Majority Of Clones Research Articles

EBV infection of B-CLL cells in vitro potentiates their allostimulatory capacity if accompanied by acquisition of the activated phenotype.

Epstein-Barr virus (EBV)-carrying immortalized lymphoblastoid cell lines (LCLs) stimulate autologous T lymphocytes in vitro. This T-cell response is independent of the EBV-specific cellular memory because it also occurs in experiments with cells of seronegative individuals. The question can be posed whether the T-cell-stimulatory potential of the LCL is coupled to its immortalized state. B-CLL cells were exploited to study this question because the majority of clones, represented by different patients, can be infected with EBV but they rarely become immortalized. We have investigated the phenotypic changes and the T-cell-stimulatory capacity of EBV-infected B-CLL cells. One aliquot of CLL cells was infected with EBV, another was activated with a mixture of Staphylococcus aureus (SAC), IL-2 and the supernatant from the T-cell hybridoma MP6 (activation mixture, AcMx) and the third aliquot received both treatments. In accordance with the individual features of the clonal populations represented by each patient, the immunophenotypic changes imposed by these treatments differed. With the samples of 3 patients the allo-stimulatory potential showed the following ranking order: EBV and AcMx-treated cells > AcMx-treated > EBV-infected. An analysis of several activation-related surface markers and adhesion molecules on the cells did not reveal any association between their expression and the EBV-imposed potentiation of allostimulatory capacity. These results may be extrapolated to EBV-genome-carrying normal B cells, suggesting that they can persist in vivo only as long as they have the resting phenotype. Once they are activated, these cells may be recognized and eliminated by T lymphocytes.

Early phenotypic choices by neuronal precursors, revealed by clonal analysis of the chick embryo hindbrain.

The mechanisms that generate diverse neuronal phenotypes within the central nervous system are thought to involve local cues or cell-cell interactions acting late in neurogenesis, perhaps as late as the last precursor cell division. We describe here a clonal analysis of neuronal development in the chick hindbrain, using an intracellular tracer to mark single precursor cells, that suggests the operation of an alternative strategy. The majority of clones, ranging from 1 to 46 cells, contained neurons of only one of several possible phenotypes. These single-phenotype clones were not positionally restricted within a rhombomere but were interspersed with other clones containing distinct phenotypes. The assignment of neuronal phenotype in this brain region may, therefore, be made in early precursors and remembered through several rounds of mitotic expansion and dispersal.

Isolation of region specific single-copy probes from human chromosome 1q23→1q25

Single-copy sequences of chromosome bands 1q23-->1q25 were enriched by subtractive hybridization of digoxigenin oligonucleotide-labeled DNA from a somatic cell hybrid containing human chromosome 1 with DNA from a somatic cell hybrid containing a deleted human chromosome 1, del(1)(q23q25). These sequences were purified with an immobilized anti-digoxigenin antibody, amplified by polymerase chain reaction (PCR) and cloned. The majority of clones contained single-copy sequences, thus allowing direct screening of human genomic DNA. With pools of probes complex hybridization patterns as well as differences in hybridization patterns between genomes were observed in Southern blotting experiments. Nine clones containing independent single-copy sequences from chromosome bands 1q23-->1q25 were isolated and sequenced to generate sequence-tagged sites (STSs). Using PCR, region specific genomic clones containing large inserts were identified in human genomic lambda phage and cosmid libraries. Thus, region specific genomic probes generated by genomic difference cloning are useful for identifying deletions and rearrangements, for generating STSs and for isolating genomic clones containing large inserts of a particular chromosomal region.

Analysis of Viral Gene Expression and Genome Persistence in CD23-Positive and -Negative Subpopulations of Epstein-Barr-Virus-lnfected BL2 Cells

Epstein-Barr virus (EBV) infection of the EBV-negative Burkitt lymphoma (BL) cell line BL2 has been studied in vitro, to assess the efficiency of stable infection and the state of the viral genome in these cells. Expression of the EBV nuclear antigen complex (EBNA 1-6) was detected by 24 h post-infection, but expression of the latent membrane protein (LMP) and of the B cell activation antigen CD23 were delayed until 8 days post-infection, and were then only detectable in a subpopulation of EBNA-positive cells. Separation of cells into CD23-positive and -negative fractions 8 days after infection showed that the CD23-positive cells expressed EBNA 1-6 and LMP and contained viral episomes, whereas the CD23-negative cells were EBNA 1-6 positive, LMP negative and had very low levels of episomal viral genome. Cloning of the CD23-positive and -negative populations showed that EBV infection in the majority of clones was lost, with only 29 out of the 164 investigated still EBNA positive after 6 months of culture. Stable infection correlated with expression of EBNA 1-6, LMP, CD23 and the presence of viral episomes. One clone stably expressed EBNA 1-6 and LMP in the absence of detectable linear or episomal viral genome; this clone was assumed to have a viral genome integrated into the cellular chromosomes. Thus integration of the viral genome following in vitro infection of BL2 cells is a rare event.

