CD117+CD44+ Stem T Cells Develop in the Thymus and Potently Suppress T-cell Proliferation by Modulating the CTLA-4 Pathway
CD117 is expressed on double-negative (DN; CD4–CD8–) cells (Nat Rev Immunol 14:529–545; 2014), but whether it is expressed in other stages and its subsequent functions are unclear. We used an improved method of flow cytometry to analyze different populations of thymocytes (Sci Rep 4:5781; 2014). The expression of CD117 and CTLA-4 were directly assayed in the early stage of thymocytes.
Flow cytometry was used to analyze different populations of thymocytes, and T-cell proliferation assays, RT-PCR, and real-time RT-PCR were used to characterize the stem cells and examine the function of CD44+CD117+ cells.
In DN cells, CD117 expression was greatest on CD44+CD25+ cells (DN2), followed by CD44+CD25– (DN1), CD44–CD25+ (DN3), and CD44–CD25– (DN4) cells. In thymocytes, CD117 expression was highest in DN cells, followed by single-positive (SP; CD4 or CD8) and double-positive (DP; CD4+CD8+) cells. Especially, CD117 expression was positively associated with CD44 and CTLA-4 expression. CTLA-4 expression was highest in DN cells, followed by SP and DP cells. CTLA-4 expression was positively associated with CD25, CD44, and Foxp3 expression. CD44+CD117+ T cells expressed more CTLA-4, which suppressed T-cell proliferation and blocked CTLA-4 to cause antibody-induced T-cell proliferation.
These results suggest that CD44+CD117+ T cells are stem cells and a specific T-cell phenotype that initially develops in the thymus, but they do not progress through DN3 and DN4 stages, lack a DP stage, and potently suppress T-cell proliferation and modulate the CTLA-4 pathway.
KeywordsCD117 CTLA-4 CD25 CD44 Stem cell Flow cytometry
Cytotoxic T lymphocyte antigen-4
Single-positive CD4 or CD8
T-cell development in mice begins with hematopoietic progenitor cells that migrate from the bone marrow to the thymus [1, 2]. The earliest precursors in the T-cell lineage are found within thymocyte populations and lack CD4 and CD8 expression, so they are referred to as CD4−CD8− or double-negative (DN) thymocytes . DN thymocytes consist of four subpopulations based on CD44 and CD25 expression . DN cells that express CD4 and CD8 are CD4+CD8+ or double-positive (DP) thymocytes before differentiating into single-positive (SP; CD4+ or CD8+) thymocytes .
Stems cells include embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, adult tissue stem/progenitor cells, and cancer stem cells. Mesenchymal stem cells (MSCs) have been shown to inhibit abnormally activated immune functions in dendritic cells, T cells, and B cells; however, preclinical efficacy studies to evaluate MSCs in autoimmune animal models have been contradictory [6, 7, 8].
Mammalian CD117 (c-kit) gene protein is a stem cell marker and a type III tyrosine kinase receptor [9, 10], widely expressed in hematopoietic stem cells (HSC), myeloid progenitor cells, as well as pro-B cells and pro-T cells [11, 12, 13]. In the hematopoietic system this protein contributes to cell growth, metabolism, proliferation, and survival [14, 15] during gametocyte development, pigmentation, and intestinal motility , and in the immune system contributes to inflammation . The four DN subsets are defined by surface expression of c-kit, CD44, and CD25. Cells at the DN3 stage are positive for c-kit loCD44−CD25+. CD25 expression is downregulated as cells transition to the DN4 cell stage [14, 18].
Nanog is an important transcription factor for maintaining self-renewal and pluripotency of ES cells and regulates the fate of inner cell mass of blastocysts in the embryo during early development. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells .
Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an inhibitory relative of the T-cell costimulatory molecule, CD28. Whereas CD28 signaling promotes T-cell activation, CTLA-4 serves an immunoregulatory function by suppressing the T-cell response [20, 21, 22]. CTLA-4 also has intrinsic functions for T-conv cells and regulates trafficking of self-reactive T cells [23, 24].
