Experimental evidence and preliminary clinical studies have demonstrated that human mesenchymal stem cells (MSCs) display important immune modulatory function of potential relevant interest in the setting of allogeneic hematopoietic stem cell (HSC) transplantation. Effectiveness of MSCs in controlling severe GVHD seems to be related to the immune-regulatory role they play in suppressing alloantigen-specific T-cell activation. Aim of the present study was to extend the analysis of the mechanisms responsible for the immune regulatory effect of interaction between MSCs and alloantigen-specific immune response elicited in vitro in primary and in secondary mixed lymphocyte culture (MLC). At difference with most previously reported studies, we decided to employ non-irradiated MSCs, reasoning that irradiation might impair, beside the proliferative capacity, also the differentiation capability of MSCs and, consequently, alter their interaction pattern with lymphocyte subsets. MSC were added to primary MLC at different doses (MLC-responder-PBMC:MSC ratios = 1:1 and 10:1). Dendritic cell (DC) differentiation, lymphocyte proliferation, alloantigen-specific cytotoxic activity and differentiation of CD4+ T-cell subsets expressing CD25 and/or CTLA4 antigens were assessed in primary and secondary MLC, comparing the effect observed using third-party MSCs with that obtained employing autologous to the MLC-responder (autologous) MSCs. Results demonstrated that human MSCs: (1) strongly inhibit alloantigen-induced DC1 differentiation; (2) down-regulate, in a dose-dependent manner, alloantigen-induced lymphocyte expansion, especially that of CD8+ T cells and of NK lymphocytes; (3) favor the differentiation of CD4+ T cells co-expressing CD25 and/or CTLA4, a phenotype associated with regulatory/suppressive function of immune response; (4) cause a dose-dependent reduction of alloantigen-specific cytotoxic capacity mediated by either cytotoxic T lymphocytes or NK cells; (5) exert more effective suppressive activity on MLC-induced T-cell activation when they are allogeneic rather than autologous with respect to responder cells. In particular, higher percentages of CD4+ and of CD4+CD25+ T cells co-expressing CTLA4+ were detected when third-party, rather than autologous, MSCs were added to MLC. These data suggest that T-cell recognition of alloantigens expressed by MSCs may further facilitate the preferential differentiation of activated CD4+ T cells expressing CTLA4, a glycoprotein, known to deliver an inhibitory signal to T cells and to mediate apoptosis of previously activated T lymphocytes. Several studies previously demonstrated that MSCs exert inhibitory effect on lymphocyte activation through the release of soluble factors. Our data suggest that the preferential differentiation of CD4+CD25+ regulatory T-cell subsets may be favored by other mechanisms of MSC-mediated inhibition of alloantigen-induced effector cell activation and expansion, and, in turn, these CD4+CD25+ cells contribute to propagate and extend suppressor activity. Altogether, our results provide immunological support to the use of MSCs for prevention of immune complications related to both HSC and solid organ transplantation and to the theory that MSCs are “universal” suppressors of immune reactivity.

