IL-7 Is Essential for the Development and the Persistence of Chronic Colitis

Animals

Female C57BL/6 mice were purchased from Japan CLEA. C57BL/6 background RAG-2^−/− mice were obtained from Taconic Farms. C57BL/6 background RAG-1^−/− mice and C57BL/6 background IL-7-deficient (IL-7^−/−) mice were provided from Dr. R. Zamoyska (National Institute for Medical Research, London, U.K.) (16). IL-7^−/− mice were intercrossed into RAG-1^−/− mice to generate IL-7^−/− × RAG-1^−/− mice in the Animal Care Facility of Tokyo Medical and Dental University. Mice were maintained under specific pathogen-free conditions in the Animal Care Facility of Tokyo Medical and Dental University. Female donors and recipients were used at 6–12 wk of age. All experiments were approved by the regional animal study committees and were done according to institutional guidelines and Home Office regulations.

Purification of T cell subsets

CD4⁺ T cells were isolated from spleen cells from C57BL/6 mice using the anti-CD4 (L3T4)-MACS system (Miltenyi Biotec) according to the manufacturer’s instruction. Enriched CD4⁺ T cells (96–97% pure, as estimated by FACSCalibur (BD Biosciences)) were then labeled with PE-conjugated anti-mouse CD4 (RM4-5; BD Pharmingen) and FITC-conjugated anti-CD45RB (16A; BD Pharmingen). Subpopulations of CD4⁺ cells were generated by two-color sorting on a FACSVantage (BD Biosciences). All populations were >98.0% pure on reanalysis. To isolate LP CD4⁺ T cells, colitis was induced in RAG-2^−/− mice by adoptive transfer of CD4⁺CD45RB^high T cells as described previously (17). The colitic CD4⁺CD45RB^high T cell-transferred RAG-2^−/− mice were sacrificed at 6–8 wk after transfer. The entire length of colon was opened longitudinally, washed with PBS, and cut into small pieces. The dissected mucosa was incubated with Ca²⁺-, Mg²⁺-free Hanks’ BSS containing 1 mM DTT (Sigma-Aldrich) for 45 min to remove mucus and then treated with 2.0 mg/ml collagenase (Worthington Biomedical) and 0.01% DNase (Worthington Biomedical) for 2 h. The cells were pelleted two times through a 40% isotonic Percoll solution and then subjected to Ficoll-Hypaque density gradient centrifugation (40/75%). Enriched LP CD4⁺ T cells were obtained by positive selection using anti-CD4 (L3T4) MACS magnetic beads. The resultant cells when analyzed by FACSCalibur contained >95% CD4⁺ cells.

In vivo experimental design

We performed a series of in vivo experiments below to investigate the role of IL-7 in the development and the persistence of murine chronic colitis. Experiment 1: To assess the necessity of IL-7 in the initial development of colitis, including the processes for T cell priming, activation, and persistence of pathogenic effector and memory CD4⁺ T cells, we performed the cell transfer experiments using IL-7^+/+ × RAG-1^−/− and IL-7^−/− × RAG-1^−/− littermate recipients. IL-7^+/+ × RAG-1^−/− mice (n = 10) and IL-7^−/− × RAG-1^−/− mice (n = 10) were injected i.p. with 3 × 10⁵ splenic CD4⁺CD45RB^high T cells from normal C57BL/6 mice. Experiment 2: To assess the necessity of IL-7 for the persistence of colitogenic memory CD4⁺ T cells, we performed the adoptive retransfer of colitogenic LP memory CD4⁺ T cells. First, colitis was induced in RAG-2^−/− mice by adoptive transfer of CD4⁺CD45RB^high T cells. The colitogenic memory CD4⁺ LP T cells were obtained from the colitic mice induced by adoptive transfer of CD4⁺CD45RB^high T cells 6–8 wk after transfer and then were injected again into the control IL-7^+/+ × RAG-1^−/− mice (IL-7^+/+→IL-7^+/+; n = 10) and IL-7^−/− × RAG-1^−/− mice (IL-7^+/+→IL-7^−/−; n = 10). Experiment 3: To assess the initial role of IL-7 for T cell priming, differentiation, and sustainment of colitogenic effector and memory CD4⁺ T cells, we performed another adoptive retransfer experiment. First, IL-7^−/− × RAG-1^−/− mice were transferred with CD4⁺CD45RB^high T cells. Six weeks after transfer, splenic CD4⁺ T cells were isolated from the transferred IL-7^−/− × RAG-1^−/− mice and retransferred into new IL-7^+/+ × RAG-1^−/− mice (IL-7^−/−→IL-7^+/+; n = 7). As a positive control, splenic CD4⁺ T cells obtained from colitic IL-7^+/+ × RAG-1^−/− mice that have been initially transferred with CD4⁺CD45RB^high T cells were also transferred into new IL-7^+/+ × RAG-1^−/− mice (IL-7^+/+→IL-7^+/+; n = 7). The recipient mice after transfer were weighed initially and then three times per week. They were also observed for clinical signs such as hunched posture, piloerection, diarrhea, and blood in the stool. Mice were sacrificed 4–8 wk after transfer and assessed for a clinical score (17) that is the sum of three parameters as follows: hunching and wasting, 0 or 1; colon thickening, 0–3 (0, no colon thickening; 1, mild thickening; 2, moderate thickening; 3, extensive thickening); and stool consistency, 0–3 (0, normal beaded stool; 1, soft stool; 2, diarrhea; 3, bloody stool) (17).

