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. 2021 Oct 12;54(10):2245–2255.e4. doi: 10.1016/j.immuni.2021.08.003

BCL6 controls contact-dependent help delivery during follicular T-B cell interactions

Dan Liu 1,2,3,7,8, Jiacong Yan 1,2,3,4,7,9, Jiahui Sun 1,2,3,7, Bo Liu 1,2,3, Weiwei Ma 2,3, Ye Li 2,3, Xingxing Shao 1,2,3,4, Hai Qi 1,2,3,4,5,6,10,∗
PMCID: PMC8528402  PMID: 34464595

Summary

BCL6 is required for development of follicular T helper (Tfh) cells to support germinal center (GC) formation. However, it is not clear what unique functions programmed by BCL6 can explain its absolute essentiality in T cells for GC formation. We found that ablation of one Bcl6 allele did not appreciably alter early T cell activation and follicular localization but inhibited GC formation and Tfh cell maintenance. BCL6 impinged on Tfh calcium signaling and also controlled Tfh entanglement with and CD40L delivery to B cells. Amounts of BCL6 protein and nominal frequencies of Tfh cells markedly changed within hours after strengths of T-B cell interactions were altered in vivo, while CD40L overexpression rectified both defective GC formation and Tfh cell maintenance because of the BCL6 haploinsufficiency. Our results reveal BCL6 functions in Tfh cells that are essential for GC formation and suggest that BCL6 helps maintain Tfh cell phenotypes in a T cell non-autonomous manner.

Keywords: BCL6, Tfh cells, germinal center, CD40L, calcium signaling, stim1, intravital imaging, T-B interactions, follicle, lymph node

Highlights

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    Both copies of Bcl6 gene are required for efficient follicular T-B cell entanglement

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    BCL6 is required for calcium signaling in Tfh cells and CD40L delivery to B cells

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    Expression of Tfh markers responds to acute perturbation of T-B cell interactions

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    Defective functions of BCL6-insufficient T cells are rescued by CD40L overexpression

BCL6 is required for Tfh cell development. Liu et al. show that BCL6 controls calcium signaling of Tfh cells, follicular T-B cell entanglement, and efficient T cell-to-B cell CD40L delivery and promotes Tfh cell maintenance partly in a T cell non-autonomous manner.

Introduction

The germinal center (GC) is the primary site in which high-affinity, long-lived plasma cells and memory B cells develop (Berek et al., 1991; Coico et al., 1983; Jacob et al., 1991; MacLennan, 1994). GC development requires activation of antigen-specific helper T cells and their provision of contact-dependent help signals such as the CD40 ligand (CD40L) to drive optimal clonal expansion, survival, and differentiation of antigen-specific B cells (Banchereau et al., 1994; Victora and Nussenzweig, 2012). While all helper T cells synthesize CD40L and can in principle promote B cell proliferation in vitro (Koguchi et al., 2012; Koguchi et al., 2007), follicular T helper (Tfh) cells, a subset of effector CD4+ T cells localized in the follicle (Campbell et al., 2001; Schaerli et al., 2000), are specialized in promoting the GC development in vivo (Crotty, 2011; Qi, 2016; Vinuesa et al., 2016b).

Following antigen activation, the initial encounter between antigen-specific helper T cells and B cells takes place in the interfollicular region and at the border between the T cell zone and the follicle (Garside et al., 1998; Kerfoot et al., 2011). Here, these cells form mobile but stable contacts that can last for tens of minutes to hours, invariably with migratory B cells leading the conjugate pair (Okada et al., 2005; Qi et al., 2008). As antigen-specific B cells clonally expand on helper signals provided by T cells, they migrate back into the follicle in preparation for GC formation (Cyster, 2010). A fraction of activated helper T cells concomitantly migrate into the follicle, in a manner that is independent of the cognate B cell partner (Vinuesa and Cyster, 2011). Follicular localization of activated helper T cells depends on the chemokine receptor CXCR5 (Arnold et al., 2007; Haynes et al., 2007), which senses the follicle-derived directional cue CXCL13 (Förster et al., 1996; Gunn et al., 1998), and inducible costimulator molecule (ICOS), which promotes follicular T cell motility upon engagement by ICOS ligand (ICOSL) displayed on bystander B cells (Xu et al., 2013).

BCL6 is a transcription repressor that is required for the development and maintenance of Tfh cells and Tfh cell-supported GC formation (Johnston et al., 2009; Nurieva et al., 2009; Yu et al., 2009). However, little is known about biological processes that are both controlled by BCL6 and absolutely essential for GC formation. Chromatin-binding and transcriptomic analyses have revealed many genes that are potentially subject to BCL6-mediated suppression (Hatzi et al., 2015; Liu et al., 2016b). These include signaling molecules and transcription factors that promote terminal effector T cell differentiation and chemotactic receptors that are incompatible with follicle-directed movement and localization. CD4+ T cells genetically deficient in BCL6 cannot normally express CXCR5 and do not relocalize into the follicle. Although the transcription factor ASCL2 instead of BCL6 directly binds to and regulates Cxcr5 gene expression (Hatzi et al., 2015; Liu et al., 2014; Liu et al., 2016b), BCL6 can suppress the expression of the transcription factor Id2 and thus release Id2-mediated suppression of E proteins, which can positively drive CXCR5 expression (Liu et al., 2014; Shaw et al., 2016). These results that indicate follicular localization is a key aspect for which BCL6 is required in order to promote Tfh cell development and function (Qi, 2016). However, whereas CXCR5 ablation or prevention of CCR7 downregulation in T cells causes a defect in follicular recruitment, GCs can still form inside the follicle with the help of CXCR5 null Tfh cells and suffer a relatively mild 2-fold reduction in magnitude (Arnold et al., 2007; Haynes et al., 2007). This is in sharp contrast to the outcome of T cell-specific Bcl6 ablation, which completely abrogates GC formation (Johnston et al., 2009; Nurieva et al., 2009; Yu et al., 2009). Therefore, beyond follicular recruitment, BCL6 must regulate additional aspects of T cell biology that are uniquely important for helping B cells (Qi et al., 2014). One unique aspect of T cell help for GC formation is its dependence on physical cell-cell contact, which takes on highly dynamic forms in vivo (Liu et al., 2015; Okada et al., 2005; Qi et al., 2008; Shulman et al., 2014). Such contacts require active signaling through the T cell antigen receptor (TCR), which is further enhanced by accessory molecules such as SAP and ICOS (Cannons et al., 2010; Chu et al., 2014; Liu et al., 2015; Qi et al., 2008). It has been found that, when deprived of B cell-mediated antigen presentation for several days, frequencies of Tfh cells would decrease, even though no unique instructional signals from B cells, other than antigen presentation, are required for maintaining these Tfh cells (Baumjohann et al., 2013; Deenick et al., 2010). The simplest explanation is that, in a cell-autonomous manner, BCL6 instructs a stable program of Tfh cell differentiation, albeit requiring periodic TCR stimulation to maintain, polarize, and/or manifest (Crotty, 2014; Vinuesa et al., 2016a).

In this study, we combined static and intravital imaging analyses to investigate how BCL6 regulates cell biology of Tfh cells and found that BCL6 impinges on calcium signaling of T cells, regulates dynamics of T-B cell interactions and efficiency of help delivery to B cells, and promotes Tfh cell maintenance partly in a T cell non-autonomous manner.

