Human CD4+ CD25hi Foxp3+ regulatory T cells are derived by rapid turnover of memory populations in vivo

Abstract

While memory T cells are maintained by continuous turnover, it is not clear how human regulatory CD4+ CD45RO+ CD25hi Foxp3+ T lymphocyte populations persist throughout life. We therefore used deuterium labeling of cycling cells in vivo to determine whether these cells could be replenished by proliferation. We found that CD4+ CD45RO+ Foxp3+ CD25hi T lymphocytes were highly proliferative, with a doubling time of 8 days, compared with memory CD4+ CD45RO+ Foxp3- CD25- (24 days) or naive CD4+ CD45RA+ Foxp3- CD25- populations (199 days). However, the regulatory population was susceptible to apoptosis and had critically short telomeres and low telomerase activity. It was therefore unlikely to be self regenerating. These data are consistent with continuous production from another population source. We found extremely close TCR clonal homology between regulatory and memory CD4+ T cells. Furthermore, antigen-related expansions within certain TCR Vbeta families were associated with parallel numerical increases of CD4+ CD45RO+ CD25hi Foxp3+ Tregs with the same Vbeta usage. It is therefore unlikely that all human CD4+ CD25+ Foxp3+ Tregs are generated as a separate functional lineage in the thymus. Instead, our data suggest that a proportion of this regulatory population is generated from rapidly dividing, highly differentiated memory CD4+ T cells; this has considerable implications for the therapeutic manipulation of these cells in vivo.

Figures

Figure 1. CD4+ CD25+ T cells remain functional with age.

(A) Freshly isolated PBMCs from younger and older donors were stained with CD4 and CD25 and the percentages of CD25int and CD25hi CD4+ T cells were determined. Statistical significance was determined by 2-tailed, unpaired Student’s t test. (B) The coexpression of CD25 and Foxp3 in CD4+ T cells in older and younger subjects was determined. The left panels show the gating on PBMCs labeled with CD4-PerCP and CD25-PE. Based on CD25 expression, the CD4 population is subdivided into CD25–, CD25int, and CD25hi populations. Histograms illustrate the Foxp3 expression in gated populations (log scale). Filled histograms represent staining with secondary antibody alone. Results are representative of 5 separate experiments performed on younger and older subjects. (C) Purified CD4+CD25– T cells were stimulated with immobilized anti-CD3, purified protein derivative (PPD), and tetanus toxoid in the presence of autologous irradiated PBMCs as APCs. CD4+CD25– T cells were cultured in the absence (black bars) or presence (white bars) of equal numbers of either CD4+CD25– or CD4+CD25+ cells. Proliferation was measured by 3H-thymidine incorporation on day 3 (for anti-CD3) and day 6 (for recall antigens), and results are expressed as mean ± SEM of triplicate wells. Results shown are representative of 5 experiments performed in younger and older subjects.

Figure 2. Analysis of in vivo kinetics of human CD4+ CD25hi T cells.

Outline of protocol is shown in A. Subjects received 0.64 g/kg 6,6-D2-glucose by half-hourly oral dosing for 10 hours. Blood samples were taken first during labeling, for analysis of deuterium content in plasma glucose, and second after labeling, to determine deuterium content in DNA. (B) Fraction of labeled cells (F, expressed relative to a labeling phase of 1 day) for each sorted cell population from 4 younger subjects (Y1–Y4) was determined. Open circles represent CD4+CD45RO+CD25hi, filled circles represent CD4+CD45RO+CD25–, and open squares represent CD4+CD45RA+ T cells. Error bars represent SD of triplicate gas chromatography mass spectrometry (GCMS) measurements (labeling of CD4+CD45RA+ T cells was too low to be detectable in individuals C16 and C17). Modeled proliferation and disappearance kinetics are best-fit curves as described in Methods. (C) The fraction of labeled cells in sorted T cell populations from t4 older subjects (O1–O2) and curve fits were analyzed and modeled in the same way.

Figure 3. Constraints on CD4+ CD25hi T cell maintenance.

(A) Based on CD25 expression, the CD4 population was subdivided into CD25–, CD25int, and CD25hi populations. Bcl-2 expression in each subset was determined by intracellular staining and expressed as median mean fluorescent intensity (MFI). Statistical significance was determined using a 2-tailed, paired Student’s t test. (B) CD4+CD25hi T cells have significantly shorter telomeres than total CD4 T cells in both younger and older donors. Telomere length was measured using a 3-color flow-FISH technique. P values were determined by 2-tailed, paired Student’s t test. (C) CD4+CD25hi T cells cannot upregulate telomerase. FACS-sorted CD4+CD45RO+CD25– and CD4+CD45RO+CD25hi cells from younger and older donors were stimulated with anti-CD3/anti-CD28 beads for 4 days. Telomerase activity was measured by a TRAP assay. An equivalent number of proliferating (Ki67+) cells were used in each reaction. The negative control (– cnt) contains the PCR mix without cell extract and the positive control (+ cnt) contains an extract of a telomerase-positive tumor cell line. TSR8 is a telomeric template, used as PCR control.

Figure 4. Regulatory CD4+ CD25hi T cells are closely related to effector CD4+ T cells.

CD4+CD25hi and CD4+CD25– cells were FACS sorted as before, and heteroduplex analysis was performed for 26 Vβ families. (A) Heteroduplex analysis of CD4+CD25– and CD4+CD25hi cells showing a representative Vβ family from 1 out of the 7 volunteers that were investigated. Solid arrows on the left of the blot indicate clonal bands that are present in both subsets. The asterisk indicates the position of the carrier homoduplexes. The schematic representation of the distribution of clones present in CD4+CD25– and CD4+CD25hi subpopulations across the 26 Vβ families in 1 individual is shown in B, where the presence of a clone in a particular Vβ family for either subset is represented by a symbol and a clone that is shared between the 2 subsets is represented by a line joining 2 symbols. (C) Schematic representation of the clones present in CD4+CD25hi population from 1 donor at different time points taken 7 months apart.

Figure 5. Antigen-driven expansion of CD4+ T cells is associated with concomitant expansion within the CD4+ CD45RO+ Foxp3+ CD25hi Treg population.

(A) PBMCs from a CMV seropositive donor were stimulated with CMV lysate and CMV-specific CD4+ T cells identified by IFN-γ secretion. TCR Vβ usage of the total CD4+ (open bars) and CD4+IFN-γ+ T cells (black bars) was analyzed by costaining with 24 different anti-TCR Vβ antibodies. (B) PBMCs from the same donor were then stained with CD4, Bcl-2, and a range of Vβ-specific antibodies. (C) CMV-specific CD4+Vβ2+ T cell population was largely CD27–CD28– compared with other Vβs. (D) CD4+ Vβ2+ T cell population has short telomeres relative to other less-expanded Vβ families. Telomere length was determined using 3-color flow-FISH. (E) Similar expansions in Vβ2 T cell population were found in CD4+CD25– CD4+CD25int and CD4+CD25hi subsets. The top panel shows fresh PBMCs gated on the basis of CD4 and CD25 expression as before. Bottom panels show the proportion of cells expressing Vβ2 in each of the subsets defined by CD25 expression. These data are representative of 3 experiments. (F) Similar expansions in Vβ2 T cell population were found in CD4+Foxp3– and CD4+Foxp3+ subsets. Fresh PBMCs were gated on the basis of CD4 and Foxp3 expression (top panel), and proportion of cells expressing Vβ2 in each subset is indicated (bottom panels). These data are representative of 3 experiments.