
==== Front
Proc Natl Acad Sci U S A
Proc Natl Acad Sci U S A
PNAS
Proceedings of the National Academy of Sciences of the United States of America
0027-8424
1091-6490
National Academy of Sciences

38261619
202304020
10.1073/pnas.2304020121
datasetDatasetresearch-articleResearch ArticleimmunImmunology and Inflammation420
Biological Sciences
Immunology and Inflammation
Targeted checkpoint control of B cells undergoing positive selection in germinal centers by follicular regulatory T cells
Ke Fang a
Benet Zachary L. a
Shelyakin Pavel b c https://orcid.org/0000-0003-0120-9319

Britanova Olga V. c d e https://orcid.org/0000-0002-6295-1392

Gupta Neetu f
Dent Alexander L. g
Moore Bethany B. a h https://orcid.org/0000-0003-3051-745X

Grigorova Irina L. igrigor@umich.edu
grig76@gmail.com
a 1 https://orcid.org/0000-0002-4963-7403

aDepartment of Microbiology and Immunology, Michigan Medicine University of Michigan, Ann Arbor, MI 48109
bAbu Dhabi Stem Cells Center, Abu Dhabi 4600, United Arab Emirates
cMolecular Technologies Division, Institute of Translational Medicine, Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Pirogov Russian National Research Medical University, Moscow 117997, Russian Federation
dGenomics of Adaptive Immunity Department, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russian Federation
eInstitute of Clinical Molecular Biology, Christian-Albrechts-University of Kiel, Kiel 24105, Germany
fDepartment of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195
gDepartment of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46123
hDepartment of Internal Medicine, Michigan Medicine University of Michigan, Ann Arbor, MI 48109
1To whom correspondence may be addressed. Email: igrigor@umich.edu or grig76@gmail.com.
Edited by Arthur Weiss, University of California San Francisco School of Medicine, San Francisco, CA; received March 10, 2023; accepted November 20, 2023

23 1 2024
30 1 2024
23 7 2024
121 5 e230402012110 3 2023
20 11 2023
Copyright © 2024 the Author(s). Published by PNAS.
2023
https://creativecommons.org/licenses/by-nc-nd/4.0/ This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

Significance

Follicular regulatory T cells (Tfr) play very important roles in the control of germinal centers (GCs), supporting high-affinity humoral immunity to foreign antigens and suppressing autoreactive antibody responses. However, understanding the duality of Tfr action on B cell response remains elusive. Our study points toward a model that explains the dichotomy of Tfr regulation of GCs. We suggest that a subset of Tfr is chemotactically targeted for direct interactions with CCL3high MYC+ centrocytes that are undergoing positive selection in GCs. Tfr can directly license transition of noncognate MYC+ centrocytes into centroblasts but suppress centrocytes that present self-antigens cognate to Tfr. The study suggests that local CCL3 gradients are critical for both positive and negative regulation of GCs by Tfr.

Follicular regulatory T cells (Tfr) can play opposite roles in the regulation of germinal center (GC) responses. Depending on the studies, Tfr suppress or support GC and B cell affinity maturation. However, which factors determine positive vs. negative effects of Tfr on the GC B cell is unclear. In this study, we show that GC centrocytes that express MYC up-regulate expression of CCL3 chemokine that is needed for both the positive and negative regulation of GC B cells by Tfr. B cell–intrinsic expression of CCL3 contributes to Tfr-dependent positive selection of foreign Ag–specific GC B cells. At the same time, expression of CCL3 is critical for direct Tfr-mediated suppression of GC B cells that acquire cognate to Tfr nuclear proteins. Our study suggests that CCR5 and CCR1 receptors promote Tfr migration to CCL3 and highlights Ccr5 expression on the Tfr subset that expresses Il10. Based on our findings and previous studies, we suggest a model of chemotactically targeted checkpoint control of B cells undergoing positive selection in GCs by Tfr, where Tfr directly probe and license foreign antigen–specific B cells to complete their positive selection in GCs but, at the same time, suppress GC B cells that present self-antigens cognate to Tfr.

follicular regulatory T cells
germinal center B cells
CCL3
CCR5
self-antigens
HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID) 100000060 AI106806 Alexander L. DentIrina L Grigorova HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID) 100000060 AI142032 Alexander L. DentIrina L Grigorova HHS | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) 100000069 AR067705 Neetu Gupta HHS | NIH | National Heart, Lung, and Blood Institute (NHLBI) 100000050 R35HL144481 Bethany B Moore HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID) 100000060 AI32771 Alexander L. DentIrina L Grigorova
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pmcTfr are FoxP3-expressing CXCR5highPD1high T cells that are present in B cell follicles and are important for regulation of humoral responses (1–11). The regulation of B cell and Ab responses by Tfr is multifaceted. On one side, Tfr prevent development of auto-Abs and autoimmunity (4, 8, 12–14), repress GC B cells that acquire some nuclear proteins (15) and may exert a modest negative effect on the foreign Ag–specific germinal center (GC) and Ab response (3, 5, 7, 8, 13). On the other side, Tfr deficiency leads to reduced somatic hypermutation (SHM), affinity maturation of foreign Ag–specific B cells, as well as diminished GC B cell, Ab, and memory responses (2, 6, 9–12). Tfr can also influence cytokine production by Tfh cells and B cell class-switching (4). While diverse modes of Tfr-dependent regulation of B cell responses may be potentially attributed to various experimental approaches, selected immunization and disease conditions, or distinct mouse models, the mechanisms of Tfr action and duality of their regulation of B cell responses have not been fully elucidated.

Tfr have been shown to support positively GC responses and B cell affinity maturation in a few mouse models of viral infection, immunization, and allergy (2, 6, 9–12). Affinity maturation of B cells takes place within GCs, where B cells undergo SHM, selection and can give rise to class-switched memory B cells and plasmablasts (PBs), some of which become long-lived plasma cells (16–18). GC B cells are composed of centroblasts (CB) that reside in the GC dark zone and centrocytes (CC) that reside in the GC light zone. In the light zone, CCs compete for acquisition of foreign Ag from follicular dendritic cells and help from CXCR5+PD1+FoxP3− follicular helper T cells (Tfh) (19–21). GC CCs selected in the light zone transiently up-regulate transcription factor MYC, move into the dark zone as CBs to undergo proliferation and SHM and then return back into the light zone for another round of competition and positive selection (22). A FoxO1-dependent transcriptional program in CBs supports their proliferation and SHM (23). Interestingly, in mice infected with LCMV as well as in the peanut allergy mouse model, Tfr support CB transcriptional program and efficient GC responses by producing IL10 that promotes FoxO1 expression and nuclear translocation in GC B cells (6, 9). While expression of IL10Rα on B cells is important for the observed regulation (2, 6, 9, 10, 12), whether direct GC B cell–Tfr interactions promote the IL10-dependent support of GC B cells and affinity maturation is not clear.

Direct GC B cell–Tfr interactions are expected to play a role in the suppression of GC B cells acquiring self-antigens cognate to Tfr. In the recent study, we demonstrated that targeting nucleosomes and other nuclear proteins (often targeted by auto-Abs in autoimmune diseases) to GC B cells promotes rapid accumulation of Tfr with up-regulated expression of the immunosuppressive genes and leads to Tfr-mediated suppression of the nuclear protein acquiring GC B cells with less pronounced inhibition of the total GC response (15).

Direct interactions between B cells and Tfr in GCs may be facilitated by CCL3 chemokine that is expressed in GC CC. In a previous study, we demonstrated that wild-type GC B cells form more frequent interaction with Tfr compared to GC B cells deficient for CCL3 (by intravital two-photon microscopy) (24). However, which GC CC subsets up-regulate production of CCL3 and the role of this chemokine in the positive or negative selection of GC B cells by Tfr is unclear.

