Publications by authors named "Sandra Nuti"

34 Publications

2020 White Paper on Recent Issues in Bioanalysis: BAV Guidance, CLSI H62, Biotherapeutics Stability, Parallelism Testing, CyTOF and Regulatory Feedback ( - Recommendations on Biotherapeutics Stability, PK LBA Regulated Bioanalysis, Biomarkers Assays, Cytometry Validation & Innovation - Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine).

Bioanalysis 2021 Mar 29;13(5):295-361. Epub 2021 Jan 29.

Regeneron Pharmaceuticals, Tarrytown, NY, USA.

The 14 edition of the Workshop on Recent Issues in Bioanalysis (14 WRIB) was held virtually on June 15-29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14 WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by LCMS were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the Global Bioanalytical Community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication covers the recommendations on (Part 2A) BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation and (Part 2B) Regulatory Input. Part 1 (Innovation in Small Molecules, Hybrid LBA/LCMS & Regulated Bioanalysis), Part 3 (Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity) are published in volume 13 of Bioanalysis, issues 4, and 6 (2021), respectively.
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http://dx.doi.org/10.4155/bio-2021-0005DOI Listing
March 2021

Formulation Design, Optimization and In Vivo Evaluations of an α-Tocopherol-Containing Self-Emulsified Adjuvant System using Inactivated Influenza Vaccine.

J Control Release 2019 12 31;316:12-21. Epub 2019 Oct 31.

GSK, Slaoui Centre for Vaccines Research, Rockville, MD, 20850, USA.

α-Tocopherol has been used as an immune supplement in humans, as an emulsion adjuvant component in several veterinary vaccines as well as an immunomodulatory component of AS03, an emulsion adjuvant that was used in an H1N1 pandemic vaccine (Pandemrix). AS03 is manufactured using microfluidization and high-pressure homogenization. Such high energy and complex manufacturing processes make it difficult and expensive to produce emulsion adjuvants on a large scale, especially in developing countries. In this study we have explored simpler, comparatively inexpensive methods, to formulate emulsion adjuvants containing α-tocopherol, that have the potential to be made in any well-established scale-up facility. This might facilitate producing and stock-piling adjuvant doses and therefore aide in pandemic preparedness. We used design of experiment as a tool to explore incorporating α-tocopherol into self-emulsified systems containing squalene oil and polysorbate 80. We created novel self-emulsified adjuvant systems (SE-AS) and evaluated their potency in vivo in BALB/c mice with inactivated quadrivalent influenza vaccine (QIV) and tested the cellular and humoral immune responses against the four vaccine strains.
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http://dx.doi.org/10.1016/j.jconrel.2019.10.042DOI Listing
December 2019

Staphylococcus aureus-dependent septic arthritis in murine knee joints: local immune response and beneficial effects of vaccination.

Sci Rep 2016 11 30;6:38043. Epub 2016 Nov 30.

GSK Vaccines, Via Fiorentina 1, Siena, 53100, Italy.

Staphylococcus aureus is the major cause of human septic arthritis and osteomyelitis, which deserve special attention due to their rapid evolution and resistance to treatment. The progression of the disease depends on both bacterial presence in situ and uncontrolled disruptive immune response, which is responsible for chronic disease. Articular and bone infections are often the result of blood bacteremia, with the knees and hips being the most frequently infected joints showing the worst clinical outcome. We report the development of a hematogenous model of septic arthritis in murine knees, which progresses from an acute to a chronic phase, similarly to what occurs in humans. Characterization of the local and systemic inflammatory and immune responses following bacterial infection brought to light specific signatures of disease. Immunization of mice with the vaccine formulation we have recently described (4C-Staph), induced a strong antibody response and specific CD4+ effector memory T cells, and resulted in reduced bacterial load in the knee joints, a milder general inflammatory state and protection against bacterial-mediated cellular toxicity. Possible correlates of protection are finally proposed, which might contribute to the development of an effective vaccine for human use.
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http://dx.doi.org/10.1038/srep38043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5128924PMC
November 2016

One Dose of Staphylococcus aureus 4C-Staph Vaccine Formulated with a Novel TLR7-Dependent Adjuvant Rapidly Protects Mice through Antibodies, Effector CD4+ T Cells, and IL-17A.

PLoS One 2016 26;11(1):e0147767. Epub 2016 Jan 26.

Novartis Vaccines and Diagnostics, S.r.l., Research Center, Siena, Italy.