Modulation of accessory cell function of immortalized bone marrow-derived macrophages by granulocyte/macrophage colony-stimulating factor

To generate cloned macrophage populations with sensitivity towards granulocyte/macrophage colony-stimulating factor (GM-CSF), bone marrow-derived macrophages (BMM phi) were immortalized by transformation with SV40. A panel of transformed clones was established. The majority of clones represented independently derived transformants, as evidenced by restriction fragment length polymorphism using genomic DNA digested with EcoRI and TaqI and the 5.2 kb SV40 DNA for hybridization analysis. The cells belong to the macrophage lineage according to several criteria, e.g. the presence of nonspecific esterase, their phagocytic capacity and their morphology. Many clones were potent antigen-presenting cells (APC), without exogenous stimulation. Two clones, which did not act efficiently as APC when used untreated, were positively responsive to GM-CSF. GM-CSF stimulation of both clones resulted in potent APC capacity. I-A alpha, I-A beta and gamma chain-specific transcripts were observed upon stimulation with GM-CSF, corresponding to detectable levels of class II surface display as revealed by cytofluorometric analysis. Thus the macrophage clones established will allow dissection of the differential effects of GM-CSF on the parameters of antigen presentation.

Use of the most JH-proximal human Ig H chain V region gene, VH6, in the expressed immune repertoire.

VH6, the most 3' human H chain V region gene, is preferentially expressed in the preimmune repertoire of the developing human fetus. To determine whether VH6 contributes to the immune repertoire, we amplified and sequenced from human spleen RNA VH6 rearrangements that were isotype-switched to IgG. Eighteen distinct in-frame VH6 clones were sequenced. Definitive assignment to C gamma 1 could be made in the majority of clones, and all had undergone extensive somatic hypermutation in the V region with an average of 14 (range, 2 to 25) mutations/281 bp in the VH6 segment. The overall frequency of somatic mutation was 5.2% of the total nucleotides sequenced. Replacement somatic mutations were targeted to the complementarity determining region, and the complementarity determining region had higher replacement to silent mutation ratios, consistent with antigenic selection. Silent somatic mutations were also significantly more frequent in the complementarity determining region compared to the framework region. Five clones were derived from the same VH6Dxp'1JH6 rearrangement, each containing unique and shared mutations. All DH segments used by independently arising clones were unique, and all DH reading frames were seen. N segments were lacking at either the 5'DH-JH junction or the 3'DH-JH junction in 7 of 13 independent rearrangements. DH-DH recombinations were frequently observed, and there was biased usage of JH3b and JH4b in the clones. The data imply that a high degree of diversity arises from use of this 3'-VH gene in the immune repertoire.

Vital dye labelling of Xenopus laevis trunk neural crest reveals multipotency and novel pathways of migration.

Although the Xenopus embryo has served as an important model system for both molecular and cellular studies of vertebrate development, comparatively little is known about its neural crest. Here, we take advantage of the ease of manipulation and relative transparency of Xenopus laevis embryos to follow neural crest cell migration and differentiation in living embryos. We use two techniques to study the lineage and migratory patterns of frog neural crest cells: (1) injections of DiI or lysinated rhodamine dextran (LRD) into small populations of neural crest cells to follow movement and (2) injections of LRD into single cells to follow cell lineage. By using non-invasive approaches that allow observations in living embryos and control of the time and position of labelling, we have been able to expand upon the results of previous grafting experiments. Migration and differentiation of the labelled cells were observed over time in individual living embryos, and later in sections to determine precise position and morphology. Derivatives populated by the neural crest are the fins, pigment stripes, spinal ganglia, adrenal medulla, pronephric duct, enteric nuclei and the posterior portion of the dorsal aorta. In the rostral to mid-trunk levels, most neural crest cells migrate along two paths: a dorsal pathway into the fin, followed by presumptive fin cells, and a ventral pathway along the neural tube and notochord, followed by presumptive pigment, sensory ganglion, sympathetic ganglion and adrenal medullary cells. In the caudal trunk, two additional paths were noted. One group of cells moves circumferentially within the fin, in an arc from dorsal to ventral; another progresses ventrally to the anus and subsequently populates the ventral fin. By labelling individual precursor cells, we find that neural tube and neural crest cells often share a common precursor. The majority of clones contain labelled progeny cells in the dorsal fin. The remainder have progeny in multiple derivatives including spinal ganglion cells, pigment cells, enteric cells, fin cells and/or neural tube cells in all combinations, suggesting that many premigratory Xenopus neural crest precursors are multipotent.

