The Normal Counterpart of IgD Myeloma Cells in Germinal Center Displays Extensively Mutated IgVH Gene, Cμ–Cδ Switch, and λ Light Chain Expression

Human myeloma are incurable hematologic cancers of immunoglobulin-secreting plasma cells in bone marrow. Although malignant plasma cells can be almost eradicated from the patient's bone marrow by chemotherapy, drug-resistant myeloma precursor cells persist in an apparently cryptic compartment. Controversy exists as to whether myeloma precursor cells are hematopoietic stem cells, pre–B cells, germinal center (GC) B cells, circulating memory cells, or plasma blasts. This situation reflects what has been a general problem in cancer research for years: how to compare a tumor with its normal counterpart. Although several studies have demonstrated somatically mutated immunoglobulin variable region genes in multiple myeloma, it is unclear if myeloma cells are derived from GCs or post-GC memory B cells. Immunoglobulin (Ig)D-secreting myeloma have two unique immunoglobulin features, including a biased λ light chain expression and a Cμ–Cδ isotype switch. Using surface markers, we have previously isolated a population of surface IgM−IgD+CD38+ GC B cells that carry the most impressive somatic mutation in their IgV genes. Here we show that this population of GC B cells displays the two molecular features of IgD-secreting myeloma cells: a biased λ light chain expression and a Cμ–Cδ isotype switch. The demonstration of these peculiar GC B cells to differentiate into IgD-secreting plasma cells but not memory B cells both in vivo and in vitro suggests that IgD-secreting plasma and myeloma cells are derived from GCs

Plasmacytoid precursor dendritic cells facilitate allogeneic hematopoietic stem cell engraftment

Bone marrow transplantation offers great promise for treating a number of disease states. However, the widespread application of this approach is dependent upon the development of less toxic methods to establish chimerism and avoid graft-versus-host disease (GVHD). CD8+/TCR− facilitating cells (FCs) have been shown to enhance engraftment of hematopoietic stem cells (HSCs) in allogeneic recipients without causing GVHD. In the present studies, we have identified the main subpopulation of FCs as plasmacytoid precursor dendritic cells (p-preDCs). FCs and p-preDCs share many phenotypic, morphological, and functional features: both produce IFN-α and TNF-α, both are activated by toll-like receptor (TLR)-9 ligand (CpG ODN) stimulation, and both expand and mature after Flt3 ligand (FL) treatment. FL-mobilized FCs, most of which express a preDC phenotype, significantly enhance engraftment of HSCs and induce donor-specific tolerance to skin allografts. However, p-preDCs alone or p-preDCs from the FC population facilitate HSC engraftment less efficiently than total FCs. Moreover, FCs depleted of preDCs completely fail to facilitate HSC engraftment. These results are the first to define a direct functional role for p-preDCs in HSC engraftment, and also suggest that p-preDCs need to be in a certain state of maturation/activation to be fully functional

Plasmacytoid Precursor Dendritic Cells Contribute to the Ability of CD8+TCR− Facilitating Cells Population of Enhance Hematopoetic Stem Cell Engraftment and HSC Clonogenicity.

Abstract The discovery of cells with facilitative property offers great therapeutic potential in hematopoietic stem cell (HSC) transplantation. CD8+/TCR− facilitating cells (FC) enhance engraftment of HSC in allogeneic recipients without causing GVHD and also significantly enhance the engraftment of limiting numbers of HSC in syngeneic recipients. We recently identified that plasmacytoid precursor dendritic cells (p-preDC), the main component of the heterogeneous FC population, contribute to facilitation, but with less efficiency than the total FC population. In the present study we determined the role p-preDC play in the ability of FC to 1) enhance syngeneic suboptimal HSC engraftment and 2) maintain HSC clonogenicity in vitro. 500 syngeneic HSC +/− 30,000 FC, or p-preDC from the FC (DC-FC) were transplanted into ablated (950 cGy) B6 mice. As expected, FC significantly increased HSC engraftment, with 87.5 % of recipients (n=19) surviving at 250 days after transplantation, while recipients of HSC alone exhibited significantly impaired survival (37.5%, P=0.0026, n=16) (Figure 1). However, DC-FC, which allowed a slight improvement of HSC engraftment (52.6% recipient survival, n=16, P=0.15), were significantly (P=0.034) less effective than total FC. These data show that DC-FC are less effective than FC total. We then evaluated the influence of p-preDC and DC-FC on HSC in vitro using the colony forming cell (CFC) assay that enumerates progenitors in the HSC population. HSC were cultured in the presence or absence of FC, p-preDC or DC-FC (at 1:3 ratio) in methylcellulose containing growth factors for 14 days. The number of CFC was significantly increased when HSC were cultured in the presence of FC (P&amp;lt;0.01) compared to HSC alone (Figure 2). In contrast, the DC-FC slightly increased clonogenicity, as did p-pre-DC, but did not replace FC total. Moreover, when we preincubated HSC with FC, DC-FC or p-pre-DC for 18 hour and assessed in the CFC assay, FC significantly increased the number of CFC although no significant effect was observed with DC-FC or p-pre-DC (Figure 2). These data suggest that the effect of FC on HSC clonogenicity and HSC engraftment is only partially due to the contribution of the DC subpopulation, and that this effect on HSC requires synergistic interactions between the cells within the FC population. Moreover these data also suggest that FC facilitation on HSC involves a combination of different mechanisms: one on host alloreactivity due likely to DC-FC and one on direct interaction with HSC due to combined effects of cells in the FC. Figure Figure Figure Figure</jats:p