This study points toward the central role of direct CCL3-mediated interaction between GC B cells and Tfr cells for both the positive and negative regulation of GC B cells by Tfr. First, we show that GC B cell–intrinsic production of CCL3 and Tfr cells are important for positive selection of the foreign Ag–specific GC B cells. Consistently with previous studies, we find that retention of B cells in GCs after the peak of GC response depends on the GC B cell–intrinsic expression of Il10R, suggesting direct Tfr-mediated IL10-driven selection of GC B cells. Our study also points to the critical role of CCL3 in the suppression of the nuclear protein-acquiring GC B cells by cognate Tfr. Finally, we find that CCL3 is up-regulated in a small subset of MYC+ GC CCs, while CCR5 (the GPCR receptor for CCL3) is expressed on the Tfr subset with elevated expression of immunosuppressive genes and Il10. Based on the previous studies and these findings, we propose a model of GC B cell checkpoint control by Tfr. We suggest that GC CCs undergoing positive selection in GCs transiently up-regulate expression of CCL3, which promotes their direct interactions with CCR5-expressing Tfr. Encountering cognate self-Ags on MYC+ GC B cells then triggers Tfr activation and proliferation leading to suppression of GC B cells. However, in the absence of cognate interactions with GC B cells, Tfr may directly license MYC+ GC CC (via IL10/IL10R signaling) to complete their transition into CBs for prolonged participation and selection in GCs. Our proposed model suggests targeted attention of Tfr to B cells undergoing positive selection in GCs and can explain previously reported controversial effects of Tfr on the B cell and Ab responses.

Results

CCL3 and Tfr Cells Are Required for Optimal Participation of B Cells in the GC and B Cell Memory Responses.

In a few mouse models of viral infection and allergy, Tfr have been shown to support GC response and antibody affinity maturation in an IL10-dependent fashion (2, 6, 9, 10, 12). However, whether Tfr-produced IL10 may also promote B cell retention in GCs and affinity maturation after a standard immunization, is unclear. In addition, whether Tfr-mediated selection of GC B cells is direct or indirect has not been addressed. Our previous studies suggested that production of CCL3 by GC B cells may facilitate their direct contacts with Tfr cells as CCL3 deficient GC B cells form 1.3-fold less frequent interactions with Tfr cells compared to CCL3 proficient cells in murine lymph nodes (LNs) (24). We therefore sought to determine whether B cell–intrinsic production of CCL3 may be required for sustained participation of B cells in GCs and for affinity maturation after a standard immunization. To address this, we generated mixed 1:1 CCL3−/− CD45.2:CCL3+/+ CD45.1 and control CCL3+/+ CD45.2:CCL3+/+ CD45.1 bone marrow chimeras (BMChs) (Fig. 1A). After BM reconstitution the BMChs were immunized with 4-Hydroxy-3-nitrophenylacetyl hapten conjugated to Keyhole Limpet Hemocyanin (NP-KLH) to track the NP-specific B cell response over time (Fig. 1A and SI Appendix, Fig. S1 A–C). In accordance with our previously published data (24), at 10 days post immunization (d.p.i.), CCL3−/− B cells expanded more in BMChs GCs compared to the CCL3+/+ counterparts. However, after the peak of GC response (days 15 to 21) CCL3−/− B cells dropped out of the mixed GCs more quickly (Fig. 1B and SI Appendix, Fig. S1D). Outcompetition of the CCL3−/− by CCL3+/+ GC B cells over time was also observed for foreign Ag NP-specific GC B cells (Fig. 1C and SI Appendix, Fig. S1E). More rapid drop of CCL3−/− B cells out of GC response led to decreased formation of NP-specific CCL3−/− memory B cells over time (Fig. 1D and SI Appendix, Fig. S1F). Importantly, analysis of the fraction of NP-specific GC B cells (NPpos) capable of binding more NP Ag (NPhigh) suggested that affinity maturation of the CCL3−/− GC B cells could be reduced compared to the CCL3+/+ GC B cells at 15 to 21 d.p.i. (Fig. 1 E–G and SI Appendix, Fig. S1 G and H). This hypothesis was based on the findings by Liu B. et al. that showed increased affinity maturation in NPhigh GC B cells (25). In accord with that, the proportion of the variable heavy-chain region 186.2 clones bearing the high-affinity W33L mutation (W33L+) in the sorted NP-positive CD45.2 GC B cells was higher in CCL3+/+ vs. CCL3−/− B cells (Fig. 1H and SI Appendix, Fig. S1I).

Fig. 1. B cell–intrinsic production of CCL3 and Tfr cells are required for sustained B cell participation in GC and memory responses and for affinity maturation (related to SI Appendix, Figs. S1 and S2). (A–H) Kinetic analysis of GC and memory responses in mixed BMChs: CCL3+/+ CD45.2: CCL3+/+ CD45.1 (black dots, Chim I) and CCL3−/− CD45.2: CCL3+/+ CD45.1 (red dots, Chim II) after immunization. (A) Experiment outline. Mixed BMChs were generated by lethally irradiating CCL3+/+ CD45.1 mice that then received a total of 4 × 106 BM cells from CD45.2 CCL3+/+ or CCL3−/− littermate control mice at 1:1 ratio with BM cells from CD45.1 CCL3+/+ mice. Eight weeks after BM transfer, recipient mice were s.c. immunized with NP-KLH in Ribi. (B–E) Flow cytometry analysis of B cells derived from CD45.2 BM donor in the dLNs including the total (B) and NP-specific (C) GC B cells, NP-specific memory B cells (D), fraction of NP-specific GC B cells that bind high levels of NP (E) at the indicated times after immunization. For the gating strategy, see SI Appendix, Fig. S1 A–C. The cell numbers were normalized to the fractions of CD45.2 B cells in the blood of each mouse at 0 d.p.i. For analysis of CD45.1 BM donor-derived control cells, see SI Appendix, Fig. S1 D–G. (F and G) Flow cytometry analysis of IgG1 NP-binding GC B cells at day 18. The gating strategy [in (F)] and fraction of CD45.2 BM donor-derived NP-specific IgG1 GC B cells that bind high levels of NP [in (G)]. For analysis of CD45.1 BM donor-derived control cells, see SI Appendix, Fig. S1H. (H) Proportion of the variable heavy-chain region 186.2 clones bearing the high-affinity W33L mutation (W33L+) in the sorted NP-positive CD45.2 GC B cells, among all sequenced clones (W33L mutants/total segments), where W33L- are clones without the W33L substitution. Also see SI Appendix, Fig. S1I. n = 3 [for (B–E)] or n = 2 [for (G) and (H)] independent experiments. Each symbol represents one mouse. Lines indicate means. *P < 0.05, **P < 0.01, and ***P < 0.001. Two-way ANOVA analysis with Sidak’s multiple comparisons test for (B–E). Unpaired Two-tailed Student’s t test for (G). The χ2 statistics with Yates correction for (H). (I–K) Kinetic analysis of CCL3+/+ and CCL3−/− Hy10 B cells competition in GCs. (I) Schematic diagram of the experiment. 5 × 104 Hy10 CCL3+/+ expressing cyan fluorescent protein (CFP) and 5 × 104 Hy10 CCL3−/− expressing green fluorescent protein (GFP) B cells were cotransferred into CD45.1 recipient mice that were then s.c immunized with DEL-OVA in Ribi. Results of the flow cytometry analysis of the CCL3+/+ and CCL3−/− Hy10 B cell participation in the GC response as a fraction of total B220+ B cells [in (J)] and their ratio in dLNs [in (K)]. (L–N) Flow cytometry analysis of CCL3+/+ CFP and CCL3−/− GFP Hy10 B cells participation in GC response in FoxP3DTR recipient mice at 15 d.p.i. and 3 d after Treg ablation with DTx or after control treatment with PBS. (L) Experimental strategy. (M and N) The numbers [in (M)] and the ratio [in (N)] of CCL3+/+ to CCL3–/– Hy10 GC B cells. Also see SI Appendix, Fig. S2 A–D. (O) Flow cytometry analysis of CCL3+/+ CFP and CCL3−/− GFP Hy10 B cells participation in GC response in Tfr-deficient Bcl6fl/fl FoxP3-cre and control Bcl6+/+ FoxP3-cre recipient mice. The ratio of CCL3+/+ to CCL3–/– Hy10 GC B cells at 15 d.p.i. Also see SI Appendix, Fig. S2 E–K. Data are representative of n = 3 to 4 independent experiments. Each symbol represents one mouse. Lines indicate means [in (J), (K), and (O)] and medians [in (M) and (N)]. *P < 0.05 and ***P < 0.001. One-way ANOVA analysis with Sidak’s multiple comparisons test [in (J)]. One-way [in (K)] and two-way [in (M)] ANOVA analysis with Tukey’s multiple comparisons test. Mann–Whitney [in (N)] and ratio paired [in (O)] two-tailed Student’s t test.