Results

BCL6 is haploinsufficient for recruitment-independent helper functions

Antigen-activated BCL6-deficient T cells exhibit an intrinsic defect in follicular localization, as demonstrated in mixed bone-marrow chimera (Yu et al., 2009), presumably because of their failure to properly shift the chemo-sensing preference by upregulating CXCR5 and downregulating CCR7 (Johnston et al., 2009; Nurieva et al., 2009; Yu et al., 2009). Lack of follicular localization of BCL6-deficient T cells precludes analysis of potentially important functions for BCL6 in the follicular phase, which appears likely given the phenotypic discrepancy in GC formation between T cell-intrinsic BCL6 deficiency and CXCR5 deficiency. To circumvent this problem, we first tested T cells isolated from heterozygous Bcl6+/− mice. As shown in Figure 1A, Bcl6+/− and Bcl6+/+ OT-II T cells expressed grossly similar amounts of CXCR5 and were recruited into the follicle with comparable efficiencies 3–4 days after NP-OVA immunization (Figures 1B and 1C), as measured by the ratio between the follicular and the T zone densities of donor OT-II T cells (the follicle recruitment index, FRI; Figure S1). These data indicate that one intact allele of the Bcl6 gene is sufficient for ensuring follicular recruitment of helper T cells. Importantly, however, Bcl6+/− OT-II T cells remained significantly less competent in promoting GC formation (Figure 1D) or maintaining the associated CXCR5+ Tfh cell phenotype over a longer term, a defect that manifested 8 days after immunization (Figure 1E). Therefore, both alleles of the Bcl6 gene must be expressed to normally orchestrate helper functions inside the follicle to maintain Tfh cell phenotype and support GC formation.

Figure 1.

Figure 1

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Haploinsufficiency of BCL6 impairs the follicular response

(A) Representative histograms and MFIs of surface CXCR5 expression on OT-II T cells of indicated genotypes in draining lymph nodes 4 days after NP-OVA immunization. Each symbol represents one experiment in which cells from 2–3 mice of each group were pooled. n.s., not significant.

(B and C) Representative distribution patterns (B) and FRIs (C) of GFP-transduced Bcl6+/+ and Bcl6+/− OT-II T cells in draining lymph nodes as in (A). Each symbol represents one follicle and its adjacent T cell zone, and lines denote means. One of 2 experiments with similar results is shown, and each group had 3 mice per experiment. n.s., not significant. Scale bar, 100 μm.

(D and E) Representative flow-cytometry profiles and frequencies of FAShi GL7hi GC B cells in total CD19+ B cells (D) or flow-cytometry profiles and frequencies of CXCR5hi PD1hi Tfh cells in OT-II T cells (E) 8 days after NP-OVA immunization in Sap−/− mice that received OT-II T cells of indicated genotypes. Each symbol represents one mouse, and lines denote the means. Data are pooled from 3 independent experiments, with at least three mice per group per experiment. ∗∗p < 0.01.

See also Figure S1.

BCL6 is not required for maintaining stable T-B cell interactions at the T zone-follicle border

Stable T-B cell interactions, in the form of long-lasting conjugate pairs at the T zone-follicle border (Okada et al., 2005; Qi et al., 2008) or entangled couples inside the follicle (Liu et al., 2015; Shulman et al., 2014), are required for GC formation and maintenance. To explore whether BCL6 would impinge on cognate T-B cell interactions in vivo, we first visualized Bcl6+/− and Bcl6+/+ OT-II T cells interacting with MD4 B cells 36–48 h after HEL-OVA immunization by 2-photon intravital microscopy. As previously reported, this is a time point when activated T cells form long-lasting conjugate pairs with cognate B cells at the T zone-follicle border (Okada et al., 2005; Qi et al., 2008). We found that both Bcl6+/− and Bcl6+/+ OT-II T cells formed comparably stable conjugates with MD4 B cells (Figure S2; Video S1 and S2). Therefore, in the early phase of the immune response at the T zone-follicle border, one intact Bcl6 allele is sufficient for activated T cells to maintain long-lasting contacts with cognate B cells.

Video S1. Comparable contacts between MD4 and dsRed- and CFP-transgenic Bcl6+/+ OT-II T cells at the T zone-follicle border, related to Figure 2

GFP-expressing MD4 (green), dsRed-expressing Bcl6+/+ (red) OT-II T cells, and CFP-expressing Bcl6+/+ (blue) OT-II T cells were visualized in the T zone-follicle border region of the draining lymph node 36 h after HEL-OVA immunization. The movie was played back twice, and representative T-B cell contacts were highlighted with arrowheads in the second playback. Scale bar, 20 μm.

Download video file (4.4MB, mp4)
Video S2. Comparable contacts between MD4 and dsRed-transgenic Bcl6+/− and CFP-transgenic Bcl6+/+ OT-II T cells at the T zone-follicle border, related to Figure 2

GFP-expressing MD4 (green), dsRed-expressing Bcl6+/− (red) OT-II T cells, and CFP-expressing Bcl6+/+ (blue) OT-II T cells were visualized in the T zone-follicle border region of the draining lymph node 36 h after HEL-OVA immunization. The movie was played back twice, and representative T-B cell contacts were highlighted with arrowheads in the second playback. Scale bar, 20 μm.

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BCL6 is required for T cells to maintain entangled contacts with cognate B cells

As the response progresses into the follicular stage, antigen-specific T-B cell interactions become shortened in duration but extensive in area of contact, frequently assuming the entangled configuration (Liu et al., 2015). To determine whether one intact Bcl6 allele is still sufficient for maintaining entangled contacts with antigen-specific B cells inside the follicle, CFP-expressing control Bcl6+/+ OT-II cells and dsRed-expressing Bcl6+/− or Bcl6+/+ OT-II cells were co-transferred together with GFP-expressing MD4 B cells into B6 recipients that were subsequently immunized with HEL-OVA. We conducted intravital imaging 96 h post immunization, when large numbers of OT-II T cells and MD4 B cells could be seen in the follicle. As shown in Videos S3 and S4 and Figures 2A–2C, contacts between Bcl6+/− OT-II T cells and MD4 B cells were comparable in duration to those between Bcl6+/+ OT-II T cells and the same cohort of MD4 B cells; however, the normalized surface area of contacts, or the extent of entanglement measured by the surface engagement index (SEI) as previously defined (Liu et al., 2015) (Figure S3), was significantly reduced for Bcl6+/− OT-II T cells, revealing an haploinsufficient role for BCL6 in maintaining efficient T-B cell entanglement.

Figure 2.

Figure 2

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BCL6 is required for efficient T-B cell entanglement inside the follicle

(A–C) GFP-expressing MD4 B cells, control CFP-expressing Bcl6+/+ and dsRed-expressing test Bcl6+/−, or test Bcl6+/+ OT-II T cells were visualized 4 days after immunization. (A) Bcl6+/+ and Bcl6+/− OT-II T cells interacting with MD4 B cells, with contacts highlighted by arrowheads. Scale bar, 20 μm. See corresponding Video S4. Duration (B) and SEI (C) of contacts of indicated types. See corresponding Videos S3 and S4. Each symbol is one contact, and lines denote the means. Data are pooled from three independent experiments. ∗∗∗p < 0.001.

See also Figure S3.

