To analyze the immunoglobulin repertoire of human IgM ϩ B cells and the CD5ϩ and CD5 Ϫ subsets, individual CD19 ϩ / IgM ϩ /CD5 ϩ or CD5 Ϫ B cells were sorted and non-productive as well as productive V H gene rearrangements were amplified from genomic DNA and sequenced. In both subsets, the V H 3 family was overrepresented largely as a result of preferential usage of a small number of specific individual family members. In the CD5 ϩ B cell subset, all other V H families were found at a frequency expected from random usage, whereas in the CD5 Ϫ population, V H 4 appeared to be overrepresented in the nonproductive repertoire, and also negatively selected since it was found significantly less often in the productive compared to the nonproductive repertoire; the V H 1 family was significantly diminished in the productive rearrangements of CD5 Ϫ B cells. 3-23/DP-47 was the most frequently used V H gene segment and was found significantly more often than expected from random usage in productive rearrangements of both CD5

To analyze the human kappa chain repertoire and the influences that shape it, a single cell PCR technique was used that amplified V J rearrangements from genomic DNA of individual human B cells. More than 350 productive and 250 nonproductive V J rearrangements were sequenced. Nearly every functional V gene segment was used in rearrangements, although six V gene segments, A27, L2, L6, L12a, A17, and O12/O2 were used preferentially. Of these, A27, L2, L6, and L12a showed evidence of positive selection based on the variable region and not CDR3, whereas A17 was overrepresented because of a rearrangement bias based on molecular mechanisms. Utilization of J segments was also nonrandom, with J 1 and J 2 being overrepresented and J 3 and J 5 underrepresented in the nonproductive repertoire, implying a molecular basis for the bias. In B cells with two V J rearrangements, marked differences were noted in the V segments used for the initial and subsequent rearrangements, whereas J segments were used comparably. Junctional diversity was generated by n-nucleotide addition in 60% and by exonuclease trimming in 75% of the V J rearrangements analyzed. Despite this large degree of diversity, a strict CDR3 length was maintained in both productive and nonproductive rearrangements. More than 23% of the productive rearrangements, but only 7% of the nonproductive rearrangements contained somatic hypermutations. Mutations were significantly more frequent in V sequences derived from CD5Ϫ as compared with CD5

We studied PTEN/MMAC1, a newly discovered candidate tumor suppressor gene at 10q23.3, for mutations in lung cancer. One hundred and thirty-six lung cancer cell line DNAs (66 small cell lung cancers, SCLC, 61 non-small cell lung cancers, NSCLC, four mesotheliomas, ®ve extrapulmonary small cell cancers) were analysed for PTEN/MMAC1 homozygous deletions and ®ve (8%) SCLC lines showed homozygous deletions interrupting the PTEN/MMAC1 gene. Using single stranded conformation polymorphism (SSCP) analysis, we screened the PTEN/MMAC1 open reading frame of 53 lung cancer cell line cDNAs for point mutations and found that 3/35 SCLCs and 3/18 NSCLCs contained homozygous amino acid sequence altering mutations. Northern blot analysis revealed that expression of the PTEN/MMAC1 gene was considerably lower in all the tumor cell lines with point mutations while no expression was detected for cell lines with PTEN/MMAC1 homozygous deletions. Mutation analysis of 22 uncultured, microdissected, primary SCLC tumors and metastases showed two silent mutations, and two apparent homozygous deletions. We also discovered a processed pseudogene (PTEN2) which has 98.5% nt identity to PTEN/MMAC1, that needs to be accounted for in cDNA mutation analysis. Our ®ndings suggest that genetic abnormalities of the PTEN/MMAC1 gene are only involved in a relatively small subset of lung cancers.

The capacity of endothelial cells (EC) to produce IL-15 and the capacity of IL-15 to influence transendothelial migration of T cells was examined. Human umbilical vein endothelial cells expressed both IL-15 mRNA and protein. Moreover, endothelial-derived IL-15 enhanced transendothelial migration of T cells as evidenced by the inhibition of this process by blocking monoclonal antibodies to IL-15. IL-15 enhanced transendothelial migration of T cells by activating the binding capacity of the integrin adhesion molecule LFA-1 (CD11a/CD18) and also increased T cell motility. In addition, IL-15 induced expression of the early activation molecule CD69. The importance of IL-15 in regulating migration of T cells in vivo was documented by its capacity to enhance accumulation of adoptively transferred human T cells in rheumatoid arthritis synovial tissue engrafted into immune deficient SCID mice. These results demonstrate that EC produce IL-15 and imply that endothelial IL-15 plays a critical role in stimulation of T cells to extravasate into inflammatory tissue.

Our data demonstrate that 2-CdA is highly effective in inducing CR in 84% of patients with MALT-type lymphoma.