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 ol.1900488 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2019/09/10/jimmunol.190048 Material 8.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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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 blood, spleens, 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+ T cell 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 plasma cell 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 B cell 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 transitional B cell (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, regulatory B cell; 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. Spleen 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).