NIH Public Access Michela Locci Michael Cho Terence ... · Bcl6 and Maf cooperate to instruct human follicular helper CD4 T cell (Tfh) differentiation Mark A. Kroenke, Danelle Eto,

Bcl6 and Maf cooperate to instruct human follicular helper CD4 Tcell (Tfh) differentiation

Mark A. Kroenke*, Danelle Eto*, Michela Locci*, Michael Cho^, Terence Davidson†, EliasHaddad§, and Shane Crotty*,1

*Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037†Division of Otolaryngology, University of California School of Medicine, San Diego, CA92103-8895§Vaccine & Gene Therapy Institute of Florida, Port St. Lucie, FL 34987^Mission Hospital and Children’s Hospital of Orange County (CHOC), Mission Viejo, CA 92691

AbstractFollicular helper CD4 T cells (Tfh) provide B cells with signals important for the generation ofhigh-affinity antibodies and immunological memory, and are therefore critical for the protectiveimmunity elicited by most human vaccines. Transcriptional regulators of human Tfh celldifferentiation are poorly understood. Here we demonstrate that Bcl6 controls specific genemodules for human Tfh differentiation. The introduction of Bcl6 expression in primary humanCD4 T cells resulted in regulation of a core set of migration genes that enable trafficking togerminal centers: CXCR4, CXCR5, CCR7, and EBI2. Bcl6 expression also induced a module ofprotein expression critical for T:B interactions, including SAP, CD40L, PD-1, ICOS, andCXCL13. This is the first direct evidence for Bcl6 control of most of these functions, and includesthree genes known to be loci of severe human genetic immunodeficiencies (CD40L, SH2D1A,ICOS). Introduction of Bcl6 did not alter expression of IL-21 or IL-4, the primary cytokines ofhuman Tfh cells. We show here that introduction of Maf (c-Maf) does induce the capacity toexpress IL-21. Surprisingly, Maf also induced CXCR5 expression. Co-expression of Bcl6 and Maftogether revealed that Bcl6 and Maf cooperate in the induction of CXCR4, PD-1, and ICOS.Altogether, these findings reveal that Bcl6 and Maf collaborate to orchestrate a suite of genes thatdefine core characteristics of human Tfh cell biology.

IntroductionFollicular helper CD4 T cells (Tfh) are a specialized subset of T cells that provide help to Bcells (1). The defining characteristics of Tfh cells are their ability to co-localize with B cellsin the follicle and their ability to provide specialized help to B cells in the form of specificcytokines and cell surface molecules. Tfh cells are required for germinal centers. In theabsence of Tfh cells, severe reductions are seen in the development of antigen-specific IgGresponses, memory B cells, and memory plasma cells (1). A long-term antibody response isthe central attribute of most successful human vaccines (2, 3), and understanding Tfh cells istherefore important for developing truly rational vaccine development strategies.Furthermore, Tfh cells are potentially useful biomarkers in human vaccine clinical trials. Inorder to harness this biology, however, the transcriptional control of human Tfh cell

1This work was supported by grants from the National Institutes of Health and LIAI institutional funds to SC.Correspondence to: Shane Crotty, Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, 9420 Athena Circle,La Jolla, CA 92037. Phone: 858-752-6816, Fax: 858-752-6993, [email protected].

NIH Public AccessAuthor ManuscriptJ Immunol. Author manuscript; available in PMC 2013 April 15.

Published in final edited form as:J Immunol. 2012 April 15; 188(8): 3734–3744. doi:10.4049/jimmunol.1103246.

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differentiation and function must be understood. Finally, several autoimmune diseases arecharacterized by the presence of increased germinal centers and autoantibodies (4, 5), whichare dependent on Tfh cells in multiple models (6, 7). Understanding the transcriptionalregulators of Tfh cells is therefore also valuable for developing therapeutics against majorautoimmune diseases.

Bcl6 was originally identified as a human B cell oncogene. A significant percentage ofindividuals with follicular lymphoma or diffuse large B cell lymphoma have mutations inBcl6 (8, 9). Bcl6 also plays an important role in acute lymphoblastic leukemia (10). Bcl6 is acritical transcription factor for germinal center B cells (8, 11, 12). Extensive studies on Bcl6in B cells have revealed that Bcl6 regulates germinal center B cell survival, cell cyclecontrol, and somatic hypermutation (8, 10, 13–15). Recently it has been determined thatBcl6 is important in mice for Tfh cell differentiation (16–18). However, it is not known ifand how Bcl6 controls Tfh differentiation of human T cells (1). Importantly, there is limitedevidence of which proteins in CD4 T cells are explicitly regulated by Bcl6 in any species, asthere are no obvious parallels between Bcl6 expressing B cells and Bcl6 expressing CD4 Tcells (13). How specific transcription factors imbue different differentiated CD4 T cell typeswith distinct functional characteristics has been a topic of great interest in immunology (19,20). Here we describe that Bcl6 instructs core modules of Tfh cell biology in human CD4 Tcells, but Bcl6 does not regulate all human Tfh cell features. The transcription factor Mafcontributes significantly to IL-21 production by Tfh cells. Furthermore, Maf and Bcl6 worktogether to maximally induce important Tfh cell characteristics.

