Antigen-specific peripheral shaping of the natural regulatory T cell population

Abstract

Although regulatory T (T reg) cells are thought to develop primarily in the thymus, the peripheral events that shape the protective T reg cell population are unclear. We analyzed the peripheral CD4(+) T cell receptor (TCR) repertoire by cellular phenotype and location in mice with a fixed TCRbeta chain. We found that T reg (Foxp3(+)) cells showed a marked skewing of TCR usage by anatomical location in a manner similar to antigen-experienced (CD44(hi)Foxp3(-)) but not naive (CD44(lo)Foxp3(-)) cells, even though CD44(hi) and T reg cells used mostly dissimilar TCRs. This was likely unrelated to peripheral conversion, which we estimate generates only a small percentage of peripheral T reg cells in adults. Conversion was readily observed, however, during the immune response induced by Foxp3(-) cells in lymphopenic hosts. Interestingly, the converted Foxp3(+) and expanded Foxp3(-) TCR repertoires were different, suggesting that generation of Foxp3(+) cells is not an automatic process upon antigen activation of Foxp3(-) T cells. Retroviral expression of these TCRs in primary monoclonal T cells confirmed that conversion did not require prior cellular conditioning. Thus, these data demonstrate that TCR specificity plays a crucial role in the process of peripheral conversion and in shaping the peripheral T reg cell population to the local antigenic landscape.

Figure 1.

The natural T reg and CD44^hi but not naive CD44^lo TCR repertoires vary by anatomical location. (A) Analysis of individual TCRs. Using the pooled TCR dataset described in Table I, we chose the most frequent TCR of a given phenotype (shown at left) at each location (shown on top). To decrease the effect of mouse-to-mouse variability, we only chose TCRs that were found in at least two independent experiments. Because the naive CD44^lo TCRs were fairly uniform across locations, the three most frequent naive TCRs, irrespective of location, are shown from left to right. Symbols represent the frequency (as a percentage of total sequences) of the TCR, denoted above by its CDR3 amino acid sequence, within the dataset of that phenotype and the location in each of the four experiments. (B) Anatomical distribution of the most abundant TCR sequences within each phenotype. Using the pooled TCR datasets from all four experiments, the top 50 TCR sequences by frequency within a given phenotype were selected. For each TCR, the relative frequency with which it is found at each anatomical location was calculated by dividing its frequency at each location by the sum of the frequencies at all locations. The frequency is represented on the heat map by decile, with the scale indicating the color at 100 (red), 75, 50, 25, and 0% (yellow) for a TCR at a given location. The data are clustered such that those TCRs predominantly found in one location are shown together. (C) Statistical analysis of similarity. The mean Morisita-Horn similarity index (±SD; n = 3–4 independent experiments) is shown for the comparison between the mesenteric (Mes) LNs versus the spleen, cervical (Cerv), axillary (Axil), and inguinal (Ing) LNs (top), and the inguinal LNs versus the spleen, Mes, Cerv, and Axil LNs (bottom).

Figure 2.

The CD44^hi, CD44^lo, and T reg TCR repertoires show little overlap. (A) Comparison of the frequencies of prevalent TCRs between the CD44^hi and Foxp3⁺ or CD44^lo datasets. In each graph, the 50 most abundant TCRs from each of the two indicated populations were selected and plotted on the x axis, with the frequency of the TCR in the indicated subset on the y axis. TCRs are arranged in order of decreasing frequency within the CD44^hi subset, and increasing frequency within the other subset. (B) Statistical analysis of similarity. (left) The Morisita-Horn similarity values between the indicated phenotypes within each experiment are shown, with each experiment represented by a different symbol. (right) The interexperiment similarity within each phenotype is shown, with each comparison represented by a unique symbol. (C) Cluster analysis of the peripheral TCR dataset. Data shown are a heatmap representation of the Morisita-Horn similarity indices calculated pairwise for all cell phenotype and location combinations using the pooled TCR dataset. Each decile is represented by distinct shades, with the scale indicating the color at 0, 0.5, and 1. The mean linkage algorithm was used to obtain a hierarchical cluster (or dendrogram; right) by sequentially grouping the two most correlated observations on the basis of a distance metric provided by the square root of 1 (Morisita-Horn index).

Figure 3.

