Performance of calibration standards for antigen quantitation with flow cytometry in chronic lymphocytic leukemia.

BACKGROUND: The fluorescence intensities of CD3, CD4 on T cells and CD20, CD22 molecules on B cells were quantitatively measured on lymphocytes from chronic lymphocytic leukemia (CLL) patients and healthy donors. METHODS: The performance of three different types of microbeads was compared, i.e. Quantum molecules of equivalent soluble fluorochrome (Q-MESF), Quantum simply cellular (QSC), and QuantiBRITE (QB). As all PE-conjugates had a F/P ratio of 1:1, the MESF units represented also the antibody binding capacity (ABC). RESULTS: The ABCs of CD4 and CD20 antigens estimated with QSC (ABC(QSC)) were higher than those assigned with QB (ABC(QB)) with an average difference 49%. Higher numbers of antigenic sites were obtained with Q-MESF than with QSC for CD20 antigen. On the contrary, CD4 antigenic sites numbers estimated with QSC were higher than those estimated with Q-MESF. ABC values estimated with Quantum MESF PE (ABC(Q-MESF)) were approximately 15% higher than ABC(QSC), whereas ABC(Q-MESF) was approximately 49% higher than ABC(QB). Statistically significant correlations were found between the values obtained using various standards. The present study is the first to report down-regulation of CD3 antigen on T cells from patients with CLL. CONCLUSIONS: This study emphasizes the relevance of quantitative measurement of fluorescence intensity by flow cytometry as a standardized approach to measure and interpret the expression of some CLL markers and reduce variability of results obtained at different sites in multi-center clinical studies.

Idiotype protein vaccination in combination with adjuvant cytokines in patients with multiple myeloma--evaluation of T-cell responses by different read-out systems.

Anti-idiotypic T cells were analyzed in myeloma patients (n=18) vaccinated with idiotypic protein together with the adjuvant cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and/or interleukin-12 (IL-12). In the group given IL-12/GM-CSF, 78% developed idiotype specific T cells as compared to 22% in the group given only IL-12 (proliferation/ELISPOT assays) (p<0.05). The percentage of immune-responding patients increased when quantitative real time polymerase chain reaction assays for cytokines were included. A predominance of a Th1 (IFN-gamma/TNF-alpha) immune response was noted in the IL-12 group while a Th2 (IL-5) response prevailed in the IL-12/GM-CSF group (p=0.053). Application of multiple read-out systems improved the characterization of the immune response.

Durable carcinoembryonic antigen (CEA)-specific humoral and cellular immune responses in colorectal carcinoma patients vaccinated with recombinant CEA and granulocyte/macrophage colony-stimulating factor.

PURPOSE: Previous studies have indicated that carcinoembryonic antigen (CEA) might be a suitable immunotherapeutic target in colorectal carcinoma (CRC). The aim of the present study was to analyze the immunological and clinical effects of vaccination with CEA together with the adjuvant granulocyte/macrophage colony-stimulating factor (GM-CSF). EXPERIMENTAL DESIGN: Twenty-four resected CRC patients without macroscopic disease were immunized seven times with recombinant CEA at four different dose levels over a 12-month period. Half of the patients received GM-CSF (80 microg/day for 4 consecutive days) at each immunization. Patients were monitored immunologically for 36 months and clinically for 76 months. T-cell response was evaluated by a [(3)H]thymidine incorporation assay, and IgG response was determined by ELISA. RESULTS: Minor local side effects were common. All 12 patients (100%) in the GM-CSF group developed a CEA-specific T-cell as well as an IgG response. The corresponding figures in the CEA alone group were 9 of 12 (75%) and 8 of 12 (66%), respectively. GM-CSF significantly augmented the amplitude of the T-cell response and the IgG titers. No dose-response relationship was noted. The immune responses at 12 months persisted 24 months after the last vaccination. Anti-CEA IgG titers were associated with increased survival (P < 0.05), whereas standard prognostic factors had no relationship, with the exception of serum CEA value. CONCLUSIONS: Vaccination with recombinant CEA and GM-CSF appears to be a nontoxic regimen inducing potent and durable antigen-specific IgG and T-cell response. The results of this study justify more extensive trials with recombinant CEA protein for immunotherapy of CRC.

Signalling molecules and cytokine production in T cells of multiple myeloma-increased abnormalities with advancing stage.