Histological examination

Tissue samples were fixed in PBS containing 6% neutral-buffered formalin. Paraffin-embedded sections (5 μm) were stained with H&E. Three tissue samples from the proximal, middle, and distal parts of the colon were prepared. The sections were analyzed without prior knowledge of the type of T cell reconstitution or treatment. The area most affected was graded by the number and severity of lesions. The mean degree of inflammation in the colon was calculated using a modification of a previously described scoring system (17), as follows: mucosa damage, 0; normal, 1; 3–10 intraepithelial cells (IEL)/high power field (HPF) and focal damage, 2; >10 IEL/HPF and rare crypt abscesses, 3; >10 IEL/HPF, multiple crypt abscesses and erosion/ulceration, submucosa damage, 0; normal or widely scattered leukocytes, 1; focal aggregates of leukocytes, 2; diffuse leukocyte infiltration with expansion of submucosa, 3; diffuse leukocyte infiltration, muscularis damage, 0; normal or widely scattered leukocytes, 1; widely scattered leukocyte aggregates between muscle layers, 2; leukocyte infiltration with focal effacement of the muscularis, 3; extensive leukocyte infiltration with transmural effacement of the muscularis.

Cytokine ELISA

To measure cytokine production, 1 × 10⁵ LP CD4⁺ T cells were cultured in 200 μl of culture medium at 37°C in a humidified atmosphere containing 5% CO₂ in 96-well plates (Costar) precoated with 5 μg/ml hamster anti-mouse CD3ε mAb (145-2C11; BD Pharmingen) and hamster 2 μg/ml anti-mouse CD28 mAb (37.51; BD Pharmingen) in PBS overnight at 4°C. Culture supernatants were removed after 48 h and assayed for cytokine production. Cytokine concentrations were determined by specific ELISA per manufacturer’s recommendation (R&D Systems).

Flow cytometry

To detect the surface expression of a variety of molecules, isolated splenocytes or LP mononuclear cells were preincubated with a FcγR-blocking mAb (CD16/32, 2.4G2; BD Pharmingen) for 20 min followed by incubation with specific FITC-, PE-, PECy5-, or biotin-labeled Abs for 30 min on ice. The following mAbs other than biotin-conjugated anti-mouse IL-7Rα (A7R34; Immuno-Biological Laboratories) were obtained from BD Pharmingen: anti-CD4 mAb (RM4-5), anti-CD25 mAb (7D4), anti-CD45RB mAb (16A), anti-CD62L mAb (MEL-14), anti-CD44 mAb (IM7), anti-CD69 mAb (H1.2F3), and anti-Bcl-2 mAb (3F11). Biotinylated Abs were detected with PE- or CyChrome-streptavidin. Standard two- or three- color flow cytometric analyses were obtained using the FACSCalibur using CellQuest software. Background fluorescence was assessed by staining with control irrelevant isotype-matched mAbs.

To analyze Bcl-2 expression in LP CD4⁺ T cells, cells were fixed and permeabilized with BD Cytofix/Cytoperm solution before intracellular cytokine staining with FITC-conjugated anti-Bcl-2 mAb (BD Pharmingen). To analyze the TCR Vβ family repertoire, splenic cells were double-stained with PE-conjugated anti-CD4 mAb (RM4-5), and the following FITC-conjugated mAbs: Vβ2; KJ25, Vβ3; KT4, Vβ4; MR9-4, Vβ5; RR4-7, Vβ6; TR310, Vβ7; MR5-2, Vβ8.1/2; B21.14, Vβ8.3; MR10-2, Vβ9; B21.5, Vβ10; RR3-15, Vβ11; MR11-1, Vβ12; IN12.3, Vβ13; 14.2, Vβ14; and KJ23, Vβ17. All Abs were purchased from BD Pharmingen.

CFSE labeling of T cells

T cell division in vivo was assessed by flow cytometry of CFSE-labeled cells. Isolated LP CD4⁺ T cells were stained in vitro with the cytoplasmic dye CFSE (Molecular Probes) before reconstitution. Briefly, cells were incubated for 7 min at 37°C with 5 μM CFSE. The reaction was quenched by washing in ice-cold DMEM supplemented with 10% FCS. The recipient mice were injected i.p. with 3.0 × 10⁶ CFSE-labeled CD4⁺ T cells for 5–10 days. At the indicated time points, isolated splenic cells were stained with allophycocyanin-labeled anti-CD4 mAb (L3T4), PerCP-labeled anti-CD3 mAb (2C11), and PE-labeled Annexin V (MBL), and the intensity of CFSE and annexin V was determined after gating on CD3⁺CD4⁺ T cells. To assess the role of commensal bacteria in cell division, a group of mice were treated with a set of four antibiotics, i.e., ampicillin (1g/L; Sigma-Aldrich), vancomycin (500 mg/L; Abbott Laboratories), neomycin sulfate (1 g/L; Pharmacia), and metronidazole (1 g/L; Sidmack) in drinking water 2 wk before beginning the adoptive transfer and during the course of experiment based on a variation of the commensal depletion protocol of Fagarasan et al. (18). At the indicated time points, isolated spleen cells were stained with APC-labeled anti-CD4 mAb and PerCP-labeled anti-CD3 mAb, and the intensity of CFSE was determined after gating on CD3⁺CD4⁺ T cells.

Statistical analysis

The results were expressed as the mean ± SD. Groups of data were compared by Mann-Whitney U test. Differences were considered to be statistically significant when p < 0.05.

