Positive and negative selection to reduce tumour contamination in peripheral blood stem cell harvests.

Peripheral blood progenitor cells used during high dose treatments for malignancy may be contaminated with tumour cells that could later contribute to recurrence. CD34+ selected harvests still contain tumour cells and an additional negative selection may be capable of reducing this contamination. We have assessed a two-stage technique in which a CD34+ selection is followed by a tumour specific depletion stage using a B cell or breast cancer specific antibody panel. Initial small-scale selections on 11 patients with NHL and breast cancer showed that cell loss was greatest following the CD34+ selection with a median yield of 38.8 per cent (range 17. 2-56.4 per cent). The addition of the depletion stage resulted in a minimal loss of CD34+ cells with a yield for this step of 94.2 per cent (range 77.5-99.3 per cent). Clinical scale selections were performed on seven patients with CLL and a median of 2.8x10(6)/kg CD34+ cells (range 1.5-6.1x10(6)/kg) were collected. Cell recovery was 53.3 per cent following CD34+ selection and 76.9 per cent following the tumour specific depletion stage, resulting in a final product containing a median of 1.0x10(6)/kg CD34+ cells (range 0. 55-2.0x10(6)/kg). All unmanipulated harvests were heavily contaminated with tumour cells (median contamination 10.2 per cent, range 2.0-83.1 per cent) as measured by flow cytometry and a median 4.7 log (range 3-5 log) tumour cell purge was produced following two-stage selection. Six of the patients have received cells manipulated in this way with median engraftment times of neutrophils>0.5x10(9)/l=16 days (range 13-20 days) and platelets>20x10(9)/l=16.5 days (range 11-42 days). At a median follow-up of 25 months, these transplanted patients remain well and in molecular complete remission.

Novel molecular mechanisms of dendritic cell-induced T cell activation.

In this study we have re-examined the molecular mechanisms involved in activation of T cells by dendritic cells (DC). Human peripheral blood DC (PBDC) were derived by 2 h adhesion followed by 7 day culture in a combination of granulocyte macrophage colony stimulating factor and IL-4, and depletion of residual T and B cells. These PBDC were used to induce autologous T cell proliferation in a CD3-dependent response, and antibodies against CD11a/18 and CD86 were used as control inhibitors of accessory function. Antibodies against five of the cell surface molecules that we have recently identified on the surface of DC, CD13, CD87, CD98, CD147 and CD148, and an antibody which recognizes a molecule that has not as yet been identified, all inhibited the CD3-induced T cell proliferation. These findings were observed not only when antibodies were present throughout the culture, but also when they were prepulsed on to the surface of the DC, suggesting the inhibition was mediated via the antigen-presenting cells rather than the T cell. The same set of antibodies also inhibited an allospecific mixed lymphocyte reaction, confirming that the inhibitory effect was not dependent on the use of a CD3 antibody as the stimulating agent. All the antibodies of known specificity inhibited both CD4 and CD8 T cells equally. Unlike CD87, CD98 and CD147 antibodies, which inhibited activation of both CD45RA (naive) T cells and CD45RO (memory) T cells, CD13 and CD148 appeared to be involved in activation of naive cells only. The molecules identified in this study have not previously been demonstrated to play a role as accessory molecules on DC, the cells that are pivotal for immune induction. Therefore they may provide new potential targets for modulation of the immune response at the APC level.

Molecular characterization of U937-dependent T-cell co-stimulation.

U937 cells provide a co-stimulatory signal for CD3-mediated T-cell activation which is independent of the CD28/CD80/CD86 interaction. This study set out to identify which molecules contribute to this co-stimulatory activity. Monoclonal antibodies (mAb) to the known accessory molecules CD11a, CD18, CD54 and CD45, all inhibited T-cell proliferation. Although CD11a/18 mAb inhibited U937/T-cell cluster formation as well as proliferation, CD45 enhanced the size of the clusters formed, suggesting that this was not the only mechanism of inhibition. The alternative co-stimulatory pathway provided by U937 cells preferentially stimulated a response in the CD18+ T-cell population, and this reflected the reduced sensitivity of CD8+ T cells to CD28-mediated activation. Monoclonal antibodies to three molecules, CD53, CD98 and CD147, also inhibited U937-dependent T-cell proliferation. The mAb to CD98 and CD147 were inhibitory when prepulsed on to the U937 cells, suggesting an effect mediated by these molecules on the antigen-presenting cell.

The role of reactive oxygen species in triggering proliferation and IL-2 secretion in T cells.

This paper examines the hypothesis that reactive oxygen species (ROS) play an important role as second messengers in T cell activation. Activation of T cells with phorbol ester in combination with either calcium ionophore, or anti-CD3 antibody results in a large rapid flux of ROS activity. In contrast, co-stimulation with CD28 does not enhance ROS activity. The ROS signal was sensitive to ascorbic acid, desferrioxamine and dimethyl sulfoxide, suggesting that the major active species being generated was the hydroxyl radical, probably by iron-catalyzed decomposition of hydrogen peroxide. The generation of ROS in T cells was regulated by an accessory population within the peripheral blood. An anti-CD2 antibody induced a strong ROS flux, suggesting that the CD2/LFA-3 interaction may be important in this regulation. T cell activation was inhibited by the same panel of anti-oxidants as ROS generation, but much higher concentrations were required for inhibition of proliferation and IL-2 release than those required to block ROS generation. These data imply that ROS are not obligate second messengers for initiation of T cell activation. The results are compatible, however, with a role for activation-dependent T cell ROS generation in modulating the overall T cell response via autocrine and paracrine signalling pathways.

