Direct evidence that leukemic cells present HLA-associated immunogenic peptides derived from the BCR-ABL b3a2 fusion protein.

The BCR-ABL oncogene is central in the pathogenesis of chronic myeloid leukemia (CML). Here, tandem nanospray mass spectrometry was used to demonstrate cell surface HLA-associated expression of the BCR-ABL peptide KQSSKALQR on class I-negative CML cells transfected with HLA-A*0301, and on primary CML cells from HLA-A3-positive patients. These patients mounted a cytotoxic T-lymphocyte response to KQSSKALQR that also killed autologous CML cells, and tetramer staining demonstrated the presence of circulating KQSSKALQR-specific T cells. The findings are the first demonstration that CML cells express HLA-associated leukemia-specific immunogenic peptides and provide a sound basis for immunization studies against BCR-ABL.

Mitosis and differentiation in T-cells under cytotoxic action of Echinococcus granulosus hydatid fluid.

In the T-cell line, D10, thymidine uptake was used to measure the proportion of cells in S-phase, and the MTT assay to measure the number of viable cells. The effect of Echinococcus granulosus hydatid fluid (HF) on the lymphocytes was assayed in 3-day cultures of the T-cell line, D10, in increasing concentrations of HF. Apparent cytotoxic effects of HF were recorded as a log-linear decline in S-phase activity, which was reduced by the presence of IL-1, IL-2, or a combination of the two. In the presence of IL-2, however, mitogenic treatment with concanavalin A increased the cytotoxic effect in 3-day cultures, while in day-2 cultures, HF itself showed mitogenic effect. HF-induced decline in S-phase activity was not matched by a parallel decline in viable cells, suggesting that the apparent cytotoxicity of HF could result from cell-cycle arrest. Depending on its origin, HF enhanced membrane expression of CD25 and CD38 on human peripheral blood lymphoblasts, and diminished that of CD28. Taken together, these changes suggest that HF can induce T-cell mitosis and reduce co-stimulation with subsequent T-cell anergy or apoptosis.

Echinococcus granulosus: regulation of leukocyte growth by living protoscoleces from horses, sheep, and cattle.

To determine whether living hydatid tissue can, like hydatid fluid, regulate leukocyte growth, T-cell, B-cell, and macrophage lines were cocultured with protoscoleces of Echinococcus granulosus and their growth was compared with that of control cultures by thymidine uptake estimates and chemiluminescent assays of cell number. Protoscoleces supported mitosis of IL-1-deprived D10 T cells, but did not increase D10 count. The action of protoscoleces was affected by the species and organ of their origin and the length of time in culture. Unusually marked mitotic reaction, unaffected by parasite age and origin, was recorded in the B-cell line, BSM, also without commensurate count increase, indicating that induced mitosis resulted in cell loss. It is concluded that protoscoleces can induce mitosis in B and T cells of particular lineages and that this is a potential means of producing the pathological proliferation and depletion of B- and T-cell areas which characterize local reaction to hydatids.

Growth kinetics of leukocyte cell lines cultured with hydatid fluid of Echinococcus granulosus equinus.

Oxidation of 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and tritiated thymidine uptake were used to measure, respectively, the viable cell count and the S-phase activity of D10, B9, A20 and p388d leukocytic cell lines cultured in the presence of varying concentrations of fluid from fertile and infertile hydatid cysts of Echinococcus granulosus equinus. Exposure to hydatid fluid raised or lowered the entry of D10 cells into S-phase depending on the concentration of the fluid and of the supporting cytokines. Enlargement of the S-phase population was unaccompanied by increase in viable count indicating that the mitotic cycles induced by hydatid products were not completed. A similar conclusion was reached in respect of the p388d monocytic line, and both changes appear consistent with those occurring in histopathology in vivo. By contrast, the main effect on B9 and A20 B cells was inhibition of entry into S-phase.

Echinococcus granulosus: assays for hydatid immunoregulatory factors using established lymphoid cell lines.

Mitosis, mitochondrial metabolic rate and proliferation were measured in established lymphoid cell lines exposed to chromatographic fractions of equine Echinococcus granulosus hydatid fluid. In several cell lines, one or more of the three parameters were modified by the exposure. As an assay for potential immunoregulatory activity, the method was simple and repeatable. The following novel observations were made: (1) Mitotic reaction was found among lines of T-cell, B-cell and macrophage origin; (2) mitosis was accompanied by proliferation in the B-cell lines, B9 and A20, and in the macrophage lines, HL-60 and P388d. With mitotically responsive T-cells, proliferation was slight in CTLL-2 and absent in D10, implying cell-cycle modification; (3) mitotic responsiveness tended to occur in cell lines with mature characteristics; (4) among cytokine-dependent cell lines, hydatid fluid FPLC fraction 1 mimicked IL-1 and several fractions mimicked IL-2 and IL-6 in the maintenance of mitosis; and (5) there was significant statistical interaction between the influences of mammalian cytokines and hydatid fluid fractions, implying that the propensity of antigenically unprimed lymphoid cells to be regulated by E. granulosus is conditioned by cytokine activity.

The influence of class II HLA type on the lymphoproliferative response of normal donors to a bcr-abl fusion peptide.

Chronic myelogenous leukemia (CML) is characterized by a t(9;22) chromosomal translocation resulting in the expression of a novel bcr-abl fusion protein. The region spanning the fusion point is novel to the immune system and hence represents a potential leukemia-specific antigen. The ability of a 21-mer b3a2 fusion peptide to induce an in vitro lymphoproliferative response in a panel of 54 normal donors has been tested. This gave a mean stimulation index of 2.73 (95% CI 2.42-3.05) and 50/54 (93%) of donors gave responses that were greater than those with bcr or abl control peptides. The mean stimulation index relative to that of the control peptides was 1.80 (95% CI 1.63-1.97; p < 0.001). Responses were optimal at concentrations ranging from 0.3-150 micrograms/mL and in most cases peaked at 9 days. There was no clear relationship between level of responsiveness to the b3a2 fusion peptide and the presence of any single HLA-A, -B, -DR, or -DQ allele. HIA-DRB1*0404 was the only allele that was not associated with responsiveness. It is therefore likely that the b3a2 fusion peptide can be presented to T cells during a primary immune response in the context of several different class II HLA allelic products, albeit at low efficiency. The implications for specific active immunotherapy of CML patients are discussed.