Flow cytometry can be used to characterize single cells in an efficient and expedient manner , and can be used to describe DN cells which are present in limited amounts in the mouse thymus. Previously, a highly reproducible multi-color flow cytometry method was developed to increase thymocytes collected from 104 to more than 106 . Using this method, we identified genes with low expression in DN cells and this permitted characterization of DN cell subsets.
In the present study, CD117, CTLA-4, CD4, CD8, CD25, and CD44 expression was measured in thymocytes and expression of CD117 and CTLA-4 was assayed directly in thymocytes and CD117+ stem T cells in the thymus and spleen. These data provide evidence of a relationship among CD117, CTLA-4, CD25, and CD44 cell surface markers on DN cells and other thymocytes.
Female C57BL/6 mice (Taconic Farms, Germantown, NY, USA) were maintained under specific pathogen-free conditions and were studied at 4–6 weeks of age, according to protocols approved by the Institutional Animal Care and Use Committee at Xi’an Jiaotong University, China.
Flow cytometric analysis and cell sorting
Thymocytes and splenocytes were obtained from naive mice and suspended in PBS plus 1% fetal calf serum (FCS). To avoid nonspecific binding to mouse Fc-γ receptors, cells were blocked with mouse CD16/CD32 mAb (0.25 μg/100 μl; BD Biosciences, Franklin Lakes, NJ, USA) for 15 min at room temperature. Cells were washed and then stained with FITC anti-CD4 (clone RM4-5), PercP5.5-anti-CD8 (clone 53-6.7), APC-CY7-anti-CD44 (clone IM7), PE-CY7-anti-CD25 (clone PC61), PE-anti-CD117 (clone 2B8), FITC-CD117 (Clone 2B8), and APC-anti-CD3e (Clone 145-2C11) antibodies for 60 min at 4 °C. For some samples, surface-stained cells were fixed/permeabilized with a Cytofix/Cytoperm Kit (BD Biosciences) and stained with PE-anti-Foxp3 (clone FJK-16 s; eBioscience) and APC-anti-CTLA-4 (clone UC10-4B9; eBioscience) antibody for 45 min at 4 °C. The corresponding isotype controls (Armenian Hamster IgG, and Rat IgG2b,k) were purchased from eBioscience. Cells were sorted and analyzed using a FACSAria II, and data were analyzed using BD FACSDiva software (BD Biosciences).
Culture medium and antibodies
RPMI 1640 medium containing 10% FCS was used for CD4 naive T-cell culture. Antibodies used for cell culture were anti-mouse CD3 (145-2C11) and anti-mouse IL2 (300 u/ml) (BD BioSciences, San Jose, CA, USA).
Coculture system development
A coculture system was developed using a six-well Transwell plate (0.4 μm pore size insert; Corning Incorporated, Corning, NY, USA).
Naive T cell and CD44+CD117+ cell sorting and stimulation
Lymphocytes from the spleen and thymus were stained with fluorochrome-conjugated antibodies, CD4 (FITC), CD8 (PerCp5.5), CD44 (APC-CY7), and CD117 (PE) for 45 min at 4 °C. Cells were then washed twice and resuspended at a density of 1 × 108 cells/ml. Naive CD4, CD4+CD44+CD117+, and CD8+CD44+CD117+ cells were sorted using BD FACS Aria II. Anti-CD3 antibody was coated onto 24-well flat-bottom plates at 1 μg/ml in PBS overnight and then washed once with PBS. Naive T cells and CD4+CD44+CD117+ (CD8+CD44+CD117+) were then placed into the wells at a density of 1 × 106 cells/ml in culture. Cells were incubated at 37 °C containing 5% CO2 for 7 days.
