Human CD8+ CTL recognition and in vitro lysis of herpes simplex virus-infected cells by a non-MHC restricted mechanism.

Cytotoxic T lymphocytes (CTL) are important for the recognition and lysis of virally infected cells, but their effectiveness can be limited by viral immune evasion mechanisms. We investigated the immunophenotype and function of human CD8+ T cells raised in response to herpes simplex virus (HSV). The expanded population contained cells of an activated and mature phenotype, as determined by the expressions of CD25, CD45RO, CD57, CD95 and HLA-DR. Cultured cells also expressed CD45RA. These cells lysed autologous and allogeneic HSV-infected lymphoblastoid cell line (LCL) targets via a non-major histocompatibility complex (MHC) restricted recognition pathway. Inhibition assays showed the mechanism of cytotoxicity to be calcium-dependent, granule exocytosis pathway, rather than the internal disintegration pathway. Cold target competition assays indicated that a common CTL population contributed to the recognition of autologous and allogeneic-infected targets. These effectors showed recognition of infected targets which was distinct from that of K562 cells. Non-MHC restricted lysis-associated molecule 2B4 (CD244) was upregulated on culturing and made a significant contribution to lysis of FcgammaR-bearing targets in a redirected killing assay. These findings suggest that CTL can recognize virally infected cells through a combination of non-MHC restricted mechanisms and may result in more efficient lysis than classical CD8+ T cells.

The use of Teflon cell culture bags to expand functionally active CD8+ cytotoxic T lymphocytes.

We have investigated the ability of Teflon cell culture (TCC) bags, compared to conventional tissue culture flasks and plates, to support the expansion of human CD8+ T cells in response to an allogeneic stimulus. TCC bags, which are compatible with good manufacturing practice (GMP), facilitated CD8+ T cell growth as well as conventional culture vessels and resulted in cytotoxic T cells which were able to kill allogeneic targets. Growth characteristics were compared by investigating the number, immunophenotype and cell cycle properties of the cells generated. The kinetics of cell growth were not significantly different over the first 14 days of culture in each vessel type, with the cell counts being highest at day 10 in all cases. However, the TCC bags resulted in a significantly higher proportion of cells with the morphology of typical lymphocytes than tissue culture flasks after 14 and 18 days in culture. There were no significant differences in the percentage of typical lymphocytes expanded in TCC bags compared to those expanded in plates. Expanded CD8+ cells maintained their initial level of expression of CD3, CD11a, CD18 and T cell receptor (alphabeta heterodimer, TCR (alphabeta)) but increased expression of CD45RO, CD95 and of activation markers HLA-DR and CD25 in each culture vessel. Studies of cell cycle parameters showed that each vessel supported CD8+ T cell stimulation, as demonstrated by significantly higher levels of S phase than fresh PBMN cells. The cells generated in TCC bags were able to kill allogeneic targets and also possessed natural killer (NK) cell activity. Thus, TCC bags are able to support the expansion of CD8+ T lymphocytes as well as flasks or tissue culture plates and are applicable to lymphocyte expansion for use in immunotherapy.

Minimal residual disease status before allogeneic bone marrow transplantation is an important determinant of successful outcome for children and adolescents with acute lymphoblastic leukemia.

The efficacy of allografting in acute lymphoblastic leukemia (ALL) is heavily influenced by remission status at the time of transplant. Using polymerase chain reaction (PCR)-based minimal residual disease (MRD) analysis, we have investigated retrospectively the impact of submicroscopic leukemia on outcome in 64 patients receiving allogeneic bone marrow transplantation (BMT) for childhood ALL. Remission BM specimens were taken 6 to 81 days (median, 23) before transplant. All patients received similar conditioning therapy; 50 received grafts from unrelated donors and 14 from related donors. Nineteen patients were transplanted in first complete remission (CR1) and 45 in second or subsequent CR. MRD was analyzed by PCR of Ig or T-cell receptor delta or gamma rearrangements, electrophoresis, and allele-specific oligoprobing. Samples were rated high-level positive (clonal band evident after electrophoresis; sensitivity 10(-2) to 10(-3)), low-level positive (MRD detected only after oligoprobing; sensitivity 10(-3) to 10(-5)), or negative. Excluding 8 patients transplanted in CR2 for isolated extramedullary relapse (all MRD-), MRD was detected at high level in 12 patients, low level in 11, and was undetectable in 33. Two-year event-free survival for these groups was 0%, 36%, and 73%, respectively (P <.001). Follow-up in patients remaining in continuing remission is 20 to 96 months (median, 35). These results suggest that MRD analysis could be used routinely in this setting. This would allow identification of patients with resistant leukemia (who may benefit from innovative BMT protocols) and of those with more responsive disease (who may be candidates for randomized trials of BMT versus modern intensive relapse chemotherapy).

Minimal residual disease status as a predictor of relapse after allogeneic bone marrow transplantation for children with acute lymphoblastic leukaemia.