T-cell immune dysfunction in patients with malignant tumours has been attributed to the altered expression of components of the T-cell receptor (TCR)/CD3 complex and their associated intracellular protein tyrosine kinases. In this study, four-colour flow cytometry was applied to study the surface bound molecules TCRalphabeta, CD28, CD152 and CD154 involved in T-cell signalling and the signal transduction molecules CD3zeta, p56lck, p59fyn, ZAP-70 and phosphatidyl-inositol-3 kinase (PI3-k) as well as the intracellular cytokines interferon-gamma (IFN-gamma), interleukin (IL)-4 and IL-2 as a functional read-out of non-stimulated and superantigen (staphylococcus enterotoxin B)-stimulated blood T cells of multiple myeloma (MM) patients at different stages of the disease. Multiple abnormalities were demonstrated in the CD4 and CD8 populations, both under non-stimulated and superantigen-stimulated conditions. There was a marked reduction, particular in advanced stage MM, in the proportion of CD4 and CD8 cells expressing CD28, CD152, CD3zeta, p56lck, ZAP-70 and PI3-k. The level of intracellular T-cell cytokines (IFN-gamma, IL-2 and IL-4) was normal or increased in non-stimulated cells but activation-induced cytokine production was impaired. These results illustrated profound and multiple T-cell signalling defects, from the surface and down-stream, consistent with involvement of a master T-cell function, especially in advanced stage MM. These data should be taken into consideration when developing immune-based therapeutic approaches and when applying new emerging technologies that aim to restore T-cell functions.

Intracellular T cell cytokines in patients with B cell chronic lymphocytic leukaemia (B-CLL).

Analysis of cytokine production is a tool to functionally characterise T cells. In this study, spontaneous and polyclonal activation induced cytokine production in T cells were assessed by flow cytometry in patients with B-CLL. Patients with progressive disease had a significantly increased number of T cells spontaneously producing IL-2, IL-4 and GM-CSF as compared to healthy donors and patients with non-progressive CLL, which was not the case for TNF-alpha and IFN-gamma producing T cells. However, no difference in the frequency of T cells producing these cytokines was seen comparing patients with non-progressive disease to control donors. Polyclonal activation of B-CLL T cells in vitro induced an increased proportion of T cells producing these five cytokines in patients as well as in control donors, indicating that T cells in CLL patients might have a relatively well preserved functional capacity. However, the increase in GM-CSF, TNF-alpha and IL-4 producing T cells was more marked in CLL patients than in controls. Furthermore, following activation, a higher frequency of cytokine-producing T cells was noted in patients with progressive disease as compared to those with non-progressive disease. The augmented number of cytokine-producing T cells in CLL may indicate an up-regulated capability of T cells to secrete cytokines, especially in patients with progressive CLL. The increased production of the T cell derived cytokines GM-CSF, TNF-alpha, IL-4 and IL-2 is interesting, as these cytokines have previously been shown to support growth of B-CLL leukaemic cells in vitro and as T cells might specifically recognise the autologous leukaemic B cells in vivo. The findings may suggest a role for T cells in the pathogenesis of B-CLL.

Significance of phosphotyrosine proteins, Bcl-2 and p53 for apoptosis in resting B-chronic lymphocytic leukemia (CLL) cells.

Signal transduction and apoptosis in B-cell chronic lymphocytic leukemia (CLL) cells with a post-germinal center (GC) phenotype were studied. Specific activation of the cells was induced by a combination of soluble anti-CD40 monoclonal antibody and interleukin-4 (CD40/IL-4) and nonspecific activation with a combination of phytohemagglutinin, phorbol-12-myristate-13-acetate and ionomycin (chemical mixture). Less than 5% of these leukemia cells entered the cell cycle after activation, as indicated by the number of cells in G0/G1 phase. The protein tyrosine phosphorylation pattern and expression of the Bcl-2 protein were specific in ex vivo CLL cells of each individual patient. Expression of the p53 protein was not detectable in these leukemia cells. Cross-linking of the CD40/IL-4 receptors on CLL cells significantly upregulated phosphotyrosine proteins and the p53 protein. In the presence of chemical mixture, downregulated phosphotyrosine proteins were detected. Alterations in Bcl-2 expression were independent of cross-linking with CD40/IL-4 or chemical mixture. A high frequency of apoptotic cells was detected in cells that had downregulated phosphotyrosine proteins and Bcl-2 protein. There was no correlation between induction of apoptosis and expression of p53 protein. Our results suggest that apoptosis in resting leukemia cells could occur prior to the cell cycle progression. Alterations in phosphotyrosine proteins and Bcl-2 but not p53 might play an important role in the regulation of apoptosis in resting G0/G1 memory post-GC B-CLL cells.