Chromatin Remodeling by the Nurd Complex Regulates Development of Follicular Helper and Regulatory T Cells

Chromatin remodeling by the NuRD complex regulates development of follicular helper and regulatory T cells

Erxia 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 30 Göteborg, 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, AL 35233

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 Tcellsubsets The Mi-2β-nucleosome-remodeling deacetylase complex (Mi- reflect an interplay between chromatin regulators and transcription 2β-NuRD) couples a histone deacetylase and a nucleosome- factors (TF). Follicular T cell development is regulated by the Bcl6 TF, stimulated ATPase to several corepressors, including a family which helps determine the phenotype and follicular localization of of metastasis-associated (MTA) proteins (11, 12), which can + repress transcription following interactions with site-specific both CD4 follicular helper T cells (TFH) and follicular regulatory DNA binding proteins (11). Previous studies have indicated Tcells(TFR). Here we show that Bcl6-dependent control of follicular β T cells is mediated by a complex formed between Bcl6 and the Mi- that B cell development may reflect recruitment of Mi-2 -NuRD β β to Bcl6 target loci by MTA3, a cell-type-specific subunit of the 2 -nucleosome-remodeling deacetylase complex (Mi-2 -NuRD). For- β mation of this complex reflects the contribution of the intracellular Mi-2 -NuRD complex (12). Recent analysis of the Bcl6 sec- ondary repression domain (Bcl6-RD2) has also suggested that isoform of osteopontin (OPN-i), which acts as a scaffold to stabilize + MTA3 may interact with Bcl6 in CD4 T cells (13). However, binding between Bcl6 and the NuRD complex that together regulate FH whether Bcl6, MTA3, and Mi-2β-NuRD form a complex in TFH the genetic program of both TFH and TFR cells. Defective assembly and T cells and the impact of a putative Bcl6–MTA3–Mi-2β- – FR of the Bcl6 NuRD complex distorts follicular T cell differentiation, NuRD complex on follicular T cell differentiation during an resultinginimpairedTFR development and skewing of the TFH line- immune response is unknown. + age toward a TH1-like program that includes expression of Blimp1, Our recent analysis of CD4 T-helper responses has revealed Tbet, granzyme B, and IFNγ. These findings define a core Bcl6-directed + that expression of the intracellular isoform of osteopontin (OPN- transcriptional complex that enables CD4 follicular T cells to regulate i) is essential for the differentiation of both follicular T cell the germinal center response. subsets –TFH and TFR cells (4). For example, analysis of TFH cells indicates that engagement of ICOS on TFH and TFR cells follicular helper T cells | follicular regulatory T cells | promotes nuclear translocation of OPN-i, binding to Bcl6 via the germinal center response | osteopontin | Bcl6 transcription factor RD2 domain and protection of the Bcl6–OPN-i complex from proteasomal degradation to allow sustained TFH/TFR responses he germinal center (GC) response is a highly dynamic pro- following initial lineage commitment (4). Tcess in tissues where high levels of dying cells provide a battery of self-antigens that can activate autoreactive antibody Significance responses (1). Generation of high-affinity antibodies and avoid- ance of autoimmune responses after microbial infection or vacci- Production of high-affinity antibody responses after infection nation requires precise control of the GC reaction, depending, to a or vaccination requires precise control of germinal center B cells + large degree, on the combined activities of CD4 follicular helper by follicular helper T cells and follicular regulatory T cells. Al- T(TFH) and follicular regulatory T (TFR)cells(2–6). TFH cells that though the Bcl6 transcription factor plays a central role in fol- + arise from naive CD4 T cells induce GC formation and help B licular T cell differentiation, the molecular basis of Bcl6 control cells to produce protective antibody responses to invading patho- has been clouded in uncertainty. Here we report that Bcl6- gens through generation of memory B cells and long-lived plasma dependent control reflects the formation of a macromolecular + β cells (2, 3, 7). TFR cells that originate from FoxP3 Treg precursors complex between Bcl6 and the Mi-2 -nucleosome remodeling β dampen TFH-driven GC responses and can prevent the emergence of deacetylase complex (Mi-2 -NuRD). The repressive activity of autoreactive B cells and consequent autoantibody production (4–6). this intranuclear complex potentiates the follicular T cell phe- While TFH and TFR cells have opposing functions, shared expression notype and inhibits alternative T cell fates. Identification of this of the Bcl6 TF serves to repress alternative differentiation pathways intracellular complex may facilitate new targeted approaches (5, 6). Although engagement of the T cell antigen receptor (TCR) to the treatment of autoimmune disorders. and costimulatory receptor inducible T cell costimulator (ICOS) has been implicated in this process (4–6, 8), the conserved genetic and Author contributions: E.S., Q.W., H.C., and J.W.L. designed research; E.S., Q.W., H.R., W.L., epigenetic mechanisms that ensure Bcl6-directed differentiation of and J.W.L. performed research; E.S., Q.W., H.R., W.L., H.C., and J.W.L. analyzed data; and E.S., H.C., and J.W.L. wrote the paper. this critical pair of follicular T cells remain largely unknown. + Reviewers: M.L.S., Duke University School of Medicine; and G.C.T., Beth Israel Deaconess Differentiation of CD4 T cells following engagement of the Medical Center and Harvard Medical School. TCR and costimulatory receptors is determined by changes in gene expression, which in part reflect chromatin modifications The authors declare no conflict of interest. that shape transcription, differentiation, and cellular replica- Published under the PNAS license. tion. Regulation of gene expression during differentiation of 1E.S. and Q.W. contributed equally to this work. 2 TFH and TFR cells depends on Bcl6-dependent recruitment of To whom correspondence may be addressed. Email: [email protected] or corepressor complexes that help shape the chromatin landscape [email protected]. surrounding Bcl6 target loci, including Prdm1 (encoding This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Blimp1) and other genes that may promote alternative T-helper 1073/pnas.1805239115/-/DCSupplemental. (TH)-cell fates (9, 10). Published online June 11, 2018.

