Imatinib inhibits the activation and proliferation of normal T lymphocytes in vitro.

The ABL tyrosine kinase inhibitor imatinib mesylate is highly effective in the treatment of CML and is increasingly used in the stem cell transplantation (SCT) setting. Since ABL-dependent intracellular signaling molecules are involved in T-cell activation, imatinib may affect T-cell responses in vivo, thus affecting T-cell function in CML patients, disrupting immune reconstitution after allogeneic SCT and/or impeding the graft-versus-leukemia effect. Here we demonstrate that imatinib inhibits PHA-induced proliferation of normal peripheral blood mononuclear cells at in vitro concentrations (1-5 micromol/l) representative of the pharmacological doses used therapeutically in vivo. The effect is not dependent on antigen-presenting cells because CD3/CD28-induced T-cell stimulation was similarly inhibited by imatinib. Dose-dependent inhibition of the proliferative response of purified CD8+ and CD4+ T lymphocytes to anti-CD3/CD28 was similarly observed and associated with reduction in IFN-gamma production. The inhibitory effect could not be ascribed to an increased rate of apoptosis but the expression of activation markers on CD3+ T cells was significantly reduced in the presence of imatinib (1-5 micromol/L). Inhibition of T-cell proliferation was reversible after removal of the drug from the cultures. Thus, imatinib inhibits T-cell proliferation in vitro, an effect that is APC-independent, reversible, and does not involve apoptosis induction.

Development of a novel flow cytometric cell-mediated cytotoxicity assay using the fluorophores PKH-26 and TO-PRO-3 iodide.

A flow cytometric (FCM) assay has been developed for the determination of cell-mediated cytotoxicity (CMC). In the assay, the target tumour cell population was labelled with a membrane dye, PKH-26, prior to incubation with splenocyte effector cells. Cell death within the target population was assessed by the addition of the viability probe TO-PRO-3 iodide (TP3) and analysed by flow cytometry. The extent of cytotoxicity was determined by the relative number of live target cells labelled with PKH-26 only and dead, permeabilised cells labelled with both PKH-26 and TP3. This CMC method allows the analysis to be conducted on a single cell basis and overcomes the need for radiochemicals. This communication indicates that the FCM assay is an accurate and reproducible experimental system capable of analysing natural killer (NK) cell and antibody-dependent cell-mediated cytotoxicity. The procedure is comparable to the chromium release assay. We believe that this is one of the first demonstrations of an FCM-based antibody-dependent cell-mediated cytotoxicity (ADCC) assay.

A transgenic mouse model for tumour immunotherapy: induction of an anti-idiotype response to human MUC1.

MUC1 is a membrane bound, polymorphic epithelial mucin expressed at the luminal surface of glandular epithelium. It is highly expressed in an underglycosylated form on carcinomas and metastatic lesions and is, therefore, a potential target for immunotherapy of cancer. The monoclonal antibody HMFG1 binds the linear core protein sequence, PDTR, contained within the immunodominant domain of the tandem repeat of MUC1. The efficacy of murine and humanized HMFG1 (Ab1) used as an anti-idiotypic vaccine was examined in mice transgenic for human MUC1 (MUC1.Tg) challenged with murine epithelial tumour cells transfected with human MUC1. Humoral idiotypic cascade through Ab2 and Ab3 antibodies was observed in MUC1.Tg mice following multiple antibody inoculations in the presence of adjuvant. Impaired tumour growth at day 35 and highest Ab3 levels were found in mice that had received mHMFG1 with RAS adjuvant. However, comparison of Ab3 levels in individual mice with tumour size in all treatment groups did not show a correlation between smaller tumours and increased levels of anti-idiotype antibody. This suggests that the anti-tumour effects of anti-idiotype vaccination are not solely related to the induction of idiotypic antibody cascades and probably involve other mechanisms.

DNA vaccination favours memory rather than effector B cell responses.

