Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience.
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Adoptive Transfer↗
Biomedical subjects
Jan W Gratama
Publications and source records attributed to Jan W Gratama.
CD38 as a prognostic factor in B cell chronic lymphocytic leukaemia (B-CLL): comparison of three approaches to analyze its expression.
BACKGROUND: Increased CD38 expression by leukemic cells has been suggested as an adverse prognostic factor in B-CLL. Several approaches have been proposed to quantify its level of expression by flow cytometry. METHODS: We compared the use of (i) the percentage of CD38 positive cells, (ii) CD38 antibodies bound per cell (ABC), and (iii) a semi-quantitative method based on the shape of the CD38 histogram, within a cohort of 78 B-CLL patients. RESULTS: A decreased overall survival was seen with >30% CD38 positivity among B-CLL cells, with CD38 ABC >100, and with bimodal or unimodal, strongly positive CD38 histograms. However, patients with unimodal weakly positive CD38 histograms also showed a significantly reduced survival as did patients with intermediate proportions (i.e. 5-30%) of CD38+ cells. Furthermore, within the group with <5% CD38 positivity among their B-CLL cells, 84% of patients showed prognostically favourable mutated IGVH gene segments and 100% had low ZAP70 gene expression. For 5-30% CD38 positivity, these proportions were 50 and 83%, while for >30% CD38 positivity, these proportion were only 28 and 56%, respectively. CONCLUSIONS: We found a simple method of quantitation of CD38 expression (i.e., >5% CD38 positivity among B-CLL cells) to be sufficient to identify patients with an unfavourable prognosis. The level of CD38 expression as defined with this method correlated well with the IGVH mutation status and ZAP70 gene expression.
ADP-ribosyl Cyclase 1↗
Resolution of EBV(+) CNS lymphoma with appearance of CSF EBV-specific T cells.
Current knowledge of Epstein-Barr virus (EBV)-specific T-cell responses in the cerebrospinal fluid (CSF) of patients with EBV-related lymphoproliferative disease (EBV-LPD) in the central nervous system (CNS) is very limited. Here, we present two recipients of hematopoietic stem cell transplants with EBV-LPD in the CNS. EBV-specific CD8(+) T lymphocytes were detected in CSF and peripheral blood using major histocompatibility complex (MHC) class I multimers loaded with EBV-derived peptides. The appearance of EBV-specific CD8(+) T cells in CSF and blood correlated with neurological improvement and disappearance of EBV-LPD. These observations suggest a role for EBV-specific CD8(+) T cells in the control of EBV-LPD in the CNS.
Aged↗
Measuring Ag-specific immune responses: understanding immunopathogenesis and improving diagnostics in infectious disease, autoimmunity and cancer.
Characterization of antigen-specific immune responses at the single-cell level has been made possible by recent advancements in reagent and technology development, combined with increasing knowledge of molecular mechanisms. Fluorescently labelled MHC-peptide multimers and antigens identify directly specific T and B cells, respectively, whereas dynamic assays exploit mediator production or secretion, or the changes in surface expression of other proteins, to identify specific lymphocytes--some techniques enabling the recovery of viable cells. Meanwhile, multiparameter flow cytometry has emerged as the most versatile platform for integrating most of these methods. As the complexity of experimental data increases, so does the level of technical sophistication required for analysis and interpretation, both in terms of basic research and modern medicine, with new applications for infectious diseases, autoimmunity and cancer.
Animals↗
Critical role of flow cytometry in evaluating peripheral blood hematopoietic stem cell grafts.
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Antigens, CD34↗
Enumeration of antigen-specific CD8+ T lymphocytes by single-platform, HLA tetramer-based flow cytometry: a European multicenter evaluation.
