Suppressed IgG4 class switching in dupilumab- and TNF inhibitor-treated patients after mRNA vaccination.

BACKGROUND: The noninflammatory immunoglobulin G4 (IgG4) is linked to tolerance and is unique to humans. Although poorly understood, prolonged antigenic stimulation and IL-4-signaling along the T helper 2-axis may be instrumental in IgG4 class switching. Recently, repeated SARS-CoV-2 mRNA vaccination has been linked to IgG4 skewing. Although widely used immunosuppressive drugs have been shown to only moderately affect humoral responses to SARS-CoV-2 mRNA vaccination, the effect on IgG4 switching has not been investigated. METHODS: Here we study the impact of such immunosuppressive drugs, including the IL-4 receptor-blocking antibody dupilumab, on IgG4 skewing upon repeated SARS-CoV-2 mRNA vaccination. Receptor-binding domain (RBD) specific antibody responses were longitudinally measured in 600 individuals, including patients with immune-mediated inflammatory diseases treated with a TNF inhibitor (TNFi) and/or methotrexate (MTX), dupilumab, and healthy/untreated controls, after repeated mRNA vaccination. RESULTS: We observed a substantial increase in the proportion of RBD-specific IgG4 antibodies (median 21%) in healthy/untreated controls after third vaccination. This IgG4 skewing was profoundly reduced in dupilumab-treated patients (<1%). Unexpectedly, an equally strong suppression of IgG4 skewing was observed in TNFi-treated patients (<1%), whereas MTX caused a modest reduction (7%). RBD-specific total IgG levels were hardly affected by these immunosuppressive drugs. Minimal skewing was observed, when primary vaccination was adenoviral vector-based. CONCLUSIONS: Our results imply a critical role for IL-4/IL-13 as well as TNF in vivo IgG4 class switching. These novel findings advance our understanding of IgG4 class switch dynamics, and may benefit humoral tolerance induction strategies, treatment of IgG4 pathologies and mRNA vaccine optimization.

T cell activation markers CD38 and HLA-DR indicative of non-seroconversion in anti-CD20-treated patients with multiple sclerosis following SARS-CoV-2 mRNA vaccination.

BACKGROUND: Messenger RNA (mRNA) vaccines provide robust protection against SARS-CoV-2 in healthy individuals. However, immunity after vaccination of patients with multiple sclerosis (MS) treated with ocrelizumab (OCR), a B cell-depleting anti-CD20 monoclonal antibody, is not yet fully understood. METHODS: In this study, deep immune profiling techniques were employed to investigate the immune response induced by SARS-CoV-2 mRNA vaccines in untreated patients with MS (n=21), OCR-treated patients with MS (n=57) and healthy individuals (n=30). RESULTS: Among OCR-treated patients with MS, 63% did not produce detectable levels of antibodies (non-seroconverted), and those who did have lower spike receptor-binding domain-specific IgG responses compared with healthy individuals and untreated patients with MS. Before vaccination, no discernible immunological differences were observed between non-seroconverted and seroconverted OCR-treated patients with MS. However, non-seroconverted patients received overall more OCR infusions, had shorter intervals since their last OCR infusion and displayed higher OCR serum concentrations at the time of their initial vaccination. Following two vaccinations, non-seroconverted patients displayed smaller B cell compartments but instead exhibited more robust activation of general CD4+ and CD8+ T cell compartments, as indicated by upregulation of CD38 and HLA-DR surface expression, when compared with seroconverted patients. CONCLUSION: These findings highlight the importance of optimising treatment regimens when scheduling SARS-CoV-2 vaccination for OCR-treated patients with MS to maximise their humoral and cellular immune responses. This study provides valuable insights for optimising vaccination strategies in OCR-treated patients with MS, including the identification of CD38 and HLA-DR as potential markers to explore vaccine efficacy in non-seroconverting OCR-treated patients with MS.

Isolation and expansion of pure and functional γδ T cells.