Lack of colitis in IL-7^−/− × RAG-1^−/− mice transferred with CD4⁺CD45RB^high T cells

We used a chronic colitis model induced by an adoptive transfer of splenic CD4⁺CD45RB^high T cells from normal C57BL/6 mice into IL-7^−/− × RAG-1^−/− and IL-7^+/+ × RAG-1^−/− littermate recipients to assess a requirement of IL-7 for the development of chronic colitis (Fig. 1⇓A). Consistent with previous reports (14), the control IL-7^+/+ × RAG-1^−/− mice manifested progressive weight loss from 3 wk after transfer (data not shown) and clinical symptoms of colitis as shown as clinical scores estimated by diarrhea with increased mucus in the stool, anorectal prolapse, hunched posture, and weight loss by 8 wk after transfer (Fig. 1⇓B). In contrast, the IL-7^−/− × RAG-1^−/− mice transferred with CD4⁺CD45RB^high T cells showed no clinical signs of colitis and weight loss throughout the entire observation periods (Fig. 1⇓B). At 8 wk after transfer, the colon from the transferred IL-7^+/+ × RAG-1^−/− mice, but not that from the transferred IL-7^−/− × RAG-1^−/− mice, was enlarged and had a greatly thickened wall (Fig. 1⇓C). In addition, the enlargement of the spleen and mesenteric lymph nodes was also present in the control IL-7^+/+ × RAG-1^−/− mice transferred with CD4⁺CD45RB^high T cells as compared with the IL-7^−/− × RAG-1^−/− mice transferred with CD4⁺CD45RB^high T cells (Fig. 1⇓C). Totally, the assessment of colitis by clinical scores showed a clear difference between the transferred IL-7^+/+ × RAG-1^−/− mice and the transferred IL-7^−/− × RAG-1^−/− mice (Fig. 1⇓B). Histological examination showed prominent epithelial hyperplasia with glandular elongation with a massive infiltration of mononuclear cells in the LP of the colon from the transferred IL-7^+/+ × RAG-1^−/− mice (Fig. 1⇓D). In contrast, the glandular elongation was mostly abrogated, and only a few mononuclear cells were observed in the LP of the colon from the transferred IL-7^−/− × RAG-1^−/− mice (Fig. 1⇓D). This difference was also confirmed by histological scoring of multiple colon sections, which was 0.16 ± 0.04 in the transferred IL-7^+/+ × RAG-1^−/− mice vs 5.16 ± 0.19 in the transferred IL-7^−/− × RAG-1^−/− mice (p < 0.01) (Fig. 1⇓E). We confirmed that the IL-7^−/− × RAG-1^−/− mice transferred with CD4⁺CD45RB^high T cells did not develop intestinal inflammation cells until 20 wk of observation after transfer (data not shown).

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FIGURE 1.

IL-7^−/− × RAG-1^−/− transferred with CD4⁺CD45RB^high T cells did not develop chronic colitis. A, IL-7^−/− × RAG-1^−/− (n = 10) and IL-7^+/+ × RAG-1^−/− (n = 10) mice were transferred with normal splenic CD4⁺CD45RB^high T cells (3 × 10⁵ cells/mouse). B, Clinical scores were determined at 8 wk after the transfer as described in Materials and Methods. Data are indicated as the mean ± SEM of seven mice in each group. ∗, p < 0.001. C, Gross appearance of the colon, spleen, and mesenteric lymph nodes from CD4⁺CD45RB^high T cell-transferred IL-7^−/− × RAG-1^−/− (top) and IL-7^+/+ × RAG-1^−/− (bottom) mice at 8 wk after the transfer. D, Histological examination of the colon from IL-7^−/− × RAG-1^+/+ and IL-7^+/+ × RAG-1^−/− recipients at 6 wk after the transfer. Original magnification: ×40 (upper); ×100 (lower). E, Histological scoring of IL-7^−/− × RAG-1^−/− and IL-7^+/+ × RAG-1^−/− mice transferred with CD4⁺CD45RB^high T cells after 8 wk after transfer. Data are indicated as the mean ± SEM of seven mice in each group. ∗, p < 0.005. LP (F) and spleen (G) CD4⁺ T cells were isolated from IL-7^−/− × RAG-1^−/− and IL-7^+/+ × RAG-1^−/− mice transferred with CD4⁺CD45RB^high T cells 8 wk after transfer, and the number of CD4⁺ cells was determined by flow cytometry. Data are indicated as the mean ± SEM of seven mice in each group. ∗, p < 0.01. H, Cytokine production by LP CD4⁺ T cells. LP CD4⁺ T cells were isolated at 8 wk after transfer and stimulated with anti-CD3 and anti-CD28 mAbs for 48 h. IFN-γ, IL-2, and TNF-α concentrations in culture supernatants were measured by ELISA. Data are indicated as the mean ± SD of seven mice in each group. ∗, p < 0.001. I, Phenotypic characterization of LP CD4⁺ T cells isolated from IL-7^−/− × RAG-1^−/− and IL-7^+/+ × RAG-1^−/− mice transferred with CD4⁺CD45RB^high T cells 8 wk after transfer.

