http://repub.eur.nl/res/aut/1483/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/18108/ 2009-02-01T00:00:00Z http://repub.eur.nl/res/pub/30471/ 2008-07-01T00:00:00Z We studied the recovery of CMV-specific CD4+and CD8+T-cell immunity in 52 recipients of allogeneic stem cell transplantation (SCT). The proportions of IFN-γ-producing CD4+and CD8+T cells upon in vitro activation using peptide pools representing the CMV pp65 and IE-1 proteins were assessed at multi-ple time points post SCT, and correlated with the occurrence of CMV reactivation. In a retrospective analysis, recurrent CMV reactivations occurred in 9 patients and were associated with low pp65-specific CD4+T-cell and low IE-1-specific CD8+T-cell reactivities, whereas patients without detectable CMV reactivation (n = 30) or a single reactivation (n = 13) showed a better recovery of these immune responses. CD4+T-cell responses to IE-1 were infrequent in most patients, whereas CD8+T-cell responses to pp65 occurred frequently, but did not correlate with protection against (recurrent) reactivation. Prospectively, CMV-specific T-cell responses could be studied prior to 14 reactivation episodes in 8 patients. CD4+T-cell responses to IE-1 and pp65 were positive in only 1 and 2 episodes, respectively. CD8+T-cell responses against IE-1 were positive in 4, but against pp65 in 12 episodes, again showing that CD8+T-cell reactivity against pp65 did not prevent CMV reactivation. Thus, monitoring of particular CMV-specific CD4+and CD8+T-cell responses after allogeneic SCT may identify patients at risk for recurrent CMV reactivations. http://repub.eur.nl/res/pub/30481/ 2008-05-01T00:00:00Z Background: Long-term stabilized blood samples are potentially useful as positive or negative procedure controls for flow cytometric HLA-B27 screening, and could serve as test samples in an external quality assessment (EQA) scheme. We evaluated long-term stabilized whole blood specimens as prepared for the UK NEQAS for Leucocyte Immunophenotyping EQA scheme (Sheffield, UK). Methods: Peripheral blood samples were obtained from nine blood bank donors with known HLA-B typing. Short-term stabilization with Trans-FIX™ was performed before shipment to Sheffield. Thereafter, long-term stabilization was performed. Commercially available HLA-B27 mAb were tested periodically between 1 week and 12 months on (i) fresh, (ii) short-term stabilized, and (iii) long-term stabilized blood samples using a stain, lyse, and wash technique. We compared the forward scatter (FSC), sideward scatter (SSC), and fluorescence signals of lymphocytes as a function of time. Furthermore, a pilot send-out with stabilized blood samples of four blood bank donors was distributed among the participants to the Benelux EQA scheme for HLA-B27 screening, and results were compared with historical EQA data obtained using nonstabilized blood samples from the same donors. Results: There were no major effects on FSC and SSC characteristics of lymphocytes. Background fluorescence of stabilized samples increased and specific fluorescence of stabilized HLA-B27 positive samples decreased as compared with fresh samples. However, discrimination between the investigated HLA-B27 positive and HLA-B27 negative samples remained feasible poststabilization. In the pilot send-out, the results obtained with stabilized samples were less concordant than with the corresponding fresh samples due to variable quality of the stabilized samples. Conclusion: Long-term stabilized whole blood samples are potentially useful as true HLA-B27 positive and true HLA-B27 negative control cells for daily and longitudinal quality control of flow cytometric HLA-B27 screening. In the same way, long-term stabilized samples may be used for EQA purposes. However, these samples are currently not feasible for reagent validation purposes. Extensive quality control of long-term stabilized samples is necessary before distribution in multicenter surveys. http://repub.eur.nl/res/pub/30457/ 2008-03-01T00:00:00Z A biannual external quality assessment (EQA) scheme for flow cytometric lymphocyte immunophenotyping is operational in the Benelux countries since 1996. We studied the effects of the methods used on assay outcome, and whether or not this EQA exercise was effective in reducing between-laboratory variation. Eighty test samples were distributed in 20 biannual send-outs. Per send-out, 50-71 participants were requested to enumerate CD3+, CD4+, and CD8+T cells, B cells, and NK cells, and