Video S3. Similar duration and surface engagement of dsRed- and CFP-transgenic Bcl6+/+ follicular OT-II T cells, related to Figure 2

CFP-expressing Bcl6+/+ (blue), dsRed-expressing Bcl6+/+ (red) OT-II T cells, and GFP-expressing MD4 B cells (green) were visualized in a follicle of the draining lymph node 96 h after HEL-OVA immunization. The movie was played back twice, and representative T-B cell contacts were highlighted with arrowheads in the second playback. Scale bar, 20 μm.

Download video file (15.7MB, mp4)
Video S4. Similar duration but reduced surface engagement of dsRed-transgenic Bcl6+/− as compared with CFP-transgenic Bcl6+/+ follicular OT-II T cells, related to Figure 2

CFP-expressing Bcl6+/+ (blue), dsRed-expressing Bcl6+/− (red) OT-II T cells, and GFP-expressing MD4 B cells (green) were visualized in a follicle of the draining lymph node 96 h after HEL-OVA immunization. The movie was played back twice, and representative T-B cell contacts were highlighted with arrowheads in the second playback. Scale bar, 20 μm. See also corresponding Figure 2A.

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T-B cell entanglement is associated with heightened calcium signaling in T cells (Liu et al., 2015). We thus conducted calcium imaging of Bcl6+/− or Bcl6+/+ OT-II T cells interacting with MD4 B cells in the follicle after HEL-OVA immunization, using a fluorescence resonance energy transfer (FRET)-based calcium reporter YC-Nano-50CD (Horikawa et al., 2010), as previously used in Liu et al. (2015). As exemplified in Figure 3A and Videos S5 and S6, contacts between Bcl6+/+ OT-II T cells and MD4 B cells were more frequently associated with pronounced calcium signaling than those between Bcl6+/− T cells and MD4 B cells. The fractional change in the normalized FRET signal intensity, ΔRt/R0, reflects the change in intracellular (Cheli et al., 2016) at time t as compared with the no-contact reference state 0 of the same T cell prior to a contact. The mean ΔRt/R0 measures the average calcium signaling intensity of a T cell in contact with a B cell. Quantitatively, Bcl6+/− OT-II T cells exhibited significantly lower mean ΔRt/R0 values than control T cells (Figure 3B; mean ± SEM of mean ΔRt/R0 values for 65 Bcl6+/+ versus 64 Bcl6+/− contacts: 29% ± 6% versus 8% ± 4%, p < 0.01). To gauge the overall productivity of contact events involving the two T cell types, we used the time-integrated calcium-response index (TICRI) as previously described (Liu et al., 2015), which is calculated by multiplying mean ΔRt/R0 of each contact with its duration. As shown in Figure 3C, TICRIs of Bcl6+/− cells were lower by ∼3-fold (Bcl6+/+: 134 ± 34; Bcl6+/−: 28 ± 13; p < 0.01). Combined together, these data reveal a haploinsufficient role for BCL6 in promoting optimal calcium signaling and T-B cell entanglement inside the follicle.

Figure 3.

Figure 3

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BCL6 is required for optimal calcium signaling during T-B cell entanglement in vivo

(A–C) dsRed-expressing MD4 B cells and YC-Nano-50CD-transduced Bcl6+/+ or Bcl6+/− OT-II T cells were visualized in B6 recipients 4 days after immunization. (A) Image sequences showing a Bcl6+/+ and a Bcl6+/− OT-II T cell interacting with MD4 B cells. CFP fluorescence from the YC-Nano-50CD reporter identifies T cells in the fluorescence overlays (columns 1 and 3), and ratiometric FRET signals are presented in the heatmap images (columns 2 and 4). Scale bar, 20 μm. See corresponding Videos S5 and S6. (B and C) Mean ΔRt/R0 (B) and time-integrated calcium index (C) of individual contacts between Bcl6+/+ (n = 65) or Bcl6+/− (n = 64) OT-II T cells and MD4 B cells. Lines denote the means. Data are pooled from four experiments. ∗∗p < 0.01.

Video S5. Calcium fluxes in Bcl6+/+ T cells are associated with contacts with cognate B cells, related to Figure 3

DsRed-expressing MD4 B cells (red) and YC-Nano-50CD-transduced Bcl6+/+ OT-II T cells (blue) were visualized in a follicle of the draining lymph node 96 h after HEL-OVA immunization. The left panel is fluorescent image sequence, and the right panel is pseudo-color FRET ratiometric image sequence of the T cell. A white circle highlights increased calcium flux in a Bcl6+/+ OT-II T cell during the contact with an MD4 B cell. Scale bar, 20 μm. See also corresponding Figure 3A.

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Video S6. Reduced calcium fluxes in Bcl6+/− T cells are associated with contacts with cognate B cells, related to Figure 3

DsRed-expressing MD4 B cells (red) and YC-Nano-50CD-transduced Bcl6+/− OT-II T cells (blue) were visualized in a follicle of the draining lymph node 96 h after HEL-OVA immunization. The left panel is fluorescent image sequence, and the right panel is pseudo-color FRET ratiometric image sequence of the T cell. A white circle highlights subdued calcium response in a Bcl6+/− OT-II T cell during the contact with an MD4 B cell. Scale bar, 20 μm. See also corresponding Figure 3A.

Download video file (1MB, mp4)

The absence of BCL6 impairs CD40L externalization

During T-B cell entanglement in the follicle or GCs, calcium-signaling-mediated CD40L externalization is a prerequisite for contact-dependent CD40L delivery from Tfh cells to cognate B cells (Liu et al., 2015). Blockade of CD40L signaling to antigen-activated B cells already in the follicular phase (e.g., as late as 50–60 h in the model of MD4 and OT-II co-transfer) markedly impaired GC formation (Figure S4). While surface CD40L expression was comparable between Bcl6+/− than Bcl6+/+ Tfh cells in vivo, the total amount of CD40L protein detected by intracellular staining was lower in Bcl6+/− cells, consistent with impaired calcium signaling in T cells when BCL6 expression is compromised (Figures 4A and 4B). Importantly, when T cells were stimulated via anti-CD3 to mobilize CD40L from the intracellular store onto the plasma membrane over a brief period of 15 min, a calcium-signaling-dependent process, Bcl6+/− Tfh cells mobilized significantly less amounts of CD40L (Figures 4C and 4D). These data suggest that, despite being able to express CD40L, BCL6-insufficient T cells could not efficiently deliver CD40L during those brief contacts with cognate B cells, most likely as a result of reduced calcium-signaling efficiency.

Figure 4.

Figure 4

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BCL6 is required for efficient CD40L delivery

OT-II T cells of indicated genotypes, 7–8 days after being activated in vivo in adoptive B6 hosts immunized with NP-OVA, were subjected to the CD40L mobilization assay as described in the STAR Methods.

(A) Representative histograms of cell surface CD40L (left) or total CD40L measured by intracellular staining (right) of Bcl6+/+ and Bcl6+/− OT-II T cells.

(B) MFIs of CD40L staining for conditions in (A); each line represents one independent experiment for each comparison setup.

(C and D) CD40L mobilization by anti-CD3 in Bcl6+/+ and Bcl6+/− OT-II T cells; representative histogram overlays (C) and changes in surface CD40L MFI (ΔMFI) following indicated stimulation (D). Each line represents one independent experiment. n.s. not significant, ∗∗p < 0.01.

