Adoptive Transfer of In Vitro-Stimulated CD4⁺CD25⁺ Regulatory T Cells Increases Bacterial Clearance and Improves Survival in Polymicrobial Sepsis

Mice, CLP, and adoptive transfer

Female BALB/c and BALB/c athymic nude mice were purchased from Harlan, acclimated for 1 wk, and used at 8–10 wk of age. Mice were housed in a room that maintained constant temperature and humidity, and were subjected to one 12-h light/dark cycle per day. Mice received normal rodent chow and water ad libitum. The experiments were performed in accordance with the National Institutes of Health guidelines and approved by the Institutional Animal Care and Use Committee.

Mice were anesthetized with an i.p. injection of ketamine HCl (87 mg/kg body weight; Ketaset; Fort Dodge Animal Health) and xylazine (13 mg/kg body weight; The Butler Company). The cecum was exposed aseptically through a 1-cm incision of the lower abdomen, tightly ligated with a 4-0 silk suture below the ileocecal valve without causing bowel obstruction, and punctured through-and-through once with a 27-gauge-diameter needle. The ligated and perforated cecum was replaced in the peritoneal cavity, and surgical incision was closed with 4-0 silk sutures and stainless-steel wound clips (BD Biosciences). All of the mice received 1 ml of prewarmed (37°C) normal saline s.c. for fluid resuscitation and were placed on a heating pad until they recovered from anesthesia. Mice were injected with 3 × 10⁵ CD4⁺ or CD4⁺CD25⁺ T cells in 0.5 ml of sterile HBSS by tail vein just before CLP surgery or 6-h post-CLP surgery. The mice were monitored four times per day for 14 days.

Cell culture reagents and condition

Supplemented RPMI 1640 consisted of RPMI 1640 with 10% FBS, 55 mM 2-ME, and 1% antibiotic-antimycotic. All tissue culture reagents were purchased from Invitrogen Life Technologies. All cells were cultured at 37°C with 5% CO₂.

Cell isolation and in vitro stimulation of CD4⁺ T cells and CD4⁺CD25⁺ Tregs

All cell populations were isolated with Ab-tagged magnetic beads and AutoMACS (Miltenyi Biotec). Mouse CD4⁺ T cells and dendritic cells were isolated from mouse splenocytes by positive selection with anti-CD4 and anti-CD11c microbeads, respectively. CD4⁺ cells isolated in this manner or CD4⁺CD25⁻ cells stimulated in vitro for 3 days (as described below) were used as control cells in CLP experiments. CD4⁺CD25⁺ Tregs were isolated from mouse splenocytes with a CD4⁺CD25⁺ Regulatory T Cell Isolation kit (Miltenyi Biotec). CD4⁺CD25⁻ T cells or Tregs (5 × 10⁵ cells per well) were stimulated by culture with 4 ng/ml recombinant mouse IL-2 (R&D Systems) for 72 h in 24-well plates, which were precoated with 1 μg/ml anti-mouse CD3 and anti-mouse CD28 Abs (BD Pharmingen) before adoptive transfer into CLP mice. The purity of isolated Tregs was >90% by flow cytometric analysis with anti-CD4 and anti-CD25 staining (data not shown).

In vitro Treg suppression assay

CD4⁺ T cells (2 × 10⁵ per well) were cultured with splenic dendritic cells (2 × 10⁴ per well), the indicated number of CD4⁺CD25⁺ Tregs, and 0.1 mg/ml anti-mouse CD3 Ab in 96-well round-bottom plates for 66 h. Cells were pulsed with 1 μCi/well [³H]thymidine during the last 16 h of incubation. T cell proliferation was determined by the incorporation of [³H]thymidine. To calculate the suppression percentage, the cpm value from each well was divided by the average cpm value from six wells without Tregs.

Peritoneal wash collection

Mice were sacrificed at 24 h post-CLP by CO₂ asphyxiation. Two milliliters of sterile PBS was injected into the peritoneal cavity and mixed by gentle squeezing of the abdomen. The peritoneal wash fluid was collected with a syringe and an 18-gauge needle and stored on ice. Samples were removed immediately for bacterial CFU analysis, and then cells were pelleted by centrifugation for differential analysis, and supernatants were aliquoted and stored at −80°C for cytokine/chemokine analysis.

Measurement of peritoneal and plasma cytokines/chemokines

Peritoneal lavage fluid was subjected to ELISA with the use of Mouse Quantikine kits (R&D Systems) according to the manufacturer’s instructions. Absorbance readings were obtained on a Versamax microplate reader (Molecular Devices), and data were analyzed with Microsoft Excel 2000 software. Cytokines/chemokines in EDTA plasma were analyzed at Rules Based Medicine by a proprietary technology similar to luminex for cytokines and chemokines.

Measurement of peritoneal bacterial CFU

Peritoneal fluid was analyzed for bacterial CFU/milliliter by dilution plating onto trypticase soy agar with 5% sheep blood (BBL; BD Biosciences) and colony counting following overnight incubation.

