HLA class I expression on human platelets is highly variable and correlates with distinct allele group frequencies.

BACKGROUND: Human leukocyte antigen (HLA) class I molecules are expressed on platelets and can represent a source of alloimmunization in recipients of platelet transfusions. HLA mismatch between donors and recipients may be associated with the induction of anti-HLA antibodies, which can culminate in refractoriness to platelet transfusions. In the present study we analyzed HLA allele group frequencies and HLA expression levels on human platelets from blood donors. MATERIALS AND METHODS: Platelet-rich plasma was collected from 139 donors to monitor platelet HLA class I expression by flow cytometry. DNA from donors with high and low platelet HLA expression was used in the genotype studies. Frequencies of large and normal-sized platelet subpopulations were determined and HLA class I expression was studied. Mean platelet volume (MPV) and platelet large-cell ratio (P-LCR) were analyzed in both groups of donors. RESULTS: The analysis showed variable platelet HLA class I expression with significant differences among donors. HLA class I allele group frequencies in donors with high and low platelet HLA expression showed distinctive genotypic features strictly related to expression level. The main allele groups found in samples with high platelet HLA class I expression were HLA-A*02, -A*68, -B*15, -B*49, and -C*03. Platelet HLA class I expression did not change over time or during freezing-thawing cycles. The analysis of platelet subpopulations showed a statistically significant higher expression of HLA class I molecules on large platelets than on normal-sized platelets. Moreover, donors with high HLA class I expression showed a higher frequency of large platelets (p<0.0001). The analysis of P-LCR in both groups of donors showed a statistically significant difference (p<0.05) within high HLA-expressing donors. DISCUSSION: Our data suggest an allele-dependent expression of HLA class I molecules on human platelets with distinct HLA allele group frequencies and different platelet subpopulation frequencies among blood donors.

A phase I, randomized, controlled, dose-ranging study of investigational acellular pertussis (aP) and reduced tetanus-diphtheria-acellular pertussis (TdaP) booster vaccines in adults.

Despite high vaccination coverage worldwide, pertussis has re-emerged in many countries. This randomized, controlled, observer-blind phase I study and extension study in Belgium (March 2012-June 2015) assessed safety and immunogenicity of investigational acellular pertussis vaccines containing genetically detoxified pertussis toxin (PT) (NCT01529645; NCT02382913). 420 healthy adults (average age: 26.8 ± 5.5 years, 60% female) were randomized to 1 of 10 vaccine groups: 3 investigational aP vaccines (containing pertussis antigens PT, filamentous hemagglutinin [FHA] and pertactin [PRN] at different dosages), 6 investigational TdaP (additionally containing tetanus toxoid [TT] and diphtheria toxoid [DT]), and 1 TdaP comparator containing chemically inactivated PT. Antibody responses were evaluated on days 1, 8, 30, 180, 365, and approximately 3 years post-booster vaccination. Cell-mediated immune responses and PT neutralization were evaluated in a subset of participants in pre-selected groups. Local and systemic adverse events (AEs), and unsolicited AEs were collected through day 7 and 30, respectively; serious AEs and AEs leading to study withdrawal were collected through day 365 post-vaccination. Antibody responses against pertussis antigens peaked at day 30 post-vaccination and then declined but remained above baseline level at approximately 3 years post-vaccination. Responses to FHA and PRN were correlated to antigen dose. Antibody responses specific to PT, toxin neutralization activity and persistence induced by investigational formulations were similar or significantly higher than the licensed vaccine, despite lower PT doses. Of 15 serious AEs, none were considered vaccination-related; 1 led to study withdrawal (premature labor, day 364; aP4 group). This study confirmed the potential benefits of genetically detoxified PT antigen. All investigational study formulations were well tolerated.

Early Rise of Blood T Follicular Helper Cell Subsets and Baseline Immunity as Predictors of Persisting Late Functional Antibody Responses to Vaccination in Humans.