Clonal expansion of lymphocytes from oral lichen planus lesions

Lymphocytes were extracted from 11 biopsy specimens of oral lichen planus (OLP) by collagenase digestion, and cell lines were expanded with repetitive cycles of stimulation (with phytohaemagglutinin) and rest in media supplemented with interleukin 2. Four OLP lines contained a majority of CD3+CD4-CD8+ cells, in six lines the CD4:CD8 ratio was between 1 and 2, and in one line the CD4:CD8 ratio was 5:1. Limiting dilution of nine lines at 0.3 and 1.0 cells/well resulted in viable wells (putative clones) with plating efficiencies ranging from 0.0 to 18.1 percent and 0.0 to 22.2 percent respectively. The majority of clones were CD3+CD4-CD8+alpha beta+gamma delta-, although three clones were CD3+CD4+CD8-alpha beta+gamma delta- and one clone was CD3+CD4-CD8- and expressed the gamma delta T cell receptor. T cell clones derived from lymphocytes extracted from OLP lesions may be generated and maintained in culture providing opportunity for their further phenotypic and functional characterisation. This strategy may facilitate the identification of a putative oral lichen planus-specific antigen and indicate the frequency of lichen planus-specific T cells within lesions of OLP.

Stimulus-Dependent Modulation of Human B Cell Function by T Cell Clones

T cells exert both positive and negative regulatory effects on B cell function. To determine whether the nature of the stimulus could alter the immunoregulatory effects of T cells, the capacity of a battery of human T cell clones to modulate B cell function after stimulation with either pokeweed mitogen or a mAb to the CD3 molecular complex was examined. It was observed that most clones induced B cell differentiation when stimulated with immobilized mAb to CD3 (64.1) regardless of their phenotype. Moreover, the majority of clones (42 of 51) augmented the generation of immunoglobulin-secreting cells (ISC) supported by anti-CD3-stimulated blood CD41 T cells. By contrast, none of the clones induced B cell differentiation when stimulated with PWM and 48 of 51 clones suppressed the generation of ISC induced by blood CD41 T cells. This suppression could not be accounted for by the depletion of essential molecules or factors or by secretion of suppressive factors. Suppressive activity of clones did not correlate with the CD4 or CD8 phenotype and was not overcome by the addition of supernatants generated from mitogen-stimulated T cells or recombinant IL-2. Suppression by most clones, however, was abrogated when the clones were treated with mitomycin C or irradiated. A number of suppressive mechanisms by individual PWM-stimulated clones was identified, including direct inhibition of B cell function by cytotoxic and non-lytic means and suppression of helper T cell function. The failure of anti-CD3-stimulated clones to suppress the differentiation of B cells appeared to reflect the capacity of this stimulus to induce apoptosis by the clones after initial activation. These results indicate that T cell clones can provide help for B cell differentiation or can suppress B cell function by a variety of mechanisms depending upon the mode of stimulation.

IL-4 and IFN (alpha and gamma) exert opposite regulatory effects on the development of cytolytic potential by Th1 or Th2 human T cell clones.