BACKGROUND: HLA class I peptide tetramers represent powerful diagnostic tools for detection and monitoring of antigen-specific CD8(+) T cells. The impetus for the current multicenter study is the critical need to standardize tetramer flow cytometry if it is to be implemented as a routine diagnostic assay. Hence, the European Working Group on Clinical Cell Analysis set out to develop and evaluate a single-platform tetramer-based method that used cytomegalovirus (CMV) as the antigenic model. METHODS: Absolute numbers of CMV-specific CD8(+) T cells were obtained by combining the percentage of tetramer-binding cells with the absolute CD8(+) T-cell count. Six send-outs of stabilized blood from healthy individuals or CMV-carrying donors with CMV-specific CD8(+) T-cell counts of 3 to 10 cells/microl were distributed to 7 to 16 clinical sites. These sites were requested to enumerate CD8(+) T cells and, in the case of CMV-positive donors, CMV-specific subsets on three separate occasions using the standard method. RESULTS: Between-site coefficients of variation of less than 10% (absolute CD8(+) T-cell counts) and approximately 30% (percentage and absolute numbers of CMV-specific CD8(+) T cells) were achieved. Within-site coefficients of variation were approximately 5% (absolute CD8(+) T-cell counts), approximately 9% (percentage CMV-specific CD8(+) T cells), and approximately 17% (absolute CMV-specific CD8(+) T-cell counts). The degree of variation tended to correlate inversely with the proportion of CMV-specific CD8(+) T-cell subsets. CONCLUSIONS: The single-platform MHC tetramer-based method for antigen-specific CD8(+) T-cell counting has been evaluated by a European group of laboratories and can be considered a reproducible assay for routine enumeration of antigen-specific CD8(+) T cells.
CD8-Positive T-Lymphocytes↗
Impaired recovery of Epstein-Barr virus (EBV)--specific CD8+ T lymphocytes after partially T-depleted allogeneic stem cell transplantation may identify patients at very high risk for progressive EBV reactivation and lymphoproliferative disease.
Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes are considered pivotal to prevent lymphoproliferative disease (LPD) in allogeneic stem cell transplantation (SCT) recipients. We evaluated the recovery of EBV-specific CD8+ T cells after partially T-cell-depleted SCT and studied the interaction between EBV-specific CD8+ T cells, EBV reactivation, and EBV-LPD. EBV-specific CD8+ T cells were enumerated using 12 class I HLA tetramers presenting peptides derived from 7 EBV proteins. Blood samples were taken at regular intervals after SCT in 61 patients, and EBV DNA levels were assessed by real-time polymerase chain reaction. Forty-five patients showed EBV reactivation, including 25 with high-level reactivation (ie, more than 1000 genome equivalents [geq] per milliliter). Nine of these 25 patients progressed to EBV-LPD. CD8+ T cells specific for latent or lytic EBV epitopes repopulated the peripheral blood at largely similar rates. In most patients, EBV-specific CD8+ T-cell counts had returned to normal levels within 6 months after SCT. Concurrently, the incidence of EBV reactivations clearly decreased. Patients with insufficient EBV-specific CD8+ T-cell recovery were at high risk for EBV reactivation in the first 6 months after SCT. Failure to detect EBV-specific CD8+ T cells in patients with high-level reactivation was associated with the subsequent development of EBV-LPD (P =.048). Consequently, the earlier defined positive predictive value of approximately 40%, based on high-level EBV reactivation only, increased to 100% in patients without detectable EBV-specific CD8+ T cells. Thus, impaired recovery of EBV-specific CD8+ T cells in patients with high-level EBV reactivation may identify a subgroup at very high risk for EBV-LPD and supports that EBV-specific CD8+ T cells protect SCT recipients from progressive EBV reactivation and EBV-LPD.
Adolescent↗
Flow cytometric HLA-B27 screening: cross-reactivity patterns of commercially available anti-HLA-B27 monoclonal antibodies with other HLA-B antigens.
BACKGROUND: Some 50 clinical laboratories in the Benelux perform flow cytometric HLA-B27 screening and participate in the Benelux external quality assessment scheme operational since 1995. Results from this scheme indicate that cross-reactivity of HLA-B27 monoclonal antibodies (mAbs) is a major problem. METHODS: We analyzed cross-reactivity patterns of commercially available mAbs for HLA-B27 screening. Three clones of HLA-B27 mAb (ABC-m3, n = 3; FD705; and GS145.2) from five manufacturers were evaluated. Test cells were selected as to express HLA-B antigens with known serologic cross-reactions (HLA-B7, B12, B13, B16, B17, B22, B37, B40, B41, B42, B47, and B48). Cells without B27 cross-reactive antigens (B5, B8, B14, B15, B21, and B35) and cells positive for B27 were included as controls. All tests were performed and interpreted as recommended by the manufacturers. Cross-reactivity was defined as increased fluorescence intensity in comparison with the baseline reactivity observed with the corresponding immunoglobulin G isotype control mAb. RESULTS AND CONCLUSIONS: All mAbs tested showed cross-reactivity, ranging from weak (+/-) to strong (+), with different antigens and different degrees of intensity-ABC-m3: (+/-) B12, B16, B17, B41, B47, and B48 and (+) B7, B13, B22, B37, B40, and B42; GS145.2: (+/-) B13, B17, B22, B40, and B47 and (+) B7, B16, B37, B42, and B48; FD705: (+/-) B12, B13, B16, and B48 and (+) B17, B37, and B47. If one mAb had been used for HLA-B27 screening, ABC-m3 would have yielded nine false-positive B27 assignments, FD705 would have yielded seven, and GS145.2 would have yielded two. This problem largely canbe avoided by the combined use of two different mAb clones. The combination of FD705 and GS145.2 yielded the best results, with one false-positive HLA-B27 assignment among the 99 HLA-B27(-) samples of this highly selected panel.
