Publications by authors named "Anja Ten Brinke"

83 Publications

Breakthrough SARS-CoV-2 infections with the delta (B.1.617.2) variant in vaccinated patients with immune-mediated inflammatory diseases using immunosuppressants: a substudy of two prospective cohort studies.

Lancet Rheumatol 2022 Apr 29. Epub 2022 Apr 29.

Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, Netherlands.

Background: Concerns have been raised regarding the risks of SARS-CoV-2 breakthrough infections in vaccinated patients with immune-mediated inflammatory diseases treated with immunosuppressants, but clinical data on breakthrough infections are still scarce. The primary objective of this study was to compare the incidence and severity of SARS-CoV-2 breakthrough infections between patients with immune-mediated inflammatory diseases using immunosuppressants, and controls (patients with immune-mediated inflammatory diseases not taking immunosuppressants and healthy controls) who had received full COVID-19 vaccinations. The secondary objective was to explore determinants of breakthrough infections of the delta (B.1.617.2) variant of SARS-CoV-2, including humoral immune responses after vaccination.

Methods: In this substudy, we pooled data collected in two large ongoing prospective multicentre cohort studies conducted in the Netherlands (Target to-B! [T2B!] study and Amsterdam Rheumatology Center COVID [ARC-COVID] study). Both studies recruited adult patients (age ≥18 years) with immune-mediated inflammatory diseases and healthy controls. We sourced clinical data from standardised electronic case record forms, digital questionnaires, and medical files. We only included individuals who were vaccinated against SARS-CoV-2. For T2B!, participants were recruited between Feb 2 and Aug 1, 2021, and for ARC-COVID, participants were recruited between April 26, 2020, and March 1, 2021. In this study we assessed data on breakthrough infections collected between July 1 and Dec 15, 2021, a period in which the delta SARS-CoV-2 variant was the dominant variant in the Netherlands. We defined a SARS-CoV-2 breakthrough infection as a PCR-confirmed or antigen test-confirmed SARS-CoV-2 infection that occurred at least 14 days after vaccination. All breakthrough infections during this period were assumed to be due to the delta variant due to its dominance during the study period. We analysed post-vaccination serum samples for anti-receptor binding domain (RBD) antibodies to assess the humoral vaccination response (T2B! study only) and anti-nucleocapsid antibodies to identify asymptomatic breakthrough infections (ARC-COVID study only). We used multivariable logistic regression analyses to explore potential clinical and humoral determinants associated with the odds of breakthrough infections. The T2B! study is registered with the Dutch Trial Register, Trial ID NL8900, and the ARC-COVID study is registered with Dutch Trial Register, trial ID NL8513.

Findings: We included 3207 patients with immune-mediated inflammatory diseases who receive immunosuppressants, and 1807 controls (985 patients with immune-mediated inflammatory disease not on immunosuppressants and 822 healthy controls). Among patients receiving immunosuppressants, mean age was 53 years (SD 14), 2042 (64%) of 3207 were female and 1165 (36%) were male; among patients not receiving immunosuppressants, mean age was 54 years (SD 14), 598 (61%) of 985 were female and 387 (39%) were male; and among healthy controls, mean age was 57 years (SD 13), 549 (67%) of 822 were female and 273 (33%) were male. The cumulative incidence of PCR-test or antigen-test confirmed SARS-CoV-2 breakthrough infections was similar in patients on immunosuppressants (148 of 3207; 4·6% [95% CI 3·9-5·4]), patients not on immunosuppressants (52 of 985; 5·3% [95% CI 4·0-6·9]), and healthy controls (33 of 822; 4·0% [95% CI 2·8-5·6]). There was no difference in the odds of breakthrough infection for patients with immune-mediate inflammatory disease on immunosuppressants versus combined controls (ie, patients not on immunosuppressants and healthy controls; adjusted odds ratio 0·88 [95% CI 0·66-1·18]). Seroconversion after vaccination (odds ratio 0·58 [95% CI 0·34-0·98]; T2B! cohort only) and SARS-CoV-2 infection before vaccination (0·34 [0·18-0·56]) were associated with a lower odds of breakthrough infections.

Interpretation: The incidence and severity of SARS-CoV-2 breakthrough infections in patients with immune-mediated inflammatory diseases on immunosuppressants was similar to that in controls. However, caution might still be warranted for those on anti-CD20 therapy and those with traditional risk factors.

Funding: ZonMw (the Netherlands Organization for Health Research and Development) and Reade foundation.
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http://dx.doi.org/10.1016/S2665-9913(22)00102-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9054068PMC
April 2022

Longitudinal T-Cell Responses After a Third SARS-CoV-2 Vaccination in Patients With Multiple Sclerosis on Ocrelizumab or Fingolimod.

Neurol Neuroimmunol Neuroinflamm 2022 Jul 6;9(4). Epub 2022 May 6.

From the Department of Immunopathology (V.P.C., L.Y.L.K., M.D., V.A.L.K., N.J.M.V., M.S., T.R., G.W., S.M.v.H., A.t.B.), Sanquin Research and Landsteiner Laboratory, Amsterdam UMC; Department of Neurology and Neurophysiology (L.K., L.W., K.P.J.v.D., E.W.S., F.E.), Amsterdam Neuroscience, Amsterdam UMC, location AMC, University of Amsterdam; Department of Hematopoiesis (R.R.H., C.E.v.d.S.), Sanquin Research and Landsteiner Laboratory, Amsterdam UMC; Department of Experimental Immunohematology (R.R.H.), Sanquin Research and Landsteiner Laboratory, Amsterdam, the Netherlands; Department of Microbiology and Immunology (C.E.v.d.S.), University of Melbourne, Peter Doherty Institute for Infection and Immunity, Victoria, Australia; Amsterdam Rheumatology and Immunology Center (L.B., G.W.), location Reade, Department of Rheumatology; Amsterdam Rheumatology and Immunology Center (S.W.T.), Amsterdam UMC, Department of Rheumatology and Clinical Immunology, University of Amsterdam; Department of Neurology (J.K., Z.L.E.v.K.), Amsterdam UMC, Vrije Universiteit; Department 32 of Pediatric Immunology (T.W.K.), Rheumatology and Infectious Disease, Amsterdam UMC, location AMC, University of Amsterdam; and Swammerdam Institute for Life Sciences (S.M.v.H.), University of Amsterdam, the Netherlands.

Objectives: To evaluate whether a third vaccination shows an added effect on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) T-cell responses in patients with multiple sclerosis treated with ocrelizumab or fingolimod.

Methods: This is a substudy of a prospective multicenter study on SARS-CoV-2 vaccination in patients with immune-mediated diseases. Patients with MS treated with ocrelizumab, fingolimod, and no disease-modifying therapies and healthy controls were included. The number of interferon (IFN)-γ secreting SARS-CoV-2-specific T cells at multiple time points before and after 3 SARS-CoV-2 vaccinations were evaluated.

Results: In ocrelizumab-treated patients (N = 24), IFN-γ-producing SARS-CoV-2-specific T-cell responses were induced after 2 vaccinations with median levels comparable to healthy controls (N = 12) and patients with MS without disease-modifying therapies (N = 10). A third vaccination in ocrelizumab-treated patients (N = 8) boosted T-cell responses that had declined after the second vaccination, but did not lead to higher overall T-cell responses as compared to immediately after a second vaccination. In fingolimod-treated patients, no SARS-CoV-2-specific T cells were detected after second (N = 12) and third (N = 9) vaccinations.

