Identifying Foxp3-expressing suppressor T cells with a bicistronic reporter - PubMed (original) (raw)
Identifying Foxp3-expressing suppressor T cells with a bicistronic reporter
Yisong Y Wan et al. Proc Natl Acad Sci U S A. 2005.
Abstract
Regulatory T cells are critical for maintaining self-tolerance and to negatively regulate immune responses. Foxp3 is a regulatory T cell-specific transcription factor that functions as the master regulator of the development and function of regulatory T cells. Here, we report the generation of a mouse model, in which a bicistronic reporter expressing a red fluorescent protein has been knocked into the endogenous Foxp3 locus. Using this mouse model, we assessed Foxp3 expression in various lymphocyte compartments and identified previously unreported Foxp3-expressing cells. In addition, we showed that de novo Foxp3 expression along with suppressive function were induced by TGF-beta in activated CD4 T cells in vitro. Finally, we demonstrated that non-Foxp3-expressing CD4 T cells could not be converted into Foxp3-expressing cells upon adoptive transfer into immunodeficient hosts. This Foxp3 bicistronic reporter knockin mouse model should greatly enhance the study of regulation and function of Foxp3-expressing regulatory T cells.
Figures
Fig. 1.
Targeting IRES-mRFP reporter into the mouse Foxp3 locus. (A) Maps for mouse Foxp3 locus, targeting DNA construct, and the targeted Foxp3 locus. An 11-kb mouse genomic DNA, including exon 13 of Foxp3 gene, was excised by using BstZ17I (B) and _Hpa_I (H) (Top) and cloned into pEasy-Flox vector adjacent to the thymindine kinase (TK) selection marker. A cassette containing IRES-mRFP and LoxP-flanked neomycin (Neo) selection marker was inserted into an _Ssp_I (S) site between the translation stop codon (UGA) and the polyadenylation signal (A2UA3) of Foxp3 gene (Middle). A correctly targeted ES cell was used to create chimeras and germ-line-transmitted mice. The Neo gene was removed in vivo by using deletor mice transgenic for Cre recombinase to generate mice bearing targeted Foxp3 locus (Lower). (B) PCR geno-typing FIR mice. Three primers (P1 to P3 as indicated) were designed to genotype FIR mice. PCR yielded 517-bp product for the wild-type (Wt) Foxp3 allele and 470-bp product for targeted Foxp3 allele.
Fig. 2.
mRFP expression faithfully marks Foxp3-expressing CD4 T cells without compromising their regulatory activity, and Foxp3 expression was detected in different lymphocyte compartments. (A) Peripheral lymphocytes from FIR mice were harvested and stained with fluorophore-conjugated anti-CD4 and anti-CD25 antibodies. mRFP expression in CD4 T cells was monitored by flow cytometry (Left). RNA was extracted from different populations of peripheral CD4 T cells (as indicated) purified from FIR mice by FACS. Relative mRNA levels of Foxp3 were determined by TaqMan real-time quantitative PCR, and combined results of two experiments were plotted. (B) CD4+mRFP+ suppressor (S) and CD4+CD25–mRFP– responder (R) T cells were purified by FACS. Suppressor and responder cells were either cultured alone or mixed at indicated ratios (R:S), whereas the number of responder cells remained the same (2 × 104). T cells were activated by soluble anti-CD3 and anti-CD28 antibodies in the presence of irradiated APCs. Three days after stimulation, T cell proliferation was measured by a [3H]thymidine incorporation assay. Combined results from two experiments are shown. (C) In FIR mice, cells from peripheral lymph nodes, bone marrow, and thymus were harvested and stained with fluorophore-conjugated anti-CD4, anti-CD8, anti-B220, and anti-TCR antibodies. By detecting mRFP, the expression of Foxp3 was assessed in CD4 T cells (CD4+TCR+), CD8 T cells (CD8+TCR+), and B cells (B220+) from peripheral lymphocytes (Top Left, Top Left Center, and Top Right Center); in CD4+ cells from bone marrow cells (Top Right with inserted histogram showing the percentage of Foxp3+ cells in CD4+TCR+ population); and in CD4 SP, double-positive (DP), CD8 SP, double-negative (DN) thymocytes (Middle). In addition, based on the expression levels of TCR, CD4+ thymocytes were divided into three groups, TCRHigh, TCRMed, and TCRLow, (Lower Left) and Foxp3 expression in each population was evaluated (Lower Left Center, Lower Right Center, and Lower Right). Typical results of three experiments are shown. (D) Thymocytes and bone marrow cells from FIR mice were stained with fluorophore-conjugated anti-CD4 and anti-CD25 antibodies and analyzed by flow cytometry. Results typical of three experiments are shown.
