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Cutting Edge: A Regulatory T Cell-Dependent Novel Function of CD25 (IL-2R) Controlling Memory CD8⁺ T Cell Homeostasis¹

Division of Rheumatology and Immunology, Department of Internal Medicine, University of Virginia, Charlottesville, VA 22908

	Abstract

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A massive systemic expansion of CD8⁺ memory T (T_M) cells and a remarkable increase in circulating IL-2 were observed only in IL-2R (CD25) knockout (KO) mice but not in IL-2 KO and scurfy mice, although all three mutants lack regulatory T (Treg) cells. However, both phenotypes were suppressed by the transfer of Treg cells. The data presented indicate that Treg cell deficiency drives naive T cells to T_M cells. The lack of high-affinity IL-2R in IL-2R KO mice increases circulating IL-2 that is then preferentially used by CD8⁺ T_M cells through its abundant low-affinity IL-2R, resulting in systemic CD8⁺ T_M cell dominance. Our study demonstrates the critical control of CD8⁺ T_M cell homeostasis by a Treg cell-dependent novel function of CD25 and resolves its mechanism of action.

	Introduction

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Under normal conditions, thymic selection generates two sets of T cells: a CD4⁺CD25⁺ regulatory T (Treg)⁴ cell set that suppresses Ag-specific responses and a CD4⁺ (and CD8⁺) CD25^– single-positive T cell set that responds to Ags in the host. The conditions required for the generation, maintenance, and functions of Treg cells have been defined (1, 2). Treg cell selection involves high-affinity TCR/peptide plus MHC interaction and is skewed toward self-Ags (3). In addition, it requires the presence of the Foxp3 transcription factor (4). In the periphery, the maintenance of Treg cells requires high-affinity IL-2/IL-2R interaction (5) and TGF- (6). Thus, mice with targeted mutation of IL-2, IL-2R, IL-2R (CD122), and TGF- are defective in Treg cell expression. The requirement for high-affinity IL-2R for Treg cell maintenance puts CD25 as the major marker for Treg cells.

The primary function of Treg cells is to keep immune responses in check by inhibiting the activation of Ag-specific T cells, including autoimmune response (1, 2). However, recent studies based on mAb treatment have suggested that Treg cells also regulate CD8⁺ memory T (T_M) cell expression, but the precise mechanism remains unclear (7, 8, 9). Indeed, the loss of CD8⁺ T_M cell homeostasis control in Treg cell-deficient strains has not been established. In this study, we describe our analysis of CD8⁺ T_M cell homeostasis in three Treg cell-deficient strains: IL-2 knockout (KO), IL-2R KO, and scurfy. Interestingly, systemic CD8⁺ T_M cell dominance was observed only in IL-2R KO mice, and only IL-2R KO mice displayed very high levels of serum IL-2, a factor critical to CD8⁺ T_M cell homeostatic expansion. Significantly, both phenotypes were suppressed by CD4⁺CD25⁺ Treg cells. Our study demonstrates the critical role of CD25 and the novel molecular mechanism in the CD25-mediated regulation of CD8⁺ T_M cell homeostasis by controlling Treg cells and IL-2 expression.

	Materials and Methods

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Mice

C57BL/6 (B6) mice, B6.Il2^+/– mice B6.Il2R^+/– mice, B6.Cg-Foxp3^sf/x/J, and B6.SJL-Ptprc^aPepc^b/BoyJ (B6.CD45.1) mice were obtained from The Jackson Laboratory. B6.Il2^+/– mice and B6.Il2R^+/– mice were bred to obtain B6.Il2^–/– (IL-2 KO) and B6.Il2R^–/– (IL-2R KO) offspring, respectively. Scurfy mice were obtained by breeding female B6.Foxp3^sf/x mice with male B6 mice.

Flow cytometric analysis

Single-cell suspensions were prepared from blood and various lymphoid and nonlymphoid organs as described (10). Cells were stained with fluorescently labeled Abs and analyzed by flow cytometry. FITC-, PE-, PE-Cy5-, or biotin-conjugated mAb against CD4 (clone GK1.5), CD8 (clone 53-6.7), Thy-1.2 (clone 30H12), CD45.1 (clone A20), CD44 (clone IM7), CD62L (clone MEL-14), CD69 (clone H1.2F3), IL-2R (clone PC61), and Ly-6C were obtained from BD Biosciences. Alexa 488-conjugated streptavidin was obtained from Invitrogen Life Technologies. Intracellular Foxp3 was detected using the Foxp3 staining kit (eBioscience). The total cell number for CD4⁺ and CD8⁺ T cells in each sample was determined as described previously (10).

