Hematopoietic Stem Cell Mobilization Is Necessary but Not Sufficient for Tolerance in Islet Transplantation

Abstract

Overcoming the immune response to establish durable immune tolerance in type 1 diabetes remains a substantial challenge. The ongoing effector immune response involves numerous immune cell types but is ultimately orchestrated and sustained by the hematopoietic stem cell (HSC) niche. We therefore hypothesized that tolerance induction also requires these pluripotent precursors. In this study, we determined that the tolerance-inducing agent anti-CD45RB induces HSC mobilization in nonautoimmune B6 mice but not in diabetes-prone NOD mice. Ablation of HSCs impaired tolerance to allogeneic islet transplants in B6 recipients. Mobilization of HSCs resulted in part from decreasing osteoblast expression of HSC retention factors. Furthermore, HSC mobilization required a functioning sympathetic nervous system; sympathectomy prevented HSC mobilization and completely abrogated tolerance induction. NOD HSCs were held in their niche by excess expression of CXCR4, which, when blocked, led to HSC mobilization and prolonged islet allograft survival. Overall, these findings indicate that the HSC compartment plays an underrecognized role in the establishment and maintenance of immune tolerance, and this role is disrupted in diabetes-prone NOD mice. Understanding the stem cell response to immune therapies in ongoing human clinical studies may help identify and maximize the effect of immune interventions for type 1 diabetes.

Figure 1

The tolerance-inducing agent anti-CD45RB promotes HSC mobilization that is necessary to establish transplant tolerance. A: HSCs were identified in 8-week-old female nondiabetic B6 and prediabetic NOD mice as LSK cells by staining with a pan-lineage kit to identify lineage-negative (Lin⁻) cells that were then analyzed for expression of c-Kit and Sca-1 (Lin⁻ cells shown in plots). Treatment with anti-CD45RB (100 μg/day on days 0, 1, 3, 5, and 7) led to an increase in LSK cells in B6 marrow relative to untreated mice. The frequency of stem cells in the control groups was set to 1 in each experimental repetition (*P < 0.005 by t test; five independent experiments; n = 14 for control and anti-CD45RB). Treatment of prediabetic NOD mice with the same regimen of anti-CD45RB led to no change in LSK frequency (P = NS by t test; three independent experiments; n = 6 for control and anti-CD45RB). B: To verify increased HSC activity in B6 mice, a CFC assay was performed. A total of 5,000 BMCs from anti-CD45RB–treated or untreated (untx) B6 mice (nondiabetic) was plated for 7 days in methylcellulose culture medium, and colonies were counted. Marrow from anti-CD45RB–treated mice produced more than double the colonies (&P < 0.0001 by one-way ANOVA; three independent samples per condition in six replicates each, repeated twice). C: HSCs were depleted by either a single 500-μg injection of monoclonal antibody ACK2 (c-Kit; Tocris Bioscience, Bristol, U.K.) 1 day prior to transplantation (administered on day −1) or weekly via 500-μg injections of ACK2 for 5 weeks (administered on days −1, 7, 14, 21, 28). On day 0, STZ-treated, chemically diabetic, B6 mice were transplanted with allogeneic C3H islets and treated with a standard 7-day course of anti-CD45RB (aCD45RB). Both transient and prolonged depletion of HSCs prior to anti-CD45RB therapy led to more rapid rejection in a portion of some islet allograft recipients.

Figure 2

The effect of anti-CD45RB is not directly on HSCs but rather on osteoblasts. A: Analysis of bone marrow–resident HSCs (top) from both nondiabetic B6 and prediabetic NOD mice showed that neither expressed significant levels of the CD45RB isoform. In comparison, a significant portion of lineage-positive (Lin⁺) BMCs in both strains expressed CD45RB (bottom). B: Cytometric analysis of cultured osteoblasts from nondiabetic B6 mice demonstrate expression of Rank-L and osteocalcin as expected. Moreover, these osteoblasts express CD45RB (gray is isotype control). C: Cultured nondiabetic B6 osteoblasts were treated with G-CSF (R&D Systems) or anti-CD45RB (aCD45RB) or left untreated (control) to determine whether the expressed CD45RB was functional (n = 4–6 mice/group, repeated in triplicate). As compared with untreated osteoblasts, G-CSF and anti-CD45RB–treated osteoblasts downregulated the expression of CXCL12, which normally retains HSCs in their niche. Other osteoblast activation markers were also slightly downregulated, but osteopontin was maintained, indicating viable cells. Alk Phos, alkaline phosphatase. Col Ia, Collagen Ia; Lin−, lineage negative.

Figure 3

Sympathectomy prevents HSC mobilization by anti-CD45RB and abrogates transplantation tolerance. A: Nondiabetic B6 mice were chemically sympathectomized with 6-OHD (Sigma-Aldrich) on days −4 (100 mg/kg) and −2 (250 mg/kg) relative to the initiation of a standard 7-day course of anti-CD45RB. The marrow LSK cell response was then measured on day 8. As previously shown, anti-CD45RB induced a significant increase in the percent of LSK cells. Sympathectomy itself had no effect, whereas it completely abolished anti-CD45RB–mediated LSK response (P < 0.01 by one-way ANOVA followed by Tukey multiple-comparisons posttest: *P < 0.05 anti-CD45RB vs. 6-OHD/CD45, *P < 0.05 anti-CD45RB vs. control; N = 6 mice/group). B: 6-OHD–sympathectomized or control STZ-treated, chemically diabetic B6 mice received allogeneic C3H islet transplants and were left untreated (untx) or treated with anti-CD45RB (aCD45RB). Sympathectomy itself had no effect on rejection kinetics. Tolerance induction by anti-CD45RB was abrogated by prior sympathectomy (P < 0.001 by log-rank test). Lin−, lineage negative.

Figure 4

Blockade of excess CXCR4 enhances NOD HSC mobilization and extends islet transplant survival. A: Nondiabetic B6 and prediabetic NOD mice were left untreated or treated with G-CSF (250 μg/kg/day for 5 days in eight divided doses). Spleens were harvested 2 to 3 h after the last dose. Whereas B6 mice demonstrated a robust increased in LSK cells following G-CSF treatment, little change was seen in NOD mice (*P < 0.001 by two-way ANOVA followed by Sidak multiple-comparisons posttest). B: Prediabetic NOD bone marrow–resident LSK cells demonstrated significantly greater CXCR4 expression than B6 LSK cells as measured by flow cytometry (&P < 0.05 by Student t test). C: Blockade of CXCR4 with AMD3100 (100 μg/day for 5 days, subcutaneous injections; Sigma-Aldrich) led to significantly increased LSK cell frequencies in the spleens of prediabetic NOD mice (*P < 0.05 by two-way ANOVA followed by Sidak multiple-comparisons posttest). D: Islet transplantation following pretreatment with AMD3100 (100 μg/day on days −4, −3, −2, −1, and 0 relative to the day of transplant) in STZ-treated, chemically diabetic NOD mice led to a significant prolongation in islet survival, which was further extended by addition of anti-CD45RB (aCD45RB), although permanent tolerance was not achieved (P values determined by log-rank test). E: A schematic of the bone marrow HSC niche indicating the presumed action of anti-CD45RB on osteoblasts and the contribution of the SNS (1), as well as the ability of CXCR4-CXCL12 blockade to bypass these steps and promote mobilization in NOD mice (2). Lin−, lineage negative; MFI, mean fluorescence intensity; untx, untreated.