CD24hiCD38hi and CD24hiCD27+ Human Regulatory B Cells Display Common and Distinct Functional Characteristics

This information is current as Md Mahmudul Hasan, LuAnn Thompson-Snipes, Goran of September 23, 2021. Klintmalm, Anthony J. Demetris, Jacqueline O'Leary, SangKon Oh and HyeMee Joo J Immunol published online 11 September 2019 http://www.jimmunol.org/content/early/2019/09/10/jimmun

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published September 11, 2019, doi:10.4049/jimmunol.1900488 The Journal of Immunology

CD24hiCD38hi and CD24hiCD27+ Human Regulatory B Cells Display Common and Distinct Functional Characteristics

Md Mahmudul Hasan,*,† LuAnn Thompson-Snipes,† Goran Klintmalm,‡ Anthony J. Demetris,x Jacqueline O’Leary,‡ SangKon Oh,*,† and HyeMee Joo*,†

Although IL-10–producing regulatory B cells (Bregs) play important roles in immune regulation, their surface phenotypes and functional characteristics have not been fully investigated. In this study, we report that the frequency of IL-10–producing Bregs in human peripheral , , and tonsils is similar, but they display heterogenous surface phenotypes. Nonetheless, CD24hiCD38hi transitional B cells (TBs) and CD24hiCD27+ B cells (human equivalent of murine B10 cells) are the major IL-10– producing B cells. They both suppress CD4+ proliferation as well as IFN-g/IL-17 expression. However, CD24hiCD27+ Bcells were more efficient than TBs at suppressing CD4+ T cell proliferation and IFN-g/IL-17 expression, whereas they both coexpress

IL-10 and TNF-a.TGF-b1 and granzyme B expression were also enriched within CD24hiCD27+ B cells, when compared with TBs. Downloaded from Additionally, CD24hiCD27+ B cells expressed increased levels of surface integrins (CD11a, CD11b, a1, a4, and b1) and CD39 (an ecto-ATPase), suggesting that the in vivo mechanisms of action of the two Breg subsets are not the same. Lastly, we also report that liver allograft recipients with hepatitis had significant decreases of both Breg subsets. The Journal of Immunology, 2019, 203: 000–000.

ccumulating evidence indicates that B cells expressing zone (MZ) (15), MZ precursor or transitional 2 (1), follicular http://www.jimmunol.org/ immunosuppressive cytokines, especially IL-10, can effi- (1, 16), CD1dhiCD5+ (B10) (17), pro‐B (10), and plasmablasts/ A ciently curtail inflammatory responses. Such B cells are plasma cells (18, 19). In addition, T cell Ig domain and mucin now collectively termed regulatory B cells (Bregs). Immune regu- domain protein 1 (TIM-1) is known to be expressed on the ma- latory functions of Bregs have also been documented in several jority of IL-10–expressing B cells in mice (20), but TIM-1+IL-10+ disease models (e.g., autoimmune and inflammatory diseases) (1–6), B cells also represent the major subpopulations, including cancer (6, 7), infectious diseases (6, 8), and transplantation (9–14). transitional, MZ, and follicular, as well as the CD1dhiCD5+ B10 Nonetheless, there are several major questions about Bregs that cells (9). need to be addressed. It is still unclear whether Bregs represent a Similar but not same as the murine Bregs, phenotypes of IL-10– developmentally specified and stable lineage comparable to FOXP3+ producing Bregs in human blood also range from early immature by guest on September 23, 2021 regulatory T cells (Tregs) or differentiated subsets of B cells that (TBs) (CD24hiCD38hi) (8, 21–24) to plasma- can display immunosuppressive functions in certain circumstances. blasts (CD27intCD38+) (18) and Br1 cells (17). The human In the latter case, any B cell subsets could potentially display im- equivalent of B10 cells (CD24hiCD27+) have also been reported mune suppressive functions, depending on not only the nature of (25–27). All these human B cell subsets are capable of expressing individual B cell subsets, but also the tissue microenvironments IL-10 and could thus suppress inflammatory responses. Compared where they are located. Indeed, murine Bregs expressing IL-10 with other subsets of Bregs, human TBs are relatively well stud- showed various surface phenotypes, including splenic marginal ied in the context of inflammatory diseases (11, 13, 21, 22, 28). Human IL-10–producing TIM-1+ B cells are also reported to

† preferentially present in TBs (29). However, it is still not known *Department of Immunology, Mayo Clinic, Scottsdale, AZ 85259; Institute of Bio- medical Studies, Baylor University, Waco, TX 76706; ‡Annette C. and Harold which subsets of human Bregs are more effective than others at C. Simmons Transplant Institute, Baylor University Medical Center, Dallas, TX x suppressing inflammatory response. Their effectiveness could be 75246; and Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15213 affected by the possibly different mechanisms of action of indi- ORCIDs: 0000-0003-3221-0240 (M.M.H.); 0000-0002-1271-5703 (L.T.-S.). vidual Breg subsets. Subsets of Bregs are known to express not Received for publication April 30, 2019. Accepted for publication August 13, 2019. only IL-10, but also other inhibitory molecules (30–36), including This work was supported by a start-up fund from Mayo Clinic (to S.O. and H.J.), PD-L1, granzyme B, and TGF-b, which can suppress inflamma- National Institutes of Health 1 R01 AI 105066 (to S.O.), and the Caruth Foundation (to S.O. and H.J.). tory responses. In this study, we examined two major human Breg subsets, M.M.H. and L.T.-S. performed experiments. G.K., A.J.D., and J.O. determined pa- hi hi hi + tients with PCH and provided patient samples. M.M.H., L.T.-S., H.J., and S.O. de- CD24 CD38 TBs and CD24 CD27 B cells (human equivalent signed experiments and analyzed data. M.M.H., S.O., and H.J wrote the manuscript. of B10 cells) (25–27), for their regulatory functions by assessing Address correspondence and reprint requests to Dr. SangKon Oh and Dr. HyeMee their ability to express IL-10, TGF-b1, granzyme B, and PD-L1 Joo, Mayo Clinic, 13400 E. Shea Boulevard, Scottsdale, AZ 85259. E-mail addresses: and to suppress T cell responses. The clinical relevance of the two [email protected] (S.O.) and [email protected] (H.J.) subsets of human Bregs was subsequently tested by examining The online version of this article contains supplemental material. their frequency in the peripheral blood of liver allograft recipients Abbreviations used in this article: Breg, ; DC, dendritic cell; MNC, mononuclear cell; MZ, marginal zone; P1, population 1; P2, population 2; P3, with plasma cell hepatitis (PCH). PCH, also known as a de novo population 3; P4, population 4; PCH, plasma cell hepatitis; TB, transitional autoimmune hepatitis, is a variant of late-onset rejection and has B cell; TIM-1, T cell Ig domain and mucin domain protein 1; Treg, regulatory been increasingly diagnosed in both pediatric and adult postliver T cell; t-SNE, t-distributed stochastic neighbor embedding. transplantation recipients in recent years (37–40). Our data sug- Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 gest a novel role of Bregs in PCH in liver allograft recipients.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900488 2 FUNCTIONAL CHARACTERISTICS OF HUMAN Breg SUBSETS