A further quantitative evaluation of CD4⁺ T cell infiltration was made by isolating LP and splenic CD4⁺ T cells. Only a few CD4⁺ T cells were recovered from the colonic tissue of the transferred IL-7^−/− × RAG-1^−/− mice as compared with the transferred IL-7^+/+ × RAG-1^−/− mice (Fig. 1⇑F). The number of CD4⁺ cells recovered from the colon of the transferred IL-7^+/+ × RAG-1^−/− mice (150.5 ± 3.23 × 10⁵) far exceeded the number of originally injected cells (3.0 × 10⁵), indicating an extensive T cell proliferation and survival in the inflamed colon, which was mostly abrogated in the transferred IL-7^−/− × RAG-1^−/− mice (0.60 ± 0.65 × 10⁵) (Fig. 1⇑F). Furthermore, the number of CD4⁺ splenocytes from the transferred IL-7^+/+ × RAG-1^−/− mice was also significantly increased as comparable to that from the transferred IL-7^−/− × RAG-1^−/− recipients (Fig. 1⇑G). We also examined the cytokine production by LP CD4⁺ T cells from the transferred IL-7^+/+ × RAG-1^−/− mice and the transferred IL-7^−/− × Rag-1^−/− mice. As shown in Fig. 1⇑H, LP CD4⁺ T cells from the transferred IL-7^−/− × RAG-1^−/− mice produced significantly less IFN-γ, IL-2, and TNF-α as compared with those from the transferred IL-7^+/+ × RAG-1^−/− mice upon in vitro stimulation. Importantly, flow cytometry analysis revealed that the LP CD4⁺ T cells isolated from both IL-7^+/+ × RAG-1^−/− and IL-7^−/− × RAG-1^−/− recipients 8 wk after an adoptive transfer of CD4⁺CD45RB^high T cells were CD44^highCD62L⁻CD69⁺CD45RB^lowIL-7Rα^high (Fig. 1⇑I), indicating that the transferred CD4⁺CD45RB^high T cells could differentiate to activated T_EM cells even in the absence of IL-7. These results suggest that the lack of IL-7 prevented the development of colitis primarily by inhibiting the expansion and/or survival of colitigenic CD4⁺ T_EM cells in the colon and secondarily by inhibiting the development of the Th1 T_EM cells.

Kinetics of development of colitis and IL-7Rα expression

When the immune system is first challenged by exposure to Ags, naive CD4⁺ T cells become activated and undergo many rounds of expansion as they differentiate into effector and memory CD4⁺ T cells. Because it is believed that IL-7 is not involved in the initial expansion of the effector cells but its role in maintaining pool size may influence either the onset or maintenance of colitis, we assessed the kinetics of the development of colitis and IL-7Rα expression to determine whether IL-7 is crucial for the onset of colitis in our model (Fig. 2⇓A). At 1 wk after an adoptive transfer of CD4⁺CD45RB^high T cells into IL-7^+/+ × RAG-1^−/− and IL-7^−/− × RAG-1^−/− mice, both mice did not develop colitis (Fig. 2⇓B), and the average histological scores in both mice were 0 (Fig. 2⇓C). At 2 wk after the transfer, however, the transferred IL-7^+/+ × RAG-1^−/− recipients develop mild colitis, characterized with the infiltration of a small number of mononuclear cells in the LP, but the transferred IL-7^−/− × RAG-1^−/− recipients did not (Fig. 2⇓B). The average histology scores revealed that the transferred IL-7^+/+ × RAG-1^−/− recipients had significantly higher colitis scores as compared with the transferred IL-7^−/− × RAG-1^−/− recipients (Fig. 2⇓C). Four weeks after the transfer, the difference between two groups was more apparent, as the transferred IL-7^+/+ × RAG-1^−/− mice developed more severe colitis, but the transferred IL-7^−/− × RAG-1^−/− mice did not (Fig. 2⇓, B and C). To examine the effects of IL-7 on the expansion of CD4⁺ T cells in the recipients, we compared the infiltration of CD4⁺ T cells by determining the number of CD4⁺ T cells in the spleen and the LP by FACS analysis. In accordance with the histological scores, the average number of LP and splenic CD4⁺ T cells recovered from the transferred IL-7^+/+ × RAG-1^−/− recipients were significantly increased from 2 wk after the transfer as compared with those from the transferred IL-7^−/− × RAG-1^−/− recipients (Fig. 2⇓D).

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FIGURE 2.

Kinetics of development of colitis and IL-7Rα expression. A, IL-7^−/− × RAG-1^−/− (n = 12) and IL-7^+/+ × RAG-1^−/− (n = 10) mice were transferred with CD4⁺CD45RB^high T cells (3 × 10⁵ cells/mouse) and assessed at the indicated time points (each n = 4). B, Histological examination of the colon from IL-7^−/− × RAG-1^−/− and IL-7^+/+ × RAG-1^−/− mice transferred with colitogenic LP CD4⁺CD44^highCD62L⁻ T_EM cells at 1, 2, and 4 wk after transfer. Colonic sections were stained with H&E and photographed at ×100 magnification. C, Histological scoring of IL-7^−/− × RAG-1^−/− and IL-7^+/+ × RAG-1^−/− recipients at the indicated time points. Data are indicated as the mean ± SEM of seven mice in each group. ∗, p < 0.05. D, Cell number of splenic and LP CD4⁺ T cells recovered from IL-7^+/+ × Rag-1^−/− and IL-7^−/− × Rag-1^−/− recipients. Data are indicated as the mean ± SEM of seven mice in each group. ∗, p < 0.05. E, Cell surface IL-7Rα expression on CD4⁺CD45RB^high T cells before transfer and LP CD4⁺ T cells from IL-7^+/+ × Rag-1^−/− and IL-7^−/− × Rag-1^−/− recipients at the indicated time points.