Materials and MethodsHuman samples

Fresh human tonsils were obtained from the University of San Diego (UCSD) HillcrestMedical Center, the National Disease Resource Interchange (NDRI), or the Children’sHospital of Orange County (CHOC), Mission Viejo. The majority of tonsils were fromadults. Informed consent was obtained from all donors. Tonsils were homogenized usingwire mesh and passed through a cell strainer to make a single cell suspension. Mononuclearcells were isolated using Histopaque 1077 (Sigma-Aldrich, St. Louis, MO). All protocolswere approved by the LIAI and NDRI, LIAI and UCSD, or LIAI and CHOC InstitutionalReview Boards.

Flow cytometry and sortingFor intracellular staining, cells were restimulated with 25 ng/mL PMA and 1 µg/mLionomycin in the presence of 10 µg/mL brefeldin A for 3 hours. Cells were fixed with BDPhosflow Fix Buffer I and permeabilized with BD Phosflow Perm/wash Buffer I.

All cells were sorted using a BD FACS Aria. All Tfh cell sorts were initially gated on CD4+

CD19− 7-AAD− cells. CD45RO− cells were confirmed to be naïve by co-staining forCD45RA. Naïve B cells were CD19+CD20+CD3−CD27−IgD+. The following anti-humanantibodies were used: CD45RO (clone UCHL1), CD45RA (clone HI100), CD19 (cloneHIB19), CD38 (clone HIT2), CD20 (clone 2H7), CD27 (clone O323), PD-1 (clone J105),ICOS (clone ISA-3), CXCR4 (clone 12G5), CD40L (clone 24–31), CD3 (clone OKT3), andCD4 (clone RPA-T4) (eBioscience, San Diego, CA); CXCR5 (clone RF8B2), CCR7 (clone3D12), Bcl6 (clone K112-91), and IgD (clone IA6-2) (BD Biosciences, Franklin Lakes, NJ);SAP (clone 1D12) (Cell Signaling Technology, Danvers, MA) (21). For intracellularstaining, the following antibodies were used: IL-4 (clone MP4-25D2) (Biolegend, SanDiego, CA); IL-21 (clone 3A3-N2), IL-17 (clone 64DEC17), and IFNγ (clone 4S.B3)

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(eBioscience); CXCL13 (clone 53610) (R&D Systems, Minneapolis, MN) (22). Anti-CD19(clone HIB19) was used as the mouse IgG1 isotype for the CXCL13 stain.

B-T Co-Culture assaySorted tonsil CD4+ T cell subsets CD45RO−CXCR5−, CD45RO+CXCR5−,CD45RO+CXCR5int and CD45RO+CXCR5hi were co-cultured with autologous naïve Bcells (4×104 cells/well) in the presence of SEB (250 ng/ml, Sigma-Aldrich) in 96-well v-bottom plates. In blocking experiments, endogenous IL-21 was neutralized by the inclusionof IL-21R/Fc (20µg/mL; R&D Systems) or an isotype-matched control. Secretion of IgMwas determined by ELISA after 7 days in culture. 96-well Polysorp microtiter plates (Nunc)were coated overnight with monoclonal anti-human IgM (1µg/ml, Abbiotech) in PBS. Allsamples were run in duplicate, and IgM from the co-culture supernatant was detected withHRP-conjugated human IgM Fc (Hybridoma Reagent Laboratory).

T cell culturesSorted cells were stimulated with anti-CD3/CD28 Dyna beads (Invitrogen, Carlsbad, CA) in96 well flat bottom plates at a starting density of 7.5×104 cells/well. Beads were used at aconcentration of 1 µl per well. RPMI medium with 10% fetal calf serum was supplementedwith 2 ng/mL recombinant human IL-7. Cells were split as necessary.

Lentiviral vectors and transductionspHAGE vector containing a PGK promoter, a multiple cloning site, and ZsGreendownstream of an IRES, was obtained from Dr. Richard Mulligan (23). Bcl6 LV (Bcl6-LV)was constructed by cloning the human Bcl6 ORF BC150184 (purchased from OpenBiosystems, Huntsville, AL) into the NotI and BamHI sites of pHAGE, with an optimalKozak sequence. A modified vector pHAGE-PGK-IRES-dNGFR (pHPN) was made byintroduction of a non-signaling version of the human NGFR gene (24). For the doubletransduction experiments, the Bcl6 ORF was cloned into pHPN. Maf LV (Maf-LV) wasconstructed by cloning a codon optimized human MAF ORF into pHAGE, with an optimalKozak sequence. 293T cells were transfected with lentivirus along with four expressionvectors encoding the packaging proteins Rev, Tat, Gag/Pol, and the G-protein of vesicularstomatitis virus (VSV). Supernatant was collected at 24 and 48 hours and passed through a0.22 µm filter. Virus was concentrated by spinning supernatant at 24,000 rpm for 90 minutesat 10 °C. Lentiviral preparations were titered on 293T cells, and CD4 T cells weretransduced at an MOI of 2. Cells were transduced 36–48 hours after the start of culture byspinning at 1,500 rpm for 90 minutes at 37° C. Analysis was performed 5 days post-transduction for each experiment. Co-transduction of both control vectors (ZsGreen-LV +dNGFR-LV) was tested in pilot experiments and no difference in cell phenotype wasobserved when compared to single transduction with either control vector alone (ZsGreen-LV or dNGFR-LV) (data not shown).