6780–6785 | PNAS | June 26, 2018 | vol. 115 | no. 26 www.pnas.org/cgi/doi/10.1073/pnas.1805239115 Downloaded by guest on September 30, 2021 Here we analyze the transcriptional events that confer commitment to the two major follicular T cell lineages. We noted a surprising and profound defect in early TFH/TFR lineage commitment by OPN-i–deficient cells despite intact Bcl6 protein levels. Analyses of the complex formed by OPN-i, Bcl6, and Mi-2β-NuRD revealed that the OPN-i protein acts as a scaffold that supports the formation of a complex between Bcl6 and MTA3 that mediates the genetic programming of TFH and TFR cells (SI Ap- pendix, Fig. S1). Additional interrogation of the biologic activity of this complex revealed that OPN-i–dependent recruitment of the Bcl6–Mi-2β-NuRD complex to Bcl6 target loci is a critical step in the transcriptional repression of the Prdm1 locus and commitment to the TFH and TFR cell genetic program. Results

OPN-i Deficiency Impairs TFH and TFR Early Commitment. To define the impact of OPN-i deficiency on early commitment of TFH and flstop TFR cells, we used Spp1 mice bearing a mutated Spp1 allele that allows expression of the OPN-i isoform after Cre-mediated + recombination. 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 cells from OPN-i-KI or OPN-KO mice that coexpress the OT-II [ovalbumin (OVA)-specific] TCR transgene. Since TFH commitment occurs as early as 72 h in vivo (8), we analyzed T cell + H differentiation at 2.5 d after transfer of these CD4 T cells along −/− −/− with B cells into Rag2 Prf1 mice followed by immunization Fig. 1. OPN-i deficiency impairs TFH and TFR early commitment. (A and B) with NP13-OVA in Complete Freunds’ Adjuvant (CFA) (Fig. 1). + FACS analysis of TH cell differentiation at day 2.5 after transfer of OT-II CD4 Bcl6 protein levels were not affected by OPN-i deficiency at this −/− −/− + T cells and B cells into Rag Prf1 mice followed by immunization with early time point (Fig. 1A) (4). However, OPN-KO CD4 T cells 2 NP13-OVA in CFA. (A) Histogram overlays of intracellular protein expression displayed a marked impairment in T commitment, as reflected – + + FH (gated on FoxP3 CD4 Tcells).(B) Plots of non-TFH and TFH phenotype – + by reduced proportions of CD4 T cells expressing CXCR5 (gated on FoxP3 CD4 T cells) and mean ratios of non-TFH to TFH cells are compared with OPN-i-KI cells (Fig. 1B). A bifurcation between shown for OPN-i-KI and OPN-KO mice (n = 3–4 for each group). GzmB, + TFH and other effector TH cells, particularly TH1 cells, occurs granzyme B. (C and D) Treg from CD45.2 WT, OPN-i-KI, or OPN-KO mice + + −/− during early TH cell fate determination (8). Analysis of the TH1- were transferred along with naive CD45.1 CD4 T cells into Tcra mice cell-associated phenotype of these differentiating cells revealed followed by immunization with NP -OVA in CFA. Analysis of CD45.2+ + 13 that a substantially increased proportion of OPN-KO CD4 Tregcells(gatedonFoxP3+) 3 d postimmunization. Histogram overlays (C) T cells expressed the Tbet, Ly6C, and granzyme B triad, which and quantitation of mean fluorescence intensity (MFI) (D) of each protein characterize a T 1-like phenotype (14). As a consequence, OPN- (n = 3 for each group). Data shown are representative of three in- + H + + KO CD4 T cells displayed an increased ratio of triad CD4 dependent experiments (*P < 0.05 and **P < 0.01). Error bars indicate + + T cells to CXCR5 CD4 T cells compared with their OPN-i-KI mean ± SEM. counterparts (Fig. 1B), suggesting that OPN-i deficiency