To obtain a more comprehensive understanding of the development of effector and memory CD4⁺ T cells over time in this setting, we carefully examined the cell surface expression of IL-7Rα on LP CD4⁺ T cells obtained from the recipients at the indicated time points. As shown in Fig. 2⇑E, the splenic CD4⁺ T cells (donor CD4⁺CD45RB^high T cells) before the transfer (0 wk) and the LP CD4⁺ T cells after 1 wk of transfer highly expressed IL-7Rα, but approximately half of CD4⁺ T cells from both the transferred IL-7^+/+ × RAG-1^−/− and IL-7^−/− × RAG-1^−/− recipients had down-regulated IL-7Rα at 2 wk after transfer. However, a large portion of LP CD4⁺ T cells from both the transferred mice at 4 wk after the transfer had regained higher expression of IL-7Rα to >80% per total CD4⁺ T cells (Fig. 2⇑E). These data indicate that IL-7Rα expression was regulated during T cell differentiation from CD4⁺CD45RB^highIL-7Rα^high naive T cells (0 wk before the transfer), CD4⁺IL-7Rα^low effector T cells (1 and 2 wk after the transfer), to CD4⁺IL-7Rα^high memory T cells (4 wk after the transfer), suggesting that naive and memory CD4⁺ T cells may be more responsive to IL-7-mediated signaling.

TCR Vβ repertoires in IL-7^+/+ × RAG-1^−/− and IL-7^−/− × RAG-1^−/− mice transferred with CD4⁺CD45RB^high T cells

Analysis of the TCR repertoire of the immunodeficient SCID/Rag-1/2^−/− recipients in the CD4⁺CD45RB^high T cell transfer model may provide an opportunity to characterize the unique T cell population present in the diseased individuals in a manner not possible in clinical studies on human patients. Furthermore, it was also possible that the adoptive transfer of CD4⁺CD45RB^high T cells to IL-7^−/− × RAG-1^−/− recipients gains more skewed and restricted clonality of CD4⁺ T cells as compared with that in IL-7^+/+ × RAG-1^−/− recipients. To assess this possibility, we next made a comparison between TCR Vβ repertoires of splenic CD4⁺ T cells from colitic CD4⁺CD45RB^high T cell-transferred and IL-7^−/− × RAG-1^−/− and IL-7^+/+ × RAG-1^−/− and normal age-matched wild-type mice. Flow cytometric analysis of these splenic CD4⁺ cells using a panel of 15 anti-Vβ mAbs showed that most major Vβ population was Vβ8.1/8.2 in all three groups, i.e., age-matched C57BL/6J and IL-7^−/− × RAG-1^−/− and IL-7^+/+ × RAG-1^−/− mice transferred with CD4⁺CD45RB^high T cells. Although only a Vβ4 ratio in colitic IL-7^+/+ × RAG-1^−/− recipients was significantly decreased as compared with that in normal C57BL/6J mice, there were no significant differences of other Vβ repertoires between these two groups (Fig. 3⇓). In noncolitic IL-7^−/− × RAG-1^−/− recipients, however, the repertoires of Vβ3 and Vβ10b were significantly decreased as compared with those in normal C57BL/6J mice and colitic IL-7^+/+ × RAG-1^−/− recipients (Fig. 3⇓), indicating that the lack of IL-7 suppressed the expansion of certain type of Vβ populations as compared with other repertoires.

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FIGURE 3.

Flow cytometric analysis of Vβ families on the surface of the splenic CD4⁺ T cells. To analyze the TCR Vβ family repertoire, splenic cells were double-stained with PE-conjugated anti-CD4 mAb (RM4-5), and the following splenic cells were double-stained with a panel of 15 FITC-conjugated Vβ mAbs. Each percentage value indicates the frequency of each Vβ pooled from three independent experiments (n = 12). ∗, p < 0.05.

Lack of colitis in IL-7^−/− × RAG-1^−/− mice transferred with colitogenic LP CD4⁺ T_EM cells

To next assess the role of IL-7 in the persistent colitis without the impact of naive T cell priming, activation, and differentiation, we first isolated LP CD4⁺ T cells as colitogenic CD4⁺ T_EM cells from CD4⁺CD45RB^high T cell-transferred colitic RAG-2^−/− mice at 4–6 wk after transfer because flow cytometry analysis revealed that the colitic LP CD4⁺ T cells were CD44^high CD62L⁻IL-7Rα^high T_EM cells (Fig. 1⇑I), and we previously demonstrated that the adoptive transfer of these cells into new IL-7-competent RAG-2^−/− mice induces chronic colitis (17). We then transferred these LP CD4⁺ T_EM cells into IL-7^+/+ × RAG-1^−/− mice (IL-7^+/+→IL-7^+/+) and the IL-7^−/− × RAG-1^−/− mice (IL-7^+/+→IL-7^−/−) to focus on the persistence of colitogenic CD4⁺ T_EM cells (Fig. 4⇓A). IL-7^+/+→IL-7^+/+ recipients developed a severe colitis 4 wk after the transfer, characterized by significant weight loss, diarrhea, and higher total clinical scores (Fig. 4⇓B) and thickening of the colonic wall with inflammation (Fig. 4⇓C). Average histological scores characterized by transmural inflammation with high numbers of lymphocytes in the LP and submucosa, and prominent epithelial hyperplasia with loss of goblet cells (Fig. 4⇓D) were 4.83 ± 0.98 in IL-7^+/+→IL-7^+/+ mice (Fig. 4⇓E). In contrast, IL-7^+/+→IL-7^−/− recipients appeared healthy and did not exhibit any signs of colitis (Fig. 4⇓B), with gradual increase of body weight and no apparent thickening of the colonic wall (Fig. 4⇓C). No evident pathological changes were observed in the colon (Fig. 4⇓D). This histological difference was also confirmed by histological scoring, which was 0 in IL-7^+/+→IL-7^−/− recipient mice (p < 0.005 as compared with IL-7^+/+→IL-7^+/+ mice) (Fig. 4⇓E). The average recovered numbers of LP and splenic CD4⁺ T cells from colitic IL-7^+/+→IL-7^+/+ recipients were 173.7 ± 11.2 × 10⁵ cells/colon (Fig. 4⇓F) and 51.5 ± 11.3 × 10⁵ cells/spleen (Fig. 4⇓G), respectively, whereas those from IL-7^+/+ → IL-7^−/− mice were 0.64 ± 0.7 × 10⁵ cells/colon (Fig. 4⇓F) (p < 0.01) and 0.54 ± 0.29 × 10⁵ cells/spleen (Fig. 4⇓G) (p < 0.05), respectively. As shown in Fig. 4⇓H, LP CD4⁺ T cells from IL- 7^+/+→IL-7^−/− recipients produced significantly less IFN-γ, IL-2, and TNF-α as compared with those from IL-7^+/+→IL-7^+/+ mice upon in vitro stimulation (Fig. 4⇓H). Furthermore, flow cytometry analysis showed that the LP CD4⁺ T cells isolated from both IL-7^+/+→IL-7^+/+ and IL-7^+/+→IL-7^−/− recipients were sustained by the phenotype of CD44^highCD62L⁻IL-7Rα^high T_EM cells (Fig. 4⇓I).