In addition to the analysis of the role of CCL3 for the endogenous B cell participation in the GC response, we explored its function using an adoptive transfer model with CCL3+/+ and CCL3−/− transgenic Hy10 B cells specific to duck egg lysozyme (DEL) (26). Recipient mice were cotransferred with 5 × 104 CFP CCL3+/+ and GFP CCL3−/− Hy10 B cells and were immunized with DEL conjugated to ovalbumin (OVA) for the time-course analysis of Hy10 B cell participation in the GC response in draining LNs (Fig. 1I). At 8 d.p.i., the participation of CCL3+/+ and CCL3−/− Hy10 B cells in the GC response was comparable. However, by 15 d.p.i., the CCL3−/− Hy10 B cells dropped out of the GCs 10-fold more than CCL3+/+ Hy10 B cells (Fig. 1 J and K). To summarize the above, both the BMChs and Hy10 adoptive transfer models suggest that CCL3 is required for sustained participation of B cells in the GC response and therefore for more optimal B cell affinity maturation and memory responses.

To determine whether the observed CCL3-mediated retention of foreign Ag–specific B cells in the GCs observed above depends on Tregs, we have examined whether transient depletion of Tregs would abolish preferential selection of CCL3+/+ vs. CCL3−/− Hy10 B cells in GCs after the peak of GC response. To that end CCL3+/+ vs. CCL3−/− Hy10 B cells were cotransferred into the FoxP3-DTR mice [that express diphtheria toxin receptor in Tregs, (27)] that were then immunized with DEL-OVA as described above. At 12 d.p.i. (after the peak of the GC response) the recipient mice were injected with diphtheria toxin at concentrations that lead to transient depletion of Tregs (24, 27) or with PBS for control (Fig. 1L). Similar to the wild-type recipient mice, FoxP3-DTR mice injected with PBS had over 10 times more CCL3+/+ compared to CCL3−/− Hy10 B cells in GCs at 15 d.p.i. (Fig. 1 J, K, M, and N). As expected, administration of diphtheria toxin led to a significant drop in the numbers of Tfr cells, but not Tfh cells (SI Appendix, Fig. S2 A–C). Interestingly, it was accompanied by the decrease in the numbers of total GC B cells (SI Appendix, Fig. S2D) and CCL3+/+ Hy10 GC B cells, while CCL3−/− Hy10 GC B cells numbers were not affected (Fig. 1M, P > 0.99). Therefore, transient depletion of Tregs leads to decreased selection of the CCL3+/+ as compared to CCL3−/− Hy10 B cells in GCs (Fig. 1N). To more specifically assess the role of Tfr cells in the persistence of CCL3+/+ GC B cells, CCL3+/+ vs. CCL3−/− Hy10 B cells were cotransferred into Tfr-deficient Bcl6fl/fl Foxp3-Yfpcre (Bcl6fl/fl Foxp3-cre) and control Bcl6+/+ Foxp3-cre mice (4) to assess their competition in GCs at 15 d.p.i. (SI Appendix, Fig. S2E). Analysis of Tfr, Tfh cells and GC B cells revealed similar effects in the Tfr-deficient mice and mice with transiently depleted Tregs (SI Appendix, Fig. S2 F–I). Moreover, the advantage for CCL3+/+ vs. CCL3−/− Hy10 B cells participation in GCs at 15 d.p.i. was also lost in Tfr-deficient mice (Fig. 1O and SI Appendix, Fig. S2 J and K).

The data suggest that production of CCL3 by foreign Ag–specific GC B cells is important for their sustained participation in the GC response in a Tfr-dependent fashion. Since CCL3 expression by GC B cells was previously shown to facilitate their encounters with Tfr, we propose that direct interactions between GC B cells and Tfr may promote prolonged participation of B cells in GCs after a standard immunization and thus contribute to GC B cell affinity maturation and memory B cell response.

GC B Cell–Intrinsic Expression of IL10R Is Important for Positive Selection of GC B Cells after the Peak of GC Response.

Previous studies demonstrated that IL10 production by Tfr was important for expression of FoxO1 in GC CBs and for sustained GC responses in a few murine models of viral infection and allergy. It was also dependent on the expression of IL10R by B cells (6, 9). However, whether Tfr-produced IL10 was directly acting on the GC B cells selection or indirectly and whether this regulation was also important for immune response after a standard immunization has not been addressed.

To verify the role of direct IL10R-mediated signaling for GC B cell selection after a standard immunization, we generated mixed BMChs with irradiated CD45.1 mice that were transferred with 1:1 ratio of BM cells from Mb1cre+/− IL10Rfl/fl CD45.2 (or Mb1cre+/− IL10R+/+ CD45.2) mice and wild-type CD45.1 mice (Fig. 2A). Reconstituted BMChs were immunized with NP-KLH and the dLNs were isolated for analysis (for gating strategy, see SI Appendix, Fig. S1 A–C and Fig. 1F) at 10 and 18 d.p.i. Based on the ratio of CXCR4highCD86low (dark zone, DZ) to CXCR4lowCD86high (light zone, LZ) GC B cells, comparable participation of IL10R-deficient and proficient GC B cells in the DZ was detected at 10 d.p.i. However, at 18 d.p.i., the frequency of IL10R-deficient DZ cells has decreased (Fig. 2 B and C). In line with that, we found no difference between the IL10R deficient and proficient NP-specific B cells in the GC response at 10 d.p.i., while at 18 d.p.i., IL10R-deficient B cells dropped out of the GCs more than IL10R-sufficient cells (Fig. 2D). This was reflected into significant decrease in the numbers of IgG1 NP-specific GC and memory B cells deficient for IL10R (Fig. 2 E and F) and in the fraction of NPhigh IgG1 GC B cells as compared to IL10R-sufficient B cells at 18 d.p.i. (Fig. 2G).

Fig. 2. B cell–intrinsic expression of IL-10R is important for sustained B cell participation in GC and memory responses. Analysis of GC and memory B cell responses in the dLNs of mixed BMChs: IL-10R+/+ Mb1Cre+/- CD45.2: CD45.1 (black dots, Chim I) and IL-10Rfl/fl Mb1Cre+/− CD45.2: CD45.1 (red dots, Chim II) after immunization. (A) Experiment outline. Mixed BMChs were generated by lethally irradiating CD45.1 mice that then received a total of 4 × 106 BM cells from CD45.2 IL-10Rfl/fl Mb1Cre+/− or IL-10R+/+ Mb1Cre+/− mice at 1:1 ratio mixed with BM cells from CD45.1 mice. Eight weeks after BM transfer, recipient mice were s.c. immunized with NP-KLH in Ribi. For gating strategy, see SI Appendix, Fig. S1 A–C and Fig. 1F. (B and C) Representative flow plots for CD45.2 GC B cells’ staining for DZ and LZ phenotype [in (B)] and CD45.2 and CD45.1 DZ/LZ GC B cell ratio at 10 and 18 d.p.i. [in (C)]. (D–G) Results of flow cytometry analysis of NP-specific GC B cells, total [in (D)], and class-switched to IgG1 [in (E)], and NP-specific IgG1 memory B cells [in (F)] from either CD45.2 or CD45.1 cells at the indicated times after immunization, normalized to the fractions of CD45.2 or CD45.1 B cells in the blood of each mouse at 0 d.p.i. (G) The ratio of NP-high to total NP-binding IgG1+ GC B cells from either CD45.2 or CD45.1 cells at the indicated times after immunization. n = 2 independent experiments. Each symbol represents one mouse. Lines indicate means. ***P < 0.001 and ****P < 0.0001. Two-way ANOVA analysis with Sidak’s multiple comparisons test.

These findings are consistent with GC B cell–intrinsic role of IL10R signaling for their reentry into the DZ from the LZ, prolonged participation in GCs, affinity maturation, and memory B cell formation. They also demonstrate that following a standard immunization IL10R signaling supports GC B cells after the peak of GC response (similarly to the kinetics of GC B cell selection mediated by CCL3) (Figs. 1 B–E and 2 C–G). To summarize the above, after a standard immunization GC B cell–intrinsic expression of CCL3 and IL10R, as well as Tfr, are important for persistent participation of foreign Ag–specific B cells in GCs.