Following priming and boosting of mice with a DNA vector pEE6DeltaS-hCGss expressing sequences encoding a transmembrane version of the beta-chain of human chorionic gonadotropin (hCGbeta), we failed to detect appreciable levels of specific antibody. However, subsequent challenge with hCG protein in Ribi adjuvant elicited a strong and rapid secondary immune response. This response was of comparable magnitude to that produced following priming, boosting and challenge with protein in adjuvant. Thus, DNA vaccination with this vector is as efficient in generating B cell memory as is conventional immunization, but the memory generation occurs in the absence of an overt effector response. Despite an overall similar level of specific antibody, the DNA-vaccinated mice produced hCG-specific antibodies biased towards IgG2a and IgG2b isotypes, whereas the protein-vaccinated mice produced higher levels of IgG1 antibodies. Both Th1 and Th2 cytokines (interferon-gamma (IFN-gamma) and IL-4) were lower in the spleens of the DNA-immunized animals compared with the protein-Ribi-immunized animals, possibly suggesting a different level of helper T cell response to the two different modes of immunization.

Humoral immune response to human aortic valve homografts.

The humoral response to homograft valves in humans is largely unknown. The anti-human lymphocyte antigen (HLA) antibody production, specificity, and immunoglobulin class were examined sequentially in 73 patients undergoing aortic valve replacement. In addition, the long-term production of antibodies was examined in a cross-sectional study of 160 patients at periods varying from 1 to 15 years postoperatively. Human lymphocyte antigen antibodies were produced in 17 of 30 antibiotic-sterilized homografts (56%) and in 15 of 15 "homovital" homograft recipients, compared with 6 of the 28 control xenograft recipients (21%) (p < 0.001). The HLA antibodies were immunoglobulin G in all 15 homovital homografts, in 11 of 17 antibiotic-sterilized homografts, and in four of the six xenograft cases. Human lymphocyte antigen specificities could be assigned to the antibodies in 21 cases. In 10 of 11 cases in which donor HLA typing data were available, the antibodies detected were directed against donor HLA class I antigens. Of six possible determinants of HLA antibody production, the type of homograft valve implanted (homovital or antibiotic sterilized) correlated with antibody formation. In the cross-sectional study, 66 of the 85 homovital homograft recipients tested for HLA antibodies after 1 year were found to have antibodies, compared with 29 of 75 antibiotic-sterilized homograft recipients (p = 0.00003). We conclude that homografts appear to stimulate a strong donor HLA-specific antibody response, particularly of the immunoglobulin G class. This is most common in homovital valve recipients. These antibodies can persist for 15 years after operation. The clinical significance of this response requires further investigation.

The effect of panel reactive antibodies and the donor specific crossmatch on graft survival after heart and heart-lung transplantation.

Data from 699 cardiac and 290 heart-lung transplants has been analysed to determine the importance of the lymphocytotoxic crossmatch result and panel reactive antibody (PRA) status on graft survival. Donor reactive crossmatching was performed for 636 cardiac transplants. One year actuarial survival for a negative crossmatch (n = 580) was 73% compared to 56% for the positive crossmatch recipients (n = 56) p = 0.0014. Where crossmatches were performed on separated T and B cells, the T cell directed crossmatch was found to be highly predictive of graft failure in 289 cardiac transplants. One year survival for a negative crossmatch was 73% (n = 258), for B cell positive crossmatch recipients 62% (n = 24), and for a positive T cell crossmatch 28% (n = 7) (p = 0.001). Patients' PRA status were grouped into those with negative, medium and high frequencies. There was a trend (not statistically significant) for patients with PRA above 50% to have poor graft survival. Patients with PRA above 50% were significantly more likely to have a positive lymphocytotoxic crossmatch against donor lymphocytes. Donor reactive crossmatching was performed for 283 heart-lung transplants. One year actuarial survival for a negative crossmatch was 61% (n = 251) and for a positive result was 50% (n = 32), p = 0.02.(ABSTRACT TRUNCATED AT 250 WORDS)