Purification and adoptive transfer of lymphocytes

The CD4⁺CD25⁺ Treg cells (89%) and CD4⁺CD25^– T cells (97%) were purified from lymph nodes of B6.CD45.1 mice using Treg cell isolation kit (Miltenyi Biotec). The cells (10⁶) were injected i.p. into 6-day-old IL-2R KO mice. Blood samples were collected 4 wk later and analyzed by flow cytometry.

Determination of IL-2

The levels of IL-2 in serum samples were determined using the OptEIA kit (BD Biosciences) with recombinant murine IL-2 as standard.

T cell response to IL-2

CD44⁺CD8⁺ T cells from IL-2R KO and B6.CD45.1 mice were purified using magnetic beads (Miltenyi Biotec). An equal number of cells from both strains were cocultured at 37°C in a 10% CO₂ incubator in the presence of varying concentrations (IU) of human rIL-2 (Hoffman La Roche). Cell viability and the total number of cells were determined 3 days later. The proportion of CD44⁺CD8⁺ T cells of donor mice was determined by flow cytometric analysis.

Lymph node cells of IL-2R KO mice were cultured for 3 days in the presence of human rIL-2. The proportion and the total number of CD4⁺ and CD8⁺ T cells, both before and after culture, were determined as described above.

	Results

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Role of Treg cells in CD8⁺ T cell homeostasis: comparison among Treg cell-deficient strains

Because recent studies have suggested that Treg cells regulate CD8⁺ T_M homeostasis, we compared CD8⁺ T cell levels in the lymph nodes between sex- and age-matched IL-2R KO and scurfy mice (3–4 wk old) and between IL-2R KO and IL-2 KO mice (12–15 wk old) (Fig. 1, A and B), all deficient in Treg cells. Sex- and age-matched normal B6 mice were included as control. As early as 3–4 wk, a significant increase in the proportion of CD8⁺ T cells was already evident in IL-2R KO mice but not in scurfy and B6 mice.⁵ At 12–15 wk, a massive expansion of CD8⁺ T cells was observed in IL-2R KO but not in IL-2 KO and B6 mice. The CD8⁺ T cell dominance is shown by both the high CD8⁺ to CD4⁺ T cell ratio and the high absolute number of CD8⁺ T cells in all secondary lymphoid organs and inflamed nonlymphoid organs (Table I).

View larger version (48K): [in this window] [in a new window]   FIGURE 1. CD8+ T cell dominance was observed in IL-2R KO mice. A, Lymph node cells from 3- to 4-wk-old male B6, IL-2R KO, and scurfy mice were stained with FITC-anti-CD8 and PE-anti-CD4 mAb. CD8+ T cell dominance was observed in IL-2R KO but not in B6 and scurfy mice. B, CD8+ T cell dominance was observed in the lymph nodes of 12- to 15-wk-old IL-2R KO mice but not in B6 and IL-2 KO mice. C, The majority of the CD8+ T cells from IL-2R KO but not B6 mice displayed the TM phenotype. Lymph node cells were used and analyzed as described in Materials and Methods. D, The majority of the CD8+ T cells of scurfy and IL-2 KO mice also expressed TM phenotype. E, Essentially all CD4+ T cells of IL-2R KO mice expressed CD44high. Approximately half of them expressed TM phenotype and half were recently activated based on CD69 expression. Data presented are representative of three experiments.

View this table: [in this window] [in a new window]   Table I. Absolute numbers of CD8+ and CD4+ T cells in various organs of B6 and IL-2R KO micea

The great majority of CD4⁺ T cells in IL-2R KO mice also expressed a CD44^highCD62L^low T_M cell phenotype and approximately half of the CD4⁺ T cells were CD69^high, indicating that they have undergone T cell activation and differentiation like the CD8⁺ T cells; yet, the dominance of CD8⁺ T_M cell but not CD4⁺ T_M cells was observed (Fig. 1E).

IL-2 accumulates in IL-2R KO mice, induces homeostatic proliferation of CD8⁺ T_M cells, and offers a proliferation advantage over CD4⁺ T_M cells

We hypothesize that IL-2, produced by the constant autoimmune stimulation in Treg cell-deficient mice, is underused in IL-2R KO mice due to the absence of high-affinity IL-2R. The IL-2 accumulated then becomes available for the low-affinity IL-2R, which is more abundantly expressed on the CD8⁺ T_M cells than on the CD4⁺ T_M cells for expansion (11). This hypothesis satisfactorily explains why systemic CD8⁺ T_M cell dominance was observed in IL-2R KO mice but not in IL-2 KO (for lacking IL-2) and scurfy mice (for having IL-2R). Indeed, IL-2R KO mice (5 wk old) expressed high levels of serum IL-2 (160 pg/ml), whereas it was extremely low (3 pg/ml) in scurfy mice (4 wk old) and undetectable in B6 mice (5 wk old) (Fig. 2A).

