IL-3 Receptor Expression on Activated Human Th Cells Is Regulated by IL-4, and IL-3 Synergizes with IL-4 to Enhance Th2 Cell Differentiation This information is current as of October 1, 2021. Anil Kumar, Lekha Rani, Suhas T. Mhaske, Satish T. Pote, Shubhanath Behera, Gyan C. Mishra and Mohan R. Wani J Immunol published online 3 January 2020 http://www.jimmunol.org/content/early/2020/01/02/jimmun ol.1801629 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2020/01/02/jimmunol.180162 Material 9.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 January 3, 2020, doi:10.4049/jimmunol.1801629 The Journal of Immunology IL-3 Receptor Expression on Activated Human Th Cells Is Regulated by IL-4, and IL-3 Synergizes with IL-4 to Enhance Th2 Cell Differentiation Anil Kumar, Lekha Rani, Suhas T. Mhaske, Satish T. Pote, Shubhanath Behera, Gyan C. Mishra,1 and Mohan R. Wani1 IL-3, a cytokine secreted by activated T lymphocytes, is known to regulate the proliferation, survival, and differentiation of hematopoietic cells. However, the role of IL-3 in regulation of T cell functions is not fully delineated. Previously, we have reported that IL-3 plays an important role in development of regulatory T cells in mice. In this study, we investigated the regulation of IL-3R expression on human Th cells and also examined the role of IL-3 in effector functions of these cells. We found that human peripheral blood Th cells in resting state do not show surface expression of IL-3R; however, its expression was observed at transcript and intracellular protein levels. The functional IL-3R expression on the surface was seen only after antigenic stimu- Downloaded from lation. When naive Th cells were activated in the presence of various cytokines, we found that IL-4 significantly increases the surface expression of IL-3R and also increases the number of IL-3R+ Th cells. Interestingly, IL-3R+ cells exhibit a Th2 cell–like phenotype and show high GATA-3 expression. Moreover, Th2 cells in presence of IL-3 show increased expression of type 2 effector cytokines, such as IL-4, IL-5, and IL-13. Furthermore, IL-3R expressing and IL-3–secreting Th cells were high in house dust mite– allergic patients. Thus, to our knowledge, we provide the first evidence that the expression of IL-3R on activated human Th cells is modulated by IL-4, and IL-3 regulates the effector functions of Th2 cells. Our results suggest that IL-3 may play an important http://www.jimmunol.org/ role in regulating allergic immune responses. The Journal of Immunology, 2020, 204: 000–000. nterleukin-3, a pleiotropic cytokine also known as multi-CSF, functions of IL-3 are mediated by the activation of downstream stimulates the proliferation, survival, and differentiation JAK2–STAT5 pathway (11). I of multipotent hematopoietic stem cells. Unlike other CSFs, In addition to hematopoiesis, the role of IL-3 has also been IL-3 has broader target range and ability to promote the growth of implicated in other biological processes, such as angiogenesis (12) progenitors of myeloid lineage (1). IL-3 is mainly produced by and proliferation and survival of neuronal progenitor cells (13). activated T lymphocytes; however, other cells, such as NK cells, Recent studies from our laboratory have demonstrated the novel by guest on October 1, 2021 cells of myeloid lineage, astrocytes, thymic epithelial cells, and role of IL-3 in regulation of bone remodeling. IL-3 inhibits mouse keratinocytes, are also known to secrete IL-3 (2–9). IL-3R is a osteoclast differentiation and diverts the osteoclast precursors membrane-bound heterodimer of two subunits, a cytokine-specific toward macrophage lineage (14). IL-3 also inhibits human oste- a-subunit, and a common b-subunit which is also shared by IL-5 oclast differentiation and diverts the cells toward dendritic cell and GM-CSF, and these three cytokines form a discrete group lineage (15). Importantly, we have demonstrated that IL-3 has known as the b common–chain cytokine family. The a-subunit anti-inflammatory and immunomodulatory properties and protects of IL-3R determines the ligand specificity and binds to IL-3 with bone and cartilage loss in vivo in arthritic mice by upregulating low affinity, whereas the b common subunit does not bind to IL-3 regulatory T (Treg) cells (16, 