RT-PCRRNA was isolated by Qiagen RNeasy spin columns and reverse-transcribed into cDNAusing Superscript II Reverse Transcriptase (Invitrogen). Quantitative real-time PCR ofGAPDH and EBI2 was performed using the following primers: GAPDH forward, 5’-ACATCGCTCAGACACCATG-3’, GAPDH reverse, 5’-TGTAGTTGAGGTCAATGAAGGG-3’, EBI2 forward, 5’-AACATGCCACAGTTAAAACTTACT-3’, EBI2 reverse, 5’-GAGGGCGGAGTAAAATTGTTTG-3’, MAF forward, 5’-CAAGCTAGAAGCGCCCC-3’, MAF reverse 5’-AGTTCTGATGCCATTCTCCTG-3’.Real-time PCR was set up with BioRad iTaq SYBR Green Supermix.



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Statistical analysisA two-tailed, paired t test was used to analyze phenotypic differences between the CXCR5hi,CXCR5int, and CXCR5− CD4 T cells, shown in Figure 1. Based on the data in Figure 1, aclear prediction of the phenotype of a Bcl6hi Tfh cell could be made and consequently, one-tailed, paired t tests were used to analyze differences in Bcl6-transduced versus control-transduced cells. For the intracellular cytokine staining done in transduced cells (Fig. 6), atwo-tailed, paired test was used. For ectopic expression of Maf + Bcl6 together, two-tailedpaired t tests were used. For correlative analyses between Bcl6 and other markers, linearregression analysis was performed and R2 was calculated using Prism 5.0 (Graphpad, CA).

ResultsBcl6 protein is expressed by human Tfh and GC Tfh

Bcl6 mRNA is present in CXCR5+ CD4 T cells isolated from human tonsil (18, 25, 26).However, Bcl6 mRNA levels frequently do not correlate with Bcl6 protein expression (13).Bcl6 protein has been detected in human germinal center CD4 T cells byimmunofluorescence (26, 27). In order to address the importance of Bcl6 in human Tfh celldifferentiation, we first examined Bcl6 protein expression in human CD4 T cells at thesingle cell level by flow cytometry. Human tonsil is a lymphoid tissue rich in germinalcenters. When tonsillar B cells were stained with a Bcl6 monoclonal antibody, expressionwas limited to CD20+ CD38+ germinal center B cells, as expected (Suppl. Fig. S1).Tonsillar CD4 T cells were gated on CD45RO+ (memory/effector) or CD45RO− (naïve),and CD45RO+ were subsequently divided into CXCR5hi, CXCR5int, and CXCR5− gates(Fig. 1A). At least two distinct populations of CXCR5-expressing Tfh cells exist, onepopulation within the germinal center (GC) and another population outside (1, 28). In mice,these two populations have been termed GC Tfh and Tfh cells, respectively, and can bedistinguished on the basis of PD-1 expression (29). Given the dynamic nature of CD4 Tcells entering and exiting germinal centers (30, 31), it is likely that Tfh and GC Tfh cellsrepresent two interconverting activation states of the same cell type captured at differentmoments in time (1). While human GC Tfh cells are sometimes defined by CD57 expression(32, 33), CD57 is not exclusively expressed on GC Tfh cells (25, 34). These CXCR5int Tfhand CXCR5hi GC Tfh cell populations are more precisely delineated by co-staining withICOS (34, 35) (Fig. 1B) or by their dramatically high level of PD-1 (18, 25)(Fig. 1A). Wefound that both CXCR5hi and CXCR5int cells expressed significantly more Bcl6 proteincompared to CXCR5− CD45RO+ cells (p=0.0014 and 0.0038, Fig. 1B,C). CXCR5hi cellsalso expressed very high levels of PD-1, ICOS, CXCR4, and SLAM-associated protein(SAP) when compared to CXCR5int or CXCR5− cells (Fig. 1B,C). CXCR5int Tfh cells alsoexpress most canonical Tfh cell markers but at lower levels than CXCR5hi cells (Fig. 1B,C).We further characterized the different tonsillar CD4 T cell subsets by comparing their B cellhelp capabilities in co-culture with B cells. When cultured in the presence of autologousnaïve B cells and SEB, both CXCR5int and CXCR5hi CD4 T cells induced substantial IgMproduction (Fig. 1D), consistent with both CXCR5int and CXCR5hi CD4 T cells beingrelated populations of Tfh cells. The majority of the antibody secretion was dependent onIL-21 (Fig. 1D), consistent with previous reports (25, 35, 36).

Bcl6 instructs the conversion of Tfh to GC TfhIn order to determine if high Bcl6 expression was sufficient to induce the human GC Tfhphenotype, we constructed a viral expression vector, Bcl6-LV, that constitutively expressesBcl6 and a fluorescent protein (ZsGreen). CXCR5int Tfh cells were sorted from tonsil (Fig.2A), stimulated with anti-CD3/CD28 coated beads, and transduced with either Bcl6expressing viral vector (Bcl6-LV) or an equivalent vector with no Bcl6 insert (Ctrl-LV)(Fig. 2B). The Bcl6-LV drove a nearly 10-fold increase in Bcl6 protein expression when



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compared to the Ctrl-LV (Fig. 2C). Importantly, this increase in Bcl6 expression led tostrong upregulation of CXCR5, the central marker of Tfh cells (p=0.0011, Fig. 2D). TheMFI of CXCR5 exhibited a strikingly strong linear relationship with the MFI of Bcl6(R2=0.97) (Fig. 2K).

