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We performed aCGH, SKY/FISH, molecular mapping and expression analyses on a permanent CD8+ NK/T cell line, 'SRIK-NKL' established from a lymphoma (ALL) patient, in attempt to define the fundamental genetic profile of its unique NK phenotypes. aCGH revealed hemizygous deletion of 6p containing genes responsible for hematopoietic functions. The SKY demonstrated that a constitutive reciprocal translocation, rcpt(5;14)(p13.2;q11) is a stable marker. Using somatic hybrids containing der(5) derived from SRIK-NKL, we found that the breakpoint in one homologue of no. 5 is located upstream of IL7R and also that the breakpoint in no. 14 is located within TRA@. The FISH analysis using a BAC which contains TRA@ and its flanking region further revealed a approximately 231kb deletion within 14q11 in the der(5) but not in the normal homologue of no. 14. The RT-PCR analysis detected mRNA for TRA@ transcripts which were extending across, but not including, the deleted region. IL7R was detected at least at mRNA levels. These findings were consistent with the immunological findings that TRA@ and IL7R are both expressed at mRNA levels and TRA@ at cytoplasmic protein levels in SRIK-NKL cells. In addition to rcpt(5;14), aCGH identified novel copy number abnormalities suggesting that the unique phenotype of the SRIK-NKL cell line is not solely due to the TRA@ rearrangement. These findings provide supportive evidence for the notion that SRIK-NKL cells may be useful for studying not only the function of NK cells but also genetic deregulations associated with leukemiogenesis.

T-cells and NK cells arise from common pluripotent stem cells, with shared early developmental pathway and surface markers. Distinguishing between them is becoming difficult, but critical for study. A large family of NK cells, including classical NK, NK-T, and NK-CTL exists. Natural cytotoxicity receptors (NKp46, NKp44, NKp30) have been proposed as specific for classical NK cells, but were expressed at protein and mRNA level by CD8+NK/T cell line SRIK-NKL, suggesting more widespread expression. We investigated and found expression of these markers at the protein and mRNA level in multiple human cell lines.

Natural killer (NK) cells, innate lymphocyte effectors, kill virus-infected host and tumor cells in non-MHC, non-TCR restricted fashion, unlike T cells. The role of NK cells in recognition and killing of pathogens remains unknown. Expression of the ten human pathogen associated molecular pattern or toll-like receptors (TLR's), associated molecules, and antimicrobial peptides alpha, beta defensins, and LL-37 by NK cells has not been investigated previously. We report our CD8+ NK/T cell line SRIK-NKL, derived from leukemic phase of acute lymphoblastic lymphoma, expresses mRNA and protein for 8/10 TLR's, associated proteins for signaling, defensins, cathelicidin/LL-37, and responds to live bacteria by cell proliferation and increased IFN-gamma, TNF-alpha, TNF-beta, and MIP-1alpha production.

The distinction between T cells and NK cells is difficult, and becoming more complex, as the diversity of the human lymphocyte repertoire is evident. We report the establishment of a permanent CD8+ NK/T cell line (SRIK-NKL) from a patient with leukemic phase of acute lymphoblastic lymphoma having characteristics of both NK and T cells, and extensively describe its phenotype, including cytotoxic activity, NK cell receptor expression, and other molecules critical for immune function. We further compare SRIK-NKL to other available NK/NK-T cell lines. SRIK-NKL may be useful for studying NK cell development, functions, and modulation, leading to novel strategies for treatment of autoimmune disease, infection, and cancer.

