Allogenic Umbilical Cord-Derived Mesenchymal Stromal Cells Sustain Long-Term Therapeutic Efficacy Compared With Low-Dose Interleukin-2 in Systemic Lupus Erythematosus

Abstract

Objectives: Mesenchymal stromal cells (MSCs) and low-dose interleukin-2 (IL-2) both have demonstrated efficacy in treating systemic lupus erythematosus (SLE). The aim of this study is to conduct a head-to-head comparison between the 2 treatments and provide insights for clinical applications.

Methods: Lupus-prone mice were treated with umbilical cord-derived MSCs (UC-MSCs), IL-2, or a combination of UC-MSCs and IL-2, respectively. The lupus-like symptoms, renal pathology, and T-cell response were assessed 1 or 4 weeks later. Modulation of IL-2 production by MSCs on immune cells was investigated by the coculture assay. Disease activity and serum IL-2 of SLE patients were determined before and after receiving UC-MSCs.

Results: Both UC-MSCs and IL-2 improved lupus symptoms in lupus-prone mice 1 week after treatment, while the effects of UC-MSCs lasted up to 4 weeks. Moreover, the UC-MSC-treated group showed better renal pathology improvement. Importantly, UC-MSCs combined with IL-2 did not provide better efficacy than UC-MSCs alone. Consistent with this, UC-MSCs alone and UC-MSCs + IL-2 resulted in similar levels of serum IL-2 and frequencies of Tregs. Neutralization of IL-2 partly reduced the promotion of Tregs by UC-MSCs, suggesting that IL-2 was involved in the upregulation of Tregs by UC-MSCs. Lastly, an increase in serum IL-2 positively correlated with the reduction of disease activity of SLE patients by UC-MSCs.

Conclusion: Both the single injection of UC-MSCs and repeated IL-2 administration exerted comparable efficacy in alleviating SLE manifestations, but UC-MSCs provided sustained alleviation and showed better improvement in renal pathology.

Keywords: interleukin-2; mesenchymal stromal cells; regulatory T cells; systemic lupus erythematosus.

Graphical Abstract

Figure 1.

UC-MSCs or low-dose IL-2 treatment alleviated lupus diseases in MRL/lpr mice. (A) Schematic illustration of experiments. (B-C) Spleen/body weight ratio (mg/g) (B) and lymph nodes/body weight ratio (mg/g) (C) were calculated. (D-G) Serum concentrations of anti-dsDNA antibody (D), ANA (E), IgG (F), IFN-γ, and IL-10 (G) in MRL/lpr mice were determined by ELISA. All the experiments were repeated 3 times. n = 5, per group. *P < .05, **P < .01, ***P < .001, ****P < .0001.

Figure 2.

UC-MSCs treatment provided long-term alleviation of renal damage. (A-C) 24 h proteinuria (A), serum creatinine (B), and BUN (C) were measured in the respective groups at 1 or 4 weeks after treatment. The level of 24 h proteinuria was expressed as micrograms of protein in 24-h urine. (D-E) Glomerular pathology quantified as glomerular, interstitial, and perivascular scores of mice receiving different treatments were determined. (D) Representative images of H&E-stained kidneys. Scale bar, 50 μm. (E) Histological scores of renal lesions were calculated by the severity of glomerulonephritis, interstitial nephritis, and vessels, graded on a 1-4 scale. All the experiments were repeated 3 times. n = 5, per group. *P < .05, **P < .01, ***P < .001, ****P < .0001.

Figure 3.

UC-MSCs treatment maintained long-term inhibition of immune complexes deposition. Kidney damage was assessed by evaluation for glomerular immunoglobulin deposition, as well as immunofluorescence intensity. (A) Representative kidney immunofluorescence staining of IgG (green) and C3 (red) deposition in the respective groups. Scale bar, 30 μm. (B-C) Quantification of IgG (B) and C3 (C) immunofluorescence intensity was normalized to PBS group after analyzed by ImageJ. All the experiments were repeated 3 times. n = 5, per group. *P < .05, **P < .01, ***P < .001, ****P < .0001.

