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Keywords:

  • CD4+ CD25+ Treg;
  • L-selectin;
  • suppression

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

CD4+ CD25+ regulatory T cells (Treg) are potent suppressors, and play important roles in autoimmunity and transplantation. Recent reports suggest that CD4+ CD25+ Treg are not a homogeneous cell population, but the differences in phenotype, function, and mechanisms among different subsets are unknown. Here, we demonstrate CD4+ CD25+ Treg cells can be divided into subsets according to cell-surface expression of CD62L. While both subsets express foxp3 and are anergic, the CD62L+ population is more potent on a per cell basis, and proliferates and maintains suppressive function far better than the CD62L– population and unseparated CD4+ CD25+ Treg. The CD62L+ population preferentially migrates to CCL19, MCP-1 and FTY720. Both CD62L+ and CD62L– subsets prevent the development of autoimmune gastritis and colitis induced by CD4+ CD25–CD45RBhigh cells in severe combined immunodeficiency (SCID) mice. Overall, these results suggest CD4+ CD25+ Treg are not a homogenous cell population, but can be divided into at least two subsets according to CD62L expression. The CD62L+ subset is a more potent suppressor than the CD62L– population or unfractionated CD4+ CD25+ Treg cells, can be expanded far more easily in culture, and is more responsive to chemokine-driven migration to secondary lymphoid organs. These properties may have significant implications for the clinical manipulation of the CD4+ CD25+ CD62L+ cells.

Tolerance is a feature of the immune system that is intimately related to discrimination between self and nonself. Clonal deletion of self-reactive T cells in the thymus is a primary tolerance mechanism, while induction of unresponsiveness or anergy in post thymic T cells may be required for the establishment of peripheral tolerance. Recent data show that Treg may play a critical role in the induction and maintenance of immune tolerance (1–5). Various types of Treg cells have been described, including Tr1 cells, Th3 cells, CD4+ CD25+ Treg cells, and others (6). CD4+ CD25+ Treg cells were first described by Sakaguchi (7). Five to 10% of CD4+ T cells constitutively expressed the α chain of IL-2-receptor (CD25) and were crucial for the control of autoreactive T cells in vivo. These cells are generated in the thymus of naïve mice, perhaps via altered negative selection by self-antigen (8). Subsequent in vitro studies showed that CD4+ CD25+ cells are typically anergic, unresponsive to TCR stimulation alone, but proliferate after addition of exogenous IL-2 (9). These cells suppress the proliferation of other CD4+ and CD8+ T cells in an antigen-nonspecific manner via a cell contact-dependent, cytokine-independent mechanism (10,11). A similar population of CD4+ CD25+ Treg cells has been defined in humans, with identical phenotypic and functional properties (12–15).

CD4+ CD25+ Treg are potent suppressors in a number of in vivo models of autoimmunity, including gastritis, thyroiditis, inflammatory bowel disease and insulin-dependent diabetes (16–19). Regulation of disease activity in vivo seems to require both suppressor cytokines (such as IL-4, IL-10 and TGF-β) and cell contact-dependent mechanisms. Recent reports addressing the role of CD4+ CD25+ Treg in allogeneic responses or transplantation show that these cells also suppress the proliferative responses of CD4+ CD25– T cells to alloantigenic stimulation in vitro (1,2), and are required for ex vivo induction of tolerance to alloantigen via costimulatory blockade (3). Alloantigen-specific CD4+ CD25+ Treg can maintain tolerance in vivo, and require IL-10 and CTLA4 for their functional activity (4,5). CD4+ CD25+ Treg can significantly delay or even prevent GVHD (20,21), and the protective effect is partially dependent on IL-10 (22). These diverse findings suggest heterogeneity in the CD4+ CD25+ Treg population or at least heterogeneity in suppressive mechanisms, depending on the experimental models and protocols. Many additional issues require further clarification, such as how many subsets of CD4+ CD25+ Treg exist, what are the functions of these subsets, what are their mechanisms of suppression, what is their relationship to Tr1 and Th3, how to expand these subsets to retain their suppressive function, and how to generate antigen-specific Treg cells for therapeutic purposes.

l-selectin (CD62L) is a member of the selectin adhesion molecule family, and is required for lymphocyte homing to peripheral lymph nodes via binding to sialylated oligosaccharide determinants on high endothelial venules in peripheral lymph nodes (23,24). CD62L is constitutively expressed on most thymocytes and leukocytes, including B and T lymphocytes, neutrophils, monocytes, and eosinophils (25–26). Unlike other leukocyte adhesion proteins, CD62L is rapidly down-regulated from the cell surface upon cell activation (27). The level of CD62L expression, along with other markers, distinguishes naïve T cells from most effector/memory T cells (28). Recent investigations show that memory T cells can be further divided into functionally distinct subsets, memory effector T cells and central memory T cells, according to the expression of CD62L and CCR7 on the cell surface. Central memory T cells express high levels of CD62L and CCR7, home to and proliferate within secondary lymphoid organs, and differentiate into CCR7– effector cells upon secondary stimulation. In contrast, memory effector T cells express low levels of CD62L and CCR7, and display immediate effector function (29–32).

As Treg are CD25+, this argues they are antigen experienced. Conversely, the fact that many Treg are CD62+ and CD45RBlow suggest that they have not undergone full activation, or have reverted back to a partially naïve phenotype. CD62L expression has also served to define certain CD4+ regulatory T-cell subpopulations (33–35). In prediabetic nonobese diabetic (NOD) mice, CD4+ splenocytes prevent transfer of diabetes to immunodeficient NOD recipients; the regulatory CD4+ T cells express high levels of CD62L, in contrast to diabetogenic T cells that reside within the CD62L– population. More recent studies demonstrate that the regulatory T-cell population in prediabetic NOD mice is CD4+ CD25+ CD62+ (36). Given these findings, we hypothesized that CD4+ CD25+ Treg cells could be divided into functionally distinct subsets according to the expression of CD62L. We found that CD4+ CD25+ Treg cells are indeed not a homogeneous cell population; CD62L is highly expressed on the surface of 50–60% of the cells. CD62+ and CD62L– populations are similar in that they are both anergic and suppressive and can be initially expanded in vitro without loss of the suppressive activity. However, there are substantial differences in their receptor expression, suppressive potency, suppressive mechanisms, and cytokine production. Of particular importance is a difference in their proliferative capacity and migration to chemokines. These results are important for defining the mechanisms of cellular activity of distinct Treg subsets.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

Mice

BALB/c (H-2d) and C57BL/6 (H-2b) female mice (8–10 weeks of age) were purchased from Harlan Sprague-Dawley (Indianapolis, IN). C57BL/6 CD62L-/- mice, C57BL/6 IL-2-/- mice, and C57BL/6 IL-10-/- mice were purchased from The Jackson Laboratories (Bar Harbor, ME). CRF2-4-/- mice (37) were a kind gift of Dr Aguet (Genentech, South San Francisco, CA). All mice were housed in a specific pathogen-free facility in microisolator cages. All experiments were performed with age- and sex-matched mice in accordance with IACUC-approved criteria.