We have analysed the behaviour of minimal residual disease (MRD) after allogeneic bone marrow transplantation (allo-BMT) in 71 children with acute lymphoblastic leukaemia (ALL). The method relied on PCR of IgH, TCRdelta and/or TCRgamma gene rearrangements followed by electrophoretic size resolution and allele-specific oligoprobing. Patients were similarly conditioned; 55 received marrow from unrelated donors and 16 from related donors. MRD was assessed at various time-points up to 24 months after BMT. Three children were not evaluable due to transplant-related mortality. MRD was detected in 28/32 patients (88%) who relapsed post-BMT; 16 were positive at all times and 12 were initially negative but became positive at a median of 3 months (range 1.5-11) prior to relapse. In contrast, only eight of 36 (22%) patients who remained in continuing complete remission (CCR) (median follow-up 43 months, range 20-94) showed MRD at any time after BMT (P<0.0001). In these eight patients MRD was found up to 9 months after transplant and at low levels (0.01-0.001%). All eight (median follow-up 39 months, range 24-87) had at least two MRD-negative samples tested subsequently and five of the eight had evidence of grade I-II acute graft-versus-host disease (GvHD), raising the possibility of a graft-versus-leukaemia effect. In general, any evidence of MRD after allo-BMT is a poor prognostic sign. However, if immunotherapy were to be targeted towards patients with evidence of persisting MRD after BMT, the method described would expose only a small proportion of patients to unnecessary additional toxicity.

Minimal residual disease analysis for the prediction of relapse in children with standard-risk acute lymphoblastic leukaemia.

We report a largely retrospective analysis of minimal residual disease (MRD) in a cohort of 66 children suffering from acute lymphoblastic leukaemia (ALL). All patients lacked high-risk features at diagnosis, i.e. the presenting white cell count was <50 x 10(9)/l, age 1-16 years and translocations t(9;22) and t(4;11) were not present. All were treated according to either the MRC protocols UKALL X or XI. PCR of IgH, TCRdelta and TCRgamma gene rearrangements and allele-specific oligoprobing were employed for the detection of MRD. Sensitivity was at least 10(-4) in 78/82 (93%) probes examined. A total of 33 patients relapsed (seven on therapy and 26 off) and 33 remain in continuing complete remission (CCR) (median follow-up 69 months from diagnosis). Of those who remain in CCR, MRD was present in the bone marrow in 32%, 10% and 0% at 1, 3 and 5 months into therapy respectively. This is in marked contrast to the presence of MRD at these times in 82%, 60% and 41% of patients who relapsed (P<0.001, P<0.005 and P<0.005). These results provide further evidence of a strong correlation between clearance of MRD early in therapy and clinical outcome in childhood ALL.

Same-day determination of chimaeric status in the immediate period following allogeneic bone marrow transplantation.

The accurate and rapid determination of the origin of haemopoietic cells may provide valuable information as to the aetiology of, and most appropriate therapy for, leucopenia following allogeneic bone marrow or peripheral blood stem cell transplantation. We describe an approach to the analysis of chimaerism post bone marrow transplantation (BMT) based on the immunomagnetic capture of white cells combined with microsatellite polymerase chain reaction (PCR) and resolution of products by polyacrylamide gel electrophoresis (PAGE). This non-isotopic method enables the chimaeric status to be determined from as little as 1.0 ml of profoundly leucopenic peripheral blood (WBC < or =0.1 x 10(9)/l) and has been applicable to all donor/recipient pairs tested so far. Results are available within 6 h of blood sampling and lineage-specific chimaerism is possible. Furthermore, blood transfusions do not interfere with the analysis.

A rapid and highly selective approach to cell separations using an immunomagnetic colloid.

Immunomagnetic colloids have the properties of solutions and, therefore, offer distinct advantages in their ability to bind to cells as compared to larger magnetic microspheres. B cells and T cells have been isolated with higher degrees of purity following incubation with monoclonal antibodies (MoAbs) and a goat anti-mouse Ig ferrofluid. This was demonstrated both with mixtures of cell lines and lines titrated into normal bone marrow. In all cases, low non-specific binding was observed. Maximal cell capture was obtained without washing out excess MoAb, resulting in cell separations that could be completed in under 30 min. The system permits not only the magnetic capture of cells onto pins in a high gradient separator, but also their recovery. Cells separated in this way appear to be highly viable and it is possible to manipulate them further as the immunocolloid is sufficiently small for it not to interfere with tests such as indirect immunofluorescence. The advantages and disadvantages of immunocolloids versus larger magnetic microspheres are discussed.

Meta-iodobenzylguanidine (mIBG) uptake and storage in the human neuroblastoma cell line SK-N-BE(2C).