BCL6 insufficiency impairs CD40L delivery to B cells in a polyclonal response

Our results have so far revealed two major defects of BCL6-insufficient T cells inside the follicle that can be explained by an impairment of TCR-triggered calcium flux: reduced T-B cell physical contact and reduced CD40L externalization. These helper defects can readily explain impairment of B cell expansion and GC formation. On the other hand, it is difficult to explain why Bcl6+/− T cells exhibited grossly normal Tfh cell development at the beginning but could not maintain their Tfh cell phenotype over time. In particular, the effect of missing one copy of Bcl6 gene in T cells appeared to have a more immediate and pronounced repercussion on B cells being helped rather than on the helper cells per se.

Our use of OT-II and MD4 receptor-transgenic cells, while necessary for intravital imaging experiments to examine cell-cell interaction dynamics, might reveal functional defects that do not normally manifest. Moreover, germline Bcl6 ablation might have unintended consequences. To ameliorate these concerns, we bred Cd4-cre and Bcl6fl/fl mice to create T cell-specific BCL6-deficient animals. In replicating our observations with the heterozygous germline Bcl6+/− (Figure 1), we found that Cd4-cre × Bcl6fl/+ OT-II T cells were able to express similar amounts of CXCR5 following 4 days of activation in vivo but were significantly less competent in promoting GC formation or maintaining the CXCR5+ phenotype by day 8 (Figure S5). Next, we transferred splenocytes from Cd4-cre, Cd4-cre × Bcl6fl/+, or Cd4-cre × Bcl6fl/fl mice into sub-lethally irradiated CD45.1 SAP-deficient mice and immunized these recipients with NP-KLH (Figure 5A). In this system, normal polyclonal B cells would respond to NP-KLH with cognate T cell help provided by exogenous wild-type (Cd4-cre), BCL6-insufficient (Cd4-cre × Bcl6fl/+), or BCL6-deficient (Cd4-cre × Bcl6fl/fl) T cells from a polyclonal repertoire. The endogenous T cells cannot support GC formation because of the SAP deficiency (Qi et al., 2008). By day 13, when the NP-KLH-induced GC response typically peaked in wild-type mice, the group with BCL6-deficient T cells had far fewer T cells exhibiting the Tfh phenotype and almost no detectable GCs, as expected; with BCL6-insufficient T cells as helper T cells, the magnitude of the GC response was halved, while the frequency of T cells exhibiting the Tfh phenotype was barely reduced (Figures 5B and 5C). These data indicate that, in a polyclonal response to immunization, both copies of the Bcl6 gene are required for normal helper T cell activities to support GC formation, which is, again, more sensitive to the BCL6 insufficiency in T cells than is T cell development into Tfh cells per se (appearance of CXCR5+PD-1+ T cells).

Figure 5.

Figure 5

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Haploinsufficiency of BCL6 impairs a polyclonal response

(A) The experimental scheme.

(B and C) Representative flow-cytometry profiles and frequencies of FAShi GL7hi GC B cells in total B220+ B cells (B) or CXCR5hiPD-1hi Tfh cells in CD44hi CD4 T cells of the donor origin (C) 13 days after NP-KLH immunization in CD45.1 Sap−/− recipients. All donors are on the CD4-Cre background, and genotypes of the Bcl6 allele are indicated. Each symbol represents one mouse, and lines denote the means. Data are pooled from 2 independent experiments. n.s., not significant. ∗p < 0.05.

Next, to look for signs that BCL6-insufficient T cells may not efficiently deliver CD40L to B cells in a polyclonal response in vivo, we transferred wild-type (Cd4-cre) or BCL6-insufficient (Cd4-cre × Bcl6fl/+) OT-II T cells into SAP-deficient mice and immunized these mice with NP-OVA (Figure S6A). By day 3 post immunization, we sort-purified NP-binding B cells that were developing into GCs (Figure S6B) and conducted RNA sequencing (RNA-seq) analyses of their transcriptome. Using a gene expression signature induced by CD40 signaling in B cells (Basso et al., 2004) for enrichment analysis, we found such CD40-induced gene set expression was significantly deprived in those NP-binding B cells helped by Cd4-cre × Bcl6fl/+ T cells (Figure S6C). These data suggest BCL6 insufficiency in T cells leads to impaired CD40L signaling to B cells.

BCL6 and other Tfh cell markers depend on acute T-B cell interactions

It has been reported that, when Tfh cells are deprived of reciprocal B cell stimulation for several days, Tfh cell maintenance is severely impaired (Baumjohann et al., 2013; Deenick et al., 2010). Based on a model of BCL6-instructed Tfh cell differentiation and maintenance (Crotty, 2014; Vinuesa et al., 2016a), one interpretation is that BCL6 may initiate an epigenetic Tfh cell program (Hatzi et al., 2015; Kroenke et al., 2012; Li et al., 2018; Liu et al., 2016a; Liu et al., 2012; Lu et al., 2011) that is inheritable but requires intermittent antigen stimulation to enforce over a long period of time. Alternatively, because markers of Tfh cells (namely CXCR5, PD-1, and BCL6) can be rapidly upregulated upon antigen stimulation in vivo, at least for naive T cells (Chen et al., 2015; Kerfoot et al., 2011), it is also plausible that, even in the follicular phase of the response, reduced T-B cell interactions have a rapid repercussion on expression of markers of Tfh cells within a short time frame. Either of these two models could explain the inability of Bcl6+/− T cells to maintain Tfh cell phenotype in the follicular phase. A key distinction, however, is the timescale by which manipulation of follicular T-B cell interactions can affect the Tfh cell phenotype.

To test whether BCL6 and other markers of Tfh cells can be rapidly up- or downregulated upon acute manipulation of follicular T-B cell interactions, we took two approaches. First, we injected αDEC205 antibody linked to the ovalbumin-derived peptide epitope OVA323-339 (αDEC-OVA) 100 h post HEL-OVA immunization of B6 mice that received OT-II T cells and MD4 B cells. Because as antigen-activated B cells upregulate DEC-205 as they develop into GC B cells, the αDEC-OVA treatment preferentially delivers OVA epitope to those B cells (Victora et al., 2010). Merely 15 h after αDEC-OVA injection, OT-II T cells in the spleen markedly upregulated CXCR5, PD-1, and BCL6 (Figure 6A), leading to doubling of the frequency of CXCR5hiPD-1hi cells (Figure 6B) without changing the overall OT-II abundance. BCL6 expression in cells already gated as CXCR5hiPD-1hi were also markedly increased by ∼3-fold after αDEC-OVA injection (Figure 6C). Second, we injected αCD40L antibody to block CD40-CD40L interactions between follicular OT-II and MD4 B cells. Within the same 15 h period, amounts of BCL6, CXCR5, and PD-1 decreased on OT-II T cells (Figure 6A), with their nominal Tfh cell frequency being reduced by approximately one-third (Figure 6B). Those CXCR5hiPD-1hi cells also showed a significant decrease in BCL6 expression (Figure 6C). These results suggest any BCL6-instructed, inheritable program that specifies and maintains the CXCR5hiPD-1hiBCL6+ Tfh cell phenotype would not be stable for longer than 15 h without exogenous input (e.g., B cell-mediated antigen presentation). Given how brief this 15 h period, it is plausible that BCL6 also helps maintain the CXCR5hiPD-1hi phenotype in a T cell non-autonomous manner by simply promoting cognate T-B cell interactions (Figures 3 and 4).

