Foxp3+ CD25- CD4 T cells constitute a reservoir of committed regulatory cells that regain CD25 expression upon homeostatic expansion

Abstract

Expression of the IL-2 receptor alpha chain (CD25) by peripheral CD4 T cells follows cellular activation. However, CD25 expression by CD4 cells is widely used as a marker to identify regulatory T cells (T(R)), although cells with regulatory properties are also found in the CD4+CD25- subset. By using in vivo functional assays and Foxp3 expression as a faithful marker of T(R) differentiation, we have evaluated the requirements for CD25 expression by peripheral T(R). We first show that in vivo depletion of CD25+ cells prevents the development of spontaneous encephalomyelitis in recombination-activating gene (RAG)-deficient anti-myelin basic protein T cell antigen receptor (TCR) transgenic mice, and allows disease induction in otherwise healthy RAG-competent transgenic mice. Similar treatment in normal thymectomized animals is followed by the fast recovery of a normal number of CD25+ T(R). Consistently, Foxp3-expressing T(R) encompassed in the CD25- cell population convert to CD25+ after homeostatic expansion and are selectable by IL-2 in vitro. Surface expression of CD25 on T(R) is controlled by the activity of conventional CD4 cells and is fully labile because it can be lost and regained without affecting the functional potential of the cells. These findings reveal that Foxp3-expressing CD25- cells constitute a peripheral reservoir of differentiated T(R), recruited to the CD25+ pool upon homeostatic expansion and/or activation. This analysis, together with the notion that physiological commitment of T(R) takes place exclusively in the thymus should help for the interpretation of experiments assessing peripheral T(R) differentiation from naive CD4 T cells, defined as CD25-.

Fig. 1.

Depletion of both activated and regulatory T cells by anti-CD25 antibody in vivo. T/R^– and T/R⁺ mice were injected with 200 μg of anti-CD25 mAb. (A) One-month-old T/R^– animals (n = 7) received five weekly injections and were followed for 4.5 months. The percentage of mice that developed EAE (score ≥2) is represented. (B) Sick T/R^– mice (EAE score = 2–3) received a continuous weekly treatment (n = 5). As control, a group of T/R^– mice was left untreated (n = 20). Plotted is the percentage of mice alive at 5 months of age and the EAE score of the survivors. Group comparison for the mean EAE score was statistically significant using Student's t test (C) Adult T/R⁺ mice were treated once with the anti-CD25 mAb (day 0) either alone or together with 200 ng of pertussis toxin (day 0 and 2). Control T/R⁺ mice were left untreated. EAE level was scored weekly, and plotted is the percentage of mice that developed EAE (score ≥2).

Fig. 2.

CD4⁺CD25⁺ T_R recover normal levels after in vivo depletion of Tx mice. Adult-Tx BALB/c(n = 7) mice were injected i.p. with 200 μg of anti-CD25 mAb or rat IgG (controls) twice at 1-week intervals. (A) Peripheral blood lymphocytes were analyzed for the frequency of CD25⁺ among CD4⁺ cells at different days after the injection. Shown is the percentage of CD25⁺ among CD4⁺ cells in treated animals relative to control animals (n = 7). (B) CD4⁺CD25⁺ cells were sort-purified from LNs of either CD25⁺-depleted (CD25⁺ Tx-depleted) or from rat IgG-injected (CD25⁺ Tx control) mice 3 months after depletion and tested for their capacity to suppress IL-2 production by stimulated CD4⁺CD25^– cells. Primary cultures consisted in 2.5 × 10³ CD4⁺CD25^– cells isolated from normal mice, stimulated alone or in the presence of different numbers of CD25⁺ cells isolated from Tx-depleted or Tx control mice. As a negative control, CD25^– cells (CD25^–) were also tested. Shown is the percentage of inhibition [(cpm in control) – (cpm in experiment)/cpm in control] is plotted versus the ratio of the population tested/CD4⁺CD25^– cell number at the origin of the primary culture.

Fig. 3.

