of three in-dependent experiments (*P < 0.05 and **P < 0.01). Error bars indicatemean ± SEM.

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proteins expressed by CD4+ T cells from OT-II×OPN-i-KI micecompared with CD4+ T cells from OT-II×OPN-KO mice 3 dafter immunization indicated that OPN-i deficiency greatly re-duced the association of Bcl6 with MTA3 and Mi-2β in OPN-KOCD4+ T cells, despite unaltered Bcl6 protein expression (Fig.2B). These results suggested that OPN-i promoted formation ofthe Bcl6–MTA3–Mi-2β-NuRD complex.The regulatory activity of the Mi-2β-NuRD complex depends

on the activity of its corepressor components, including MTA3family members, which may demarcate distinct forms of Mi-2β-NuRD that control cell-type-specific transcription (11, 16). Wenoted that MTA3 bound to both Bcl6 and OPN-i within the nu-cleus of CD4+ T cells (Fig. 2C). We further defined the OPN-iinteraction with MTA3 according to mutational analysis (Fig. 2D).We found that a specific interaction between OPN-i and theELM2 domain of MTA3 promoted binding of the complex toBcl6. Thus, MTA3-ELM2 deletion mutants (but not MTA3-WTor MTA3-BAH mutants) failed to bind to Bcl6, as judged by anti-Flag (Bcl6) immunoprecipitation (Fig. 2E). These findings suggestthat binding of OPN-i to both Bcl6 and MTA3 allows OPN-i tofunction as a scaffold or bridge to promote the association ofBcl6 with the Mi2β-NuRD complex (SI Appendix, Fig. S1).

OPN-i Promotes Bcl6–MTA3-Dependent Repression of Prdm1/IfnγExpression by TH1 Cells. Repression of Blimp1 and other non-TFHgenes by Bcl6 plays a central role in TFH commitment and mainte-nance of the TFH phenotype (9, 10). To determine whether theOPN-i–dependent association between Bcl6 and MTA3–Mi-2β-NuRD noted above contributed to Bcl6 transcriptional repression ofcanonical TH1 genes, we asked whether forced expression ofBcl6 alone or with MTA3 in TH1 cells [which do not express sig-nificant levels of Bcl6 or MTA3 (4)], might reprogram this CD4+ THsubset. We therefore infected in-vitro–differentiated TH1 cells [after5 d culture as described previously (17)] with retroviruses expressingBcl6, MTA3, or both Bcl6 and MTA3. Quantitative RT-PCRanalysis of TH1-associated gene expression showed that retroviralcoexpression of Bcl6 and MTA3, but not expression of either ret-rovirus alone, substantially repressed both Prdm1 and Ifnγ expression(Fig. 3A). The specificity of this response was confirmed by thefinding that transduction of these TH1 cells with a retrovirusexpressing the N-terminal Bcl6-RD2 deletion mutant (Δ120–300),which impairs theMTA3–Bcl6 interaction (Fig. 2A), failed to repressPrdm1 or Ifnγ expression even at the highest dose tested (Fig. 3B).In view of our findings that localized the interaction of OPN-i

with MTA3 to the MTA3-ELM2 domain (Fig. 2 D and E), wetested the functional impact of this interaction on the CD4+ T cellgenotype in vitro. We observed that transduction of TH1 cells (afterdifferentiation from CD25–CD4+ T cells from OT-II×OPN-i-KImice) with a retrovirus expressing Bcl6 and the MTA3 protein butnot the MTA3-ELM2 deletion mutant suppressed Prdm1 or Ifnγexpression (Fig. 3C). Moreover, limiting concentrations of Bcl6and MTA3, which did not repress Prdm1 or Ifnγ, fully repressedthese genes in the presence of OPN-i (Fig. 3D), consistent withearlier findings that coexpression of OPN-i enhances the repressiveefficiency of Bcl6–MTA3. These findings together are consistentwith the ability of OPN-i to promote the biochemical association ofBcl6 with MTA3–Mi-2β-NuRD (Fig. 2).