A rapidly acting, single dose vaccine against Staphylococcus aureus would be highly beneficial for patients scheduled for major surgeries or in intensive care units. Here we show that one immunization with a multicomponent S. aureus candidate vaccine, 4C-Staph, formulated with a novel TLR7-dependent adjuvant, T7-alum, readily protected mice from death and from bacterial dissemination, both in kidney abscess and peritonitis models, outperforming alum-formulated vaccine. This increased efficacy was paralleled by higher vaccine-specific and α-hemolysin-neutralizing antibody titers and Th1/Th17 cell responses. Antibodies played a crucial protective role, as shown by the lack of protection of 4C-Staph/T7-alum vaccine in B-cell-deficient mice and by serum transfer experiments. Depletion of effector CD4+ T cells not only reduced survival but also increased S. aureus load in kidneys of mice immunized with 4C-Staph/T7-alum. The role of IL-17A in the control of bacterial dissemination in 4C-Staph/T7-alum vaccinated mice was indicated by in vivo neutralization experiments. We conclude that single dose 4C-Staph/T7-alum vaccine promptly and efficiently protected mice against S. aureus through the combined actions of antibodies, CD4+ effector T cells, and IL-17A. These data suggest that inclusion of an adjuvant that induces not only fast antibody responses but also IL-17-producing cell-mediated effector responses could efficaciously protect patients scheduled for major surgeries or in intensive care units.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0147767PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4727907PMC
July 2016

MF59- and Al(OH)3-Adjuvanted Staphylococcus aureus (4C-Staph) Vaccines Induce Sustained Protective Humoral and Cellular Immune Responses, with a Critical Role for Effector CD4 T Cells at Low Antibody Titers.

Front Immunol 2015 7;6:439. Epub 2015 Sep 7.

Research Center, Novartis Vaccines and Diagnostics S.r.l. , Siena , Italy.

Staphylococcus aureus (S. aureus) is an important opportunistic pathogen that may cause invasive life-threatening infections, like sepsis and pneumonia. Due to the increasing antibiotic resistance, the development of an effective vaccine against S. aureus is needed. Although a correlate of protection against staphylococcal diseases is not yet established, several findings suggest that both antibodies and CD4 T cells might contribute to optimal immunity. In this study, we show that adjuvanting a multivalent vaccine (4C-Staph) with MF59, an oil-in-water emulsion licensed in human vaccines, further potentiated antigen-specific IgG titers and CD4 T-cell responses compared to alum and conferred protection in the peritonitis model of S. aureus infection. Moreover, we showed that MF59- and alum-adjuvanted 4C-Staph vaccines induced persistent antigen-specific humoral and T-cell responses, and protected mice from infection up to 4 months after immunization. Furthermore, 4C-Staph formulated with MF59 was used to investigate which immune compartment is involved in vaccine-induced protection. Using CD4 T cell-depleted mice or B cell-deficient mice, we demonstrated that both T and B-cell responses contributed to 4C-Staph vaccine-mediated protective immunity. However, the role of CD4 T cells seemed more evident in the presence of low-antibody responses. This study provides preclinical data further supporting the use of the adjuvanted 4C-Staph vaccines against S. aureus diseases, and provides critical insights on the correlates of protective immunity necessary to combat this pathogen.
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http://dx.doi.org/10.3389/fimmu.2015.00439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4561515PMC
October 2015

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

J Immunol 2015 Aug 13;195(4):1617-27. Epub 2015 Jul 13.

Research Center, Novartis Vaccines and Diagnostics, 53100 Siena, Italy; and

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.
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http://dx.doi.org/10.4049/jimmunol.1402604DOI Listing
August 2015

The Human Pathogen Streptococcus pyogenes Releases Lipoproteins as Lipoprotein-rich Membrane Vesicles.

Mol Cell Proteomics 2015 Aug 27;14(8):2138-49. Epub 2015 May 27.