Coulter count, cytospin, and differential determination

The peritoneal cell suspension was centrifuged at 350 × g for 10 min at 4°C in a Beckman GPR centrifuge. The supernatant was discarded, and the cell pellet resuspended in 1 ml of Dulbecco’s PBS (DPBS; Invitrogen Life Technologies). From this cellular suspension, the number of infiltrating leukocytes was enumerated with a Z2 Coulter Particle Counter and Size Analyzer (Beckman Coulter). The average of two counts was recorded. Instrument settings were as follows: aperture size, 100 μm; lower threshold (TL), 3 μm; count mode above TL. Differentiation of cell populations was determined by microscopic morphology of the cells. Cell concentration was adjusted in a cytofunnel (Thermo Electron Corporation) to ∼1 × 10⁵ cells/funnel with the addition of DPBS. Cells were centrifuged at 700 rpm for 10 min (Cytospin 2; Shandon/Thermo Electron Corporation). Slides were stained in HEMA 3 staining set (Fisher Scientific), rinsed in Millipore water, and dried at room temperature. Leukocytes were classified as neutrophils, eosinophil, lymphocytes, macrophage, or mast cells. A percentage of each population was estimated by counting 100 cells, twice, in various random fields by two blinded independent observers. The mean for each cell type was then calculated.

Statistical analysis

Data are expressed as the mean ± SEM. Survival analysis was done by the Kaplan-Meier method, and statistical comparisons between groups were performed by a log rank test with JMP 4.0.4 software (SAS Institute). Statistical comparisons for all other data were conducted by ANOVA analysis with JMP 4.0.4 software. Data that were skewed were transformed by Box Cox transformation and analyzed by ANOVA with JMP 4.0.4 software. A value of p < 0.05 was considered significant.

Adoptive transfer of in vitro-stimulated Treg just before CLP improves survival in a dose-dependent fashion

To examine the effect of Tregs on septic outcome, we adoptively transferred 3 × 10⁵ naive syngeneic Tregs into BALB/c mice just before CLP and followed the mice for survival up to 2 wk. Mice that received unstimulated Tregs exhibited only 53% mortality at 2 wk, whereas mice that received either vehicle or naive CD4⁺ cells exhibited 67 and 70% mortality, respectively (data not shown; n = 30, p = 0.0507 for naive CD4⁺CD25⁺ vs naive CD4⁺). It has been shown that both polyclonal and Ag-specific stimulation of Tregs results in increased suppressive activity in vitro and in vivo (19, 20, 21). To determine whether in vitro-stimulated Tregs might have a greater effect on CLP survival, we adoptively transferred 3 × 10⁵ Tregs stimulated in vitro with IL-2, anti-CD3, and anti-CD28 for 3 days just before CLP. An in vitro Treg suppression assay was used to measure their suppressive activity (11). As expected, the in vitro-stimulated Tregs exhibited significant enhanced suppression of CD4⁺ T cell proliferation in vitro (Fig. 1⇓A, p < 0.05). Adoptive transfer of in vitro-stimulated Tregs resulted in a significant improvement in 2-wk survival of CLP mice compared with groups receiving either naive CD4⁺ or nonstimulated Tregs (Fig. 1⇓B; p < 0.005 and 0.02, respectively). Furthermore, the improvement in survival with stimulated Tregs was dose dependent with a minimum of 3 × 10⁵ cells required per mouse to observe the effect (Fig. 1⇓C, p < 0.05). In addition, the improved CLP survival was only observed with adoptive transfer of stimulated Tregs, because in vitro-stimulated CD4⁺CD25⁻ cells under the same conditions did not result in improved survival (Fig. 1⇓D). Because in vitro-stimulated Tregs exhibited a significant improvement in survival, all subsequent experiments were done with these cells in our studies.

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FIGURE 1.

In vitro-stimulated Tregs improve survival dose dependently in a mouse CLP model. A, In vitro Treg suppression of CD4⁺ T cell proliferation is greater with Tregs previously stimulated with IL-2, anti-CD3/anti-CD28 for 3 days. The data represent mean ± SEM of triplicate wells. B, Survival curves of mice receiving either 3 × 10⁵ naive CD4⁺, naive CD4⁺CD25⁺, or in vitro-stimulated CD4⁺CD25⁺ cells just before CLP. The data are a composite of two independent experiments, each with similar results. C, Survival curves of mice receiving either 3 × 10⁵ naive CD4⁺ cells or 3 × 10⁵, 3 × 10⁴, or 3 × 10³ in vitro-stimulated CD4⁺CD25⁺ cells just before CLP. D, Survival curves of mice receiving either vehicle, 3 × 10⁵ in vitro-stimulated CD4⁺CD25⁻ cells, or 3 × 10⁵ in vitro-stimulated CD4⁺CD25⁺ cells just before CLP.

