Henny G. Otten

Tolerogenic immune responses to novel T-cell epitopes from heat-shock protein 60 in juvenile idiopathic arthritis

BACKGROUND Juvenile idiopathic arthritis is a heterogeneous autoimmune disease characterised by chronic inflammation of one or more joints. In patients with this disease, T-cell reactivity to autologous heat-shock protein 60 (HSP60) is associated with a favourable prognosis. We sought to identify HSP60 T-cell epitopes to find potential targets for HSP60 immunotherapy and to assess whether immune responses to these epitopes contribute to the distinct clinical outcome of this disease. METHODS We identified eight potential epitopes using a computer algorithm from both self and microbial HSP60 binding to many HLA-DR molecules. We analysed the pattern of T-cell responses induced by these HSP60 peptides in peripheral-blood mononuclear cells (PBMC) of 57 patients with juvenile idiopathic arthritis, 27 healthy controls, and 20 disease controls. We undertook in-vitro MHC binding studies with the identified peptides, and HLA class II typing of a subset of patients with juvenile idiopathic arthritis. FINDINGS Five of the eight peptides identified yielded proliferative T-cell responses in 50-70% of PBMC from patients with juvenile idiopathic arthritis irrespective of MHC genotype, but not in PBMC from healthy or disease controls. Although PBMC from both patients with juvenile idiopathic arthritis and healthy controls produced interferon gamma in response to these peptides, only PBMC from patients with the disease produced interleukin 10. INTERPRETATION The recorded T-cell-induction in juvenile idiopathic arthritis is tolerogenic. In patients with oligoarticular disease, the immune responses to the HSP60 epitopes identified could contribute to disease remission. RELEVANCE TO PRACTICE The broad recognition of these HSP60 epitopes in a population of patients with polymorphic MHC genotypes opens the way for HSP60-peptide immunotherapy, representing a novel treatment option to specifically modulate the immune system in patients with juvenile idiopathic arthritis.

CD8+ T Cell Responses in Bronchoalveolar Lavage Fluid and Peripheral Blood Mononuclear Cells of Infants with Severe Primary Respiratory Syncytial Virus Infections

A protective role for CD8+ T cells during viral infections is generally accepted, but little is known about how CD8+ T cell responses develop during primary infections in infants, their efficacy, and how memory is established after viral clearance. We studied CD8+ T cell responses in bronchoalveolar lavage (BAL) samples and blood of infants with a severe primary respiratory syncytial virus (RSV) infection. RSV-specific CD8+ T cells with an activated effector cell phenotype: CD27+CD28+CD45RO+CCR7−CD38+HLA-DR+Granzyme B+CD127− could be identified in BAL and blood. A high proportion of these CD8+ T cells proliferated and functionally responded upon in vitro stimulation with RSV Ag. Thus, despite the very young age of the patients, a robust systemic virus-specific CD8+ T cell response was elicited against a localized respiratory infection. RSV-specific T cell numbers as well as the total number of activated effector type CD8+ T cells peaked in blood around day 9–12 after the onset of primary symptoms, i.e., at the time of recovery. The lack of a correlation between RSV-specific T cell numbers and parameters of disease severity make a prominent role in immune pathology unlikely, in contrast the T cells might be involved in the recovery process.

Selective depletion of major and minor histocompatibility antigen reactive T cells: towards prevention of acute graft-versus-host disease

Development of acute graft‐versus‐host disease (aGVHD) following HLA‐identical sibling bone marrow transplantation (BMT) remains a serious complication. Aselective depletion of T cells has proved to be effective in preventing aGVHD but is associated with relapse and increased incidence of infection. As aGVHD is directed mainly against epithelial tissues we examined whether it would be feasible to selectively deplete T cells reactive with epithelial cells whilst preserving other specificities. Donor T cells which express HLA‐DR, CD25, CD69 and CD71 activation markers after cocultivation with patient keratinocytes were depleted using magnetic cell separation techniques. Depletion of major as well as minor histocompatibility antigen activated T cells revealed a significant (P = 0.004 and P = 0.031, respectively) 10‐fold decrease in the frequency of donor T lymphocyte precursors reactive with patient keratinocytes. The frequency reactive with third‐party and patient peripheral blood mononuclear cells, including leukaemia cells, remained unchanged, supporting the notion that aGVHD and graft‐versus‐leukaemia (GVL) may be separable. This alloantigen‐specific depletion may be used in matched unrelated as well as HLA‐identical sibling BMT for reducing aGVHD whilst conserving GVL.