From sentinel to messenger: an extended phenotypic analysis of the monocyte to dendritic cell transition.

The transitional stages in the relationship between sentinel monocytes and messenger dendritic cells that are active in adaptive immunity, are, as yet, unclear. To explore these events, 2-hr adherent peripheral blood mononuclear cells were used either as monocytes, or cultured for 7 days with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) to generate dendritic cells, and the phenotypic features and relationship of the two cell populations was investigated using an extensive panel of monoclonal antibodies (mAbs). The features of the shift from monocyte to dendritic cell were also examined by daily phenotyping during the 7-day culture period. Twenty-five mAbs, most of which recognized known CD molecules, bound both monocytes and dendritic cells equally, whereas 19 mAbs exhibited differential staining. Four molecules not previously reported on dendritic cells were documented: CD87, CD98, CD147 and CD148. Seven cell-surface molecules (HLA-DQ, CD1a, CD13, CD30, CD43, CD63 and CD86) were expressed either at very low levels or not at all on monocytes, but had a strikingly increased expression on dendritic cells, suggesting a role in antigen presentation. The kinetics of monocyte to dendritic cell transition revealed a rapid activation phase within the first 24 hr, with a considerable increase in expression of the activation markers HLA-DR, CD13, CD14 and CD98; this was followed by a down-regulation of CD14 and a more gradual development of the other dendritic cell features over the remaining 6 days, with steady increases in CD1a, CD18, CD43, CD86, HLA-DR and HLA-DQ. Thus, these studies have demonstrated four novel components of the dendritic cell, and have documented the dynamic multistep nature of the process whereby an antigen-presenting dendritic cell phenotype may emerge from a monocyte precursor.

Mobilization of Philadelphia-negative peripheral blood mononuclear cells in chronic myeloid leukaemia using hydroxyurea and G-CSF (filgrastim).

A relatively simple and non-toxic out-patient-based regimen for the mobilization of Philadelphia-negative (Ph-ve) mononuclear cells in chronic myeloid leukaemia (CML) was evaluated in 10 patients, nine in stable chronic phase and one in accelerated phase. They received oral hydroxyurea at a mean dose of 3.5 g/m2 daily for 7 d, followed by 300 micrograms of G-CSF daily until the last day of harvesting. In the nine chronic-phase patients the mean number of days from the end of hydroxyurea to the commencement of harvesting was 14.5 (range 10-18). The patient in accelerated phase recovered and was harvested after 6 d. The mean number of aphereses performed was 3.4. Adequate numbers of stem cells were obtained in 9/10 patients judged by our usual criteria. Side-effects were mild in comparison to published intravenous schedules. No patients lost their hair. Five (50%) patients required admission with neutropenic fever which responded to antibiotics in all cases. Four (40%) patients developed a transient rash and four (40%) experienced mild oral mucostis. This level of toxicity enabled half of the patients to be treated entirely on an out-patient basis. The harvest products were analysed for cells belonging to the leukaemic clone by conventional cytogenetics, FISH and PCR. All were PCR positive. The mean Ph positivities by cytogenetics and FISH were comparable at 18.1% and 15% respectively. Half the patients had > 98% normal metaphases. We conclude that this approach is comparable in efficacy to published intravenous regimens and significantly less toxic. It can be safely used at diagnosis before interferon therapy commences.

Peripheral blood stem cell transplantation in myeloma using CD34 selected cells.

We have performed nine CD34 selection procedures on peripheral blood stem cells harvested from eight patients with myeloma using the Cellpro avidin-biotin immunoaffinity column (Ceprate). They all received CVAMP chemotherapy to maximum response prior to mobilisation. Six of the patients have been transplanted using these cells, one receiving successive autografts. Median absolute cell numbers processed and retrieved were: 31.1 x 10(9) pre-column, 2.07 x 10(8) in the final product and 30.4 x 10(9) in the column waste. Mean CD34 positivity in the product was 49% (range 18.4-98) with a median CD34+ yield of 31.4% (range 21-37.8). IgH PCR was performed and seven of the eight patients were amplifiable. Of these, two were positive in the pre-column product and both of these were successfully purged with a negative result in the final, post-column product. Patients were transplanted with a median of 2.0 x 10(6) CD34+ cells/kg (range 1.5-9.4) following conditioning with melphalan 200 mg/m2. The mean time to recovery of neutrophils to > 0.5 x 10(9)/l and platelets to > 20 x 10(9)/l was 16 and 17 days, respectively. At a mean follow-up of 9 months, four of the six patients transplanted are alive, three of them in complete remission and one in a clinically stable relapse. One has died of disease relapse and one of progressive neurological problems the aetiology of which was uncertain but there was no sign of progression of their myeloma. We conclude that PBSCT using CD34 selected cells is safe and practical in myeloma following remission induction with CVAMP chemotherapy.