The effect of Treg transfer on CLP survival is host T cell dependent and occurs with therapeutic administration

Treg activity on suppression of immune responses has been demonstrated to be mediated through suppression of T cell activation both in vitro and in vivo (3, 7, 10, 11). To determine whether the improved survival of CLP mice by Treg transfer is also mediated through host T cells in vivo, we adoptively transferred syngeneic Tregs into BALB/c athymic nu/nu mice just before CLP. Interestingly, unlike our observations in wild-type mice, we did not observe any effect of Tregs on improved survival in CLP nude mice, suggesting that host T cells are required for the protective effect (Fig. 2⇓A). To test whether therapeutic treatment with Tregs could also improve survival, we adoptively transferred syngeneic Tregs to BALB/c mice at 6 h post-CLP and monitored survival for 2 wk. The delayed administration of 3 × 10⁵ Tregs also resulted in a significant improvement in survival compared with transferred naive CD4⁺ T cells (Fig. 2⇓B; p < 0.05). To determine whether the protective effect on survival was MHC restricted, we adoptively transferred 3 × 10⁵ allogeneic Tregs from C57BL/6 mice into BALB/c mice at 6 h post-CLP. The administration of allogeneic Tregs also resulted in an improvement in 2-wk survival, although the effect did not reach significance (Fig. 2⇓C; n = 10, p = 0.0823).

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FIGURE 2.

Improved survival with Treg transfer is host T cell dependent and observed with administration at 6 h post-CLP. A, Survival curves of athymic nu/nu female BALB/c mice receiving either 3 × 10⁵ naive CD4⁺ or in vitro-stimulated CD4⁺CD25⁺ cells just before CLP. B, Survival curves of female BALB/c mice receiving either 3 × 10⁵ naive CD4⁺ or in vitro-stimulated CD4⁺CD25⁺ cells at 6 h post-CLP. The data are a composite of two independent experiments, each with similar results. C, Survival curves of female BALB/c mice receiving either 3 × 10⁵ naive CD4⁺ or in vitro-stimulated CD4⁺CD25⁺ cells derived from C57BL/6 mice at 6 h post-CLP.

Adoptive transfer of stimulated Tregs leads to improved bacterial clearance and increased recruitment of peritoneal mast cells and TNF-α production

To understand the mechanism of action of adoptively transferred Tregs on CLP survival, we examined peritoneal bacterial load in CLP mice at 24 h post-CLP. Mice that received Tregs just before CLP exhibited significantly lower peritoneal bacterial load compared with mice that received naive CD4⁺ T cells (205 ± 108 vs 1891 ± 520 (×10³ CFU/ml); Fig. 3⇓A; p < 0.002). To determine whether the increased bacterial clearance was associated with increased leukocyte infiltration into the peritoneum, peritoneal lavages from mice at 24 h post-CLP were examined by cytospin and differential cell counts. Significant cell infiltration of mononuclear cells, neutrophils, eosinophils, and lymphocytes was clearly evident in both the Treg and naive CD4⁺ treated groups (Table I⇓). The majority of infiltrated cells were neutrophils in both groups, which constituted ∼70% of total cells. Interestingly, animals that received Tregs were found to have significantly greater numbers of mast cells in peritoneal lavages (Table I⇓; Fig. 3⇓B; p < 0.02), with no other notable differences.

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FIGURE 3.

Improved bacterial clearance, increased peritoneal mast cells, and enhanced peritoneal TNF-α production with adoptive transfer of in vitro-stimulated Tregs. A, Individual values for peritoneal bacterial CFU/milliliter at 24 h post-CLP are plotted as vertical dot plots for mice that received either 3 × 10⁵ naive CD4⁺ or in vitro-stimulated CD4⁺CD25⁺ cells just before CLP. The data are a composite of three independent experiments, each with similar results. B, Enumeration of peritoneal mast cells at 24 h post-CLP by cytospin and differential counting for mice that received either 3 × 10⁵ naive CD4⁺ or in vitro-stimulated CD4⁺CD25⁺ cells just before CLP. The data are represented as mean ± SEM. There were no detectable mast cells in mice that received naive CD4⁺ cells. C, Peritoneal TNF-α levels at 24 h post-CLP in mice that received either 3 × 10⁵ naive CD4⁺ or in vitro-stimulated CD4⁺CD25⁺ cells just before CLP. The data are represented as mean ± SEM.

To determine whether adoptive transfer of Tregs suppressed systemic or peritoneal cytokine/chemokine production, plasma and peritoneal lavage fluids were analyzed for either TNF-α, IL-6, IL-2, IL-10, eotaxin-1, or MCP-1 at 24 h post-CLP. There were no significant differences between the groups in plasma levels of IL-6, IL-2, IL-10, or TNF-α, although both groups exhibited elevated plasma levels relative to control mice as expected (Table I⇑). Analysis of TNF-α, IL-6, MCP-1, and eotaxin in peritoneal lavage fluids indicated that these markers were significantly elevated above control naive mice in both groups as expected (Table I⇑). However, only TNF-α was significantly higher in the group that received Tregs (Fig. 3⇑C; p < 0.03).