Discussion: In ocrelizumab-treated patients with MS, a third SARS-CoV-2 vaccination had no additive effect on the maximal T-cell response but did induce a boost response. In fingolimod-treated patients, no T-cell responses could be detected following both a second and third SARS-CoV-2 vaccination.
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http://dx.doi.org/10.1212/NXI.0000000000001178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9082763PMC
July 2022

Humoral responses after second and third SARS-CoV-2 vaccination in patients with immune-mediated inflammatory disorders on immunosuppressants: a cohort study.

Lancet Rheumatol 2022 May 17;4(5):e338-e350. Epub 2022 Mar 17.

Department of Rheumatology and Clinical Immunology, University Medical Center Groningen, University Groningen, Groningen, Netherlands.

Background: Disease-specific studies have reported impaired humoral responses after SARS-CoV-2 vaccination in patients with immune-mediated inflammatory disorders treated with specific immunosuppressants. Disease-overarching studies, and data on recall responses and third vaccinations are scarce. Our primary objective was to investigate the effects of immunosuppressive monotherapies on the humoral immune response after SARS-CoV-2 vaccination in patients with prevalent immune-mediated inflammatory disorders.

Methods: We did a cohort study in participants treated in outpatient clinics in seven university hospitals and one rheumatology treatment centre in the Netherlands as well as participants included in two national cohort studies on COVID-19-related disease severity. We included patients aged older than 18 years, diagnosed with any of the prespecified immune-mediated inflammatory disorders, who were able to understand and complete questionnaires in Dutch. Participants with immune-mediated inflammatory disorders who were not on systemic immunosuppressants and healthy participants were included as controls. Anti-receptor binding domain IgG responses and neutralisation capacity were monitored following standard vaccination regimens and a three-vaccination regimen in subgroups. Hybrid immune responses-ie, vaccination after previous SARS-CoV-2 infection-were studied as a proxy for recall responses.

Findings: Between Feb 2 and Aug 1, 2021, we included 3222 participants in our cohort. Sera from 2339 participants, 1869 without and 470 participants with previous SARS-CoV-2 infection were analysed (mean age 49·9 years [SD 13·7]; 1470 [62·8%] females and 869 [37·2%] males). Humoral responses did not differ between disorders. Anti-CD20 therapy, sphingosine 1-phosphate receptor (S1P) modulators, and mycophenolate mofetil combined with corticosteroids were associated with lower relative risks for reaching seroconversion following standard vaccination (0·32 [95% CI 0·19-0·49] for anti-CD20 therapy, 0·35 [0·21-0·55] for S1P modulators, and 0·61 [0·40-0·90] for mycophenolate mofetil combined with corticosteroids). A third vaccination increased seroconversion for mycophenolate mofetil combination treatments (from 52·6% after the second vaccination to 89·5% after the third) but not significantly for anti-CD20 therapies (from 36·8% to 45·6%) and S1P modulators (from 35·5% to 48·4%). Most other immunosuppressant groups showed moderately reduced antibody titres after standard vaccination that did not increase after a third vaccination, although seroconversion rates and neutralisation capacity were unaffected. In participants with previous SARS-CoV-2 infection, SARS-CoV-2 antibodies were boosted after vaccination, regardless of immunosuppressive treatment.

Interpretation: Humoral responses following vaccination are impaired by specific immunosuppressants. After standard vaccination regimens, patients with immune-mediated inflammatory disorders taking most immunosuppressants show similar seroconversion to controls, although antibody titres might be moderately reduced. As neutralisation capacity and recall responses are also preserved in these patients, this is not likely to translate to loss of (short-term) protection. In patients on immunosuppressants showing poor humoral responses after standard vaccination regimens, a third vaccination resulted in additional seroconversion in patients taking mycophenolate mofetil combination treatments, whereas the effect of a third vaccination in patients on anti-CD20 therapy and S1P modulators was limited.

Funding: ZonMw (The Netherlands Organization for Health Research and Development).
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http://dx.doi.org/10.1016/S2665-9913(22)00034-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8930018PMC
May 2022

Platelet concentrates in platelet additive solutions generate less complement activation products during storage than platelets stored in plasma.

Blood Transfus 2022 Mar 7. Epub 2022 Mar 7.

Department Immunopathology Sanquin Blood Supply, Amsterdam, the Netherlands.

Background: Platelet transfusions can be associated with adverse reactions, such as febrile non-haemolytic transfusion reaction (FNHTR). It has been suggested that damage-associated molecular patterns (DAMP) and complement play a role in FNHTR. This study investigated the nature of DAMPs and complement activation products contained in platelet concentrates during storage, with a specific focus on different platelet storage solutions.

Materials And Methods: Buffy coats (BC) from healthy donors were pooled (15 BC per pool) and divided into three groups of the same volume. After addition of different storage solutions (plasma, platelet additive solutions [PAS]-C or PAS-E; n=6 for each group), BC pools were processed to platelet concentrates (PC). Leukoreduced PCs were stored on a shaking bed at 20-24°C and sampled on days 1, 2, 6 and 8 after collection for selected quality parameters: platelet activation, DAMPs (High Mobility Group Box 1 [HMGB1], nucleosomes), and complement activation products.

Results: During storage, equal levels of free nucleosomes and increasing concentrations of HMGB1 were present in all groups. Complement activation was observed in all PC. However, by day 8, the use of PAS had reduced C3b/c levels by approximately 90% and C4b/c levels by approximately 65%.

Discussion: Nucleosomes and HMGB1 were present in PCs prepared in plasma and PAS. Complement was activated during storage of platelets in plasma and in PAS. The use of PAS is associated with a lower amount of complement activation products due to the dilution of plasma by PAS. Therefore, PC in PAS have less complement activation products than platelets stored in plasma. These proinflammatory mediators in PC might induce FNHTR.
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http://dx.doi.org/10.2450/2022.0323-21DOI Listing
March 2022

Associations Between Symptoms, Donor Characteristics and IgG Antibody Response in 2082 COVID-19 Convalescent Plasma Donors.

Front Immunol 2022 28;13:821721. Epub 2022 Feb 28.

Department of Experimental Immunohematology, Sanquin Research and Landsteiner Laboratory Amsterdam University Medical Centre, Amsterdam, Netherlands.

Many studies already reported on the association between patient characteristics on the severity of COVID-19 disease outcome, but the relation with SARS-CoV-2 antibody levels is less clear. To investigate this in more detail, we performed a retrospective observational study in which we used the IgG antibody response from 11,118 longitudinal antibody measurements of 2,082 unique COVID convalescent plasma donors. COVID-19 symptoms and donor characteristics were obtained by a questionnaire. Antibody responses were modelled using a linear mixed-effects model. Our study confirms that the SARS-CoV-2 antibody response is associated with patient characteristics like body mass index and age. Antibody decay was faster in male than in female donors (average half-life of 62 versus 72 days). Most interestingly, we also found that three symptoms (headache, anosmia, nasal cold) were associated with lower peak IgG, while six other symptoms (dry cough, fatigue, diarrhoea, fever, dyspnoea, muscle weakness) were associated with higher IgG concentrations.
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http://dx.doi.org/10.3389/fimmu.2022.821721DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8918483PMC
March 2022

Risk factors associated with short-term adverse events after SARS-CoV-2 vaccination in patients with immune-mediated inflammatory diseases.

