T follicular helper (TFH) cells are central to the development and regulation of T cell-dependent humoral immune responses. The transcriptional repressor BCL6 is required for TFH responses, but the kinetics of BCL6 protein expression in activated CD4+ T cells have not been established. We measured BCL6 expression during TFH cell development at the single-cell level using intracellular staining and YFP-BCL6 fusion protein reporter mice. BCL6 was immediately upregulated in all dividing T cells during dendritic cell–T cell interactions. A second wave of early BCL6 expression coincided with the induction of CXCR5, resulting in a distinct and stable TFH cell population. Cognate B cells were not required for the induction of BCL6, but supported the expansion of TFH cells. These data suggest that BCL6 participates in very early events in TFH cell development, and that repeated encounters with APCs reinforce BCL6 expression, thereby establishing the TFH cell phenotype.
T follicular helper (TFH) cells are the prototypical Th cells involved in the initiation and maintenance of germinal center (GC) responses that generate memory B cells and long-lived plasma cells (1–4). TFH cells express molecules that facilitate their functional interaction with Ag-specific B cells (1), including the chemokine receptor CXCR5 (5); the costimulatory molecules PD-1, ICOS, and BTLA; and the cytokine IL-21 (6, 7). In addition, TFH cells shape the humoral immune response by producing CD40L and cytokines that influence the Ig isotype produced by B cells (8–10). The transcriptional repressor BCL6 is essential for TFH cell development (11–13). Bcl6 mRNA is induced in activated T cells (14) and is abundant in TFH cells (15). However, BCL6 expression is subject to several modes of posttranslational regulation (16, 17), and single-cell analysis of BCL6 protein expression has been hampered by a lack of reliable detection reagents. In this study, we define the precise kinetics of BCL6 protein expression during TFH cell development in vivo. Early BCL6 upregulation occurs at the first cell division and can be induced by Ag-presenting dendritic cells (DCs). A second wave of BCL6 expression coincides with the induction of CXCR5 expression and results in the development of a distinct BCL6+CXCR5+ TFH cell population, even in the absence of cognate B cell interactions.
Materials and Methods
TCR-transgenic (tg) OT-II (004194) mice were purchased from The Jackson Laboratory (JAX) and crossed with B6.SJL-Ptprca Pepcb/BoyJ (002014) mice to obtain congenic CD45.1/2 offspring for adoptive transfers. TCR-tg SMARTA/BoyJ mice were provided by M. Matloubian and BCR-tg MD4/BoyJ mice by A. DeFranco (both from University of California, San Francisco). TCR-tg Do11.10/Rag2−/− mice were purchased from Taconic Farms. C57BL/6 (Charles River) or congenic B6-LY5.2/Cr (National Cancer Institute) mice were used as recipients. BALB/c (JAX) mice were recipients for Do11.10 cells. YFP-BCL6 knock-in reporter mice (18), in which a yfp construct was inserted into exon 1 of the Bcl6 gene resulting in the expression of a chimeric YFP-BCL6 protein, were crossed with OT-II and SMARTA mice to obtain heterozygous Bcl6YFP/+ offspring for adoptive transfers. All experiments were done according to the Institutional Animal Care and Use Committee guidelines of the University of California, San Francisco.
Adoptive cell transfers and immunizations
Naive CD4+ T cells (CD4+CD8−CD25−CD44lowCD62Lhigh) were enriched from spleens and lymph nodes (LNs) with the CD4+ negative isolation kit (Invitrogen) and further purified on a FACS Aria II cell sorter (BD Biosciences). T cells were labeled with 5 μM CellTrace Violet (Invitrogen) and injected i.v. into recipient mice. NP16-OVA (Biosearch Technologies) was mixed with Imject Alum (Pierce), and 5 μg NP16-OVA was injected s.c. into each footpad or 50 μg s.c. in the base of tail and flank. Bone marrow-derived DCs were generated, as previously described (19), using rGM-CSF (PeproTech). Maturation was induced with 0.5 μg/ml LPS (Sigma-Aldrich) overnight. Mature DCs were loaded with 3 μM OVA323–339 peptide (Anaspec) and washed extensively to remove free peptide. OVA-DCs or 5 μg OVA protein plus 2 μg LPS were injected s.c. into each footpad. For viral infections, 5 × 105 PFU lymphocytic choriomeningitis virus Armstrong were injected s.c. into each footpad of SMARTA cell recipients.