Figure 4.

UC-MSCs regulated T-cell subtypes. (A-B) Flow cytometric analysis of T_H1 and T_H2 cells in the lymph nodes of MRL/lpr mice in the respective groups. (A) Representative staining profiles of T_H1 and T_H2 cells and (B) percentages of T_H1 and T_H2 cells were shown. (C-D) Flow cytometric analysis of Tregs in the spleens, PBMCs, and lymph nodes of MRL/lpr mice in the respective groups. (C) Representative staining profiles of Tregs in the lymph nodes and (D) percentages of Tregs in the lymph nodes, PBMCs, and spleens were shown. (E) The ratio of T_H1/Treg in the lymph nodes, PBMCs, and spleens was shown. PBS group in 1-week treatment (A-B, E), n = 4. Other groups, n = 5. All the experiments were repeated 3 times. *P < .05, **P < .01, ***P < .001, ****P < .0001. w, week.

Figure 5.

IL-2 was involved in MSCs-induced Tregs. (A) Serum IL-2 of MRL/lpr mice in the respective groups was determined by ELISA. (B) Splenocytes of C57BL/6 mice were co-cultured with MSCs in vitro at different ratios and 12 h later, fold change of il-2 mRNA was determined by qRT-PCR. (C-D) Splenocytes of C57BL/6 mice were co-cultured with MSCs at 1:30 (as MSCs: splenocytes) and 24 h later, (C) concentrations of IL-2 in culture supernatants were measured by ELISA and (D) percentages of Tregs were determined by FACS. (E) Splenocytes of C57BL/6 mice were co-cultured with MSCs in the presence of IL-2 neutralizing antibodies or not, then were harvested 24 h later. Percentages of Tregs were determined. (F-G) Frequencies of CD3⁺IL-2⁺, B220⁺IL-2⁺, and CD11b⁺IL-2⁺cells in splenocytes from mice treated with MSCs and PBS for 4 weeks. All the experiments were repeated 3 times. n = 3-6, per group. *P < .05, **P < .01, ***P < .001, ****P < .0001.

Figure 6.

Neutralization of IL-2 significantly impaired the therapeutic effects of MSCs on SLE. After receiving MSCs, MRL/lpr mice were treated with IL-2 neutralizing antibodies or istotype IgG. Mice that received PBS only were set as control. One week later, lymph nodes, PBMCs, and spleens were collected. (A-B) Percentages of Tregs were determined by FACS and representative staining profiles of lymph nodes were shown. (C) Spleens were weighed and spleen/body weight ratio (mg/g) was calculated. (D) Serum anti-dsDNA antibody, ANA, and IgG were determined by ELISA. (E) T_H1 in the lymph nodes, PBMCs, and spleens were determined by FACS. (F) 24 h proteinuria, serum creatinine, and BUN were measured. The level of 24 h proteinuria was expressed as micrograms of protein in 24-h urine. (G) Representative images of H&E-stained kidneys. Scale bar, 100 μm. (H) Glomerular pathology quantified as glomerular, interstitial, and perivascular scores of mice receiving different treatments were determined. All the experiments were repeated 3 times. n = 5, per group. *P < .05, **P < .01, ***P < .001, ****P < .0001.

Figure 7.

Higher IL-2 maintaining positively correlated with therapeutic efficacy of UC-MSCs treatment in lupus nephritis patients. (A-C) PBMCs were isolated from 4 SLE patients, then were co-cultured with MSCs at the ratio of 1:30 (as MSCs: PBMCs) for 12 h (A) and 24 h (B-C), respectively. (A) PBMCs were then harvested for further examination of their il-2 mRNA expression by qRT-PCR. (B) IL-2 concentration in the culture supernatant was measured by ELISA. (C) Frequencies of Tregs (CD4⁺CD25⁺FoxP3⁺cells) for the indicated subsets were determined. (D-E) Serum IL-2 concentration (D) and SLEDAI-2K scores (E) of 5 lupus nephritis patients treated with MSCs were collected. *P < .05, **P < .01, ***P < .001, ****P < .0001.