BMC Med 2022 03 2;20(1):100. Epub 2022 Mar 2.

Department of Neurology, Leiden University Medical Center, Leiden, The Netherlands.

Background: Studies have suggested incremental short-term adverse events (AE) after repeated vaccination. In this report, we assessed occurrence and risk factors for short-term AEs following repeated SARS-CoV-2 vaccination in patients with various immune-mediated inflammatory diseases (IMIDs).

Methods: Self-reported daily questionnaires on AEs during the first 7 days after vaccination were obtained of 2259 individuals (2081 patients and 178 controls) participating in an ongoing prospective multicenter cohort study on SARS-CoV-2 vaccination in patients with various IMIDs in the Netherlands (T2B-COVID). Relative risks were calculated for potential risk factors associated with clinically relevant AE (rAE), defined as AE lasting longer than 2 days or impacting daily life.

Results: In total, 5454 vaccinations were recorded (1737 first, 1992 second and 1478 third vaccinations). Multiple sclerosis, Crohn's disease and rheumatoid arthritis were the largest disease groups. rAEs were reported by 57.3% (95% CI 54.8-59.8) of patients after the first vaccination, 61.5% (95% CI 59.2-63.7) after the second vaccination and 58% (95% CI 55.3-60.6) after the third vaccination. At day 7 after the first, second and third vaccination, respectively, 7.6% (95% CI 6.3-9.1), 7.4% (95% CI 6.2-8.7) and 6.8% (95% CI 5.4-8.3) of patients still reported AEs impacting daily life. Hospital admissions and allergic reactions were uncommon (<0.7%). Female sex (aRR 1.43, 95% CI 1.32-1.56), age below 50 (aRR 1.14, 95% CI 1.06-1.23), a preceding SARS-CoV-2 infection (aRR 1.14, 95% CI 1.01-1.29) and having an IMID (aRR 1.16, 95% CI 1.01-1.34) were associated with increased risk of rAEs following a vaccination. Compared to the second vaccination, the first vaccination was associated with a lower risk of rAEs (aRR 0.92, 95% CI 0.84-0.99) while a third vaccination was not associated with increased risk on rAEs (aRR 0.93, 95% CI 0.84-1.02). BNT162b2 vaccines were associated with lower risk on rAEs compared to CX-024414 (aRR 0.86, 95% CI 0.80-0.93).

Conclusions: A third SARS-CoV-2 vaccination was not associated with increased risk of rAEs in IMID patients compared to the second vaccination. Patients with an IMID have a modestly increased risk of rAEs after vaccination when compared to controls. Most AEs are resolved within 7 days; hospital admissions and allergic reactions were uncommon.

Trial Registration: NL74974.018.20 , Trial ID: NL8900. Registered on 9 September 2020.
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http://dx.doi.org/10.1186/s12916-022-02310-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8889379PMC
March 2022

Minimalistic In Vitro Culture to Drive Human Naive B Cell Differentiation into Antibody-Secreting Cells.

Cells 2021 05 12;10(5). Epub 2021 May 12.

Department of Immunopathology, Sanquin Research and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, 1066 CX Amsterdam, The Netherlands.

High-affinity antibody-secreting cells (ASC) arise from terminal differentiation of B-cells after coordinated interactions with T follicular helper (Tfh) cells in germinal centers (GC). Elucidation of cues promoting human naive B-cells to progress into ASCs is challenging, as this process is notoriously difficult to induce in vitro while maintaining enough cell numbers to investigate the differentiation route(s). Here, we describe a minimalistic in vitro culture system that supports efficient differentiation of human naive B-cells into antibody-secreting cells. Upon initial stimulations, the interplay between level of CD40 costimulation and the Tfh cell-associated cytokines IL-21 and IL-4 determined the magnitude of B-cell expansion, immunoglobulin class-switching and expression of ASC regulator . In contrast, the B-cell-specific transcriptional program was maintained, and efficient ASC formation was hampered. Renewed CD40 costimulation and Tfh cytokines exposure induced rapid secondary STAT3 signaling and extensive ASC differentiation, accompanied by repression of B-cell identity factors and and further induction of Our work shows that, like in vivo, renewed CD40L costimulation also induces efficient terminal ASC differentiation after initial B-cell expansion in vitro. This culture system for efficient differentiation of human naive B-cells into ASCs, while also maintaining high cell numbers, may form an important tool in dissecting human naive B-cell differentiation, thereby enabling identification of novel transcriptional regulators and biomarkers for desired and detrimental antibody formation in humans.
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http://dx.doi.org/10.3390/cells10051183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8151070PMC
May 2021

Dynamics of antibodies to SARS-CoV-2 in convalescent plasma donors.

Clin Transl Immunology 2021 16;10(5):e1285. Epub 2021 May 16.

Department of Immunopathology Sanquin Research Amsterdam The Netherlands.

Objectives: Characterisation of the human antibody response to SARS-CoV-2 infection is vital for serosurveillance purposes and for treatment options such as transfusion with convalescent plasma or immunoglobulin products derived from convalescent plasma. In this study, we longitudinally and quantitatively analysed antibody responses in RT-PCR-positive SARS-CoV-2 convalescent adults during the first 250 days after onset of symptoms.

Methods: We measured antibody responses to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and the nucleocapsid protein in 844 longitudinal samples from 151 RT-PCR-positive SARS-CoV-2 convalescent adults. With a median of 5 (range 2-18) samples per individual, this allowed quantitative analysis of individual longitudinal antibody profiles. Kinetic profiles were analysed by mixed-effects modelling.

Results: All donors were seropositive at the first sampling moment, and only one donor seroreverted during follow-up analysis. Anti-RBD IgG and anti-nucleocapsid IgG levels declined with median half-lives of 62 and 59 days, respectively, 2-5 months after symptom onset, and several-fold variation in half-lives of individuals was observed. The rate of decline of antibody levels diminished during extended follow-up, which points towards long-term immunological memory. The magnitude of the anti-RBD IgG response correlated well with neutralisation capacity measured in a classic plaque reduction assay and in an in-house developed competitive assay.

Conclusion: The result of this study gives valuable insight into the long-term longitudinal response of antibodies to SARS-CoV-2.
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http://dx.doi.org/10.1002/cti2.1285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8126762PMC
May 2021

Inhibition of Dendritic Cell Activation and Modulation of T Cell Polarization by the Platelet Secretome.

Front Immunol 2021 25;12:631285. Epub 2021 Feb 25.

Department of Immunopathology, Sanquin Research and Landsteiner Laboratory, Amsterdam University Medical Center, University of Amsterdam, Amsterdam, Netherlands.