Antibodies, Monoclonal↗
Clinical utility of tetramer-based immune monitoring in allogeneic stem cell transplantation.
The construction and use of Class I human leucocyte antigen (HLA) tetramers has, for the first time, allowed the direct enumeration of CD8(+) T lymphocytes specific for the antigen of interest. Tetramer staining can be combined with functional assays of antigen-specific T cells measuring their production of intracellular cytokines after short-term stimulation with antigen. The advantages of flow cytometric tetramer-based assays are their short turn-around time and their amenability to standardisation. Currently, their main limitation is that only a limited number of Class I HLA tetramers are available. This situation may bias the information derived from such studies. Nevertheless, clinically useful information has been obtained in tetramer-based studies of the regeneration of cytomegalovirus (CMV) and Epstein-Barr virus (EBV)-specific CD8(+) T cells after allogeneic stem cell transplantation (SCT). Following myeloablative cytoreductive therapy and SCT, a period of deep cellular immunodeficiency follows until the donor-derived immune system has sufficiently regenerated. As a result of the lack of immunosurveillance, endogenous viruses such as CMV and EBV may reactivate and cause disease. In order to prevent these complications, pre-emptive therapeutic interventions are made, in which antiviral treatment is administered based on frequent viral load measurements. In this way, overtreatment associated with prophylactic strategies is reduced; however, a subgroup of patients developing recurrent reactivations and/or disease remains. Interventions aimed at the prevention of graft versus host disease (GVHD) and the reduction of its severity greatly reduce the rate of (virus-specific) T-cell reconstitution and hence, increase the frequency and severity of viral reactivations. Specifically, T-cell depletion of SCT and the use of antithymocyte globulin as part of the conditioning regimen reduces the (virus-specific) T lymphocytes transferred with the graft, which otherwise would have contributed to the first phase of T-cell regeneration post-SCT. Consequently, patients whose CMV- and EBV-specific CD8(+) T cells fail to regenerate to levels detectable by tetramer-based flow cytometry during the first 3-6 months post-SCT were at highly increased risk for CMV disease and EBV(+) B-lymphoproliferative disease, respectively. Furthermore, delayed reconstitution of the CD4(+) T-cell compartment results in the lack of adequate help for the generation of sufficient antiviral CD8(+) T cells. Conversely, adoptive immunotherapy using CMV- or EBV-specific T cells results in the swift restoration of virus-specific T-cell immunity and the reduction of viral load, unless corticosteroids have to be administered to treat concurrent GVHD. Studies have shown the clinical utility of tetramer-based immune monitoring in the setting of allogeneic SCT and indicate that such assays, extended by functional studies of the virus-specific T cells, may constitute a valuable extension of current viral load monitoring strategies.
Histocompatibility Antigens Class I↗
Repeated administrations of interleukin (IL)-12 are associated with persistently elevated plasma levels of IL-10 and declining IFN-gamma, tumor necrosis factor-alpha, IL-6, and IL-8 responses.