Single-cell suspensions were prepared by mincing and filtering LN cells through fine mesh. Abs were purchased from eBioscience, BD Biosciences, or Biolegend. Biotinylated anti-CXCR5 (BD Biosciences) was visualized with streptavidin-allophycocyanin. The anti-BCL6 Ab (clone K112-91) was from BD Biosciences. Unspecific binding was blocked with anti-CD16/CD32 plus 2% normal mouse/rat serum. Intracellular BCL6 staining was performed with the Foxp3 Staining Set (eBioscience). Samples were acquired on a LSR II cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star), gating out doublets and B cells. Dead cells were excluded with 7-aminoactinomycin D or Fixable Viability Dye eFluor780 (eBioscience).
Results and Discussion
BCL6 protein expression in TFH cells
We investigated the expression of BCL6 protein in TFH cells at the single-cell level using an optimized protocol for intracellular BCL6 staining. Wild-type (wt) mice were seeded with naive OVA-specific TCR-tg CD4+ (OT-II) T cells, and then immunized with OVA/alum in the hind footpads. By day 6.5, immunization provoked the generation of IgDlow FAS+ GC B cells that also stained brightly for BCL6. Because BCL6 is strongly upregulated in GC B cells, these data validate the specificity of the anti-BCL6 mAb (Fig. 1A). OT-II cells with a TFH phenotype (CXCR5+PD-1+) were already detectable at day 3.5, peaked around day 6.5, and declined by day 10.5 (Fig. 1B). TFH cells expressed higher levels of BCL6 than CXCR5lowPD-1low non-TFH cells at all time points analyzed (Fig. 1B), consistent with previous findings on the mRNA level (15). Furthermore, BCL6 expression positively correlated with the expression level of the TFH cell markers CXCR5 and PD-1, as shown by intracellular staining and visualization of a transgenic YFP-BCL6 fusion protein in OT-II and SMARTA T cells after protein immunization and viral infection, respectively (Supplemental Fig. 1). In summary, these data clearly showed that TFH cells expressed significant amounts of BCL6 protein and that TFH cells developed before the appearance of GC B cells.
Two waves of BCL6 expression during naive CD4+ T cell activation
Next, we combined adoptive transfer of CellTrace-labeled naive OT-II cells with FACS-based analysis of intracellular BCL6 protein expression to visualize the expression kinetics of BCL6 during early stages of TFH cell development. By 48 h after immunization, OT-II cells had started to divide in the draining, but not in the distal LNs (Fig. 2A). We observed a strong increase in BCL6 expression in dividing OT-II cells after the first cell division (Fig. 2A). Because CXCR5 was not yet induced at this early time point (Fig. 2B), BCL6 expression preceded the emergence of the TFH cell surface phenotype.
A strong increase in BCL6 expression at the first cell division stage was also detectable on day 3.5 (Fig. 2C). BCL6 expression waned with successive cell divisions in most of the dividing cells. After approximately five cell divisions, BCL6 was further upregulated in a subset of cells, although a clear boundary between BCL6high and BCL6low cells was not evident. Increased BCL6 expression coincided with the induction of a population of CXCR5+ cells. Plotting CXCR5 versus BCL6 expression at each cell division confirmed that BCL6 expression preceded the induction of CXCR5 (Fig. 2D). Importantly, this culminated in the emergence of a distinct BCL6+CXCR5+ TFH cell population among the most divided cells. The transgenic YFP-BCL6 fusion protein was expressed with similar kinetics (Fig. 2E, Supplemental Fig. 2). Divided OT-II cells recovered from distal LNs only expressed marginal levels of BCL6 and CXCR5, and most likely represented emigrants that were primed in the draining LNs (Fig. 2C, Supplemental Fig. 2).
Consistent with our previous reports, the chemokine receptors CXCR5 and CCR7 underwent reciprocal regulation during the first 3.5 d after immunization (Fig. 2B, Supplemental Fig. 2), reflecting their roles in positioning activated T cells in proximity to activated B cells (20, 21). CXCR5 was gradually upregulated on dividing OT-II cells, whereas CCR7 was rapidly lost upon T cell activation. The costimulatory molecules PD-1, ICOS, and BTLA were rapidly upregulated during OT-II cell priming (Fig. 2B). PD-1 levels further increased at later divisions, correlating with the increase in BCL6 and CXCR5 levels (Fig. 2C, Supplemental Fig. 2). ICOS expression was maintained at high levels during ensuing divisions. Taken together, these data demonstrate that BCL6 is induced very early during the priming of naive T cells, and that a subset of dividing Ag-specific T cells undergoes a second, stronger wave of BCL6 expression.