Fig. 2. Interaction of OPN-i with MTA3 increases Bcl6–MTA3–NuRD complexformation. (A) Cotransfection of 293T cells with vectors expressing Flag-Bcl6WT or Flag-Bcl6-RD2 deletion mutant and HA-MTA3 without or with in-creasing concentrations of OPN-i was followed by immunoprecipitation (IP)with anti-Flag antibody (Ab) and immunoblotting (WB) with indicated Abs.(B) Cell lysates of purified CD44hiCD25–CD4+ T cells from OT-II×OPN-i-KI orOT-II×OPN-KO mice 3 d after NP13-OVA immunization were immunopreci-pitated with anti-Bcl6 Ab or rabbit IgG control before immunoblotting withAbs to Bcl6, MTA3, OPN, and Mi-2β. (C) Interaction of OPN-i and theMTA3 component of NuRD complex in CD4+ T cells. Nuclear lysates of

purified CD4+CD44+ T cells from mice at day 5 postimmunization with NP26-KLH in CFA were immunoprecipitated with anti-MTA3 and immunoblottedwith Abs to Bcl6 and OPN. Input, immunoblot analysis of an aliquot of lysatewithout IP. (D) MTA3–OPN-i interaction depends on ELM2 domain of MTA3.293T cells were cotransfected with vectors expressing HA-MTA3 WT or itsdeletion mutants (diagramed above) with or without OPN-i, followed by IPwith anti-HA and immunoblotting with anti-HA and anti-OPN Abs. (E) De-letion of the ELM2 domain impairs the Bcl6–MTA3 interaction. 293T cellswere cotransfected with plasmids expressing Flag-Bcl6, HA-MTA3 WT, or itsdeletion mutants with or without OPN-i, followed by IP with anti-Flag Aband immunoblotting with the indicated Abs. Data shown are representativeof three independent experiments.

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Promotion of TFH and TFR Differentiation in Vivo Requires an InteractionBetween OPN-i and MTA3. The specificity of the OPN-i–Bcl6–MTA3 interaction described above is supported by findings thatdeletion of the ELM2 domain of MTA3 disrupts binding of OPN-ito MTA3 (Fig. 2D), impairs MTA3 binding to Bcl6 (Fig. 2E), anddecreases Bcl6–MTA3-dependent repression of Prdm1/Ifnγ ex-pression (Fig. 3C). We then tested the physiological relevance ofthe OPN-i–Bcl6–MTA3 interaction to TFH and TFR differentiationin vivo using a retroviral reconstitution system (4). We transducedin-vitro–activated OT-II CD4+ T cells with a retroviral vectorexpressing GFP alone (empty vector, EV) or GFP plus either WTMTA3 (MTA3) or the MTA3-ELM2 deletion mutant (delELM2).Since MTA3 and delELM2 are expressed within the same bicis-tronic IRES retroviral vector as GFP, their expression is correlatedwith GFP levels. We transferred sorted GFP+ cells into Tcra−/−

hosts followed by immunization with NP13-OVA in CFA (Fig. 4Aand SI Appendix, Fig. S3A). TFH differentiation and associated GCB cell formation were increased for OT-II CD4+ T cells transducedto express WT MTA3 compared with CD4+ T cells transducedwith EV (Fig. 4B). In contrast, transduction of OT-II CD4+ T cellswith the delELM2 mutant resulted in decreased numbers of TFHand GC B cells (Fig. 4B). Consequently, both the total (anti-NP23)and high-affinity (anti-NP4) NP-specific antibody responses weremarkedly impaired (Fig. 4C). Although Bcl6 levels were not altered