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FIGURE 4.

IL-7^−/− × RAG-1^−/− transferred with colitogenic LP CD4⁺CD44^highCD62L⁻ T_EM cells did not develop chronic colitis. A, Colitogenic LP CD4⁺CD44^highCD62L⁻ T_EM cells obtained from colitic mice transferred with CD4⁺CD45RB^high T cells were transferred into new IL-7^+/+ × RAG-1^−/− (IL-7^+/+→IL-7^+/+; n = 10) and IL-7^−/− × RAG-1^−/− (IL-7^+/+→IL-7^−/−; n = 10) mice. B, Clinical scores were determined 4 wk after transfer as described in Materials and Methods. Data are indicated as the mean ± SEM of seven mice in each group. ∗, p < 0.005. C, Gross appearance of the colon, spleen, and mesenteric lymph nodes from IL-7^+/+→IL-7^−/− (top) and IL-7^+/+→IL-7^+/+ (bottom) mice 4 wk after transfer. D, Histological examination of the colon from IL-7^+/+→IL-7^−/− and IL- 7^+/+→IL-7^+/+ mice 4 wk after transfer. Original magnification: ×40 (upper); ×100 (lower). E, Histological scoring of IL-7^+/+→IL-7^−/− and IL-7^+/+→IL-7^+/+ mice 4 wk after transfer. Data are indicated as the mean ± SEM of seven mice in each group. ∗, p < 0.005. LP (F) and spleen (G) CD4⁺ T cells were isolated from IL-7^+/+→IL-7^−/− and IL-7^+/+→IL-7^+/+ mice 4 wk after transfer, and the number of CD4⁺ cells was determined by flow cytometry. Data are indicated as the mean ± SEM of seven mice in each group. ∗, p < 0.01. H, Cytokine production by LP CD4⁺ T cells. CD4⁺ LPL were isolated from each mouse at 4 wk after transfer and stimulated with anti-CD3 and anti-CD28 mAbs for 48 h. IFN-γ, IL-2, and TNF-α concentrations in culture supernatants were measured by ELISA. Data are indicated as the mean ± SD of seven mice in each group. ∗, p < 0.005. I, Phenotypic characterization of LP CD4⁺ T cells isolated from IL- 7^+/+→IL-7^−/− and IL-7^+/+→IL-7^+/+ mice 4 wk after transfer.

IL-7 is essential for the survival of colitogenic CD4⁺ T_EM cells

To further examine the effect of IL-7 on the proliferation and the survival of the colitogenic LP CD4⁺ T_EM cells in vivo, we used the CFSE dilution method since dilution of CFSE provided a division history of the cells, allowing us to examine cells undergoing proliferation. First, the LP CD4⁺ T_EM cells obtained from CD4⁺CD45RB^high-transferred colitic mice were labeled with CFSE and adoptively transferred into IL-7^+/+ × RAG-1^−/− or IL-7^−/− × RAG-1^−/− mice. Although the relatively delayed division of expanded CD4⁺ T cells in IL-7^−/− × RAG-1^−/− mice at the indicated time points, the LP CD4⁺ T cells had markedly divided until 10 days after transfer both in the IL-7^−/− × RAG-1^−/− mice and the IL-7^+/+ × RAG-1^−/− recipients, indicating that it appears IL-7 is not essential for colitogenic CD4⁺ T_EM cells to undergo lymphopenia-driven rapid proliferation (19). Interestingly, consistent with the markedly and significantly decreased cell numbers of splenic and LP CD4⁺ T cells in CD4⁺CD45RB^high T cell (Fig. 1⇑, F and G)- or colitogenic LP CD4⁺ T cell (Fig. 4⇑, F and G)-transferred IL-7^−/− × RAG-1^−/− mice as compared with those in the paired transferred IL-7^+/+ × RAG-1^−/− mice, CFSE⁻ cells that have divided more than eight times (19) in IL-7^−/− × RAG-1^−/− mice were stained with annexin V with higher percentages at day 7 after transfer as compared with those in IL-7^+/+ × RAG-1^−/− mice (Fig. 5⇓A), indicating that rapidly dividing cells in IL-7^−/− × RAG-1^−/− mice could not survive to maintain their cell number. To further address the survival checkpoint, we next assessed whether regulation of Bcl-2 requires IL-7 at 7 days after the transfer since induction of the anti-apoptotic protein, Bcl-2, is a hallmark of responses to IL-7 (20). As expected, the splenic CD4⁺ T cells in the transferred IL-7^−/− × RAG-1^−/− mice expressed significantly less level of Bcl-2 as compared with those in the transferred IL-7^+/+ × RAG-1^−/− mice (Fig. 5⇓, B and C).