TBs (CD19+CD24hiCD38hi), CD19+CD24hiCD27+,CD19+CD24+CD272, Materials and Methods + 2 2 Blood and tissue samples and CD19 CD24 CD27 were sorted using FACSAria II (BD Biosciences). Blood samples from healthy donors and liver transplant patients with PCH Measurement of cytokines, granzyme B, and PD-L1 expressed were acquired in accordance with the protocols approved by the Institu- by B cells tional Review Board. Blood samples from all liver transplant patients were A total of 4 3 105 purified B cells, PBMCs, tonsil MNCs, or splenocytes collected prospectively at prespecified time points posttransplant and, were cultured in complete RPMI 1640 plus 10% FBS for indicated time at the time of indication, liver biopsies were collected and stored in the Baylor University Medical Center Transplant Biorepository. Patients periods. Cell stimulation mixture containing PMA, ionomycin, brefeldin with a confirmed diagnosis of PCH were selected for this study, and A, and monensin (eBioscience) was added for the last 5 h. Human blood samples from the time of their diagnosis or within 1 mo of con- Fc-blocker (Miltenyi Biotec) was added, and dead cells were excluded by firmatory liver biopsies (re-evaluated for this study by a pathologist staining them with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life A.J.D.) were analyzed. tissue from deceased organ donors and Technologies). For surface staining, cells were stained with indicated Abs. tonsils from tonsillectomy were obtained from the tissue bank at the Cells were then washed, fixed, and permeabilized using Cytofix/Cytoperm Baylor University Medical Center with approval from the Institutional (BD Biosciences) followed by intracellular cytokines and granzyme B Review Board. staining. Cells were analyzed with a BD LSRFortessa (BD Biosciences). Data were analyzed with FlowJo v10 (FlowJo). After gating on IL-10+ Cell isolation and stimulation B cells or sorted B cell subsets (P1-P4), t-distributed stochastic neighbor embedding (t-SNE) (41) clustering was performed using FlowJo. The PBMCs, mononuclear cells (MNCs) from tonsils and splenocytes were amount of IL-10–, IL-6–, and TNF-a–secreted by B cells was quantified isolated by density gradient centrifugation using Ficoll-Paque PLUS (GE by bead-based multiplex assays (Millipore Sigma). Healthcare). B cells were purified by negative selection using a Pan-B Cell Enrichment Kit (STEMCELL Technologies). Cells were incubated in In vitro functional assay Downloaded from complete RPMI 1640 (Invitrogen) supplemented with 25 mM HEPES + hi hi + hi + L FACS-sorted CD19 CD24 CD38 population 1 (P1) CD19 CD24 CD27 (Invitrogen), 1% nonessential amino acids, 2 mM -glutamate (Sigma- 2 m m population 2 (P2), CD19+CD24+CD27 population 3 (P3), and Aldrich), 50 g/ml penicillin, and 50 g/ml streptomycin (Life Tech- 2 2 nologies), and 10% FBS (Life Technologies). During incubation, cells CD19+CD24 CD27 population 4 (P4) B cells were stimulated with in- were stimulated with indicated B cell activators, including recombinant dicated stimulators and cocultured for 6 d with CFSE (Invitrogen)–labeled human CD40L (R&D Systems) at 0.5 mg/ml, class B CpG oligodeox- CD4+ T cells. CD4+ T cells were further stimulated with allogeneic den- ynucleotides (ODN 2006) (Invivogen) at 2.5 mg/ml, anti-BCR (poly- dritic cells (DCs). CD4+ T cell proliferation was assessed by measuring clonal goat anti-human IgG plus IgM) (Jackson ImmunoResearch) at CFSE dilution. Human Fc-blocker was added, and live-dead staining was http://www.jimmunol.org/ 2.5 mg/ml, or combinations of activators. Blood B cell subsets, including performed. PMA, ionomycin, brefeldin A, and monensin was added for the by guest on September 23, 2021