DCs are potent inducers of BCL6 expression in naive CD4+ T cells
The kinetics of early BCL6 upregulation suggested that BCL6 might be induced by DCs during T cell priming. To restrict Ag presentation to DCs, we immunized mice with LPS-matured and OVA323–339 peptide-loaded bone marrow-derived DCs. As a control, we immunized mice with OVA/LPS. Using LPS or alum as adjuvant yielded similar results (compare Figs. 3A, 2C). DC immunization induced significantly higher early BCL6 expression as compared with whole protein immunization (Fig. 3A, 3B). However, at later cell divisions, the percentage of BCL6+ cells rapidly declined in OVA-DC–primed Do11.10 cells, whereas BCL6 expression stabilized in the cells that had divided the most following protein immunization. Again, BCL6 expression preceded expression of CXCR5 in both immunization regimens. The inability of OVA-DCs to stabilize the CXCR5+BCL6+ TFH phenotype at this early time point correlated with a decrease in TFH cells on day 6 after immunization (Fig. 3C). As expected from the DC-restricted peptide immunization design, which omitted both specific Ag presentation by and BCR triggering of cognate B cells, DC immunization failed to induce potent GCs as compared with protein immunization (Fig. 3D). Interestingly, DC immunization led to elevated ICOS levels, but reduced PD-1 levels on proliferating T cells at all time points analyzed (Fig. 3A, 3C). It was recently reported that peptide-based booster immunization can induce a TFH phenotype regardless of cognate interactions with B cells, possibly due to sustained interaction with Ag-presenting DCs (22). Our data indicate that DCs are indeed able to induce a TFH phenotype, including early expression of BCL6, but that other mechanisms are required to sustain BCL6 expression in developing TFH cells following protein immunization.
Cognate interactions with B cells are dispensible for early BCL6 expression
Because B cells are required for the development of TFH cells (11, 20), we tested whether they were also responsible for inducing BCL6 expression in activated T cells early in the immune response. We transferred CellTrace-labeled naive OT-II cells into wt mice with a normal B cell population or MD4 BCR-tg mice that harbor a nearly monoclonal B cell repertoire that cannot respond to OVA. We observed similar kinetics of BCL6, CXCR5, PD-1, and ICOS expression in dividing cells in both groups of recipients (Fig. 4A). However, OT-II cells divided slightly less in MD4 recipients (Fig. 4B). Because the most divided cells are enriched for BCL6+CXCR5+ cells, MD4 recipients produced less TFH cells (Fig. 4C). These data clearly demonstrate that cognate interactions with B cells are not required for BCL6 expression at the initial priming stage, but that they begin to play an important role in sustaining and reinforcing TFH cell expansion and phenotype as early as 3.5 d into the primary response, prior to the formation of GCs. At later time points, B cells become the major APCs for follicle-homing T cells, and are then required to maintain TFH cells (9, 11, 20, 22).
In summary, we have shown that BCL6 is induced at the protein level in CD4+ T cells very early in primary immune responses. An initial transient wave of BCL6 expression occurs during the priming of naive CD4+ T cells by DCs. Although BCL6 expression subsequently wanes in most of the dividing cells, a second wave of BCL6 expression in a subset of cells coincides with upregulation of CXCR5, resulting in the emergence of a distinct BCL6+CXCR5+ TFH cell population. Early cognate interactions with B cells enhanced the development of TFH cells by supporting increased T cell division, but were not directly required for the upregulation of BCL6 or CXCR5 in responding cells. Thus, B cells apparently function primarily as amplifiers of early TFH development, although they may play a more direct role in maintaining BCL6 expression and the TFH gene expression program after GCs are established. These data provide evidence for tightly regulated BCL6 expression starting very early in the development of TFH cells in vivo and clarify the potential for BCL6 to induce the TFH cell gene expression program even prior to interaction with Ag-specific B cells and the development of GCs. A better understanding of TFH cell development may facilitate the development of more effective vaccines, or novel therapies that target this CD4+ T cell subset in the context of autoimmune diseases and allergies.
We thank Marisella Panduro for technical assistance, Mehrdad Matloubian, Anthony DeFranco, Chris Allen, and Abul Abbas for sharing mice and reagents, and Erika O'Donnell (BD Biosciences) for providing the anti-BCL6 Ab.
This work was supported by the Burroughs Wellcome Fund (to K.M.A.), the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to T.O.), and a Swiss National Science Foundation postdoctoral fellowship (to D.B.).
The online version of this article contains supplemental material.
Abbreviations used in this article:
T follicular helper
The authors have no financial conflicts of interest.