might impair early TFH commitment and precede the reduced – – Bcl6 protein levels that occur later in the response (4). OPN-i Interacts with MTA3 to Promote the Formation of a Bcl6 MTA3 NuRD Complex. An interaction between Bcl6 and the Mi-2β-NuRD Bcl6-dependent differentiation of TFH cells includes repression complex via the MTA3 corepressor contributes to Bcl6 transcrip- INFLAMMATION of an alternative Blimp1-associated non-TFH program (Fig. 1) (9, IMMUNOLOGY AND 15). We therefore asked whether OPN-i deficiency altered the tional repressive activity and B cell fate (12). Previous studies have + also indicated that, in response to TCR and ICOS signals, a Bcl6−Blimp1 balance during early CD4 TH cell differentiation. We used Blimp1-YFP reporter mice to generate Blimp1-YFP×OPN-KO pool of OPN-i translocates into the nucleus to interact with Bcl6 via the Bcl6-RD2 domain (4). The above findings that a mice and Blimp1-YFP×OPN-i-KI mice. Analysis of TFH differentiation at day 2.5 postimmunization revealed that the proportions of complex formed by OPN-i and Bcl6 might regulate early TFH + − – Blimp1 CD4 effector T cells (FoxP3 ) were considerably higher in and TFR commitment led us to ask whether OPN-i dependent OPN-KO mice than OPN-i-KI mice, despite unimpaired Bcl6 pro- formation of a Bcl6–MTA3–Mi-2β-NuRD complex might me- tein expression (SI Appendix,Fig.S2). Moreover, higher frequen- diate Bcl6-dependent TFH and TFR differentiation. We first ana- + + + cies of Blimp1 FoxP3 CD4 Treg were also noted in OPN-KO lyzed 293T cells transfected with vectors expressing HA-tagged mice compared with OPN-i-KI mice (SI Appendix,Fig.S2), opening MTA3, OPN-i, and Flag-tagged Bcl6 followed by immunopre- the possibility that OPN-i deficiency might impair Bcl6-dependent cipitation with anti-Flag antibody. Consistent with previous re- repression of Blimp1 transcription in both TFH and TFR cells. ports (4, 12), immunoblot analysis revealed that Bcl6 interacted We then asked whether early TFR differentiation was also affected with both MTA3 and OPN-i, either directly or indirectly. We by OPN-i deficiency using the approach described above (Fig. 1 A also noted that the Bcl6–MTA3 association was enhanced in and B). We transferred CD25hi Treg cells from WT, OPN-KO, or direct proportion to increasing concentrations of OPN-i (Fig. 2A, + OPN-i-KI mice along with naive CD4 T cells from CD45.1 con- Left). Deletion of the N-terminal portion of the Bcl6-RD2 region −/− Δ – – genic mice into Tcra mice followed by immunization with NP13- ( 120 300), which disrupts the Bcl6 OPN-i interaction (4), OVAinCFA.After2.5d,OPN-KObutnotOPNWTorOPN-i-KI prevented OPN-i–dependent enhancement of the Bcl6–MTA3 Treg displayed elevated expression of Blimp1 and Tbet but reduced association (Fig. 2A, Right). Moreover, enhanced binding of + expression of CXCR5 by FoxP3 T cells (Fig. 1 C and D), suggesting Bcl6 to MTA3, a component of the Mi-2β-NuRD complex, was that OPN-i deficiency skewed Treg away from the conventional associated with increased binding of Bcl6 to Mi-2β (Fig. 2A), a follicular phenotype. Taken together, these results suggested that central component of the Mi-2β-NuRD complex (11, 12). – OPN-i might regulate early TFH and TFR commitment, in part We then asked whether OPN-i mediated enhancement of through enhanced Bcl6-dependent repression of alternative genetic Bcl6–MTA3–Mi-2β-NuRD complex formation noted above might + programs that might depend on Blimp1 expression. be apparent in primary CD4 T cells. Analysis of Bcl6-associated