FIGURE 1. Heterogeneity of human IL-10–producing B cells in the blood, spleens, and tonsils. Cells from blood, spleens, and tonsils were incubated with 2.5 mg/ml CpG-B for 16 or 48 h. B cells were then activated for 5 h with PMA/ionomycin in the presence of monensin and brefeldin A before staining them for intracellular IL-10 expression. (A) Gating strategy for IL-10+CD19+ B cells in the blood. (B) Frequency of IL-10+CD19+ B cells in the blood, spleens, and tonsils upon 48 h CpG-B stimulation. Each dot represents data generated with cells from a single donor. Error bars represent mean 6 SD. (C) t-SNE analysis of IL-10+ B cells from blood, spleens, and tonsils upon 16-h CpG-B stimulation. Cells were stained with anti-IgD (IA6-2), anti-CD5 (UCHT2), anti-CD9 (M-L13), anti-CD10 (HI10a), anti-CD19 (SJ25C1), anti-CD24 (ML5), anti-CD27 (M-T271), anti-CD38 (HB7), anti-CD43 (L60), and anti–TIM-1 (1D12), all from BD Biosciences; anti-IgM (MHM-88) from BioLegend. Cells were then washed, fixed, and permeabilized using Cytofix/Cytoperm (BD Biosciences), followed by intracellular staining with anti–IL-10 (JES3-9D7) from eBiosciences. Heatmap statistics were usedto show the expression of each surface markers tested. Data generated with cells from four donors were compiled. The p values were calculated with nonparametric ANOVA. ns, not significant. The Journal of Immunology 3 last 5 h. Cells were then washed, fixed, and permeabilized using Statistical analysis Cytofix/Cytoperm (BD Biosciences), followed by intracellular stain- ing with anti–IFN-g (B27), anti–IL-17A (BL168), and anti–TNF-a Statistical analysis was performed with Prism Software (GraphPad) by (MAb11) purchased from BioLegend. Intracellular cytokine expres- Student t test (paired or unpaired) or ANOVA, as appropriate. All data 6 , sions were assessed with a BD LSRFortessa. Percent suppression was are shown as mean SD. A p value 0.05 was considered significant; 2 + , , , , calculated by the formula: (percentage of CFSE CD4 T cells cultured *p 0.05, **p 0.01, ***p 0.005, ****p 0.001. with unstimulated B cells – percentage of CFSE2CD4+ T cells cul- 2 tured with activated B cells)/percentage of CFSE CD4+ T cells cul- Results tured with unstimulated B cells 3 100% (11). Heterogeneity of IL-10–producing B cells in the blood, spleens, Blocking experiments and tonsils CpG-B–activated total CD19+ B cells or FACS-sorted CD19+CD24hiCD38hi (P1), Because of the lack of lineage-specific markers or transcription + hi + + + 2 + 2 2 CD19 CD24 CD27 (P2), CD19 CD24 CD27 (P3), and CD19 CD24 CD27 factors, IL-10 expression is used as a major readout to determine (P4) B cells were preincubated with the indicated reagents, anti–IL-10 at Bregs in both mice and human (42–45). We thus examined the 10 mg/ml (JES3-9D7; Miltenyi Biotec), anti–IL-10R at 10 mg/ml (3F9; + BioLegend), anti–PD-L1 at 10 mg/ml (MIH-1; eBioscience), anti–IL-6 frequency of IL-10 B cells in the blood, spleens, and tonsils (6708; R & D systems), anti–IL-6R (17506; R & D systems), TNF-a blocker (Fig. 1A, 1B). MNCs from human blood, spleens, and tonsils were etanercept (Enbrel; Amgen), anti–TGF-b1 (21C11: BioLegend), granzyme B incubated for 48 h with CpG-B and then further restimulated with blocker (Ac-IEPD-CHO; Abcam), or isotypes for 12 h. B cells were then PMA/ionomycin to induce IL-10 expression. Flow cytometry washed and cocultured for 6 d with CFSE-labeled CD4+ T cells upon stimu- lation with allogenic DCs or 4 d with CFSE-labeled CD4+ T cells upon stim- gating strategy is shown in Fig. 1A. Summarized data (Fig. 1B) ulation with anti-CD3/anti-CD28 beads (Dynabeads; Life Technologies). T cell show that human blood, spleens, and tonsils have comparable proliferation and cytokine expression were assessed as described above. frequencies of IL-10+ B cells. Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 2. Surface phenotypes of CD24hiCD38hi TBs and CD24hiCD27+ human equivalent of B10 cells in the blood. (A) Gating strategy for dividing B cell subsets P1 (TBs), P2 (human equivalent of B10), P3 (CD24+CD272), and P4 (CD242CD272) B cells in the blood of healthy individuals. B cells were stained with anti-IgD (IA6-2), anti-CD19 (SJ25C1), anti-CD24 (ML5), anti-CD27 (M-T271), and anti-CD38 (HB7), all from BD Biosciences. (B) t-SNE clustering, showing the distribution of the four B cell subsets (P1 to P4). (C) Representative histograms of IgD, IgM, CD1c, CD1d, CD5, CD9, CD10, CXCR3, CD11a, CD11b, CD11c, integrin a1, integrin a4, integrin b1, integrin b7, CD21, CD23, CD24, CD27, CD38, CD39, and CD43. B cells were labeled with anti-IgD (IA6-2), anti-CD1c (F10/21A3), anti-CD5 (UCHT2), anti-CD9 (M-L13), anti-CD10 (HI10a), anti-CD21 (B-ly4), anti-CD24 (ML5), anti-CD27 (M-T271), anti-CD38 (HB7), anti-CD39 (Tu¨66), anti-CD43 (L60), anti-CD11a (Hl111), anti-CD11c (B-ly6), anti-CD49d/integrin a4(9F10),integrina-b7 (FIB504), anti-CD365/TIM-1 (1D12), anti-CD19 (SJ25C1), and anti-CD3 (UCHT1), all from BD Biosciences; anti-IgM (MHM-88), anti-CD1d (51.1), anti-CD23 (EBVCS-5), anti-CD183/CXCR3 (G025H7), anti-CD11b (CBRM1/5), anti-CD49a/integrin a1 (TS2/7), anti-CD29/integrin b1 (TS2/16), and anti-CD274/PD-L1 (29E.2A3) from BioLegend. Data generated from three independent experiments with cells from five donors were compiled. 4 FUNCTIONAL CHARACTERISTICS OF HUMAN Breg SUBSETS

We next performed a t-SNE clustering analysis using a series of CD24hiCD38hi TBs (P1), CD24hiCD27+ human equivalent of B cell surface markers tested in Fig. 1C. In line with previously B10 (P2), CD24+CD272 (P3), and CD242CD272 B cells (P4). published data (8, 11, 17, 18, 21–25), IL-10+ B cells in the blood Clustering analysis further demonstrated that P1 (red), P2 (blue), were highly heterogenous (Fig. 1C, upper panel). Indeed, they P3 (pink), and P4 (green) have distinct phenotypic properties were populated in groups of B cells expressing any of the surface (Fig. 2B). markers tested. Although TIM-1+ B cells were reported to be a We next characterized surface phenotypes of the four B cell major subset of IL-10–producing Bregs in mice (9, 20), our data subsets (P1 to P4) (Fig. 2C). P1 expressed higher levels of sur- indicated that TIM-1 does not represent human IL-10+ blood face IgD, IgM as well as CD1d, CD5, CD10, CD24, and CD38 but B cells. Fractions of IL-10+ cells coexpress CD9 (46), CD24, and lower levels of CD21 and CD27 than P2 and P3. A large fraction CD38, suggesting the existence of CD9+ TBs. The expression of of CD9hi B cells (46) were enriched in P1. P1 also expressed CD24, CD27, CD5 on IL-10+ B cells also suggested that there increased levels of integrin b1, when compared with P3, but P2 could be the human equivalent of B10 cells that also expressed expressed the highest levels of integrin a1, a4, and b1. P2 high level of CD1d (25). expressed high levels of both CD24 and CD27 that distinguished IL-10+ B cells from both spleens and tonsils were also highly them from the other three subsets. P2 also expressed increased heterogenous (Fig. 1C, middle and bottom panel). The expression levels of CD1c, CXCR3, CD11a, CD11b, and CD43. Moreover, of IgD, IgM, CD24, CD27, and CD38 on IL-10+ B cells further P2 expressed the highest level of CD39 that is known to be expressed indicates that such IL-10+ B cells are comprised of both imma- by both Tregs and myeloid-derived suppressor cells (47–50). P3 ture TBs (CD19+CD24hiCD38hi) and human-equivalent B10 cells were separated from P4 mainly by expressing increased levels of