To date no hematopoietic progenitors of dendritic Langerhans' cells (DLC), which represent an highly efficient class of antigen presenting cells, have been identified or the cytokines they elaborate have been defined. Here we describe an acute leukemia patient whose blasts (90-96% in peripheral blood and bone marrow) had a phenotype consistent with putative progenitors of DLC. The patient was treated with ara-C and VP-16 but did not achieve remission. The blasts had lobulated nuclei, no cytoplasmic vacuolation or Auer rods and were weakly positive for acid phosphatase and non-specific esterase and negative for PAS, granzyme A, dipeptidyl aminopeptidase IV, ATPase/ADPase and lysozyme production. The blasts were positive for CD1a, CD4, CD16, CD35, HLADR, HLADQ, CD11b, CD11c, CD14, CD33, CD34, CD11a, CD71, CD19, CD25, IL-2R beta and negative for CD2, CD7, CD8, CD10, CD22, CD56, CD57, surface or cytoplasmic CD3, TCR delta and TCR beta, HTLV-1p19 and P-glycoprotein. On liquid culture with or without 5 x 10(-9) M 12-O-tetradecanoylphorbol-13-acetate (TPA) for 3 days, the blasts formed aggregates of proliferating and elongating cells on the wall of the flasks with a decline in CD34, numerous dendritic processes appeared on the cells and there was strong positivity for ATPase/ADPase, but no other changes in phenotype. No macrophages were observed, indicating derivation from separate DLCs. Cytogenetic analysis showed chromosomal abnormalities and electron microscopy showed Birbeck granules. Southern blotting of DNA showed rearrangement of one allele for both JH and TCR beta but no HTLV-1 related sequences. Culture supernatants from blasts cultured with or without TPA showed the production of large amounts of IL-8, IL-6, TNF-alpha, MIP-1 alpha, IL-10 and interferon gamma and modest amounts of IL-1 alpha, GM-CSF and stem cell factor. The presence not only of CD1a, HLADR, HLADQ and many other characteristics including Birbeck granules, but also differentiation along the lines of DLC with appearance of dendritic processes on the cells and expression of ATPase/ADPase activity, indicate that the leukemic blasts in our patient represented a leukemic counterpart of normal progenitors of DLC and the leukemia a new entity which could possibly be classified as AML-M8. Lastly, many pro-inflammatory cytokines produced by DLC could contribute to inflammation and IL-10 to immunosuppression.

The culture supernatants from 43 human cell lines obtained during log phase and from purified normal peripheral blood B-lymphocytes cultured at 10(6) cells ml-1 for 48 h in RPMI 1640-5% fetal calf serum were examined for interleukin-8 (IL-8) using Elisa kits. Constitutive IL-8 production was found for 14/15 B-cell lines (5 derived from normal persons and 2 from AML patients, 1 pre-B-ALL, 2 CLL with trisomy 12, 2 HTLV-I+, 1 HTLV-II+, 1/2 Burkitt lymphoma), 4/16 T-cell lines (3/6 HTLV-I+, 1 HTLV-II+, 0/9 T-ALL), myeloid line HL-60, monocytoid line U937, 3/3 ovarian carcinoma, 1/1 endometriosis, 2/2 normal fibroblast, 0/2 C-ALL, 0/1 pre-erythroid line K562, as well as for normal B-lymphocytes. Later, cells examined by indirect immunofluorescence using IL-8 antibodies gave a positive reaction. DNA from 4 IL-8 producing and 3 non-producing cell lines, when probed with IL-8 cDNA gave the same 3.5 kb EcoRI fragment indicating similarities of the IL-8 gene in these cells. Two B-cell lines examined showed the expression of 1.8 kb IL-8 mRNA. These results indicate IL-8 production by a greater variety of cells than previously believed which open possibilities for new IL-8-mediated immune functions by such cells as B-cells.

We have examined alterations in terminal deoxynucleotidyl transferase (TdT) immunofluorescence (IF) in MOLT-4 cells during changes in growth conditions. Subsequently, we used cells from 48 human hematopoietic cell lines of different cell lineages and maturation stages to compare the IF and biochemical assays for expression of TdT. In addition, we have attempted to correlate the expression of TdT, adenosine deaminase (ADA), thymidine phosphorylase (TP) and immunological markers with maturation stages in these different cell lines. The results indicate that TdT positive cells remain TdT positive when assayed by either the biochemical or IF tests during growth or early plateau phase, but that cells under poor growth conditions, such as in old cultures, may give a negative TdT IF reaction. Otherwise, biochemical and IF assays for TdT gave comparable results in the 48 cell lines tested, testifying to the reliability of the IF test. Based on the comparisons of the various cell lines studied, it appears that both ADA and TdT decrease progressively as maturation of T cells from Blast I to Blast IV to mature T cells increases. TP was deficient in all T-cell lines compared to normal peripheral blood T cells, which in turn had lower activity compared to normal peripheral blood B cells. Pre-B cells, although indistinguishable from each other by immunological markers and all having low TP and ADA activity, showed heterogeneity, with TdT activity high in some and low in others. All non-T, non-B lines had high TdT activity, but low ADA and TP activity. B- and myelocytic cell lines had low ADA and TdT activity, and showed an increase in TP activity as the maturation of cells increased. These results indicate that the TdT IF test is a reliable procedure for detecting TdT positive cells, and that TdT, ADA and TP could be useful markers for studying the differentiation of human hematopoietic cells.