Ara h 2 is the best predictor for peanut allergy in adults

BACKGROUND Specific IgE (sIgE) to Ara h 2 as a clinical predictor for peanut allergy in children has a diagnostic value comparable with a prediction model that contains sex, skin prick test (SPT), sIgE to peanut extract, and total IgE minus sIgE. In adults, the diagnostic value of peanut components has not yet been studied. OBJECTIVE To validate a pediatric prediction model in an adult population; to define the diagnostic value of sIgE to peanut components. METHODS Validation was performed by discrimination with an area under the receiver operating characteristic curve (AUC) and calibration with the Hosmer-Lemeshow test. The diagnostic value of the peanut components was assessed with the AUC. RESULTS Validation of the pediatric model in 94 adults showed poor discrimination (AUC, 0.64) but good calibration (P = .48); sIgE to Ara h 2 was the best diagnostic predictor (AUC, 0.76). By using a cutoff value with a 100% positive predictive value (≥1.75 kU/L), 28% of patients could be diagnosed with 100% accuracy. The highest negative predictive value was 63%. A higher negative predictive value could not be calculated for any other test. Although sIgE to Ara h 2 was significantly correlated with severity, it did not discriminate between mild and severe allergy in individual patients (AUC < 0.65). CONCLUSION sIgE to Ara h 2 has the best discriminative ability of all diagnostic tests. It can accurately diagnose peanut allergy in 28% of patients but cannot be used to exclude a peanut allergy in an adult population.

Predicted indirectly recognizable HLA epitopes presented by HLA-DR correlate with the de novo development of donor-specific HLA IgG antibodies after kidney transplantation

BACKGROUND HLA class-I mismatches selectively induce antibody formation after kidney transplantation. The de novo development of donor-specific IgG HLA class-I antibodies may be dependent on the HLA class-II background of the patient by presenting T-helper epitopes within the recognized HLA class-I antigens. METHODS The correlation between antibody production against mismatched donor human leukocyte antigens (HLA) class I and the number of HLA class II-restricted predicted indirectly recognizable HLA epitopes (PIRCHE-II) in the respective HLA class-I mismatches was investigated. To this end, we analyzed sera taken after nephrectomy from a cohort of 21 non-immunized individuals that received a renal transplant. RESULTS Fourty-nine HLA class-I mismatches were found which all contained immunogenic eplets according to HLAMatchmaker. Donor specific HLA antibody responses were detected against 38 HLA class-I mismatches after nephrectomy. These mismatches were found to contain a larger number of PIRCHE-II when compared to mismatches which did not induce donor specific HLA antibodies. Most PIRCHE-II (68%) were not part of an eplet as defined by HLAMatchmaker. CONCLUSIONS Our data suggest that presentation of donor-derived HLA class-I peptides by recipient HLA class-II molecules plays a significant role in de novo development of donor-specific IgG HLA antibodies.

Cartilage proteoglycan aggrecan epitopes induce proinflammatory autoreactive T cell responses in rheumatoid arthritis and osteoarthritis

Objectives: To explore potential T-cell epitopes of the core protein of human cartilage proteoglycan aggrecan (PG) in patients with rheumatoid arthritis (RA) or osteoarthritis. Methods: Peptide-specific T-cell proliferation and cytokine/chemokine production in response to PG-specific peptides were measured in RA and osteoarthritis patients and in healthy controls. Results: Peptides representing amino acid regions 16–39 and 263–282 of PG were most frequently recognised by T cells in a subset of patients with RA or osteoarthritis. Peripheral blood mononuclear cells from these PG-reactive RA and osteoarthritis patients showed increased production of proinflammatory cytokines/chemokines in response to PG peptide stimulation. As PG p263–282 was found to show high sequence homology with Yersinia Yop protein, the corresponding bacterial (Yersinia) peptide was also tested. Remarkably, RA and osteoarthritis patients responding to the Yersinia peptide also responded to p263–282 of PG suggesting a possibility of molecular mimicry in these patients. Conclusions: These results indicate that PG-specific peptides, located in the G1 domain of PG, can induce (auto)antigenic T-cell responses in RA and osteoarthritis patients. These peptides might thus be involved in the immune pathogenesis and/or cartilage degradation in RA and osteoarthritis.

Dynamics of Human Respiratory Virus-Specific CD8+ T Cell Responses in Blood and Airways during Episodes of Common Cold

We determined the dynamics of CD8+ T cells specific for influenza virus and respiratory syncytial virus in blood and tracheostoma aspirates of children during the course of respiratory infections. We showed that during localized respiratory infections the ratio of activated effector CD8+ T cells to resting memory/naive CD8+ T cells in peripheral blood increased significantly. Furthermore, the number of effector/memory T cells specific for respiratory viruses declined in blood and increased in the airways, suggesting that these T cells redistributed from blood to airways. T cells specific for the infecting virus were present in the airways for longer periods at increased levels than nonspecifically recruited bystander T cells. After clearance of the infection, the ratio of resting memory and naive CD8+ T cells normalized in peripheral blood and also memory T cell numbers specific for unrelated viruses that declined during the infection due to bystander recruitment were restored. Taken together, these results showed a significant systemic T cell response during relatively mild secondary infections and extensive dynamics of virus-specific and nonspecific Ag-experienced T cells.