Antibodies and reagents

The 145–2C11 hamster antimurine CD3 hybridoma was a gift of Dr J. A. Bluestone (UCSF, CA). CyChrome-anti-CD4 (RM4-5), FITC-anti-CD25 (7D4), APC-anti-CD62L (l-selectin), PE-anti-CD4 (RM4-5), PE-anti-CD62L (l-selectin), PE-anti-CTLA4 (UC10–4F10-11), PE-anti-IL-10R, PE-anti-CD44 (Pgp-1), PE-anti-CD45RB (16 A), PE-anti-CD69 (H1.2F3), PE-anti-CD28 (37.51), PE-anti-CD80 (B7-1, 16–10A1), and PE-anti-CD86 (B7-2, GL1) were purchased from BD PharMingen (San Diego, CA). Purified anti-IL-10, anti-IL-10R, anti-TGF-β, and anti-CTLA4 were purchased from BD PharMingen (San Diego, CA).

Murine CCL19, MCP-1, CCL1, CCL5, CCL22 and CXCL9 were purchased from R & D System (Minneapolis, MN). FTY720 (38) was a kind gift from Dr V. Brinkmann (Novartis Pharma AG, Basel, SW).

Lymphocyte preparation and FACS analysis

Mice were sacrificed, spleens removed and gently dissociated into single-cell suspensions, and RBCs were removed by tris-NH4Cl lysis. Cells were stained with four-color fluorescence including CyChrome-anti-CD4, FITC-anti-CD25, APC-anti-CD62L, and PE-anti-IL-10R1, anti-CD44, anti-CD45RB, anti-CD69, anti-CD28, anti-CD80, or anti-CD86. Intracellular expression of CTLA-4 was determined by staining with PE-anti-CTLA4 using Cytofix/Cytoperm kit (BD PharMingen). Data were analyzed with a FACSCalibur flow cytometer using CELLQuest™ software (BD Biosciences, Mountain View, CA).

Purification of cell subsets

BALB/c or C57BL/6 CD4+ T-cell subsets were isolated from splenocyte suspensions using the Mouse T-cell CD4 Subset Column Kit (R & D System) according to the manufacturer's protocol, and the purity of the enriched CD4+ cells ranged between 85 and 90%. Afterwards, the enriched CD4+ cells were stained with PE-anti-CD4 and FITC-anti-CD25, or FITC-anti-CD25 and PE-anti-CD62L, and sorted into CD4+ CD25+ cells and CD4+ CD25– cells, or CD25+ CD62+ cells and CD25+ CD62L– cells using MoFlo (DakoCytomation, Fort Collins, CO). For IL-10R1 subsets, the enriched CD4+ cells were stained with FITC-anti-CD25 and PE-anti-IL-10R1, and sorted into CD25+ IL-10R1+ cells and CD25+ IL-10R1– cells. The purity of the sorted cells ranged between 94 and 98%.

In vitro expansion of T cells

Two × 104 CD4+ CD25– cells, CD4+ CD25+ cells, CD4-enriched CD25+ CD62+ cells, or CD4-enriched CD25+ CD62L– cells from BALB/c mice were plated in 96-well flat-bottom plates (Costar) in a final volume of 200 μL of complete RPMI medium (RPMI 1640 supplemented with 10% fetal calf serum, 1 mM sodium pyruvate, 2 mM l-glutamine, 100 IU/mL penicillin, 100 μg/mL streptomycin, 1 × nonessential amino acids, and 2 × 10−5M 2-mercaptoethanol) in the presence of 1 μg/mL of anti-CD3  mAb and 50 U/mL of IL-2, with 1 × 105 irradiated (500 Rads) syngeneic splenocytes as feeder cells that had been depleted of T cells by negative selection using Mouse pan T Dynabeads according to the manufacturer's protocol (Dynal ASA, Oslo, Norway). The medium was changed, and anti-CD3 and IL-2 were added every 3–4 days, and fresh feeder cells were added every 10–14 days. Cell numbers were counted every 3–4 days, culture supernatants were collected, and the suppressive function of these cells was tested on days 4, 7, 10, 14, 20, and 30.

ELISA

Cytokine production in the culture supernatants was tested by ELISA. For IL-4, IL-10, and IFN-γ, cytokines were measured by BD OptEIA™ ELISA kit (BD PharMingen) according to the manufacturer's protocol. For TGF-β1 assay, samples were acidified by addition of 1 N HCl for 60 min at 4 °C, neutralized by 1 N NaOH, and the TGF-β1 concentration was measured by two-antibody capture ELISA as recommended by the manufacturer (BD Pharmingen) using rat antimouse, human, and pig TGF-β1 capture mAb and biotinylated rat antimouse, human, and pig TGF-β1-detecting mAb. Optical densities were measured at 405 nm using an FL600 microplate ELISA reader (Bio-Tek Inc, Windoski, VT).

Proliferation assay

To analyze proliferation in response to anti-CD3 activation, 2 × 104 freshly isolated CD4+ CD25– cells, CD4+ CD25+ cells, CD4-enriched CD25+ CD62+ cells, or CD4-enriched CD25+ CD62L– cells from BALB/c mice with 1 × 105 irradiated (500 Rads) syngeneic T-depleted splenocytes were plated in triplicated in 96-well round-bottomed plates (Costar) in a final volume of 200 μL RPMI 1640–10% FBS, in the presence of 1 μg/mL of anti-CD3 mAbs. Wells were pulsed with 1 μCi [3H]thymidine (PerkinElmer, Gaithersburg, MD) for the last 18 h of the 72-h culture, harvested onto filter membranes using a Wallac harvester (Tomtec, Hamden, CT), and incorporated [3H]thymidine measured with a Wallac Betaplate counter (PerkinElmer).

Suppression assay

To test the suppressive function of CD4+ CD25+ CD62+ and CD4+ CD25+ CD62L– cells to anti-CD3 stimulation, 2 × 104 freshly isolated CD4+ CD25– cells from BALB/c mice were stimulated in triplicate with 1 × 105 irradiated (500 Rads) syngeneic T-depleted splenocytes in the presence of 1 μg/mL anti-CD3 mAbs. Graded numbers (2.5 × 103, 5 × 103, 1 × 104, or 2 × 104) of freshly isolated or cultured CD4+ CD25– cells, CD4+ CD25+ cells, CD4+ CD25+ CD62+ cells, or CD4+ CD25+ CD62L– cells from BALB/c mice were used as Treg and added to the cultures. Cells were cocultured in a final volume of 200 μL of complete medium in 96-well round-bottomed plates for 3 days. Ten μg/mL control IgG, anti-IL-10, anti-IL-10R, anti-TGF-β, and/or anti-CTLA4 mAbs were added as indicated. Wells were pulsed with 1μ of Ci [3H]thymidine 18 h before harvesting.