The findings described above point toward an important role of direct encounters between Tfr and foreign Ag–specific GC B cells for the positive selection of B cells within GCs after a standard immunization. However, a previous study suggested that cognate interactions between GC B cells and Tfr cells promote suppression of the GC responses with the predominant effect on the GC B cells that acquire self-Ags cognate to Tfr (15). In that study, we demonstrated that in mice immunized with streptavidin conjugated to DEL (SA-DEL) and boosted with SA conjugated to nucleosomes and other selected nuclear proteins (SA-NucPr), the accumulation of the NucPr-acquiring GC B cells was rapidly abolished. The observed suppression of GC B cells was dependent on Tfr cells, as it was completely reversed in Tfr-deficient Bcl6fl/fl Foxp3-cre mice (15). However, whether this process is also dependent on the CCL3-dependent direct interactions between GC B cells and Tfr has not been addressed.

B Cell–Intrinsic Production of CCL3 Is Required for Suppression of Nuclear Protein–Acquiring GC B Cells by Tfr.

In this study, we have undertaken to examine whether in addition to the role of CCL3 expression by GC B cells in the positive selection of foreign Ag–specific B cells, it may be also important for the Tfr-mediated negative control of the NucPr-acquiring GC B cells. To address this, we generated mixed BMChs, where lethally irradiated mice were transferred with CD45.2 CCL3+/+ or CCL3−/− BM cells (from littermate controls) mixed with CD45.1 CCL3+/+ BM cells at 1:1 ratio (as in Fig. 1A). After the BM reconstitution, mixed BMChs were immunized with SA-DEL and boosted with SA-NucPr or SA for control as is shown in Fig. 3A. Boosting both types of mixed BMChs with SA-NucPr led to a significant increase in the frequency and numbers of Tfr cells in the dLNs (SI Appendix, Fig. S3), similarly to what has been shown before in wild-type mice (15). Interestingly, the accumulation of Tfr in the CCL3−/− CD45.2: CCL3+/+ CD45.1 BMChs was reduced as compared to the control CCL3+/+ CD45.2: CCL3+/+ CD45.1 BMChs (SI Appendix, Fig. S3 B and C). Analysis of the GC responses revealed no significant difference in the frequency of CCL3-proficient and deficient GC B cells in the BMChs boosted with SA (Fig. 3 B–D). However, while boosting with SA-NucPr reduced the SA-specific (and to a smaller extent the total) CCL3+/+ GC B cells (both CD45.1 and CD45.2, Fig. 3 B–I), no significant decrease in the CCL3−/− GC B cells within the same dLNs was detected (Fig. 3 D–F). These data suggest that CCL3 expression by GC B cells is critical for direct Tfr-mediated suppression of their NucPr-acquiring GC B cells.

Fig. 3. B cell–intrinsic production of CCL3 is required for suppression of NucPr-acquiring GC B cells (related to SI Appendix, Fig. S3). Analysis of GC response in mixed BMChs: CCL3+/+ CD45.2: CCL3+/+ CD45.1 (black dots, Chim I) and CCL3−/− CD45.2: CCL3+/+ CD45.1 (red dots, Chim II). (A) Experiment outline. Mixed BMChs were s.c. immunized with SA-DEL in Ribi. At day 12 mice were s.c. reimmunized with SA or SA-NucPr in Ribi for analysis 3 d later. (B and C) Representative flow plots showing SA-specific B cells from the CD45.2 and CD45.1 GC B cells in Chim I (B) and Chim II (C). (D–I) The total number of GC B cells, the SA-specific B cells percentage of GC B cells, and the total number of SA-specific GC B cells for CD45.2 cells [in (D), (E), and (F) correspondingly] and CD45.1 cells [in (G), (H), and (I) correspondingly] in Chim I and Chim II. n = 2 independent experiments. Each symbol represents one mouse. Lines indicate means. **P < 0.01 and ***P < 0.001. Two-way ANOVA analysis with Tukey’s multiple comparisons test.

Based on the above and previously published data (15), we suggest that production of CCL3 by GC B cells is important both for the positive selection of foreign Ag–specific GC B cells by IL10-producing Tfr, as well as for the suppression of GC B cells that acquire and present self-Ags cognate to Tfr.

Given that Tfr are less abundant in B cell follicles (and GCs) than Tfh cells (24), one of the central questions to the suggested direct regulation of GC B cells by Tfr is the mechanism that ensures physical encounters between GC B cells and Tfr. Chemokines secreted by GC B cells have been previously shown to promote their interactions with Tfh or Tfr cells in vivo via chemotaxis (24, 25). However, the expression of Ccl3/4 is only slightly up-regulated in bulk murine GC CC (LZ cells) compared to CB (DZ cells) (24, 28–30). Moreover, intravital two-photon imaging of murine LNs detected a robust but relatively small (1.3-fold) decrease in Tfr encounters with CCL3-deficient GC B cells as compared to CCL3-proficient cells (24). Given that CCL3 deficiency in GC B cells leads to striking defects in the regulation, we hypothesized that imaging of bulk GC B cells and Tfr may have significantly underestimated the contribution of CCL3-mediated chemotaxis of Tfr to GC B cells if CCL3 was produced by a small subset of GC B cells. For example, if only 10% of GC B cells were to promote chemoattraction of Tfr, then a 1.3-fold increase detected for bulk encounters would indicate ~fourfold increase in the rate of Tfr encounters with this GC B cell subset.

CCL3 Is Up-Regulated in MYC+ GC Centrocytes.

To determine whether GC CC have uniform expression of Ccl3 and Ccl4 or only a few CCs up-regulate Ccl3/4 production in vivo, we performed single-cell qPCR analysis of GC CC, CB, and non-GC B cells at 10 d.p.i. We found that the majority of CC and CB had comparable low expression of Ccl3 and Ccl4 (SI Appendix, Fig. S4). However, a small fraction of GC CC (12% in experiment 1 and 9% in experiment 2) had up-regulated expression of Ccl3/4 (SI Appendix, Fig. S4 C and F). The increased Ccl3/4 expression in a subset of cells was observed regardless of whether B2m or HPRT1 housekeeping genes were used for normalization (SI Appendix, Fig. S4 D, E, G, and H). In addition, while CCL3/4 expression in CB and naïve B cells were best described by a single Gaussian distribution, based on the maximal likelihood analysis two or more Gaussian distributions were required to fit GC CC data (SI Appendix, Fig. S4I). The data suggest that increased expression of Ccl3/4 detected in GC CC vs. CB occurs due to a small subset of GC CC that have up-regulated expression of Ccl3/4 in vivo.

Previous studies demonstrated that GC CC that undergo positive selection in GCs (~10% of CC) transiently up-regulate MYC (22). Because Ccl3/4 expression is also up-regulated in about 10% of CC, we examined whether MYC+ CC are enriched for Ccl3/4high cells. To this end, we have utilized GFP-c-Myc reporter mice that express N-terminal EGFP-MYC fusion protein (31). MYC-GFPhigh and MYC-GFPlow GC CC, as well as CB were sorted from the dLNs of immunized Myc-gfp mice at 10 d.p.i. for qPCR analysis (SI Appendix, Fig. S4 A and B). As expected, Myc expression was up-regulated in MYC-GFPhigh CC but not in CBs or MYC-GFPlow GC CC (Fig. 4A). Importantly, MYC-GFPhigh CC exhibited increased expression of Ccl3 (~eightfold) and Ccl4 (~twofold) (Fig. 4 B and C).