View larger version (36K): [in this window] [in a new window]   FIGURE 2. IL-2 accumulates in high concentrations in IL-2R KO mice, giving CD8+ TM cells a proliferation advantage over CD4+ TM cells. A, Serum IL-2 levels were determined by ELISA (standard curve in inset). IL-2R KO mice but not IL-2 KO, scurfy, and B6 mice show high serum IL-2 levels (n = 3). B, Purified CD44+CD8+ cells from IL-2R KO and B6.CD45.1 mice were cocultured in various concentrations (IU) of human rIL-2. Viable CD8+ T cells were counted 3 days later using allele-specific mAbs. The data are presented as fold changes in viable cells. A representative of three experiments is shown. C, Lymph node cells from IL-2R KO mice were cultured for 3 days in the presence of human rIL-2 (1000 IU). Viable cells were counted and stained for CD8+ and CD4+ T cells. The total number and percentage of CD8+ and CD4+ T cells were indicated. A representative of three experiments is shown.

We also cultured lymph node cells of IL-2R KO mice in the presence of rIL-2 (Fig. 2C). At 3 days after culture, the proportion of CD8⁺ T_M cells increased from 56 to 81% whereas CD4⁺ T_M cells dropped from 15 to 5%. The total number of CD8⁺ T_M cells increased from 1.6 x 10⁵ to 6.5 x 10⁵ (4-fold), whereas essentially no expansion was observed for CD4⁺ T_M cells (Fig. 2C).

Treg cells inhibit the CD8⁺ T_M cell dominance and IL-2 accumulation in IL-2R KO mice

To determine whether and how Treg cell deficiency is required for CD8⁺ T_M cell dominance in IL-2R KO mice, we transferred purified CD4⁺CD25⁺ Treg cells i.p. (10⁶ cells; 97% CD4⁺ and 92% CD25⁺) of B6.CD45.1 mice into 6-day-old IL-2R KO neonates. The expansion of CD8⁺ T cells was followed 4 wk later by examining blood samples. Dominance of CD8⁺ T cells was observed in untreated mice but not in mice transferred with CD4⁺CD25⁺ Treg cells (Fig. 3A). In addition, the number of total lymphocytes per milliliter of blood was reduced to 50–66% of untreated mice. Moreover, the proportions of CD8⁺ T_N to CD8⁺ T_M cells (Fig. 3B, top) and CD4⁺ T_N to CD4⁺ T_M cells (Fig. 3B, bottom) based on CD44 expression became similar to that of B6 control. The data indicate that Treg cells prevent the formation of both CD4⁺ T_M and CD8⁺ T_M cells mainly by inhibiting T_N cell activation, although they may also act at least in part as an IL-2 sink. Along with the inhibition of T_N cell activation, serum IL-2 became undetectable and CD8⁺ T_M cell dominance was prevented (Fig. 3C). Finally, CD4⁺CD25⁺ and CD4⁺Foxp3⁺ Treg cells (2% of CD4⁺ T cells) were detected in the IL-2R KO recipients of CD4⁺CD25⁺ Treg cells (Fig. 3D).

View larger version (35K): [in this window] [in a new window]   FIGURE 3. CD4+CD25+ Treg cells suppress CD8+ TM cell dominance and serum IL-2 expression in IL-2R KO mice. A, Blood lymphocytes from B6 mice, IL-2R KO mice, and IL-2R KO recipients of CD4+CD25+ Treg cells were stained with the indicated mAbs at 4 wk after transfer. Treg cells inhibited CD8+ T cell dominance in IL-2R KO mice. A representative of three experiments is shown. B, Gated CD8+ (top) and CD4+ (bottom) T cells were analyzed with FITC-anti-CD44 and PE-anti-CD62L mAbs. For both T cell subsets, Treg cells maintained the TN phenotype and prevented TM cell expression. C, Serum IL-2 expression was inhibited in the IL-2R KO mice by Treg cells. D, Gated CD4+ T cells were stained with anti-CD25 and anti-Foxp3 mAbs. CD4+CD25+Foxp3+ cells were present in B6 mice and IL-2R KO mice that had received CD4+CD25+ T cells from B6 mice. Isotype control stain was <0.5% of CD4+ T cells in all cases.

The IL-2R KO recipients of CD4⁺CD25⁺ Treg cells continue to maintain a normal lymphocyte subset phenotype (>12 wk) without any clinical signs of autoimmune diseases such as anemia, body weight loss, and colitis (10). Interestingly, they become fertile and all their offspring are of the IL2R^–/– genotype.