Fig. 3. OPN-i promotes Bcl6–MTA3-dependent repression of Prdm1 and Ifnγexpression in TH1 cells, which requires the MTA3 ELM2 domain. (A) OT-IITH1 cells were transduced with retroviral vectors expressing either Flag-Bcl6 or HA-MTA3 alone [multiplicity of infection (MOI) = 10]; or a mixturecontaining constant Flag-Bcl6 concentrations (MOI = 10) combined with in-creasing concentrations of HA-MTA3 (wedge: 2.5, 5, 10). (B) OT-II TH1 cellswere transduced with a mixture of retroviral vectors expressing constant HA-MTA3 (MOI = 10) combined with Flag-Bcl6 at increased MOI (wedge: 2.5, 5,10), or with Flag-Bcl6-RD2 deletion mutant (MOI = 10). (C) OT-II TH1 cellswere transduced with a mixture of retroviral vectors expressing constantconcentrations of Flag-Bcl6 (MOI = 10) with HA-MTA3 ELM2 deletion mutant(MOI = 10). (D) OT-II TH1 cells were transduced with retroviral vectorsexpressing [Flag-Bcl6 (MOI = 5) + HA-MTA3 (MOI = 5)] or OPN-i (MOI = 5), orFlag-Bcl6 + HA-MTA3 + OPN-i (each at a suboptimal MOI of 5). qRT-PCR wasperformed after 2.5 d. Gene expression was normalized to expression of thecontrol gene Rps18 (encoding ribosomal protein S18) and presented as rel-ative to cells transduced with control virus, set as 1. Data shown are repre-sentative of three independent experiments (*P < 0.05, **P < 0.01, and***P < 0.001). Error bars indicate mean ± SEM.

Fig. 4. OPN-i–mediated promotion of TFH differentiation requires intactOPN-i−MTA3 interaction. (A and F) Schematic diagrams of experimentalprotocols. Purified naive OT-II×OPN-i-KI CD4+ T cells were activated in vitro,transduced with retroviral vector encoding GFP alone (EV) or GFP plus WTMTA3 (MTA3) or deletion mutant MTA3 (delELM2). GFP+ CD4+ T cells (A) andGFPhi or GFPmed-lo CD4+ T cells (F) were then sorted and transferred intoTcra−/− hosts followed by immunization with NP13-OVA in CFA. B–E wereanalyzed from protocol A, and G and H from protocol F. (B and G) FACSanalysis of TFH cells (gated on FoxP3– CD4+ T cells) and GC B cells (gated onB220+ cells) 10 d postimmunization. (D and H) Histogram overlays of Bcl6,Tbet, Ly6C, and Blimp1 expression in donor CD4+ T cells (gated on FoxP3–

CD4+ T cells). (E) Quantitation of MFI of each protein and frequency ofLy6C+CD4+FoxP3– cells in D. Data shown are representative of two in-dependent experiments. (C) Ectopic expression of the delELM2 mutant inOT-II CD4+ T cells impairs the Ab response postimmunization. Anti-NP23 oranti-NP4 antibody titers were determined from mice transferred with OT-IICD4+ T cells expressing empty control, WT MTA3, or delELM2 mutant fol-lowed by immunization, as in A. ***P < 0.001, and ns, no significance. Errorbars indicate mean ± SEM.

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among cells expressing GFP alone, MTA3, or the delELM2 mu-tant, transduction with the delELM2 mutant exerted a “dominantnegative” impact on TFH cell function, as judged by increased Tbet,Ly6C, and Blimp1 expression (Fig. 4 D and E).The above finding that CD4+ T cells expressing the delELM2

mutant failed to differentiate into functional TFH cells led us to askwhether the delELM2 MTA3 mutant could compete with endog-enous MTA3 to interfere with TFH differentiation. To address this,we transduced OT-II CD4+ T cells with a retroviral vectorexpressing GFP alone or GFP plus the delELM2 mutant, and thentransferred sorted GFPhi or GFPmed-lo CD4+ T cells separatelyinto Tcra−/− mice followed by immunization with NP13-OVA inCFA (Fig. 4F and SI Appendix, Fig. S3B). Consistent with theseresults (Fig. 4B), TFH differentiation was decreased for OT-IICD4+ T cells transduced to express the delELM2 mutant com-pared with cells expressing GFP alone, which was associated withreduced GC B cells (Fig. 4G). The frequencies of TFH and GC Bcells were also not affected by GFP levels in OT-II CD4+ T cellsexpressing GFP alone. In contrast, the extent of TFH differentia-tion and GC B cell formation in mice transferred with OT-II CD4+T cells expressing delELM2 was negatively correlated with levels ofdelELM2 (GFP) and associated with increased expression of non–TFH-associated markers (Tbet, Ly6C, and Blimp1) (Fig. 4 G andH). These results suggest that expression of the ectopic delELM2mutant might impede TFH differentiation by competing with theendogenous MTA3 in a dose-dependent manner.Using a retroviral reconstitution system similar to that described