T-cell proliferation assays
Cell proliferation was quantified using a Cell Trace CFSE Cell Proliferation Kit (Invitrogen, Carlsbad, CA, USA). Splenic T cells were prepared by passing splenocytes through nylon-wool columns (108 splenocytes were incubated in the column for 1 h at 37 °C in 5% CO2 before eluting with RPMI medium), in which CD4+CD117+ cells (from flow cytometric cell sorting) were added to effecter cells (splenic T cells) at ratios of 1:20, 1:10, and 1:5. Cells were incubated with 2.5 μM CFSE (Invitrogen, USA) in PBS for 20 min at 37 °C. An excess of ice-cold RPMI 1640 with 10% FBS was added to the cells to quench the reaction. CFSE-labeled cells (1 × 106/well) were washed twice with RPMI 1640 culture medium and then cultured in complete RPMI 1640 medium with 2 mM l-glutamine (Gibco), supplemented with 10 mM HEPES, 1% penicillin–streptomycin solution (Gibco), and 10% FBS (Gibco). A total 3 μg/ml of ConA or IL-2 and anti-CD3 antibody was added for polyclonal stimuli. Samples not stimulated with ConA or IL-2 and anti-CD3 were used as negative controls. Cells were then incubated for 7 days and stained with CD4 and CD8. T-cell proliferation was measured by quantifying CFSE fluorescence intensity via flow cytometry.
CTLA-4 block assay
To block CTLA-4, 8 μl anti-mouse CD152 (CTLA-4) or isotype Ctrl (Armenian Hamster IgG) antibody was added in medium before incubation during the T-cell proliferation assay.
RT-PCR and real-time RT-PCR
Primers used for RT-PCR to detect CTLA-4, Nanog mRNAs
GAA ATC GTG CGT GAC ATC AAA G
TGT AGT TTC ATG GAT GCC ACA G
Mean and SD values were calculated with Microsoft Excel. At least three independent experiments were performed. A Tukey–Kramer post test was used to compare three or more means and a two-tailed unpaired Student’s t test was used to compare two groups (p ≤ 0.05 was considered statistically significant).
CD117 and CD44 expression in different thymocyte subpopulations
Thymocytes from naive mice were stained with antibody cocktail to confirm whether CD117 expression is associated with CD44 expression in the other thymocyte subpopulations. In three experiments performed independently, more DN, CD4, and CD8 CD44+ cells expressed CD117 compared with DN, CD4 and CD8 CD44– cells (Fig. 1e). There was also a positive correlation between CD25 and CD117 expression among CD4+CD44+ cells and CD8+CD44+ cells (Fig. 1f). Data (Fig. 1g, h) indicate that CD117 expressed in early stage T cells in the thymus positively correlates with CD44 in thymocytes.
CTLA-4 expression correlates with CD25 and CD44 expression in different thymocyte populations
CTLA-4 correlates with Foxp3 expression in thymocytes
CTLA-4 expression in CD44+CD117+cells in the thymus
CTLA-4 expression correlates with CD44 expression in different populations of CD117-positive thymocytes
CD44+CD117+ stem T cells from the thymus and spleen suppressed T-cell proliferation
CTLA-4 suppressed T-cell proliferation and blocking CTLA-4 led to antibody-induced T-cell proliferation
CD44+CD117+ stem T cells can self-renew
CD44+CD117+ cells in blood and lymph nodes
Using an advanced flow cytometry method, we researched the characteristic of T-cell development in the thymus by measuring expression of CD117 and CTLA-4 in thymocytes. Surprisingly, CD117+ stem T cells were found in mature thymocytes and CD117 and CTLA-4 expression occurred at each stage of T-cell development. Changes in CD25, CD44, CD117, and CTLA-4 expression were examined in DN, DP, and SP cells using multi-color flow cytometry. CD117 was expressed in the early stages (DN1 and DN2) of T cells (Fig. 1a, c), and DN cells expressed more CD117 than did DP and SP cells (Fig. 1b, d). CD117 expression was positively correlated with CD44 in thymocytes (Fig. 1e–h). CD44+CD117+ T cells could be also found in blood and lymph nodes (Fig. 11). CD4SP and CD8SP cells contain many CD117+ cells, most of which are CD44+CD117+ cells. CD44+CD117+ cells have been used to label tumor stem cells [8, 10, 23, 28]. In the current study, CD117 and CD44 were used to label stem T cells and development stages were identified in the thymus.