PURPOSE: Repeated administrations of recombinant human interleukin-12 (rHuIL-12) to cancer patients are characterized by a reduction of side effects during treatment. Induction of IFN-gamma, considered a key mediator of antitumor effects of IL-12, is known to decline on repeated administrations. We studied whether other immunological effects of rHuIL-12 are tapered in the course of treatment. EXPERIMENTAL DESIGN: In a Phase I study of 26 patients with advanced renal cell cancer, rHuIL-12 was administered s.c. on day 1, followed by 7 days rest and six injections administered over a 2-week time period. Plasma concentrations of various cytokines were monitored, as well as absolute counts of circulating leukocyte and lymphocyte subsets. RESULTS: The first injection of IL-12 was accompanied by rapid, transient, and dose-dependent increments of plasma levels IFN-gamma, tumor necrosis factor-alpha, IL-10, IL-6, IL-8, but not IL-4, as well as rapid, transient, and dose-dependent reductions of lymphocyte, monocyte, and neutrophil counts. The major lymphocyte subsets, i.e., CD4+ and CD8+ T cells, B cells, and natural killer cells, followed this pattern. On repeated rHuIL-12 injections, IL-10 concentrations increased further, whereas the transient increments of IFN-gamma, tumor necrosis factor-alpha, IL-6, and IL-8 concentrations, as well as the fluctuations of the leukocyte subset counts, were tapered. Dose escalation of IL-12 within clinically tolerable margins did not reduce the decline of these immunological effects. CONCLUSIONS: Induction of pro-inflammatory cytokines and associated fluctuations in leukocyte subset counts decrease on repeated administrations of rHuIL-12. The steady increment of IL-10 plasma levels may mediate the observed down-regulation of clinical and immunological effects.
Adult↗
Quantification of CD34 epitopes in AML.
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Acute Disease↗
Long wavelength fluorophores and cell-by-cell correction for autofluorescence significantly improves the accuracy of flow cytometric energy transfer measurements on a dual-laser benchtop flow cytometer.
BACKGROUND: Flow cytometric fluorescence resonance energy transfer (FCET) is an efficient method to map associations between biomolecules because of its high sensitivity to changes in molecular distances in the range of 1-10 nm. However, the requirement for a dual-laser instrument and the need for a relatively high signal-to-noise system (i.e., high expression level of the molecules) pose limitations to a wide application of the method. METHODS: Antibodies conjugated to cyanines 3 and 5 (Cy3 and Cy5) were used to label membrane proteins on the cell surface. FCET measurements were made on a widely used benchtop dual-laser flow cytometer, the FACSCalibur, by using cell-by-cell analysis of energy transfer efficiency.ResultsTo increase the accuracy of FCET measurements, we applied a long wavelength donor-acceptor pair, Cy3 and Cy5, which beneficially affected the signal-to-noise ratio in comparison with the classic pair of fluorescein and rhodamine. A new algorithm for cell-by-cell correction of autofluorescence further improved the sensitivity of the technique; cell subpopulations with only slightly different FCET efficiencies could be identified. The new FCET technique was tested on various direct and indirect immunofluorescent labeling strategies. The highest FCET values could be measured when applying direct labeling on both (donor and acceptor) sides. Upon increasing the complexity of the labeling scheme by introducing secondary antibodies, we detected a decrease in the energy transfer efficiency. CONCLUSIONS: We developed a new FCET protocol by applying long wavelength excitation and detection of fluorescence and by refining autofluorescence correction. The increased accuracy of the new method makes cells with low receptor expression amenable to FCET investigation, and the new approach can be implemented easily on a commercially available dual-laser flow cytometer, such as a FACSCalibur.
Algorithms↗
Reduction of variation in T-cell subset enumeration among 55 laboratories using single-platform, three or four-color flow cytometry based on CD45 and SSC-based gating of lymphocytes.
BACKGROUND: Enumeration of CD4(+) and CD8(+) T-cell subsets provides relevant information for diagnosis and monitoring of patients with cellular immunodeficiencies. As a result, an external quality assurance scheme was implemented in Belgium, The Netherlands, and Luxembourg in 1995. A workshop was held to train the participants in state-of-the art technology for assessment of absolute T-cell subset counts (i.e., a three or four-color, single-platform assay with lymphocyte gating based on CD45 and sideward light scatter) with the aim to achieve between-site coefficients of variation (CVs) <10% and within-site CVs <5% for > or =75% of the participants. METHODS: Three send-outs of stabilized blood from a healthy donor were distributed to 55 laboratories, each with the request to perform the standard assay on three occasions. For comparison, each laboratory performed its local technique in parallel. RESULTS: With the standard technique, between-site CVs of approximately 8% (CD3+ T cells), approximately 9% (CD4+ T cells), and approximately 10% (CD8+ T cells) were achieved. Within-site CVs were <5% for 82% (CD3+ T cells) and approximately 70% (CD4+ and CD8+ subsets) of the participants. Local techniques yielded between-site CVs of 13%-17% for CD3+, CD4+, and CD8+ T cells. CONCLUSIONS: The state-of-the-art technology for T-cell subset enumeration was implemented successfully among 55 Belgian-Dutch laboratories and resulted in significant reductions of between-site variation of absolute CD3+, CD4+, and CD8+ T-cell counts.
CD3 Complex↗