Congenic mesenchymal stem cell therapy reverses hyperglycemia in experimental type 1 diabetes

Abstract

Objective: A number of clinical trials are underway to test whether mesenchymal stem cells (MSCs) are effective in treating various diseases, including type 1 diabetes. Although this cell therapy holds great promise, the optimal source of MSCs has yet to be determined with respect to major histocompatibility complex matching. Here, we examine this question by testing the ability of congenic MSCs, obtained from the NOR mouse strain, to reverse recent-onset type 1 diabetes in NOD mice, as well as determine the immunomodulatory effects of NOR MSCs in vivo.

Research design and methods: NOR MSCs were evaluated with regard to their in vitro immunomodulatory function in the context of autoreactive T-cell proliferation and dendritic cell (DC) generation. The in vivo effect of NOR MSC therapy on reversal of recent-onset hyperglycemia and on immunogenic cell subsets in NOD mice was also examined.

Results: NOR MSCs were shown to suppress diabetogenic T-cell proliferation via PD-L1 and to suppress generation of myeloid/inflammatory DCs predominantly through an IL-6-dependent mechanism. NOR MSC treatment of experimental type 1 diabetes resulted in long-term reversal of hyperglycemia, and therapy was shown to alter diabetogenic cytokine profile, to diminish T-cell effector frequency in the pancreatic lymph nodes, to alter antigen-presenting cell frequencies, and to augment the frequency of the plasmacytoid subset of DCs.

Conclusions: These studies demonstrate the inimitable benefit of congenic MSC therapy in reversing experimental type 1 diabetes. These data should benefit future clinical trials using MSCs as treatment for type 1 diabetes.

FIG. 1.

Characterization of NOR MSCs. A: Immunohistochemical staining of NOR MSC cultures demonstrates fibroblast cell morphology by hematoxylin–eosin staining, substantial expression of the MSC markers CD44 and CD105, moderate expression of CD29, and lack of expression for the hematopoietic stem cell marker CD34. B: Flow cytometric analysis of NOR MSC P4 cultures (n = 5, data shown as mean ± SEM) shows abundant expression of the classical MSC markers CD29, CD44, and CD105, while MSCs were negative for the hematopoietic lineage-restricted markers CD45 and CD90.2. Sca-1, CD73, and VCAM (CD106) were expressed at moderate levels. C: NOR MSCs were shown to undergo osteogenesis and chondrogenesis after exposure to differentiation factors. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 2.

NOR MSC suppression of diabetogenic autoreactive T-cells via PD-L1. A: Cytokine studies of NOR MSC cultures revealed considerable levels of IL-6, with M-CSF and Flt3L produced at lesser but substantial levels in comparison to other growth factors (n = 4). B: NOR MSCs suppressed TCR-stimulated proliferation of NOD CD4⁺ cells in a dose-dependent manner, in which increasing numbers of IFN-γ-stimulated NOR MSCs were added to 1 × 10⁵ NOD CD4⁺ cells in the presence of 1 μg/ml anti-CD3 and anti-CD28 (n = 5, P < 0.027 for 1 × 10⁴ MSCs, P < 0.0001 for 2 × 10⁴ and 4 × 10⁴ MSCs), and IFN-γ challenge enhanced the suppressive effect of NOR MSCs. (C) 2 × 10⁴ NOR MSCs were shown to significantly reduce autoreactive T-cell proliferation (n = 5, P = 0.047), as evaluated by CFSE dilution and calculation of proliferation index when added to a BDC2.5 autoreactive assay containing BDC2.5 CD4⁺ T-cells, NOD DCs, and 100 ng/ml BDC2.5 islet peptide. D: IFN-γ production was similarly suppressed in the presence of 2 × 10⁴ NOR MSCs by ELISPOT in the BDC2.5 autoreactive assay (n = 5, P = 0.0024), while IL-6 levels were enhanced (P = 0.0005). E: Addition of 1, 2, or 4 × 10⁴ NOR splenocytes had no suppressive effect on anti-CD3/-CD28 T-cell proliferation as compared with addition of identical numbers of NOR MSCs (n = 4, P < 0.0001 for NOR MSCs, not significant for NOR splenocytes). F: NOR MSCs stimulated with 0.05, 0.5, or 5 ng/ml recombinant murine IFN-γ show dose-dependent upregulation of PD-L1 expression by flow cytometric analysis (n = 3, P < 0.008 for 0.5 and 5 ng/ml), a minor increase in PD-1 (p = not significant), and no increase in expression of PD-L2. G: NOR MSCs after coculture with BDC2.5 CD4⁺ T-cells, NOD DCs, and BDC2.5 peptide exhibited marked upregulation of PD-L1 expression at 72 h by flow cytometric analysis (n = 5, P < 0.0001). H: siRNA knockdown of PD-L1 in MSCs abrogated the suppressive effect observed on autoreactive T-cell proliferation when 2 × 10⁴ MSCs were added (n = 6, P = 0.0034 for control versus nontargeting siRNA, not significant for control versus PD-L1 siRNA for representative experiment shown), and (I) gene expression analysis of MSCs demonstrated efficient suppression of PD-L1 transcripts in response to siRNA treatment (n = 4, P = 0.016). Experiments were performed between 3 and 6 times, and data are displayed with means and SEM.

FIG. 3.