CD4+ T follicular helper cells (T(FH)) have been identified as the T-cell subset specialized in providing help to B cells for optimal activation and production of high affinity antibody. We recently demonstrated that the expansion of peripheral blood influenza-specific CD4(+)IL-21(+)ICOS1(+) T helper (T(H)) cells, three weeks after vaccination, associated with and predicted the rise of protective neutralizing antibodies to avian H5N1. In this study, healthy adults were vaccinated with plain seasonal trivalent inactivated influenza vaccine (TIIV), MF59(®)-adjuvanted TIIV (ATIIV), or saline placebo. Frequencies of circulating CD4(+) T(FH)1 ICOS(+) T(FH) cells and H1N1-specific CD4(+-)IL-21(+)ICOS(+) CXCR5(+) T(FH) and CXCR5(-) T(H) cell subsets were determined at various time points after vaccination and were then correlated with hemagglutination inhibition (HI) titers. All three CD4(+) T cell subsets expanded in response to TIIV and ATIIV, and peaked 7 days after vaccination. To demonstrate that these T(FH) cell subsets correlated with functional antibody titers, we defined an alternative endpoint metric, decorrelated HI (DHI), which removed any correlation between day 28/day 168 and day 0 HI titers, to control for the effect of preexisting immunity to influenza vaccine strains. The numbers of total circulating CD4(+)T(FH)1 ICOS(+) cells and of H1N1-specific CD4(+)IL-21(+)ICOS(+) CXCR5(+), measured at day 7, were significantly associated with day 28, and day 28 and 168 DHI titers, respectively. Altogether, our results show that CD4(+) T(FH) subsets may represent valuable biomarkers of vaccine-induced long-term functional immunity.

Ultrastructural Visualization of Vaccine Adjuvant Uptake In Vitro and In Vivo.

Adjuvants are substances that enhance adaptive immune responses when formulated in a vaccine. Alum and MF59 are two vaccine adjuvants licensed for human vaccination. Their mode of action has not been completely elucidated. Here we show the first ultrastructural visualization of Alum and MF59 interaction with immune cells in vitro and in vivo. We observed that Alum is engulfed by cells as inclusions of laminae that are detectable within draining lymph nodes. MF59 is instead engulfed by cells in vitro as low-electron-dense lipid-like inclusions that display a vesicle pattern, as confirmed by confocal microscopy using fluorescently labeled MF59. However, lipid-like inclusions with different high- and low-electron-dense content are detected within cells of draining lymph nodes when injecting MF59. As high-electron-dense lipid-like inclusions are also detected upon injection of Alum, our results suggest that the low-electron-dense inclusions are formed by engulfed MF59, whereas the high-electron-dense inclusions are proper lipid inclusions. Thus, we demonstrated that vaccine adjuvants are engulfed as inclusions by lymph node cells and hypothesize that adjuvant treatment may modify lipid metabolism.

Oil-in-Water Emulsion MF59 Increases Germinal Center B Cell Differentiation and Persistence in Response to Vaccination.

Induction of persistent protective immune responses is a key attribute of a successful vaccine formulation. MF59 adjuvant, an oil-in-water emulsion used in human vaccines, is known to induce persistent high-affinity functional Ab titers and memory B cells, but how it really shapes the Ag-specific B cell compartment is poorly documented. In this study, we characterized the Ab- and Ag-specific B cell compartment in wild-type mice immunized with HlaH35L, a Staphylococcus aureus Ag known to induce measurable functional Ab responses, formulated with MF59 or aluminum salts, focusing on germinal centers (GC) in secondary lymphoid organs. Taking advantage of single-cell flow cytometry analyses, HlaH35L-specific B cells were characterized for the expression of CD38 and GL-7, markers of memory and GC, respectively, and for CD80 and CD73 activation markers. We demonstrated that immunization with MF59-, but not aluminum salt-adjuvanted HlaH35L, induced expanded Ag-specific CD73(+)CD80(-) GC B cells in proximal- and distal-draining lymph nodes, and promoted the persistence of GC B cells, detected up to 4 mo after immunization. In addition to increasing GC B cells, MF59-adjuvanted HlaH35L also increased the frequency of T follicular helper cells. This work extends previous knowledge regarding adaptive immune responses to MF59-adjuvanted vaccines, and, to our knowledge, for the first time an adjuvant used in human licensed products is shown to promote strong and persistent Ag-specific GC responses that might benefit the rational design of new vaccination strategies.

Innate Response Activator (IRA) B Cells Reside in Human Tonsils and Internalize Bacteria In Vitro.