Abstract The cytolytic potential of a total number of 118 CD4+ human T cell clones specific for purified protein derivative (PPD) from Mycobacterium tuberculosis, tetanus toxoid, Lolium perenne group I allergen (Lol p I), Poa pratensis group IX allergen (Poa p IX), or Toxocara canis excretory/secretory antigen(s) (TES) was assessed by both a lectin (PHA)-dependent and a MHC-restricted lytic assay and compared with their profile of cytokine secretion. The majority of clones with Th1 or Th0 cytokine profile exhibited cytolytic activity in both assays, whereas Th2 clones usually did not. There was an association between the cytolytic potential of T cell clones and their ability to produce IFN-gamma, even though IFN-gamma produced by T cell clones was not responsible for their cytolytic activity. IL-4 added in bulk culture before cloning inhibited not only the differentiation of PPD-specific T cells into Th1-like cell lines and clones, but also the development of their cytolytic potential. The depressive effect of IL-4 on the development of PPD-specific T cell lines with both Th1 cytokine profile and cytolytic potential was dependent on early addition of IL-4 in bulk cultures. In contrast, the addition in bulk culture of IFN-gamma enhanced both the cytolytic activity of PPD-specific T cell lines, as well as the proportion of PPD-specific T cell clones with cytolytic activity. The addition in bulk cultures before cloning of IFN-gamma or IFN-alpha favored the development of TES-specific and Poa p IX-specific T cells into T cell clones showing a Th0 or even a Th1, rather than a Th2, cytokine profile. Accordingly, most of TES- and Poa p IX-specific T cell clones derived from cultures containing IFN-gamma or IFN-alpha displayed strong cytolytic activity. These data indicate that the majority of human T cell clones that produce IFN-gamma, but not IL-4 (Th1-like), as well as of T cell clones that produce IFN-gamma in combination with IL-4 (Th0-like) are cytolytic. More importantly, they demonstrate that the addition of IFN (alpha and gamma) or IL-4 in bulk cultures before cloning may influence not only the cytokine profile of human CD4+ T cell clones but also their cytolytic potential.

Changes in the precursor frequencies of IL-4 and IFN-gamma secreting CD4+ cells correlate with resolution of lesions in murine cutaneous leishmaniasis.

Limiting dilution analysis was used to estimate the frequency of clonogenic Ag-specific CD4+ T lymphocytes in draining lymph nodes of mice over the course of infection with Leishmania major, and to measure the production of IL-2, IL-3, IL-4, IFN-gamma, and TNF by the resultant clones. Infection of both genetically susceptible BALB/c ("non-healer") and resistant C57BL/6 ("healer") mice resulted in at least a fourfold increase in the frequency (to about 0.3%) and at least a 10-fold increase in the total number of lymph node CD4+ cells that formed clones when cultured with L. major Ag in vitro. At 1 wk after infection, the majority of clones from BALB/c mice secreted IL-4 (precursor frequency 0.15%) and fewer secreted IFN-gamma (0.05%); this pattern remained constant for at least 8 wk after infection. In C57BL/6 mice, however, a high precursor frequency of IL-4-secreting clones was measured in the first 1 to 2 wk when the mice had lesions, but resolution of infection was associated with a decrease in the frequency of IL-4-secreting clones (from 0.13% at 2 wk to 0.03% at 4 wk) and an increase in the frequency of IFN-gamma-secreting clones (from 0.08% to 0.22%). At all stages of infection, most clones from either mouse strain secreted IL-3 and very few secreted TNF. Analysis of PCR-amplified cDNA from draining lymph nodes of infected mice also revealed that IL-4 and IFN-gamma mRNA were expressed in both mouse strains early in infection. IL-4 mRNA was the major species at 2 and 6 wk after infection in BALB/c mice, but declined relative to IFN-gamma mRNA over this time in C57BL/6 lymph nodes. Precursor frequency estimates of lymphokine-secreting CD4+ cells in draining lymph nodes therefore correlated with lymphokine expression patterns in vivo. Analysis of a panel of individual short term clones derived from mice 1 wk after infection revealed marked heterogeneity in lymphokine production patterns. In BALB/c mice, 49% secreted IL-4 without IFN-gamma, 18% secreted IFN-gamma without IL-4, and 14% secreted both IL-4 and IFN-gamma. Similarly in C57BL/6 mice, 39% secreted IL-4, 20% secreted IFN-gamma, and 17% secreted both lymphokines. Many of the clones also produced IL-3 and/or IL-2. Together the data suggest that both IL-4 and IFN-gamma are synthesized early in infection of susceptible and resistant mice as assessed by mRNA and precursor frequency analyses.(ABSTRACT TRUNCATED AT 400 WORDS)

Haplotypic origin of beta-chain genes expressed by human T-cell clones.