Shen et al. PNAS | June 26, 2018 | vol. 115 | no. 26 | 6781 Downloaded by guest on September 30, 2021 + proteins expressed by CD4 T cells from OT-II×OPN-i-KI mice + compared with CD4 T cells from OT-II×OPN-KO mice 3 d after immunization indicated that OPN-i deficiency greatly reduced the association of Bcl6 with MTA3 and Mi-2β in OPN-KO + CD4 T cells, despite unaltered Bcl6 protein expression (Fig. 2B). These results suggested that OPN-i promoted formation of the Bcl6–MTA3–Mi-2β-NuRD complex. The regulatory activity of the Mi-2β-NuRD complex depends on the activity of its corepressor components, including MTA3 family members, which may demarcate distinct forms of Mi-2β- NuRD that control cell-type-specific transcription (11, 16). We noted that MTA3 bound to both Bcl6 and OPN-i within the nu- + cleus of CD4 T cells (Fig. 2C). We further defined the OPN-i interaction with MTA3 according to mutational analysis (Fig. 2D). We found that a specific interaction between OPN-i and the ELM2 domain of MTA3 promoted binding of the complex to Bcl6. Thus, MTA3-ELM2 deletion mutants (but not MTA3-WT or MTA3-BAH mutants) failed to bind to Bcl6, as judged by anti- Flag (Bcl6) immunoprecipitation (Fig. 2E). These findings suggest that binding of OPN-i to both Bcl6 and MTA3 allows OPN-i to function as a scaffold or bridge to promote the association of Bcl6 with the Mi2β-NuRD complex (SI Appendix,Fig.S1).

OPN-i Promotes Bcl6–MTA3-Dependent Repression of Prdm1/Ifnγ Expression by TH1 Cells. Repression of Blimp1 and other non-TFH genes by Bcl6 plays a central role in TFH commitment and mainte- nance of the TFH phenotype (9, 10). To determine whether the OPN-i–dependent association between Bcl6 and MTA3–Mi-2β- NuRD noted above contributed to Bcl6 transcriptional repression of canonical TH1 genes, we asked whether forced expression of Bcl6 alone or with MTA3 in T 1 cells [which do not express sig- H + nificant levels of Bcl6 or MTA3 (4)], might reprogram this CD4 TH subset. We therefore infected in-vitro–differentiated TH1 cells [after 5 d culture as described previously (17)] with retroviruses expressing Bcl6, MTA3, or both Bcl6 and MTA3. Quantitative RT-PCR analysis of TH1-associated gene expression showed that retroviral coexpression of Bcl6 and MTA3, but not expression of either retrovirus alone, substantially repressed both Prdm1 and Ifnγ expression (Fig. 3A). The specificity of this response was confirmed by the finding that transduction of these TH1 cells with a retrovirus expressing the N-terminal Bcl6-RD2 deletion mutant (Δ120–300), which impairs the MTA3–Bcl6 interaction (Fig. 2A), failed to repress Prdm1 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), we + tested the functional impact of this interaction on the CD4 Tcell genotype in vitro. We observed that transduction of T 1cells(after – + H differentiation from CD25 CD4 T cells from OT-II×OPN-i-KI mice) with a retrovirus expressing Bcl6 and the MTA3 protein but not the MTA3-ELM2 deletion mutant suppressed Prdm1 or Ifnγ expression (Fig. 3C). Moreover, limiting concentrations of Bcl6 and MTA3, which did not repress Prdm1 or Ifnγ, fully repressed these genes in the presence of OPN-i (Fig. 3D), consistent with earlier findings that coexpression of OPN-i enhances the repressive efficiency of Bcl6–MTA3. These findings together are consistent with the ability of OPN-i to promote the biochemical association of Bcl6 with MTA3–Mi-2β-NuRD (Fig. 2).

+ + purified CD4 CD44 T cells from mice at day 5 postimmunization with NP26- KLH in CFA were immunoprecipitated with anti-MTA3 and immunoblotted Fig. 2. Interaction of OPN-i with MTA3 increases Bcl6–MTA3–NuRD complex with Abs to Bcl6 and OPN. Input, immunoblot analysis of an aliquot of lysate formation. (A) Cotransfection of 293T cells with vectors expressing Flag-Bcl6 without IP. (D) MTA3–OPN-i interaction depends on ELM2 domain of MTA3. WT or Flag-Bcl6-RD2 deletion mutant and HA-MTA3 without or with in- 293T cells were cotransfected with vectors expressing HA-MTA3 WT or its creasing concentrations of OPN-i was followed by immunoprecipitation (IP) deletion mutants (diagramed above) with or without OPN-i, followed by IP with anti-Flag antibody (Ab) and immunoblotting (WB) with indicated Abs. with anti-HA and immunoblotting with anti-HA and anti-OPN Abs. (E) De- – + (B) Cell lysates of purified CD44hiCD25 CD4 T cells from OT-II×OPN-i-KI or letion of the ELM2 domain impairs the Bcl6–MTA3 interaction. 293T cells

OT-II×OPN-KO mice 3 d after NP13-OVA immunization were immunopreci- were cotransfected with plasmids expressing Flag-Bcl6, HA-MTA3 WT, or its pitated with anti-Bcl6 Ab or rabbit IgG control before immunoblotting with deletion mutants with or without OPN-i, followed by IP with anti-Flag Ab Abs to Bcl6, MTA3, OPN, and Mi-2β.(C) Interaction of OPN-i and the and immunoblotting with the indicated Abs. Data shown are representative + MTA3 component of NuRD complex in CD4 T cells. Nuclear lysates of of three independent experiments.