γδ T cells are important components of the immune system due to their ability to elicit a fast and strong response against infected and transformed cells. Because they can specifically and effectively kill target cells in an MHC independent fashion, there is great interest to utilize these cells in anti-tumor therapies where antigen presentation may be hampered. Since only a small fraction of T cells in the blood or tumor tissue are γδ T cells, they require extensive expansion to allow for fundamental, preclinical and ex vivo research. Although expansion protocols can be successful, most are based on depletion of other cell types rather than γδ T cell specific isolation, resulting in unpredictable purity of the isolated fraction. Moreover, the primary focus only lies with expansion of Vδ2+ T cells, while Vδ1+ T cells likewise have anti-tumor potential. Here, we investigated whether γδ T cells directly isolated from blood could be efficiently expanded while maintaining function. γδ T cell subsets were isolated using MACS separation, followed by FACS sorting, yielding >99% pure γδ T cells. Isolated Vδ1+ and Vδ2+ T cells could effectively expand immediately after isolation or upon freeze/thawing and reached expansion ratios between 200 to 2000-fold starting from varying numbers using cytokine supported feeder stimulations. MACS/FACS isolated and PHA stimulated γδ T cells expanded as good as immobilized antibody mediated stimulated cells in PBMCs, but delivered purer cells. After expansion, potential effector functions of γδ T cells were demonstrated by IFN-γ, TNF-α and granzyme B production upon PMA/ionomycin stimulation and effective killing capacity of multiple tumor cell lines was confirmed in killing assays. In conclusion, pure γδ T cells can productively be expanded while maintaining their anti-tumor effector functions against tumor cells. Moreover, γδ T cells could be expanded from low starting numbers suggesting that this protocol may even allow for expansion of cells extracted from tumor biopsies.

mRNA-1273 vaccinated inflammatory bowel disease patients receiving TNF inhibitors develop broad and robust SARS-CoV-2-specific CD8+ T cell responses.

SARS-CoV-2-specific CD8+ T cells recognize conserved viral peptides and in the absence of cross-reactive antibodies form an important line of protection against emerging viral variants as they ameliorate disease severity. SARS-CoV-2 mRNA vaccines induce robust spike-specific antibody and T cell responses in healthy individuals, but their effectiveness in patients with chronic immune-mediated inflammatory disorders (IMIDs) is less well defined. These patients are often treated with systemic immunosuppressants, which may negatively affect vaccine-induced immunity. Indeed, TNF inhibitor (TNFi)-treated inflammatory bowel disease (IBD) patients display reduced ability to maintain SARS-CoV-2 antibody responses post-vaccination, yet the effects on CD8+ T cells remain unclear. Here, we analyzed the impact of IBD and TNFi treatment on mRNA-1273 vaccine-induced CD8+ T cell responses compared to healthy controls in SARS-CoV-2 experienced and inexperienced patients. CD8+ T cells were analyzed for their ability to recognize 32 SARS-CoV-2-specific epitopes, restricted by 10 common HLA class I allotypes using heterotetramer combinatorial coding. This strategy allowed in-depth ex vivo profiling of the vaccine-induced CD8+ T cell responses using phenotypic and activation markers. mRNA vaccination of TNFi-treated and untreated IBD patients induced robust spike-specific CD8+ T cell responses with a predominant central memory and activated phenotype, comparable to those in healthy controls. Prominent non-spike-specific CD8+ T cell responses were observed in SARS-CoV-2 experienced donors prior to vaccination. Non-spike-specific CD8+ T cells persisted and spike-specific CD8+ T cells notably expanded after vaccination in these patient cohorts. Our data demonstrate that regardless of TNFi treatment or prior SARS-CoV-2 infection, IBD patients benefit from vaccination by inducing a robust spike-specific CD8+ T cell response.

Distinct dynamics of antigen-specific induction and differentiation of different CD11c+Tbet+ B-cell subsets.

BACKGROUND: CD11c+Tbet+ B cells are enriched in autoimmunity and chronic infections and also expand on immune challenge in healthy individuals. CD11c+Tbet+ B cells remain an enigmatic B-cell population because of their intrinsic heterogeneity. OBJECTIVES: We investigated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen-specific development and differentiation properties of 3 separate CD11c+ B-cell subsets-age-associated B cells (ABCs), double-negative 2 (DN2) B cells, and activated naive B cells-and compared them to their canonical CD11c- counterparts. METHODS: Dynamics of the response of the 3 CD11c+ B-cell subsets were assessed at SARS-CoV-2 vaccination in healthy donors by spectral flow cytometry. Distinct CD11c+ B-cell subsets were functionally characterized by optimized in vitro cultures. RESULTS: In contrast to a durable expansion of antigen-specific CD11c- memory B cells over time, both ABCs and DN2 cells were strongly expanded shortly after second vaccination and subsequently contracted. Functional characterization of antibody-secreting cell differentiation dynamics revealed that CD11c+Tbet+ B cells were primed for antibody-secreting cell differentiation compared to relevant canonical CD11c- counterparts. CONCLUSION: Overall, CD11c+Tbet+ B cells encompass heterogeneous subpopulations, of which primarily ABCs as well as DN2 B cells respond early to immune challenge and display a pre-antibody-secreting cell phenotype.