to provide methodological details. Participants received written debriefings with personalized recommendations after each send-out. For this report, data were analyzed using robust multivariate regression. Five variables were associated with significant positive or negative bias of absolute lymphocyte subset counts: (i) platform methodology (i.e., single-platform assays yielded lower CD4+and CD8+T-cell counts than did dual-platform assays); (ii) sample preparation technique (i.e., assays based on mononuclear cells isolation yielded lower T-cell counts than those based on red cell lysis); (iii) gating strategies based on CD45 and sideward scatter gating of lymphocytes yielded higher CD4+T-cell counts than those based on "backgating" of lymphocytes guided by CD45 and CD14); (iv) stabilized samples were generally associated with higher lymphocyte subset counts than nonstabilized samples; and (v) laboratory. Platform methodology, sample stabilization, and laboratory also affected assay variability. With time, assay variability tended to decline; this trend was significant for B-cell counts only. In addition, significant bias and variability of results, independent of the variables tested for in this analysis, were also associated with individual laboratories. In spite of our recommendations, participants tended to standardize their techniques mainly with respect to sample preparation and gating strategies, but less with absolute counting techniques. Failure to fully standardize protocols may have led to only modest reductions in variability of results between laboratories. http://repub.eur.nl/res/pub/36067/ 2007-07-01T00:00:00Z Aim: In paraneoplastic neurological syndromes (PNS) associated with small cell lung cancer (SCLC) and Hu antibodies (Hu-PNS), Hu antigens expressed by the tumour hypothetically trigger an immune response that also reacts with Hu antigens in the nervous system, resulting in tumour suppression and neuronal damage. To gain more insight into the hypothesized CD8+T cell-mediated immune pathogenesis of these syndromes, we searched for circulating HuD-specific CD8+T cells in a large cohort of Hu-PNS patients and controls. Patients and methods: Blood was tested from 43 Hu-PNS patients, 31 Hu antibody negative SCLC patients without PNS and 54 healthy controls. Peripheral blood mononuclear cells (PBMC) were stimulated with HuD protein-spanning peptide pools (15-mers) and individual HuD-derived peptides (9-mers) and analysed by cytokine flow cytometry and interferon-γ ELISPOT-assays. Additionally, HuD-based Class I HLA multimers were used to visualize HuD-specific CD8+T cells. Results: No HuD-specific CD8+T cells could be detected in the blood of Hu-PNS patients or controls. Conclusions: Our results do not support a role for HuD-specific CD8+T cells in Hu-PNS. Further studies should focus on the detection of circulating HuD-specific CD4+T cells and examine the antigen specificity of T cells in affected tissues. http://repub.eur.nl/res/pub/37020/ 2007-05-15T00:00:00Z Background: A biannual external quality assurance (EQA) scheme for flow cytometric CD34+ haematopoietic stem cell enumeration has been operational in the Benelux countries since 1996. In an evaluation of the results of 16 send-outs, we studied the effects of the methods used on assay outcome and whether or not this exercise was effective in reducing between-laboratory variation. Methods: Data were analyzed using robust multivariate regression. This approach is relatively insensitive to outliers and is used to assess the effect of methodological aspects of CD34+ cell counting on the bias and variability. Results: Five variables were associated with significant bias of absolute CD34+ cell counts: (i) unique laboratory number (ULN), (ii) gating strategy; (iii) CD34 mAb fluorochrome; (iv) type of flow cytometer, and (v) method of sample preparation. In addition, ULN and platform methodology (i.e., single vs. dual) contributed significantly to the variability of this assay. Overall, the variability in results of CD34+ cell enumeration has declined with time; in particular, after a practical workshop in which participants were trained to use the "single platform ISHAGE protocol." Conclusions: Between-laboratory variation in CD34+ cell enumeration can be reduced by standardization of methodologies between centres. Our approach, i.e., EQA with targeted training and feedback in response to reported results, has been successful in reducing the variability of CD34+ cell enumeration between participants. http://repub.eur.nl/res/pub/36831/ 2007-01-01T00:00:00Z Multiple sclerosis (MS) is a complex disease with a substantial, yet poorly identified, genetic influence. We estimated the pattern of familial aggregation of MS in a recent genetically isolated population in the Netherlands. Forty-eight MS patients were identified. Their relationship was evaluated by tracing extended pedigrees, making use of municipal and church records. Of the 48 MS patients, 24 could be linked to a common ancestor in 14 generations. However, multiple relationships exist between patients and, to take these into account, we calculated inbreeding and kinship coefficients. We found that MS patients from the isolate were significantly more often related to each other and significantly more often inbred than a non-MS control group, drawn from the same isolate. There was no clustering of Type I diabetes and autoimmune thyroid diseases in families of MS patients from this isolate. Finally, HLA typing was performed. Although there was a trend towards a higher prevalence of the HLA DRB1*15 allele in patients compared to controls, differences did not reach significance. This study suggests familial aggregation in the genetically isolated population. The high level of inbreeding makes this population valuable for finding novel genes involved in MS. http://repub.eur.nl/res/pub/39662/ 2006-12-01T00:00:00Z The cellular immune response to respiratory syncytial virus (RSV) is considered important in both protection and immunopathogenesis. We have studied the HLA class I- and class II-restricted T cell responses to RSV fusion (F) and attachment (G) proteins in peripheral blood mononuclear cells (PBMCs) obtained from healthy young adults. PBMCs were stimulated with autologous cells infected with recombinant modified vaccinia virus Ankara (rMVA) expressing RSV F (rMVA-F) or G (rMVA-G). In rMVA-F-stimulated bulk cultures F-specific CD4+and CD8+T cell responses were demonstrated, whereas in rMVA-G-stimulated cultures only G-specific CD4+T cell responses were detected. Using a set of overlapping peptides spanning the F protein, a number of the F-specific T cell responses could be mapped to different antigenic regions, whereas for the G protein only CD4+T cell responses recognizing the central conserved domain could be detected. These results suggest that the RSV glycoprotein-specific T cell response is directed to a number of different epitopes. Further studies must be performed to confirm the apparent inability of the RSV G protein to induce CD8+T cell responses. The rMVA-based in vitro stimulation protocol will be useful to define protein-specific T cell responses in different viral systems. http://repub.eur.nl/res/pub/10304/ 2004-01-01T00:00:00Z The cellular immune response to respiratory syncytial virus (RSV) is important in both protection and immunopathogenesis. In contrast to HLA class I, HLA class II-restricted RSV-specific T-cell epitopes have not been identified. Here, we describe the generation and characterization of two human RSV-specific CD4(+)-T-cell clones (TCCs) associated with type 0-like cytokine profiles. TCC 1 was specific for the matrix protein and restricted over HLA-DPB1*1601, while TCC 2 was specific for the attachment protein G and restricted over either HLA-DPB1*0401 or -0402. Interestingly, the latter epitope is conserved in both RSV type A and B viruses. Given the high allele frequencies of HLA-DPB1*0401 and -0402 worldwide, this epitope could be widely recognized and boosted by recurrent RSV infections. Indeed, peptide stimulation of peripheral blood mononuclear cells from healthy adults resulted in the detection of specific responses in 8 of 13 donors. Additional G-specific TCCs were generated from three of these cultures, which recognized the identical (n = 2) or almost identical (n = 1) HLA-DP4-restricted epitope as TCC 2. No significant differences were found between the capacities of cell lines obtained from infants with severe (n = 41) or mild (n = 46) RSV lower respiratory tract infections to function as antigen-presenting cells to the G-specific TCCs, suggesting that the severity of RSV disease is not linked to the allelic frequency of HLA-DP4. In conclusion, we have identified an RSV G-specific human T helper cell epitope restricted by the widely expressed HLA class II alleles DPB1*0401 and -0402. Its putative role in protection and/or immunopathogenesis remains to be determined. http://repub.eur.nl/res/pub/10311/ 