Antigen-specific T-cell immunity in multiple myeloma patients is restored following high-dose therapy: implications for timing of vaccination.

The present study analyses the influence of high-dose chemotherapy (HD) and autologous stem cell transplantation on natural and vaccine induced specific immunity in multiple myeloma patients. Peripheral blood was collected from six multiple myeloma (MM) patients at serial time points in connection with treatment and during a follow-up period of 3 months. T-cell response to cytomegalovirus (CMV), varicella zoster virus (VZV) and tetanus toxoid (TT) was determined by flow cytometry analysis for CD69, TNFalpha, IFNgamma, IL-4 expression and cell proliferation. At diagnosis and prior to induction chemotherapy TNFalpha expressing T cells in 5/6 patients were found specific for CMV, 3/6 for VZV and 4/6 for TT. Serial analyses during treatment conclude impaired immune response, however, 3 months post-transplantation all but one patient had regained cytokine expressing CD8(+) T cells specific for CMV, VZV and TT. The highest percentages of cytokine responding T cells were observed after stimulation with CMV antigen. A striking observation was the low cytokine reactivity (close to zero) measured in G-CSF mobilized blood at the time of leukapheresis. In spite of a general reduction of the CD4/CD8 ratio following transplantation, recovery of antigen specific CD4(+) T cells reactivity generally occurred prior to CD8(+) recovery and often to a higher level. In conclusion, the study demonstrates that natural as well as vaccine induced specific immunity present prior to HD was regained after stem cell transplantation, hence identifying a possible window for future vaccination trials.

Characterization of monocyte-derived dendritic cells maturated with IFN-alpha.

Dendritic cells (DC) are promising candidates for cancer immunotherapy. These cells can be generated from peripheral blood monocytes cultured with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). In order to obtain full functional capacity, maturation is required, but the most potent reagents such as LPS or polyriboinosinic polyribocytidylic acid (Poly I:C) are not approved for clinical use. We tested the ability of type I interferon (IFN) to induce such maturation. We found that 24-h IFN-alpha co-culture of day 7 monocyte-derived DC generated with GM-CSF and IL-4 induces increased numbers of DC positive for CD54 and CD40 together with the co-stimulatory molecule CD80 but not the activation marker CD83. Also, IFN-alpha maturation leads to an increase in IP-10 and MCP-1 chemokine secretion, but only a minor increase in IL-12p40 secretion. In line with this, maturation with IFN-alpha has only a small effect on induction of autologous T-cell stimulatory capacity of the DC. However, an increase in DC allogeneic T-cell stimulatory capacity was observed. These data suggest that IFN-alpha has a potential as a maturation agent used in DC-based cancer vaccine trials, but not as a single reagent.

Phenotypic and functional characterization of clinical grade dendritic cells generated from patients with advanced breast cancer for therapeutic vaccination.

Dendritic cells (DC) are promising candidates for cancer immunotherapy. However, it is not known whether in vitro-generated monocyte-derived DC from cancer patients are altered compared with DC from healthy donors. In a clinical phase I/II study, monocyte-derived DC were generated in vitro utilizing granulocyte macrophage colony-stimulating factor and rh-interleukin-4 (IL-4) and used for cancer immunotherapy. In this study, we tested the effect of various maturation cocktails and performed a comparative evaluation of the DC phenotype and functional characteristics. Polyriboinosinic polyribocytidylic acid (Poly I:C) + tumour necrosis factor-alpha (TNF-alpha) induced significant IL-12 p70 secretion, which was increased after addition of a decoy IL-10 receptor. The lymph node homing chemokine receptor CCR-7 expression was induced by TNF-alpha + IL-1beta + IL-6 + prostaglandin E2 but was not induced by Poly I:C + TNF-alpha. In general, DC from patients had an intermediate maturity phenotype with a significantly higher expression of CD40 and CD54 compared with healthy donors. In vitro analyses showed an unimpaired capacity of the patient-derived DC for antigen-specific (cytomegalovirus, tetanus and keyhole limpet haemocyanin) T-cell stimulation, whereas the allostimulatory capacity of patient-derived DC was significantly decreased. These data suggest that patient-derived DC are more differentiated but are less sensitive to maturation-inducing agents than DC obtained from healthy individuals.