Fig. 4. Ccl3/4 expression in MYC+ GC CC and contribution of CCR5/CCR1 receptors to Tfr migration to CCL3 (related to SI Appendix, Figs. S4–S7). (A–C) qPCR analysis of CCL3 and CCL4 expression in GC CC. Myc-GFP+ and Myc-GFP− CC and CB were sorted from dLNs and spleens of Myc-GFP+/+ and control B6 mice at 10 d.p.i. with OVA in Ribi. For flow sorting gating strategy of Myc-GFP+ CC, Myc-GFP- CC, and total CB, see SI Appendix, Fig. S4 A and B. qPCR analysis of Myc (A), Ccl3 (B), and Ccl4 (C) expression in Myc-GFP+ and Myc-GFP− CC and CB. Results are normalized to Myc-GFP− CC cells and are presented relative to expression of the control gene B2m (2−ΔΔCt) and normalized to the average value for Myc-GFP− CC. Data are representative of n = 3 independent experiments. ***P < 0.001. One-way ANOVA analysis with Tukey’s multiple comparisons test. (D and E) qPCR analysis of Ccr5 (D) and Ccr1 (E) expression on Tfr, Tfh, CXCR5intPD1int FoxP3+ (Tint) and CXCR5lowPD1low FoxP3+ (Tlow) Treg cells sorted from dLNs of FoxP3-GFP mice and CD4+CD25+ T cells from CCR5−/− mice at day 10 post s.c immunization with OVA in Ribi as shown in SI Appendix, Fig. S6. Results are normalized to Tfh cells and are presented relative to expression of the control gene B2m (2−ΔΔCt). n = 3 independent experiments. **P < 0.01 and ***P < 0.001. One-way ANOVA analysis with Tukey’s multiple comparisons test. (F) Single-cell UMAP analysis of Ccr5, Il10, and Foxp3 expression in follicular T cells from the publicly available single-cell dataset (GSE216236). In the study, mice were immunized with SA-DEL in Ribi and boosted with SA or SA-NucPr, dLNs were collected and CXCR5highPD1highCD4+ T cells were sorted for 10× analysis. (G–I) Migration of CD4 T cells (purified from dLNs of WT or CCR5−/− mice at 10 d after s.c. immunization with OVA in Ribi) to CCL3 in the presence of CCR1 antagonist BL5923 or vehicle control. (G) Experiment outline and the gating strategy. (H and I) The ratio of Tfr (H) or Tfh cells (I) that transmigrated to CCL3 compared to no chemokine CCL3 control. n = 3 independent experiments. **P < 0.01 and ***P < 0.001. Two-way ANOVA analysis with Tukey’s multiple comparisons test.

These data suggest that murine GC CC that undergo positive selection in GCs and up-regulate Myc also transiently up-regulate expression of Ccl3 (and to some extent Ccl4). Interestingly, analysis of the publicly available human GC single-cell data (32, 33) also suggests enrichment of CCL3+ cells in the MYC+ GC B cells (SI Appendix, Fig. S5 and Table S1). Analysis of the human tonsillar GC B cells single-cell data suggests some CCL3 expression in the LZ (CD86highCXCR4low cluster 0) and more extensive enrichment in the MYC+ cluster 4 (that is characterized by the increased expression of genes associated with CD40 and NFkB signaling) (SI Appendix, Fig. S5 B and C). As expected, based on the previous studies, the MYC+ cluster is positioned between the LZ (cluster 0) and DZ (CD86low CXCR4high PCNAhigh cluster 1) states (SI Appendix, Fig. S5 C and D). Interestingly, the MYC+ CCL3+ cells are concentrated closer to the interface of MYC+ cluster 4 with the LZ (SI Appendix, Fig. S5D, red arrow). Based on this analysis, the human GC data are consistent with our hypothesis of transient upregulation of CCL3 in the positively selected GC B cells. However, in addition to this, human tonsillar single-cell data reveal that high expression of CCL3 is in the plasma cells/plasmablasts (cluster 6) (SI Appendix, Fig. S5 B and D).

CCR5 Is Expressed in a Subset of Tfr with Immunosuppressive Transcriptional Signature and Together with CCR1 Promotes Tfr Chemotaxis to CCL3.

In the previous study, we showed that CCL3 and CCL4 promote transmigration of murine Tfr and other Tregs (but not Tfh cells) in transwell assays (24). However, the contribution of CCR5 and CCR1 (the established GPCR receptors for CCL3 and CCL4) to the observed migration has not been assessed. We therefore performed qPCR analysis of Ccr5 and Ccr1 expression in Tfr, CXCR5int PD1int, and CXCR5low PD1low FoxP3+ Tregs as compared to Tfh cells sorted from the immunized FoxP3-GFP mice and CD4+CD25+ cells sorted from CCR5 KO mice for control (SI Appendix, Fig. S6 and Fig. 4 D and E). Expression of CCR5 was significantly up-regulated on bulk Tfr and CXCR5int PD1int Tregs as compared to Tfh cells (Fig. 4D). We have also assessed Ccr5 expression in murine follicular T cells using the previously published single-cell dataset for CD4+CXCR5highPD1high T cells sorted from the dLNs of C57BL/6 mice immunized with SA-DEL in Ribi and at day 8 boosted with SA or SA-NucPr for 10× analysis 3 d later (15) (GSE216236). Examination of the single-cell data revealed elevated expression of Ccr5 in many Foxp3+ follicular T cells (Fig. 4F). According to the previously performed clustering and gene expression analysis, Ccr5+ cells are enriched within Tfr cluster 10 (that have elevated expression of multiple Treg-associated immunosuppressive genes, including Il10) (Fig. 4F and SI Appendix, Fig. S7 A–C) (15). While a few Ccr5+ cells can be detected within Foxp3− Tfh-like cell subsets, their frequency is much lower compared to Tfr (Fig. 4F and SI Appendix, Fig. S7B). Importantly, the majority of the Ccr5+ cells have little to no expression of Tfh cell–associated CD40l and Il21 (SI Appendix, Fig. S7D). In addition to the analysis of CCR5 expression, we also detected increased expression of CCR1 on the Tfr by qPCR (Fig. 4E) that was not revealed by the 10× genomics approach.

To assess the contribution of CCR5 and CCR1 to the observed chemotaxis of Tfr to CCL3, we isolated CD4 T cells from the dLNs of immunized WT and CCR5 KO mice and examined their transmigration to CCL3 in the presence of the specific CCR1 inhibitor BL5923 or vehicle control (Fig. 4G). We found that Tfr transmigration to CCL3 was fully abolished for CCR5-KO Tfr in the presence of the CCR1 inhibitor (Fig. 4 G and H). As before, no transmigration of Tfh cells to CCL3 was detected (Fig. 4I) (24).

These data suggest that both CCR5 and CCR1 receptors contribute to Tfr migration to the CCL3 chemokine ex vivo and are likely to be responsible for the increased probing of the CCL3-producing GC CC by the Tfr reported in vivo (24).

Discussion

Tfr play an important role in the regulation of GC response, with predominantly negative control of the autoreactive Ab responses and nuclear protein acquiring GC B cells reported in some studies, and contribution to the positive selection and affinity maturation of foreign Ag–specific GC B cells described in the other studies (3–5, 7, 8, 12–15). However, i) which factors determine Tfr negative vs. positive roles in the GC B cell selection and ii) the contribution of direct (GC B cell–Tfr contact dependent) vs. indirect (contact-independent) Tfr action on the regulation has been unclear. The role of direct regulation of GC B cells by Tfr has been especially questionable, given that Tfr are significantly less abundant in B cell follicles than Tfh cells, with the majority of Tfr reported to be at the outer borders of GCs or beyond (24, 34).

Our study provides evidence that direct regulation of GC B cells by Tfr could be facilitated by chemotactic targeting of Tfr to the subset of GC B cells that are undergoing positive selection in GCs. In this work, we show that in mice CCL3 (and to some extent CCL4) is up-regulated in only a small subset of GC CCs (<10%), most of which express MYC, a transcription factor that is transiently up-regulated in GC B cells undergoing positive selection (22). In addition, based on the scRNAseq analysis, we found elevated expression of CCR5 (GPCR receptor for CCL3) on the subset of Tfr cells with immunosuppressive gene expression profile. This subset constitutes about 20% of CXCR5highPD1highFoxP3+ T cells and is enriched for Il10 expressing cells (15). We also show that CCR5 and to some extent CCR1 receptors promote Tfr chemotaxis to CCL3 ex vivo. Based on the above, we suggest that CCL3 is transiently up-regulated by GC B cells undergoing positive selection in the light zone to promote CCR5-mediated sampling by the “immunosuppressive” Tfr. Of note, previous studies showed that GC B cells undergoing positive selection in the light zone first move to the outer edge of GCs before becoming CBs and moving into the dark zone for another round of proliferation and somatic hypermutation (35). However, the functional significance of this complex spatial movement was unclear. We speculate that relocalization of the selected GC B cells to the outer edge of GCs may further increase their chances of multiple encounters with Tfr that are more abundant in these regions than within GCs (24, 34, 36).