Figure 6.

Figure 6

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Maintenance of BCL6 and other Tfh cell markers requires constant T-B cell interactions

(A–C) CXCR5, PD-1, and BCL6 expression after acute antigen stimulation and T-B cell cooperation blockade in vivo. (A) Histograms and MFI statistics of indicated markers on transferred OT-II T cells 15 h after PBS, αDEC-OVA, or αCD40L injection. (B) Frequencies of OT-II Tfh cells gated as CXCR5hi PD1hi cells. (C) BCL6 expression by CXCR5hi PD1hi OT-II Tfh cells as gated in (B). Each symbol in scatterplots denotes one mouse. One of four experiments with similar results is shown. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Defects in GC formation and Tfh cell maintenance associated with BCL6-compromised T cells can be rescued

If there is a T cell non-autonomous mechanism of BCL6-dependent Tfh cell maintenance as deduced above, one would predict that if CD40L is made more abundantly available in BCL6-insufficient T cells to compensate for the impaired delivery of CD40L, both the failure of normal GC formation and Tfh cell maintenance would be rescued. On the other hand, if BCL6 only instructs and maintains a Tfh cell fate in a strictly T cell autonomous manner, CD40L overexpression should not rescue the Tfh cell defect of BCL6-insufficient cells, even if it might rescue defective GC formation. To test this, we overexpressed CD40L in Bcl6+/− and Bcl6+/+ OT-II T cells by retroviral transduction, transferred these cells together with MD4 B cells into SAP-deficient mice, and examined MD4 GC formation and OT-II Tfh cell development. As shown in Figure 7A, while control-transduced Bcl6+/− OT-II T cells were not able to support normal GC formation, Bcl6+/− OT-II T cells overexpressing CD40L now supported a similar magnitude of GC formation as compared with the Bcl6+/+ counterpart. Importantly, the reduced CXCR5hiPD1hi Tfh cell frequency of Bcl6+/− cells was rectified to a comparable magnitude of control-transduced Bcl6+/+ cells (Figure 7B). Taken together, these data suggest that, in the follicular phase, the key biological process that BCL6 controls is contact-dependent help delivery to B cells, which is not only essential for GC formation but also for Tfh cell maintenance in a T cell non-autonomous manner.

Figure 7.

Figure 7

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Rescue of BCL6-insufficient Tfh cells secondary to GC rectification

(A) Representative flow-cytometry profiles (left) and frequencies of IgMa+ MD4 GC B cells in total CD19+ B cells (right) 5 days after HEL-OVA immunization of Sap−/− mice that received transfer of MD4 B cells and Bcl6+/+ or Bcl6+/− OT-II T cells transduced with a control GFP or CD40L-expressing vector. Each symbol in the scatterplot (right) represents one mouse, and lines denote the means. One of three independent experiments with similar results is shown. ∗∗p < 0.01, ∗∗∗p < 0.001.

(B) Representative flow-cytometry profiles (left) and frequencies of CXCR5hi PD1hi Tfh cells in OT-II T cells as in (A). Each symbol shape represents one mouse, and lines denote the means. One of three independent experiments with similar results is shown. ∗p < 0.05.

BCL6 is required for normal expression of calcium-signaling-related genes

Finally, in trying to understand how BCL6 may regulate calcium signaling in Tfh cells, which is important for T-B cell entanglement and for CD40L delivery, we noted that regulatory regions of human STIM1 gene are bound by BCL6 in human Tfh cells (Hatzi et al., 2015). We conducted mRNA sequencing of BCL6-sufficient (Cd4-cre) and BCL6-insufficient (Cd4-cre × Bcl6fl/+) OT-II T cells sort-purified 3 days after activation in vivo by NP-OVA immunization. As shown in Figure S7, when one copy of the Bcl6 gene was missing, CXCR5+PD-1+ Tfh cells differentially expressed many genes; among those downregulated were Stim1 and Plcg1 (which code for STIM1 and PLCγ1, respectively) that both impinge on calcium signaling downstream of TCR activation. Moreover, KEGG pathway analyses revealed that the calcium-signaling-related pathway was generally upregulated in wild-type as compared with BCL6-insufficient Tfh cells (Figure S7). Together, these data support the notion that BCL6 controls follicular T-B cell interactions by regulating multiple target genes involved in antigen-triggered calcium signaling in T cells.

Discussion

Our study reveals a previously unappreciated role for BCL6 in enhancing antigen-triggered calcium signaling in Tfh cells and promoting their entangled interactions with and CD40L delivery to cognate B cells, all specifically in the follicular phase of a primary response. Combined with its obligate role in regulating CXCR5 expression and associated follicular recruitment (Liu et al., 2014; Shaw et al., 2016), our findings provide a more complete picture for biological processes that BCL6 actually controls in T cells to render its essentiality for GC formation. At least in the model of protein immunization used here, early CXCR5 upregulation and Tfh cell development proceeded normally as far as one copy of Bcl6 allele is intact, whereas follicular functions of BCL6 in T cells required both alleles intact. Given the fact that BCL6 is very abundantly expressed in normal Tfh cells, we speculate those target genes involved in BCL6-dependent calcium signaling might utilize low-affinity binding sites such that only a high concentration of nuclear BCL6, achievable with expression from both alleles, can ensure productive engagement and regulation.

Although the capacity to produce CD40L is not unique to the Tfh cell subset, by definition only Tfh cells are tasked to deliver CD40L to B cells inside the follicle. CD40L delivery requires cell-cell contacts of sufficient stability, strength, and quality. The follicular microenvironment is highly dynamic, with all cells being in constant and fast migration, increasing the threshold for any T-B cell contacts to be functionally productive. This is probably the main reason why two copies of the Bcl6 gene are not required for stable T-B cell interactions at the T zone-follicle border but become essential for those entangled contacts inside the follicle.

Expression of BCL6, CXCR5, and PD-1—markers universally used to identify Tfh cells and to assess the underlying developmental program—could markedly change within 15 h, depending the status of follicular T-B cell interactions in vivo. In additional work, αDEC-OVA was found to drive such changes within 7 h (unpublished data). Our results set the upper time limit by which manifestation of a Tfh cell program can be maintained in the follicular phase of a primary response following protein immunization. These results are also consistent with the fact that none of Tfh cell-associated genes are known to be epigenetically rendered open or shut across multiple mitoses in single Tfh cells by a BCL6-mediated mechanism, even though transcription of Tfh cell-associated genes may depend on BCL6, directly or indirectly.

It can be argued that because only three main markers of Tfh cells are examined in the current study, other Tfh cell-associated molecules or pathways, such as those underlying BCL6 follicular functions reported here, might be programmed in a more long-lasting mode. This possibility warrants further investigation. However, our results do caution interpreting changes in CXCR5, PD-1, and BCL6 markers, either in terms of expression intensities or frequencies of marker-positive cells (universally used to report Tfh cell frequencies in the literature), as demonstration of differentiation, trans-differentiation, or plasticity events being induced by any experimental perturbation—particularly when the perturbation is applied days or even weeks before reading the outcome. Such perturbation could be simply changing the strength of TCR stimulation and T-B cell interactions hours before the assay readout. At the same time, we should also note the fact that T cell CXCR5 expression, besides its extreme flexibility that is conditional upon T-B cell interactions in the follicle, might be subjected to long-lasting epigenetic regulation, as implied by the previous observation that some CXCR5+ T cells do not completely lose CXCR5 expression a month after being transferred into antigen-free hosts (Lüthje et al., 2012).

