Human CD4+ Memory T Cells Can Become CD4+IL-9+ T Cells

Citation: Putheti P, Awasthi A, Popoola J, Gao W, Strom TB ( + + + Human CD4 Memory T Cells Can Become CD4 IL-9 T Cells Prabhakar Putheti 0 Amit Awasthi 0 Joyce Popoola 0 Wenda Gao 0 Terry B. Strom 0 Derya Unutmaz, New York University, United States of America 0 1 Department of Medicine, Transplant Institute, Beth Israel Deaconess Medical Center, Harvard Medical School , Boston , Massachusetts, United States of America, 2 Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School , Boston, Massachusetts , United States of America Background: IL-9 is a growth factor for T- and mast-cells that is secreted by human Th2 cells. We recently reported that IL4+TGF-b directs mouse CD4+CD252CD62L+ T cells to commit to inflammatory IL-9 producing CD4+ T cells. Methodology/Principal Findings: Here we show that human inducible regulatory T cells (iTregs) also express IL-9. IL-4+TGF-b induced higher levels of IL-9 expression in plate bound-anti-CD3 mAb (pbCD3)/soluble-anti-CD28 mAb (sCD28) activated human resting memory CD4+CD252CD45RO+ T cells as compared to nave CD4+CD252CD45RA+ T cells. In addition, as compared to pbCD3/sCD28 plus TGF-b stimulation, IL-4+TGF-b stimulated memory CD4+CD252CD45RO+ T cells expressed reduced FOXP3 protein. As analyzed by pre-amplification boosted single-cell real-time PCR, human CD4+IL-9+ T cells expressed GATA3 and RORC, but not IL-10, IL-13, IFNc or IL-17A/F. Attempts to optimize IL-9 production by pbCD3/sCD28 and IL-4+TGF-b stimulated resting memory CD4+ T cells demonstrated that the addition of IL-1b, IL-12, and IL-21 further enhance IL-9 production. Conclusions/Significance: Taken together these data show both the differences and similarities between mouse and human CD4+IL9+ T cells and reaffirm the powerful influence of inflammatory cytokines to shape the response of activated CD4+ T cells to antigen. - Funding: This work was supported by the grants to Terry B. Strom from the Juvenile Diabetes Research Foundation grant 7-2005-1329 and National Institute of Health grant 5P01 A1041521-1. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Cytokines present within the milieu in which human nave CD4+ T cells are activated via stimulation of the TCR complex and co-stimulatory molecules dictate whether these cells commit to Th1, Th2, Th17 or regulatory (Treg) T cell phenotype [14]. The commitment of mouse and human CD4+ T cells to such distinct phenotypes is directed through expression of lineage specific transcription factors, e.g., Tbet for Th1, GATA3 for Th2, and RORct for Th17 cells [57]. While IL-4 and TGF-b respectively direct activated mouse and human nave CD4+ T cells to commit to the Th2 or inducible Treg (iTreg) phenotypes, the presence of IL-4+TGF-b directs mouse CD4+CD252CD62L+ T cells or CD4+CD252CD44low T cells to commit to supposedly a novel inflammatory IL-9 producing CD4+ T cell subset (Th9) [810]. Mouse studies did not examine the effects of IL-4+TGF-b in fostering commitment of nave CD4+CD252CD62LhighCD44low versus resting memory CD4+CD252CD62LlowCD44high T cells to CD4+IL-9+ T cells [8,10]. IL-9 was initially cloned as a T-cell growth factor and IL-9 receptor shares the common c-chain with other IL-2 family members IL-2, -4, -7, -15, and -21 [11,12]. It is yet to be examined if IL-4+TGF-b induce a Th9 like subset in humans. We hypothesized that IL-4+TGF-b in the setting of plate bound-anti-CD3 mAb (pbCD3)/soluble-anti-CD28 mAb (sCD28) stimulation fosters commitment of human nave and resting memory CD4+CD252 T cells to express IL-9. Our analysis which includes a successful effort to optimize IL-9 expression or frequency of CD4+IL-9+ T cells and their molecular phenotype reveals both similarities and distinctions between human and murine IL-9+ T cells. In addition, we have determined that resting and memory CD4+ T cells are not equal in their frequency to express IL-9. While examining the requirements to optimize generation of human CD4+IL-9+ T cells, we found that IL-1b, IL12, or IL-21, that foster commitment to Th1 or Th17 subsets, enhance IL-4+TGF-b induced CD4+IL-9+ T cell generation in presence of pbCD3/sCD28 stimulation. Materials and Methods Ethics Statement This study was conducted according to the principles expressed in the Declaration of Helsinki. The study was approved by the Institutional Review Board of Beth Israel Deaconess Medical Center. All adult healthy volunteers provided written informed consent for the collection of samples at Childrens Hospital Blood Donor Center, Boston, MA, and for subsequent analysis. Media, Monoclonal Antibodies, and Cytokines RPMI 1640 (Gibco, Paisley, Scotland) with L-glutamine was supplemented with 1% non-essential amino acids, 10% FCS, 50 U/ml penicillin and 50 mg/ml streptomycin (all from Gibco BRL, Maryland, USA) and used as culture medium. The following anti-human mAbs were used: Anti-CD3 (UCHT1) and anti-CD28 (CD28.2); Peridinin chlorophyll protein (PerCP, red fluorescence) cy5.5 labeled anti-CD4 (BD biosciences); phycoerythrin (PE, orange fluorescence)-labeled anti-IL-10, anti-IFNc, anti-IL-17, anti-IL-9 (Biolegend); Alexa-488- or fluorescence isothiocyanate (FITC, green fluorescence)-labelled anti-FOXP3 and anti-Annexin-V (Biolegend). Irrelevant isotype-matched mouse mAbs were purchased from Biolegend: FITC-IgG1, PE-IgG2a, PE-cy5IgG2a, and PerCP-IgG1. Recombinant IL-1b, IL-2, IL-4, IL-6, IL-12, IL-21, TGF-b, and neutralizing anti-IFNc, -IL-2, and -IL-4 mAb were purchased from R&D. Preparation of Mononuclear Cells Leukofilters were obtained from fifteen adult healthy volunteers. Leukocytes were obtained by back-washing cells from leukofilters with Dulbeccos phosphate buffered saline (PBS) (Gibco). CD4+ T cells were collected from leukocytes using Rosette-sep Human CD4+ T cell isolation kit (Stemcell technologies) and density gradient centrifugation on Lymphoprep (Nycomed, Oslo, Norway). The cells from the interphase were collected and washed three times with PBS, and trace erythrocytes were removed by hypotonic lysis. Cell viability as measured by Trypan blue exclusion and the purity of CD4+ T cell isolation as analyzed by flow cytometry always exceeded 95%. Isolation of CD4+CD252 T Cells Isolated CD4+ T cells were then stained with biotinylated-antiCD25 mAb, and CD25+ T cells were depleted using Easy-sep biotin selection kit (Stemcell technologies). The purity of CD4+CD252 T cell isolation was always .99%, as analyzed by flow-cytometry. To isolate nave CD4+ T cells, CD4+CD252 T cells were stained with biotinylatedanti-CD45RO mAb and then CD45RO+ T cells were depleted using Easy-sep biotin selection kits; similarly to isolate resting memory CD4+ T cells, CD4+CD252 T cells were stained with biotinylatedanti-CD45RA mAb and CD45RA+ T cells were depleted using Easy-sep biotin selection k (...truncated)