Bcl6 expression also drove the expression of another major chemokine receptor, CXCR4(p=0.0016) (Fig. 2E). Detailed analysis revealed that CXCR4 levels exhibited a tight linearcorrelation with Bcl6 expression (R2=0.96, Fig. 2K). Cells that expressed intermediate levelsof ZsGreen and Bcl6 exhibited the same changes as the top 5–10%, but to a lesser extent(Suppl. Fig. S2). CCR7 was consistently downregulated in the presence of Bcl6 (p=0.0172,Fig. 2F; R2=0.81, Fig. 2L). Downregulation of CCR7 is critical for T cell entry to the B cellfollicle (37). Bcl6 also negatively regulated the G-protein coupled receptor EBI2 (Fig. 2M,p=0.004). EBI2 downregulation is critical for movement of B cells from the outer follicleinto the germinal center (38–41), but there is no known role for EBI2 in CD4 T cells. Herewe show that Bcl6 mediates repression of EBI2, which is likely important for entry of Tfhcells into the germinal center. CXCR4 expression is important for correct germinal centerarchitecture and is associated with localization of GC B cells to the dark zone (42).Therefore, Bcl6 regulates the migratory capacity of human Tfh cells for appropriatelocalization to, and within, germinal centers by controlling CXCR5, CXCR4, EBI2, andCCR7 expression.

SAP is an intracellular SH2 domain signaling molecule that transmits signals for SLAMfamily receptors, and mutations in the gene encoding SAP (SH2D1A) are the cause of thesevere human genetic immunodeficiency X-linked lymphoproliferative disease (XLP) (43).SAP is critically required for germinal center and memory B cell development in both miceand man (30, 44, 45), and murine GC Tfh require SAP expression (29). No connection hasbeen made between Bcl6 and SAP. Therefore we examined whether SAP expression isregulated by Bcl6 in human CD4 T cells. SAP was uniformly upregulated by Bcl6expression (p=0.0002, Fig. 2G). SAP mRNA was also upregulated in Bcl6 expressing cells(data not shown). The data here now directly connect these two central regulators of T cellhelp to B cells.

In addition to Bcl6 directing the upregulation of SAP, the cell surface signaling moleculePD-1 was upregulated by Bcl6 (p=0.0013, Fig. 2H). PD-1 expression by Tfh cells may beimportant both for regulating Tfh cell proliferation and signaling to GC B cells. ICOS is alsocentral to Tfh cell interactions with B cells. Humans with ICOS deficiencies exhibit severeTfh cell and GC defects and have minimal response to vaccines (46–48). Tfh cells dependon ICOSL signals from B cells to maintain Bcl6 expression (49). Therefore, we examinedwhether ICOS expression is regulated by Bcl6 in human CD4 T cells. ICOS expression wasmodestly increased by expression of Bcl6 (p<0.04, Fig. 2I). Surprisingly, CD40L wasupregulated by expression of Bcl6 (p=0.04) (Figure 2J). CD40L is one of the most criticalmolecules for T cell help to B cells. Mice deficient in CD40-CD40L signaling have neithergerminal centers nor plasma cells (50–55). Defective CD40-CD40L signaling in humanscauses the severe immunodeficiency hyper IgM syndrome (HIGM) (56). Human Bcl6therefore controls both the central components of Tfh cell migration (CXCR5, CXCR4,CCR7, EBI2) and multiple central components of T:B interactions (SAP, PD-1, ICOS,CD40L). Collectively, these data indicate that Bcl6 instructs Tfh differentiation byregulating a program of gene expression that allows the T cells to co-localize with B cellsand provide critical help functions.

Bcl6 can convert non-Tfh cells to Tfh cellsNext, we set out to determine if Bcl6 expression was sufficient to drive the differentiation ofeffector CD4 T cells (CD45RO+ CXCR5−) to Tfh cells. We transduced CD45RO+ CXCR5−



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CD4 T cells with either Bcl6-LV or Ctrl-LV and analyzed the cells at day 7 (Fig. 3A andSuppl. Fig. 2A). Expression of Bcl6 was confirmed by flow cytometry (Fig. 3B). Activationof human CD4 T cells was sufficient to induce some expression of CXCR5 (57) (this isdifferent than murine CD4 T cells (58)). Nevertheless, expression of Bcl6 in humanCXCR5− effector Th cells led to a much greater and sustained increase in CXCR5expression (p=0.0099, Fig. 3C). Furthermore, Bcl6+ cells specifically exhibited significantlyincreased expression of SAP, PD-1, and CD40L (p=0.001, 0.002, and 0.02 respectively. Fig.3F,G,I), and downregulation of CCR7 (p=0.01, Fig. 3E; R2=0.80, Fig. 3K). A small increasein ICOS was observed (Fig. 3H, p<0.05). A robust correlation between Bcl6 and bothCXCR5 and CXCR4 expression was observed (R2=0.88 and R2=0.97, Fig. 3J). Similareffects were seen when starting with naïve (CD45RO−CXCR5−) CD4 T cells (Sup Fig. 4).Overall, these data demonstrate that Bcl6 controls a program of Tfh gene expression inhuman CD4 T cells, and Bcl6 expression is sufficient to convert previously antigen-experienced human CD45RO+ CXCR5− CD4 T cells into Tfh cells.