Phagocytosis of platelets opsonized with differently glycosylated anti-HLA hIgG1 by monocyte-derived macrophages.

Immune-mediated platelet refractoriness (PR) remains a significant problem in the setting of platelet transfusion and is predominantly caused by the presence of alloantibodies directed against class I human leukocyte antigens (HLA). Opsonization of donor platelets with these alloantibodies can result in rapid clearance after transfusion via multiple mechanisms, including antibody dependent cellular phagocytosis (ADCP). Interestingly, not all alloimmunized patients develop PR to unmatched platelet transfusions, suggesting variation in HLA-specific IgG responses between patients. Previously, we observed that the glycosylation profile of anti-HLA antibodies was highly variable between PR patients, especially with respect to Fc galactosylation, sialylation and fucosylation. In the current study, we investigated the effect of different Fc glycosylation patterns, with known effects on complement deposition and FcγR binding, on phagocytosis of opsonized platelets by monocyte-derived human macrophages. We found that the phagocytosis of antibody- and complement-opsonized platelets, by monocyte derived M1 macrophages, was unaffected by these qualitative IgG-glycan differences.

Fcα Receptor-1-Activated Monocytes Promote B Lymphocyte Migration and IgA Isotype Switching.

Patients with inflammatory bowel disease (IBD) produce enhanced immunoglobulin A (IgA) against the microbiota compared to healthy individuals, which has been correlated with disease severity. Since IgA complexes can potently activate myeloid cells via the IgA receptor FcαRI (CD89), excessive IgA production may contribute to IBD pathology. However, the cellular mechanisms that contribute to dysregulated IgA production in IBD are poorly understood. Here, we demonstrate that intestinal FcαRI-expressing myeloid cells (i.e., monocytes and neutrophils) are in close contact with B lymphocytes in the lamina propria of IBD patients. Furthermore, stimulation of FcαRI-on monocytes triggered production of cytokines and chemokines that regulate B-cell differentiation and migration, including interleukin-6 (IL6), interleukin-10 (IL10), tumour necrosis factor-α (TNFα), a proliferation-inducing ligand (APRIL), and chemokine ligand-20 (CCL20). In vitro, these cytokines promoted IgA isotype switching in human B cells. Moreover, when naïve B lymphocytes were cultured in vitro in the presence of FcαRI-stimulated monocytes, enhanced IgA isotype switching was observed compared to B cells that were cultured with non-stimulated monocytes. Taken together, FcαRI-activated monocytes produced a cocktail of cytokines, as well as chemokines, that stimulated IgA switching in B cells, and close contact between B cells and myeloid cells was observed in the colons of IBD patients. As such, we hypothesize that, in IBD, IgA complexes activate myeloid cells, which in turn can result in excessive IgA production, likely contributing to disease pathology. Interrupting this loop may, therefore, represent a novel therapeutic strategy.

Termination of CD40L co-stimulation promotes human B cell differentiation into antibody-secreting cells.

Human naïve B cells are notoriously difficult to differentiate into antibody-secreting cells (ASCs) in vitro while maintaining sufficient cell numbers to evaluate the differentiation process. B cells require T follicular helper (TFH ) cell-derived signals like CD40L and IL-21 during germinal center (GC) responses to undergo differentiation into ASCs. Cognate interactions between B and TFH cells are transient; after TFH contact, B cells cycle between GC light and dark zones where TFH contact is present and absent, respectively. Here, we elucidated that the efficacy of naïve B cells in ACS differentiation is dramatically enhanced by the release of CD40L stimulation. Multiparameter phospho-flow and transcription factor (TF)-flow cytometry revealed that termination of CD40L stimulation downmodulates NF-κB and STAT3 signaling. Furthermore, the termination of CD40 signaling downmodulates C-MYC, while promoting ASC TFs BLIMP1 and XBP-1s. Reduced levels of C-MYC in the differentiating B cells are later associated with crucial downmodulation of the B cell signature TF PAX5 specifically upon the termination of CD40 signaling, resulting in the differentiation of BLIMP1 high expressing cells into ASCs. The data presented here are the first steps to provide further insights how the transient nature of CD40 signaling is in fact needed for efficient human naïve B cell differentiation to ASCs.

