CCL2 deficient mesenchymal stem cells fail to establish long-lasting contact with T cells and no longer ameliorate lupus symptoms

Abstract

Systemic lupus erythematosus (SLE) is a multi-organ autoimmune disease characterized by autoantibody production. Mesenchymal stem cells (MSCs) ameliorate SLE symptoms by targeting T cells, whereas the mechanisms of their efficacy remain incompletely understood. In this study, we show that transfer of human MSCs increased MRL.Fas^lpr mouse survival, decreased T cell infiltration in the kidneys, and reduced T cell cytokine expression. In vitro, allogeneic mouse MSCs inhibited MRL.Fas^lpr T cell proliferation and cytokine production. Time-lapse imaging revealed that MSCs recruited MRL.Fas^lpr T cells establishing long-lasting cellular contacts by enhancing T cell VCAM-1 expression in a CCL2-dependent manner. In contrast, CCL2 deficient MSCs did not induce T cell migration and VCAM-1 expression, resulting in insufficient cell-cell contact. Consequently, CCL2 deficient MSCs did not inhibit IFN-γ production by T cells and upon transfer no longer prolonged survival of MRL.Fas^lpr mice. Taken together, our imaging study demonstrates that CCL2 enables the prolonged MSC-T cell interactions needed for sufficient suppression of autoreactive T cells and helps to understand how MSCs ameliorate symptoms in lupus-prone MRL.Fas^lpr mice.

Figure 1. MSCs ameliorate SLE development in MRL.Fas^lpr mice (multiple injections).

MRL.Fas^lpr mice were intravenously injected with vehicle (control, n = 5), MSCs (1 × 10⁶ cells/injection, n = 6), or cyclophosphamide (CP, 50 mg/kg, n = 6) 6 times (arrows) every two weeks from the age of 10 weeks. (a,b) Survival (a) and body weight (b) were measured every week. (c–e) Serum and urine were collected every two weeks. The levels of anti-dsDNA IgG (c) and total IgG (d) in serum and the level of proteinuria (e) were measured. *p < 0.01 versus control.

Figure 2. MSCs ameliorate SLE development in MRL.Fas^lpr mice (single injection).

MRL.Fas^lpr mice were intravenously injected with vehicle (control, n = 5), MSCs (4 × 10⁴ cells, 4 × 10⁵ cells, or 4 × 10⁶ cells/injection, n = 5), or cyclophosphamide (CP, 50 mg/kg, n = 5) once at the age of 12 weeks. (a–c) Survival (a) and body weight (b) were measured every week. The serum levels of anti-dsDNA IgG were measured every two weeks (c). (d,e) Mice were sacrificed at the age of 20 weeks and spleens were isolated. Total RNA was isolated from spleen cells and the expression of inflammatory cytokine genes was examined by RT-PCR (d). Subset ratios were analyzed by flow cytometry (e). *p < 0.01 versus control.

Figure 3. MSCs inhibit T cells via soluble factor- and contact-dependent mechanisms.

(a,b) T cells (1 × 10⁵ cells/well) were activated by the T cell-specific mitogen concanavalin A (Con A, 1 μg/ml) in the presence or absence of MSCs (0.1 × 10⁵ cells/well) for 72 h (n = 3). The levels of IFN-γ and IL-2 in culture medium were measured by ELISA (a) and T cell proliferation was measured by the mitogen assay (b). *p < 0.01. (c) MSCs (0.1 × 10⁵ cells/well) were added to the upper wells and T cells (1 × 10⁵ cells/well) to the lower wells to avoid cell–cell contact in transwell plates having a 5 μm insert. MSCs (0.1 × 10⁵ cells/well) and T cells (1 × 10⁵ cells/well) were also added to the lower wells to ensure cell–cell contact. After incubation with Con A for 72 h, the mitogen assay was performed (n = 3). *p < 0.01. (d) The levels of NO, TGF-β, and PGE₂ accumulated in culture medium of MSCs for 24 h were measured. *p < 0.01 (n = 3). (e) MSCs (0.1 × 10⁵ cells/well) and T cells (1 × 10⁵ cells/well) were co-cultured in the presence of Con A for 24 h. Another experiment, MSCs were treated with only IFN-γ (100 U/ml) for 24 h. The levels of IDO were measured with ELISA (n = 3). *p < 0.01.

Figure 4. T cell migration towards CCL2-KD MSCs.

(a–c) Balb/c MSCs were transfected with negative (control), CCL2, CCL3, CCL4, CXCL10, or CXCL12 siRNA. T cells were purified from spleen cells of MRL.Fas^lpr mice. For time-lapse imaging, MSCs (70 μl of 0.3 × 10⁶ cells/ml) were seeded into the left chamber and T cells (70 μl of 3 × 10⁶ cells/ml) into the right chamber of culture-insert μ-Dish^35mm culture dishes. Images were acquired every 2 min for 6 h (n = 3). Representative snapshots (a), the tracks of T cells in white boxes (b), and the number of T cells passing through the white boxes are shown (c). *p < 0.01 versus control. (d) Control or chemokine-knockdown (KD) MSCs (0.3 to 3 × 10⁴ cells/well) were added to the lower wells and MRL.Fas^lpr T cells (1 × 10⁴ cells/well) to the upper wells of transwell plates having a 5 μm insert. After 1.5 h, the number of T cells migrating to the lower well was determined (n = 3). *p < 0.01 versus control.