To test the suppressive function of CD4+ CD25+ CD62+ and CD4+ CD25+ CD62L– cells to alloantigen stimulation, 1 × 105 freshly isolated CD4+ CD25– cells from BALB/c mice were stimulated in triplicate with 1 × 105 irradiated (500 Rads) allogeneic splenocytes from C57BL/6 mice. Graded numbers (1.25 × 104, 2.5 × 104, 5 × 104, or 1 × 105) of freshly isolated or cultured CD4+ CD25+ cells, CD4+ CD25+ CD62+ cells, or CD4+ CD25+ CD62L– cells from BALB/c mice were used as Treg and added to the cultures. Cells were cocultured in a final volume of 200 μL of complete medium in 96-well round-bottomed plates for 5 days. Wells were pulsed with 1μ Ci [3H]thymidine 18 h before harvesting.

Transwell assay

Transwell experiments were performed in 5-μm pore size, polycarbonate, 24-well tissue culture plates (Costar, Cambridge, MA). 2 × 105 freshly isolated CD4+ CD25– cells from BALB/c mice were stimulated in triplicate with 5 × 105 irradiated (500 Rads) syngeneic T-depleted splenocytes in the presence of 1 μg/mL of anti-CD3 mAbs in the lower wells of the plates. 1 × 105 CD4+ CD25– cells, CD4+ CD25+ cells, CD4+ CD25+ CD62+ cells, or CD4+ CD25+ CD62L– cells from BALB/c mice were either added directly to the lower wells or placed in the upper wells of the plates with 2.5 × 105 irradiated (500 Rads) syngeneic T-depleted splenocytes. Ten μg/mL anti-CTLA4 mAbs were added to the lower wells as indicated. Wells were pulsed with 5μ Ci [3H]thymidine 18 h before harvesting.

Chemotaxis assay

T-cell subsets were isolated from splenocyte suspensions using the Mouse T-cell Enrichment Column Kit (R & D System) according to the manufacturer's protocol, and the purity of the enriched T cells ranged between 85 and 90%. 5 × 105 T cells were incubated with or without 0.5 μg/mL FTY720 at 37 °C for 15 min, washed, resuspended in RPMI-1640 containing 0.5% bovine serum albumin, and added in a volume of 100 μL to the upper wells of a 24-well transwell plate. Lower wells contained 0.5 μg/mL of CCL19, MCP-1, CCL5, or CXCL9, or 0.1 μg/mL of CCL1 or CCL22 in 600 μL of RPMI-1640/0.5% BSA. Control wells received medium without chemoattractant. The number of T cells migrating to the lower wells following 2 h of incubation at 37 °C was determined. The migrated T cells were then stained with CyChrome-anti-CD4, FITC-anti-CD25, and PE-anti-CD62L, and analyzed with a FACSCalibur flow cytometer. The percentage of each cell population chemotaxing was determined by dividing the number of cells of that subset migrating to lower wells by the number of cells of that subset in the starting population before migration.

In vivo migration to FTY720

FTY720 was dissolved in distilled water, and 1 mg/kg administered orally by gavage in a 200-μL volume. Control mice received water alone. Eighteen hours later, mice were sacrificed, spleen, and the thymus and lymph node were removed and made into single-cell suspensions. Peripheral blood was aspirated, and red blood cells lysed with PharM Lyse (BD PharMingen). Viable cells were counted by trypan blue exclusion, and stained with CyChrome-anti-CD4, FITC-anti-CD25, and PE-anti-CD62L. Data were analyzed with a FACSCalibur flow cytometer using CELLQuest™ software.

Real-time RT-PCR for foxp3 expression

Total cellular RNA was extracted from 5 × 105 sorted cells using Trizol reagent (Invitrogen, Carlsbad, CA) digested with RNase-free DNase I (Invitrogen), and reverse-transcribed into cDNA using Sensiscript RT Kit (Qiagen, Valencia, CA) and random primers (Invitrogen) according to the manufacturer's protocol. Foxp3 mRNA levels were quantified by real-time PCR using QuantiTect SYBR Green PCR Kit (Qiagen) with the LightCycler (Roche, Indianapolis, IN). PCR consisted of a 15-min 95 °C denaturation step followed by 45 cycles of 15 s at 94 °C, 20 s at 56 °C and 15 s at 72 °C. Foxp3 primers: 5′−CCC AGG AAA GAC AGC AAC CTT– 3′ and 5′–TTC TCA CAA CCA GGC CAC TTG– 3′; Cyclophilin A primers: 5′–AGG GTG GTG ACT TTA CAC GC- 3′ and 5′–ATC CAG CCA TTC AGT CTT GG- 3′. Normalized values for foxp3 mRNA expression were calculated as the relative quantity of foxp3 divided by the relative quantity of cyclophilin A. All samples were run in triplicate.

Induction of colitis and histological examination

The well-defined CD4+ CD25–CD45RBhigh-induced SCID colitis model was used (39). In brief, C.B-17 SCID mice were injected intraperitoneally with 5 × 105 CD4+ CD25–CD45RBhigh cells from BALB/c mice either alone or together with 2.5 × 105 freshly isolated CD4+ CD25+ CD62+ or CD4+ CD25+ CD62L– cells from BALB/c mice. Colitis development was monitored by body weight every 3–5 days. Histological analysis was performed at the termination of the experiments. Colons and stomachs were removed from mice 9–10 weeks after T-cell reconstitution and fixed in 10% buffered formalin, and paraffin-embedded sections (6 μm) were cut and stained with H&E. The sections were scanned with a Nikon Eclipse E800 microscope (Nikon, Japan), and pictures were taken with a Spot Insight digital camera (Diagnostic Inc., Sterling Heights, MI) at a magnification of ×40 or ×100.

Statistical analysis

Analysis for statistically significant differences was performed with a Student's t-test. P < 0.05 was considered significant. Results are expressed as mean ± SEM.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

Phenotype of murine CD4+ CD25+ CD62L+ and CD4+ CD25+ CD62L– cells

Given previous indications that CD4+ CD25+ Treg are heterogenous and that CD62L may distinguish between functional subsets, four-color staining was used to analyze the phenotype of murine CD4+ CD25+ CD62+ and CD4+ CD25+ CD62L– cells (Figure 1). In 12 experiments with both BALB/c and C57BL/6 strains, CD4+ CD25+ cells represented approximately 2% of total splenocytes and approximately 10% of CD4+ T cells. Among CD4+ CD25+ Treg, 50–60% of the cells expressed high levels of CD62L. The cell-surface phenotype of the two subsets was further explored and noted to be very different. The majority of CD4+ CD25+ CD62+ cells expressed high levels of CD45RB (85.1% high), but the majority of CD4+ CD25+ CD62L– cells expressed low levels of CD45RB (84.9% low). The percentage of CD4+ CD25+ CD62+ cells expressing IL-10R1 (50.7%) was much higher than that of CD4+ CD25+ CD62L– cells (13.7%). In addition, more CD4+ CD25+ CD62+ cells expressed CD80 and CD86 (34.3% and 31.4%, respectively) in comparison with CD4+ CD25+ CD62L– cells (21.1% and 15.8%, respectively). However, cell-surface expression of CD28, CD44, and CD69 and intracellular expression of CTLA4 were equivalent for both subsets of T cells. Another phenotypic characteristic of Treg that has been recently described is expression of the foxp3 transcription factor (40–42). Real-time PCR for foxp3 in freshly isolated Treg subsets showed that both the CD62+ and CD62L– subsets expressed foxp3 (Figure 1D), but there was no consistent difference in the level of foxp3 expression between the two subsets in three experiments. Previous reports suggested that the quantitative level of foxp3 expression did not correlate with degree of suppressive activity. These results suggest that both subsets are bona fide Treg.