CD45RB Glycosylation and Ig Isotype Define Maturation of Functionally Distinct B Cell Subsets in Human Peripheral Blood.

Glycosylation of CD45RB (RB+) has recently been identified to mark antigen-experienced B cells, independent of their CD27 expression. By using a novel combination of markers including CD45RB glycosylation, CD27 and IgM/IgD isotype expression we segregated human peripheral blood B cell subsets and investigated their IGHV repertoire and in vitro functionality. We observed distinct maturation stages for CD27-RB+ cells, defined by differential expression of non-switched Ig isotypes. CD27-RB+ cells, which only express IgM, were more matured in terms of Ig gene mutation levels and function as compared to CD27-RB+ cells that express both IgM and IgD or cells that were CD27-RB-. Moreover, CD27-RB+IgM+ cells already showed remarkable rigidity in IgM isotype commitment, different from CD27-RB+IgMD+ and CD27-RB- cells that still demonstrated great plasticity in B cell fate decision. Thus, glycosylation of CD45RB is indicative for antigen-primed B cells, which are, dependent on the Ig isotype, functionally distinct.

Soluble FAS Ligand Enhances Suboptimal CD40L/IL-21-Mediated Human Memory B Cell Differentiation into Antibody-Secreting Cells.

Differentiation of Ag-specific B cells into class-switched, high-affinity, Ab-secreting cells provides protection against invading pathogens but is undesired when Abs target self-tissues in autoimmunity, beneficial non-self-blood transfusion products, or therapeutic proteins. Essential T cell factors have been uncovered that regulate T cell-dependent B cell differentiation. We performed a screen using a secreted protein library to identify novel factors that promote this process and may be used to combat undesired Ab formation. We tested the differentiating capacity of 756 secreted proteins on human naive or memory B cell differentiation in a setting with suboptimal T cell help in vitro (suboptimal CD40L and IL-21). High-throughput flow cytometry screening and validation revealed that type I IFNs and soluble FAS ligand (sFASL) induce plasmablast differentiation in memory B cells. Furthermore, sFASL induces robust secretion of IgG1 and IgG4 Abs, indicative of functional plasma cell differentiation. Our data suggest a mechanistic connection between elevated sFASL levels and the induction of autoreactive Abs, providing a potential therapeutic target in autoimmunity. Indeed, the modulators identified in this secretome screen are associated with systemic lupus erythematosus and may also be relevant in other autoimmune diseases and allergy.

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

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 PRDM1. 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 PAX5, BACH2 and IRF8 and further induction of PRDM1. 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.

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

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 ex vivo stimulated myeloid CD1c+ conventional DC2. Moreover, platelets and platelet-derived soluble mediators inhibited T cell priming and T helper differentiation toward an IFNγ+ 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.

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

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.

The Role of Autoimmunity-Related Gene CLEC16A in the B Cell Receptor-Mediated HLA Class II Pathway.

C-type lectin CLEC16A is located next to CIITA, the master transcription factor of HLA class II (HLA-II), at a susceptibility locus for several autoimmune diseases, including multiple sclerosis (MS). We previously found that CLEC16A promotes the biogenesis of HLA-II peptide-loading compartments (MIICs) in myeloid cells. Given the emerging role of B cells as APCs in these diseases, in this study, we addressed whether and how CLEC16A is involved in the BCR-dependent HLA-II pathway. CLEC16A was coexpressed with surface class II-associated invariant chain peptides (CLIP) in human EBV-positive and not EBV-negative B cell lines. Stable knockdown of CLEC16A in EBV-positive Raji B cells resulted in an upregulation of surface HLA-DR and CD74 (invariant chain), whereas CLIP was slightly but significantly reduced. In addition, IgM-mediated Salmonella uptake was decreased, and MIICs were less clustered in CLEC16A-silenced Raji cells, implying that CLEC16A controls both HLA-DR/CD74 and BCR/Ag processing in MIICs. In primary B cells, CLEC16A was only induced under CLIP-stimulating conditions in vitro and was predominantly expressed in CLIPhigh naive populations. Finally, CLIP-loaded HLA-DR molecules were abnormally enriched, and coregulation with CLEC16A was abolished in blood B cells of patients who rapidly develop MS. These findings demonstrate that CLEC16A participates in the BCR-dependent HLA-II pathway in human B cells and that this regulation is impaired during MS disease onset. The abundance of CLIP already on naive B cells of MS patients may point to a chronically induced stage and a new mechanism underlying B cell-mediated autoimmune diseases such as MS.