Peripheral conversion in nonlymphopenic hosts is infrequent. (A) Adoptive transfer of peripheral Foxp3⁻ cells into congenic hosts. FACS-purified Foxp3⁻CD45.2⁺ (10⁷) and Foxp3⁺CD45.1⁺ (1–2 × 10²) T cells were intravenously transferred into normal Thy1.1 hosts. After 3–4 wk, acquisition of Foxp3 was analyzed by flow cytometry. Pooled spleen and LN cells were gated on CD4⁺ and Thy1.2⁺ (donor cells; left) to determine the frequency of Foxp3⁺ cells (middle). Within the Foxp3⁺ cells, the outgrowth of the contaminating cells in the original CD45.2⁺Foxp3⁻ population was estimated by the spike of Foxp3⁺CD45.1⁺ cells (right). Data shown are representative of three independent experiments (n = 5 mice), and percentages are shown. (B) The frequency of conversion varies by anatomical location. The experiment was performed as in A, and after 4 wk the LNs or spleens from three animals were pooled for analysis. Data shown are representative of two independent experiments, and percentages are shown. (C) Summary of the flow cytometric data described in A and B. The frequency of Foxp3⁺ cells is shown. Each symbol represents data from an individual mouse (“pooled”; as in A) or an experiment in which cells from the organs of three mice were pooled (as in B) from the spleen, Mes LNs, and a pool of Cerv/Axil/Ing LNs. (D) Adoptive transfer of mature Foxp3⁻ thymocytes. (top) Mature HSA^loCD62L^hiFoxp3⁻CD25⁻ CD4SP thymocytes or Foxp3⁻CD4⁺ peripheral T cells were adoptively transferred into congenic hosts. Percentages are shown. (bottom) The frequency of Foxp3⁺ cells in pooled spleen and LN preparations was assessed at 3 wk by flow cytometry. Each symbol represents data from an individual recipient from two experiments in which 3.4 × 10⁶ (closed circles) or 9 × 10⁶ (open circles) cells were injected per mouse. The horizontal bars represent mean values.

Figure 4.

TCR specificity is important for peripheral conversion in lymphopenic hosts. (A) The converted and nonconverted TCR repertoires differ. TCRα chain sequences were obtained as described in Materials and methods. The 50 most abundant TCRs from each of the converted Foxp3⁺ and nonconverted Foxp3⁻ pooled datasets are shown, with the frequency in the indicated subset on the y axis. The TCRs are ordered from the most abundant in the Foxp3⁺ subset (left) to the most abundant in the Foxp3⁻ subset (right). (B) TCRs are preferentially skewed toward either the Foxp3⁺ or Foxp3⁻ subset. For prevalent TCRs in the pooled dataset (summed frequencies >0.5%), the percentage of each TCR in the Foxp3⁺ subset (%Foxp3⁺/%Foxp3⁺ + %Foxp3⁻) is plotted in decreasing order. Arbitrary cutoffs (vertical dashed lines) are shown for TCRs highly (>80% Foxp3⁺), poorly (<20%), or not (<1%) associated with conversion. (C) Assessment of mouse-to-mouse variability. (left) Morisita-Horn index values comparing the Foxp3⁺ and Foxp3⁻ TCR datasets within each individual recipient are shown. (right) The Foxp3⁺ TCR datasets from individual animals within the same experiment are compared in green, and the Foxp3⁻ datasets are compared in orange. There were four and two recipients for the first and second independent experiments, respectively.

Figure 5.

TCRs that facilitate peripheral conversion are found in the normal peripheral and thymic T reg cell TCR repertoires. (A) Comparison with the normal peripheral TCR repertoire. Prevalent TCRs recovered after the transfer of Foxp3⁻ cells into lymphopenic hosts that are also found in the normal peripheral dataset (Table I) at a summed frequency of >0.15% were identified (Fig. S9, available at http://www.jem.org/cgi/content/full/jem.20081359/DC1). The frequencies of these overlapping TCRs in the normal dataset are plotted. The TCRs are sorted by their ability to facilitate conversion, as designated by the vertical dashed lines (Fig. 4B). (B) Comparison with the normal thymic TCR repertoire. The frequency of the TCR in the normal thymic dataset (reference 38) is shown as in A.

Figure 6.

TCR specificity is sufficient to direct peripheral conversion in lymphopenic hosts. (A) Frequency distribution of TCRs tested. Data shown are the frequencies (percentages) of the TCRs in the datasets from thymocytes (reference 38), normal peripheral T cells (Table I), and T cells recovered after adoptive transfer into lymphopenic hosts (Fig. S6, available at http://www.jem.org/cgi/content/full/jem.20081359/DC1). (B) Retroviral manipulation of TCR specificity in peripheral T cells. Monoclonal TCli αβTCR transgenic T cells were transduced with TCRα chains, as described in Materials and methods. (top) The frequency of Foxp3⁺ cells in the transduced (Vα2⁺Vβ6⁺CD4⁺) T cell population was assessed by flow cytometry 2.5–3 wk after adoptive transfer into Tcrb^−/− mice. (bottom) The absolute number of transduced (Vα2⁺) cells recovered was calculated using the cell count obtained via either a Coulter counter (Beckman Coulter) or hemacytometer (Hausser), and the frequency of Vα2⁺Vβ6⁺CD4⁺ T cells within the live gate, as determined by flow cytometry. Data shown are from individual mice and were obtained from five independent experiments constituting at least two independent retroviral transductions per TCR. Each symbol represents data from an individual recipient (n = 4–6), and the horizontal bars represent mean values.