TCR repertoire and Foxp3 expression define functionally distinct subsets of CD4+ regulatory T cells

Abstract

Despite extensive research efforts to characterize peripheral regulatory T (T(reg)) cells expressing transcription factor Foxp3, their subset complexity, phenotypic characteristics, TCR repertoire and Ag specificities remain ambiguous. In this study, we identify and define two subsets of peripheral T(reg) cells differing in Foxp3 expression level and TCR repertoires. T(reg) cells expressing a high level of Foxp3 and TCRs not used by naive CD4(+) T cells present a stable suppressor phenotype and dominate the peripheral T(reg) population in unmanipulated mice. The second T(reg) subset, expressing a lower level of Foxp3 and using TCRs shared with naive CD4(+) T cells constitutes a small fraction of all T(reg) cells in unmanipulated mice and enriches T(reg) population with the same Ag specificities as expressed by activated/effector T cells. This T(reg) subset undergoes extensive expansion during response to Ag when it becomes a major population of Ag-specific T(reg) cells. Thus, T(reg) cells expressing TCRs shared with naive CD4(+) T cells have a flexible phenotype and may down-regulate Foxp3 expression which may restore immune balance at the conclusion of immune response or convert these cells to effector T cells producing inflammatory cytokines.

FIGURE 1

Analysis of CD4⁺ T_reg cell subsets defined by Foxp3 expression level. (A) The level of the Foxp3 transcript is proportional to the expression of the GFP reporter. Foxp3^GFP− and Foxp3^GFP+ CD4⁺ T cells expressing various levels of GFP were sorted and Foxp3 transcript was quantitated by RT-PCR with primers specific for endogenous Foxp3. Sorting gates are shown as rectangles on the histogram. The plot shows relative intensities of DNA bands after scanning the gel image (nd – not detectable). The Foxp3 mRNA level in the cell subset labeled “a” was set as “1”. Experiment was repeated two times. (B) GFP reporter is expressed only in cells expressing endogenous Foxp3 transcript. Single cell analysis of the Foxp3 expression in populations of Foxp3^GFP− and Foxp3^GFPlo and Foxp3^GFPhi CD4⁺ T cells (sorting gates are shown in C). Actin and Foxp3 were amplified in sorted single cells. (C) Flow cytometry analysis of peripheral lymph node cells stained with CD4. Gates used to sort CD4⁺ Foxp3^GFP− (continuous line), Foxp3^GFPlo (broken line) and Foxp3^GFPhi (dotted line) T_reg cells are shown. (D) Foxp3^GFPlo and Foxp3^GFPhi T_reg cells express low and high levels of Foxp3 transcript and protein. RT-PCR and Western blot analysis of Foxp3 expression in sorted Foxp3^GFP−, Foxp3^GFPlo and Foxp3^GFPhi T_reg cells. Experiment was repeated two times. (E) Foxp3^GFPlo (open rectangles) and Foxp3^GFPhi (crosses) T_reg cells suppress proliferation of effector T cells. Typical experiment of three is shown. (F) Foxp3^GFPlo and Foxp3^GFPhi T_reg cells differ in the expression of cell surface markers. Flow cytometry analysis of intracellular Foxp3 expression and cell surface expression of CD25, GITR and CD44. Analysis gates are shown in C. Numbers represent percentage of cells in each quadrant. Representative experiment of three is shown. (G) Weight of the TCRα⁻ recipients receiving adoptive transfer of Foxp3^GFPlo (□, ◊, Δ, x, *) and Foxp3^GFPhi (■, ▪, ▲, ●) cells and co-transferred with Foxp3^GFP− cells into TCRα⁻ mice. Each line represents individual mouse. All recipients receiving Foxp^GFPhi cells remained healthy while two (marked by x, Δ) of the recipients of Foxp3^GFPlo cells succumbed to wasting disease.