2004-01-01T00:00:00Z In the present study, the recognition of epitope variants of influenza A viruses by human CTL was investigated. To this end, human CD8(+) CTL clones, specific for natural variants of the HLA-B*3501-restricted epitope in the nucleoprotein (NP(418-426)), were generated. As determined in (51)Cr release assays and by flow cytometry with HLA-B*3501-peptide tetrameric complexes, CTL clones were found to be specific for epitopes within one subtype or cross-reactive with heterosubtypic variants of the epitope. Using eight natural variants of the epitope, positions in the 9-mer important for T cell recognition and involved in escape from CTL immunity were identified and visualized using multidimensional scaling. It was shown that positions 4 and 5 in the 9-mer epitope were important determinants of T cell specificity. The in vivo existence of CD8(+) cells cross-reactive with homo- and heterosubtypic variants of the epitope was further confirmed using polyclonal T cell populations obtained after stimulation of PBMC with different influenza A viruses. Based on the observed recognition patterns of the clonal and polyclonal T cell populations and serology, it is hypothesized that consecutive infections with influenza viruses containing different variants of the epitope select for cross-reactive T cells in vivo. http://repub.eur.nl/res/pub/10325/ 2004-01-01T00:00:00Z To study whether individual HLA class I alleles are used preferentially or equally in human virus-specific CTL responses, the contribution of individual HLA-A and -B alleles to the human influenza virus-specific CTL response was investigated. To this end, PBMC were obtained from three groups of HLA-A and -B identical blood donors and stimulated with influenza virus. In the virus-specific CD8(+) T cell population, the proportion of IFN-gamma- and TNF-alpha-producing cells, restricted by individual HLA-A and -B alleles, was determined using virus-infected C1R cells expressing a single HLA-A or -B allele for restimulation of these cells. In HLA-B*2705- and HLA-B*3501-positive individuals, these alleles were preferentially used in the influenza A virus-specific CTL response, while the contribution of HLA-B*0801 and HLA-A*0101 was minor in these donors. The magnitude of the HLA-B*0801-restricted response was even lower in the presence of HLA-B*2705. C1R cells expressing HLA-B*2705, HLA-A*0101, or HLA-A*0201 were preferentially lysed by virus-specific CD8(+) T cells. In contrast, the CTL response to influenza B virus was mainly directed toward HLA-B*0801-restricted epitopes. Thus, the preferential use of HLA alleles depended on the virus studied. http://repub.eur.nl/res/pub/10337/ 2004-01-01T00:00:00Z Viruses can exploit a variety of strategies to evade immune surveillance by cytotoxic T lymphocytes (CTL), including the acquisition of mutations in or adjacent to CTL epitopes. Recently, an amino acid substitution (R384G) in an HLA-B*2705-restricted CTL epitope in the influenza A virus nucleoprotein (nucleoprotein containing residues 383 to 391 [NP(383-391)]; SRYWAIRTR, where R is the residue that was mutated) was associated with escape from CTL-mediated immunity. The effect of this mutation on the in vitro influenza A virus-specific CTL response was studied. To this end, two influenza A viruses, one with and one without the NP(383-391) epitope, were constructed by reverse genetics and designated influenza viruses A/NL/94-384R and A/NL/94-384G, respectively. The absence of the HLA-B*2705-restricted CTL epitope in influenza virus A/NL/94-384G was confirmed by using (51)Cr release assays with a T-cell clone specific for the NP(383-391) epitope. In addition, peripheral blood mononuclear cells (PBMC) stimulated with influenza virus A/NL/94-384G failed to recognize HLA-B*2705-positive target cells pulsed with the original NP(383-391) peptide. The proportion of virus-specific CD8+ gamma interferon (IFN-gamma)-positive T cells in in vitro-stimulated PBMC was determined by intracellular IFN-gamma staining after restimulation with virus-infected autologous B-lymphoblastoid cell lines and C1R cell lines expressing only HLA-B*2705. The proportion of virus-specific CD8+ T cells was lower in PBMC stimulated in vitro with influenza virus A/NL/94-384G obtained from several HLA-B*2705-positive donors than in PBMC stimulated with influenza virus A/NL/94-384R. This finding indicated that amino acid variations in CTL epitopes can affect the virus-specific CTL response