Platelet transfusions are a frequently administered therapy for especially hemato-oncological patients with thrombocytopenia. Next to their primary function in hemostasis, currently there is increased attention for the capacity of platelets to affect the function of various cells of the immune system. Here, we investigate the capacity of platelets to immuno-modulate monocyte-derived dendritic cells (moDC) as well as primary dendritic cells and effects on subsequent T cell responses. Platelets significantly inhibited pro-inflammatory (IL-12, IL-6, TNFα) and increased anti-inflammatory (IL-10) cytokine production of moDCs primed with toll-like receptor (TLR)-dependent and TLR-independent stimuli. Transwell assays and ultracentrifugation revealed that a soluble factor secreted by platelets, but not microvesicles, inhibited DC activation. Interestingly, platelet-derived soluble mediators also inhibited cytokine production by human stimulated myeloid CD1c+ conventional DC2. Moreover, platelets and platelet-derived soluble mediators inhibited T cell priming and T helper differentiation toward an IFN+ Th1 phenotype by moDCs. Overall, these results show that platelets are able to inhibit the pro-inflammatory properties of DCs, and may even induce an anti-inflammatory DC phenotype, with decreased T cell priming capacity by the DC. The results of this study provide more insight in the potential role of platelets in immune modulation, especially in the context of platelet transfusions.
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http://dx.doi.org/10.3389/fimmu.2021.631285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7961920PMC
September 2021

Flow Cytometric Methods for the Detection of Intracellular Signaling Proteins and Transcription Factors Reveal Heterogeneity in Differentiating Human B Cell Subsets.

Cells 2020 12 8;9(12). Epub 2020 Dec 8.

Department of Immunopathology, Sanquin Research and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, 1066 CX Amsterdam, The Netherlands.

The flow cytometric detection of intracellular (IC) signaling proteins and transcription factors (TFs) will help to elucidate the regulation of B cell survival, proliferation and differentiation. However, the simultaneous detection of signaling proteins or TFs with membrane markers (MMs) can be challenging, as the required fixation and permeabilization procedures can affect the functionality of conjugated antibodies. Here, a phosphoflow method is presented for the detection of activated NF-κB p65 and phosphorylated STAT1, STAT3, STAT5 and STAT6, together with the B cell differentiation MMs CD19, CD27 and CD38. Additionally, a TF-flow method is presented that allows the detection of the B cell TFs PAX5, c-MYC, BCL6 and AID and antibody-secreting cell (ASC) TFs BLIMP1 and XBP-1s, together with MMs. Applying these methods on in vitro-induced human B cell differentiation cultures showed significantly different steady-state levels, and responses to stimulation, of phosphorylated signaling proteins in CD27-expressing B cell and ASC populations. The TF-flow protocol and Uniform Manifold Approximation and Projection (UMAP) analysis revealed heterogeneity in TF expression within stimulated CD27- or CD38-expressing B cell subsets. The methods presented here allow for the sensitive analysis of STAT, NF-κB p65 signaling and TFs, together with B cell differentiation MMs, at single-cell resolution. This will aid the further investigation of B cell responses in both health and disease.
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http://dx.doi.org/10.3390/cells9122633DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7762542PMC
December 2020

Guidelines for analysis of low-frequency antigen-specific T cell results: Dye-based proliferation assay vs H-thymidine incorporation.

J Immunol Methods 2020 12 3;487:112907. Epub 2020 Nov 3.

Department of Immunopathology, Sanquin Research and Landsteiner Laboratory, AMC, Amsterdam, the Netherlands. Electronic address:

It is generally recognized that dysregulation of the immune system plays a critical role in many diseases, including autoimmune diseases and cancer. T cells play a crucial role in maintaining self-tolerance, while loss of immune tolerance and T cell activation can lead to severe inflammation and tissue damage. T cell responses have a key role in the effectiveness of vaccination strategies and immunomodulating therapies. Immunomonitoring methods have the ability to elucidate immunological processes, monitor the development of disease and assess therapeutic effects. In this respect, it is of particular interest to evaluate antigen (Ag)-specific T cells by determining their frequency, type and functionality in cellular assays. Nevertheless, Ag-specific T cells are detected infrequently in most diseases using current techniques. Many efforts have been made to develop more sensitive, reproducible, and reliable methods for Ag-specific T cell detection. It has been found that analysis of cellular proliferation can be a useful tool to determine the presence and frequency of Ag-specific T cell and to provides insight into modulation of the T cell response by a specific antigen or therapy. However, the selection of a cut-off value for a positive response and therefore a more accurate interpretation of the data, continues to be a major concern. Here, we provide guidelines to select a proper cut-off for monitoring of Ag-specific CD4 T cell responses. In vitro Ag-stimulation has been assessed with two methods; a dye-based proliferation assay and H-thymidine-based assay. Two cut-off approaches are compared; mean and variance of control wells, and the stimulation index. By evaluating the proliferative response to the in vitro Ag-stimulation using these two methods, we demonstrate the importance of taking into consideration the variability of the control wells to distinguish a positive from a false positive response.
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http://dx.doi.org/10.1016/j.jim.2020.112907DOI Listing
December 2020

Performance of monocyte activation test supplemented with human serum compared to fetal bovine serum.

ALTEX 2021 28;38(2):307-315. Epub 2020 Oct 28.

Sanquin Research, Dept Immunopathology, Amsterdam, The Netherlands.

The monocyte activation test (MAT) is used to detect pyrogens in pharmaceutical products and serves as replacement of the rabbit pyrogen test. The peripheral blood mononuclear cell-based MAT assay requires the addition of serum to the medium and is performed with either fetal bovine serum (FBS) or human serum (HS). Since the capacity to detect non-endotoxin pyrogens (NEPs) in a sensitive manner is an important strength of MAT compared to the bacterial endo­toxin test, the performance of the MAT using FBS and HS was compared using endotoxin and several NEPs. The MAT was more sensitive for endotoxin when FBS was used, however for most NEPs the MAT was more sensitive when per­formed in HS. Furthermore, heat-inactivation of FBS affected the performance of the MAT for endotoxin to some extent but not for the NEPs. Interestingly, heat-inactivation of HS led to an almost complete loss of reactivity towards endotoxin, reduced the response towards heat-killed Staphylococcus aureus and peptidoglycan, but had minor or no effects on the responses towards R848, flagellin, and Pam3CSK4. Product testing of a human blood-derived product in MAT using HS was beneficial since endotoxin spike recoveries were improved. This product is therefore currently batch released with the HS-based MAT assay. Overall, to guarantee optimal performance of MAT, heat-inactivated serum should be avoided. The HS-based MAT appears to be the first choice to replace the rabbit pyrogen test, while in some cases the FBS-based MAT may be favored.
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http://dx.doi.org/10.14573/altex.2008261DOI Listing
October 2021

Divergent SARS-CoV-2-specific T- and B-cell responses in severe but not mild COVID-19 patients.

Eur J Immunol 2020 Dec 16;50(12):1998-2012. Epub 2020 Nov 16.