image

Figure 1. Phenotype of murine CD4+ CD25+ CD62L+ and CD4+ CD25+ CD62L– cells. BALB/c murine splenocytes were stained with four-color fluorescence and analyzed by fluorescent flow cytometry. (A) CD4+ CD25+ Treg in murine splenocytes. (B) Cell-surface expression of CD62L on CD4+ CD25+ Treg cells. (C) Surface phenotype of CD4+ CD25+ CD62+ cells (heavy line) and CD4+ CD25+ CD62L– cells (thin line) for cell-surface CD45RB, IL-10R, CD80, CD86, CD28, CD44, CD69, and intracellular CTLA4. (isotype control as dotted line). (D) Foxp3 expression by real-time PCR. Cells of the indicated subsets were freshly isolated, mRNA obtained, and real-time PCR for foxp3 and cyclophilin A performed as described in Methods. (A–C) Data representative of 12 experiments; and (D) data representative of three experiments.

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Both CD62L+ and CD62L+ subpopulations are anergic and suppressive in vitro; the CD62+ subset is more potent than the CD62L– subset

To determine if the proliferative capacities of CD62+ and CD62L– subsets were the same as the unseparated CD4+ CD25+ Treg population, 2 × 104 freshly isolated cells were stimulated with anti-CD3 mAb plus syngeneic T-depleted splenocytes as antigen presenting cells (APCs). Similar to CD4+ CD25+ Treg, neither CD4+ CD25+ CD62+ nor CD4+ CD25+ CD62L– cells proliferated in response to immobilized anti-CD3 mAbs. When exogenous IL-2 was added to the culture at a concentration of 50 U/mL, both subsets proliferated in response to anti-CD3 mAbs; the CD62+ subset responded better than the CD62L– subset or the unseparated CD4+ CD25+ population (Figure 2A). These results indicate that both CD62+ and CD62L– subsets of CD4+ CD25+ Treg are anergic in vitro. Other concentrations of anti-CD3 mAbs and IL-2 revealed the same differences between the two subsets (not shown).

image

Figure 2. Both CD62L+ and CD62L– subpopulations are anergic and suppress proliferation to anti-CD3 mAbs and alloantigen. (A) Freshly isolated BALB/c CD4+ CD25+ CD62+ or CD4+ CD25+ CD62L– cells were stimulated with anti-CD3 mAbs (1 μg/mL) and syngeneic T-depleted splenocytes (1 × 105 cells per well) with or without IL-2 (50 U/mL). Wells were pulsed with [3H] thymidine for the last 18 h of the 72-h culture. (B) Graded numbers of freshly isolated CD62+ or CD62L– cells were added to cultures of syngeneic CD4+ CD25– responders stimulated with anti-CD3 mAb plus APC. Wells were pulsed with [3H] thymidine for the last 18 h of the 72-h culture. *p < 0.05 compared with CD4+ CD25– control group. (C) Graded numbers of freshly isolated CD62+ or CD62L– cells were added to cultures of syngeneic CD4+ CD25– responders stimulated with C57BL/6 allogeneic spleen cells. Wells were pulsed with [3H] thymidine for the last 18 h of the 5-day culture. *p < 0.05 compared with CD4+ CD25– control group. Data representative of six experiments.

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As others have shown that Treg require IL-2 and IL-2R chains for their development and function (43), it was important to determine the role of IL-2 in the development and function of these subsets. As noted by others, very few CD4+ CD25+ cells were recovered from the spleens of IL-2-/- mice, and none of the separate populations inhibited the proliferation of CD4+ CD25– responder cells from wild-type mice (not shown). Therefore, IL-2 is not only important for the expansion of bulk Treg cells, but also critical for the development and function of the CD62+ and CD62L– subsets.

The suppressive properties of the subsets were examined in cultures of CD4+ CD25– responder cells stimulated with anti-CD3 mAb plus syngeneic T-depleted APCs. Increasing numbers of CD4+ CD25+, CD4+ CD25+ CD62+ or CD4+ CD25+ CD62L– cells were added to the cultures (Figure 2B). As few as 2.5 × 103 CD4+ CD25+ CD62+ cells (Treg : responder ratio = 1 : 8) resulted in a 70% reduction of the proliferative response, more than that by CD4+ CD25+ Treg. The suppressive effect was enhanced in a dose-dependent fashion, and the CD62+ subset was far more potent than the CD62L– subset.

The suppressive properties of the subsets in alloantigen-stimulated cultures also demonstrated that the CD62+ subset was more potent than the CD62L– subset (Figure 2C). As few as 2.5 × 104 CD4+ CD25+ CD62+ cells (Treg : responder ratio = 1 : 4) resulted in an 83% reduction of the proliferative response, similar to the 77% suppression caused by the CD4+ CD25+ Treg, and the suppressive effect was dose-dependent.

CD62L+ and CD62L– subsets differ in their requirements for cell contact-dependent suppression

It has been shown repeatedly that the suppressive function of CD4+ CD25+ Treg is cell contact-dependent and cytokine-independent in vitro (9,10,44). We conducted a similar analysis in our system. The results in Figure 3A show that if the CD62+ subset is separated from CD4+ CD25– responders in a transwell plate, suppression is not observed, similar to CD4+ CD25+ Treg. However, if the CD62L– subset is separated from CD4+ CD25– responders by a transwell plate, 59% suppression of the proliferative response is observed, compared with 68% suppression in the cell-contact culture. These results indicate that the mechanism(s) of suppression by the CD62+ and CD62L– subsets are different. Suppression by the CD62+ subpopulation is cell contact-dependent, while suppression by the CD62L– subpopulation is mostly cell contact-independent. Alternatively there may be multiple subsets within the CD62L– subpopulation, so that suppression by some subsets is contact-independent, but for other subsets suppression is contact-dependent. Although the CD62L– subset demonstrates some contact-independent suppression, the whole CD4+ CD25+ T cells show only contact-dependent suppression, although approximately 40–50% of these cells are CD62L–. The discrepancy is likely owing to the fact that the CD62L– subset is weaker than the CD62+ subset, and its effects rapidly diluted out in the culture (Figure 2B).