FIGURE 2

Stability of the T_reg phenotype of Foxp3^GFPlo and Foxp3^GFPhi cells. (A) Flow cytometry analysis of Foxp3^GFP expression in in vitro cultured sorted CD4⁺ Foxp3^GFP− (dashed line), Foxp3^GFPlo (continuous line) and Foxp3^GFPhi (dotted line) cells (upper left panel). Cells were stimulated with plate-bound anti-CD3/anti-CD28 antibodies either alone (continuous line) or in the presence of TGFβ and Il-2 (broken line). Foxp3^GFP expression in unstimulated CD4⁺ Foxp3^GFP− is shown on the upper right panel (dashed line). Numbers in each plot indicate percentage of Foxp3^GFP+ cells. Experiment was repeated three times. (B) Purity of the sorted populations shown in (A). Histograms showing purity of sorted fractions were analyzed on flow cytometer and sorting was done on a cell sorter so the x-axes of the dot plot and histograms have different scale. (C) Foxp3^GFP expression in adoptively transferred Foxp3^GFPlo (left panel) and Foxp3^GFPhi (right panel) cells. Ly5.1⁺CD4⁺ Foxp3^GFPlo or Ly5.1⁺Foxp3^GFPhi cells were co-transferred with Ly5.1⁻CD4⁺Foxp3^GFP− cells into TCRα chain knockout mice and recipient mice were analyzed after 4 weeks. Gates used for sorting donor populations are shown in (A). At least three recipient mice were analyzed for each adoptive transfer. (D) Cytokine and transcription factor expression in unstimulated or in vitro stimulated populations of Foxp3^GFP−, Foxp3^GFPlo and Foxp3^GFPhi cells. Sorted cells were lysed directly or were stimulated with plate-bound anti-CD3/anti-CD28 antibodies alone for Il-2 detection or in conditions favoring differentiation of Th1, Th2 or Th17 cells. Population of total CD4⁺ T cells stimulated like experimental samples served as positive control (P). Cytokine expression was analyzed by RT-PCR. PCR reaction without added template was used as a negative control (N).

FIGURE 3

Regulation of Foxp3 expression in Foxp3^GFP−, Foxp3^GFPlo and Foxp3^GFPhi cells in in vitro co-culture assays. Foxp3^GFP− T_eff cells (Ly5.1⁺, 30×10³/well) were stimulated in vitro with soluble anti-CD3 antibody in the presence of low (5×10³/well, A, C) or high (30×10³/well, B, D) number of Foxp3^GFPlo (A, B) or Foxp3^GFPhi (C, D) T_reg cells (both Ly5.1⁻) and irradiated splenocytes from TCRα⁻ mice. Panels on the left show CD4 and Ly5.1 expression on cultured cells and gates used to define Ly5.1⁺ T_eff or Ly5.1⁻ T_reg cells. Histograms on the right show Foxp3 expression in Foxp3^GFPlo (A, B) and Foxp3^GFPhi (C, D) cells (upper histogram in each pair) and in T_eff cells (lower histograms in each pair).

FIGURE 4

Flow cytometry analysis of lymph node CD4⁺ T cells from TCR^mini-Foxp3^GFP mice. The fraction of CD4⁺ T cells expressing Foxp3^GFP and expression of CD44 and CD62L on Foxp3^GFP− cells is shown. Gates used to define Foxp3^GFP− cells and subsets of naive CD44⁻CD62L⁺ and activated CD44⁺CD62L⁻ cells as well as Foxp3^GFPlo and Foxp3^GFPhi T_reg cells for TCR repertoire studies are shown as rectangles. Figure shows representative data of at least five mice analyzed.

FIGURE 5

Analysis of the TCR repertoire in TCR^mini-Foxp3^GFP mice. (A) Estimation of the TCR repertoire diversity and clonal abundance (upper table) and TCR repertoire overlap (lower table) of naive and activated/memory T_eff cells and Foxp3^GFPlo and Foxp3^GFPhi T_reg cells. (B) Comparison of the frequencies (%) of most abundant TCRs in naive T_eff (purple bars) and T_reg (brown bars – Foxp^GFPhi T_reg, yellow bars – Foxp3^GFPlo T_reg) subsets (upper panel) and in activated/memory (blue bars) subset (lower panel). (C) Partitioning of the most abundant TCRs sequenced from in vitro stimulated Foxp3^GFP+ cells (combined Foxp3^GFPlo and Foxp3^GFPhi cells) between T cells that retained and lost Foxp3 expression. Percentages of a particular TCR in Foxp3^GFP− and Foxp3^GFP+ subsets are shown on the plot (numbers above and below bars show percentages for the most abundant clones). Sorted Foxp3^GFP+ cells were stimulated in vitro. After 5 days, single cells were sorted (sorting gates are shown on the plot) into Foxp3^GFP+ and Foxp3^GFP− subsets and TCRα chains were amplified and sequenced. Sequences of the TCR α chain CDR3 regions are shown in (B) and (C), red sequences mark T cell clones specific for Ep63K peptide.