Department of Hematopoiesis, Sanquin Research and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. Understanding the immune response that provides specific immunity but may also lead to immunopathology is crucial for the design of potential preventive and therapeutic strategies. Here, we characterized and quantified SARS-CoV-2-specific immune responses in patients with different clinical courses. Compared to individuals with a mild clinical presentation, CD4 T-cell responses were qualitatively impaired in critically ill patients. Strikingly, however, in these patients the specific IgG antibody response was remarkably strong. Furthermore, in these critically ill patients, a massive influx of circulating T cells into the lungs was observed, overwhelming the local T-cell compartment, and indicative of vascular leakage. The observed disparate T- and B-cell responses could be indicative of a deregulated immune response in critically ill COVID-19 patients.
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http://dx.doi.org/10.1002/eji.202048908DOI Listing
December 2020

Personalized extended interval dosing of natalizumab in MS: A prospective multicenter trial.

Neurology 2020 08 20;95(6):e745-e754. Epub 2020 Jul 20.

From the Department of Neurology, Amsterdam MS Center (Z.L.E.v.K., B.W.v.O., B.M.J.U., J.K.), Department of Radiology (M.P.W., F.B.), and Neurochemistry Lab and Biobank, Department of Clinical Chemistry (C.E.T.), Amsterdam Neuroscience, and Department of Epidemiology and Biostatistics (B.I.L.-W.), Amsterdam University Medical Centers, Vrije Universiteit; Department of Neurology (E.L.J.H.), St. Antonius Hospital, Utrecht, the Netherlands; Department of Diagnostic and Interventional Neuroradiology (M.P.W.), Hannover Medical School, Germany; Department of Neurology (N.F.K.), OLVG Hospital, Amsterdam; Department of Neurology (J.P.M.), Rijnstate Hospital, Arnhem; Biologics Lab, Bioanalysis (A.d.V.), Sanquin Diagnostic Services; Department of Immunopathology (A.t.B., T.R.), Sanquin Research, Amsterdam; Landsteiner Laboratory (A.t.B., T.R.), Academic Medical Centre, University of Amsterdam, the Netherlands; and UCL Institutes of Neurology & Healthcare Engineering (F.B.), Queen Square, London, UK.

Objective: To determine whether natalizumab efficacy is maintained when switching to personalized extended interval dosing based on individual natalizumab trough concentrations in patients with relapsing-remitting multiple sclerosis (RRMS).

Methods: This was a prospective multicenter single-arm trial with 1 year follow-up and a 1-year extension phase. Participants were adult persons with RRMS treated with natalizumab without disease activity in the year prior to enrollment. The natalizumab treatment interval was based on longitudinal natalizumab trough concentrations. Patients received 3 monthly MRI scans, relapse assessments, and disability scoring during follow-up. The primary endpoint was the occurrence of gadolinium-enhancing lesions on MRI. Secondary endpoints were new/enlarging T2 lesions on MRI and relapses and progression on the Expanded Disability Status Scale (EDSS) during follow-up and extension phase.

Results: Sixty-one patients were included. Eighty-four percent extended the interval from a 4-week interval to a 5- to 7-week interval. No patient developed gadolinium-enhancing lesions (95% confidence interval [CI] 0%-7.4%) during follow-up. No new/enlarging T2 lesions (95% CI 0%-7.4%) or relapses (95% CI 0%-7.4%) were reported during follow-up and in the extension phase. Median EDSS was comparable at baseline (3.0, interquartile range [IQR] 2.0-5.0) and after follow-up (3.0, IQR 2.0-5.0).

Conclusion: Personalized extended interval dosing did not induce recurrence of MS disease activity. Natalizumab efficacy was maintained in stable patients with RRMS receiving personalized extended interval dosing based on individual natalizumab concentrations.

Classification Of Evidence: This study provides Class IV evidence that personalized extended interval dosing of natalizumab does not result in recurrence of disease activity in stable patients with RRMS.
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http://dx.doi.org/10.1212/WNL.0000000000009995DOI Listing
August 2020

Eomes broadens the scope of CD8 T-cell memory by inhibiting apoptosis in cells of low affinity.

PLoS Biol 2020 03 17;18(3):e3000648. Epub 2020 Mar 17.

Department of Histology and Embryology, University of Rijeka, Rijeka, Croatia.

The memory CD8 T-cell pool must select for clones that bind immunodominant epitopes with high affinity to efficiently counter reinfection. At the same time, it must retain a level of clonal diversity to allow recognition of pathogens with mutated epitopes. How the level of diversity within the memory pool is controlled is unclear, especially in the context of a selective drive for antigen affinity. We find that preservation of clones that bind the activating antigen with low affinity depends on expression of the transcription factor Eomes in the first days after antigen encounter. Eomes is induced at low activating signal strength and directly drives transcription of the prosurvival protein Bcl-2. At higher signal intensity, T-bet is induced which suppresses Bcl-2 and causes a relative survival advantage for cells of low affinity. Clones activated with high-affinity antigen form memory largely independent of Eomes and have a proliferative advantage over clones that bind the same antigen with low affinity. This causes high-affinity clones to prevail in the memory pool, despite their relative survival deficit. Genetic or therapeutic targeting of the Eomes/Bcl-2 axis reduces the clonal diversity of the memory pool, which diminishes its ability to respond to pathogens carrying mutations in immunodominant epitopes. Thus, we demonstrate on a molecular level how sufficient diversity of the memory pool is established in an environment of affinity-based selection.
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http://dx.doi.org/10.1371/journal.pbio.3000648DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7077837PMC
March 2020

The natalizumab wearing-off effect: End of natalizumab cycle, recurrence of MS symptoms.

Neurology 2019 10 24;93(17):e1579-e1586. Epub 2019 Sep 24.

From the Amsterdam UMC (Z.L.E.v.K., D.D., I.D., B.I.L.-W., J.K.), Vrije Universiteit Amsterdam; Departments of Neurology (Z.L.E.v.K., D.D., I.D., J.K.) and Radiology & Nuclear Medicine (I.D.), MS Center Amsterdam, Amsterdam Neuroscience; and Departments of Epidemiology and Biostatistics (B.I.L.-W.) and Immunology (A.d.V., I.A.C., A.t.B., T.R.), Landsteiner Laboratory Sanquin Research, Amsterdam, the Netherlands.

Objective: Natalizumab is effective in treating relapsing-remitting multiple sclerosis (RRMS). However, many patients report an increase of multiple sclerosis symptoms at the end of the natalizumab cycle: a wearing-off effect. The objective of this study was to evaluate the prevalence of the wearing-off effect in patients with standard and extended intervals and to study possible associations with pharmacokinetic/dynamic measurements and patient characteristics in a prospective, monocenter, cross-sectional cohort study.

Methods: Patients with RRMS, with a minimum of 6 natalizumab infusions, were asked to complete 3 questionnaires: the Multiple Sclerosis Impact Scale, the 36-Item Short Form Health Survey, and a general questionnaire regarding the wearing-off effect. Natalizumab concentration and α4-integrin receptor saturation were measured before redosing.

Results: Ninety-three patients were included. A total of 54% experienced a wearing-off effect during natalizumab treatment and 32% experienced a current wearing-off effect at time of measurement. The self-reported wearing-off effect was not associated with natalizumab concentration nor with α4-integrin receptor saturation. The wearing-off effect was more frequently reported in the standard interval group (39%) than in the extended interval group (19%); the duration of symptoms was comparable between both groups. The wearing-off effect was not associated with number of infusions, disease duration, age, or sex.