A contribution of allelic variation of T-cell receptor (Tcr) genes to the immune response has not been studied. Here we report that the presence of insertion-deletion-related polymorphisms (IDRP) of the Tcr beta chain (Tcrb) can be utilized to distinguish the parental origin of the gene complex that undergoes rearrangement in individual T-cell clones. Phytohemagglutinin stimulated clones from an individual heterozygous for an IDRP located between the variable (V) and diversity (D)-joining (J) region genes were studied for the presence of V to DJ rearrangements in each of the two parental chromosomes. Results indicate that single rearrangements were present in the majority of clones, in contrast to the double rearrangements of D to J genes that were generally present. In this individual, V to DJ rearrangement also occurred with different frequencies on each of the two germline genes. IDRP clonotyping of the Tcrb complex should prove generally applicable to the study of the influence of allelic variation of Tcrb genes in selection of the expressed T-cell repertoire.

Isolation of chromosome 21-specific yeast artificial chromosomes from a total human genome library.

A new approach for the isolation of chromosome-specific subsets from a human genomic yeast artificial chromosome (YAC) library is described. It is based on the hybridization with an Alu polymerase chain reaction (PCR) probe. We screened a 1.5 genome equivalent YAC library of megabase insert size with Alu PCR products amplified from hybrid cell lines containing human chromosome 21, and identified a subset of 63 clones representative of this chromosome. The majority of clones were assigned to chromosome 21 by the presence of specific STSs and in situ hybridization. Twenty-nine of 36 STSs that we tested were detected in the subset, and a contig spanning 20 centimorgans in the genetic map and containing 8 STSs in 4 YACs was identified. The proposed approach can greatly speed efforts to construct physical maps of the human genome.

T‐Cell Receptor Gene Expression by Human γδ T‐Cell Clones from Peripheral Blood and Reproductive Tissues in Relation to Non‐MHC‐Restricted Cytotoxic Function

T-cell receptor gamma and delta gene expression was determined using V-region-specific monoclonal antibodies in conjunction with Southern blot analysis in panels of gamma delta T-cell clones from human peripheral blood (n = 77) and reproductive tissue (n = 9). Whereas 53 out of 77 (69%) clones from peripheral blood expressed V gamma 9 and V delta 2J1, only 2 out of 9 (22%) from reproductive tissues expressed V delta 2J1. Two out of eight decidual clones expressed both V gamma 9 and V delta 1J1, while this configuration was rare in clones from peripheral blood. The majority of clones from the peripheral blood of one donor expressed V gamma 8 and V delta 3J1. Clones were identified which expressed V delta 1J1 in the disulphide-linked C gamma 1 form of the receptor and which expressed a gene other than V delta 1 in the non-disulphide-linked C gamma 2 form, indicating incomplete concordance between expression of V delta 1 and C gamma 2. V delta 3 could be expressed in the disulphide-linked or non-disulphide-linked form of the receptor. At least 5 out of 77 peripheral clones were expressing V delta genes other than V delta 1, V delta 2, or V delta 3 in conjunction with C gamma 1 or C gamma 2. There was a strong but incomplete correlation between high non-NHC-restricted cytotoxic function and C gamma 1 expression. Clones from the same donor expressing both V gamma 9JPC gamma 1 and V delta 2J1 showed either high or negligible cytotoxicity, and cytotoxic clones expressing C gamma 2 were found. Thus no complete correlation between cytotoxic function and expression of a particular form of the gamma delta heterodimer was identified. The results also suggest that gamma delta T cells from reproductive tissues are less likely to express V delta 2J1 than those from peripheral blood.

Generation of T cell clones binding F(ab')2 fragments of the idiotypic immunoglobulin in patients with monoclonal gammopathy.

Lymphocytes from two patients with multiple myeloma stage I and one patient with monoclonal gammopathy of undetermined significance were found to proliferate specifically in response to low concentrations of F(ab')2 fragments of the autologous M component. T cell clones isolated from repeatedly stimulated cultures bound specifically the autologous idiotype and proliferated after addition of soluble idiotype and exogenous interleukin-2. The majority of clones were CD8+ and showed negligible staining for CD4. Idiotype-binding clones could not be isolated from cultures of lymphocytes from a healthy control stimulated under the same conditions. The study provides support for the existence of idiotype-reactive T cells in monoclonal gammopathies. Such cells might have a regulatory role on the tumor cell clone and may be important for a future therapeutic approach.

