Residual malignant and normal plasma cells shortly after high dose melphalan and stem cell transplantation. Highlight of a putative therapeutic window in Multiple Myeloma?

Multiple Myeloma (MM) is an incurable malignant plasma cell disorder. We have evaluated the counts of Multiple Myeloma Cells (MMCs) and normal plasma cells (N-PCs), seven days after high-dose melphalan (HDM) and autologous stem transplantation (ASCT). Two third of patients had detectable minimal residual disease (MRD+) (71.7 MMCs/µL) after induction treatment with dexamethasone and proteasome inhibitor. MMC counts were reduced by 92% (P ≤ .05) but not eradicated 7 days after HDM+ASCT. Post-HDM+ASCT MMCs were viable and bathed in a burst of MMC growth factors, linked with post-HDM aplasia. In one third of patients (MRD- patients), MMCs were not detectable after induction treatment and remained undetectable after HDM+ASCT. Major difference between MRD- and MRD+ patients is that N-PC counts were increased 3 fold (P〈.05) by HDM+ASCT in MRD- patients, but were unaffected in MRD+ patients. Possible explanation could be that clearance of MMCs in MRD- patients makes more niches available for N-PCs. Thus, MMCs are not fully eradicated shortly after HDM, are bathed in high concentrations of MMC growth factors in an almost desert BM, are viable in short-term culture, which suggests providing additional therapies shortly after HDM to kill resistant MMCs before full repair of lesions.

Characterization of a transitional preplasmablast population in the process of human B cell to plasma cell differentiation.

The early steps of differentiation of human B cells into plasma cells are poorly known. We report a transitional population of CD20(low/-)CD38(-) preplasmablasts along differentiation of human memory B cells into plasma cells in vitro. Preplasmablasts lack documented B cell or plasma cell (CD20, CD38, and CD138) markers, express CD30 and IL-6R, and secrete Igs at a weaker level than do plasmablasts or plasma cells. These preplasmablasts further differentiate into CD20(-)CD38(high)CD138(-) plasmablasts and then CD20(-)CD38(high)CD138(+) plasma cells. Preplasmablasts were fully characterized in terms of whole genome transcriptome profiling and phenotype. Preplasmablasts coexpress B and plasma cell transcription factors, but at a reduced level compared with B cells, plasmablasts, or plasma cells. They express the unspliced form of XBP1 mRNA mainly, whereas plasmablasts and plasma cells express essentially the spliced form. An in vivo counterpart (CD19(+)CD20(low/-)CD38(-)IL-6R(+) cells) of in vitro-generated preplasmablasts could be detected in human lymph nodes (0.06% of CD19(+) cells) and tonsils (0.05% of CD19(+) cells). An open access "B to Plasma Cell Atlas," which makes it possible to interrogate gene expression in the process of B cell to plasma cell differentiation, is provided. Taken together, our findings show the existence of a transitional preplasmablast population using an in vitro model of plasma cell generation and of its in vivo counterpart in various lymphoid tissues.

Mobilization of plasma cells in healthy individuals treated with granulocyte colony-stimulating factor for haematopoietic stem cell collection.

In mice, the plasma cell (PC) niche in the bone marrow is close to the haematopoietic stem cell (HSC) niche. We investigated whether PCs can be mobilized into the peripheral blood (PB) in healthy donors receiving granulocyte colony-stimulating factor (G-CSF) for the induction of HSC mobilization into the PB. G-CSF increased the count of circulating PCs 6-fold, that of circulating B lymphocytes 4-fold and that of circulating HSCs 44-fold. Mobilized circulating PCs comprised CD138(-) (62·2%) and CD138(+) (37·8%) PCs, the latter being more mature based on increased CD27, CD38 and cytoplasmic immunoglobulin expression. Mobilized PCs had a phenotype close to that of steady-state PB PCs or in vitro generated PCs, but they expressed L-selectin only weakly. Finally, a median value of 0·4 × 10(6) /kg donor PCs - one-thirtieth of the overall PC count in a healthy adult - was grafted into patients, which could contribute to immune memory recovery.