Conclusion: The wearing-off effect is a frequently reported phenomenon but is unlikely to reflect a nonoptimal pharmacokinetic/dynamic state. We did not find risk factors predicting the wearing-off effect.
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http://dx.doi.org/10.1212/WNL.0000000000008357DOI Listing
October 2019

Tolerogenic dendritic cell-based treatment for multiple sclerosis (MS): a harmonised study protocol for two phase I clinical trials comparing intradermal and intranodal cell administration.

BMJ Open 2019 09 9;9(9):e030309. Epub 2019 Sep 9.

Interuniversity Institute for Biostatistics and statistical Bioinformatics (I-BioStat), Universiteit Hasselt, Hasselt, Belgium.

Introduction: Based on the advances in the treatment of multiple sclerosis (MS), currently available disease-modifying treatments (DMT) have positively influenced the disease course of MS. However, the efficacy of DMT is highly variable and increasing treatment efficacy comes with a more severe risk profile. Hence, the unmet need for safer and more selective treatments remains. Specifically restoring immune tolerance towards myelin antigens may provide an attractive alternative. In this respect, antigen-specific tolerisation with autologous tolerogenic dendritic cells (tolDC) is a promising approach.

Methods And Analysis: Here, we will evaluate the clinical use of tolDC in a well-defined population of MS patients in two phase I clinical trials. In doing so, we aim to compare two ways of tolDC administration, namely intradermal and intranodal. The cells will be injected at consecutive intervals in three cohorts receiving incremental doses of tolDC, according to a best-of-five design. The primary objective is to assess the safety and feasibility of tolDC administration. For safety, the number of adverse events including MRI and clinical outcomes will be assessed. For feasibility, successful production of tolDC will be determined. Secondary endpoints include clinical and MRI outcome measures. The patients' immune profile will be assessed to find presumptive evidence for a tolerogenic effect in vivo.

Ethics And Dissemination: Ethics approval was obtained for the two phase I clinical trials. The results of the trials will be disseminated in a peer-reviewed journal, at scientific conferences and to patient associations.

Trial Registration Numbers: NCT02618902 and NCT02903537; EudraCT numbers: 2015-002975-16 and 2015-003541-26.
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http://dx.doi.org/10.1136/bmjopen-2019-030309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6738722PMC
September 2019

Pharmacodynamic assessment of cell-bound natalizumab on PBMC samples stored in liquid nitrogen.

J Immunol Methods 2019 10 12;473:112632. Epub 2019 Jul 12.

Department of Immunopathology, Sanquin Research, Amsterdam, the Netherlands; Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands. Electronic address:

Natalizumab is a monoclonal IgG4 antibody used for treatment of relapsing remitting MS. Natalizumab interferes with lymphocyte migration by blocking alpha-4 integrin (CD49d). Saturation levels of alpha-4 integrin on circulating T cells by natalizumab have been associated with clinical effectiveness of therapy. However, in most cases, measurements have been carried out using freshly isolated PBMCs. The aim of this study was to set up and evaluate a method to measure relative levels of cell-bound natalizumab using frozen PBMC samples. A new method was set up to measure cell-bound natalizumab by flow cytometry on T cell subsets using fully saturated cells as a 100% reference. A comparison was made between spike samples and samples of natalizumab-treated MS patients freshly isolated and stored in liquid nitrogen. Cell-bound natalizumab could be measured (using an anti-IgG4 antibody) on cells stored in liquid nitrogen. Natalizumab was found to slowly dissociate from the cells during isolation and subsequent sample work-up. This dissociation was more pronounced for monovalent natalizumab resulting from Fab arm exchange (the predominant isoform in patients) than bivalent natalizumab straight from the vial. We established a correction factor to account for this phenomenon. The resulting method has good accuracy compared to assessing fresh cells. The inter-assay precision (%CV) is ca. 12% using frozen cells. In conclusion, we established a method to assess relative levels of cell-bound natalizumab on cells obtained from frozen PBMC samples.
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http://dx.doi.org/10.1016/j.jim.2019.07.004DOI Listing
October 2019

and Are Biomarkers of Vitamin D3-Induced Tolerogenic Dendritic Cells in Multiple Sclerosis Patients.

Front Immunol 2019 19;10:1251. Epub 2019 Jun 19.

Division of Immunology, LCMN, Germans Trias i Pujol University Hospital and Research Institute, Barcelona, Spain.

The administration of autologous tolerogenic dendritic cells (tolDC) has become a promising alternative for the treatment of autoimmune diseases, such as multiple sclerosis (MS). Specifically, the use of vitamin D3 for the generation of tolDC (vitD3-tolDC) constitutes one of the most widely studied approaches, as it has evidenced significant immune regulatory properties, both and . In this article, we generated human vitD3-tolDC from monocytes from healthy donors and MS patients, characterized in both cases by a semi-mature phenotype, secretion of IL-10 and inhibition of allogeneic lymphocyte proliferation. Additionally, we studied their transcriptomic profile and selected a number of differentially expressed genes compared to control mature and immature dendritic cells for their analysis. Among them, qPCR results validated and genes as biomarkers of vitD3-tolDC in both healthy donors and MS patients. Furthermore, we constructed a network of protein interactions based on the literature, which manifested that and genes are both closely connected between them and involved in immune-related functions. In conclusion, this study evidences that and constitute robust and potentially functional biomarkers of the generation of vitD3-tolDC, opening the window for their use as quality controls in clinical trials for MS.
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http://dx.doi.org/10.3389/fimmu.2019.01251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6598738PMC
September 2020

Neutrophils acquire antigen-presenting cell features after phagocytosis of IgG-opsonized erythrocytes.

Blood Adv 2019 06;3(11):1761-1773

Department of Blood Cell Research, Sanquin Research and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.

Neutrophils are particularly well known for their antimicrobial function. Although historically they are regarded as strictly a phagocyte of the innate immune system, over time it has become clear that neutrophils are versatile cells with numerous functions including innate and adaptive immune regulation. We have previously described a role for human neutrophils in antibody-mediated red blood cell (RBC) clearance. Under homeostatic conditions, neutrophils do not take up RBCs. Yet, when RBCs are immunoglobulin G (IgG) opsonized, which can occur in alloimmunization or autoimmunization reactions, neutrophils can effectively phagocytose RBCs. In the present study, we show that human neutrophils acquire an antigen-presenting cell (APC) phenotype following RBC phagocytosis. Subsequent to RBC phagocytosis, neutrophils expressed major histocompatibility complex class II (MHC-II) and costimulatory molecules such as CD40 and CD80. Moreover, in classical APCs, the respiratory burst is known to regulate antigen presentation. We found that the respiratory burst in neutrophils is reduced after IgG-mediated RBC phagocytosis. Additionally, following RBC phagocytosis, neutrophils were demonstrated to elicit an antigen-specific T-cell response, using tetanus toxoid (TT) as an antigen to elicit an autologous TT-specific CD4 T-cell response. Lastly, although the "don't eat me" signal CD47 is known to have a powerful restrictive role in the activation of immunity against RBCs in dendritic cells, CD47 does not seem to have a significant effect on the antigen-presenting function of neutrophils in this context. Overall, these findings reveal that besides their classical antimicrobial role, neutrophils show plasticity in their phenotype.
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http://dx.doi.org/10.1182/bloodadvances.2018028753DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6560339PMC
June 2019

Anti-natalizumab antibodies during 8 years of natalizumab treatment: effect on natalizumab concentration and α4-integrin receptor saturation.