Our study suggests that CCL3-dependent direct GC-Tfr encounters could play a role in both the positive and negative regulation of GC B cells by Tfr. We show that after a standard immunization CCL3-deficient B cells specific to foreign Ag are outcompeted by CCL3-proficient B cells in GCs leading to reduced affinity maturation and B cell memory development. Importantly, transient ablation of Tregs after the peak of the GC response, or selective Tfr deficiency, reduces CCL3-dependent positive selection of GC B cells. Finally, we demonstrate that in mixed Mb1cre+/− IL10Rfl/fl: IL10R+/+ BMChs, IL10R-deficient GC B cells’ DZ/LZ ratio, persistence, binding to foreign antigen and B cell memory formation is strongly compromised, suggesting that direct IL10R signaling in GC B cells is critical for prolonged GC B cell selection. The timing of GC B cell–intrinsic IL10R- and CCL3-dependent positive selection is consistent and occurs after the peak of GC response.

Of note, targeting NucPr to GC B cells even at this later time point of the GC response reversed CCL3-driven positive selection of GC B cells. Consistent with an important role of CCL3-mediated encounters with Tfr for the negative regulation of NucPr-acquiring GC B cells, we found that in mixed Ccl3−/−: Ccl3+/+ BMChs (immunized with SA-DEL and boosted with SA-NucPr as described above) suppression of the SA-specific GC B cells deficient for CCL3 was abolished as opposed to the CCL3 proficient cells within the same LNs. Of note, the accumulation of the NucPr-induced Tfr in Ccl3−/−: Ccl3+/+ BMChs was reduced as compared to control Ccl3+/+: Ccl3+/+ BMChs. These observations are in line with a suggested role of CCL3-driven Tfr-GC B cell–specific interactions for both suppression of the NucPr-acquiring GC B cells as well as the expansion of cognate Tfr (15). Overall, the analysis of mixed Ccl3−/−: Ccl3+/+ BMChs and our previous studies point toward a critical role of direct encounters between GC B cells and Tfr for the negative control of the NucPr-acquiring GC B cells.

Based on the experimental analysis described above and on the preceding studies, we propose a model of GC B cell checkpoint control by Tfr (SI Appendix, Fig. S8). We suggest that GC CC that receive sufficient positive selection signals (through BCR signaling and Tfh cell help) in the light zone transiently up-regulate CCL3/4, which then increases their probing by CCR5 (and possibly CCR1)-expressing Tfr cells. Tfr cells directly “sample” selected GC B cells and provide them with IL10 that through IL10R signaling promotes their transition into FoxO1-expressing CB for another round of proliferation in GCs. However, when Tfr encounter cognate MHCII/self-peptides on GC B cells, they suppress these B cells, preventing or reducing their chances of further participation in the GC response.

To summarize all of the above, the model of GC B cell checkpoint control proposed in this study suggests that only GC B cells that are undergoing positive selection in GCs are extensively screened by Tfr for cognate interactions, enabling Tfr to act as the gatekeepers supporting or limiting CC transitioning into CBs and therefore limiting expansion of the potentially autoreactive B cells in GCs. The model explains the controversial dual role that Tfr play in the regulation of GC response and suggests that this mechanism may be general for immunoregulation under various GC-inducing conditions.

Limitations of the Study

Overall our data are consistent with the hypothesis that direct Tfr cells interactions with CCL3-expressing GC B cells are important for GC B cells persistence or repression, if they present cognate to Tfr cells self-Ags. However, it cannot be ruled out that deletion of Tregs and deficiency of Tfr cells leads to more complex physiological alterations that dysregulate GC B cells via an alternative mechanism(s), e.g., by affecting other cells that respond to CCL3 and/or produce IL10. In addition, while we show that CCR5 is predominantly expressed on Tfr cells with immunosuppressive gene expression profile, many of which coexpress IL10, a few Tfh cells also express CCR5. It therefore cannot be fully ruled out that these Tfh cells or Tfh cells that up-regulate FoxP3 expression in late GCs (37) contribute to the CCL3-dependent positive selection of GC CC.

Previous studies suggested that IL10 signaling promotes FoxO1 expression in B cells (6, 9). While FoxO1 directs the CB program in GC B cells, further studies may be needed to establish how IL-10 signaling promotes foreign antigen–specific LZ GC cells to cycle into the DZ.

The exact interplay between the negative and positive signals provided to GC B cells by Tfr in the immunoregulation is not yet fully understood. Cognate recognition by Tfr of the GC-presented MHCII/self-peptides likely leads to more prolonged interactions between the cells (37), potentially increasing the duration of inhibitory signals received by GC B cells from cognate Tfr. Moreover, it cannot be ruled out that in this context IL10-dependent regulation of cognate GC B cells may shift from immunostimulatory to immunosuppressive. Future studies should dissect the contribution of distinct molecular pathways to the complex interplay between the negative and positive signals provided to GC B cells by Tfr.

Methods

Mice.

C57BL/6 (B6, 000664), B6-CD45.1 (002014), CCL3 KO (002687), CCR5 KO (005427), IL10Rafl/fl (028146), and Mb1-cre (020505) mice were purchased from The Jackson Laboratory. FoxP3-EGFP mice (38) were a gift from Weiping Zou Lab (University of Michigan). Myc-GFP mice (JAX: 021935) were a gift from Ivan Millard Lab (University of Pennsylvania). BCR transgenic (Ig-Tg) Hy10 mice, β-actin-CFP, and UBC-GFP mice (26) were a gift from Jason Cyster Lab (University of California San Francisco). Foxp3DTR mice were a gift from Alexander Rudensky Lab (27). Bcl6fl/fl (39) and Foxp3-Yfpcre mice (40) were crossed in Alexander Dent’s Lab (4). All mice were bred and maintained under specific pathogen-free conditions. Relevant mice were interbred to obtain Hy10 CFP+, Hy10 GFP+ CCL3-KO, IL10Rafl/fl Mb1-cre, and Bcl6fl/fl Foxp3-Yfpcre mice. All the animal experiments were conducted in compliance with the protocols reviewed and approved by the Institutional Animal Care and Use Committee of the University of Michigan.

Antigen Preparation.

Duck egg lysozyme (DEL) purification and conjugation to ovalbumin.

Duck eggs were locally purchased and DEL was purified as previously described (26). DEL was conjugated to OVA (Sigma) via glutaraldehyde cross-linking as previously described (26). For detailed information, please see SI Appendix.

Generation of SA-DEL and SA-NucPr.

SA-DEL was generated as previously described (41). Purified DEL was conjugated to biotin at a 1:2 molar ratio using Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific) according to the manufacturer’s directions and incubated for 3 h on ice. DEL-biotin was then dialyzed three times in PBS and after that incubated with streptavidin (Sigma) at a 10:1 molar ratio for 30 min on ice. Unbound DEL-bio was removed by passage through a 30 kDa molecular weight cutoff desalting column (Bio-Rad). Nucleosome, RNP/Sm, Jo-1, Scl-70, and Ro (SSA) (AROTEC DIAGNOSTICS) were conjugated to biotin at a 1:50 molar ratio using Sulfo-NHS-LC-Biotin and incubated for 3 h on ice. After dialyzing three times in PBS, nucleosome-biotin, RNP/Sm-biotin, Jo-1-biotin, Scl-70-biotin, and Ro (SSA)-biotin were incubated with streptavidin at a 1:1 molar ratio for 30 min on ice. Antigen conjugation was verified by SDS gel electrophoresis. After incubation, antigens were aliquoted and kept at −20 °C.

Immunization.

In some experiments, mice were s.c. immunized with 50 µg NP-KLH (Biosearch), OVA, or DEL-OVA in Ribi adjuvant (Sigma). In some experiments, mice were immunized with OVA in Ribi both s.c. and interperitoneally (i.p.). In some experiments, mice were s.c. immunized with 50 µg SA-DEL and reimmunized with SA or SA-NucPrs (containing 10 µg SA) at day 12 after immunization. Lymphoid cells from the dLNs were analyzed at the indicated time points.

Flow Cytometry Analyses and FACS Sorting.