Circulating human B and plasma cells. Age-associated changes in counts and detailed characterization of circulating normal CD138- and CD138+ plasma cells.

Generation of B and plasma cells involves several organs with a necessary cell trafficking between them. A detailed phenotypic characterization of four circulating B-cell subsets (immature-, naïve-, memory- B-lymphocytes and plasma cells) of 106 healthy adults was realized by multiparametric flow cytometry. We show that CD10, CD27 and CD38 is the minimal combination of subsetting markers allowing unequivocal identification of immature (CD10(+)CD27(-)CD38(+), 6+/-6 cells/microL), naïve (CD10(-)CD27(-)CD38(-), 125+/-90 cells/microL), memory B lymphocytes (CD10(-)CD27(+)CD38(-), 58+/-42 cells/microL), and plasma cells (CD10(-)CD27(++)CD38(++), 2.1+/-2.1 cells/microL) within circulating CD19(+) cells. From these four subsets, only memory B lymphocytes and plasma cells decreased with age, both in relative and absolute counts. Circulating plasma cells split into CD138(-) (57+/-12%) and CD138(+) (43+/-12%) cells, the latter displaying a more mature phenotypic profile: absence of surface immunoglobulin, lower CD45 positivity and higher amounts of cytoplasmic immunoglobulin, CD38 and CD27. Unlike B lymphocytes, both populations of plasma cells are KI-67(+) and show weak CXCR4 expression.

gammadelta T lymphocytes count is normal and expandable in peripheral blood of patients with follicular lymphoma, whereas it is decreased in tumor lymph nodes compared with inflammatory lymph nodes.

gammadelta T lymphocytes are attractive effector cells for immunotherapy. In vitro, they can be expanded and kill efficiently a variety of tumor cells. The frequency and distribution of gammadelta T lymphocytes were compared in tumor lymph nodes of 51 patients with follicular lymphoma lymph nodes (FL-LNs) and 28 patients with inflammatory lymph nodes (I-LNs). gammadelta and CD8 T lymphocytes were less abundant in FL-LNs than in I-LNs (p

An in vitro model of differentiation of memory B cells into plasmablasts and plasma cells including detailed phenotypic and molecular characterization.

Human plasma cells (PCs) and their precursors play an essential role in humoral immune response but are rare and difficult to harvest. We report the generation of human syndecan-1(+) and immunoglobulin secreting PCs starting from memory B cells in a 3-step and 10-day (D) culture, including a 6-fold cell amplification. We report the detailed phenotypic and Affymetrix gene expression profiles of these in vitro PCs as well as of intermediate cells (activated B cells and plasmablasts) compared with memory B cells and bone marrow PCs, which is accessible through an open web ATLAS (http://amazonia.transcriptome.eu/). We show this B cell-to-PC differentiation to involve IRF4 and AICDA expressions in D4 activated B cells, decrease of PAX5 and BCL6 expressions, and increase in PRDM1 and XBP1 expressions in D7 plasmablasts and D10 PCs. It involves down-regulation of genes controlled by Pax5 and induction of genes controlled by Blimp-1 and XBP1 (unfold protein response). The detailed phenotype of D10 PCs resembles that of peripheral blood PCs detected after immunization of healthy donors. This in vitro model will facilitate further studies in PC biology. It will likewise be helpful to study PC dyscrasias, including multiple myeloma.

Natural killer cell differentiation driven by Tyro3 receptor tyrosine kinases.

Although understanding of the function and specificity of many natural killer (NK) cell receptors is increasing, the molecular mechanisms regulating their expression during late development of NK cells remain unclear. Here we use representational difference analysis to identify molecules required for late NK cell differentiation. Axl protein tyrosine kinase, together with the structurally related receptors Tyro3 and Mer, were essential for NK cell functional maturation and normal expression of inhibitory and activating NK cell receptors. Also, all three receptors were expressed in maturing NK cells, the ligands of these receptors were produced by bone marrow stromal cells, and recombinant versions of these ligands drove NK cell differentiation in vitro. These results collectively suggest that Axl, Tyro3 and Mer transmit signals that are essential for the generation of a functional NK cell repertoire.