J Neurol 2019 07 19;266(7):1804-1805. Epub 2019 Apr 19.

Department of Neurology, Neuroscience Amsterdam, Amsterdam MS Center, Amsterdam University Medical Center, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands.

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http://dx.doi.org/10.1007/s00415-019-09327-8DOI Listing
July 2019

Differential effects of anaphylatoxin C5a on antigen presenting cells, roles for C5aR1 and C5aR2.

Immunol Lett 2019 05 5;209:45-52. Epub 2019 Apr 5.

Department of Immunopathology, Sanquin Research, Amsterdam, the Netherlands and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands. Electronic address:

The anaphylatoxin C5a is well-known for its role as chemoattractant and contributes to immune cell recruitment into inflamed tissue and local inflammation. C5a has recently been implicated in modulation of antigen presenting cell function, such as macrophages and dendritic cells, which are pivotal for T cell activation and final T cell effector function. The published data on the effect of C5a on APC function and subsequent adaptive immune responses are in part conflicting, as both pro and anti-inflammatory effects have been described. In this review the opposing effects of C5a on APC function in mice and human are summarized and discussed in relation to origin of the involved APC subset, being either of the monocyte-derived lineage or dendritic cell lineage. In addition, the current knowledge on the expression of C5aR1 and C5aR2 on the different APC subsets is summarized. Based on the combined data, we propose that the differential effects of C5a on APC function may be attributed to absence or presence of co-expression of C5aR2 and C5aR1 on the specific APC.
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http://dx.doi.org/10.1016/j.imlet.2019.03.014DOI Listing
May 2019

Human B Cells Engage the NCK/PI3K/RAC1 Axis to Internalize Large Particles via the IgM-BCR.

Front Immunol 2019 13;10:415. Epub 2019 Mar 13.

Department of Immunopathology, Sanquin Research and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands.

Growing evidence indicate that large antigen-containing particles induce potent T cell-dependent high-affinity antibody responses. These responses require large particle internalization after recognition by the B cell receptor (BCR) on B cells. However, the molecular mechanisms governing BCR-mediated internalization remain unclear. Here we use a high-throughput quantitative image analysis approach to discriminate between B cell particle binding and internalization. We systematically show, using small molecule inhibitors, that human B cells require a SYK-dependent IgM-BCR signaling transduction via PI3K to efficiently internalize large anti-IgM-coated particles. IgM-BCR-mediated activation of PI3K involves both the adaptor protein NCK and the co-receptor CD19. Interestingly, we here reveal a strong NCK-dependence without profound requirement of the co-receptor CD19 in B cell responses to large particles. Furthermore, we demonstrate that the IgM-BCR/NCK signaling event facilitates RAC1 activation to promote actin cytoskeleton remodeling necessary for particle engulfment. Thus, we establish NCK/PI3K/RAC1 as an attractive IgM-BCR signaling axis for biological intervention to prevent undesired antibody responses to large particles.
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http://dx.doi.org/10.3389/fimmu.2019.00415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6425997PMC
July 2020

Ways Forward for Tolerance-Inducing Cellular Therapies- an AFACTT Perspective.

Front Immunol 2019 22;10:181. Epub 2019 Feb 22.

Division of Immunology, Germans Trias i Pujol University Hospital, LCMN, IGTP, Badalona, Spain.

Clinical studies with cellular therapies using tolerance-inducing cells, such as tolerogenic antigen-presenting cells (tolAPC) and regulatory T cells (Treg) for the prevention of transplant rejection and the treatment of autoimmune diseases have been expanding the last decade. In this perspective, we will summarize the current perspectives of the clinical application of both tolAPC and Treg, and will address future directions and the importance of immunomonitoring in clinical studies that will result in progress in the field.
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http://dx.doi.org/10.3389/fimmu.2019.00181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6395407PMC
January 2020

The role of pathogen-reduced platelet transfusions on HLA alloimmunization in hemato-oncological patients.

Transfusion 2019 02 30;59(2):470-481. Epub 2018 Nov 30.

Center for Clinical Transfusion Research, Sanquin Research, Leiden, The Netherlands.

Background: Platelet transfusions can induce alloimmunization against HLA antigens. The use of pathogen-reduced platelet concentrates (PCs) was suggested to reduce HLA alloimmunization and concomitant transfusion refractoriness.

Methods: This study investigated HLA alloimmunization in available samples from 448 hemato-oncological patients who were randomized for the Pathogen Reduction Evaluation and Predictive Analytical Rating Score (PREPAReS) trial to receive either untreated or pathogen-reduced PCs (Mirasol, Terumo BCT Inc.). Anti-HLA Class I and II antibodies were determined before the first platelet transfusion and weekly thereafter using multiplex assay with standard cutoffs to detect low- as well as high-level antibodies.

Results: When using the lower cutoff, in patients who were antibody negative at enrollment, 5.4% (n = 12) developed anti-HLA Class I antibodies after receiving untreated PCs, while this was significantly higher in patients receiving pathogen-reduced PCs, 12.8% (n = 29; p = 0.009, intention-to-treat [ITT] analysis). A similar but nonsignificant trend was observed in the per-protocol (PP) analysis (5.4% vs. 10.1%; p = 0.15). HLA class II antibody formation was similar between both types of PCs in the ITT analysis, while the PP analysis showed a trend toward lower immunization after receiving pathogen-reduced PCs. Multivariate analysis identified receiving pathogen-reduced platelets as an independent risk factor for HLA Class I alloimmunization (ITT: odds ratio [95% confidence interval] = 3.02 [1.42-6.51], PP: odds ratio [95% confidence interval] = 2.77 [1.00-5.40]), without affecting HLA Class II alloimmunization. When using the high cutoff value, the difference in HLA Class I alloimmunization between study arms remained significant in the ITT analysis and again was not significant in the PP analysis.

Conclusion: Our data clearly indicate that Mirasol pathogen inactivation does not prevent HLA Class I or II alloimmunization after platelet transfusions.
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http://dx.doi.org/10.1111/trf.15056DOI Listing
February 2019

-Induced Human IL-10 B Cells Do Not Show a Subset-Defining Marker Signature and Plastically Co-express IL-10 With Pro-Inflammatory Cytokines.

Front Immunol 2018 5;9:1913. Epub 2018 Sep 5.

Department of Immunopathology, Sanquin Research, Amsterdam, Netherlands.

Regulatory B cells (Breg) have been described as a specific immunological subsets in several mouse models. Identification of a human counterpart has remained troublesome, because unique plasma membrane markers or a defining transcription factor have not been identified. Consequently, human Bregs are still primarily defined by production of IL-10. In this study, we sought to elucidate if induced human IL-10 producing B cells are a dedicated immunological subset. Using deep immune profiling by multicolor flow cytometry and t-SNE analysis, we show that the majority of cells induced to produce IL-10 co-express pro-inflammatory cytokines IL-6 and/or TNFα. No combination of markers can be identified to define human IL-10TNFαIL-6 B cells and rather point to a general activated B cell phenotype. Strikingly, upon culture and restimulation, a large proportion of formerly IL-10 producing B cells lose IL-10 expression, showing that induced IL-10 production is not a stable trait. The combined features of an activated B cell phenotype, transient IL-10 expression and lack of subset-defining markers suggests that -induced IL-10 producing B cells are not a dedicated subset of regulatory B cells.
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http://dx.doi.org/10.3389/fimmu.2018.01913DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6143818PMC
September 2019

The quality of platelet concentrates related to corrected count increment: linking in vitro to in vivo.