Multiscale Modeling of Germinal Center Recapitulates the Temporal Transition From Memory B Cells to Plasma Cells Differentiation as Regulated by Antigen Affinity-Based Tfh Cell Help.

Germinal centers play a key role in the adaptive immune system since they are able to produce memory B cells and plasma cells that produce high affinity antibodies for an effective immune protection. The mechanisms underlying cell-fate decisions are not well understood but asymmetric division of antigen, B-cell receptor affinity, interactions between B-cells and T follicular helper cells (triggering CD40 signaling), and regulatory interactions of transcription factors have all been proposed to play a role. In addition, a temporal switch from memory B-cell to plasma cell differentiation during the germinal center reaction has been shown. To investigate if antigen affinity-based Tfh cell help recapitulates the temporal switch we implemented a multiscale model that integrates cellular interactions with a core gene regulatory network comprising BCL6, IRF4, and BLIMP1. Using this model we show that affinity-based CD40 signaling in combination with asymmetric division of B-cells result in switch from memory B-cell to plasma cell generation during the course of the germinal center reaction. We also show that cell fate division is unlikely to be (solely) based on asymmetric division of Ag but that BLIMP1 is a more important factor. Altogether, our model enables to test the influence of molecular modulations of the CD40 signaling pathway on the production of germinal center output cells.

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

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.

Correction to: Autologous tumor cell vaccination combined with systemic CpG-B and IFN-α promotes immune activation and induces clinical responses in patients with metastatic renal cell carcinoma: a phase II trial.

In the original publication of the article the following abstract and keywords were inadvertently omitted.

Autologous tumor cell vaccination combined with systemic CpG-B and IFN-α promotes immune activation and induces clinical responses in patients with metastatic renal cell carcinoma: a phase II trial.

Background: In this study the toxicity and efficacy of an irradiated autologous tumor cell vaccine (ATV) co-injected with a class-B CpG oligodeoxynucleotide (CpG-B) and GM-CSF, followed by systemic CpG-B and IFN-α administration, were examined in patients with metastatic renal cell carcinoma (mRCC). Methods: A single-arm Phase II trial was conducted, in which patients with mRCC were intradermally injected with a minimum of three whole-cell vaccines containing 0.7­1.3 × 107 irradiated autologous tumor cells (ATC), admixed with 1 mg CpG-B and 100 µg GM-CSF, followed by bi-weekly s.c. injections with 8 mg CpG-B and s.c. injections with 6 MU IFN-α three times per week. Results: Fifteen patients were treated according to the protocol. Treatment was well tolerated. Objective clinical responses occurred in three patients, including one long-term complete response. Disease stabilization occurred in another three patients. Positive delayed type hypersensitivity (DTH) responses to ATC were absent before treatment but present in 13 out of 15 patients during treatment. Immune monitoring revealed activation of plasmacytoid dendritic cells, non-classical monocytes and up-regulation of both PD-1 and CTLA4 on effector T cells upon treatment. Moreover, a pre-existing ex vivo IFN-γ response to ATC was associated with clinical response. Conclusions: ATV combined with systemic CpG-B and IFN-α is tolerable, safe, immunogenic and able to elicit anti-tumor responses in patients with mRCC. Immune activation and treatment-induced up-regulation of PD-1 and CTLA4 on circulating T cells further suggest an added benefit of combining this approach with immune checkpoint blockade [added]

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

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.

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

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 in vitro-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-10+TNFα-IL-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 in vitro-induced IL-10 producing B cells are not a dedicated subset of regulatory B cells.