Whilst many human neuroblastoma cell lines have been studied to see if they are capable of taking up mIBG, few appear to have this ability. This contrasts markedly to the situation in vivo, where uptake has been demonstrated in the majority of tumours investigated. Here we report on the human neuroblastoma cell line SK-N-BE(2C) and demonstrate that mIBG uptake can occur in this cell line through 2 mechanisms. At low concentrations of mIBG (approximately 10(-8) M) an active transport process predominates, whereas at non-physiological levels (10(-4) M) uptake occurs through passive diffusion. The active transport process is ATP-, Na(+)- and temperature-dependent. Uptake is blocked by 10(-6) M desipramine, an inhibitor of the uptake-I mechanism involved in amine transport. In contrast, desipramine has no effect on the passive diffusion of mIBG into cells. The active transport mechanism for mIBG uptake appears rather promiscuous for biogenic amines, as dopamine, tyramine and nor-adrenaline were highly efficient at blocking mIBG entry to the cell. Serotonin and histamine were capable of interfering with mIBG uptake only at much higher concentrations. Electron microscopy of SK-N-BE(2C) cells revealed a paucity of neurosecretory granules. Biochemical investigations demonstrated the majority of mIBG to be present in the cytoplasm of cells. The availability of a human neuroblastoma cell line that grows well, both as xenograft and in culture, should further our understanding of the cytotoxic effects of mIBG and thus enhance its clinical usefulness.

Cellular senescence involves stochastic processes causing loss of expression of differentiated function genes: visualization by in situ hybridization for steroid 17 alpha-hydroxylase in bovine adrenocortical cells.

When grown for long periods in culture, bovine adrenocortical cells lose the expression of a differentiated function gene, steroid 17 alpha-hydroxylase. Previously, we documented a decline in 17 alpha-hydroxylase mRNA with increasing culture passage level after induction with cyclic AMP (P. J. Hornsby et al., 1987, Proc. Natl. Acad. Sci. USA 84, 1580). We used in situ hybridization to investigate the loss of expression of this gene during cellular senescence at an individual cell level. In primary cultures, cells were uniformly positive for hybridization with cDNA for 17 alpha-hydroxylase after cyclic AMP induction. After two passages, cultures comprised a mixture of hybridizing and nonhybridizing cells. Cells appeared either to hybridize at a level comparable to that in primary cultures or to be nonhybridizing. When in situ hybridization was combined with immunofluorescence, cells positive for immunofluorescence were also positive for hybridization. Senescing mass cultures showed decreasing numbers of positive cells, and after 30 passages cultures comprised entirely nonhybridizing cells. Thus, the previously observed decline in overall 17 alpha-hydroxylase mRNA levels results from a decline in the fraction of expressing cells in the culture, and the rate of loss of expressing cells is in agreement with the rate of loss of total 17 alpha-hydroxylase mRNA. Primary clones, even when isolated at an early stage of clonal expansion, had mixtures of subclones of hybridizing and nonhybridizing cells. On recloning, hybridizing subclones usually produced uniformly nonhybridizing sub-subclones. Some subclones within primary clones had a morphology associated with replicative senescence (flattened cells with sparse intercellular contacts), yet had high numbers of hybridizing cells. We conclude that, in both mass and clonal populations, cells initially expressing 17 alpha-hydroxylase rapidly give rise to clones of nonexpressing cells. Such cells are continually derived by a stochastic process from cells originally expressing the gene.

Loss of expression of a differentiated function gene, steroid 17 alpha-hydroxylase, as adrenocortical cells senescence in culture.

Senescence in cultured adrenocortical cells involves changes in expression of differentiated functions as well as changes in responses to mitogenic stimulation. Steroid 17 alpha-hydroxylase (steroid 17 alpha-monooxygenase, EC 1.14.99.9) is an adrenal-specific enzyme, the expression of which is dependent on the presence of stimulators of cyclic AMP production, such as cholera toxin. Dot-blot hybridization of RNA from bovine adrenocortical cells that had been incubated with cholera toxin showed a marked decline in 17 alpha-hydroxylase mRNA levels as a function of population doubling level, closely paralleling the decline in induction of 17 alpha-hydroxylase enzyme activity. The lower levels of 17 alpha-hydroxylase induction did not result from a requirement for a longer time period for induction or from a specific defect in response to cholera toxin and were not caused by a general failure of enzyme induction in response to cyclic AMP. The decreased growth rate in older cells results from a general decline in response to several growth factors. However, the decline in 17 alpha-hydroxylase induction did not result from a loss of response of the cells to mitogens, since quiescent cells at a low population doubling level showed stimulation of 17 alpha-hydroxylase mRNA by cholera toxin to levels similar to those in nonquiescent cultures and added mitogens either had no effect on 17 alpha-hydroxylase mRNA levels or decreased them. There was, however, a specific posttranscriptional effect of insulin on 17 alpha-hydroxylase. The loss of 17 alpha-hydroxylase induction is unlikely to result from overgrowth of a minority cell type lacking the ability to induce 17 alpha-hydroxylase, because adrenocortical cell clones that had high levels of 17 alpha-hydroxylase induction gave rise to cells with lower levels of induction on subcloning. Thus, loss of 17 alpha-hydroxylase activity in adrenocortical cellular senescence results from a primary failure of accumulation of 17 alpha-hydroxylase mRNA after incubation with the inducing agent.