and that the NP(383-391) epitope is the most important HLA-B*2705-restricted epitope in the nucleoprotein of influenza A viruses. http://repub.eur.nl/res/pub/9812/ 2002-01-01T00:00:00Z The repertoire of human cytotoxic T-lymphocytes (CTL) in response to influenza A viruses has been shown to be directed towards multiple epitopes, with a dominant response to the HLA-A2-restricted M1(58-66) epitope. These studies, however, were performed with peripheral blood mononuclear cells (PBMC) of individuals selected randomly with respect to HLA phenotype or selected for the expression of one HLA allele without considering an influence of other HLA molecules. In addition, little information is available on the influence of HLA makeup on the overall CTL response against influenza viruses. Here, the influenza A virus-specific CTL response was investigated in groups of HLA-A and -B identical individuals. Between groups the individuals shared two or three of the four HLA-A and -B alleles. After in vitro stimulation of PBMC with influenza virus, the highest CTL activity was found in HLA-A2(+) donors. A similar pattern was observed for the precursor frequency of virus-specific CTL (CTLp) ex vivo, with a higher CTLp frequency in HLA-A2-positive donors than in HLA-A2-negative donors, which were unable to recognize the immunodominant M1(58-66) epitope. In addition, CTL activity and frequency of CTLp for the individual influenza virus epitopes were determined. The frequency of CTLp specific for the HLA-B8-restricted epitope NP(380-388) was threefold lower in HLA-B27-positive donors than in HLA-B27-negative donors. In addition, the frequency of CTLp specific for the HLA-A1-restricted epitope NP(44-52) was threefold higher in HLA-A1-, -A2-, -B8-, and -B35-positive donors than in other donors, which was confirmed by measuring the CTL activity in vitro. These findings indicate that the epitope specificity of the CTL response is related to the phenotype of the other HLA molecules. Furthermore, the magnitude of the influenza virus-specific CTL response seems dependent on the HLA-A and -B phenotypes. http://repub.eur.nl/res/pub/9846/ 2002-01-01T00:00:00Z Here, we describe a new HLA-B*3501-restricted cytotoxic T lymphocyte (CTL) epitope in the influenza A virus (H3N2) nucleoprotein, which was found to exhibit a high degree of variation at nonanchor residues. The influenza virus variants emerged in chronological order, and CTLs directed against old variants failed to recognize more recent strains of influenza A virus, indicating an escape from CTL immunity. http://repub.eur.nl/res/pub/12918/ 2001-03-13T00:00:00Z Early after seroconversion, macrophage-tropic human immunodeficiency virus type 1 (HIV-1) variants are predominantly found, even when a mixture of macrophage-tropic and non-macrophage-tropic variants was transmitted. For virus contracted by sexual transmission, this is presently explained by selection at the port of entry, where macrophages are infected and T cells are relatively rare. Here we explore an additional mechanism to explain the selection of macrophage-tropic variants in cases where the mucosa is bypassed during transmission, such as blood transfusion, needle-stick accidents, or intravenous drug abuse. With molecularly cloned primary isolates of HIV-1 in irradiated mice that had been reconstituted with a high dose of human peripheral blood mononuclear cells, we found that a macrophage-tropic HIV-1 clone escaped more efficiently from specific cytotoxic T-lymphocyte (CTL) pressure than its non-macrophage-tropic counterpart. We propose that CTLs favor the selective outgrowth of macrophage-tropic HIV-1 variants because infected macrophages are less susceptible to CTL activity than infected T cells. http://repub.eur.nl/res/pub/22298/ 1996-01-10T00:00:00Z Thrombocytopenia as a result of decreased platelet production may be treated with platelet transfusions. Decreased bone marrow function resulting in thrombocytopenia may be seen in hematopoietic diseases as leukemia, myelodysplastic syndrome or conditions of infiltration of bone marrow with solid tumor metastases. Thrombocytopenia also is a frequent side effect of cancer therapy. The application of high doses of chemotherapy and/or radiotherapy may result in periods of severe bone marrow suppression. This may be more outspoken when the bone marrow function is already compromised by the underlying disease of previous cytotoxic treatment, resulting in periods of life-threatening thrombocytopenia.