No association between 12 dopaminergic genes and schizophrenia in a large Dutch sample

It has been suggested that genes involved in dopamine neurotransmission contribute to the pathogenesis of schizophrenia. However, reported associations of the disorder with genetic markers in dopaminergic genes have yielded inconsistent results. Possible explanations are differences in phenotyping, genetic heterogeneity, low marker informativity, and the use of small sample sizes. Here, we present a two‐stage analysis of 12 dopaminergic genes in a large sample of Dutch schizophrenic patients. To reduce genetic heterogeneity, only patients with at least three Caucasian grandparents of Dutch ancestry were ascertained. An efficient genotyping strategy was used, in which polymorphic microsatellite markers were first screened for association in DNA pools. Promising results were followed up by individual genotyping in an extended sample. The pooled samples consisted of 208 schizophrenic patients and 288 unmatched control individuals. For each of the genes, more than one microsatellite marker was selected where possible, either intragenic or close to the gene. After correcting for multiple testing, significantly different allele frequencies were detected for DRD5 marker D4S615. Subsequently, we individually genotyped this particular marker and another DRD5 marker, as well as a DRD3 marker that could not be analyzed using the pooling strategy. This was done in an extended sample of 282 schizophrenic patients and a control sample of 585 individuals. In this second stage of the study, we found no association between these three markers and schizophrenia. The results of our comprehensive analysis provide no evidence for association between schizophrenia and 12 dopaminergic genes in a large Dutch sample.

Identification of Immunodominant Epitopes Derived from the Respiratory Syncytial Virus Fusion Protein That Are Recognized by Human CD4 T Cells

ABSTRACT Memory CD4 T-cell responses against respiratory syncytial virus (RSV) were evaluated in peripheral blood mononuclear cells of healthy blood donors with gamma interferon enzyme-linked immunospot (Elispot) assays. RSV-specific responses were detected in every donor at levels varying between 0.05 and 0.3% of CD4 T cells. For all donors tested, a considerable component of the CD4 T-cell response was directed against the fusion (F) protein of RSV. We characterized a set of 31 immunodominant antigenic peptides targeted by CD4 T cells in the context of the most prevalent HLA class II molecules within the Caucasian population. Most antigenic peptides were HLA-DR restricted, whereas two dominant DQ peptides were also identified. The antigenic peptides identified were located across the entire sequence of the F protein. Several peptides were presented by more than one major histocompatibility complex class II molecule. Furthermore, most donors recognized several F peptides. Detailed knowledge about immunodominant antigenic peptides will facilitate the ability to monitor CD4 T-cell responses in patients and the measurement of correlates of protection in vaccinated subjects.

Interference of daratumumab in monitoring multiple myeloma patients using serum immunofixation electrophoresis can be abrogated using the daratumumab IFE reflex assay (DIRA)

Abstract Daratumumab is a fully human anti-CD38 IgG1-κ monoclonal antibody (mAb) currently being evaluated in several Phase 2 and 3 clinical studies for the treatment of multiple myeloma (MM). In this clinical case study we demonstrate that daratumumab can be detected as an individual monoclonal band in serum immunofixation electrophoresis (IFE). M-protein follow-up by IFE is part of the International Myeloma Working Group (IMWG) criteria to assess treatment response. Therefore, it is crucial that the daratumumab band is not confused with the endogenous M-protein of the patient during IFE interpretation. Moreover, a significant number of IgG-κ M-proteins co-migrate with daratumumab. Co-migration introduces a bias in the M-protein quantification since pharmacokinetic studies show that daratumumab peak plasma concentrations reach up to 1 g/L. More importantly, co-migration can mask clearance of the M-protein by IFE which is necessary for classification of complete response by IMWG criteria (negative serum IFE). For optimal M-protein monitoring the laboratory specialist needs to be informed when patients receive daratumumab, and it is essential that the laboratory specialist is aware that a slow migrating band in the γ-region in those patients may be derived from the daratumumab. A daratumumab specific IFE reflex assay (DIRA) has been developed and can be utilized to abrogate interference. The here described mAb interference is not limited to daratumumab, and as therapeutic antibodies gain approval and enter into common clinical practice, laboratory specialists will need additional processes to characterize IFE interference and distinguish endogenous M-protein from therapeutic antibodies.