6782 | www.pnas.org/cgi/doi/10.1073/pnas.1805239115 Shen et al. Downloaded by guest on September 30, 2021 Fig. 3. OPN-i promotes Bcl6–MTA3-dependent repression of Prdm1 and Ifnγ

expression in TH1 cells, which requires the MTA3 ELM2 domain. (A) OT-II TH1 cells were transduced with retroviral vectors expressing either Flag- Bcl6 or HA-MTA3 alone [multiplicity of infection (MOI) = 10]; or a mixture containing constant Flag-Bcl6 concentrations (MOI = 10) combined with in-

creasing concentrations of HA-MTA3 (wedge: 2.5, 5, 10). (B) OT-II TH1 cells were 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 cells were transduced with a mixture of retroviral vectors expressing constant concentrations of Flag-Bcl6 (MOI = 10) with HA-MTA3 ELM2 deletion mutant

(MOI = 10). (D) OT-II TH1 cells were transduced with retroviral vectors expressing [Flag-Bcl6 (MOI = 5) + HA-MTA3 (MOI = 5)] or OPN-i (MOI = 5), or Flag-Bcl6 + HA-MTA3 + OPN-i (each at a suboptimal MOI of 5). qRT-PCR was performed after 2.5 d. Gene expression was normalized to expression of the control gene Rps18 (encoding ribosomal protein S18) and presented as relative to cells transduced with control virus, set as 1. Data shown are representative of three independent experiments (*P < 0.05, **P < 0.01, and ***P < 0.001). Error bars indicate mean ± SEM. INFLAMMATION IMMUNOLOGY AND

Promotion of TFH and TFR Differentiation in Vivo Requires an Interaction Between OPN-i and MTA3. The specificity of the OPN-i–Bcl6– MTA3 interaction described above is supported by findings that deletion of the ELM2 domain of MTA3 disrupts binding of OPN-i to MTA3 (Fig. 2D), impairs MTA3 binding to Bcl6 (Fig. 2E), and decreases Bcl6–MTA3-dependent repression of Prdm1/Ifnγ expression (Fig. 3C). We then tested the physiological relevance of – – – Fig. 4. OPN-i mediated promotion of TFH differentiation requires intact the OPN-i Bcl6 MTA3 interaction to TFH and TFR differentiation OPN-i−MTA3 interaction. (A and F) Schematic diagrams of experimental in vivo using a retroviral reconstitution system (4). We transduced × + + protocols. Purified naive OT-II OPN-i-KI CD4 T cells were activated in vitro, in-vitro–activated OT-II CD4 T cells with a retroviral vector transduced with retroviral vector encoding GFP alone (EV) or GFP plus WT expressing GFP alone (empty vector, EV) or GFP plus either WT MTA3 (MTA3) or deletion mutant MTA3 (delELM2). GFP+ CD4+ T cells (A)and MTA3 (MTA3) or the MTA3-ELM2 deletion mutant (delELM2). GFPhi or GFPmed-lo CD4+ T cells (F) were then sorted and transferred into −/− – Since MTA3 and delELM2 are expressed within the same bicis- Tcra hosts followed by immunization with NP13-OVA in CFA. B E were tronic IRES retroviral vector as GFP, their expression is correlated analyzed from protocol A, and G and H from protocol F.(B and G) FACS + − − – + / analysis of TFH cells (gated on FoxP3 CD4 T cells) and GC B cells (gated on with GFP levels. We transferred sorted GFP cells into Tcra + B220 cells) 10 d postimmunization. (D and H) Histogram overlays of Bcl6, hosts followed by immunization with NP13-OVA in CFA (Fig. 4A + – Tbet, Ly6C, and Blimp1 expression in donor CD4 T cells (gated on FoxP3 and SI Appendix,Fig.S3A). T differentiation and associated GC + FH + CD4 T cells). (E) Quantitation of MFI of each protein and frequency of B cell formation were increased for OT-II CD4 T cells transduced + + – + Ly6C CD4 FoxP3 cells in D. Data shown are representative of two in- to express WT MTA3 compared with CD4 T cells transduced + dependent experiments. (C) Ectopic expression of the delELM2 mutant in with EV (Fig. 4B). In contrast, transduction of OT-II CD4 T cells + OT-II CD4 T cells impairs the Ab response postimmunization. Anti-NP23 or with the delELM2 mutant resulted in decreased numbers of TFH anti-NP4 antibody titers were determined from mice transferred with OT-II + and GC B cells (Fig. 4B). Consequently, both the total (anti-NP23) CD4 T cells expressing empty control, WT MTA3, or delELM2 mutant fol- and high-affinity (anti-NP4) NP-specific antibody responses were lowed by immunization, as in A.***P < 0.001, and ns, no significance. Error markedly impaired (Fig. 4C). Although Bcl6 levels were not altered bars indicate mean ± SEM.