(CD19+CD24hiCD27+). CD23 and CD24. Downloaded from CD24hiCD38hi TBs and CD24hiCD27+ human equivalent of P1 and P2 B cells express IL-10 and PD-L1 B10 cells exhibit common and distinct cell surface phenotypes In addition to IL-10, PD-L1 can contribute to immune suppressive Based on the expression levels of surface molecules in Fig. 1C, functions of Bregs (51, 52). We thus measured intracellular IL-10 we divided blood B cells into four putative subsets (Fig. 2A), and surface PD-L1 expression by FACS-sorted P1, P2, P3, and P4 http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 3. CD24hiCD38hi TBs and CD24hiCD27+ human equivalent of B10 cells can express both IL-10 and PD-L1. (A) Representative FACS plots, showing the frequency of IL-10+ and PD-L1+ cells in P1, P2, P3, and P4 B cells. FACS-sorted B cell subsets were incubated for 48 h with indicated stimuli (2.5 mg/ml CpG-B ODN 2006 from Invivogen; 0.5 mg/ml recombinant human CD40L from R&D Systems; 2.5 mg/ml anti-BCR, polyclonal goat anti- human IgG + IgM, from Jackson ImmunoResearch). Cells were further stimulated for 5 h with PMA/ionomycin in the presence of monensin and brefeldin A before staining them with anti–IL-10 (JES3-9D7) from eBiosciences. B cells were also stained with anti-CD274/PD-L1 (29E.2A3) from BioLegend. (B) Summarized data for the frequency of IL-10+ B cells. (C) Summarized data for the frequency of PD-L1+ B cells. (D) Summarized data for the frequency of IL-10+PD-L1+ B cells. Data in (B)–(D) were obtained from three independent experiments using cells from five donors. Error bars represent mean 6 SD. ****p , 0.0001, ***p , 0.001, **p , 0.01, *p , 0.05, two-way ANOVA with the Tukey multiple-comparison test. ns, not significant. The Journal of Immunology 5

B cells. As shown in Fig. 3A, 3B, both P1 and P2 had higher per- P1 and P2 secreted greater amount of IL-10 (Fig. 4A, left panel) as centages of IL-10+ B cells than P3 and P4 in response to the stimuli well as TNF-a (Fig. 4A, middle panel) and IL-6 (Fig. 4A, right tested. Moreover, P2 was more efficient than P1 at expressing IL-10 panel) than P3 and P4. There was no significant difference in in response to CpG-B alone or CpG-B plus anti-BCR. IL-10 ex- the amount of IL-6 secreted by P1 and P2 B cells. However, P2 pression by P1 and P2 was also significantly enhanced by CD40L secreted a greater amount of TNF-a than P1 when they were treatment. Both P1 and P2 expressed similar levels of surface PD-L1 stimulated with a combination of CpG-B, CD40L, and anti-BCR. in response to all stimuli tested. However, PD-L1 expression was We next assessed the frequency of IL-10+, TNF-a+, IL-6+ higher on both P1 and P2 than others (P3-P4) when they were B cells as well as IL-10+TNF-a+, IL-10+IL-6+, TNF-a+IL-6+ stimulated with CpG-B, CD40L, and anti-BCR together (Fig. 3C). B cells after 48-h incubation with CpG-B (Fig. 4B, 4C). Both P1 The frequency of IL-10+PD-L1+ B cells were higher in P1 and P2 and P2 had increases of IL-10+ B cells than P3 and P4 (Fig. 4C, than P3 and P4 (Fig. 3D), and this was mainly due to the increase of upper panel). The highest frequency of TNF-a+ and IL-6+ B cells IL-10+ cells in P1 and P2. We also found that P2 was more efficient were observed in P2. We also found that large fractions of IL-10+ than P1 at coexpressing IL-10 and PD-L1 when they were stim- P2 B cells coexpressed TNF-a and IL-6. Fractions of P1 B cells ulated with CpG-B alone or with a combination of CpG-B, soluble also coexpressed IL-10 and TNF-a, but P2 B cells were more CD40L, and anti-BCR, but this difference was not statistically efficient than P1 B cells at expressing both TNF-a and IL-6. Taken significant when they were stimulated with CpG-B plus CD40L. together, substantial fractions of IL-10–expressing B cells in both We thus concluded that both P1 and P2 are superior to P3 and P4 P1 and P2 can coexpress TNF-a and IL-6. for IL-10 expression, but P2 B cells were more efficient than P1 P2 B cells are more efficient than P1 B cells at suppressing B cells at expressing IL-10. PD-L1 expression levels were also Downloaded from CD4+ T cell proliferation higher on P1 and P2 than P3 and P4 overall. We next tested the ability of individual B cell subsets to suppress Substantial fractions of P1 and P2 B cells coexpress IL-10 CD4+ T cell proliferation. Representative flow cytometry data were and TNF-a presented in Fig. 5A, showing that P1 and P2 B cells were more We next investigated the amount of IL-10 secreted by FACS-sorted efficient than P3 and P4 B cells at suppressing CD4+ Tcellpro-