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FIGURE 5.

IL-7 is essential for the survival, but not the cell turnover, of colitogenic LP CD4⁺ T_EM cells. A, Colitogenic LP CD4⁺ T_EM cells obtained from colitic mice transferred with CD4⁺CD45RB^high T cells were labeled with CFSE and adoptively transferred into IL-7^+/+ × RAG-1^−/− (n = 16) or IL-7^−/− × RAG-1^−/− (n = 16) mice. At the indicated time points after transfer (days 4, 8, and 12), CFSE incorporation was determined by flow cytometry. Histograms are gated on CD4⁺ T cells. These data were representative from six experiments. At day 8 after transfer, the gated CFSE⁻ cells represent rapid proliferation, and CFSE⁺ cells were also stained with annexin V. B, Representative flow cytometric histograms showing the expression of Bcl-2 in LP CD4⁺ T cells from IL-7^−/− × RAG-1^−/− or IL-7^+/+ × RAG-1^−/− mice transferred with the colitogenic LP CD4⁺ T cells 7 days after the transfer. C, Average percentage of LP Bcl-2⁺CD4⁺ T cells from IL-7^−/− × RAG-1^−/− (n = 6) or IL-7^+/+ × RAG-1^−/− (n = 6) recipients 7 days after the transfer. ∗, p < 0.05.

Commensal bacteria-partially dependent rapid proliferation and IL-7-dependent slow proliferation

Because we found that IL-7 is essential for the development and persistence of colitis, and it has been previously demonstrated that the presence of commensal bacteria are needed to develop and sustain chronic colitis in various models of colitis (1), we finally addressed this point in the adoptive transfer setting using IL-7^−/− × RAG-1^−/− and IL-7^+/+ × RAG-1^−/− recipients treated with or without antibiotics treatment by CFSE dilution assay at 7–21 days after transfer. In this experiment, we used normal splenic CD4⁺CD25⁻ T cells, including CD4⁺CD45RB^high naive T cells and CD4⁺CD45RB^low T_EM cells, but not CD4⁺CD25⁺ regulatory T cells, rather than colitogenic LP CD4⁺ T cells as donor cells (Fig. 5⇑) to assess two types of cell division, rapid (spontaneous, endogenous) proliferation and slow (homeostatic) proliferation (19, 21, 22). As shown in Fig. 6⇓, slow proliferation in IL-7^+/+ × RAG-1^−/− recipients at days 7–14 after transfer was observed as two to three peaks of dividing cells regardless of the antibiotic treatment, whereas none of slowly dividing cells was observed in IL-7^−/− × RAG-1^−/− recipients, indicating that slow proliferation is dependent on the presence of IL-7. In contrast, rapid proliferation in IL-7^−/− × RAG-1^−/− and IL-7^+/+ × RAG-1^−/− recipients treated with antibiotics at days 7 and 14 after transfer was partially but not completely impaired as compared with that in both IL-7^−/− × RAG-1^−/− and IL-7^+/+ × RAG-1^−/− recipients without antibiotics treatment (Fig. 6⇓), indicating that rapid proliferation in these recipients is driven not only by the presence of commensal bacterial Ags, but also presumably by other environmental Ags, such as food and bedding, and/or self-Ags. Furthermore, although rapid-proliferating cells in IL-7^−/− × RAG-1^−/− recipients should have divided over eight times (19) as is in a similar manner with IL-7^+/+ × RAG-1^−/− recipients, rapid-dividing area of IL- 7^−/− × RAG-1^−/− recipients was markedly decreased as compared with that of IL-7^−/− × RAG-1^−/− recipients, indicating that rapid-proliferating cells in IL-7^−/− × RAG-1^−/− recipients were subjected to undergo apoptosis or the rate of rapid-proliferating cells in IL-7^+/+ × RAG-1^−/− recipients were significantly faster as undetectable as for cells divided over eight times (19) by this CSFE method.

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FIGURE 6.

Commensal bacteria-partially dependent rapid proliferation and IL-7-dependent slow proliferation. Aliquots (3 × 10⁶/mouse) of CFSE-labeled CD4⁺CD25⁻ cells obtained from normal spleen were injected into IL-7^−/− × RAG-1^−/− and IL-7^+/+ × RAG-1^−/− mice treated with or without a set of four antibiotics (ampicillin, vancomycin, neomycin sulfate, and metronidazole) and analyzed at the indicated time points. Data are representative of five independent experiments.