Shen et al. PNAS | June 26, 2018 | vol. 115 | no. 26 | 6783 Downloaded by guest on September 30, 2021 binding of the Bcl6−MTA3−NuRD complex reflected increased binding to Bcl6 target loci. We focused on Prdm1, since Bcl6-dependent repression of Prdm1 is a key element in the determination of TFH and TFR cell fate (Fig. 3 and SI Appendix,Fig.S2). We noted that the mRNA levels of Prdm1 were substantially up-regulated in OPN-KO TFH and TFR cells compared with OPN-i-KI cells 3 d postimmunization, despite unaltered Bcl6 mRNA levels in these cells (Fig. 6 A and B). We performed a chromatin immunoprecipitation (ChIP)-qPCR analysis of the Bcl6 and MTA3 occupancy on the conserved Bcl6 response element (BRE) within Prdm1. We observed that Bcl6 binding to the Prdm1 BRE region was substantially decreased in OPN-KO Fig. 5. OPN-i–mediated promotion of T differentiation requires intact FR TFH cells, consistent with a failure of Bcl6 to repress Prdm1 in OPN-i−MTA3 interaction. (A) Schematic diagram of experimental procedure. OPN-i–deficient TFH cells (Fig. 6A). Moreover, analysis of OPN- Purified CD45.2+ Treg were activated in vitro, transduced with retroviral KO TFH cells revealed an almost complete loss of MTA3 bound to vector encoding GFP alone (EV), or GFP plus WT MTA3 (MTA3) or deletion + the Prdm1 BRE locus (Fig. 6C), further suggesting severely im- mutant MTA3 (delELM2). GFP Treg were then sorted and transferred into −/− + + paired recruitment to this canonical Bcl6 target gene. Since Tcra hosts along with CD45.1 naive CD4 T cells followed by immunization Bcl6 transcriptional repression is mediated in part by recruiting with NP13-OVA in CFA. (B) Frequency of TFR,TFH,andGCBcellsinSI Appendix, β Fig. S4. Data shown are representative of two independent experiments. *P < histone deacetylases to target loci via the Mi-2 -NuRD complex, we 0.05, **P < 0.01, ns, no significance. Error bars indicate mean ± SEM. asked whether OPN-i deficiency influenced the histone acetylation status surrounding Bcl6-bound loci. There was almost no acetylated H3 (AcH3) at the Prdm1 BRE locus of OPN-i-KI TFH cells, con- among cells expressing GFP alone, MTA3, or the delELM2 mu- sistent with a repressive chromatin status. In contrast, these loci tant, transduction with the delELM2 mutant exerted a “dominant displayed increased levels of AcH3 in OPN-KO TFH cells (Fig. 6C), consistent with an active chromatin locus in the absence of OPN-i. negative” impact on TFH cell function, as judged by increased Tbet, Ly6C, and Blimp1 expression (Fig. 4 D and E). Taken together, these results indicate that OPN-i is required for + The above finding that CD4 T cells expressing the delELM2 efficient binding of Bcl6 and MTA3 to a major Bcl6 target gene as well as associated repression of this locus. mutant failed to differentiate into functional TFH cells led us to ask whether the delELM2 MTA3 mutant could compete with endogenous MTA3 to interfere with T differentiation. To address this, Discussion + FH we transduced OT-II CD4 T cells with a retroviral vector Specification of T cell fate reflects the concerted action of chro- expressing GFP alone or GFP plus the delELM2 mutant, and then matin regulators and transcription factors in response to signals + transferred sorted GFPhi or GFPmed-lo CD4 T cells separately emanating mainly from the TCR and costimulatory receptors. Our −/− into Tcra mice followed by immunization with NP13-OVA in studies suggest that the functional differentiation of TFH and TFR CFA (Fig. 4F and SI Appendix,Fig.S3B). Consistent with these cells is mediated, in part, by recruitment of the Mi-2β-NuRD results (Fig. 4B), T differentiation was decreased for OT-II complex to specific Bcl6 target loci. The formation of this complex + FH + CD4 T cells transduced to express the delELM2 mutant com- in differentiating CD4 T cells requires the scaffold-like contribu- pared with cells expressing GFP alone, which was associated with tion of OPN-i to the binding of Bcl6 to the MTA3–Mi-2β-NuRD reduced GC B cells (Fig. 4G). The frequencies of T and GC B complex and formation of a biologically active corepressor complex. FH + cells were also not affected by GFP levels in OT-II CD4 Tcells The transcriptional activity of Bcl6 in other cell types may also expressing GFP alone. In contrast, the extent of T differentia- reflect recruitment of different corepressor complexes to different FH + tion and GC B cell formation in mice transferred with OT-II CD4 Bcl6 domains and the