Impact of high-dose chemotherapy on antigen-specific T cell immunity in breast cancer patients. Application of new flow cytometric method.

The present study analyses the influence of high-dose chemotherapy (HD) and autologous stem cell transplantation on natural and vaccine-induced specific immunity in breast cancer patients. Peripheral blood was collected from five breast cancer patients at serial time points in connection with treatment and in a follow-up period of 1 year. The frequencies of CD8+ and CD4+ T cells responsive to cytomegalovirus (CMV), varicella zoster virus (VZV), and tetanus in antigen-activated whole blood were determined by flow cytometric analysis of CD69, TNF alpha, IFN gamma and IL-4 expression. Mononuclear cells were labelled with PKH26 dye and the CMV, VZV, and tetanus toxoid-specific proliferation of T cell subpopulations was analysed by flow cytometry. In none of the patients did the treatment result in loss of overall T cell reactivity for any of the antigens. Prior to chemotherapy 5/5 patients possessed TNF alpha expressing T cells specific for CMV, 4/5 for VZV, and 3/5 for tetanus. One year after stem cell transplantation all patients possessed TNF alpha expressing T cells specific for CMV, VZV and tetanus. The highest percentages of cytokine-responding T cells were seen after stimulation with CMV antigen. In general, the lowest reactivity (close to zero) was measured in G-CSF-mobilised blood at the time of leukapheresis. In spite of a continuously reduced CD4 to CD8 ratio after transplantation, recovery of CD4+ T cells usually occurred prior to CD8+ recovery and often to a higher level. The study demonstrates that natural as well as vaccine-induced specific immunity established prior to HD can be regained after stem cell transplantation. These data indicate that introduction of a preventive cancer vaccination in combination with intensive chemotherapy may be a realistic treatment option.

Flow cytometric detection of growth factor receptors in autografts and analysis of growth factor concentrations in autologous stem cell transplantation: possible significance for platelet recovery.

In order to improve prediction of hematopoietic recovery, we conducted a pilot study, analyzing the significance of growth factor receptor expression in autografts as well as endogenous growth factor levels in blood before, during and after stem cell transplantation. Three early acting (stem cell factor (SCF), Flt3 ligand (Flt3) and fetal antigen 1 (FA1)) and three lineage-specific growth factors (EPO, G-CSF and thrombopoietin (Tpo)) were analyzed by ELISA in 16 patients with multiple myeloma (MM) and 16 patients with non-Hodgkin's lymphoma (NHL). The relative number of SCF, Flt3, Tpo and G-CSF receptor positive, CD34+ progenitor cells were measured by flow cytometry in the leukapheresis product used for transplantation in a subgroup of 15 patients (NHL, n = 8, MM, n = 7). Three factors were identified as having a significant impact on platelet recovery. First, the level of Tpo in blood at the time of the nadir (day +7). Second, the percentage of re-infused thrombopoietin receptor positive progenitors and finally, the percentage of Flt3 receptor positive progenitors. On the other hand, none of the analyzed factors significantly predicted myeloid or erythroid recovery. These findings need to be confirmed in prospectively designed studies.

Validation of the Nordic flow cytometry standard for CD34+ cell enumeration in blood and autografts: report from the third workshop. Nordic Stem Cell Laboratory Group.

Following two workshops on standardization of enumeration of CD34+ cells in blood and leukapheresis products, the Nordic Stem Cell Laboratory Group (NSCL-G) evaluated the Milan/Mulhouse/Nordic standard in clinical practice during the third workshop (WS-III). This report documents an acceptable interlaboratory variation in the most clinically active laboratories, with a coefficient of variation (CV) below 0.19 in 7 of 8 analyses performed. The introduction of a pan-CD45 antibody in the analysis did not improve the CV. Comparison of two different CD34 class II antibodies on a total of 99 samples and procedures with and without washing on a total of 96 samples revealed a significant correlation (r2 >0.99) for all analyses. Finally, subset analysis of uncommitted and lineage-specific progenitors revealed major gating difficulties, indicating that further improvements are necessary. In an analysis of more than 600 patients undergoing mobilization and harvest of blood progenitors, with about 500 patients autografted, we found a significant correlation between blood levels of CD34+ cells and recovery of CD34+ cells from each harvest as well as between CD34+ cell number reinfused and time to neutrophil and platelet recovery. This report documents for the first time that the very simple Milan/Mulhouse method (termed The Nordic Standard) can be used by a group of laboratories to obtain important clinical information. Consequently, we consider this method as the conventional method in quality assessment of autografts, which should provide a benchmark for development of second-generation improvements.