While how BCL6 precisely controls STIM1 and PLCγ1 expression and promotes calcium signaling in Tfh cells remains to be further investigated, it is notable that BCL6 expression in T cells partly depends on the nuclear factor of activated T cell (NFAT) transcription factor (Martinez et al., 2016; Vaeth et al., 2016), which is activated by calcium signaling (Gwack et al., 2007). This pathway potentially explains how the amount of BCL6 protein in normal Tfh cells can rapidly increase in response to acute antigen stimulation in vivo. A positive feedback might operate inside Tfh cells between BCL6-promoted calcium signaling downstream of TCR stimulation and NFAT-facilitated BCL6 expression. When BCL6 becomes insufficient, even though the calcium signaling is not totally abolished, a reduced efficiency in T-B cell interactions would compromise help delivery, hamper Tfh cell maintenance, and reduce GC formation. In this scenario, development and maintenance of full Tfh cell features require intrinsic BCL6 expression but invoke both cell-autonomous and non-autonomous modes of action.

The role of BCL6 in regulating calcium signaling and T-B cell interactions is reminiscent of ICOS, which co-stimulates TCR-mediated calcium signaling and thereby promotes T-B cell interactions (Liu et al., 2015). It is also reminiscent of SAP, which protects proximal TCR signaling (Chu et al., 2014; Kageyama et al., 2012; Qi, 2012) and thereby promotes T-B cell interactions (Qi et al., 2008). It is probably not a coincidence that these three T cell-expressed factors essential for GC formation all impinge on antigen-triggered signaling, all regulate physical T-B cell interactions, and none demonstrably instruct a unique signaling pathway dedicated for Tfh cell development or maintenance, with the potential exception for ICOS-TBK1-associated signaling (Pedros et al., 2016), perhaps because sufficient CD40L delivery through physical T-B cell contacts is likely the only help factor that is absolutely essential and irreplaceable for GC formation in a T-dependent B cell response (Wan et al., 2019).

Limitations of study

Our multiple attempts to rescue the helper activity of BCL6-insufficient T cells with STIM1 overexpression did not yield consistent results, potentially because of strict requirement for appropriate stoichiometry of STIM and ORAI proteins, which is difficult to achieve using retroviral overexpression. The current study has not established a causal link between reduced STIM1 and/or PLC-γ1 expression and defective T-B cell interactions when T cells are BCL6 insufficient.

STAR★Methods

Key resources table

REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
anti-CD19 APC-Cy7 (clone 1D3) eBioscience CAT# 25-0193-82
anti-B220 APC-Cy7 (clone RA3-6B2) eBioscience CAT# 47-0452-82
anti-CD4 AF700 (clone RM 4-5) eBioscience CAT# 56-0042-82
anti-CD4 APC (clone GK1.5) eBioscience CAT# 17-0041-82
anti-CD4 FITC (clone GK1.5) eBioscience CAT# 11-0041-82
anti-CD44 AF700 (clone 1M7) eBioscience CAT# 56-0441-82
anti-CD45.1 APC (clone A20) eBioscience CAT# 17-0453-82
anti-CD45.2 eFlour450 (clone 104) eBioscience CAT# 48-0454-82
anti-CD95 PE-Cy7 (clone Jo2) BD Biosciences CAT# 557653
anti-GL7 eFlour450 (clone GL7) eBioscience CAT# 48-5902-82
anti-PD-1 PE-Cy7 (clone RMP1-30) Biolegend CAT# 109110
anti-IgMa PE (clone DS-1) BD Biosciences CAT# 553517
anti-IgD FITC (clone 11-26c) eBioscience CAT# 11-5993-82
anti-CXCR5 biotinylated (clone 2G8) BD Biosciences CAT# 551960
anti-CD40L PE (clone MR1) BD Biosciences CAT# 553658
Streptavidin PE BD Biosciences CAT# 554061
Streptavidin APC BioLegend CAT# 554067
NP-PE Biosearch Technologies CAT# N-5070-1
7-AAD Biotium CAT# 40084
Anti-CD40L (clone MR1) Bio X Cell CAT# BP0017-1
Anti-CD3ε (clone 145-2C11) Bio X Cell CAT# BP0001-1
Anti-CD28 (clone 37.51) Bio X Cell CAT# BE0015-1
Commercial reagents
Lipopolysaccharides (Escherichia coli, serotype O111:B4) Sigma-Aldrich CAT# L2630-25MG
NP-OVA Biosearch Technologies CAT# N-5051-10
NP-KLH Biosearch Technologies CAT# N-5060
Imject Alum Adjuvant Thermo Fisher CAT# 77161
Lysozyme from chicken egg white Sigma-Aldrich CAT# L4919
Albumin from chicken egg white Sigma-Aldrich CAT# A2512
RNase Inhibitor Takara CAT# 2313B
TE buffer Invitrogen CAT# 12090015
Triton X-100 Invitrogen CAT# 15596018
Oligo-dT Takara CAT# 3806
dNTP Invitrogen CAT# 18427013
SuperScript II reverse transcriptase Invitrogen CAT# 18064071
Superscript II first-strand buffer Invitrogen CAT# 18064014
DTT Solarbio CAT# D1070-5
Betaine Sigma CAT# 107-43-7
MgCl2 Invitrogen CAT# AM9530G
KAPA HiFi HotStart ReadyMix KAPA CAT# Kk2601
DNA clean beads Vazyme CAT# N411-02
Nuclease-free water Invitrogen CAT# 10977015
TruePrep DNA Library Prep Kit V2 for Illumina Vazyme CAT# TD501-02
SDS Takara CAT# 3806
Q5 HF master mix NEB CAT# M0492L
Tris Amresco N/A
Critical commercial assays
Cytoperm/Cytofix kit BD Biosciences CAT# 554714
Foxp3/Transcription Factor Staining Buffer Set eBioscience CAT# 00-5523-00
Deposited data
mRNA sequencing data of pre-GC B cells helped by Cd4-cre or Cd4-cre × Bcl6fl/+ T cells This paper GSE181081
mRNA sequencing data of activated Cd4-cre or Cd4-cre × Bcl6fl/+ T cells This paper GSE181081
Recombinant DNA
Plasmid pMSCV N/A
Plasmid pIE N/A
Plasmid for DEC205-OVA Gift from Dr. M. Nussenzweig N/A
Plasmid for YC-nano50CD reporter N/A
Experimental models: Organisms/strains
Mouse: C57BL/6J Jax 664
Mouse: Sap−/− Gift from Dr. P. Schwartzberg (Czar et al., 2001) N/A
Mouse: Bcl6−/− Gift from Dr. L. Staudt (Dent et al., 1997) N/A
Mouse: Bcl6fl/fl Gift from Dr. T. Takemori (Kaji et al., 2012) N/A
Mouse: CD45.1 Jax 002014
Mouse: CD4-Cre Jax 022071
Mouse: GFP-expressing Jax 4353
Mouse: CFP-expressing Jax 4218
Mouse: dsRed-expressing Jax 6051
Mouse: OT-II Jax 4194
Mouse: MD4 Jax 2595
Software and algorithms
FlowJo v10 Tree Star N/A
ImageJ Wayne Rasband N/A
Imaris v9 Bitplane N/A
Prism v6 Graphpad N/A
GSEA Broad Institute N/A
Adobe AfterEffect Adobe N/A
Adobe PhotoShop Adobe N/A
FastQC v0.11.9
Hisat2 version 2.2.1
Samtools version 1.10
HTSeq-count version 0.12.4
DESeq2 software R N/A
Other
HiSeq × Ten sequencer Illumina N/A
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Resource availability

Lead contact

Further information and requests for resources and reagents should be directed and will be fulfilled by the lead contact Hai Qi (qihai@tsinghua.edu.cn).