CD45RB^lowCD25^– cells that acquire in vivo CD25 expression display a regulatory phenotype. RAG2^–/– mice received 3 × 10⁵ CD4⁺CD25^– cells either Thy1.2⁺CD45RB^low or Thy1.1⁺CD45RB^high (Single), or an equal number of both cell subsets (Co), and were analyzed 12 days later. For the sequential transfer (Seq), mice that received 3 × 10⁵ Thy1.1⁺CD45RB^high cells at day 0 were injected with 3 × 10⁵ Thy1.2⁺CD4⁺CD25^– cells at day 12 and analyzed at day 24. (A) FACS analysis of splenocytes. Shown is the percentage of CD25⁺ cells inside a gate either CD4⁺Thy1.2⁺ (originally CD25^–45RB^lo) or CD4⁺ Thy1.1⁺ (originally CD25^–45RB^lo). (B) Foxp3 mRNA levels determined by real-time PCR. (Left) cDNA prepared from freshly isolated (Fresh) CD4⁺cells, either CD25⁺ (25⁺), CD25^–CD45RB^high (25^–45^hi), or CD25^–CD45RB^low (25^–45^low) served as controls. (Right) cDNA was prepared from CD4⁺CD25⁺ or CD4⁺CD25^– cells sort-purified from pooled spleen and LN of either of the single-transfer recipients (Post-Transfer). (C) IL-2 production upon TCR triggering. Thy1.2⁺CD4⁺ cells (originally CD25^–CD45RB^low) were sort-purified from co-transferred recipients and fractionated as CD25⁺ or CD25^–. Shown is the proliferation of CTLL-2 cells exposed to supernatants of primary cultures that contained 2 × 10³ of either cell subset. Control was CD4⁺CD25⁺ cells purified from normal C57BL/6 animals. The background (CTLL-2) and the maximum proliferation (CTLL-2 IL-2) are those of CTLL-2 cells maintained in medium either alone or containing saturating amounts of IL-2. (D) Suppression of IL-2 production. As in C except that primary cultures consisted in 2.5 × 10³ CD4⁺CD25^– cells isolated from normal mice, stimulated alone (Targets) or in the presence of 1.25 × 10³ CD4⁺ cells (filled bars) either CD25⁺ or CD25^– purified as in C.

Fig. 4.

Activation of CD45RB^lowCD25^– cells in vitro reveals their regulatory properties. (A) Sorted CD45RB^highCD25^– and CD45RB^lowCD25^– cells (25^–45^hi and 25^–45^lo, respectively) maintained for 6 days in culture containing anti-CD3 mAb and IL-2 (IL-2) were washed and cultured with the same number of untreated CD4⁺CD25^– cells, and cultured for another 3 days in the presence of anti-CD3 mAb and antigen-presenting cells. Freshly isolated (Fresh) CD4⁺CD25⁺ (25⁺), CD45RB^lowCD25^– (25^–45RB^lo), and CD45RB^highCD25^– (25^–45^hi) cells were used as controls. (B) Sorted CD45RB^lowCD25^– and CD45RB^highCD25^– cells (25^–45RB^lo and 25^–45RB^hi, respectively) were stimulated with plate-bound anti-CD3 mAb according to Materials and Methods. The evaluation of their suppressor function was performed by adding them at various ratios to untreated CD4⁺CD25^– cells as in A. Freshly isolated CD4⁺CD25⁺ cells (25⁺fresh) were used as control. The percentage of inhibition of naive T cell proliferation is plotted versus the ratio of the population tested/CD4⁺CD25^– cell number at the origin of the culture, as in Fig. 2B.

Fig. 5.

Functional CD4⁺CD25⁺ cells can lose and regain CD25 expression. RAG2^–/– mice received CD4 cells either Thy1.2⁺CD25⁺ or Thy1.1⁺CD25^– alone (Single), together in same number (Co), at a 100:1 ratio (1%), or sequentially (Seq) and were analyzed 12 days after the last transfer. (A) Efficient control of Thy1.1⁺ expansion by Thy1.2⁺ cells. The number of Thy1.1⁺ (Left) and Thy1.2⁺ (Right) cells recovered from the spleen is shown for each animal. (B) Loss of CD25 expression upon adoptive transfer. CD4⁺ cells from mice recipient of 3 × 10⁵ Thy1.2⁺CD25⁺ cells transferred alone (Single) or together with 3 × 10³ Thy1.1⁺CD25^– cells are analyzed for CD25 and Thy1.1 expression. (C) In cotransfer experiments, CD4⁺CD25^– cells maintain and restore CD25 expression on Thy1.2⁺ CD4⁺CD25⁺ cells. Shown is the CD25 expression on Thy1.2⁺ cells recovered from mice that received an equal number of Thy1.2⁺CD4⁺CD25⁺ and Thy1.1⁺CD4⁺CD25^– cells either at the same time (Co) or 12 days apart (Seq).

Fig. 6.

CD25 is an activation marker of both T_R and conventional CD4 cell subsets. In the thymus, conventional CD4 cells undergo positive selection, and these CD4⁺Foxp3^–cells exit the thymus as naive CD45RB^highCD25^–. Once in the periphery, if they receive activating signals, they acquire an activated phenotype characterized by a low level of CD45RB and high level of CD25 expression. Exit from the environment where the activation signals are provided associates with the loss of CD25 expression but maintenance of an antigen experienced CD45RB^low phenotype. Thymic T_R commitment and differentiation require interaction with activating ligands (gray area), and T_R exit the thymus with a CD25⁺CD45RB^low phenotype. Once in the periphery, Foxp3 expression is maintained. If specific activating signals are absent or below a certain threshold, CD25 expression is lost but CD45RB expression remains low. Reexposure to activation signals reverts this phenotype, and T_R may reacquire a CD25 surface expression.