Transient low-dose methotrexate generates B regulatory cells that mediate antigen-specific tolerance to alglucosidase alfa.

Biologic drugs, including enzyme-replacement therapies, can elicit anti-drug Abs (ADA) that may interfere with drug efficacy and impact patient safety. In an effort to control ADA, we focused on identifying regimens of immune tolerance induction that may be readily available for clinical use. Data generated in both wild-type mice and a Pompe disease mouse model demonstrate that single-cycle, low-dose methotrexate can be as effective as three cycles of methotrexate in providing a long-lived reduction in alglucosidase alfa-specific ADA. In addition, we show that methotrexate induces Ag-specific tolerance as mice generate similar Ab responses to an irrelevant Ag regardless of prior methotrexate treatment. Methotrexate-induced immune tolerance does not seem to involve cell depletion, but rather a specific expansion of IL-10- and TGF-ß-secreting B cells that express Foxp3, suggesting an induction of regulatory B cells. The mechanism of immune tolerance induction appears to be IL-10 dependent, as methotrexate does not induce immune tolerance in IL-10 knockout mice. Splenic B cells from animals that have been tolerized to alglucosidase alfa with methotrexate can transfer tolerance to naive hosts. We hypothesize that methotrexate induction treatment concomitant with initial exposure to the biotherapeutic can induce Ag-specific immune tolerance in mice through a mechanism that appears to involve the induction of regulatory B cells.

Circulating CD31+ leukocyte frequency is associated with cardiovascular risk factors.

OBJECTIVES: CD31 identifies a heterogeneous population of cells in the blood, consisting of mature leukocytes and platelets, as well as smaller numbers of endothelial and progenitor cells. Because unfractionated CD31+ blood cells have demonstrated angiogenic properties in vivo, we hypothesized that circulating CD31+ cells would be related to the presence of cardiovascular risk factors in humans. METHODS AND RESULTS: We studied 1487 participants, free of cardiovascular disease, from the Framingham Offspring Study. Using anti-human CD31 and CD45 antibodies, distinct CD31+/CD45+ leukocyte populations were enumerated in blood samples by FACS analysis. We used linear regression analyses to investigate the relation of each cell phenotype with cardiovascular risk factors. We identified 3 distinct leukocyte populations: CD31-, CD31 dim, and CD31 bright cells. Using forward/side scatter analyses, CD31- and CD31 dim cells mapped to lymphoid gates while CD31 bright cells were monocytoid. In multivariable analyses, higher frequency of CD31 bright cells was associated with older age, male sex, HDL cholesterol, and CRP (all P < 0.01). In contrast, CD31 dim was inversely associated with age, male sex, CRP, and smoking (all P < 0.01). Framingham Risk Score was positively associated with CD31 bright frequency (P = 0.002), and negatively associated with CD31 dim frequency (P = 0.020). CONCLUSIONS: CD31+ staining identifies 2 major leukocyte populations, CD31 bright and CD31 dim, which demonstrated significant and opposite associations with cardiovascular risk in humans. Further research is needed to define the biological and potential therapeutic roles of CD31+ subpopulations in vascular disease.

Circulating CD34(+) progenitor cell frequency is associated with clinical and genetic factors.

Circulating blood CD34(+) cells consist of hematopoietic stem/progenitor cells, angiogenic cells, and endothelial cells. In addition to their clinical use in hematopoietic stem cell transplantation, CD34(+) cells may also promote therapeutic neovascularization. Therefore, understanding the factors that influence circulating CD34(+) cell frequency has wide implications for vascular biology in addition to stem cell transplantation. In the present study, we examined the clinical and genetic characteristics associated with circulating CD34(+) cell frequency in a large, community-based sample of 1786 Framingham Heart Study participants.Among subjects without cardiovascular disease (n = 1595), CD34(+) frequency was inversely related to older age, female sex, and smoking. CD34(+) frequency was positively related to weight, serum total cholesterol, and statin therapy. Clinical covariates accounted for 6.3% of CD34(+) variability. CD34(+) frequency was highly heritable (h(2) = 54%; P < .0001). Genome-wide association analysis of CD34(+) frequency identified suggestive associations at several loci, including OR4C12 (chromosome 11; P = 6.7 × 10(-7)) and ENO1 and RERE (chromosome 1; P = 8.8 × 10(-7)). CD34(+) cell frequency is reduced in older subjects and is influenced by environmental factors including smoking and statin use. CD34(+) frequency is highly heritable. The results of the present study have implications for therapies that use CD34(+) cell populations and support efforts to better understand the genetic mechanisms that underlie CD34(+) frequency.