Sustained CD4⁺ T cells in IL-7^−/− × RAG-1^−/− recipients do not have a potential to induce colitis when transferred to IL-7^+/+ × RAG-1^−/− recipients

Finally, we address a question whether sustained CD4⁺CD44^highCD62L⁻ effector-memory type of T cells in IL-7^−/− × RAG-1^−/− mice transferred with CD4⁺CD45RB^high T cells (Fig. 1⇑) have a potential to induce colitis if they were transferred to new IL-7-competent IL-7^+/+ × RAG-1^−/− mice. Because it was very important to assess a possibility that a small but substantial number of CD4⁺ T cells would be maintained by other factors, such as commensal bacterial Ag-driven TCR signaling and IL-15, as suggested by others (16, 23, 24, 25) in CD4⁺CD45RB^high T cell-transferred IL-7^−/− × RAG-1^−/− mice, but not enough to expand to induce colitis due to the absence of IL-7, we isolated splenic CD4⁺ T cells from colitic CD4⁺CD45RB^high T cell-transferred colitic IL-7^+/+ × RAG-1^−/− mice and noncolitic IL-7^−/− × RAG-1^−/− mice at 8 wk after transfer (Fig. 7⇓A). We next retransferred these splenic CD4⁺ T cells into new IL-7^+/+ × RAG-1^−/− mice (Fig. 7⇓A). Expectedly and similarly with the result from the adoptive transfer of colitic LP CD4⁺ T cells (Fig. 4⇑), IL-7^+/+ × RAG-1^−/− recipients transferred with splenic CD4⁺ T cells from colitic CD4⁺CD45RB^high T cell-transferred IL-7^+/+ × RAG-1^−/− (IL-7^+/+→ IL-7^+/+) mice developed a severe colitis until 4–6 wk after the transfer, characterized by significant weight loss, diarrhea, and higher total clinical scores (Fig. 7⇓B) and thickening of the colonic wall with inflammation (Fig. 7⇓, C and D). In contrast, IL-7^+/+ × RAG-1^−/− recipient (IL-7^−/−→IL-7^+/+) mice appeared healthy and did not exhibit any signs of colitis until 9 wk after transfer (Fig. 7⇓B), and no apparent thickening of the colonic wall (Fig. 7⇓C). No evident pathological changes were observed in the colon (Fig. 7⇓D). Average histological scores characterized by severe inflammation and epithelial hyperplasia (Fig. 7⇓D) were 4.90 ± 0.87 in those IL-7^+/+→IL-7^+/+ mice in contrast to 0.42 ± 1.13 in those IL-7^−/−→IL-7^+/+ mice (p < 0.001) (Fig. 7⇓E). The average recovered numbers of LP and splenic CD4⁺ T cells from colitic IL-7^+/+→IL-7^+/+ mice were 190.8 ± 77.3 × 10⁵ cells/colon (Fig. 7⇓F) and 31.3 ± 18.3 × 10⁵ cells/spleen (Fig. 7⇓G), respectively, whereas those from noncolitic IL-7^−/−→IL-7^+/+ mice were 29.7 ± 39.9 × 10⁵ cells/colon (Fig. 7⇓F) (p < 0.005) and 6.23 ± 13.68 × 10⁵ cells/spleen (Fig. 7⇓G) (p < 0.05), respectively. As shown in Fig. 7⇓H, LP CD4⁺ T cells from noncolitic IL-7^−/−→IL-7^+/+ mice produced significantly less IFN-γ, IL-2, and TNF-α as compared with those from colitic IL-7^+/+→IL-7^+/+ mice (Fig. 7⇓H). Furthermore, flow cytometry analysis showed that the LP CD4⁺ T cells isolated from both colitic IL-7^+/+→IL-7^+/+ recipients and noncolitic IL-7^−/−→IL-7^+/+ mice were sustained the phenotype of CD44^highCD62L⁻IL-7Rα^high T_EM cells (Fig. 7⇓I).

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FIGURE 7.

Sustained CD4⁺ T cells in IL-7^−/− × RAG-1^−/− recipients do not have a potential to induce colitis when transferred to IL-7^+/+ × RAG-1^−/− recipients. A, IL-7^+/+ × RAG-1^−/− mice were transferred with splenic CD4⁺ T cells obtained from colitic CD4⁺CD45RB^high T cell-transferred IL-7^+/+ × RAG-1^−/− mice (IL-7^+/+→IL-7^+/+, n = 7) and noncolitic CD4⁺CD45RB^high T cell-transferred IL-7^−/− × RAG-1^−/− mice (IL-7^−/−→IL-7^+/+, n = 7) . B, Clinical scores were determined 4 wk after transfer as described in Materials and Methods. Data are indicated as the mean ± SEM of seven mice in each group. ∗, p < 0.0005. C, Gross appearance of the colon, spleen, and mesenteric lymph nodes from IL-7^−/−→IL-7^+/+ (top) and IL-7^+/+→IL-7^+/+ (bottom) mice 9 wk after transfer. D, Histological examination of the colon from IL-7^−/−→IL-7^+/+ and IL-7^+/+→IL-7^+/+ mice 9 wk after transfer. Original magnification: ×40 (upper); ×100 (lower). E, Histological scoring of IL-7^−/−→IL-7^+/+ and IL-7^+/+→IL-7^+/+ mice 4 wk after transfer. Data are indicated as the mean ± SEM of seven mice in each group. ∗, p < 0.001. LP (F) and spleen (G) CD4⁺ T cells were isolated from IL-7^−/−→IL-7^+/+ and IL-7^+/+→IL-7^+/+ mice 4 wk after transfer, and the number of CD4⁺ cells was determined by flow cytometry. Data are indicated as the mean ± SEM of seven mice in each group. LP, ∗, p < 0.005; spleen, ∗, p < 0.05. H, Cytokine production by LP CD4⁺ T cells. CD4⁺ LPL were isolated from each mouse at 4 wk after transfer and stimulated with anti-CD3 and anti-CD28 mAbs for 48 h. IFN-γ, IL-2, and TNF-α concentrations in culture supernatants were measured by ELISA. Data are indicated as the mean ± SD of seven mice in each group. ∗, p < 0.005. I, Phenotypic characterization of LP CD4⁺ T cells isolated from IL- 7^−/−→IL-7^+/+ and IL-7^+/+→IL-7^+/+ mice 9 wk after transfer.