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The absolute content of CD34 ؉ cells in the peripheral blood of 84 patients with myelofibrosis with myeloid metaplasia (MMM) and 23 patients with other Philadelphia-negative (Ph ؊ ) chronic myeloproliferative disorders (CMDs) was investigated. In MMM, the median absolute number of circulating CD34 ؉ cells was consistently high (91.6 ؋ 10 6 /L; range, 0-2460 ؋ 10 6 /L). Receiver operating characteristic curve analysis showed that 15 ؋ 10 6 /L as a decision criterion for CD34 ؉ cells produced an almost complete discrimination between MMM patients out of therapy and other Ph ؊ CMDs (positive predictive value, 98.4%; negative predictive value, 85.0%). MMM patients with higher numbers of CD34 ؉ cells had a significantly longer disease duration (P ‫؍‬ .019) and higher spleen volume index (P ‫؍‬ .014), liver volume (P ‫؍‬ .000), percentage of circulating immature myeloid cells (P ‫؍‬ .020), and percentage of myeloid blasts (P ‫؍‬ .000). When CD34 ؉ cells were correlated with the use of Dupriez risk stratification, CD34 ؉ cells increased significantly from low-risk (median, 68.1 ؋ 10 6 /L) to intermediate-risk (median, 112.8 ؋ 10 6 /L) and high-risk patients (median 666.1 ؋ 10 6 /L) (F ‫؍‬ 4.95; P ‫؍‬ .009). When CD34 ؉ cells were correlated with a severity score on the basis of both myeloproliferative and myelodepletive characteristics of the disease, only the myeloproliferation index was significantly associated with CD34 ؉ cell level (F ‫؍‬ 5.7; P ‫؍‬ .000). Overall survival and interval to blast transformation from the time of CD34 ؉ cell analysis were significantly shorter in patients with more than 300 ؋ 10 6 /L CD34 ؉ cells (P ‫؍‬ .005 and .0005, respectively). In conclusion, the absolute number of CD34 ؉ circulating cells allows MMM to be distinguished from other Ph ؊ CMDs; it is strongly associated with the extent of myeloproliferation and predicts evolution toward blast transformation. (Blood. 2001;98:3249-3255)

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IntroductionMultiple myeloma (MM) remains an incurable disease, despite conventional and high-dose chemotherapies. 1 Molecules targeting not only plasma cells, but also the bone marrow (BM) microenvironment are needed to overcome drug resistance.Pathologic angiogenesis is a constant component of the MM microenvironment. 2 The vascular endothelial growth factor (VEGF)/ VEGF receptor-2 (VEGFR-2) pathway greatly contributes to MM angiogenesis and growth, 3 and mediates proliferation and capillarogenesis in MM endothelial cells (MMECs) through an autocrine loop. 4 VEGF 165 is the most abundant and effective isoform. 5 It binds simultaneously to its cognate receptors VEGFR-1 and VEGFR-2 and to the coreceptor neuropilin-1 (NRP1), a cellsurface glycoprotein expressed on axons in the developing nervous system as well. 6 NRP1 is the ligand-binding subunit of the receptor complex for class 3 semaphorins, a family of secreted proteins that mediate neuronal guidance. 7 Binding of secreted semaphorin 3A (SEMA3A) to NRP1 induces the collapse of neuronal growth cones 8 by activating the signal-transducing subunit(s) of the receptor complex, that is, class A plexins, a family of transmembrane proteins whose cytoplasmic domain is endowed with an R-Ras GAP activity that inhibits integrin function. 9 NRP1 is also expressed on ECs, 10 where it acts as an isoform-specific receptor for VEGF 165 , and enhances by 4-to 6-fold its affinity for VEGFR-2, as well as VEGF 165 -induced cell chemotaxis and proliferation. 11 By competing with VEGF 165 for binding to NRP1 12 and by activating class A plexins, 13 SEMA3A inhibits integrinbased EC adhesion and migration and capillary sprouting. Autocrine loops of endothelial SEMA3A play a self-limiting role in angiogenesis and regulate EC behavior during its physiologic development. 13 Here we analyze the expression levels of VEGF 165 , SEMA3A, and their receptors NRP1 and plexin-A1 in BM ECs isolated from patients with MM and MGUS (MMECs and MGECs), and from the human umbilical vein (HUVECs). We show that overangiogenic MMECs display a high VEGF 165 /SEMA3A ratio and behave like MGECs and HUVECs upon exposure to exogenous SEMA3A, which seems as effective as an anti-VEGFR-2 antibody. Our observations point to SEMA3A as a potential antiangiogenic Patients, materials, and methods PatientsThirty-two patients fulfilling the International Myeloma Working Group diagnostic criteria 14 for MM (n ϭ 18) and MGUS (n ϭ 14) were studied at diagnosis. The MM patients (12 male, 6 female) were aged 44 to 81 years (median 68.5 years) and staged 15 as IIA (n ϭ 4), IIB (n ϭ 2), IIIA (n ϭ 10), and IIIB (n ϭ 2); the M-component was IgG (n ϭ 12), IgA (n ϭ 4), and or (n ϭ 2). The MGUS patients (8 male, 6 female) were aged 42 to 79 years (median 70.6 years) and were IgG (n ϭ 10) or IgA (n ϭ 4). The study was approved by the local ethics committee of the University of Bari Medical School, Italy, and all patients gave their informed consent in accordance with the Declaration of Helsinki. Separation and culture of ECs and...