Innate response activator (IRA) B cells have been described in mice as a subset of B-1a B cells that produce granulocyte/macrophage colony-stimulating factor (GM-CSF) and have been found in the spleen upon activation. In humans, identification, tissue localization and functionality of these lymphocytes are poorly understood. We hypothesized that IRA B cells could reside in human palatine tonsils, which are a first line of defense from infection of the upper respiratory tract. In the present work, we used flow cytometry and confocal microscopy to identify and characterize human IRA (hIRA) B cells in tonsils. We show that CD19⁺CD20⁺GM-CSF⁺ B cells are present in the tonsils of all the subjects studied at a frequency ranging between ~0.2% and ~0.4% of the conventional CD19⁺CD20⁺GM-CSF⁻ B cells. These cells reside within the B cell follicles, are mostly IgM⁺IgD⁺, express CD5 and show phagocytic activity. Our results support a role for hIRA B cells in the effector immune response to infections in tonsils.

Vaccine adjuvant MF59 promotes retention of unprocessed antigen in lymph node macrophage compartments and follicular dendritic cells.

Ag retention within lymph nodes (LNs) upon vaccination is critical for the development of adaptive immune responses, because it facilitates the encounter of the Ag with cognate lymphocytes. During a secondary exposure of the immune system to an Ag, immune complexes (ICs) that contain the unprocessed Ag are captured by subcapsular sinus macrophages and are transferred onto follicular dendritic cells, where they persist for weeks, facilitating Ag presentation to cognate memory B cells. The impact of adjuvants on Ag retention within the draining LNs is unknown. In this article, we provide the first evidence, to our knowledge, that the oil-in-water emulsion adjuvant MF59 localizes in subcapsular sinus and medullary macrophage compartments of mouse draining LNs, where it persists for at least 2 wk. In addition, we demonstrate that MF59 promotes accumulation of the unprocessed Ag within these LN compartments and facilitates the consequent deposition of the IC-trapped Ag onto activated follicular dendritic cells. These findings correlate with the ability of MF59 to boost germinal center generation and Ag-specific Ab titers. Our data suggest that the adjuvant effect of MF59 is, at least in part, due to an enhancement of IC-bound Ag retention within the LN and offer insights to improve the efficacy of new vaccine adjuvants.

Surface molecules on stimulated plasmacytoid dendritic cells are sufficient to cross-activate resting myeloid dendritic cells.

Human plasmacytoid dendritic cells (pDCs) and myeloid dendritic cells (mDCs) are 2 types of antigen-presenting cells that exert complementary roles in innate immune responses. We demonstrated previously that in the presence of suboptimal stimulation or when only 1 dendritic cell type is directly stimulated, contact-dependent crosstalk between mDCs and pDCs leads to the activation of both cell types and thus provides them with the ability to induce an optimal T-cell response. The precise mechanism is currently unknown. Here we demonstrate that pDCs, unable to secrete soluble factors because of previous stimulation, induce optimal mDC maturation, indicating that resting immature mDCs are fully competent to respond to Toll-like receptor-9-engaged pDCs in the absence of soluble factors. Thus, we conclude that immature mDCs already express receptors recognized by ligands that are upregulated on the surface of activated pDCs. Intercellular adhesion molecule-1 upregulated by activated pDCs may play a role in a donor-dependent manner.

B-cell differentiation in EBV-positive Burkitt lymphoma is impaired at posttranscriptional level by miRNA-altered expression.

Endemic, sporadic and HIV-associated Burkitt lymphoma (BL) all have a B-cell phenotype and a MYC translocation, but a variable association with the Epstein-Barr virus (EBV). However, there is still no satisfactory explanation of how EBV participates in the pathogenesis of BL. A recent investigation suggested that EBV-positive and EBV-negative BL have different cells of origin. In particular, according to immunoglobulin gene mutation analysis, EBV-negative BLs may originate from early centroblasts, whereas EBV-positive BLs seem to arise from postgerminal center B cells or memory B cells. The appearance of a germinal center phenotype in EBV-positive cells might thus derive from a block in B-cell differentiation. The exit from the germinal center involves a complex series of events, which require the activation of BLIMP-1, and the consequent downregulation of several target genes. Here, we investigated the expression of specific miRNAs predicted to be involved in B-cell differentiation and found that hsa-miR-127 is differentially expressed between EBV-positive and EBV-negative BLs. In particular, it was strongly upregulated only in EBV-positive BL samples, whereas EBV-negative cases showed levels of expression similar to normal controls, including microdissected germinal centers (GC) cells. In addition, we found evidence that hsa-miR-127 is involved in B-cell differentiation process through posttranscriptional regulation of BLIMP1 and XBP1. The overexpression of this miRNA may thus represent a key event in the lymphomagenesis of EBV positive BL, by blocking the B-cell differentiation process.