Figure 5. T cell migration towards Ccl2^-/- MSCs.

(a,b) For time-lapse imaging, MSCs (70 μl of 0.3 × 10⁶ cells/ml) were seeded into the left chamber and T cells (70 μl of 3 × 10⁶ cells/ml) into the right chamber of culture-insert μ-Dish^35mm culture dishes. MSCs were generated from bone marrow cells of C57BL/6 (Ccl2^+/+) and Ccl2^−/− mice and T cells were purified from spleen cells of MRL.Fas^lpr mice. T cells were also pretreated with 30 μg/ml RS102895 for 1 h. Images were acquired every 2 min for 6 h (n = 3). Representative snapshots (a) and the number of T cells passing through the white boxes (b) are shown. *p < 0.01 versus control. (c) Ccl2^+/+ or Ccl2^−/− MSCs (0.3 to 3 × 10⁴ cells/well) were added to the lower wells and MRL.Fas^lpr T cells (3 × 10⁴ cells/ml) to the upper wells of transwell plates having a 5 μm insert. T cells were also pretreated with RS102895 (RS, 30 μg/ml) for 1 h. After 1.5 h, the number of T cells migrating to the lower well were determined (n = 3). *p < 0.01 versus Ccl2^+/+ MSC and T cell group.

Figure 6. Contact dynamics between MSCs and T cells.

Control, CXCL12-KD, and CCL2-KD MSCs were labeled with CMTMR (red). MRL.Fas^lpr T cells were labeled with CFSE (green). MSCs (0.1 × 10⁵ cells/well) and T cells (1 × 10⁵ cells/well) were mixed and added onto 35-mm culture dishes. Dishes were preincubated for 1 h under the microscope and images were acquired in three channels (phase contrast; CFSE, green filter; and CMTMR, red filter) every 2 min for 6 h (six movies from three independent experiments per group). (a) Representative images (original magnification, 100x; zoomed, 2.3×). (b) Instantaneous velocities of non-contacting T cells (n = 78, 76, and 74 from the left) and contacting T cells (n = 58, 53, and 48 from the left). (c) Representative profiles of T cell velocity. (d) The number of T cell contacts per MSC (n = 39, 31, and 32 from the left). (e) Contact duration between T cells and MSCs (n = 281, 240, and 190 from the left). *p < 0.01 versus control. Bars represent the mean of the data.

Figure 7. CCL2 deficient MSCs did not ameliorate SLE development in MRL.Fas^lpr mice.

(a) MRL.Fas^lpr T cells were activated with concanavalin A (Con A) and were co-cultured with control, CCL2-KD, or Ccl2^−/− MSCs for 72 h, and the levels of IFN-γ in culture medium were measured by ELISA (n = 3). *p < 0.01. (b–d) MRL.Fas^lpr mice were intravenously injected with vehicle (control, n = 5), Ccl2^+/+ MSCs (1 × 10⁶ cells/injection, n = 5), Ccl2^−/− MSCs (1 × 10⁶ cells/injection, n = 5), or cyclophosphamide (CP, 10 mg/kg, n = 5) once at the age of 12 weeks. Survival (b) was measured every week and total IgG level in serum (c) and protein level in urine (d) was measured at the age of 22 weeks. *p < 0.01 versus control.

Figure 8. Binding rates of MSCs and T cells.

(a) Binding rates of CMTMR-labeled MSCs (0.1 × 10⁵ cells/tube) and CMFDA-labeled T cells (1 × 10⁵ cells/tube) were analyzed by flow cytometry (n = 3). The conjugation ratio was calculated as the portion of CMFDA/CMTPX double-positive events. T cells were treated with 30 μg/ml RS102895 for 1 h. *p < 0.01. (b,c) MSCs (0.1 × 10⁵ cells/well) were added to the lower wells of transwell plates having a 0.4 μm insert and preincubated for 2 h. T cells (1 × 10⁵ cells/well) were then added to the upper wells and plates were incubated for 2 h (b). T cells were pretreated with vehicle (DMSO) or RS102895 (RS) for 1 h, washed twice and then added (1 × 10⁵ cells/well) to the lower wells (c). Adhesion molecule expression in T cells was assessed by RT-PCR and western blotting (n = 3). (d) T cells were directly treated with CCL2 for 2 h in 6-well plates, and VCAM-1 expression was assessed by RT-PCR and western blotting (n = 3). (e) MSCs were stained with anti-mouse VLA4 antibody conjugated with phycoerythrin and analyzed with a flow cytometer (n = 3). (f,g) MSCs were incubated with anti-mouse VLA4-neutralizing antibody for 24 h, washed twice, and mixed with T cells. Imaging was performed every 2 min for 6 h (six movies from three independent experiments). The numbers of T cell contacts per MSC (n = 36 and 37 from the left) (f). Contact duration between T cells and MSCs (n = 267 and 270 from the left) (g). *p < 0.01. (h) MSCs were incubated with anti-mouse VLA4 antibody for 24 h, washed twice, and mixed with T cells and 1 μg/ml Con A (added to activate T cells). After 72 h, the levels of IFN-γ accumulated in culture medium were determined by ELISA (n = 3). *p < 0.01. Bars represent the mean of the data.