P1, P2, P3 and P4 B cells. Consistent with the data in Fig. 3, both liferation. Summarized data generated with three different B:T cell http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 4. Substantial fractions of IL-10–producing B cells in CD24hiCD38hi TBs (P1), and CD24hiCD27+ human equivalent of B10 cells (P2) also express TNF-a.(A) Summarized data for the amount of IL-10, TNF-a, and IL-6 secreted by P1, P2, P3, and P4 B cells stimulated for 48 h in the presence of indicated stimuli. The amount of cytokines was measured by bead-based multiplex cytokine assay. Data from five independent experiments using cells from five donors are presented. Error bars represent mean 6 SD. ****p , 0.0001, **p , 0.01, *p , 0.05, two-way ANOVA with Dunnett multiple comparisons test. (B) Representative FACS plots showing the frequency of IL-10+ and TNF-a+ and IL-6+ B cells. B cells in (A) were further stimulated for 5 h with PMA/ionomycin in the presence of monensin and brefeldin A. they were then stained with anti–IL-10 (JES3-9D7) and anti–IL-6 (MQ2-13A5) from eBiosciences; anti–TNF-a (MAb11) from BioLegend. (C) Summarized data for the frequency of IL-10+,TNF-a+,IL-6+,IL-10+TNF-a+,IL-10+IL-6+,and TNF-a+IL-6+ B cells. Each dot indicates data generated with B cells from one donor. Data were obtained from three independent experiments using cells from five donors. Error bars represent mean 6 SD. ****p , 0.0001, ***p , 0.001, **p , 0.01, *p , 0.05, nonparametric ANOVA. ns, not significant. 6 FUNCTIONAL CHARACTERISTICS OF HUMAN Breg SUBSETS ratios further demonstrated that P2 B cells were more efficient P1 and P2 contribute to the suppression of CD4+ T cell re- than P1 B cells at inhibiting CD4+ T cell proliferation elicited by sponses. Importantly, P2 B cells were more efficient than P1 allogeneic DCs. Blocking IL-10 (Fig. 5B) and PD-L1 (Fig. 5C) at suppressing CD4+ T cell proliferation in the two different resulted in partial recoveries of CD4+ T cell proliferation that was experimental systems. suppressed by P1 and P2 B cells. IL-10 and PD-L1 expressed by P1 and P2 B cells contribute to The suppressive functions of P1 and P2 B cells were further the suppression of IFN-g and IL-17 expression by CD4+ T cells assessed by measuring CD4+ T cell responses elicited with anti- + CD3/anti-CD28 beads (Supplemental Fig. 1A). Both P1 and P2 We next tested whether P1 and P2 B cells could also control CD4 were more efficient than P3 and P4 at suppressing CD4+ T cell T cell expression of IFN-g and IL-17. Representative flow cytom- proliferation. In support of the data in Fig. 3, Fig. 4 as well as etry data of IFN-g expression was presented in Fig. 5D. Both P1 previously published data (11, 21, 22, 25, 53), blocking IL-10 also and P2 B cells were able to suppress IFN-g expression.Fig.5E,5F resulted in the recovery (∼75–80%) of CD4+ T cell proliferation further demonstrated that blocking either IL-10 or PD-L1 resulted suppressed by P1 and P2 B cells (Supplemental Fig. 1B). Blocking in the restoration of IFN-g expression by CD4+ T cells activated PD-L1 also resulted in the recovery of CD4+ T cell proliferation with allogeneic DCs. P1 and P2 B cells were also capable of that was suppressed by P1 and P2 B cells (Supplemental Fig. 1C). suppressing IL-17 expression by CD4+ T cells (Fig. 5G). Blocking We thus concluded that both IL-10 and PD-L1 expressed by either IL-10 (Fig. 5H) or PD-L1 (Fig. 5I) resulted in significant Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 5. CD24hiCD38hi (P1) and CD24hiCD27+ human equivalent of B10 cells (P2) suppress CD4+ T cell proliferation and IFN-g/IL-17 expression. (A) Representative FACS data of T cell proliferation assay. FACS-sorted subsets of B cells (P1-P4) were stimulated for 48 h with CpG-B and then cocultured for 6 d with CFSE-labeled CD4+ T cells stimulated with allo-DCs. CD4+ T cell proliferation was assessed by measuring CFSE dilution. Right panel shows summarized data from five independent experiments performed with cells from seven healthy individuals. Error bars are mean 6 SD. ****p , 0.0001, ***p , 0.001, two-way ANOVAwith Dunnett multiple comparisons test. (B) Anti–IL-10 and anti–IL-10R Abs (10 mg/ml) were added to the B and T cell cocultures. CD4+ T cell proliferation was assessed as in (A). (C) Anti–PD-L1 Ab (10 mg/ml) was added to the B and T cell cocultures. (D) Representative FACS data showing CD4+ T cell proliferation and IFN-g expression. FACS-sorted subsets of B cells (P1–P4) were stimulated for 48 h with CpG-B. After washing, they were cocultured for 6 d with CFSE-labeled CD4+ T cells stimulated with allo-DCs. T cells were then restimulated with PMA/ionomycin for 5 h in the presence of monensin and brefeldin A before staining them for intracellular IFN-g. Experiment in (A) was performed in the presence of anti–IL-10/IL-10R or control Abs (10 mg/ml for each) (E) and 10 mg/ml anti–PD-L1 or control Ab (F). (G) Representative FACS data showing CD4+ T cell proliferation and IL-17 expression. Experiment in (G) was performed in the presence of anti–IL-10/IL-10R or control Abs (10 mg/ml for each) (H) and 10 mg/ml anti–PD-L1 or control Ab (I). Error bars are mean 6 SD of six independent experiments using cells from six donors. ****p , 0.0001, ***p , 0.001, **p , 0.01, *p , 0.05, two-way ANOVA with Sida ´k correction. The Journal of Immunology 7 increases of IL-17 expression. We thus concluded that both P1 and tonsils. We were able to detect TIM-1+ B cells in the blood, spleens, P2 B cells can suppress IFN-g and IL-17 expression by CD4+ and tonsils, but the frequency of TIM-1+ B cells was lower than T cells in IL-10– and PD-L1–dependent manners. We also tested other Breg subsets tested in this study. whether TNF-a and IL-6 expressed by P2 B cells affected IFN-g P2 B cells express increased levels of both TGF-b1 and and IL-17 expression by CD4+ T cells. Interestingly, blocking granzyme B TNF-a or IL-6 increased IFN-g (Supplemental Fig. 2A, 2B) and IL-17 (Supplemental Fig. 3C, 3D) expression by CD4+ T cells B cell–derived TGF-b (56) and granzyme B (55) are also known cocultured with P2 B cells. This suggests that IL-6 and TNF-a to suppress inflammatory responses. We found that both P2 B cells expressed by P2 cells could also contribute to the suppression express higher levels of both TGF-b1 (Fig. 7A, 7B) and granzyme of IFN-g and IL-17 expression by CD4+ T cells cocultured with B (Fig. 7C, 7D) than P1, P3, and P4 B cells. Furthermore, neu- P2 Bregs. tralizing TGF-b1 resulted in a significant increase of IFN-g ex- pression by CD4+ T cells cocultured with P2 B cells (Fig. 7E and Frequency of human Breg subsets in the blood, tonsils, left panel in Fig. 7F), suggesting that TGF-b1 expressed by P2 and spleens B cells contributes to the inhibition of IFN-g expression. Blocking To date, a number of human Breg subsets have been reported. These granzyme B activity also increased IFN-g expression by CD4+ include TBs (P1) (21), human equivalent of murine B10 (P2) (25), T cells cocultured with P2 and P1 B cells, but such blocking effect CD27+CD38hi plasmablast Bregs (18), CD25hiCD71hiCD73lo was more significant for P2 than P1 B cells (right panel, Fig. 7F). (35), CD39+CD73+ (54), CD38+CD1d+IgM+CD147+ (55), and Neutralizing TGF-b1 also increased IL-17 expression by CD4+ + TIM-1 Bregs (29). We thus compared the frequency of individual T cells (Fig. 7G and left panel in Fig. 7H). However, TGF-b1 Downloaded from subsets of Bregs in the blood, tonsils and spleens (Fig. 6A). As blocking effect was also more significant when CD4+ T cells were shown in Fig. 6B, the frequency of P2 B cells was greater than cocultured with P2 B cells compared with those cocultured with P1 B cells in the blood as well as in spleens and tonsils. We also P1 B cells (left panel, Fig. 7H). Blocking granzyme B activity also found that tonsils contain greater numbers of both plasmablast resulted in a significant increase of IL-17 expression by CD4+ Bregs (CD27+CD38+) and Br1 Bregs (CD25hiCD71hiCD73lo) than T cells cocultured with P2 B cells. We thus concluded that both P1

blood and spleens. A large fraction of circulating B cells was and P2 B cells can express TGF-b1 and granzyme B that con- http://www.jimmunol.org/ CD39+CD73+ (Fig. 6A), and they were also found in both spleens tribute to the suppression of IFN-g and IL-17 expression by CD4+ and tonsils similarly. The frequency of CD38+CD1d+IgM+CD147+ T cells. However, P2 B cells are more efficient than P1 B cells Bregs was similar in the blood and spleens but slightly lower in at suppressing CD4+ T cell expression of IFN-g and IL-17. by guest on September 23, 2021