formation of target-specific complexes. For T cells expressing delELM2 was negatively correlated with levels of example, an interaction between the Bcl6-BTB domain and the delELM2 (GFP) and associated with increased expression of non– BCOR/SMART corepressors promotes GC B cell differentiation TFH-associated markers (Tbet, Ly6C, and Blimp1) (Fig. 4 G and without a significant effect on the TFH cell response (18). In con- H). These results suggest that expression of the ectopic delELM2 trast, previous studies of TFH differentiation have underlined the mutant might impede TFH differentiation by competing with the significance of an interaction between the Bcl6-RD2 domain and endogenous MTA3 in a dose-dependent manner. MTA3 (13) as well as a second interaction with OPN-i (4). Here we Using a retroviral reconstitution system similar to that described identify OPN-i as a critical bridging intermediary that facilitates above, we evaluated the physiological contribution of the OPN-i– binding between Bcl6 and MTA3 and promotes the formation of a Bcl6–MTA3 interaction to the formation of T cells. We trans- + + + FR duced CD45.2 WT CD25 CD4 T cells with retroviral vectors expressing GFP alone (EV) or GFP plus WT MTA3 (MTA3) or delELM2 mutant (delELM2), then transferred each group of cells + – + − − together with CD45.1 CD25 CD4 T cells into Tcra / mice, followed by immunization of hosts with NP -OVA in CFA (Fig. 13 + 5A). TFR differentiation was increased for CD45.2 Treg transduced to express WT MTA3 compared with cells transduced with EV (Fig. 5B and SI Appendix,Fig.S4). In contrast, expression of + the delELM2 mutant in CD45.2 Treg decreased TFR differentiation to levels comparable to cells expressing GFP alone, which Fig. 6. OPN-i promotes binding of Bcl6 and MTA3 to Bcl6 target genes and regulates Bcl6 transcriptional activity. (A and B) qRT-PCR analysis of Prdm1 was associated with increased TFH differentiation and GC B cell > formation (Fig. 5B and SI Appendix,Fig.S4). These results indicate and Bcl6 in sorted pure ( 95%) TFH cells (A)orTFR cells (B) from OPN-i-KI and − − − OPN-KO mice at day 3 postimmunization with NP13-OVA in CFA. Gene ex- that an OPN-i MTA3 interaction required for Bcl6 MTA3 pression was normalized to expression of the control gene Rps18 (encoding NuRD complex formation in vitro is also essential for TFH and ribosomal protein S18) and expressed as relative to T or T cells from OPN- T differentiation in vivo. FH FR FR i-KI mice, set as 1. (C) In-vitro–differentiated TFH cells from OT-II×OPN-i-KI or OT-II×OPN-KO mice were cross-linked, chromatin prepared, and ChIP-PCR OPN-i Promotes Binding of Bcl6 and MTA3 to Bcl6 Target Genes and analyses performed for Bcl6, MTA3, and Acetylated H3 (AcH3) at the BRE of Regulates Bcl6 Transcriptional Activity. To gain insight into the genetic Prdm1 gene. Data, shown as the percent of input, reflecting enriched mechanisms that underpinned OPN-i–mediated enhancement of binding at the indicated loci, are representative of three independent ex- TFH and TFR differentiation, we asked whether OPN-i–promoted 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. Downloaded by guest on September 30, 2021 Bcl6–NuRD complex that is equipped to direct both TFH and TFR predominantly to promoter regions (23), while analysis of cell differentiation. The extended and flexible structure of OPN-i, a murine TFH cells has revealed about 5,100 Bcl6 binding peaks member of the SIBLING protein family (19), may permit inter- localized mainly to intron and intergeneic regions (24). It is actions with a variety of partners, including the Mi-2β-NuRD likely that a more precise identification of the key target genes macromolecule in the nucleus, as described here, as well as with that control TFH and TFR differentiation may come from proteasomal complexes in the cytosol, as noted previously (20), to identification of the genetic loci that are cooccupied by both promote Bcl6-directed differentiation of follicular T cells. Bcl6 and the partner Mi-2β-NuRD complex identified in These findings provide insight into the epigenetic mechanisms this study. that govern lineage commitment of the follicular T cell pair that Our findings are also relevant to understanding pathways that regulates GC antibody and autoantibody responses. Our findings lead to autoimmunity. Aberrant or altered interactions with target also help clarify the differentiative relationship between the TFH gene loci by the Bcl6–OPN-i–Mi-2β-NuRD complex