From the ‡Novartis Vaccines and Diagnostics (a GSK company), Via Fiorentiina 1, 53100 Siena, Itlay;

Bacterial lipoproteins are attractive vaccine candidates because they represent a major class of cell surface-exposed proteins in many bacteria and are considered as potential pathogen-associated molecular patterns sensed by Toll-like receptors with built-in adjuvanticity. Although Gram-negative lipoproteins have been extensively characterized, little is known about Gram-positive lipoproteins. We isolated from Streptococcus pyogenes a large amount of lipoproteins organized in vesicles. These vesicles were obtained by weakening the bacterial cell wall with a sublethal concentration of penicillin. Lipid and proteomic analysis of the vesicles revealed that they were enriched in phosphatidylglycerol and almost exclusively composed of lipoproteins. In association with lipoproteins, a few hypothetical proteins, penicillin-binding proteins, and several members of the ExPortal, a membrane microdomain responsible for the maturation of secreted proteins, were identified. The typical lipidic moiety was apparently not necessary for lipoprotein insertion in the vesicle bilayer because they were also recovered from the isogenic diacylglyceryl transferase deletion mutant. The vesicles were not able to activate specific Toll-like receptor 2, indicating that lipoproteins organized in these vesicular structures do not act as pathogen-associated molecular patterns. In light of these findings, we propose to name these new structures Lipoprotein-rich Membrane Vesicles.
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http://dx.doi.org/10.1074/mcp.M114.045880DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4528243PMC
August 2015

Four-component Staphylococcus aureus vaccine 4C-staph enhances Fcγ receptor expression in neutrophils and monocytes and mitigates S. aureus infection in neutropenic mice.

Infect Immun 2015 Aug 26;83(8):3157-63. Epub 2015 May 26.

Novartis Vaccines and Diagnostics, s.r.l., a GSK Company, Siena, Italy

Staphylococcus aureus is a human bacterial pathogen causing a variety of diseases. The occurrence of multidrug-resistant strains of Staphylococcus aureus underlines the need for a vaccine. Defining immune correlates of protection may support the design of an effective vaccine. We used a murine Staphylococcus aureus infection model, in which bacteria were inoculated in an air pouch generated on the back of the animal. Analysis of the air-pouch content in mice immunized or not with an adjuvanted multiantigen vaccine formulation, four-component S. aureus vaccine (4C-Staph), prior to infection allowed us to measure bacteria, cytokines, and 4C-Staph-specific antibodies and to analyze host immune cells recruited to the infection site. Immunization with 4C-Staph resulted in accumulation of antigen-specific antibodies in the pouch and mitigated the infection. Neutrophils were the most abundant cells in the pouch, and they showed the upregulation of Fcγ receptor (FcγR) following immunization with 4C-Staph. Reduction of the infection was also obtained in mice immunized with 4C-Staph and depleted of neutrophils; these mice showed an increase in monocytes and macrophages. Upregulation of the FcγR and the presence of antigen-specific antibodies induced by immunization with 4C-Staph may contribute to increase bacterial opsonophagocytosis. Protection in neutropenic mice indicated that an effective vaccine could activate alternative protection mechanisms compensating for neutropenia, a condition often occurring in S. aureus-infected patients.
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http://dx.doi.org/10.1128/IAI.00258-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4496606PMC
August 2015

Evaluating the efficiency of isotope transmission for improved panel design and a comparison of the detection sensitivities of mass cytometer instruments.

Cytometry A 2015 Apr 20;87(4):357-68. Epub 2015 Feb 20.

CEA, Division of Immuno-Virology, IDMIT Center, Institute for Emerging Diseases and Innovative Therapies (iMETI), DSV, Fontenay-aux-Roses, France; Université Paris-Sud, UMR E1, Orsay, France; Vaccine Research Institute (VRI), Créteil, France.

The recent introduction of mass cytometry, a technique coupling a cell introduction system generating a stream of single cells with mass spectrometry, has greatly increased the number of parameters that can be measured per single cell. As with all new technology there is a need for dissemination of standardization and quality control procedures. Here, we characterize variations in sensitivity observed across the mass range of a mass cytometer, using different lanthanide tags. We observed a five-fold difference in lanthanide detection over the mass range and demonstrated that each instrument has its own sensitivity pattern. Therefore, the selection of lanthanide combinations is a key step in the establishment of a staining panel for mass cytometry-based experiments, particularly for multicenter studies. We propose the sensitivity pattern as the basis for panel design, instrument standardization and future implementation of normalization algorithms.
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http://dx.doi.org/10.1002/cyto.a.22648DOI Listing
April 2015

Optimized fluorescent labeling to identify memory B cells specific for Neisseria meningitidis serogroup B vaccine antigens ex vivo.

Immun Inflamm Dis 2013 Oct 30;1(1):3-13. Epub 2013 Oct 30.

Novartis Vaccines & Diagnostics Siena, Italy.