FIGURE 6. Comparison of the frequency of human Breg subsets in the blood, tonsils, and spleens. (A) FACS plot showing gating strategy for different Breg subsets, including CD24hiCD38hi, CD24hiCD27+, CD27+CD38hi, CD25hiCD71hiCD73lo, CD39+CD73+, CD38+CD1d+IgM+CD147+, and TIM+ B cells. B cells were stained with anti-CD19 (SJ25C1), anti-CD24 (ML5), anti-CD25 (M-A251), anti-CD27 (M-T271), and anti-CD38 (HB7), all from BD Biosciences; anti-IgM (MHM-88), anti-CD1d (51.1), anti-CD71 (CY1G4), anti-CD73 (AD2), anti-CD147 (HIM6), and anti–TIM-1 (1D12) from BioLegend. (B) Data generated from five donors were compiled. Error bars represent mean 6 SD. 8 FUNCTIONAL CHARACTERISTICS OF HUMAN Breg SUBSETS Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 7. CD24hiCD27+ human equivalent of B10 cells (P2) are more efficient than others at expressing TGF-b1 and granzyme B. (A) Representative FACS data for the frequency of TGF-b1+ B cells. FACS-sorted B cell subsets (P1, P2, P3, and P4) were incubated for 48 h in the presence of CpG-B. B cells were then stimulated for 5 h with PMA and ionomycin in the presence of monensin and brefeldin A before staining cells with anti–TGF-b1Ab. (B) Summary of three independent experiments using cells from five donors. (C) Granzyme B expression by B cells stimulated as in (A) and (B). (D) Summary of three independent experiments using cells from five donors. (E) Intracellular IFN-g expression by CD4+ T cells cocultured for 4 d with FACS- sorted B cell subsets (P1-P4). B cells were cultured for 48 h with CpG-B, and anti–TGF-b1 (10 mg/ml), granzyme B inhibitor (20 mmol/ml), or isotype control Ab (10 mg/ml) was added before mixing with CD4+ T cells. T cells were then restimulated with PMA/ionomycin for 5 h in the presence of monensin and brefeldin A before staining them for intracellular IFN-g expression. (F) Summarized data from three independent experiments performed with cells from four healthy individuals. (G) Intracellular IL-17 expression by CD4+ T cells cocultured with B cell subsets in (E). (H) Summarized data from three independent experiments performed with cells from four healthy individuals. Error bars represent mean 6 SD. The p values were determined by two- way ANOVA with Dunnett multiple comparisons test or nonparametric ANOVA as appropriate. ****p , 0.0001, ***p , 0.001, **p , 0.01, *p , 0.05.

Alterations in P1 and P2 B cells in liver allograft recipients they had a significantly decreased frequency of IL-10+ B cells in with PCH peripheral blood. The frequency of both P1 and P2 in the blood of We next examined the frequency of P1 and P2 B cells in a rare healthy individuals was not affected by the ages (Supplemental cohort of liver allograft recipients with PCH. PCH patient infor- Fig. 3). We thus concluded that liver allograft recipients with PCH mation was summarized in Fig. 8A. Blood samples from six pa- have a significantly altered B cell repertoires, specifically they are tients were collected as indicated. Age- and sex-matched healthy deficient in P1 and P2 B cells that serve as the most potent Bregs. individuals were used as controls. As shown in Fig. 8B, there was no significant difference in the numbers of circulating B cells in Discussion the two groups. However, the frequency of P1 (TBs) was lower in Because of the lack of lineage-specific markers or transcription PCH patients than control subjects (Fig. 8C). In addition, PCH factors for Bregs, IL-10 expression is still considered as one of the patients had a decrease of P2 (human equivalent of B10 cells). In major characteristics of Bregs in both humans and mice (42–45). line with the decreases of both P1 and P2 B cells in PCH patients, By assessing cell surface phenotypes of IL-10–expressing B cells, The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/