Variation in Pied Flycatcher (Ficedula hypoleuca) Mitochondrial DNA

-Pied Flycatchers (Ficedula hypoleuca) in Sweden have low levels of variation in nuclear genes relative to most other bird species. This lack of variation has been attributed to population bottlenecks caused by Pleistocene glaciations. We studied mitochondrial DNA (mtDNA) variation in 20 Pied Flycatchers from four Swedish localities. Eight restriction endonucleases yielded a total of 207-212 DNA fragments (approximately 5% of the mtDNA genome). The mean pairwise divergence between the individuals was 0.35 ? 0.16% (range 0.00-0.82%), which suggests that the 18 identified mtDNA clones diverged within the past million years, and that the majority of clones evolved within the last 100,000 years. If genetic variation was reduced by prolonged bottlenecking during the last glacial period, low protein heterozygosity and high variability in mtDNA can be explained by a difference in rates of recovery of nuclear and mtDNA variation. The Pied Flycatcher in northern Europe may have only recently begun to regain variation in nuclear genes, whereas considerable variation in mtDNA has already accumulated through mutation. Received 7 December 1989, accepted 11 May 1990. GENETIC variability and relationships among clones of mitochondrial DNA (mtDNA) can provide background data to hypotheses on biogeographical history and population structure. As a clonally transmitted marker, the distribution of the maternally inherited mtDNA will reflect founder or rare immigration events more directly than nuclear DNA (Wilson et al. 1985). The study of mtDNA variability is highly relevant for avian species because of the relative lack of observed differentiation in proteins as assessed by electrophoresis (Barrowclough

Human CD4‐8‐ ‐Derived Clones Phenotypic and Functional Characteristics and Variation between Donors in Patterns of T‐Cell Receptor y Gene Rearrangements

Clones were derived from highly purified human CD4-8- lymphocytes from three different donors and maintained in the presence of interleukin 2 and phytohaemagglutinin. Considerable variation was noted between donors in the phenotype and T-cell receptor (TCR) gamma gene rearrangements of CD4-8- -derived clones. In one donor, most clones remained CD4-8- and all were CD3+WT31- and therefore expressed gamma/delta heterodimers. TCR gamma gene rearrangements almost all involved C gamma 1. In contrast, most clones from a second donor were CD3+WT31+, and therefore expressed alpha/beta heterodimers, and many were positive for CD4 or CD8. Most clones from a third donor were CD3+WT31- with a high proportion of TCR gamma gene rearrangements involving C gamma 2. The V gamma 9JP rearrangement was exclusively confined to CD3+WT31- clones and was present in the majority of clones. Almost all CD3+WT31- clones showed TCR beta as well as gamma gene rearrangements. Most CD3+WT31- clones with at least one chromosome rearranged to C gamma 1 exhibited high non-major histocompatibility complex (MHC)-restricted cytotoxic activity, while most of those with two C gamma 2 rearrangements, and therefore expressing a non-disulphide-linked gamma/delta heterodimer, had low activity. Preincubation of effector cells with anti-CD3 strongly inhibited the cytotoxicity of CD3+WT31- clones while that of CD3+WT31+ clones was enhanced. This implicates the CD3-gamma/delta complex in target cell recognition by cytotoxic gamma/delta-bearing T-cell clones. The results show that there is heterogeneity between donors in the relative proportions of CD4-8- -derived clones expressing alpha/beta heterodimers and the different forms of the gamma/delta heterodimer.