Materials availability

Supply of the following reagents and mice are subject to MTA agreements: DEC205-OVA (Dr. M. Nussenzweig); Bcl6−/− mice (Dr. L. Staudt); Bcl6fl/fl mice (Dr. T. Takemori).

Experimental model and subject details

Animal experimental models

C57BL/6 (Jax 664), CD45.1 (Jax 002014), CD4-Cre (Jax 022071), GFP-expressing (Jax 4353), CFP-expressing (Jax 4218), dsRed-expressing (Jax 6051), OVA323-339-specific TCR transgenic OT-II (Jax 4194) and HEL-specific Ig-transgenic MD4 (Jax 2595) mice were originally purchased from the Jackson Laboratory. Bcl6−/− mice (Dent et al., 1997), Bcl6fl/fl mice (Kaji et al., 2012) and Sap−/− mice (Czar et al., 2001) were gifts from Drs. L. Staudt, T. Takemori and P. Schwartzberg, respectively. Relevant alleles were interbred to obtain desired genotypes. All mice were maintained under specific-pathogen free conditions, and used according to institutional and governmental guidelines for animal welfare.

Method details

Cell isolation, culture and retroviral transduction

Naive OT-II T cells, polyclonal T cells, MD4 B cells or polyclonal B cells were isolated using the negative CD4 T cell or B cell isolation kit (Miltenyi Biotec). To obtain activated T cells in vitro, naive OT-II or polyclonal B6 CD4 T cells were cultivated on plates coated with anti-CD3 (8 μg/mL) and anti-CD28 (8 μg/mL). Retroviral vectors that express relevant target genes were packaged with the Plat-E system, as previously described (Xu et al., 2013). For transduction, activated T cells were spin-infected at 2200 rpm with viral supernatants in the presence of 1 μg/mL polybrene (Sigma) for 2 h at 32°C. Infected T cells were then cultivated in the presence of 10 ng/mL human IL-2 until use for in vitro experiments or for adoptive transfer.

Adoptive transfer, immunization, and perturbation of T-B cell interactions

For various in vivo experiments, naive or in vitro activated and retrovirally transduced OT-II T cells were intravenously injected into B6 or SAP-deficient mice, alone or in combination with MD4 B cells. For certain experiments, total splenocytes were intravenously injected into sub-lethally irradiated CD45.1 SAP-deficient mice. In such experiments, the number of CD4 T cells in the splenocyte inoculum was kept constant among different groups. Typically, splenocytes from one donor mouse were split and transferred into three recipients. For subcutaneous immunization, each recipient mouse was given either 30 μg NP-OVA (Biosearch Technology) or 30 μg HEL-OVA antigen plus 0.5 μg LPS in alum (Thermo Scientific). For intraperitoneal immunization, each recipient mouse was given 100 μg NP-OVA (Biosearch Technology) or 100 μg NP-KLH (Biosearch Technologies) or 50 μg HEL-OVA antigen plus 1 μg LPS in alum (Thermo Scientific). For CD40L blockade in the early follicular phase, HEL-OVA-immunized B6 recipients of OT-II T cells and MD4 B cells were intravenously given 200 μg αCD40L antibody or PBS 50-60 h post immunization. For αDEC-OVA and CD40L blockade experiments, HEL-OVA-immunized B6 recipients of OT-II T cells and MD4 B cells were intravenously given 50 μg αDEC-OVA or 100 μg αCD40L antibody or PBS 100 h post immunization, a time point well into the follicular phase of the response.

Flow cytometry

Single-cell suspension of splenic or lymph node cells were incubated with 10% 2.4G2 culture supernatants to block Fc receptors and then stained with indicated antibodies in MACS buffer (PBS supplemented with 1% FBS and 5mM EDTA). Staining reagents include: APC-Cy7 anti-CD19 (1D3), APC-Cy7 anti-B220 (RA3-6B2), AF700 anti-CD4 (RM 4-5), APC anti-CD4 (GK1.5), FITC anti-CD4 (gk1.5), AF700 anti-CD44 (IM7), APC anti-CD45.1 (A20), eF450 anti-CD45.2 (104), PE-Cy7 anti-CD95 (Jo2), eFlour450 anti-GL7 (GL7), PE-Cy7 anti-PD-1 (RMP1-30), PE anti-IgMa (DS-1), FITC anti-IgD (11-26c) from eBioscience; biotinylated anti-CXCR5 (2G8), PE anti-CD40L (MR1), streptavidin PE (Cat 554061) and streptavidin APC (Cat 554067) from BD Biosciences. Dead-cell exclusion was based on 7-AAD staining (Biotium), and non-singlet events were excluded with FSC-H/FSC-W and SSC-H/SSC-W. Isotype-matched non-specific antibodies were purchased from the corresponding companies. Cells were stained with primary antibodies for 60-90 min, washed, and then with secondary reagents for 30 min on ice. For intracellular staining, cells were stained using Cytoperm/Cytofix kit (BD Biosciences) according to the manufacturer’s protocol. All flow-cytometry data were collected on an LSR-II or Aria III (BD).

Immunohistochemistry

To examine T cell distribution in the draining lymph node, tissues were fixed with paraformaldehyde, and sections were stained with EF450-anti-IgD and APC-anti-CD3, mounted with the ProlongGold Antifade reagent (Invitrogen), and imaged with an Olympus FV1000 upright microscopy using 20 × lens.

CD40L mobilization of T cells ex vivo

As described previously (Liu et al., 2015), OT-II T cells of indicated genotypes were transferred into B6 mice, which were subsequently immunized with NP-OVA intraperitoneally. 7 to 8 days later, splenocytes were stimulated by anti-CD3 (2 μg/mL) for 15 min in the presence of an anti-CD40L staining antibody (clone MR1) to capture externalized CD40L on the cell surface. Cells were further stained for additional surface markers before analyzed by flow cytometry.

Intravital imaging

Intravital two-photon imaging of mouse inguinal lymph nodes were done as described previously (Liu et al., 2015), including measurement of T-B cell contact extents by contact duration and SEI. To visualize T cell calcium signaling during T-B cell contacts in vivo, YC-nano50CD reporter was transduced into Bcl6+/+ or Bcl6+/− OT-II T cells. Ratiometric images of calcium signals were analyzed with the RatioPlus plugin of ImageJ. Quantitative analyses were conducted by the method established previously (Liu et al., 2015). Adobe Photoshop and AfterEffect were used to annotate and prepare image sequences and to make playback videos.