Bcl6 induces CXCL13 productionTfh cells are well known as important producers of the helper cytokines IL-4 and IL-21 (1).Human Tfh cells but not murine, also specifically express the chemokine CXCL13 (32, 34,36), a B cell attracting molecule usually made by stromal cells (59). We hypothesized thatCXCL13 expression by Tfh cells may be regulated by Bcl6. Unmanipulated human GC Tfhcells are a significant source of CXCL13 protein, as determined at the single cell level (Fig.4A). Impressively, when Bcl6 was constitutively expressed in human CXCR5int Tfh cells,thereby inducing a GC Tfh phenotype (Fig. 2), greater than 10-fold increases in CXCL13protein were detected in culture supernatants when compared to control cells (60–100 pg/mL; p<0.05, Fig. 4B). CXCL13 mRNA was also upregulated in Bcl6 expressing cells (datanot shown). This specialized production of CXCL13 by GC Tfh cells may help properlyorganize the germinal center, and may also provide cytokine-like signals to GC B cells.

Helper cytokines IL-4 and IL-21A population of human GC Tfh cells produce both IL-4 and IL-21 (Fig. 5). These cytokineshave been implicated in the maintenance of GC B cells and the differentiation of long-livedplasma cells (1, 60). IL-4 is known to provide prosurvival signals to B cells (61, 62). Micedually deficient in both IL-21 and IL-4 signals had significantly reduced B cell responses(63). Human GC Tfh cells exhibited considerably more IL-4 expression than Tfh cells, oreven the effector CD4 T cell (effector Th) population that includes Th2 cells (Fig. 5).Virtually all of the GC Tfh IL-4+ cells also expressed IL-21, the canonical Tfh cytokine(Fig. 5) (6, 26, 49, 64). The frequency of IL-4+ IL-21+ cells in the CXCR5hi GC Tfh cellpopulation was significantly higher than in the CXCR5int cell population (p=0.005) or theeffector Th subsets (p=0.003, Fig. 5).

The Tfh program is perhaps best conceptualized as a developmental program that can co-exists with Th1, Th2, or Th17 gene expression to a limited extent (1). Therefore, weexamined the capacity of human Tfh and GC Tfh cells to produce Th1 or Th17 cytokines.GC Tfh cells secreted significantly less IL-17 when compared to Tfh or effector Th cells(Fig. 5). A modest reduction in the IFNγ+ cells was observed for GC Tfh cells in comparisonto effector Th cells (p=0.225 and p=0.239, respectively) (Fig. 5). A considerable percentageof human CXCR5int Tfh cells produced IL-17 or double produced IFNγ and IL-17,consistent with CXCR5int Tfh cells being a less polarized and more heterogeneous cell typethan CXCR5hi GC Tfh cells (Fig. 5).

Given that Bcl6 controls both Tfh cell migration and Tfh:B interaction genes, we set out todetermine if Bcl6 also instructs GC Tfh cell cytokine expression. Unexpectedly, constitutive



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expression of Bcl6 did not significantly increase expression of IL-4 or IL-21 in any CD4 Tcell subset examined (Fig. 6). Furthermore, the IL-4+ IL-21+ population characteristic of GCTfh cells was not observed (Fig. 6). Therefore, while Bcl6 instructs many aspects of thehuman Tfh cell phenotype, the production of B cell help cytokines requires additionalsignals.

Maf induces IL-21 secretion in CD4 T cellsMaf is a transcription factor involved in IL-21 (65, 66) and IL-4 (67) production in mouseCD4 T cells. Maf is expressed at higher level in CXCR5hi and CXCR5int human Tfh cellsthan in naïve or CXCR5−CD45RO+ cells (Fig. 7A). Maf expression can be induced byICOS signaling (68, 69). Consequently, we set out to test the hypothesis that Maf contributesa complementary role to Bcl6 in Tfh cell differentiation by inducing secretion of helpercytokines. In order to test this hypothesis, we generated a viral expression vector containinghuman Maf. Transduced cells were visualized by their expression of ZsGreen (Maf) or atruncated, non-signaling human nerve growth factor receptor (dNGFR; Bcl6). Transductionof CD45RO− CXCR5− CD4 T cells with Maf did not result in a notable change in thepercentage of cells secreting IL-4 upon restimulation (Suppl. Figure S3). However, Mafexpression resulted in a marked increase in the percentage of IL-21 producing CD4 T cellscompared to control transduced or Bcl6 transduced cells (p=0.0009 and 0.016, respectively,Fig. 7B and 7C). Dual transduction with both Maf and Bcl6 (Fig. 7D), did not increaseIL-21 production above the % induced by Maf alone (Fig. 7C). Dual transduction with bothMaf and Bcl6 did not enhance IL-4 production (Suppl. Figure S3). Maf is expressed byTh17 cells (65, 69); however, no significant change in IL-17 expression was observed inMaf transduced human CD45RO+CXCR5int CD4 cells (data not shown). Naïve CD4 T cellsthat were activated and then transduced with Maf also did not express IL-17 (data notshown). These data demonstrate that while Bcl6 is critical for T cell positioning within thefollicle and direct T:B interactions, Maf but not Bcl6 can enhance IL-21 production by CD4T cells.