above, we evaluated the physiological contribution of the OPN-i–Bcl6–MTA3 interaction to the formation of TFR cells. We trans-duced CD45.2+ WT CD25+CD4+ T cells with retroviral vectorsexpressing GFP alone (EV) or GFP plus WT MTA3 (MTA3) ordelELM2 mutant (delELM2), then transferred each group of cellstogether with CD45.1+ CD25–CD4+ T cells into Tcra−/− mice,followed by immunization of hosts with NP13-OVA in CFA (Fig.5A). TFR differentiation was increased for CD45.2+ Treg trans-duced to express WT MTA3 compared with cells transduced withEV (Fig. 5B and SI Appendix, Fig. S4). In contrast, expression ofthe delELM2 mutant in CD45.2+ Treg decreased TFR differenti-ation to levels comparable to cells expressing GFP alone, whichwas associated with increased TFH differentiation and GC B cellformation (Fig. 5B and SI Appendix, Fig. S4). These results indicatethat an OPN-i−MTA3 interaction required for Bcl6−MTA3−NuRD complex formation in vitro is also essential for TFH andTFR differentiation in vivo.

OPN-i Promotes Binding of Bcl6 and MTA3 to Bcl6 Target Genes andRegulates Bcl6 Transcriptional Activity. To gain insight into the geneticmechanisms that underpinned OPN-i–mediated enhancement ofTFH and TFR differentiation, we asked whether OPN-i–promoted

binding of the Bcl6−MTA3−NuRD complex reflected increasedbinding to Bcl6 target loci. We focused on Prdm1, since Bcl6-de-pendent repression of Prdm1 is a key element in the determinationof TFH and TFR cell fate (Fig. 3 and SI Appendix, Fig. S2). We notedthat the mRNA levels of Prdm1 were substantially up-regulated inOPN-KO TFH and TFR cells compared with OPN-i-KI cells 3 dpostimmunization, despite unaltered Bcl6 mRNA levels inthese cells (Fig. 6 A and B). We performed a chromatin im-munoprecipitation (ChIP)-qPCR analysis of the Bcl6 andMTA3 occupancy on the conserved Bcl6 response element(BRE) within Prdm1. We observed that Bcl6 binding to thePrdm1 BRE region was substantially decreased in OPN-KOTFH cells, consistent with a failure of Bcl6 to repress Prdm1 inOPN-i–deficient TFH cells (Fig. 6A). Moreover, analysis of OPN-KO TFH cells revealed an almost complete loss of MTA3 bound tothe Prdm1 BRE locus (Fig. 6C), further suggesting severely im-paired recruitment to this canonical Bcl6 target gene. SinceBcl6 transcriptional repression is mediated in part by recruitinghistone deacetylases to target loci via the Mi-2β-NuRD complex, weasked whether OPN-i deficiency influenced the histone acetylationstatus surrounding Bcl6-bound loci. There was almost no acetylatedH3 (AcH3) at the Prdm1 BRE locus of OPN-i-KI TFH cells, con-sistent with a repressive chromatin status. In contrast, these locidisplayed increased levels of AcH3 in OPN-KO TFH cells (Fig. 6C),consistent with an active chromatin locus in the absence of OPN-i.Taken together, these results indicate that OPN-i is required forefficient binding of Bcl6 and MTA3 to a major Bcl6 target gene aswell as associated repression of this locus.