in TFH and and TFR cell lineages. Although TFH and TFR cells coexpress TFR cells are likely to be associated with dysregulated differenti- Bcl6 as well as several surface receptors, the shared genetic el- ation of these cells, and potential autoimmune or inflammatory ements responsible for follicular differentiation of these two + sequelae. Analysis of the chromatin landscape surrounding genes CD4 T cell lineages have been obscure. We have reported targeted by the Bcl6–OPN-i–Mi-2β-NuRD complex in follicular + previously that TFH and TFR cells may share an ICOS-dependent CD4 T cells from autoimmune-prone and autoimmune-resistant pathway that promotes the formation of an intranuclear complex mouse strains may reveal new disease susceptibility loci and a between Bcl6 and OPN-i that protects the Bcl6 protein from molecular foothold for new approaches to these disorders. proteasomal degradation (4). Here we identify an additional role for OPN-i in TFH differentiation, i.e., integration of Bcl6 with Methods β Mi-2 -NuRD to form biologically active complexes that enhance Mice. C57BL/6J (B6), Tcra−/−, OT-II transgenic [B6.Cg-Tg(TcraTcrb)425Cbn/J], TFH and TFR lineage differentiation. Regulation of Prdm1 and Blimp1-YFP reporter [B6.Cg-Tg(Prdm1-EYFP)1Mnz/J] (Jackson Labs), Rag2−/− other canonical target genes by this complex may account for Prf1−/−, B6SJL (CD45.1) (Taconic Farms), Spp1flstopCre+, and Cre– littermates core features shared by differentiated TFH and TFR cells that (4) were housed in pathogen-free conditions and used at 7–12 wk of age. reside in the germinal centers and lymphoid tissue follicles. Experiments were performed in an unblinded fashion, with both sexes in- Formation of this Bcl6 complex may represent a critical down- cluded for all experiments. All experiments were performed in compliance stream consequence of the ICOS-dependent pathway that favors + with federal laws and institutional guidelines as approved by Dana-Farber the differentiation of follicular T cells from CD4 precursors (4, Cancer Institute’s Animal Care and Use Committee. 8). Since the ratio of TFH to TFR cells has a direct impact on the intensity and quality of GC antibody responses (4, 21), a detailed Statistical Analyses. Statistical analyses were performed using two-tailed, correlation between the TFH/TFR ratio and the intensity and unpaired Student’s t test or Mann–Whitney test with the assumption of quality of the B cell response at defined intervals after immu- equal sample variance, with GraphPad Prism V6 software. Error bars indicate nization is necessary to fully evaluate the impact of this Bcl6- mean ± SEM. A P value < 0.05 was considered to be statistically significant containing complex on the immune response. (*≤ 0.05, **≤ 0.01, ***≤ 0.001). No exclusion of data points was used. Our finding that Bcl6 transcriptional activity in TFH cells de- Sample size was not specifically predetermined, but the number of mice pends on its association with the complex described here also used was consistent with previous experience with similar experiments. suggests that anti-Bcl6-based ChIP-seq analysis of TFH cells may Additional methods are provided in SI Appendix. lack the specificity necessary to precisely define the genetic program of TFH (and TFR) cells. A precedent for this comes from ACKNOWLEDGMENTS. We thank H.-J. Kim for critical reading and insightful analysis of early B cell differentiative steps that are regulated by comments, and A. Angel for manuscript/figure preparation. These studies Ikaros–Mi-2β-NuRD complexes. These studies indicate that were supported in part by research grants from the National Institutes of combined occupation of target loci in early B cells by Ikaros and Health (AI48125 and AI37562) and LeRoy Schecter Research Foundation (to β H.C.), and the University of Alabama at Birmingham start-up funds (to Mi-2 -NuRD, but not by Ikaros alone, is essential for func- J.W.L.), the National Natural Science Foundation of China (31500712) and tional control of early B cell differentiation genes (22). Recent Science and Technology Program of Guangzhou (201707010350) (to E.S.), genomewide Bcl6 ChIP-seq analysis of human GC TFH cells has and a Fellowship from the Sahlgrenska Academy, University of Gothenburg and

indicated that Bcl6 binds to over 8,500 target loci that localize Foundation Blanceflor Boncompagni Ludovisi, née Bildt (to H.R.). INFLAMMATION IMMUNOLOGY AND

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