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Figure 3. Suppressive effect is independent of IL-10 and IL-10R; the CD62L+ subset is cell contact-dependent, cytokine-independent and CTLA4-independent; and the CD62L– subset is cell contact-independent, cytokine-independent and partially CTLA4-dependent. (A) 2 × 105 CD4+ CD25– BALB/c responder T cells were stimulated with anti-CD3 mAb plus syngeneic T-depleted APCs in the lower wells of the transwell plate. 1 × 105 CD4+ CD25+ CD62+ or CD4+ CD25+ CD62L– cells were either added directly to the lower wells or were placed in the upper wells of the transwell plate with 2.5 × 105 syngeneic T-depleted APCs. Wells were pulsed with [3H] thymidine for the last 18 h of the 72-h culture. (B) Freshly isolated CD4+ CD25+ CD62+ or CD4+ CD25+ CD62L– BALB/c subsets (1 × 104 cells per well) were tested for their ability to inhibit the proliferation of CD4+ CD25– T cells (2 × 104 cells per well) in response to anti-CD3 mAbs in the presence of the indicated mAbs (10 μg/mL). Wells were pulsed with [3H] thymidine for the last 18 h of the 72-h culture. (C) CD4+ CD25– cells, CD4+ CD25+ cells, and the CD62+ and the CD62L– subsets from IL-10-/- or wild-type C57BL/6 mice were tested for their ability to inhibit the proliferation of CD4+ CD25– T cells from wild-type mice in response to anti-CD3 mAbs. (D) IL-10R1 + and IL-10R1- BALB/c subsets were tested for their suppressive function. (E) CRF2-4-/-, wild-type C57BL/6, and BALB/c Treg can suppress syngeneic and allogeneic responder cells. Data representative of three experiments.

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Several studies have shown that some regulatory cells, such as Tr1 and Th3, suppress immune responses via the production of IL-10 and/or TGF-β (45,46). Hence, the role of these cytokines in the suppressive function of CD62+ and CD62L– subsets was assessed (Figure 3B). The addition of anti-IL-10, anti-IL-10R, anti-TGF-β or anti-CTLA4 failed to block suppression by either the CD62+ subset or the CD4+ CD25+ Treg, as reported many times by others (10,11). For the CD62L– subset, addition of anti-IL-10, anti-IL-10R, or anti-TGF-β did not influence suppression, but addition of anti-CTLA4 partially inhibited suppression. In four separate experiments anti-CTLA4 inhibited the suppression of the CD62L– subset by 50–80%. The combination of anti-CTLA4 plus anti-IL-10, anti-IL-10R1 or anti TGF-β did not have any further effects on the suppressive function of any of the subsets; and the blocking effect of anti-CTLA4 was seen only in cell contact but not transwell cultures (data not shown). To further evaluate the role of IL-10 in Treg function, CD62+ and CD62L– subsets were isolated from IL-10-/- mice. The results in Figure 3(C) show that CD62+ or CD62L– subsets from IL-10-/- mice suppress similar to wild-type cells. While the results in Figure 1 show that the CD62+ subset expresses high levels of IL-10R1, selection of IL-10R1+ and IL-10R1– subsets by the flow cytometry demonstrates that the suppressive activity resides only in the CD4+ CD25+ IL-10R1– subset (Figure  3D). CRF2-4-/- mice do not express the IL-10R2 chain (37) and thus cannot respond to IL-10 or the cytokines IL-22, IL-28, or IL-29, which also utilize this chain in their heterodimeric receptor complexes (47,48). Treg subsets from CRF2-4-/- mice on the C57BL/6 background were able to suppress CRF2-4-/- responder cells, wild-type C57BL/6 responders, and allogeneic BALB/c responders (Figure 3E). Likewise, Treg from these other strains could suppress CRF2-4-/- responders. Thus, IL-10 and the IL-10R are not critical for Treg development or function or for receptivity of responder cells to Treg suppression. These results indicate that suppression by the CD62+ subset is cytokine- and CTLA4-independent, while suppression by the CD62L– subset is partially CTLA4-dependent, but independent of the tested cytokines. As the CD62L– subset does function across a transwell plate, there may be other suppressive soluble mediators.

Treg function of CD4+ CD25+ cells is not dependent on CD62L expression

As CD62L seems to differentiate between functional subsets of Treg, we determined whether CD62L expression is critical to the suppressive activity. First, CD4+ CD25+ cells from CD62L-/- mice were used as Treg and compared with CD4+ CD25+ Treg from wild-type mice (Figure 4A). The results show that at a Treg : responder ratio of 1 : 2, CD4+ CD25+ cells from CD62L-/- mice suppress similar to wild-type cells. Thus, at least at the level of the unseparated CD4+ CD25+ population, CD62L expression is not required for Treg function.

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Figure 4. Treg function of CD4+ CD25+ cells is not dependent on CD62L expression. (A) CD4+ CD25– or CD4+ CD25+ cells from l-selectin-/- or wild-type C57BL/6 mice (1 × 104 cells/well) were tested for their ability to inhibit the proliferation of CD4+ CD25– T cells from wild-type mice (2 × 104 cells per well) in response to anti-CD3 mAbs. (B) CD4+ CD25– cells, CD4+ CD25+ cells or CD62+ or CD62L– BALB/c subsets (0.25–2 × 104 cells/well) were tested for their ability to inhibit the proliferation of CD4+ CD25– T cells (2 × 104 cells/well) in response to anti-CD3 mAbs with or without 10 μg/mL anti-CD62L mAb. Data representative of three experiments.

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Second, the MEL-14 anti-CD62L mAb was added to cultures to assess its effect on CD4+ CD25+, CD4+ CD25+ CD62+, and CD4+ CD25+ CD62L– subset suppressive function in the presence of CD4+ CD25– responders stimulated with anti-CD3 mAb and syngeneic T-depleted APCs (Figure 4B). After the addition of saturating concentrations of the anti-CD62L mAb, the various subsets of Treg still inhibited the proliferation of CD4+ CD25– cells in a dose-dependent fashion that was unaffected by the mAb. The anti-CD62L mAb did cause an increase in the baseline control proliferation in all groups, but did not prevent suppression. While CD62L is an important phenotypic marker for functionally distinct Treg subsets, it is not critical for their in vitro function. Because CD62L is the major lymph node homing receptor, it may be critical for in vivo migration and compartmentalization of regulatory function. It should also be noted in Figures 3(C) and 4(A,B) that the overall suppression was less than that measured in other experiments. This is likely owing to the fact that these experiments involved C57BL/6 strain mice, which show less Treg activity than the BALB/c used in the other experiments.

CD62L+ subset has greater proliferative and expansion potential

One key to understanding the molecular immunology of Treg and using them for meaningful clinical protocols is to expand them in culture. CD4+ CD25+ Treg have been expanded in vitro when activated with anti-CD3 mAbs, syngeneic APCs and exogenous IL-2 (2,21). Using a similar protocol, we stimulated CD62+ and CD62L– subsets with anti-CD3 mAbs, syngeneic APC and exogenous IL-2 (Figure 5A). The CD62+ subset proliferated well in vitro and could be expanded 20–30-fold (from 2.5 × 104 cells at day 0, to 7.2 × 105 cells at day 14 with 70–80% viability, and 5.0 × 105 cells at day 30 with 60–70% viability), which was similar to CD4+ CD25+ Treg (from 2.5 × 104 cells at day 0, to 6.0 × 105 cells at day 14 with 60–70% viability, and 4.0 × 105 cells at day 30 with 30–40% viability). In contrast, the CD62L– subset proliferated less well and only for a short period of time (from 2.5 × 104 cells at day 0, 1.5 × 105 cells at day 7 with 50–60% viability, and down to 7.5 × 104 cells at day 14 with 20–30% viability). These data indicate that although both subpopulations could be expanded in vitro initially, the proliferative capacities were different, with CD62+ cells proliferating far better than CD62L– cells.