Single-cell suspensions from dLNs and spleens were prepared and filtered through a 70-µm nylon cell strainer (BD). Red blood cells were lysed. Cells were washed in FACS buffer (2% FBS, 1 mM EDTA, 0.1% NaN3 in PBS) and followed by surface staining for the indicated markers for 20 min at 4 °C. NP-specific B cells or SA-specific B cells were detected with BCR-specific binding with NP-PE (Biosearch Technologies) or SA-PE (BioLegend). For intracellular staining, after surface markers were stained, cells were fixed and stained by using regulatory T cell Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer’s instructions. All samples were acquired on a BD FACSCanto flow cytometer. For cell sorting, enriched B cells and T cells were incubated with antibodies in sorting buffer (0.5% FBS and 2 mM EDTA in PBS) and were performed on a BD FACSAria III cell sorter. CB, Myc-GFP+ CC, and Myc-GFP− CC cells were sorted from Myc-GFP mice. Tfr, PD-1int Treg, PD-1low Treg, and Tfh cells were sorted from FoxP3GFP mice, and CD4+CD25+ T cells were sorted from B6 and CCR5KO mice. All data were analyzed with FlowJo (version 10.6.0) software.

Real-Time PCR Analysis.

Total RNA was extracted from sorted cell populations using the RNeasy Plus Micro Kit (QIAGEN), and complementary DNA (cDNA) was made using the SuperScript III First-Strand Synthesis System (Life Technologies). cDNA was preamplified with 10 cycles of target genes using the TaqMan PreAmp Master Mix (Applied Biosystems). The preamplified cDNA was then analyzed by using the TaqMan Probes specific for Ccl3, Ccl4, Ccr1, Ccr5, and the housekeeping gene B2m with the TaqMan Gene Expression Master Mix (Applied Biosystems). To measure Myc expression, SYBR Green PCR mix (Applied Biosystem) was used. Real-time quantitative (qPCR) was performed on an Applied Biosystems 7500 Fast machine. The relative amount of each target gene mRNA was calculated using the 2−ΔΔCt method.

Single-Cell qPCR Analysis.

Single-cell RT-PCR was performed using the method described before (42). For detailed information, please see SI Appendix.

Bone Marrow Chimeras.

Bone marrow chimeric mice were generated by lethally irradiating the recipient CD45.1 mice with 960 Rads. Following irradiation, mice were intravenously injected with 4 × 106 total bone marrow cells derived from indicated types. For some experiments, bone marrow cells contained 1:1 mix of CD45.1 and CD45.2 CCL3+/+ or CD45.2 CCL3−/− littermate control mice. For some experiments, bone marrow cells contained 1:1 mix of CD45.1 and CD45.2 IL10Rfl/fl Mb1Cre+ or CD45.2 IL10R+/+ Mb1Cre+ littermate control mice. Chimeric mice were given antibiotics in drinking water and were allowed to reconstitute for 8 wk before immunization.

Adoptive Transfer Experiments.

For competition experiments, purified 5 × 104 Hy10 CCL3+/+ CFP and 5 × 104 Hy10 CCL3−/− GFP B cells were injected intravenously (i.v.) into CD45.1 recipient mice that were then s.c immunized with DEL-OVA in Ribi. At days 6, 8, 12, and 15 following immunization dLNs were collected for cell assessment by flow cytometry. For Treg depletion experiments, purified 5 × 104 Hy10 CCL3+/+ CFP and 5 × 104 Hy10 CCL3−/− GFP B cells were injected i.v. into FoxP3DTR recipient mice that were then s.c immunized with DEL-OVA in Ribi. FoxP3+ cells were deleted by administering 50 µg per kg body weight of diphtheria toxin in 0.9% NaCl intraperitoneally (i.p.) into FoxP3DTR mice at day 12 following immunization. Three days later, dLNs were collected, and single-cell suspensions were then analyzed by flow cytometry.

Migration Assays.

Migration assays were done in 6.5-mm diameter, 5.0-µm pore size polycarbonate membrane filter transwell plates (Corning). CD4 T cells were isolated and enriched from dLNs of WT or CCR5KO mice s.c. immunized with OVA in Ribi at day 10 following immunization. WT or CCR5KO CD4 T cells were resuspended with DMEM (Corning) supplemented with 0.5% fatty acid–free BSA (Sigma), 10 mM HEPES, 100 U/mL of penicillin, and 100 µg/mL of streptomycin (HyClone). First, 0.6 mL of the same medium that contained or without 800 ng/mL CCL3 (PeproTech) was placed in the lower chamber. Transwell plates were then incubated at 37 °C 5% CO2 for 10 min. At the meantime, WT or CCR5 KO CD4 T cells were incubated with 100 nM CCR1 inhibitor BL5923 (Novartis) or vehicle control (0.5% hydroxyethyl cellulose) at 37 °C 5% CO2 for 10 min. After that, WT or CCR5 KO CD4 T cell suspension (0.1 mL at 4 × 106 cells/mL) was added to the upper filters. After incubation at 37 °C and 5% CO2 for 3 h, the cells migrated into the lower chamber were stained and analyzed by FACS. Three replicates per condition have been performed per experiment. The chemotactic index was calculated as the ratio of cells that transmigrated to chemokine compared to no chemokine control.

VH186.2 Sequence Analysis of GC B Cells.

Genomic DNA was extracted using the QIAamp DNA Micro Kit (Qiagen) from sorted NP-specific GC B cells (B220+ Fas+GL7+IgDloNP+). The isolated genomic DNA was used for PCR amplification of variable heavy chain region 186.2-joining heavy chain region 2 segments (VH186.2-JH2). PCR was performed using 25 ng of genomic DNA with a previously described seminested PCR protocol (43). For detailed information, please see SI Appendix.

Human and Mouse scRNA-Seq Analysis.

For scRNA-seq analysis of human GC B cells, publicly available raw reads of B cells from full tonsillar GCs (SRR11827034 and SRR11827035) and from GC light zone (SRR11827038 and SRR11827039) were downloaded from the GSE139891 dataset (32). As an unrelated dataset, we also downloaded the preprocessed data on the germinal center (GC) from Human Tonsils Atlas (33). Raw reads were processed with STARsolo (44) that accounts for multimapping reads (soloMultiMappers EM). All subsequent analysis was done in Seurat (45). Because of the low number of cells with detected expression of CCL3, we used ALRA, the method designed to impute technical zeroes in expression data, while preserving true biological zeroes (46). For murine scRNA-seq analysis, the publicly available dataset for follicular T cells was downloaded (GSE216236) (15). Barcodes with mitochondrial reads >5% were removed. Preprocessing, clustering, and dimensionality reduction were performed using Loupe Browser (10× genomics, Cell Ranger). Graph-based clustering of Tfr was visualized in 2D using the UMAP algorithm.

Statistics.

Statistical tests were performed as indicated using Prism 8 (GraphPad). No blinding or randomization was performed for animal experiments, and no animals or samples were excluded from analysis. All the statistical details of experiments and statistical analysis can be found in figure legends. Differences between groups not annotated by an asterisk did not reach statistical significance.

Supplementary Material

Appendix 01 (PDF)

Click here for additional data file.

Code S01 (PDF)

Click here for additional data file.

Code S02 (PDF)

Click here for additional data file.

We thank Prof. Jason Cyster (UCSF) and Laura Santambrogio (Cornell University) for useful discussions and comments on the manuscript. We thank Prof. Anukul Shenoy (University of Michigan) for technical assistance. We thank Novartis for provision of the CCR1 inhibitor BL5923. This work was supported by the NIH RO1 AI106806 and R21 AI142032 (I.L.G.), RO1 AR067705 (N.G.), R35HL144481 (B.B.M.), and RO1 AI32771 (A.L.D.).

Author contributions

F.K., Z.L.B., and I.L.G. designed research; F.K. and Z.L.B. performed research; N.G., A.L.D., and B.B.M. contributed new reagents/analytic tools; F.K., Z.L.B., P.S., O.V.B., and I.L.G. analyzed data; and F.K., O.V.B., and I.L.G. wrote the paper.

Competing interests

The authors declare no competing interest.

Data, Materials, and Software Availability

Flow cytometry data have been deposited in (47).