MSC suppression of DC differentiation. A: Using an established model of DC generation from NOD bone marrow mononuclear cells, coculture with NOR MSCs was shown to markedly reduce the CD11c⁺CD11b⁺ population, so that the predominant cell phenotype was CD11c^lowCD11b^low (CD11c⁺CD11b⁺ cells = 40.7 ± 2.6% and 22.1 ± 2.4% for control and NOR MSC-treated, respectively, n = 5, P = 0.0007), whereas treatment with anti-IL-6 in large part abrogated this effect (CD11c⁺CD11b⁺ cells = 30.9 ± 4.7%, not significant in comparison to control [−/−] and MSCs alone [+/−]). Analysis of expression of Ly-6c in the CD11b⁺ fraction demonstrated that coculture with NOR MSCs resulted in a decrease in both the CD11b⁺Ly-6c^high and CD11b⁺Ly-6c^int populations (n = 4, P = 0.0053 and P = 0.02, respectively), which was fully abrogated by blockade of IL-6 (p = not significant). B: The population of lineage-negative cells was evaluated in DC culture as a function of progenitor frequency; coculture with MSCs increased the percentage of Lin⁻ cells (n = 4, Lin⁻ cells = 8.94 ± 0.87% and 13.73 ± 1.08% for control and NOR MSC-treated, respectively, P = 0.004), which was in part rescued by addition of anti-IL-6 (p = not significant). Similarly, Sca-1 expression within the lineage-negative population was markedly increased in the presence of MSCs (n = 4, Lin⁻ Sca-1⁺ cells = 6.98 ± 1.27% and 30.53 ± 6% for control and NOR MSC-treated, respectively, P = 0.0085). Although treatment with anti-IL-6 resulted in loss of significance of this effect, IL-6 blockade appeared to be incompletely effective in reducing Sca-1 expression in response to MSCs. C: Cytokine analysis of cocultures of DCs and NOR MSCs demonstrated marked IL-6 production in the presence of MSCs (n = 4, P = 0.0074) as well as efficient blockade of IL-6 in response to treatment with anti-IL-6. Both Flt3L and M-CSF levels were substantially increased in response to MSC coculture (P = 0.03 and P = 0.04, respectively), and IL-6 blockade had no effect on these growth factors (P = 0.013 and P = 0.018, respectively, in comparison to DCs alone). Conversely, TNF-α production was reduced in the presence of MSCs (P = 0.0056), and anti-IL-6 treatment resulted in abrogation of this effect (p = not significant). D: Giemsa staining of DC culture cytospins demonstrated a lower nuclear/cytoplasmic ratio in response to coculture with MSCs, and IL-6 blockade appeared to in large part abrogate this effect. Experiments were performed between 3 and 5 times, and data are displayed with means and SEM. (A high-quality color representation of this figure is available in the online issue.)

FIG. 4.

NOR MSC therapy induces long-term reversal of recent-onset hyperglycemia. A: NOD mice were monitored beginning at 10 weeks of age, and on day 2 of hyperglycemia (blood glucose > 240 mg/dl), an insulin pellet was inserted subcutaneously for maintenance of normal glycemia during treatment. Mice were randomized to control or NOR MSC-treated groups; for NOR MSC treatment, 1 × 10⁶ cells were injected intravenously twice per week for 4 weeks, and blood glucose measurements were taken daily. Eight of nine NOD mice treated with NOR MSCs exhibited reversal of diabetes. Five of six treated mice followed for 12 weeks maintained their reversal, whereas controls reverted to hyperglycemia (>600 mg/dl) soon after dissolution of the insulin pellet. B: Means of cumulative blood glucose measurements demonstrate no difference in level of hyperglycemia at days −1 and 0 (p = not significant), while weekly mean measurements beginning at week 2 after the initiation of treatment show significant decreases in blood glucose in response to NOR MSC therapy (P < 0.001 for all from week 2 to week 12). Data are displayed with means and SEM.

FIG. 5.

NOR MSC therapy alters DC phenotype, cytokine profile, and effector cell frequency in vivo. A: Serum cytokine studies of NOR MSC-treated and control mice were performed at days 0, 7, 14, and 21 after completion of our treatment protocol at 4 weeks (n = 3–5 samples). NOR MSC therapy increased circulating levels of IL-6 at days 7 and 14 (P < 0.0065), increased IL-7 levels at days 0 and 7 (P < 0.00021), increased levels of IL-10 at days 0 and 7 (P < 0.0052), and increased levels of IL-12(p40) at days 0 and 7 (P < 0.00091). B: CD4 and CD8 effector cell frequency (identified as CD44^highCD62L^low) was reduced in the PLN of NOR MSC-treated mice, while no difference was detected in Treg frequency (n = 5, P = 0.041 and P = 0.0022 for CD4 and CD8 effectors, respectively). C: CD11c single-positive cells and CD11c^highCD11b⁺ cells were reduced in response to NOR MSC therapy (n = 5, P = 0.004 and P = 0.015, respectively) while the CD11c^lowCD11b⁺ population was increased (n = 5, 1.12 ± 0.1% and 2.06 ± 0.34% for control and NOR MSC-treated, respectively, P = 0.029). Analysis of the CD11c single-positive population showed a marked increase in the plasmacytoid DC subset in NOR MSC-treated mice (gated on CD11c⁺CD11b⁻, followed by analysis of B220 expression, n = 5, 21.9 ± 3.18% and 37.5 ± 3.31% for control and NOR MSC-treated, respectively, P = 0.0094). The frequencies of CD11b⁺Ly-6c⁺ monocytes (n = 3–5, P = 0.042) and F4/80⁺ macrophages (n = 5, P = 0.015) were also found to be reduced in response to NOR MSCs. All data are displayed with means and SEM.