Genetic and clinical correlates of early-outgrowth colony-forming units.

BACKGROUND: Several bone marrow-derived cell populations may have angiogenic activity, including cells termed endothelial progenitor cells. Decreased numbers of circulating angiogenic cell populations have been associated with increased cardiovascular risk. However, few data exist from large, unselected samples, and the genetic determinants of these traits are unclear. METHODS AND RESULTS: We examined the clinical and genetic correlates of early-outgrowth colony-forming units (CFUs) in 1799 participants of the Framingham Heart Study (mean age, 66 years; 54% women). Among individuals without cardiovascular disease (n = 1612), CFU number was inversely related to advanced age (P = 0.004), female sex (P = 0.04), and triglycerides (P = 0.008) and positively related to hormone replacement (P = 0.008) and statin therapy (P = 0.027) in stepwise multivariable analyses. Overall, CFU number was inversely related to the Framingham risk score (P = 0.01) but not with prevalent cardiovascular disease. In genome-wide association analyses in the entire sample, polymorphisms were associated with CFUs at the MOSC1 locus (P = 3.3 × 10(-7)) and at the SLC22A3-LPAL2-LPA locus (P = 4.9 × 10(-7)), a previously replicated susceptibility locus for myocardial infarction. Furthermore, alleles at the SLC22A3-LPAL2-LPA locus that were associated with decreased CFUs were also related to increased risk of myocardial infarction (P = 1.1 × 10(-4)). CONCLUSIONS: In a community-based sample, early-outgrowth CFUs are inversely associated with select cardiovascular risk factors. Furthermore, genetic variants at the SLC22A3-LPAL2-LPA locus are associated with both decreased CFUs and an increased risk of myocardial infarction. These findings are consistent with the hypothesis that decreased circulating angiogenic cell populations promote susceptibility to myocardial infarction.

Association of colony-forming units with coronary artery and abdominal aortic calcification.

BACKGROUND: Certain bone marrow-derived cell populations, called endothelial progenitor cells, have been reported to possess angiogenic activity. Experimental data suggest that depletion of these angiogenic cell populations may promote atherogenesis, but limited data are available on their relation to subclinical atherosclerotic cardiovascular disease in humans. METHODS AND RESULTS: We studied 889 participants of the Framingham Heart Study who were free of clinically apparent cardiovascular disease (mean age, 65 years; 55% women). Participants underwent endothelial progenitor cell phenotyping with an early-outgrowth colony-forming unit assay and cell surface markers. Participants also underwent noncontrast multidetector computed tomography to assess the presence of subclinical atherosclerosis, as reflected by the burden of coronary artery calcification and abdominal aortic calcification. Across decreasing tertiles of colony-forming units, there was a progressive increase in median coronary artery calcification and abdominal aortic calcification scores. In multivariable analyses adjusting for traditional cardiovascular risk factors, each 1-SD increase in colony-forming units was associated with a ≈16% decrease in coronary artery calcification (P=0.02) and 17% decrease in abdominal aortic calcification (P=0.03). In contrast, neither CD34(+)/KDR(+) nor CD34(+) variation was associated with significant differences in coronary or aortic calcification. CONCLUSIONS: In this large, community-based sample of men and women, lower colony-forming unit number was associated with a higher burden of subclinical atherosclerosis in the coronary arteries and aorta. Decreased angiogenic potential could contribute to the development of atherosclerosis in humans.

Therapeutic targeting of a stem cell niche.

The specialized microenvironment or niche where stem cells reside provides regulatory input governing stem cell function. We tested the hypothesis that targeting the niche might improve stem cell-based therapies using three mouse models that are relevant to clinical uses of hematopoietic stem (HS) cells. We and others previously identified the osteoblast as a component of the adult HS cell niche and established that activation of the parathyroid hormone (PTH) receptor on osteoblasts increases stem cell number. Here we show that pharmacologic use of PTH increases the number of HS cells mobilized into the peripheral blood for stem cell harvests, protects stem cells from repeated exposure to cytotoxic chemotherapy and expands stem cells in transplant recipients. These data provide evidence that the niche may be an attractive target for drug-based stem cell therapeutics.