Immature B cells from human and mouse bone marrow can change their surface light chain expression

The capacity of bone marrow-derived surface immunoglobulin-positive (sIg⁺) human and mouse immature B cells, generated either in vitro or in vivo, to change their light (L) chain expression, has been assayed by the number of cells which change in vitro from one type of L chain to the other type, or to no sIg at all. Immature sIg⁺ B cells were generated in vitro from sIg^? precursor cells from human or mouse bone marrow. The immature sIg⁺ cells expressed RAG-1. Human sIg⁺ cells expressed xfr; and λ L chains in ratios between 1:1 and 3:1, whereas in mouse cells, this ratio ranged from 10:1 to 20:1. Upon reculture of the human and mouse xfr;⁺sIg⁺ cells, about half of them remained xfr;⁺, a quarter became λ⁺, and another quarter became sIg^?. Between 1 and 3% expressed both xfr; and λ chains. Of the human λ⁺ cells, about two-thirds remained λ⁺, only 1 to 2% became xfr;⁺, while the other third became sIg^?. Again, between 1 and 3% expressed both xfr; and λ L chains. These results indicate that expression of sIgM in the B cell membrane does not terminate L chain gene rearrangement, and that some order exists in xfr; versus λ gene rearrangements. Hence, human and mouse xfr;⁺ immature B cells can become λ⁺, but very few of the λ⁺ cells can become xfr;⁺, and both can become sIg^?. Further, human CD10⁺/sIg⁺ xfr;⁺ and λ⁺ cells and mouse B220^low/sIg^low xfr;⁺ cells enriched from bone marrow, i.e. immature B cells differentiated in vivo, changed their Ig phenotype upon in vitro culture, but in lower frequencies. By contrast, human and mouse mature B cells did not change their L chain or Ig phenotype. Hence, at least a part of the sIg⁺ immature B cells in bone marrow retain the capacity to change their L chain and Ig phenotype, and this capacity is lost when they become mature, peripheral B cells.     

In vitro growth of human multiple myeloma: implications for biology and therapy.

In vitro data allow presentation of a plausible scenario for the in vivo growth, progression, and dissemination of human multiple myeloma (MM) that involves the interactions between the monoclonal B-cell clone and the bone marrow (BM) microenvironment. A large series of adhesion and extracellular matrix molecules allow trapping of circulating plasma cell precursors within the BM, and a battery of locally released cytokines promote their growth and final differentiation. Malignant B cells establish close contacts with BM stromal cells and release a host of cytokines that recruit and activate BM stromal cells and also T lymphocytes to produce other cytokines. All these cytokines might conceivably act in concert in a self-perpetuating mechanism of mutual help between malignant plasma cells and BM stromal cells to favor the progressive expansion of the malignant clone through a sort of an "avalanche effect." Also, most cytokines produced by malignant B cells, stromal cells, and activated T lymphocytes, including IL-1 beta, TNF-beta, M-CSF, IL-3, and IL-6, have osteoclast-activating properties, thus explaining why the expansion of the B-cell clone is matched by the activation and numeric increase of osteoclasts.     

Targeted next-generation sequencing in chronic lymphocytic leukemia: a high-throughput yet tailored approach will facilitate implementation in a clinical setting

Next-generation sequencing has revealed novel recurrent mutations in chronic lymphocytic leukemia, particularly in patients with aggressive disease. Here, we explored targeted re-sequencing as a novel strategy to assess the mutation status of genes with prognostic potential. To this end, we utilized HaloPlex targeted enrichment technology and designed a panel including nine genes: ATM, BIRC3, MYD88, NOTCH1, SF3B1 and TP53, which have been linked to the prognosis of chronic lymphocytic leukemia, and KLHL6, POT1 and XPO1, which are less characterized but were found to be recurrently mutated in various sequencing studies. A total of 188 chronic lymphocytic leukemia patients with poor prognostic features (unmutated IGHV, n=137; IGHV3-21 subset #2, n=51) were sequenced on the HiSeq 2000 and data were analyzed using well-established bioinformatics tools. Using a conservative cutoff of 10% for the mutant allele, we found that 114/180 (63%) patients carried at least one mutation, with mutations in ATM, BIRC3, NOTCH1, SF3B1 and TP53 accounting for 149/177 (84%) of all mutations. We selected 155 mutations for Sanger validation (variant allele frequency, 10–99%) and 93% (144/155) of mutations were confirmed; notably, all 11 discordant variants had a variant allele frequency between 11–27%, hence at the detection limit of conventional Sanger sequencing. Technical precision was assessed by repeating the entire HaloPlex procedure for 63 patients; concordance was found for 77/82 (94%) mutations. In summary, this study demonstrates that targeted next-generation sequencing is an accurate and reproducible technique potentially suitable for routine screening, eventually as a stand-alone test without the need for confirmation by Sanger sequencing.     

Functional genomic screen of human stem cell differentiation reveals pathways involved in neurodevelopment and neurodegeneration

Human embryonic stem cells (hESCs) can be induced and differentiated to form a relatively homogeneous population of neuronal precursors in vitro. We have used this system to screen for genes necessary for neural lineage development by using a pooled human short hairpin RNA (shRNA) library screen and massively parallel sequencing. We confirmed known genes and identified several unpredicted genes with interrelated functions that were specifically required for the formation or survival of neuronal progenitor cells without interfering with the self-renewal capacity of undifferentiated hESCs. Among these are several genes that have been implicated in various neurodevelopmental disorders (i.e., brain malformations, mental retardation, and autism). Unexpectedly, a set of genes mutated in late-onset neurodegenerative disorders and with roles in the formation of RNA granules were also found to interfere with neuronal progenitor cell formation, suggesting their functional relevance in early neurogenesis. This study advances the feasibility and utility of using pooled shRNA libraries in combination with next-generation sequencing for a high-throughput, unbiased functional genomic screen. Our approach can also be used with patient-specific human-induced pluripotent stem cell-derived neural models to obtain unparalleled insights into developmental and degenerative processes in neurological or neuropsychiatric disorders with monogenic or complex inheritance.     

CD100/Plexin-B1 interactions sustain proliferation and survival of normal and leukemic CD5+ B lymphocytes

Growth and survival of chronic B-cell tumors are favored by the malignant cell's capacity to respond to selected microenvironmental stimuli provided by nontumoral bystander cells. To investigate which mechanisms operate in these crosstalks and whether they are malignancy-related or reproduce the mechanisms used by normal B cells we have studied the expression and functional role of semaphorin CD100 (now called Sema4D) in chronic lymphocytic leukemia (CLL) cells and normal CD5+ B cells. We demonstrate here that (1) leukemic and normal CD5+ B lymphocytes uniformly express CD100; (2) the CD100 high-affinity receptor Plexin-B1 is expressed by bone marrow stromal cells, follicular dendritic cells, and activated T lymphocytes, and is thus available to CD100+ lymphocytes in different specific microenvironments; and (3) upon interaction between CD100 and Plexin-B1 both CLL and normal CD5+ B cells increase their proliferative activity and extend their life span. These findings establish that Plexin-B1 is an easily accessible receptor for CD100 within the immune system. The encounter of CD100+ leukemic cells with Plexin-B1 may promote the proliferation and survival of malignant cells. The crosstalk operated by the CD100/Plexin-B1 interaction is not malignancy related but reproduces a mechanism used by normal CD5+ B cells.