Cmv4, a new locus linked to the NK cell gene complex, controls innate resistance to cytomegalovirus in wild-derived mice.

CMV can cause life-threatening disease in immunodeficient hosts. Experimental infection in mice has revealed that the genetically determined natural resistance to murine CMV (MCMV) may be mediated either by direct recognition between the NK receptor Ly49H and the pathogen-encoded glycoprotein m157 or by epistatic interaction between Ly49P and the host MHC H-2D(k). Using stocks of wild-derived inbred mice as a source of genetic diversity, we found that PWK/Pas (PWK) mice were naturally resistant to MCMV. Depletion of NK cells subverted the resistance. Analysis of backcrosses to susceptible BALB/c mice revealed that the phenotype was controlled by a major dominant locus effect linked to the NK gene complex. Haplotype analysis of 41 polymorphic markers in the Ly49h region suggested that PWK mice may share a common ancestral origin with C57BL/6 mice; in the latter, MCMV resistance is dependent on Ly49H-m157 interactions. Nevertheless, PWK mice retained viral resistance against m157-defective mutant MCMV. These results demonstrate the presence of yet another NK cell-dependent viral resistance mechanism, named Cmv4, which most likely encodes for a new NK activating receptor. Identification of Cmv4 will expand our understanding of the specificity of the innate recognition of infection by NK cells.

Phospholipase C-gamma2 is essential for NK cell cytotoxicity and innate immunity to malignant and virally infected cells.

Phospholipase C-gamma2 (PLC-gamma2) is a key component of signal transduction in leukocytes. In natural killer (NK) cells, PLC-gamma2 is pivotal for cellular cytotoxicity; however, it is not known which steps of the cytolytic machinery it regulates. We found that PLC-gamma2-deficient NK cells formed conjugates with target cells and polarized the microtubule-organizing center, but failed to secrete cytotoxic granules, due to defective calcium mobilization. Consequently, cytotoxicity was completely abrogated in PLC-gamma2-deficient cells, regardless of whether targets expressed NKG2D ligands, missed self major histocompatibility complex (MHC) class I, or whether NK cells were stimulated with IL-2 and antibodies specific for NKR-P1C, CD16, CD244, Ly49D, and Ly49H. Defective secretion was specific to cytotoxic granules because release of IFN-gamma on stimulation with IL-12 was normal. Plcg2-/- mice could not reject MHC class I-deficient lymphoma cells nor could they control CMV infection, but they effectively contained Listeria monocytogenes infection. Our results suggest that exocytosis of cytotoxic granules, but not cellular polarization toward targets, depends on intracellular calcium rise during NK cell cytotoxicity. In vivo, PLC-gamma2 regulates selective facets of innate immunity because it is essential for NK cell responses to malignant and virally infected cells but not to bacterial infections.

PLCgamma2 regulates Bcl-2 levels and is required for survival rather than differentiation of marginal zone and follicular B cells.

B cells from phospholipase C (PLC)gamma2-deficient mice express reduced levels of the pro-survival protein Bcl-2 and show a defect in the development of transitional T3 and marginal zone (MZ) B cells that reflects reduced B cell survival. Introduction of a bcl-2 transgene restored the numbers of MZ, T3 and follicular B cells in PLCgamma2(-/-) mice. Restricting the B cell repertoire in PLCgamma2-deficient mice by the introduction of a BCR transgene resulted in a striking reduction in the number of IgM-positive B cells and a paucity of IgD-expressing cells in the spleen which was also rescued by the bcl-2 transgene. BCR-stimulated ERK and IkappaBalpha phosphorylation were PLCgamma2 dependent, while calcium flux was reduced, but not abrogated, in the absence of PLCgamma2, suggesting an ancillary role for PLCgamma1. The bcl-2 transgene rescued development of PLCgamma2(-/-) B cells and serum IgM levels but did not restore BCR-mediated signaling, proliferation or serum IgG3 levels. These data suggest that PLCgamma2 performs a critical role in B cell development through regulation of survival rather than differentiation.