	Discussion

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To our knowledge, IL-2R KO is the first and only strain that spontaneously loses CD8⁺ T_M cell homeostatic control and develops a massive CD8⁺ T_M cell dominance systemically. A major point of the present study is that we have resolved the molecular and cellular mechanisms of this process. Treg cell deficiency allows continuous activation of T_N and T_M cells to produce IL-2 as well as the activation of T_N cells to differentiate into T_M cells. Without the high-affinity IL-2R, IL-2 is underutilized and accumulated. Accumulated IL-2 is then preferentially used by the CD8⁺ T_M cells that abundantly express the low affinity IL-2R, resulting in the massive and systemic dominance of CD8⁺ T_M cells.

A relevant question is why the targeted mutation of IL-2R does not induce CD4⁺ T cell dominance. The CD4⁺ T cells in IL-2R KO mice consist mainly of T_M cells and recently activated T cells (Fig. 1E). However, CD4⁺ T_M cells express significantly less IL-2R than CD8⁺ T_M cells and the recently activated CD4⁺ T cells of IL-2R KO mice lack the high-affinity IL-2R (11), giving CD8⁺ T_M cells a proliferation advantage in response to IL-2 (Fig. 2C). Indeed, the total number of CD4⁺ T cells in IL-2R KO mice is not increased in comparison with B6 mice. By contrast, the total number of CD8⁺ T cells in the various organs of IL-2R KO mice is 2–20 times that of B6 mice (Table I). This disadvantage in the proliferation of CD4⁺ T cells contributed to the systemic CD8⁺ T_M cell dominance observed in IL-2R KO mice.

IL-2 and IL-15 are major regulators for CD8⁺ T_M cell homeostasis (13). However, only IL-2 accumulation was observed in IL-2R KO mice, whereas IL-15 and IL-15R mRNA expression levels were comparable among IL-R KO, IL-2 KO, and scurfy mice (data not shown). In addition, transfer of CD4⁺CD25⁺ Treg cells into IL-2R KO neonates inhibited IL-2 accumulation and prevented CD8⁺ T_M cell dominance, suggesting strongly that IL-2 accumulation is necessary for CD8⁺ T_M cell dominance. A recent study suggests that IL-2 signals during priming are required for the secondary expansion of CD8⁺ T_M cells and that this priming event is defective in IL-2R KO mice (14). Our data indicate that CD8⁺ T_M cells of IL-2R KO mice are capable of homeostatic proliferation and even dominant expansion. Whether this priming defect was overridden in the absence of Treg cells or in the abundant presence of IL-2 remains to be determined.

The results from the present investigation have important practical implications in antiviral and antitumor immunity. Although Treg cells control the magnitude of recall reaction in antiviral and antitumor responses (7, 8, 9), our data obtained from IL-2 KO and scurfy mice suggests that deleting Treg cells alone has little or only mild and transient impact on the overall CD8⁺ T_M cell homeostasis and that a key second factor, i.e., IL-2 consumption linked to IL-2R, is critical to the CD8⁺ T_M cell expansion. In normal mice, Ab-mediated deletion of Treg cells with a concurrent antigenic challenge may transiently increase IL-2 production and Ag-specific CD8⁺ T_M cells expansion, but the competition for IL-2 by the high-affinity IL-2R on Ag-activated CD4⁺ T cells and/or Treg cells may effectively dampen this process, making it hard to achieve long and sustained Ag-specific CD8⁺ T_M cell presence. Nevertheless, this mechanism for CD8⁺ T_M cell expansion may be exploited experimentally. In this respect, treatment of mice with a mixture of anti-IL-2 and anti-CD25 mAbs has been shown to effectively expand CD8⁺ T_M cells (15). This treatment has the potential to deplete Treg cells, increase IL-2 levels, block high affinity IL-2R, and efficiently present an anti-IL-2/IL-2 complex to CD8⁺ T_M cells for expansion (16). It will be of great significance if these approaches, coupled with antigenic challenge, could induce a sustained increase in Ag-specific CD8⁺ T_M cells.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grants AI-036938, DE-017579 and AR-051203 (to S.-T.J.), AR-045222, AR-047988 and AR-049449 (to S.M.F.), HL-70065 (to S.-S.J.S.), AR-051391 (to U.S.D.), and DK-059850 (to W.N.J.).

² S.M.F. and S.-T.J. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. Shyr-Te Ju, Division of Rheumatology and Immunology, Department of Internal Medicine, P.O. Box 800412, Old Medical School Building, Room 5777, University of Virginia, Charlottesville, VA 22908-0412. E-mail address: sj8r{at}virginia.edu

⁴ Abbreviations used in this paper: Treg, regulatory T; KO, knockout; T_M, memory T; T_N, naive T.

⁵ Both CD8⁺ and CD4⁺ T cells were increased in scurfy mice but their ratios were maintained in a normal range.

Received for publication September 27, 2006. Accepted for publication November 29, 2006.

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