Next, CTLA-4 expression was measured at different stages of T-cell development. CTLA-4 expression was higher in DN cells compared with SP and DP cells (Fig. 2a, b) and was positively correlated with CD25 and CD44 (Fig. 2c, d). CTLA-4 and Foxp3 overlapped in DN and CD4+cells (Fig. 3). CD44+CD117+ cells and nTreg cells are two different populations of cells, although parts of them overlap (Fig. 12). CD44+CD117+ cells had more CTLA-4 expression (Figs. 4 and 5). CD44 expression was positively correlated with CTLA-4 in CD117+ thymocytes (Fig. 6). CD117+CD44+ T cells are stem T cells that expressed more Nanog than other T cells in thymocytes (Fig. 7) and have the ability to self-renew (Fig. 10), confirming that CD44+CD117+ stem T cells suppressed T-cell proliferation and CD4+CD117+CD44+ thymus cells had a potent suppressive function (Fig. 8). Because there is more CTLA-4 expression in stem T cells, we studied the effect of CTLA-4 on stem T cells. CD117+ stem T cells were incubated with CTLA-4 blocking antibodies to confirm whether blocking CTLA-4 abrogated the suppressive mechanism of CD117+ stem T cells. CD44+CD117+ stem T cells and T cells proliferated, and when cultured separately this proliferation ceased. Blocking CTLA-4 abrogated the suppressive function of CD44+CD117+ stem T cells, and induced T-cell proliferation (Fig. 9). The suppressive activity of stem T cells was partly aborted by anti-CTLA-4 antibodies and the ability of CD44+CD117+ cells to inhibit T cells was diminished in a Transwell plate, suggesting that this inhibition depends on cell-to-cell contact.
Stem T cells in the immune system differ from other stem cells with respect to development and why this is true is unclear. Research for study the effects of MSCs on lymphocyte proliferation and their immune modulation has provided invaluable data . Stem T cells are analogous to MSCs; however, they highly express abundant CTLA-4 and have a greater ability to modulate the immune system. How stem T cells interact with, and regulate, T cells, B cells, and natural killer cells, DCs, or neutrophils is unclear, as is whether stem T cells alter lymphocyte phenotype or induce development of T regulatory cells in vitro and/or in vivo, or whether stem T cells are anti-inflammatory in vivo. Tumor cells are associated with tumor stem cells , but CD44+CD117+ stem T cells regulate T-cell proliferation. The difference between tumor stem cells and CD44+CD117+ stem T cells is worthy of exploration.
Previous work indicates that CD117 cells are present in DN1 and DN2, but not in SP cells . We observed that CD117 cells such as NK T cells and Tregs develop in the thymus, and do not follow the theory of T-cell or DN T-cell development. CD117 cell development may begin from CD44+ DN cells and proceed directly to CD44+ SP cells. It is unclear why NK T cells, Tregs and CD44+CD117+ cells are T cells in phenotype but do not share the T-cell development pathway. We propose that CD44+CD117+ T cells may not have been derived from a common precursor as T cells, but rather pass through a different pathway.
In summary, CD44+CD117+ T-cell development occurred in the thymus and spleen. CD44+CD117+ T cells expressed abundant CTLA-4 and suppressed T-cell proliferation. Blocking CTLA-4 reduced the suppression of T-cell proliferation. CD44+CD117+ T cells are stem cells that expressed more Nanog and CTLA-4 and potently suppressed T-cell proliferation via modulating the CTLA-4 pathway. Finally, CD44+CD117+ T cells may have developed from CD4–CD8–CD44+CD25– T progenitor cells, without the involvement of DN3, DN4, and CD4+CD8+ (DP) stages in the thymus.
The authors gratefully acknowledge The Xi'an Jiaotong University Cardiovascular Research Center for providing their flow cytometry services.
This study was supported by The First Affiliated Hospital, Xi'an Jiaotong University and Natural Science Foundation of Shaanxi Province of China (No. 2016JM8039).
Availability of data and materials
The data that support the findings of this study are available from the corresponding author on reasonable request.
TL contributed to the design of the study and writing the manuscript, performed experiments, and analyzed data. YW, ZSH, WG, GL, BYS, and EQL participated in the experimental design and manuscript preparation, performed some experiments, and analyzed data. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
All protocols were approved by the Institutional Animal Care and Use Committee at Xi’an Jiaotong University (Permit Number: 2016-278). All surgeries and sacrifices were performed under chloral hydrate anesthesia. Every effort was made to minimize animal suffering.
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