Fig. 3.
TGF-β induces de novo Foxp3 expression and regulatory function in CD4 T cells after antigenic stimulation. (A) CD4+CD25–Foxp3– T cells were purified by FACS (Upper Left). With or without TGF-β treatment, purified T cells were activated with soluble anti-CD3 and anti-CD28 in the presence of irradiated APCs. Three days after stimulation, cells were stained with fluorophore-conjugated anti-CD4, and the percentage of Foxp3 expressing CD4 T cells were assessed by flow cytometry (Upper Center and Upper Right). In similar experiments, titrated amount of TGF-β were used, and the correlation between the percentage of Foxp3+ cells and the amount of TGF-β used was plotted (Lower). Typical results of two experiments are shown. (B) FACS-purified CD4+CD25–Foxp3– cells were labeled with CFSE. Under the treatment of 5 ng/ml TGF-β, purified T cells were either left inactivated or activated by soluble anti-CD3 and anti-CD28 antibodies in the presence of irradiated APCs. Three days after stimulation, cells were stained with fluorophore-conjugated anti-CD4 antibodies and analyzed by flow cytometry. CD4+Foxp3+ cells were gated, and the CFSE fluorescence intensity of gated cells was assessed and plotted as histograms. Results typical of two experiments are shown. (C) After 3 days of activation in the presence of 5 ng/ml TGF-β, CD4+Foxp3+, and CD4+Foxp3– cells from the experiments described in A were purified by FACS and restimulated with soluble anti-CD3 and anti-CD28 in the presence of irradiated APCs. Three days after restimulation, T cell proliferation was measured by [3H]thymindine incorporation. Combined results from three experiments are shown. (D) FACS-purified CD4+Foxp3+ (Foxp3+) and CD4+Foxp3– (Foxp3–) T cells as described in A were combined with equal amounts of FACS-purified CD4+CD25– T cells bearing the CD45.1 congenic marker. Cocultured T cells were stimulated with soluble anti-CD3 and anti-CD28 in the presence of irradiated APCs. Four days later, T cells were restimulated with phorbol 12-myristate 13-acetate and ionomycin, and IFN-γ production in CD45.1+CD4+ T cells were monitored by intracellular staining (Left). Combined results from two experiments were plotted (Right).
Fig. 4.
CD4+Foxp3– T cells are not converted into CD4+Foxp3+ cells in the immunodeficient hosts after adoptive transfer. (A) From FIR mice, CD4+CD25–Foxp3– (CD4) peripheral T cells and Foxp3– bone marrow cells (BM) were purified by FACS and then transferred into multiple sublethally irradiated (600 rad) Rag-deficient syngeneic hosts with 1 × 106 cells per recipient. At various time points after transfer, recipient mice were killed, and the peripheral T cells were harvested and stained with fluorophore-conjugated anti-CD4 antibodies; the percentage of Foxp3+ cells among CD4 T cells was determined by flow cytometry, and combined results were plotted with each dot represents one recipient mouse. (B) Six weeks after transfer, hosts that received CD4+CD25–Foxp3– peripheral T cells or Foxp3– bone marrow cells were killed. Lymphocytes from peripheral lymph nodes, mesenteric lymph nodes, and the spleens were harvested and stained with fluorophore-conjugated CD4 antibodies. Foxp3-expressing CD4 T cells were detected by flow cytometry.
References
- Nishizuka, Y. & Sakakura, T. (1969) Science 166, 753–755. - PubMed
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