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Figure 5. Both CD62L+ and CD62L– subpopulations can be expanded in vitro without loss of the suppressive function. (A) CD4+ CD25–, CD4+ CD25+, CD4+ CD25+ CD62+, or CD4+ CD25+ CD62L– BALB/c cells (2 × 104 cells per well) were stimulated with anti-CD3 mAbs (1 μg/mL) and syngeneic T-depleted APCs (1 × 105 cells per well) in the presence of IL-2 (50 U/mL). Anti-CD3 mAbs and IL-2 were added every 3–4 days, and T cells were restimulated by feeder cells every 10–14 days. Cells were counted on days 4, 7 and 14. (B–E) In vitro cultured CD4+ CD25–, CD4+ CD25+, CD4+ CD25+ CD62+ or CD4+ CD25+ CD62L– cells were tested for their ability to inhibit the proliferation of fresh CD4+ CD25– T cells in response to anti-CD3 mAbs. (B,C) Number of responder cells was fixed while the number of Treg was varied to the indicated ratio. After (B) 4 days of culture, (C) 7 days of culture, (D) 14 days of culture, and (E) 30 days of culture. Data representative of six experiments.

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Fluorescent flow cytometry was used to determine if the phenotype of the expanded subsets remained stable or had altered. The results demonstrate that after 7 days in stimulation culture, most CD62+ cells retained their phenotype: 97.8% 7-day-cultured cells express CD4, 94.1% 7-day-cultured cells express CD25, and 85.0% 7-day-cultured cells express CD62L. After 30 days in culture, the majority of CD62+ cells still retained their phenotype: 80.6% 30-day-cultured cells express CD4, 75.4% 30-day-cultured cells express CD25, and 58.3% 30-day-cultured cells express CD62L (Table 1). The stability of CD62L expression in Treg is in contrast to CD4+ CD25– naïve T cells that rapidly lose CD62L expression under similar culture conditions (72% CD62+ at day 0, 25% CD62+ at day 7, 11% CD62+ at day 20, and 2% CD62+ at day 30). CD4+ CD25+ CD62L– cells also retained their phenotype after 7 days in culture: 93.4% cells express CD4, 90.8% cells express CD25, and 3.6% cells express CD62L. However, their proliferation was so poor that by day 14 not enough cells could be harvested for reliable phenotyping.

Table 1. Phenotype of cultured CD4+ CD25+ CD62+ and CD4+ CD25+ CD62L– cells
 Day of culture 
CellsCD4+CD25+CD62+Phenotype
CD4+ CD25+ CD62+
 097.5%94.8%95.4% 
 797.8%94.1%85.0% 
 1493.8%90.1%65.3% 
 2082.4%78.6%55.7% 
 3080.6%75.4%58.3% 
CD4+ CD25+ CD62L−
 096.8%95.6%0.5% 
 793.4%90.8%3.6% 

It was also important to determine whether the expanded CD62+ and CD62L– subpopulations retained their suppressive properties. Suppressor assays tested the function of the expanded subsets after 4, 7, 14, and 30 days of culture (Figure 5B–E). After 4 days of culture (Figure 5B), both CD62+ and CD62L– subsets inhibited the proliferation of CD4+ CD25– cells by 78.7% and 75.2%, respectively, at a Treg : responder ratio of 1 : 8, whereas the freshly isolated subsets did not inhibit proliferation at this ratio. At a ratio of 1 : 2, 4-day-cultured CD62+ and CD62L– subsets inhibited proliferation by 97.4% and 98.4%, and the freshly isolated subpopulations inhibited proliferation by 90.9% and 54.4%. After 7 days of culture (Figure 5C), both subsets still retained their suppressive function, but were similar in potency to the freshly isolated subsets. After 14 days of culture (Figure 5D), the CD62+ subset still retained the suppressive function. The CD62L– subset did not proliferate well, and not enough cells could be harvested to test reliably at this time point. After 30 days of culture (Figure 5E), the CD62+ subset still retained suppressive activity, but the unseparated CD4+ CD25+ cells lost their suppressive activity. By 45 days of culture, the CD62+ subset had stopped proliferating well and lost most suppressive activity (not shown). These data indicate that the CD62+ subset can be expanded in vitro and retain the suppressive properties for an extended time (longer than the CD62L– subset and the unseparated CD4+ CD25+ cells), and may be more potent than freshly isolated subsets.

Cytokine production by CD62L+ and CD62L– subsets

To further investigate the differences between the CD62+ and CD62L– subsets, and whether this may signify their relationship to each other or additional Treg populations, both subsets were analyzed for their capacity to produce cytokines after activation with anti-CD3 mAbs and IL-2. Supernatants were collected after 2, 4, 7, and 14 days in culture, and the cytokine content (IL-4, IL-10, IFN γ, TGF-β) was determined by ELISA (Table 2). CD4+ CD25– cells produced significant amounts of IL-4, IL-10, IFN γ, and TGF-β, suggesting a mixed lineage of Th1 and Th2 cells and perhaps other cell types. CD4+ CD25+ Treg failed to produce detectable levels of IL-4, IL-10, and IFN γ, but did produce TGF-β, in keeping with earlier reports (49). The CD62+ subset produced significant amounts of TGF-β, but no IL-4, IL-10, and IFN γ, which is similar to the profile of TGF-β-producing Th3 cells (50,51). The CD62L– subset produced IL-10, IFN γ and TGF-β, similar to IL-10-producing Tr1 cells (52,53).

Table 2. Cytokine production by CD62+ and CD62L– subsets after in vitro expansion
PopulationDay of cultureIL-4 (ng/mL)IL-10 (ng/mL)IFN γ (ng/mL)TGF-β (ng/mL)
  1. ND = not detectable.