Selective loss of progenitor subsets following clinical CD34+ cell enrichment by magnetic field, magnetic beads or chromatography separation.

In this preclinical evaluation we have compared the efficacy of three clinical CD34+enrichment procedures with respect to purity, yield and recovery, as well as risk of selective loss of CD34+ lineage-specific subsets. The three devices work by different principles and have several different manipulation steps: The magnetic field separator uses paramagnetic iron-dextran particles; the magnetic microbead selection is based on the advantage of a large surface area for immobilisation of the monoclonal antibody within a very small volume; the original immunoabsorption technique is based on the use of biotinylated antibody applied to a column of avidin-coated sephadex beads. The results of this evaluation gave a median purity 96% (88-98%), 86% (62-97%), and 49% (18-85%), and median yield of 65% (54-100%), 40% (21-74%), and 30% (8-55%), respectively. Subset analysis recognised a selective loss of CD34+/61+ after enrichment, most likely due to class I-II antibodies used for the enrichment step or, alternatively, nonspecific binding of megakaryocytic progenitors. Tumour cell spiking experiments on a clinical scale documented an expected 2-4 log reduction resulting in a number of potentially malignant cells in the CD34 enriched product. Our data support four major conclusions: First, that magnetic field separation is superior to magnetic beads and chromatography selection, mainly due to the risk of cell loss and insufficient recovery with the two latter methods. Second, that late differentiated progenitors with CD34 class III epitopes present are lost during the enrichment procedures. The third major conclusion is that chromatography selection results in a selective loss of CD34bright cells, which are most likely uncommitted early progenitors. This was an unexpected finding which may be a consequence of an imbalance between the strong forces between biotin-avidin and insufficient physical manipulation for CD34+ cell release. Finally, the data document that CD34 selection alone is an inappropriate way to eliminate tumour cells due to the uncontrolled variables and the inconsistent outcome. The only products which can be expected to be purged free of tumour cells are the ones with very minimal (<10-5) contamination in the starting products, ie products documented tumour free with the most sensitive techniques for quantitation. If this is not the case, the optimal purging strategy may be a two-step procedure including CD34 selection and subsequent depletion of the tumour cells in question.

CD34+ megakaryoblastic leukaemic cells are CD38-, but CD61+ and glycophorin A+: improved criteria for diagnosis of AML-M7?

The aim of this flow cytometry study in acute megakaryoblastic leukaemia (AML-M7) was to describe the membrane phenotype of CD34+ progenitor subsets and compare these with the phenotypes expressed by other AML FAB types. Following conventional histopathological diagnosis mononuclear cells from bone marrow and blood were examined in seven patients with AML-M7 and compared with results from 26 sequential patients with AML-M0 to AML-M6. The CD34+ subsets in AML-M7 patients differed from that of patients with AML-M0 to AML-M6 as the CD34+ CD61+ and the CD34+ Glycophorin A+ subsets were median 31% and 20%, respectively, compared to 4% and 2% in the AML-M0 to AML-M6 (P = 0.0005). Only 1% of the CD34+ progenitors were CD34+ CD38+ in AML-M7 compared to 72% in other AML subtypes (P < 0.000). These findings suggest that the CD34+ cell compartment in AML-M7 consists of early lineage-specific progenitors. In conclusion, flow cytometry analysis of CD34+ subsets may improve the diagnostic safety in AML-M7 and consequently the prognostic significance of immunophenotyping in acute leukaemia.

Different membrane expression of CD11b and CD14 on blood neutrophils following in vivo administration of myeloid growth factors.