Maf cooperates with Bcl6 to instruct human Tfh cell differentiationWe examined whether Maf regulates additional Tfh cell associated proteins. Surprisingly,Maf induced a significant increase in CXCR5 expression, and this effect was particularlynotable when starting with CD45RO− CXCR5− naïve CD4 T cells (p=0.0002, Fig. 8A,B).This was not due to Maf induction of Bcl6 (Fig. 8C). Of note, ectopic expression of Bcl6induced a modest but significant increase in Maf expression (Fig. 8D). Co-transduction ofnaïve CD4 T cells with Bcl6 and Maf did not further enhance CXCR5 expression. Incontrast, ICOS and PD1 expression both increased significantly when Bcl6 and Maf wereectopically expressed together compared to cells expressing Bcl6 alone (p=0.02 and 0.002,Fig. 8E,F). CXCR4 was also significantly induced by Maf at levels comparable to that seenwith Bcl6, and further increased CXCR4 levels in doubly transduced cells was observed(p=0.03, Fig. 8G). Unlike Bcl6, Maf did not have an impact on CD40L expression (Fig. 8H).Taken together, these data show that Bcl6 and Maf both play a role in human Tfhdifferentiation. Bcl6 controls modules of genes important for Tfh cell localization and T:Binteractions. Complementing Bcl6, Maf induces IL-21 secretion and CXCR5 expression andenhances expression of a number of Bcl6 regulated Tfh associated molecules such as ICOSand PD1.

DiscussionIn depth knowledge of the pathways of human CD4 T cell differentiation is critical forunderstanding how to accomplish rational vaccine design against the myriad pathogens thathumans remained unprotected against. In addition, understanding human CD4 T cell



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differentiation is critical for understanding most human autoimmune diseases. These featscan only be accomplished with a clear understanding of the transcription factors thatregulate human CD4 T cell differentiation. Here we demonstrate that Bcl6 is a centralregulator of human Tfh cell differentiation, and we demonstrate that Bcl6 accomplishes thistask by controlling two major modules of Tfh gene expression. In human B cells, Bcl6 hasgenerally been shown to repress gene transcription (14, 15). It is likely that upregulation ofmany Tfh genes is an indirect effect brought about by Bcl6-mediated repression of specifictranscription factors and chromatin remodelers such as histone modification enzymes (9,10). However, the possibility that Bcl6 binding to some genes may positively influence geneexpression must also be considered.

To date there has been only a limited understanding of how Bcl6 controls CD4 T cells, andlittle data has been available on Bcl6 function in human CD4 T cells (18, 25). Ectopicexpression of Bcl6 in murine CD4 T cells in vivo induced a Tfh cell phenotype (17), but hadsurprisingly limited activity in purified murine CD4 T cells in vitro, with minimal Tfhassociated protein changes (17, 18, 70). Importantly, our in vitro human CD4 T cell systemhas allowed us to identify downstream targets of Bcl6 regulation, without the confoundingeffects of non-Bcl6 signals present in the in vivo mouse models that also contribute to Tfhcell differentiation. We demonstrate that introduction of Bcl6 into human CD45RO+ CD4 Tcells converts those cells to a Tfh-like cell phenotype in vitro, and the degree of conversionstrongly correlates with the level of Bcl6 expression (Figs. 2–3). Here we have shown forthe first time that Bcl6 regulates distinct modules of the Tfh program: one Bcl6-dependentmodule is genes critical for Tfh cell migration (CXCR5, CXCR4, CCR7, EBI2) and thesecond Bcl6-dependent module is a set of genes important for T:B interactions (SAP, PD1,CD40L, ICOS, CXCL13), including two genes known to be critical for contact dependent Bcell help (SAP and CD40L). Therefore, Bcl6 is a true nexus for human Tfh differentiationand functions. Perhaps the most striking finding from this study is that Bcl6 specificallyregulates CD40L, SAP and ICOS. From the perspective of human immunology, the CD40L,SH2D1A and ICOS genes are three loci of severe immunodeficiencies of adaptive immunity.Genetic lesions in CD40L and SH2D1A are lethal due to a resulting susceptibility to a rangeof infectious diseases. Extreme losses in responsiveness to vaccines and failure to develop Bcell memory are prominent characteristics of these genetic diseases. Deletion of the humanICOS gene also results in immunodeficiency, susceptibility to infections, and a failure torespond to vaccines (46–48), consistent with the importance of ICOS for Tfh differentiation(49). Here we find that Bcl6 regulates all three of these critical human genes, highlightingthe powerful role of Bcl6 in defining Tfh functionality.

The data here also show that PD-1 is explicitly regulated by Bcl6. Therefore the high levelof PD-1 on Tfh cells is not simply a byproduct of TCR stimulation but is a specificcomponent of the Tfh gene program. In the absence of PD-1 in mice, increased GC B celldeath and a defective plasma cell response were observed in one study (71), while excessiveTfh cell proliferation was seen in another study (72). PD-1 is a potent negative regulator ofT cell proliferation. We propose that PD-1 is an important negative regulator of Tfh cellsmost likely by dissociating Tfh cell TCR signaling from proliferation. The purpose ofgerminal centers is the rapid evolution of BCR affinity through rapid GC B cell proliferationand hypermutation. Tfh cells are critical for this process and must preferentially select the"best" GC B cells for further rounds of proliferation and mutation via sensing quantitativedifferences in peptide:MHC complexes between different GC B cells. This must requirehighly sensitive TCR signaling. At the same time, GC Tfh cell TCR signaling should bedissociated from proliferation, for while the GC B cells must undergo rapid proliferation forselection of novel mutations, GC Tfh cell numbers remain relatively static for the durationof the germinal center. Therefore, Bcl6 induction of PD-1 on Tfh cells is likely important forrestriction of Tfh cell proliferation in the presence of continuous antigen stimulation.