17). These results suggest that IL-3 itself, but coexpression of this subunit is critical for the formation has a regulatory role in Th cells. of the high-affinity receptor for IL-3 (10). Most of the biological Although activated T lymphocytes are the major source of IL-3, there is currently no convincing evidence to show that IL-3 has any direct or indirect influence on the development and functions of National Centre for Cell Science, Savitribai Phule Pune University, Pune 411 007, human T lymphocytes. IL-3 was initially purified from WEHI-3B India leukemia cell line (1), and IL-3 showed its ability to induce 20-a 1G.C.M. and M.R.W. share senior authorship. hydroxyl-steroid-dehydrogenase (20-A-SDH) in Thy1.2+ and 2 ORCIDs: 0000-0003-2287-5388 (A.K.); 0000-0002-5672-1355 (S.T.M.); 0000-0002- Lyt-1+,2 spleen cells in mice (18, 19). Earlier Thy1.2 and 9839-3425 (S.B.); 0000-0003-1040-3631 (M.R.W.). 20-A-SDH were considered as T lymphocyte–specific markers. Received for publication December 14, 2018. Accepted for publication November However, this claim was refuted because it was established that 21, 2019. Thy-1 Ag was not specific to T lymphocytes and its expression This work was supported by the Department of Biotechnology under Government of India Grant BT/HRD/35/01/04/2018 (to M.R.W.). A.K. received a Senior Research was also observed on macrophages and polymorphonuclear cells 125 Fellowship from the Council of Scientific and Industrial Research, New Delhi, India. (20). In addition, I-labeled IL-3 failed to bind to thymus or Address correspondence and reprint requests to Dr. Mohan R. Wani, National Centre spleen lymphocytes, supporting the fact that it is unlikely for IL-3 for Cell Science, Savitribai Phule Pune University Campus, Ganeshkhind, Pune to induce 20-A-SDH in T lymphocytes unless this is done through 411007, India. E-mail address: [email protected] some ambiguous process (21). The online version of this article contains supplemental material. We have recently shown that mice Treg cells express IL-3R, and Abbreviations used in this article: 20-A-SDH, 20-a hydroxyl-steroid-dehydrogenase; IL-3 in the presence of TGF-b1 and IL-2 enhances the differen- MFI, median fluorescence intensity; rh, recombinant human; Treg, regulatory T. tiation of naive Th cells into induced Treg cells in a STAT-5– Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 dependent manner (17). All these observations were made in mice, www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801629 2 REGULATION OF IL-3R EXPRESSION ON Th CELLS and there are no reports on the role of IL-3 in development and anti-RORgt (AFKJS-9) Abs were from eBiosciences. For immunofluo- functions of human Th cells. Sato et al. (22) analyzed human rescence staining, anti-CD123 (V-18) primary Ab was purchased from hematopoietic cells for IL-3R expression and found that CD3+ Santa Cruz Biotechnology and secondary TRITC-tagged Ab was from Bangalore Genei. Anti-GAPDH (rabbit polyclonal) Ab was from Sigma- T cells lack IL-3R expression. The examination of specific cyto- Aldrich. Dynabeads Human T-Activator CD3/CD28 for T cell expansion kine receptor expression on the cell surface is crucial for under- and activation, CSFE, cell stimulation mixture, and FOXP3 staining standing cytokine function. In the current study, we investigated buffers were obtained from Thermo Fisher Scientific. BD Cytofix/ the regulation of IL-3R expression on human Th cells and also Cytoperm and Perm Buffer III, used for intracellular cytokines and p–STAT-5 staining, respectively, were obtained from BD Biosciences. examined the role of IL-3 in the effector functions of Th cells. Recombinant human (rh) cytokines, such as IL-2, IL-3, IL-4, IL-5, IL-7, We found that resting human Th cells constitutively express IL-9, IL-10, IL-12, and TGF-b1, were purchased from R&D Systems. IL-3R at transcript and intracellular protein levels but not on X-VIVO 15 serum-free media was from Lonza. the surface. The surface expression of IL-3R was detected only Isolation of PBMCs and enrichment of CD4+ T lymphocytes after antigenic stimulation, which was significantly increased by IL-4. We further demonstrate that IL-3R+ Th cells show PBMCs were isolated using the Ficoll-Paque density gradient centri- fugation method.