During the administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF) or granulocyte-macrophage CSF (rhGM-CSF) we studied the early and late changes of membrane antigen density on neutrophils. RhG-CSF and rhGM-CSF both caused an early transient reduction in blood neutrophilic granulocyte-concentration within the first 30 min after treatment followed by a marked later increase during the subsequent 24 h. During the early neutropenia quantitative flow cytometry showed an associated marked increase in the density of membrane CD11b from 169 x 10(3) before to 568 x 10(3) A.U. per cell induced by rhGM-CSF but a non-significant change by rhG-CSF, suggesting that different mechanisms may be responsible for the transient neutropenia. The subsequent neutrophil granulocytosis was followed by a significantly (P < 0.05) increased density of the CD14 antigen from 6.1 x 10(3) before to 15.9 x 10(3) A.U. per cell during treatment with rhG-CSF, but not by rhGM-CSF administration. These results demonstrate that the two cytokines may affect the function of neutrophilic granulocytes in different ways. The increased expression of CD11b could explain some of the side-effects during treatment with rhGM-CSF. The upregulation of CD14 induced by rhG-CSF may be clinically relevant, as CD14 is an opsonic receptor for lipopolysaccharide binding proteins, acting in the defence against Gram-negative bacterial infections.

Structured modeling of a microbial system: I. A theoretical study of lactic acid fermentation.

Most fermentation models presented in the literature are unstructured, i.e., the biomass composition is assumed constant during all operating conditions. These models are unable to simulate experiments carried out at widely different operating conditions. It is therefore interesting to examine simple structured models where knowledge of the cell physiology is taken into account in the modeling phase. In this article, a simple structured model is presented. The model is based on experimental work with the lactic acid bacteria Streptococcus cremoris, but due to the similarities in basic metabolism for many microorganisms it is applicable also for other fermentation system. The basic assumption in the model is that the biomass can be divided into two parts (compartments)-an active part and a mainly inactive structural part. The size of the active part has a pivotal role in the model.

Structured modeling of a microbial system: II. Experimental verification of a structured lactic acid fermentation model.

A two-compartment model for the lactic acid fermentation with Streptococcus cremoris is experimentally verified. The seven parameters of the model are determined using steady-state chemostat data at varying values of dilution rate, D, but with a constant feed concentration, s(f), of a single carbohydrate source (glucose, lactose, or galactose), and a constant feed concentration of s(Nf) of the N source. Steady-state measurements of the RNA content at different exit concentrations, s, of the carbohydrate are included to calculate kinetic parameters that determine the cell composition for varying operating conditions. The model is tested using data from a large set of steady-state and non-steady-state experiments: batch fermentations and step and pulse experiments in a chemostat. Both qualitatively and quantitatively the major features of the model are confirmed: the external substrates enter into intracellular high-energy building blocks, and lactic acid is formed as a by-product of these reactions. Cell growth depends on the fraction of active components (X(A)) of the cell and is not accompanied by lactic acid production. Possible model modifications are discussed, primarily to obtain a better description of lactic acid fermentation at nongrowth conditions.

Structured modeling of a microbial system: III. Growth on mixed substrates.

A two-compartment model is extended to describe growth and product formation on a mixture of different carbohydrate sources. The open-ended structure of the compartment model is utilized to include description of the basic mechanisms of catabolite repression. The model is used to simulate batch triauxic, steady-state, and transient fermentation, and the simulations are compared with experimental results obtained with growth of the lactic acid bacteria Streptococcus cremoris on mixtures of glucose, galactose, and lactose. The model adequately describes characteristic experimental observations such as substrate preference and carbohydrate adoption.

FIA for on-line monitoring of important lactic acid fermentation variables.

An automated system with semi on-line monitoring of glucose, lactic acid, protein, and optical density during lactic acid fermentations, is set up to study the dynamics of lactic acid bacteria. The analyzers for glucose, lactic acid, and protein are based on flow injection analysis (FIA). The system consists of a laboratory fermentor with a continuous withdrawal system and an analysis system where glucose, lactic acid, and protein concentration are measured together with the optical density of the fermentor sample. The system is controlled by a personal computer.The system response is fast, and it yields a large number of reliable and precise analytical data, whoch is of great importance for mathematical model building. Some premliminary results are shown.