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The role of Bcl6 in human Tfh differentiation differs from murine Tfh differentiation inseveral key ways. Direct induction of CXCR5 protein expression on purified CD4 T cells isone example. Bcl6 regulation of CXCL13 is a second example. CXCL13 is expressed byhuman Tfh cells but not mouse Tfh cells. Production of CXCL13 by Tfh cells may serve twopurposes. First, CXCL13 is a chemoattractant and will recruit B cells to the location of theTfh cells. This may be important in the face of significant reductions in CXCL13 expressionby lymphoid tissue stromal cells during viral infections (73). Second, CXCL13 binding toCXCR5 on B cells induces LTα1β2 expression (74). As such, Tfh cell expression ofCXCL13 may orchestrate germinal center architecture. This CXCL13 expression by Tfhcells may also drive tertiary lymphoid neogenesis in autoimmune diseases (4). CXCL13 andLTα1β2 are important components of lymphoid organ neogenesis and the formation ofectopic lymphoid aggregates (75). It is feasible that CXCL13 binding to CXCR5 on GC Bcells induces other non-chemotactic signaling responses that are important for GC B cellsurvival or differentiation.

IL-21 is the most important known cytokine for T cell help to B cells, both throughmaintenance of germinal centers and for induction of plasma cell differentiation. Here wefind that Bcl6 has minimal influence on IL-21 production, whereas Maf does drive IL-21production. More surprisingly, Maf also controls other components of human Tfh cellfunction, including expression of CXCR5. Intriguingly, co-expression of Bcl6 and Mafexhibited additive effects on gene expression for some Tfh associated genes (e.g., PD-1 andICOS), whereas in other cases Bcl6 and Maf appear to have parallel, non-additive, capacities(e.g., CXCR5). Altogether these finding reveal that Bcl6 and Maf collaborate to orchestratea suite of genes that define nearly all of the core characteristics of human Tfh cell biology.

We have demonstrated that Bcl6 and Maf synergistically induce human Tfh differentiationand function. As such, manipulation of Bcl6 and/or Maf activity in a positive way in humanCD4 T cells may have an important impact for human vaccines, resulting in enhanced Tfhcell number or functionality and long-term B cell immunity. Alternatively, inhibition ofBcl6 and Maf in CD4 T cells is an outstanding candidate for ameliorating autoimmunediseases associated with autoantibody production.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsWe thank Dr. Erika O’Donnell and Dr. David Ernst at BD Biosciences for providing Bcl6 monoclonal antibody.We thank Anthony Jose for technical assistance. We acknowledge use of human tonsils provided by the NationalDisease Research Interchange (NDRI).

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Figure 1.Two stages of human Tfh cell differentiation, associated with two levels of Bcl6 and Tfhassociated genes. A, Human tonsil cells were gated on CD45RO+ memory/effector orCD45RO− naïve CD4 T cells (top). CD45RO+ cells were subsequently divided intoCXCR5−, CXCR5int, and CXCR5hi gates (bottom). B, Representative phenotypic analysisof CD4 T cell subsets based on CXCR5 expression, compared to naïve CD4 cells. C, Datafrom 5 individual donors. Data are shown as fold change in MFI comparing CXCR5−,CXCR5int, or CXCR5hi to naïve CD4+ T cells from the same donor. D, Tonsillar naïve,CXCR5−, CXCR5int, and CXCR5hi CD4 T cell subsets were co-cultured with autologousnaïve B cells in the presence of SEB (250 ng/mL). 20 µg/mL IL-21R/Fc or an isotype-matched antibody control was added to each well. Secretion of IgM was determined after 7



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days of culture. Duplicate samples from each of two donors is shown. * p < 0.05, ** p <0.005, *** p < 0.0005.



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Figure 2.Human Tfh cell differentiation is instructed by Bcl6 expression. A, CD45RO+ CXCR5int

CD4 T cells were sorted from human tonsil. B, Cells were stimulated with anti-CD3/CD28beads and either left unmanipulated or transduced on day 2 with Bcl6-LV or Control-LV(Ctrl-LV). Data shown is representative. C, Expression of Bcl6 was measured using amonoclonal antibody to Bcl6 based on the gates shown in B. D–J, Expression of Tfhassociated molecules by Bcl6-LV and Ctrl-LV groups, based on the gates shown in B. Arepresentative FACS plot is shown (left) alongside the complete data set with 5–6 individualdonors (right). K–L, ZsGreen expressing cells were divided into 5 equal gates based onZsGreen MFI. Bcl6, CXCR5, CXCR4, PD1 (K), and CCR7 (L) MFIs were determined foreach ZsGreen gate. Data shown in K and L are representative of 6 individual donors. M,EBI2 mRNA levels, normalized to GAPDH. * p < 0.05, ** p < 0.005