FIGURE 6

Foxp3 is upregulated in Foxp3^GFP− cells in peripheral lymph nodes of lymphoreplete mice. (A) TCR^mini-Foxp3^GFP and wild type C57BL6 Foxp3^GFP mice have similar number of cells in peripheral lymph nodes. The plot shows average number of lymph node cells isolated from axillary, brachial and inguinal lymph nodes. Twelve mice in each group was analyzed. (B) CD4⁺ Foxp3^GFP− cells upregulate Foxp3^GFP expression when transferred into lymphoreplete TCR^mini-Foxp3^GFP mice. Flow cytometer sorted CD4⁺ Foxp3^GFP− cells (3×10⁶/mouse) from Ly5.1⁺Foxp3^GFP mice expressing wild type TCR repertoire were transferred into Ly5.1⁻ TCR^mini-Foxp3^GFP expressing restricted TCR repertoire. Recipient mice were analyzed 8 days after transfer. Three recipient mice were analyzed. (C) CD4⁺ T cells in TCR^mini mice reconstituted with sorted CD4⁺ Foxp3^GFP− cells do not undergo homeostatic expansion. Percentage of CD4⁺ T cells in peripheral blood of recipient mice is shown before (left column) and after (middle column) adoptive transfer. Three recipient mice were examined. For comparison percentage of CD4⁺ T cells in peripheral blood of Foxp3^GFP mice expressing wild type TCR repertoire is shown (right column).

FIGURE 7

Analysis of the TCR repertoire in TCR^mini-Foxp3^GFP mice immunized with Ep63K peptide. (A) Naive TCR repertoire in TCR^mini-Foxp3^GFP mice is enriched in CD4⁺ cells specific for Ep63K peptide. Lymph node cells from unmanipulated mice proliferate in response to Ep63K peptide (●, ■) but not to a control peptide derived from IgGVH (○, □). Two TCR^mini mice were analyzed. (B) Amino-acid sequences of TCRα chain CDR3 regions of Ep63K-specific CD4⁺ T cell hybridomas obtained from TCR^mini-Foxp3^GFP mice. (C) Flow cytometry analysis of the draining lymph node cells from TCR^mini-Foxp3^GFP mice immunized with Ep63K and CFA. CD4⁺ T cells expressing Foxp3^GFP are shown. Gates used to define Foxp3^GFP− cells (continuous black line) and subsets of naive CD44⁻CD62L⁺ (yellow continuous line) and activated CD44⁺CD62L⁻ cells (red dotted line) as well as Foxp3^GFPlo (dashed line) and Foxp3^GFPhi T_reg cells (dotted line) for TCR repertoire studies are shown as rectangles. (D) Frequencies (%) of the 25 most abundant T cell clones from activated subset (blue bars) are shown with the frequencies of the T_reg clones expressing the same TCR. Foxp^GFPhi (brown bars) and Foxp3^GFPlo T_reg (yellow bars) subsets (upper panel) are shown. (E) Frequencies (%) of the most abundant Foxp3^GFP+ T_reg clones (combined Foxp3^GFPlo and Foxp3^GFPhi clones) in the draining lymph nodes of immunized (grey bars) TCR^mini-Foxp3^GFP mice are compared with frequencies of the same TCRs in T_reg subset of unmanipulated mice (light blue bars). TCRs shared with T_eff cells and exclusively expressed by T_reg cells are indicated by the arrows under the plot. Sequences of the TCRα chain CDR3 regions are shown in (D) and (E), red sequences mark T cell clones specific for Ep63K peptide. (F) Clonal abundance (%) of Ep63K-specific T cell clones in naive (purple bars) and activated (blue bars) subsets of T_eff cells and Foxp3^GFPlo (yellow bars) and Foxp3^GFPhi (brown bars) T_reg cells in control TCR^mini-Foxp3^GFP mice (upper panel) and mice immunized with Ep63K peptide (lower panel). Names of hybridomas expressing a particular TCRα chain are shown above upper panels.