Transfusion 2019 02 18;59(2):697-706. Epub 2018 Sep 18.

Center for Clinical Transfusion Research, Sanquin Research, Leiden University Medical Center, Leiden, the Netherlands.

Background: Storage of platelet concentrates (PCs) results in reduced recovery and survival of transfused platelets (PLTs). Upon storage PLTs develop storage lesion that can be monitored by several laboratory tests. However, correlation of these descriptive tests with corrected count increments (CCIs), a marker frequently used to establish the effectiveness of PLT transfusions, is limited or unknown. This study investigated to what extent a functional test or a combined in vitro rating score improves the correlation of laboratory tests with 1-hour CCI.

Study Design And Methods: PCs were analyzed using six different laboratory tests (n = 123) before transfusion in a prophylactic setting to 74 hematooncologic patients. Linear regression and Spearman correlation were used to determine associations between descriptive (either separately or combined in an in vitro rating score) or functional test results and 1-hour CCIs obtained after transfusion.

Results: CD62P expression (r = -0.45), annexin V binding (r = -0.36), the updated in vitro rating score (r = 0.50), and PLT responsiveness after thrombin receptor activator for peptide-6 (TRAP) (r = 0.43-0.57) or adenosine diphosphate stimulation (r = 0.11-0.51) significantly correlated to 1-hour CCIs obtained after transfusion, whereas lactate concentration, ThromboLUX score, and thromboelastography measurements did not. The strongest correlations were observed for in vitro rating score and PLT responsiveness after TRAP stimulation and these tests could explain 24 and 33% of the observed variation in 1-hour CCI, respectively.

Conclusion: Combining descriptive markers in one in vitro rating score improved correlation to 1-hour CCI compared to the tests separately. Of all tests investigated, mean PLT responsiveness after TRAP stimulation showed the strongest clinical correlation and was best able to predict the 1-hour CCI.
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http://dx.doi.org/10.1111/trf.14868DOI Listing
February 2019

Storage-Induced Platelet Apoptosis Is a Potential Risk Factor for Alloimmunization Upon Platelet Transfusion.

Front Immunol 2018 5;9:1251. Epub 2018 Jun 5.

Department of Immunopathology, Sanquin Research, Amsterdam, Netherlands.

Platelet transfusion can elicit alloimmune responses leading to alloantibody formation against donor-specific polymorphic residues, ultimately resulting in platelet transfusion refractoriness. Universal leukoreduction significantly reduced the frequency of alloimmunization after platelet transfusion, thereby showing the importance of white blood cells (WBCs) in inducing this alloresponse. It is, however, unknown if the residual risk for alloimmunization is caused by WBCs remaining after leukoreduction or if alloimmunization can be induced by platelets themselves. This study investigated the capacity of platelets to induce alloimmunization and identified potential product-related risk factors for alloimmunization. First, internalization of allogeneic platelets by dendritic cells (DCs) was demonstrated by confocal microscopy. Second, after internalization, presentation of platelet-derived peptides was shown by mass spectrometry analysis of human leukocytes antigen (HLA)-DR eluted peptides. Third, platelet-loaded DCs induced platelet-specific CD4 T cell responses. Altogether, this indicates a platelet-specific ability to induce alloimmunization. Therefore, factors enhancing platelet internalization may be identified as risk factor for alloimmunization by platelet concentrates. To investigate if storage of platelets is such a risk factor, internalization of stored platelets was compared with fresh platelets and showed enhanced internalization of stored platelets. Storage-induced apoptosis and accompanied phosphatidylserine exposure seemed to be instrumental for this. Indeed, DCs pre-incubated with apoptotic platelets induced the strongest IFN-γ production by CD4 T cells compared with pre-incubation with untreated or activated platelets. In conclusion, this study shows the capacity of platelets to induce platelet-specific alloimmune responses. Furthermore, storage-induced apoptosis of platelets is identified as potential risk factor for alloimmunization after platelet transfusions.
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http://dx.doi.org/10.3389/fimmu.2018.01251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6008548PMC
August 2019

Mass spectrometry-assisted identification of ADAMTS13-derived peptides presented on HLA-DR and HLA-DQ.

Haematologica 2018 06 22;103(6):1083-1092. Epub 2018 Mar 22.

Department of Plasma Proteins, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, the Netherlands.

Formation of microthrombi is a hallmark of acquired thrombotic thrombocytopenic purpura. These microthrombi originate from insufficient processing of ultra large von Willebrand factor multimers by ADAMTS13 due to the development of anti-ADAMTS13 autoantibodies. Several studies have identified the major histocompatibility complex class II alleles HLA-DRB1*11, HLA-DQB1*03 and HLA-DQB1*02:02 as risk factors for acquired thrombotic thrombocytopenic purpura development. Previous research in our department indicated that ADAMTS13 CUB2 domain-derived peptides FINVAPHAR and LIRDTHSLR are presented on HLA-DRB1*11 and HLA-DRB1*03, respectively. Here, we describe the repertoire of ADAMTS13 peptides presented on HLA-DQ. In parallel, the repertoire of ADAMTS13-derived peptides presented on HLA-DR was monitored. Using HLA-DR- and HLA-DQ-specific antibodies, we purified HLA/peptide complexes from ADAMTS13-pulsed monocyte-derived dendritic cells. Using this approach, we identified ADAMTS13-derived peptides presented on HLA-DR for all 9 samples analyzed; ADAMTS13-derived peptides presented on HLA-DQ were identified in 4 out of 9 samples. We were able to confirm the presentation of the CUB2 domain-derived peptides FINVAPHAR and LIRDTHSLR on HLA-DR. In total, 12 different core-peptide sequences were identified on HLA-DR and 8 on HLA-DQ. For HLA-DR11, several potential new core-peptides were found; 4 novel core-peptides were exclusively identified on HLA-DQ. Furthermore, an analysis was performed using the EpiMatrix and JanusMatrix tools to evaluate the eluted peptides, in the context of HLA-DR, for putative effector or regulatory T-cell responses at the population level. The results from this study provide a basis for the identification of immuno-dominant epitopes on ADAMTS13 involved in the onset of acquired thrombotic thrombocytopenic purpura.
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http://dx.doi.org/10.3324/haematol.2017.179119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6058777PMC
June 2018

Corrigendum: Monitoring T-Cell Responses in Translational Studies: Optimization of Dye-Based Proliferation Assay for Evaluation of Antigen-Specific Responses.

Front Immunol 2018 22;9:343. Epub 2018 Feb 22.

San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), Division of Regenerative Medicine, Stem Cells and Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy.

[This corrects the article on p. 1870 in vol. 8, PMID: 29312346.].
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http://dx.doi.org/10.3389/fimmu.2018.00343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5827416PMC
February 2018
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