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Figure 3.Bcl6 converts human CXCR5− CD4 T cells to Tfh cells. A, CD45RO+ CXCR5− CD4 Tcells were sorted from human tonsil. B, Expression of Bcl6 was measured based on the topgates shown in Supplemental Figure S2A. C–I, Expression of Tfh associated molecules byBcl6-LV and Control-LV (Ctrl-LV) groups based on gates used in Supplemental FigureS2A. A representative plot is shown (left) alongside the complete data set with 5–6individual donors (right). J–K, ZsGreen expressing cells were divided into 5 equal gatesbased on ZsGreen MFI. Bcl6, CXCR5, CXCR4, PD1 (J), and CCR7 (K) MFIs weredetermined for each ZsGreen gate. Data shown in J and K are representative of 6 individualdonors. * p < 0.05, ** p < 0.005



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Figure 4.Bcl6 instructs CXCL13 production by human Tfh cells. A, Selective production of CXCL13by human Tfh cells. Unstimulated tonsil cells were incubated for 3 hours with brefeldin Abefore intracellular staining for CXCL13. (left) CXCL13 expression shown in total CD4 Tcells. (center and right) Activated CD4 T cell subsets were gated (center) and CXCL13levels for each population are shown versus isotype control (right). Isotype control wasgated on total CD4 T cells. MFI for each population is indicated. B, CXCR5int Tfh cellswere sorted from tonsil, stimulated with anti-CD3/CD28 beads, and transduced with Bcl6-LV or Control-LV. 5 days post-transduction, CXCL13 present in the supernatant wasanalyzed by ELISA. Data is representative of 4 total donors from 2 independentexperiments. * p < 0.05, ** p < 0.01.



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Figure 5.IL-21 and IL-4 cytokine production by tonsillar CD4 T cells. A, Representative intracellularstaining of CD4 T cells isolated from a human tonsil and restimulated for 3 hours with PMAand ionomycin. B, Intracellular staining data from 5 individual donors. * p < 0.05, ** p <0.005.



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Figure 6.Bcl6 expression is not sufficient for helper cytokine regulation. CD45RO+ CXCR5int (A),CD45RO+ CXCR5− (B), or CD45RO− CXCR5− (C) CD4 T cells were sorted from humantonsil. Cells were stimulated with anti-CD3/CD28 beads and either left unmanipulated ortransduced on day 2 with Bcl6-LV or Control-LV (Ctrl-LV). Five days post-transduction,cells were restimulated with PMA and ionomycin in the presence of brefeldin A and stained.Representative data (left) and pooled data from 5 individual donors (right) are gated on thetop 5–10% of ZsGreen+ or dNGFR+ CD4 T cells in either the Bcl6-LV or Ctrl-LV groups.Quadrants are based on cells cultured with brefeldin A alone. * p < 0.05.



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Figure 7.Maf instructs IL-21 production by CD45RO− CXCR5− CD4 T cells. A, Maf mRNAexpression was measured by qPCR on different tonsillar CD4 T cell subsets (defined as inFig. 1A). B–D, CD45RO− CXCR5− CD4 T cells were sorted from human tonsil, stimulatedwith anti-CD3/CD28 beads and either left unmanipulated or transduced on day 2 with Bcl6-LV (‘Bcl6’), Maf-LV (‘Maf’), both (‘both’, Bcl6-LV and Maf-LV), or a Control-LV(dNGFR-LV, ‘C1’; ZsGreen -LV, ‘C2’). Five days post-transduction, cells wererestimulated with PMA and ionomycin in the presence of brefeldin A and stained. B,Representative plots showing IL-21 expression. C, IL-21 expression data from 8 donors. D,Representative plot of Bcl6-dNGFR and Maf-ZsGreen RV double transfection (left). * p <0.05, *** p < 0.001.



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Figure 8.Maf enhances Tfh-associated gene expression in CD4 T cells. CD45RO− CXCR5− CD4 Tcells were sorted from human tonsil, stimulated with anti-CD3/CD28 beads, and transducedwith Bcl6-LV (‘Bcl6’), Maf-LV (‘Maf’), Bcl6-LV & Maf-LV (‘both’), or Control-LV(dNGFR-LV, ‘C1’. ZsGreen-LV, ‘C2’). 5 days post-transduction, top 5% of transduced cellswere assessed by FACS for expression of Tfh-associated molecules. A, CXCR5 surfaceexpression in naïve CD4 T cells. B, A representative CXCR5 histogram from a single donor.C, Naïve CD4 T cells transduced with Maf-LV, Bcl6-LV, or Ctrl-LV were stained for Bcl6protein and percent of Bcl6+ cells is shown. D, Maf mRNA from Ctrl-LV and Bcl6-LVtransduced cells. E–H, ICOS (E), PD1 (F), CXCR4 (G), and CD40L (H) expression innaïve CD4 T cells transduced with Ctrl-LV, Bcl6-LV, Maf-LV, or both. Data isrepresentative of 6–9 individual donors. * p < 0.05, ** p < 0.005, *** p < 0.0005.



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NIH Public Access Michela Locci Michael Cho Terence ... · Bcl6 and Maf cooperate to instruct human follicular helper CD4 T cell (Tfh) differentiation Mark A. Kroenke*, Danelle Eto*,

NIH Public Access Michela Locci Michael Cho Terence ... · Bcl6 and Maf cooperate to instruct human follicular helper CD4 T cell (Tfh) differentiation Mark A. Kroenke, Danelle Eto,