  2. Lower limits of detection of the ELISAs were: IL-4 0.1 ng/mL; IL-10 0.1 ng/mL; IFN γ 0.1 ng/mL; TGF-β 0.05 ng/mL.

CD4+ CD25−
 2NDND1.2420.146 
 40.2230.1800.6160.161 
 70.7674.8207.6800.199 
 140.2350.208>40.000.312 
CD4+ CD25+
 2NDNDND0.108 
 4NDNDND0.275 
 7NDNDND0.237 
 14NDNDND0.191 
CD4+ CD25+ CD62+
 2NDNDNDND 
 4NDNDND0.169 
 7NDNDND0.199 
 14NDNDNDND 
CD4+ CD25+ CD62L−
 2NDNDNDND 
 4ND1.4620.3610.199 
 7ND1.0530.7640.449 
 14ND0.4360.5280.191 

CD4+ CD25+ CD62L+ cells preferentially migrate to CCR2 and CCR7 agonists and FTY720

As memory effector and central memory T-cell subsets differ in CD62L and CCR7 expression, and as there is contradictory information about the chemotactic activity of CD4+ CD25+ Treg (54–56), the chemotactic activity of the CD62+ and CD62L– subsets was assessed. Previous work from our lab and others showed that the sphingosine-1-phosphate receptor antagonist FTY720 enhanced both CCL19/CCR7 and MCP-1 (CCL2)/CCR2 driven T-cell migration (57–59). Unseparated T cells were placed in migration chambers and stimulated with CCL19, MCP-1, CCL1, CCL5, CCL22, CXCL9, and/or FTY720. Both the initial starting population and the migrated populations were phenotyped by fluorescent flow cytometry to determine the extent of migration of each subset. The unseparated population was used in the migration assay to avoid crosslinking effects of mAbs during the isolation procedure and their subsequent acute perturbation of migration. The results in Figure 6A,B demonstrate that the CD62+ subset preferentially migrated to CCL19 and MCP-1 in comparison with either unseparated CD4+ CD25+ Treg or the CD62L– subset, which showed little chemotactic activity. FTY720 accentuated these differences even more. No migration was observed to CCL1, CCL5, CCL22 and CXCL9 (agonists for CCR8, CCR5, CCR4 and CXCR3) (data not shown). Thus, CCR7 and CCR2 agonists cause preferential chemotaxis of the CD62+ subset. Additional controls demonstrated that the cell migration was chemotactic and not chemokinetic (not shown). To determine further the physiologic relevance of these findings, a single dose of FTY720 was administered to mice in vivo and T cells harvested 18 h later from separate lymphoid compartments. The results in Figure 6(C) demonstrate that in vivo FTY720 shifted the distribution of total T cells, CD4+ T cells, CD4+ CD25+ Treg, and CD4+ CD25+ CD62+ Treg from spleen, thymus and peripheral blood to lymph nodes, but did not cause CD4+ CD25+ CD62L– Treg homing to lymph nodes.

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Figure 6. CD62L+ subset preferentially migrates to CCL19, MCP-1 and FTY720. (A) In vitro migration. Fluorescent flow cytometric analysis of unmigrated total and CD4+ CD25+ BALB/c T cells, and migrated cells gated on total or CD4+ CD25+ cells. Numbers in upper right are percentage of cells in that quadrant in comparison to the entire gated population. (B) In vitro migration. The percentage chemotaxis for each cell population, determined by dividing the number of cells of that subset migrating to the lower wells by the number of cells of that subset before migration. (C) In vivo migration. The percentage migration to FTY720 for each population, determined by dividing the number of cells of that subset in FTY720-treated mice by the number of cells of that subset in control BALB/c mice. (A,B) Data representative of three experiments. For in vivo study, data were analyzed for six individual mice.

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Both CD62L+ and CD62L– subsets prevent the development of autoimmune gastritis and colitis

Finally, we examined whether the CD62+ and CD62L– subsets could suppress autoimmune disease that developed in the absence of T-cell regulation using the well-defined CD4+ CD25–CD45RBhigh-induced colitis model (39). Autoimmune disease was induced in SCID mice by the transfer of CD4+ CD25–CD45RBhigh cells from normal BALB/c mice. Autoimmunity is characterized by weight loss, diarrhea, and histological changes of gastritis and colitis. For the mice that also received CD62+ or CD62L– subsets, colitis and gastritis did not develop as noted by weight gain, rather than weight loss, and a marked reduction of histologic change, which were identical in both experimental groups (Figure 7). Thus, both CD62+ and CD62L– subsets can prevent the development of autoimmune gastritis and colitis.

imageimage

Figure 7. Both CD62L+ and CD62L– subsets prevent autoimmune gastritis and colitis. (A) C.B-17 SCID mice were injected intraperitoneally with 5 × 105 CD4+ CD25–CD45RBhigh cells from BALB/c mice either alone or together with 2.5 × 105 freshly isolated CD4+ CD25+ CD62+ or CD4+ CD25+ CD62L– cells (n = 3 for each group). Body weight was monitored every 3–5 days. *p < 0.05 compared with unreconstituted mouse (control). (B) Histopathology of the stomach and colon in each group and in an unreconstituted mouse (control). Magnification ×40 or ×100.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

Several reports show that CD4+ CD25+ Treg cells can be further defined as subpopulations expressing CD62+, CD45RBlow (in mice) or CD45RO+ (in human) (2,10,36). Our data support the conclusion that CD4+ CD25+ Treg are not a homogeneous cell population, but can be divided into at least two subsets according to the cell-surface expression of CD62L. CD62+ and CD62L– populations are similar in that they are both anergic and suppressive, express foxp3, and can be expanded in vitro without initial loss of suppressive function. However, there are substantial differences in their cell-surface phenotype, suppressive capacity, functional mechanisms, proliferative capacity, cytokine production, and migration. The majority of CD4+ CD25+ CD62+ cells also expressed high levels of CD45RB (CD45RBhi), while the majority of CD4+ CD25+ CD62L– cells expressed low levels of CD45RB (CD45RBlo). Additional studies demonstrated that the CD45RBloCD4+ CD25+ CD62+ subset was more suppressive than the CD45RBhiCD4+ CD25+ CD62+ population (data not shown). Thus, these subsets are also likely to be heterogenous and further significant subdivisions may be defined in the future. The CD4+ CD25+ CD62+ cells also express higher levels of IL-10R1, CD80 and CD86 in comparison with CD4+ CD25+ CD62L– cells, suggesting other functional and lineage differences. Thornton and Shevach (11) earlier concluded that the CD62+ and CD62L– subsets were not different, however, in their studies the CD62+ subset was a more potent suppressor and they did not analyze these cells for the same range of variables as in the current study. Kuniyasu et al. (60) reached a similar conclusion, but again did not analyze the range of variables in the current study and only assessed Treg function at a high Treg : responder ratio of 1 : 1.

Although CD4+ CD25+ cells secrete more IL-10 than CD4+ CD25– cells (61), the role of IL-10 in the suppressive function of CD4+ CD25+ Treg cells remains controversial. Most in vitro studies show that the suppressive function of CD4+ CD25+ cells is independent of IL-10. CD4+ CD25+ cells from IL-10-/- mice retain suppressive capacity in vitro (9), and neutralization of IL-10 or IL-10R1 has no effect on the suppressive function (2). However, several in vivo studies suggest that IL-10 is required for the regulatory function of CD4+ CD25+ cells. CD4+ CD25+ CD45RBlow cells prevent inflammatory bowel disease via an IL-10-dependent mechanism, and CD4+ CD25+ CD45RBlow cells from IL-10-/- mice fail to protect from colitis (45,62). Alloantigen-specific CD4+ CD25+ Treg cells responsible for the maintenance of tolerance to donor alloantigens in vivo require IL-10 and CTLA4 for their functional activity (4,5). CD4+ CD25+ T cells that delay or prevent GVHD are partially dependent on IL-10 (20,22). In our investigations, we showed that neither IL-10, IL-10R1, nor IL-10R2 were important for Treg development or function or for the ability of responder T cells to be subjected to the suppressive function of Treg. By implication, the cytokines IL-22, IL-28, and IL-29 are also not important for Treg, as they rely on the IL-10R2 chain (47,48). As these in vitro assays are APC-dependent, IL-10 and IL-10R are not required at the level of either T cells or APCs. The requirement for IL-10 in vivo suggests that other cell types may be subject to the influences of Treg and IL-10 (63).