DiscussionSpecification of T cell fate reflects the concerted action of chro-matin regulators and transcription factors in response to signalsemanating mainly from the TCR and costimulatory receptors. Ourstudies suggest that the functional differentiation of TFH and TFRcells is mediated, in part, by recruitment of the Mi-2β-NuRDcomplex to specific Bcl6 target loci. The formation of this complexin differentiating CD4+ T cells requires the scaffold-like contribu-tion of OPN-i to the binding of Bcl6 to the MTA3–Mi-2β-NuRDcomplex and formation of a biologically active corepressor complex.The transcriptional activity of Bcl6 in other cell types may also

reflect recruitment of different corepressor complexes to differentBcl6 domains and the formation of target-specific complexes. Forexample, an interaction between the Bcl6-BTB domain and theBCOR/SMART corepressors promotes GC B cell differentiationwithout a significant effect on the TFH cell response (18). In con-trast, previous studies of TFH differentiation have underlined thesignificance of an interaction between the Bcl6-RD2 domain andMTA3 (13) as well as a second interaction with OPN-i (4). Here weidentify OPN-i as a critical bridging intermediary that facilitatesbinding between Bcl6 and MTA3 and promotes the formation of a

Fig. 5. OPN-i–mediated promotion of TFR differentiation requires intactOPN-i−MTA3 interaction. (A) Schematic diagram of experimental procedure.Purified CD45.2+ Treg were activated in vitro, transduced with retroviralvector encoding GFP alone (EV), or GFP plus WT MTA3 (MTA3) or deletionmutant MTA3 (delELM2). GFP+ Treg were then sorted and transferred intoTcra−/− hosts along with CD45.1+ naive CD4+ T cells followed by immunizationwith NP13-OVA in CFA. (B) Frequency of TFR, TFH, and GC B cells in SI Appendix,Fig. S4. Data shown are representative of two independent experiments. *P <0.05, **P < 0.01, ns, no significance. Error bars indicate mean ± SEM.

Fig. 6. OPN-i promotes binding of Bcl6 and MTA3 to Bcl6 target genes andregulates Bcl6 transcriptional activity. (A and B) qRT-PCR analysis of Prdm1and Bcl6 in sorted pure (>95%) TFH cells (A) or TFR cells (B) from OPN-i-KI andOPN-KO mice at day 3 postimmunization with NP13-OVA in CFA. Gene ex-pression was normalized to expression of the control gene Rps18 (encodingribosomal protein S18) and expressed as relative to TFH or TFR cells from OPN-i-KI mice, set as 1. (C) In-vitro–differentiated TFH cells from OT-II×OPN-i-KI orOT-II×OPN-KO mice were cross-linked, chromatin prepared, and ChIP-PCRanalyses performed for Bcl6, MTA3, and Acetylated H3 (AcH3) at the BRE ofPrdm1 gene. Data, shown as the percent of input, reflecting enrichedbinding at the indicated loci, are representative of three independent ex-periments (mean ± SEM). *P < 0.05, **P < 0.01, ns, no significance.

6784 | www.pnas.org/cgi/doi/10.1073/pnas.1805239115 Shen et al.

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Bcl6–NuRD complex that is equipped to direct both TFH and TFRcell differentiation. The extended and flexible structure of OPN-i, amember of the SIBLING protein family (19), may permit inter-actions with a variety of partners, including the Mi-2β-NuRDmacromolecule in the nucleus, as described here, as well as withproteasomal complexes in the cytosol, as noted previously (20), topromote Bcl6-directed differentiation of follicular T cells.These findings provide insight into the epigenetic mechanisms

that govern lineage commitment of the follicular T cell pair thatregulates GC antibody and autoantibody responses. Our findingsalso help clarify the differentiative relationship between the TFHand TFR cell lineages. Although TFH and TFR cells coexpressBcl6 as well as several surface receptors, the shared genetic el-ements responsible for follicular differentiation of these twoCD4+ T cell lineages have been obscure. We have reportedpreviously that TFH and TFR cells may share an ICOS-dependentpathway that promotes the formation of an intranuclear complexbetween Bcl6 and OPN-i that protects the Bcl6 protein fromproteasomal degradation (4). Here we identify an additional rolefor OPN-i in TFH differentiation, i.e., integration of Bcl6 withMi-2β-NuRD to form biologically active complexes that enhanceTFH and TFR lineage differentiation. Regulation of Prdm1 andother canonical target genes by this complex may account forcore features shared by differentiated TFH and TFR cells thatreside in the germinal centers and lymphoid tissue follicles.Formation of this Bcl6 complex may represent a critical down-stream consequence of the ICOS-dependent pathway that favorsthe differentiation of follicular T cells from CD4+ precursors (4,8). Since the ratio of TFH to TFR cells has a direct impact on theintensity and quality of GC antibody responses (4, 21), a detailedcorrelation between the TFH/TFR ratio and the intensity andquality of the B cell response at defined intervals after immu-nization is necessary to fully evaluate the impact of this Bcl6-containing complex on the immune response.Our finding that Bcl6 transcriptional activity in TFH cells de-