FIGURE 8. Liver allograft recipients with PCH have decreased P1 and P2 B cells in the blood. (A) PCH patient information. (B) The frequency of total CD19+ B cells in the blood of patients and control subjects. (C) Representative FACS data for the frequency of P1 (CD24hiCD38hi TBs) B cells. Summarized data of PCH patients (n = 6, but with two-time blood draws for patients 1 and 4 as indicated) and healthy controls (n = 9) are presented by guest on September 23, 2021 in right panel. (D) Representative FACS data for the frequency of P2 (CD24hiCD27+ human equivalent of B10 cells) B cells. Summarized data of PCH patients and healthy controls are presented in right panel. (E) Representative FACS data for the frequency of IL-10+ B cells in the blood of PCH patients and controls. PBMCs from PCH+ liver allograft recipients (n = 6, from six patients) and healthy donors (n = 9) were stimulated with CpG-B for 48 h. Cells were then restimulated for 5 h withPMAandionomycininthepresence of monensin and brefeldin A. Cells were harvested and labeled with anti-CD19 (SJ25C1), anti-CD24 (ML5), anti-CD27 (M-T271), and anti-CD38 (HB7), all from BD Biosciences. Cells were then washed, fixed, and permeabilized using Cytofix/Cytoperm (BD Biosciences), followed by intracellular staining with anti–IL-10 (JES3-9D7) from eBiosciences. Summarized data of PCH patients and controls are presented in right panel. The p values were determined with unpaired Student t test (A–E). ***p , 0.001, *p , 0.05. ns, not significant. we demonstrated a great heterogeneity of IL-10+ B cells in human A recent study reported that TIM-1+ B cells comprise of more blood, spleens, and tonsils. However, our data demonstrated that than 70% of IL-10+ B cells in a murine islet transplantation model CD24hiCD38hi TBs and CD24hiCD27+ human equivalent of B10 (9). They, along with murine B10 (CD1dhiCD5+), were also reported cells were the major Breg subsets that could display immune to play important roles in the induction and/or maintenance of im- regulatory functions by expressing not only IL-10 and PD-L1, but mune tolerance in the mice. In humans, however, our data showed also TGF-b1 and granzyme B. We also found that CD24hiCD27+ that IL-10 expression was not greatly enriched in TIM-1+ B cells. In human equivalent of B10 cells seemed to be more effective than addition, the frequency of TIM-1+ B cells in the blood of healthy CD24hiCD38hi TBs at suppressing CD4+ T cell responses. This subjects was variable (0.1–3% of B cells), as recently reported (62). was further supported by the increased expression of IL-10, IL-10 expression was rather enriched in B cells expressing surface TGF-b1, and granzyme B by CD24hiCD27+ human equivalent IgD and IgM, CD5, CD10, CD24, and CD27, which are expressed of B10 cells, suggesting that the in vivo mechanisms of action on both TBs and CD24hiCD27+ human equivalent of B10 cells. of the two Breg subsets may not be the same. Furthermore, in- CD9 was also identified as a marker of murine CD1dhiCD5+ B10 creased expression of CD11a, CD11b, a1, a4, b1, and b7by cells (46). Interestingly, however, our data (Fig. 2C) showed CD24hiCD27+ human equivalent of B10 cells further suggested that a large fraction of human blood CD9+ cells (63) are within that effector sites of the two Breg subsets also not be the same CD24hiCD38hi TBs. either. For example, integrin a4 (CD49d) plays an important role In line with the enrichment of IL-10 expression in IgD+,IgM+, in the recruitment of leukocytes to CNS (57) and in the Breg- CD5+,CD10+,CD24+, and CD27+ blood B cells, both CD24hiCD38hi mediated controlling of experimental autoimmune encephalomy- TBs (21) and CD24hiCD27+ B cells (25, 45) were far more efficient elitis (58). Integrin b1 and integrin b7 represent the expression of than CD24+CD272 and CD242CD272 B cells at expressing IL-10 the heterodimer integrin a4b1 (59) and a4b7 (60, 61), leading, (Fig. 3). More importantly, CD24hiCD27+ B cells were more ef- respectively, to homing potential to no-intestinal (a4b72, a4b1+) ficient than TBs at expressing IL-10 when they were stimulated and intestinal tissues (a4b7+). with CpG-B alone or CpG-B, CD40L, and anti-BCR. Consistent 10 FUNCTIONAL CHARACTERISTICS OF HUMAN Breg SUBSETS with the previously published data (13, 35), CD24hiCD38hi and the in vivo mechanisms of action of the two human Breg subsets CD24hiCD27+ Bregs expressed similar but higher level of surface might not be the same. PD-L1 than the other two B cell subsets. Such increases of PD-L1 expression could be via the IL-10–mediated STAT3 activation Disclosures (64). Further enhancement of surface PD-L1 expression on The authors have no financial conflicts of interest. anti-BCR–activated Bregs could be due to the BCR-mediated NFATc1 activation followed by the induction of IL-10/STAT3/ PD-L1 expression (51). References The majority of IL-10+ B cells expressed surface PD-L1, 1. Evans, J. G., K. A. Chavez-Rueda, A. Eddaoudi, A. Meyer-Bahlburg, + D. J. Rawlings, M. R. Ehrenstein, and C. Mauri. 2007. Novel suppressive whereas only a fraction of PD-L1 B cells expressed IL-10. We function of transitional 2 B cells in experimental arthritis. J. 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Ko, X. Wang, Z. Jiao, S. Wang, Z. Hua, L. Sun, hi hi hi + G. Srivastava, et al. 2012. IL-10-producing regulatory B10 cells ameliorate The difference between CD24 CD38 TBs and CD24 CD27 collagen-induced arthritis via suppressing Th17 cell generation. Am. J. Pathol. B cells was further observed when we assessed the frequency of 180: 2375–2385. Downloaded from TGF-b1 (56, 65) and granzyme B+ cells (55) (Fig. 6). In addition, 6. Candando, K. M., J. M. Lykken, and T. F. Tedder. 2014. B10 cell regulation of hi + health and disease. Immunol. Rev. 259: 259–272. CD24 CD27 B cells expressed increased levels of integrins as well 7. Schioppa, T., R. Moore, R. G. Thompson, E. C. Rosser, H. Kulbe, as CD39, an ecto-ATPase that is expressed on Tregs and myeloid- S. Nedospasov, C. Mauri, L. M. Coussens, and F. R. Balkwill. 2011. B regulatory cells and the tumor-promoting actions of TNF-a during squamous carcinogen- derived suppressor cells (48–50). esis. Proc. Natl. Acad. Sci. USA 108: 10662–10667. 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B cells with immune-regulating more efficient than CD24hiCD38hi TBs at suppressing CD4+ T cell function in transplantation. Nat. Rev. Nephrol. 10: 389–397. 11. Khoder, A., A. Sarvaria, A. Alsuliman, C. Chew, T. Sekine, N. Cooper, proliferation as well as IFN-g and IL-17 expression (Fig. 5). These S. Mielke, H. de Lavallade, M. Muftuoglu, I. Fernandez Curbelo, et al. 2014. + + were in line with the increases of IL-10 , TGF-b1 , and granzyme Regulatory B cells are enriched within the IgM memory and transitional subsets B+ cells in CD24hiCD27+ when compared with CD24hiCD38hi in healthy donors but are deficient in chronic GVHD. Blood 124: 2034–2045. 12. Chesneau, M., L. Michel, N. Degauque, and S. Brouard. 2013. Regulatory by guest on September 23, 2021 TBs. In addition, we found that neutralizing TNF-a or IL-6 in the B cells and tolerance in transplantation: from animal models to human. Front. cocultures of CD24hiCD38hi TBs and CD4+ T cells resulted in the Immunol. 4: 497. enhanced IFN-g– and IL-17–producing CD4+ T cell responses. 13. Nova-Lamperti, E., G. Fanelli, P. D. Becker, P. Chana, R. Elgueta, P. C. Dodd, hi + G. M. Lord, G. Lombardi, and M. P. Hernandez-Fuentes. 2016. IL-10-produced This suggested that TNF-a and IL-6 expressed by CD24 CD27 by human transitional B-cells down-regulates CD86 expression on B-cells B cells might contribute to the suppression of IFN-g and IL-17 leading to inhibition of CD4+T-cell responses. Sci. Rep. 6: 20044. + 14. Cherukuri, A., D. M. Rothstein, B. Clark, C. R. Carter, A. Davison, expression by CD4 T cells. However, it is also important to note M. Hernandez-Fuentes, E. Hewitt, A. D. Salama, and R. J. Baker. 2014. Im- that not only IL-6 and TNF-a, but also other cytokines, including munologic human renal allograft injury associates with an altered IL-10/TNF-a IL-10, can be expressed by activated T cells in the cocultures. expression ratio in regulatory B cells. J. Am. Soc. Nephrol. 25: 1575–1585. 15. Lenert, P., R. Brummel, E. H. Field, and R. F. Ashman. 2005. TLR-9 activation Nonetheless, it will be important to understand the mechanisms of marginal zone B cells in mice regulates immunity through increased by which IL-6 and TNF-a contribute to the CD24hiCD27+ Breg- IL-10 production. J. Clin. Immunol. 25: 29–40. mediated immune suppression in future. 16. Gray, M., K. Miles, D. Salter, D. Gray, and J. Savill. 2007. Apoptotic cells protect mice from autoimmune inflammation by the induction of regulatory B cells. Proc. PCH is a variant of late-onset rejection and has been increasingly Natl. Acad. Sci. USA 104: 14080–14085. diagnosed in both pediatric and adult postliver transplantation 17. Yanaba, K., J. D. Bouaziz, K. M. Haas, J. C. Poe, M. Fujimoto, and T. F. Tedder. 2008. A regulatory B cell subset with a unique CD1dhiCD5+ phenotype controls recipients in recent years (37–39). Moreover, developing PCH in T cell-dependent inflammatory responses. Immunity 28: 639–650. the setting of recurrent HCV has a negative impact on allograft 18. Matsumoto, M., A. Baba, T. Yokota, H. Nishikawa, Y. Ohkawa, H. Kayama, survival (40, 66). Consistent with previously published data in A. Kallies, S. L. Nutt, S. Sakaguchi, K. Takeda, et al. 2014. Interleukin-10-producing plasmablasts exert regulatory function in autoimmune inflammation. Immunity renal transplant recipients and autoimmune disease patients, a rare 41: 1040–1051. group of PCH patients had significantly decreased frequency of 19. Neves, P., V. Lampropoulou, E. Calderon-Gomez, T. Roch, U. Stervbo, P. Shen, both CD24hiCD38hi TBs and CD24hiCD27+ B cells in their blood, A. A. Ku¨hl, C. Loddenkemper, M. Haury, S. A. Nedospasov, et al. 2010. Sig- naling via the MyD88 adaptor protein in B cells suppresses protective immunity suggesting that the absence of two Breg subsets might also play a during Salmonella typhimurium infection. Immunity 33: 777–790. permissive role in the development of PCH. 20. Xiao, S., C. R. Brooks, R. A. Sobel, and V. K. Kuchroo. 2015. Tim-1 is essential for induction and maintenance of IL-10 in regulatory B cells and their regulation In summary, this study demonstrated that the frequency of of tissue inflammation. J. Immunol. 194: 1602–1608. IL-10–producing B cells in human peripheral blood, spleens, and 21. Blair, P. A., L. Y. Noren˜a, F. Flores-Borja, D. J. Rawlings, D. A. Isenberg, tonsils is similar, but they display highly heterogenous surface M. R. Ehrenstein, and C. Mauri. 2010. CD19(+)CD24(hi)CD38(hi) B cells ex- hi hi hi + hibit regulatory capacity in healthy individuals but are functionally impaired phenotypes. Although both CD24 CD38 TBs and CD24 CD27 in systemic Lupus Erythematosus patients. Immunity 32: 129–140. B cells are capable of suppressing T cell responses, CD24hiCD27+ 22. Flores-Borja, F., A. Bosma, D. Ng, V. Reddy, M. R. Ehrenstein, D. A. Isenberg, human equivalent of B10 cells were more efficient than CD24hiCD38hi and C. Mauri. 2013. CD19+CD24hiCD38hi B cells maintain regulatory T cells while limiting TH1 and TH17 differentiation. Sci. Transl. Med. 5: 173ra23. TBs at suppressing T cell responses. This is supported by their ability 23. Latorre, I., A. Esteve-Sole, D. Redondo, S. Giest, J. Argilaguet, S. Alvarez, to express IL-10, PD-L1, TGF-b1, and granzyme B. Increased C. Peligero, I. Forstmann, M. Crespo, J. Pascual, and A. Meyerhans. 2016. hi + Calcineurin and mTOR inhibitors have opposing effects on regulatory T cells expression of surface integrins on CD24 CD27 B cells further while reducing regulatory B cell populations in kidney transplant recipients. distinguished themselves from TBs and other B cells. Therefore, Transpl. Immunol. 35: 1–6. The Journal of Immunology 11