Supporting Information

This article is a PNAS Direct Submission.
==== Refs
1 H. W. Lim, P. Hillsamer, C. H. Kim, Regulatory T cells can migrate to follicles upon T cell activation and suppress GC-Th cells and GC-Th cell-driven B cell responses. J. Clin. Invest. 114 , 1640–1649 (2004).15578096
2 M. A. Linterman , Foxp3+ follicular regulatory T cells control the germinal center response. Nat. Med. 17 , 975–982 (2011).21785433
3 Y. Chung , Follicular regulatory T cells expressing Foxp3 and Bcl-6 suppress germinal center reactions. Nat. Med. 17 , 983–988 (2011).21785430
4 H. Wu , Follicular regulatory T cells repress cytokine production by follicular helper T cells and optimize IgG responses in mice. Eur. J. Immunol. 46 , 1152–1161 (2016).26887860
5 P. T. Sage , Suppression by TFR cells leads to durable and selective inhibition of B cell effector function. Nat. Immunol. 17 , 1436–1446 (2016).27695002
6 B. J. Laidlaw , Interleukin-10 from CD4(+) follicular regulatory T cells promotes the germinal center response. Sci. Immunol. 2 , eaan4767 (2017).29054998
7 I. Wollenberg , Regulation of the germinal center reaction by Foxp3+ follicular regulatory T cells. J. Immunol. 187 , 4553–4560 (2011).21984700
8 R. L. Clement , Follicular regulatory T cells control humoral and allergic immunity by restraining early B cell responses. Nat. Immunol. 20 , 1360–1371 (2019).31477921
9 M. M. Xie , T follicular regulatory cells and IL-10 promote food antigen-specific IgE. J. Clin. Invest. 130 , 3820–3832 (2020).32255767
10 Y. Lu , CD4+ follicular regulatory T cells optimize the influenza virus-specific B cell response. J. Exp. Med. 218 , e20200547 (2021).33326020
11 C. B. Cavazzoni , Follicular T cells optimize the germinal center response to SARS-CoV-2 protein vaccination in mice. Cell Rep. 38 , 110399 (2022).35139367
12 D. Botta , Dynamic regulation of T follicular regulatory cell responses by interleukin 2 during influenza infection. Nat. Immunol. 18 , 1249–1260 (2017).28892471
13 W. Fu , Deficiency in T follicular regulatory cells promotes autoimmunity. J. Exp. Med. 215 , 815–825 (2018).29378778
14 P. Gonzalez-Figueroa , Follicular regulatory T cells produce neuritin to regulate B cells. Cell 184 , 1775–1789.e1719 (2021).33711260
15 F. Ke , Germinal center B cells that acquire nuclear proteins are specifically suppressed by follicular regulatory T cells. Elife 12 , e83908 (2023).36862132
16 S. L. Nutt, P. D. Hodgkin, D. M. Tarlinton, L. M. Corcoran, The generation of antibody-secreting plasma cells. Nat. Rev. Immunol. 15 , 160–171 (2015).25698678
17 T. Kurosaki, H. Shinohara, Y. Baba, B cell signaling and fate decision. Annu. Rev. Immunol. 28 , 21–55 (2010).19827951
18 R. Dhenni, T. G. Phan, The geography of memory B cell reactivation in vaccine-induced immunity and in autoimmune disease relapses. Immunol. Rev. 296 , 62–86 (2020).32472583
19 G. D. Victora , Germinal center dynamics revealed by multiphoton microscopy with a photoactivatable fluorescent reporter. Cell 143 , 592–605 (2010).21074050
20 W. Luo, F. Weisel, M. J. Shlomchik, B cell receptor and CD40 signaling are rewired for synergistic induction of the c-Myc transcription factor in germinal center B cells. Immunity 48 , 313–326.e315 (2018).29396161
21 J. S. Turner, F. Ke, I. L. Grigorova, B cell receptor crosslinking augments germinal center B cell selection when T cell help is limiting. Cell Rep. 25 , 1395–1403.e1394 (2018).30403996
22 D. Dominguez-Sola , The proto-oncogene MYC is required for selection in the germinal center and cyclic reentry. Nat. Immunol. 13 , 1083–1091 (2012).23001145
23 D. Dominguez-Sola , The FOXO1 transcription factor instructs the germinal center dark zone program. Immunity 43 , 1064–1074 (2015).26620759
24 Z. L. Benet , CCL3 promotes germinal center B cells sampling by follicular regulatory T cells in murine lymph nodes. Front. Immunol. 9 , 2044 (2018).30271404
25 B. Liu , Affinity-coupled CCL22 promotes positive selection in germinal centres. Nature 592 , 133–137 (2021).33597749
26 C. D. Allen, T. Okada, H. L. Tang, J. G. Cyster, Imaging of germinal center selection events during affinity maturation. Science 315 , 528–531 (2007).17185562
27 J. M. Kim, J. P. Rasmussen, A. Y. Rudensky, Regulatory T cells prevent catastrophic autoimmunity throughout the lifespan of mice. Nat. Immunol. 8 , 191–197 (2007).17136045
28 G. Caron, S. Le Gallou, T. Lamy, K. Tarte, T. Fest, CXCR4 expression functionally discriminates centroblasts versus centrocytes within human germinal center B cells. J. Immunol. 182 , 7595–7602 (2009).19494283
29 G. D. Victora , Identification of human germinal center light and dark zone cells and their relationship to human B-cell lymphomas. Blood 120 , 2240–2248 (2012).22740445
30 M. Compagno , Mutations of multiple genes cause deregulation of NF-kappaB in diffuse large B-cell lymphoma. Nature 459 , 717–721 (2009).19412164
31 C. Y. Huang, A. L. Bredemeyer, L. M. Walker, C. H. Bassing, B. P. Sleckman, Dynamic regulation of c-Myc proto-oncogene expression during lymphocyte development revealed by a GFP-c-Myc knock-in mouse. Eur. J. Immunol. 38 , 342–349 (2008).18196519
32 A. B. Holmes , Single-cell analysis of germinal-center B cells informs on lymphoma cell of origin and outcome. J. Exp. Med. 217 (2020).
33 R. Massoni-Badosa An atlas of cells in the human tonsil. bioRxiv [Preprint] (2022). 10.1101/2022.06.24.497299 (Accessed 26 June 2022).
34 J. B. Wing , A distinct subpopulation of CD25(-) T-follicular regulatory cells localizes in the germinal centers. Proc. Natl. Acad. Sci. U.S.A. 114 , E6400–E6409 (2017).28698369
35 D. Liu , T-B-cell entanglement and ICOSL-driven feed-forward regulation of germinal centre reaction. Nature 517 , 214–218 (2015).25317561
36 I. Sayin , Spatial distribution and function of T follicular regulatory cells in human lymph nodes. J. Exp. Med. 215 , 1531–1542 (2018).29769249
37 J. T. Jacobsen , Expression of Foxp3 by T follicular helper cells in end-stage germinal centers. Science 373 , eabe5146 (2021).34437125
38 E. Bettelli , Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 441 , 235–238 (2006).16648838
39 K. Hollister , Insights into the role of Bcl6 in follicular Th cells using a new conditional mutant mouse model. J. Immunol. 191 , 3705–3711 (2013).23980208
40 Y. P. Rubtsov , Regulatory T cell-derived interleukin-10 limits inflammation at environmental interfaces. Immunity 28 , 546–558 (2008).18387831
41 J. S. Turner, M. Marthi, Z. L. Benet, I. Grigorova, Transiently antigen-primed B cells return to naive-like state in absence of T-cell help. Nat. Commun. 8 , 15072 (2017).28429719
42 W. Zhao, J. Wang, L. Wang, The unsaturated bistable stochastic resonance system. Chaos 23 , 033117 (2013).24089953
43 D. Chen , Coupled analysis of transcriptome and BCR mutations reveals role of OXPHOS in affinity maturation. Nat. Immunol. 22 , 904–913 (2021).34031613
44 B. Kaminow, D. Yunusov, A. Dobin, STARsolo: Accurate, fast and versatile mapping/quantification of single-cell and single-nucleus RNA-seq data. bioRxiv [Preprint] (2021). 10.1101/2021.05.05.442755 (Accessed 5 May 2021).
45 Y. Hao , Integrated analysis of multimodal single-cell data. Cell 184 , 3573–3587.e3529 (2021).34062119
46 G. C. Linderman , Zero-preserving imputation of single-cell RNA-seq data. Nat. Commun. 13 , 192 (2022).35017482
47 I. Grigorova, F. Ke, Data from: Targeted checkpoint control of B cells undergoing positive selection in germinal centers by follicular regulatory T cells [Dataset]. Dryad. 10.5061/dryad.v15dv4215. Deposited 6 January 2024.