RNA-seq and Gene Set Enrichment Analysis (GSEA)

CD4-Cre × Bcl6fl/+ or control CD4-Cre OT-II T cells were transferred into CD45.1 Sap−/− mice (5 × 105 per recipient), which were subcutaneously immunized with 40 μg NP-OVA plus 0.4 μg LPS in alum one day after the cell transfer. At day 3 post immunization, CXCR5hiPD-1hi Tfh cells were sort-purified from the draining lymph node and prepared for RNA-seq analysis. Two technical repeats of ∼200 cells per sample from each of 3 mice were included. To analyze NP+IgDlowB220+ pre-GC B cells, cells from recipients of the same group were pooled to sort-purify 4 200-cell repeats. The SMART-Seq2 protocol for single-cell analysis was adapted and optimized for analyzing ∼200 cells. All libraries were sequenced on a HiSeq × Ten sequencer (Illumina). After pre-processed using FastQC (v0.11.9), raw sequences were aligned to the Mus musculus reference genome (GRCm38) using Hisat2 (version 2.2.1) and then sorted with Samtools (version 1.10). Reads were counted in genes with the utilization of HTSeq -count (version 0.12.4). Gene expression was calculated with the DESeq2 software in R. Heatmaps and volcano plots were generated with pheatmap and ggplot2 R functions, respectively. GSEA analysis was carried out using the GSEA software from the Broad Institute.

Statistical analysis

Statistics and graphing were done with Prism (Graphpad). Unless indicated otherwise, two-tailed Student’s t test was used to compare end-point means of different groups.

Acknowledgments

The authors thank Drs. L. Staudt and P. Schwartzberg for providing the germline Bcl6−/− and Sap−/− mice, respectively; Dr. T. Takemori for providing Bcl6fl/fl mice; and Dr. M. Nussenzweig for providing the αDEC-OVA reagent. This work was supported in part by the National Key R&D Program of China (Ministry of Science and Technology, 2018YFE0200300), the Natural Science Foundation of China (8162002 and 81761128019), and the Beijing Municipal Science and Technology Commission. H.Q. is an HHMI International Research Scholar, and this work was also funded in part by the Bill & Melinda Gates Foundation and the Howard Hughes Medical Institute. The findings and conclusions within are those of the authors and do not necessarily reflect positions or policies of the Bill & Melinda Gates Foundation or the Howard Hughes Medical Institute.

Author contributions

H.Q. conceptualized and supervised the study. D.L., J.Y., and J.S. conducted a majority of experiments. B.L. and X.S. conducted imaging and animal experiments, Y.L. and J.S. conducted RNA-seq analysis. H.Q. wrote the paper together with D.L. and B.L. All authors contributed to data interpretation.

Declaration of interests

The authors declare no competing interests.

Published: August 30, 2021

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.immuni.2021.08.003.

Supplemental information

Document S1. Figures S1–S7
mmc1.pdf (4.1MB, pdf)
Document S2. Article plus supplemental information
mmc8.pdf (7.6MB, pdf)

Data and code availability

mRNA sequencing data have been deposited at GEO and are publicly available as of the date of publication. Accession numbers are listed in the key resources table. Microscopy data reported in this paper will be shared by the lead contact upon request. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Video S1. Comparable contacts between MD4 and dsRed- and CFP-transgenic Bcl6+/+ OT-II T cells at the T zone-follicle border, related to Figure 2

GFP-expressing MD4 (green), dsRed-expressing Bcl6+/+ (red) OT-II T cells, and CFP-expressing Bcl6+/+ (blue) OT-II T cells were visualized in the T zone-follicle border region of the draining lymph node 36 h after HEL-OVA immunization. The movie was played back twice, and representative T-B cell contacts were highlighted with arrowheads in the second playback. Scale bar, 20 μm.

Download video file (4.4MB, mp4)
Video S2. Comparable contacts between MD4 and dsRed-transgenic Bcl6+/− and CFP-transgenic Bcl6+/+ OT-II T cells at the T zone-follicle border, related to Figure 2

GFP-expressing MD4 (green), dsRed-expressing Bcl6+/− (red) OT-II T cells, and CFP-expressing Bcl6+/+ (blue) OT-II T cells were visualized in the T zone-follicle border region of the draining lymph node 36 h after HEL-OVA immunization. The movie was played back twice, and representative T-B cell contacts were highlighted with arrowheads in the second playback. Scale bar, 20 μm.

Download video file (7.3MB, mp4)
Video S3. Similar duration and surface engagement of dsRed- and CFP-transgenic Bcl6+/+ follicular OT-II T cells, related to Figure 2

CFP-expressing Bcl6+/+ (blue), dsRed-expressing Bcl6+/+ (red) OT-II T cells, and GFP-expressing MD4 B cells (green) were visualized in a follicle of the draining lymph node 96 h after HEL-OVA immunization. The movie was played back twice, and representative T-B cell contacts were highlighted with arrowheads in the second playback. Scale bar, 20 μm.

Download video file (15.7MB, mp4)
Video S4. Similar duration but reduced surface engagement of dsRed-transgenic Bcl6+/− as compared with CFP-transgenic Bcl6+/+ follicular OT-II T cells, related to Figure 2

CFP-expressing Bcl6+/+ (blue), dsRed-expressing Bcl6+/− (red) OT-II T cells, and GFP-expressing MD4 B cells (green) were visualized in a follicle of the draining lymph node 96 h after HEL-OVA immunization. The movie was played back twice, and representative T-B cell contacts were highlighted with arrowheads in the second playback. Scale bar, 20 μm. See also corresponding Figure 2A.

Download video file (16.1MB, mp4)
Video S5. Calcium fluxes in Bcl6+/+ T cells are associated with contacts with cognate B cells, related to Figure 3

DsRed-expressing MD4 B cells (red) and YC-Nano-50CD-transduced Bcl6+/+ OT-II T cells (blue) were visualized in a follicle of the draining lymph node 96 h after HEL-OVA immunization. The left panel is fluorescent image sequence, and the right panel is pseudo-color FRET ratiometric image sequence of the T cell. A white circle highlights increased calcium flux in a Bcl6+/+ OT-II T cell during the contact with an MD4 B cell. Scale bar, 20 μm. See also corresponding Figure 3A.

Download video file (1MB, mp4)
Video S6. Reduced calcium fluxes in Bcl6+/− T cells are associated with contacts with cognate B cells, related to Figure 3

DsRed-expressing MD4 B cells (red) and YC-Nano-50CD-transduced Bcl6+/− OT-II T cells (blue) were visualized in a follicle of the draining lymph node 96 h after HEL-OVA immunization. The left panel is fluorescent image sequence, and the right panel is pseudo-color FRET ratiometric image sequence of the T cell. A white circle highlights subdued calcium response in a Bcl6+/− OT-II T cell during the contact with an MD4 B cell. Scale bar, 20 μm. See also corresponding Figure 3A.

Download video file (1MB, mp4)
Document S1. Figures S1–S7
mmc1.pdf (4.1MB, pdf)
Document S2. Article plus supplemental information
mmc8.pdf (7.6MB, pdf)

Data Availability Statement

mRNA sequencing data have been deposited at GEO and are publicly available as of the date of publication. Accession numbers are listed in the key resources table. Microscopy data reported in this paper will be shared by the lead contact upon request. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

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