Human thymocytes expressing gamma/delta T-cell receptors

Expression of T-cell receptors of gamma/delta type characterizes a small subset of peripheral T lymphocytes which is homogeneously composed of cytolytic cells and, in most instances, lack CD4 and CD8 differentiation antigens. By the use of anti-TCR gamma/delta MAbs it is possible to identify two distinct subsets of TCR gamma/delta+ cells that are characterized by a C gamma 1 or C gamma 2-encoded forms of gamma-chain, respectively. While the BB3 MAb-reactive (C gamma 1 encoded) cell subset is prevalent in peripheral blood (PB), these cells represent less than 10% in TCR gamma/delta+ thymocyte populations. In thymus, the majority of cells was found to react with delta-TCSI (or A13) MAbs. Culture of CD4-8- thymocytes (highly enriched in TCR gamma/delta+ cells) in IL-2 resulted in the de novo expression of CD8 surface antigen and of non MHC-restricted cytolytic activity. Cloning of CD4-8- thymocytes resulted, for the most part, in CD3+ TCR gamma/delta+ cells. Moreover, the majority of clones expressed the unusual delta-TCSI+ CD8+ phenotype and lysed the NK-sensitive K562 target cells. Analysis of the immunoprecipitated TCR molecules showed the existence of the (rare) heavy (55 kDa) form of gamma-chain. A redirected killing assay using murine P815 target cells and appropriate "stimulatory" antibodies was further employed for functional analysis of thymus-derived TCR gamma/delta+ clones. While anti-CD3 MAbs efficiently triggered the cytolytic activity of all clones irrespective of their phenotype, MAbs directed to TCR gamma/delta induced efficient lysis only of BB3+ or delta-TCSI+CD8 clones, but not of delta-TCSI+ CD8+ clones.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential lysis of melanoma clones by autologous recombinant interleukin 2-activated lymphocytes. Relationship with spontaneous resistance to doxorubicin (Dx).

To investigate whether human melanoma cells intrinsically resistant to autologous LAKs do exist, and whether a relationship between the level of lysis of LAKs and spontaneous drug resistance can be identified at the clonal level, we studied 44 clones obtained from a metastatic melanoma lesion. The antigenic phenotype of clones revealed a marked heterogeneity in the expression of HLA antigens of classes I and II. The clones were subsequently tested for sensitivity to autologous LAK and for spontaneous resistance to Dx. No clone resistant to autologous LAK was found, although a considerable range of lysis was noted with a normal frequency distribution. Growth in agar of the 2 clones in which lysis was least pronounced (6 and 26) was completely inhibited after co-culture with LAKs, indicating a lack of absolute resistance to these effectors. Spontaneous resistance to Dx, evaluated as ID50, revealed instead that the majority of clones had a low ID50. The frequency distribution of clones showed a left-skewed curve. The percentage of specific 51Cr-release and the ID50 for Dx could be correlated in 25 clones by linear regression. Sensitivity to LAK did not correlate with HLA classes I or II or melanoma-associated antigen expression. These results support the contention that increased LAK sensitivity of tumor cells is associated with drug resistance.

Tomato genome is comprised largely of fast-evolving, low copy-number sequences

Fifty random clones (350–2300 bp), derived from sheared, nuclear DNA, were studied via Southern analysis in order to make deductions about the organization and evolution of the tomato genome. Thirty-four of the clones were mapped genetically and determined to represent points on 11 of the 12 tomato chromosomes. Under moderate stringency conditions (≤80% homology required) 44% of the clones were classified as single copy. Under higher stringency, the majority of the clones (78%) behaved as single copy. Most of the remaining clones belonged to multicopy families containing 2–20 copies, while a few contained moderately or highly repeated sequences (10% at moderate stringency, 4% at high stringency). Divergence rates of sequences homologous to the 50 random genomic clones were compared with those corresponding to 20 previously described cDNA (coding sequence) clones. Rates were measured by probing each clone (random genomics and cDNAs) onto filters containing DNA from various species from the family Solanaceae (including potato, Datura, petunia and tobacco) as well as one species (watermelon) from another plant family, Cucurbitaceae. Under moderate stringency conditions, the majority of the random clones (single copy and repetitive) failed to detect homologous sequences in the more distantly related species, whereas approximately 90% of the 20 coding sequences analyzed could still be detected in all solanaceous species. The most highly repeated sequences appear to be the fastest evolving and homologous copies could be detected only in species most closely related to tomato. Dispersion of repetitive sequences, as opposed to tandem clustering, appears to be the rule for the tomato genome. None of the repetitive sequences discovered by this random sampling of the genome were tandemly arranged — a finding consistent with the notion that the tomato genome contains only a small fraction of satellite DNA. This study, along with a companion paper (Ganal et al. 1988), provides the first general sketch of the tomato genome at the molecular level and indicates that it is comprised largely of single copy sequences and these sequences, together with repetitive sequences are evolving at a rate faster than the coding portion of the genome. The small genome and paucity of highly repetitive DNA are favourable attributes with respect to the possibilities of conducting chromosome walking experiments in tomato and the fact that coding regions are well conserved among solanaceous species may be useful for distinguishing clones that contain coding regions from those that do not.