2D array of high aspect ratio TiO2 nano-pillars using direct photo-patternable TiO2 sol-gel process and colloidal photolithography

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CD8+/TCR− Graft Facilitating Cells Enhance HSC Engraftment and Survival Via TNF-α Mediated Prevention of Apoptosis.

Abstract The use of accessory cells to enhance hematopoietic stem cell (HSC) engraftment could have a significant therapeutic impact, especially when stem cell numbers are limited. The bone marrow (BM) microenvironment is involved in regulation of HSC, allowing production of mature blood cells while maintaining HSC self renewal. To date, the precise identity of specific cells in the microenvironment that exert this regulatory effect on HSC has not been defined. We recently reported that CD8+/TCR− facilitating cells (FC), a subpopulation of BM cells containing predominantly B220+/CD11c+/CD11b− tolerogenic precursor-plasmacytoid dendritic cells, enhance HSC engraftment in allogeneic recipients. Additionally, FC significantly enhance engraftment of limiting numbers of HSC in syngeneic recipients. In the present studies, we investigated the mechanism of FC-mediated enhancement of HSC engraftment. We show for the first time that FC significantly increase HSC survival in vitro and exert an anti-apoptotic effect on HSC via TNF-α. Co-culture of FC with HSC induces production of physiologically relevant low levels of TNF-α by FC. FC from TNF-α−/− mice are impaired in function in vitro and in facilitating HSC engraftment in vivo. Furthermore, neutralization of TNF-α on FC using anti-TNF antibody results in loss of FC function in vitro, confirming a major role for TNF-α in FC function. Notably, co-culture of FC with HSC prevents HSC apoptosis and is associated with significant upregulation of the anti-apoptotic I-κB family member Bcl-3 in HSC. Blocking of TNF-α on FC abrogates the anti-apoptotic effect of FC on HSC and prevents upregulation of Bc1-3 in HSC. Taken together, these findings demonstrate that TNF-α-induced in FC affects highly primitive HSC and identify Bcl-3 as a possible pathway for TNF-α in regulating HSC survival.</jats:p

Promotion of Chimerism and Tolerance by Flt3 Ligand-Mobilized Facilitating Cells Is Associated with Upregulation of CXCR4 and SDF-1.

Abstract Facilitating cells (FC) are a CD8+/TCR− subpopulation of bone marrow (BM) cells that enhance engraftment of hematopoietic stem cells (HSC) in allogeneic recipients without causing graft-versus-host disease. They also significantly enhance engraftment of limiting numbers of HSC in syngeneic recipients. Treatment of mice with Flt3 ligand (Flt3-L) induces significant expansion of FC and HSC in the BM and their mobilization into peripheral blood (PB). In the present study, we evaluated the function of Flt3-L expanded FC and potential mechanism of action. Flt3-L treatment resulted in an 8.5-fold increase of FC in BM and a 100-fold increase in PB compared to untreated mice. When FC from the PB and BM of Flt3-L-treated donors were transplanted with HSC into allogeneic recipient mice, BM FC exhibited significantly impaired function while PB FC were potently functional (Fig. a). Strikingly, this correlated with an increase in mRNA for the chemokine SDF-1 and its receptor, CXCR4, which were significantly increased in PB FC (≥10 fold) and decreased in Flt3-L-expanded BM FC as compared to untreated control BM FC (Fig. b). The impaired function for BM FC was restored within 5 days after cessation of Flt3-L-treatment. Taken together, these data suggest that FC can be mobilized efficiently with Flt3-L and their engraftment facilitating function is associated with upregulation of transcripts for SDF-1 and CXCR4, suggesting that FC may chemoattract HSC by secreting SDF-1 and enhance their homing to the new microenvironment after transplantation. The fact that FC express CXCR4 suggests that they may co-migrate with HSC to the hematopoietic microenvironment after transplantation, being chemoattracted by BM-derived SDF-1. In summary, we provide evidence that FC are mobilized with HSC; the SDF-1-CXCR4 axis is involved in FC:HSC interaction; and FC co-migrate with HSC in response to an SDF-1 gradient. Figure Figure</jats:p