pends on its association with the complex described here alsosuggests that anti-Bcl6-based ChIP-seq analysis of TFH cells maylack the specificity necessary to precisely define the geneticprogram of TFH (and TFR) cells. A precedent for this comes fromanalysis of early B cell differentiative steps that are regulated byIkaros–Mi-2β-NuRD complexes. These studies indicate thatcombined occupation of target loci in early B cells by Ikaros andMi-2β-NuRD, but not by Ikaros alone, is essential for func-tional control of early B cell differentiation genes (22). Recentgenomewide Bcl6 ChIP-seq analysis of human GC TFH cells hasindicated that Bcl6 binds to over 8,500 target loci that localize

predominantly to promoter regions (23), while analysis ofmurine TFH cells has revealed about 5,100 Bcl6 binding peakslocalized mainly to intron and intergeneic regions (24). It islikely that a more precise identification of the key target genesthat control TFH and TFR differentiation may come fromidentification of the genetic loci that are cooccupied by bothBcl6 and the partner Mi-2β-NuRD complex identified inthis study.Our findings are also relevant to understanding pathways that

lead to autoimmunity. Aberrant or altered interactions with targetgene loci by the Bcl6–OPN-i–Mi-2β-NuRD complex in TFH andTFR cells are likely to be associated with dysregulated differenti-ation of these cells, and potential autoimmune or inflammatorysequelae. Analysis of the chromatin landscape surrounding genestargeted by the Bcl6–OPN-i–Mi-2β-NuRD complex in follicularCD4+ T cells from autoimmune-prone and autoimmune-resistantmouse strains may reveal new disease susceptibility loci and amolecular foothold for new approaches to these disorders.

MethodsMice. C57BL/6J (B6), Tcra−/−, OT-II transgenic [B6.Cg-Tg(TcraTcrb)425Cbn/J],Blimp1-YFP reporter [B6.Cg-Tg(Prdm1-EYFP)1Mnz/J] (Jackson Labs), Rag2−/−

Prf1−/−, B6SJL (CD45.1) (Taconic Farms), Spp1flstopCre+, and Cre– littermates(4) were housed in pathogen-free conditions and used at 7–12 wk of age.Experiments were performed in an unblinded fashion, with both sexes in-cluded for all experiments. All experiments were performed in compliancewith federal laws and institutional guidelines as approved by Dana-FarberCancer Institute’s Animal Care and Use Committee.

Statistical Analyses. Statistical analyses were performed using two-tailed,unpaired Student’s t test or Mann–Whitney test with the assumption ofequal sample variance, with GraphPad Prism V6 software. Error bars indicatemean ± SEM. A P value < 0.05 was considered to be statistically significant(*≤ 0.05, **≤ 0.01, ***≤ 0.001). No exclusion of data points was used.Sample size was not specifically predetermined, but the number of miceused was consistent with previous experience with similar experiments.

Additional methods are provided in SI Appendix.

ACKNOWLEDGMENTS. We thank H.-J. Kim for critical reading and insightfulcomments, and A. Angel for manuscript/figure preparation. These studieswere supported in part by research grants from the National Institutes ofHealth (AI48125 and AI37562) and LeRoy Schecter Research Foundation (toH.C.), and the University of Alabama at Birmingham start-up funds (toJ.W.L.), the National Natural Science Foundation of China (31500712) andScience and Technology Program of Guangzhou (201707010350) (to E.S.),and a Fellowship from the Sahlgrenska Academy, University of Gothenburg andFoundation Blanceflor Boncompagni Ludovisi, née Bildt (to H.R.).

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