It has been shown repeatedly that the suppressive function of CD4+ CD25+ Treg is cell contact-dependent in vitro. When the suppressors were separated from responders by a semipermeable membrane, no suppression was observed. The molecular nature of the contact-dependent interaction is not fully established. Nakamura and colleagues showed that stimulated CD4+ CD25+ cells produce TGF-β1 and express TGF-β1 on the cell surface, suggesting that CD4+ CD25+ cells may exert immunosuppression by cell–cell interaction involving cell-surface TGF-β1 (43), although this association has been contradicted by others (64). CTLA4 is a negative regulator of T-cell activation, and is expressed constitutively by CD4+ CD25+ cells (65), although most investigators have failed to demonstrate inhibitory effects of CTLA4 blockade on Treg function (12–15). Nonetheless, some reports show that the addition of anti-CTLA-4 antibody abrogates suppression in vitro and in vivo in colitis and transplantation models (5,65,66), suggesting that in some situations Treg cell-surface CTLA4 is functional. Our experiments show that suppression by the CD62L– subset is partially CTLA4-dependent, different from unseparated CD4+ CD25+ Treg or the CD4+ CD25+ CD62+ subset. Further, the CD62L– subset expresses lower levels of the CD80 and CD86 ligands. These results suggest that it is the CD62L– subset that depends on CTLA4 for some of its suppressive function. The result might suggest that the cell-surface CTLA4 does not necessarily regulate intrinsic Treg activation, but rather focus these cells on CD80+ or CD86+ targets.

CD4+ CD25+ Treg cells account for only 5–10% of the total CD4+ population in both mice and humans. Therefore, the administration of sufficient numbers of freshly isolated CD4+ CD25+ cells is not therapeutically practical, and the expansion of CD4+ CD25+ Treg is critically important for clinical cellular therapy. Several groups have tried to expand CD4+ CD25+ Treg in vitro with limited success (1,2,11,20,21,49). In most situations, CD4+ CD25+ Treg could be expanded 10–30-fold after 7–14 days in culture, remained anergic and retained their suppressive properties. However, after several more rounds of restimulation, CD4+ CD25+ T cells failed to suppress CD4+ CD25– T cells and were responsive to anti-CD3 in the absence of exogenous IL-2, suggesting that CD4+ CD25+ Treg cells might lose their anergic and suppressive properties after long-term culture (67). In our system, we stimulated the CD62+ and CD62L– subsets with anti-CD3 mAbs, syngeneic T-depleted APCs and IL-2. The CD62+ subset proliferated well in vitro, could be expanded 20–30-fold, and retained suppressive function for at least 30 days in culture; far better than the CD62L– subset, which proliferated less well and for only a short period of time. Interestingly, unseparated CD4+ CD25+ Treg also lost suppressive function after 30 days in culture. This suggests that the CD62+ subset can be expanded for longer periods while retaining suppressive properties, and that stimulation of this subset within the unseparated CD4+ CD25+ population may even impede their development. Thus, previous failed attempts to sustain Treg in culture may have resulted from improper selection of the correct subset at culture initiation. These findings are relevant for the use of these cells as cellular therapy in immune diseases. Future studies must define additional culture conditions that allow further cellular expansion with retention of suppressive function.

Lymphocyte trafficking through the endothelium involves a sequence of events relying on adhesion molecules (such as CD62L), chemokine receptors, and integrins. Lymphoid chemokines are important for trafficking into and within lymphoid compartments, whereas inflammatory chemokines attract lymphocytes into peripheral tissue (68). There is contradictory information about the chemotactic activity of CD4+ CD25+ Treg. Some reports indicate that Treg express CCR5; and that its ligand, CCL4, is the most potent chemoattractant for these cells (69). CD4+ CD25+ Treg also express CCR4 and CCR8, and are chemoattracted by their respective ligands, CCL17/TARC, CCL22/MDC, and CCL1/I-309 (55,56). However, Rudensky et al. showed that CD4+ CD25+ Treg are refractory to the lymphoid chemokines CCL19/ELC, CCL21/SLC, CCL2, CCL4, CCL22 and CXCL12 (54). A recent report shows that CD4+ CD25+ CD62+ splenocytes express CCR7 at high levels and migrate toward the lymphoid chemokine ligands CCL19 and CCL21 (ligands for CCR7); whereas CD4+ CD25+ CD62L– splenocytes preferentially express CCR2, CCR4, and CXCR3 and migrate toward the corresponding inflammatory chemokine ligands CXCL9/MIG and CXCL10/IP-10 (ligands for CXCR3), and MDC and TARC (ligands for CCR4; 36). In our experiments, the CD62+ population preferentially migrated to both CCL19 and MCP-1 (ligands for CCR7 and CCR2, respectively), but not to CCL1, CCL5/RANTES, CCL22 and CXCL9 (agonists for CCR8, CCR5, CCR4 and CXCR3). Further, FTY720, a sphingosine-derived immune modulator that causes increased T-cell homing to peripheral lymph nodes from peripheral blood and spleen (38) enhanced CD62+ Treg subset migration to CCL19 and MCP-1 (57–59). The discrepancy among the various reports may be because of small variations in the precise experimental details or mouse strains used, but a more important reason may be that distinct Treg subsets have unique chemokine responses and most reports did not examine discrete subsets. Because the CD62+ subset by definition expresses l-selectin and because it preferentially responds to CCL19, it may actively home in vivo to secondary lymphoid organs and exert suppressive functions at these sites. In this regard, FTY720 could act as an immunosuppressant by promoting Treg migration to secondary lymphoid organs. The CD62L– subset did not respond to many different chemokines and thus may be sessile in the spleen (from which they were isolated) or respond to other chemokines not yet tested. As both subsets inhibited the development of gastritis and colitis, this might mean that they can both migrate to the stomach and colon. Alternatively, they could migrate to different locations in vivo, yet interrupt distinct loci in the progression of autoimmunity, resulting in similar clinical outcomes.

CD4+ CD25+ Treg are not a homogeneous cell population, but can be divided into at least two subsets according to CD62L expression. To our knowledge, this is the first report to demonstrate systematically differences in phenotype, function, and mechanism between the two subsets of CD4+ CD25+ Treg. As the CD62+ population is a more potent suppressor than the CD62L– population or unfractionated CD4+ CD25+ Treg cells, can be expanded far more easily in culture, and is more responsive to chemokine driven migration to secondary lymphoid organs, these cells may be important for clinical use.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
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