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A + (CD4 T cells + anti-CD3/CD28 beads ) BC * * None P1 P2 *** *** 100 100 + = Blocking Abs **** P1 **** - = Isotype Abs P2 75 75 T cells T cells P3 + + P4

CD4 50

50 lo CD4

P3 P4 lo 25 25 %CFSE %CFSE T cells Counts

+ 0 0 - + - + - + - + - + - + - + - + CD4 CFSE anti-IL-10/IL-10R anti-PD L-1

Supplementary Figure 1. CD24hiCD38hi transitional B cells (TBs, P1) and CD24hiCD27+ human equivalent of B10 cells (P2) suppress CD4+ T cell proliferation. (A) Representative FACS data of T cell proliferation assay. FACS-sorted subsets of B cells (P1-P4) were incubated for 48h with CpG-B. After washing, they were co-cultured for 4 days with CFSE-labeled CD4+ T cells. CD4+ T cells were stimulated with anti-CD3/anti-CD28 beads at 1 to 1 T cells and beads ratio. CD4+ T cell proliferation was assessed by measuring CFSE dilution. (B) Both anti-IL-10 (10 mg/ml) and anti-IL- 10R (10 mg/ml) were added to the B and T cell co-cultures. CD4+ T cell proliferation was assessed as in A. (C) Anti-PD-L1 antibody (10 mg/ml) was added to the B and T cell cocultures. Error bars in B and C are mean ± SD of 4 independent experiments using cells from 4 donors. ****P <0.0001, ***P<0.001, *P< 0.05. Two way ANOVA with Šidák correction or Dunnett's multiple comparisons test. Supplemental Figure 2 Hasan et al.

A B ** 45 ** Isotype TNF blocker anti-IL-6/IL-6R 48

30

32 T cells T cells + + 15 16 IFN-γ IFN-γ

IFN-γ 0 0 CD4

C D ** 12 Isotype TNF blocker anti-IL-6/IL-6R 12 **

10 8 T cells T cells + + 5 4 IL-17 IL-17

IL-17 0 0

CD4

Supplementary Figure 2. Neutralizing IL-6 and TNFα enhances IFNγ and IL-17 expression by CD4+ T cells co-cultured with P2 B cells. (A) Representative FACS data of IFNγ+CD4+ T cells. FACS-sorted P2 B cells were stimulated for 48h with CpG-B and co-cultured for 4 days with purified CD4+ T cells stimulated with anti-CD3/anti-CD28 beads in the presence of 10 μg/ml of TNF blocker, anti-IL-6/IL-6R, or control antibody. T cells were restimulated for 5h with PMA/ionomycin in the presence of brefeldin A before staining them for intracellular IFNγ and IL-17 expression. (B) Summarized data of (A) from 3 independent experiments performed with cells from 5 healthy individuals. (C) Representative FACS data of IL-17+CD4+ T cells. (D) Summarized data of (C) from 3 independent experiments performed with cells from 5 healthy individuals. **P<0.01 by paired two-tailed t-test. Hasan et al. Supplemental Figure 3

A B

12 45 B cells B cells + 30

hi 8 CD27 hi CD38 hi 4 15 % CD24 % % CD24 % 0 0 20’s 30’s 40’s 50’s 60’s 20’s 30’s 40’s 50’s 60’s

Supplementary Figure 3. Frequency of blood CD19+CD24hiCD38hi (TBs) and CD19+CD24hiCD27+ (human equivalent B10) are not age dependent. (A-B) Scattered plots illustrating the frequency of human CD19+CD24hiCD38hi (A) and CD19+CD24hiCD27+ (B) cells in different age groups. Bars indicate means values with SD.