Antigen-specific memory B cells generate anamnestic responses and high affinity antibodies upon re-exposure to pathogens. Attempts to isolate rare antigen-specific memory B cells for in-depth functional analysis at the single-cell level have been hindered by the lack of tools with adequate sensitivity. We applied two independent methods of protein labeling to sensitive and specific ex vivo identification of antigen-specific memory B cells by flow cytometry: stringently controlled amine labeling, and sortagging, a novel method whereby a single nucleophilic fluorochrome molecule is added onto an LPETG motif carried by the target protein. We show that sortagged NadA, a major antigen in the meningococcal serogroup B vaccine, identifies NadA-specific memory B cells with high sensitivity and specificity, comparable to NadA amine-labeled under stringent reaction parameters in a mouse model of vaccination. We distinguish NadA-specific switched MBC induced by vaccination from the background signal contributed by splenic transitional and marginal zone B cells. In conclusion, we demonstrate that protein structural data coupled with sortag technology allows the development of engineered antigens that are as sensitive and specific as conventional chemically labeled antigens in detecting rare MBC, and minimize the possibility of disrupting conformational B cell epitopes.
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http://dx.doi.org/10.1002/iid3.3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4217542PMC
October 2013

Ex vivo analysis of human memory B lymphocytes specific for A and B influenza hemagglutinin by polychromatic flow-cytometry.

PLoS One 2013 15;8(8):e70620. Epub 2013 Aug 15.

Novartis Vaccines and Diagnostics srl, Siena, Italy.

Understanding the impact that human memory B-cells (MBC), primed by previous infections or vaccination, exert on neutralizing antibody responses against drifted influenza hemagglutinin (HA) is key to design best protective vaccines. A major obstacle to these studies is the lack of practical tools to analyze HA-specific MBCs in human PBMCs ex vivo. We report here an efficient method to identify MBCs carrying HA-specific BCR in frozen PBMC samples. By using fluorochrome-tagged recombinant HA baits, and vaccine antigens from mismatched influenza strains to block BCR-independent binding, we developed a protocol suitable for quantitative, functional and molecular analysis of single MBCs specific for HA from up to two different influenza strains in the same tube. This approach will permit to identify the naive and MBC precursors of plasmablasts and novel MBCs appearing in the blood following infection or vaccination, thus clarifying the actual contribution of pre-existing MBCs in antibody responses against novel influenza viruses. Finally, this protocol can allow applying high throughput deep sequencing to analyze changes in the repertoire of HA⁺ B-cells in longitudinal samples from large cohorts of vaccinees and infected subjects with the ultimate goal of understanding the in vivo B-cell dynamics driving the evolution of broadly cross-protective antibody responses.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0070620PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3744578PMC
April 2014

A new flow-cytometry-based opsonophagocytosis assay for the rapid measurement of functional antibody levels against Group B Streptococcus.

J Immunol Methods 2012 Apr 30;378(1-2):11-9. Epub 2012 Jan 30.

Novartis Vaccines and Diagnostics, Via Fiorentina 1, 53100 Siena, Italy.

Opsonophagocytosis is the primary mechanism for the clearance of Group B Streptococcus (GBS) by the host, and levels of opsonic antibodies may correlate with protection in preclinical models. A killing-based opsonophagocytosis assay (OPA), can be used to determine the functional activity of vaccine-induced GBS-specific antibodies. The assay, which measures the number of bacterial colonies surviving phagocytic killing in the presence of specific antibodies and complement, is rather expensive, time-consuming and poorly standardized. Here we describe a rapid, sensitive and reproducible fluorescent OPA assay (fOPA) based on flow cytometry analysis (FACS), which allows internalized bacteria to be distinguished from those associated to the plasma membrane of phagocytic cells. Fixed GBS were labeled with pHrodo™, a fluorescent dye which dramatically increases the emitted fluorescence at the acidic conditions present in the phagocytic endosomal compartment. Labeled bacteria were incubated with HL-60 cells differentiated to phagocytes, antibodies and complement, and then analyzed by FACS. A further improvement to our method, allowing to reduce assay variability, consisted on a step of selection of effector cells among the HL-60 population. Analysis of sera from mice immunized with different GBS vaccines revealed comparable sensitivity and specificity with the traditional killing OPA assay (kOPA), and a good correlation between the fluorescent signal of bacteria internalized by HL-60 phagocytes and killing. Remarkably, the pHrodo-based approach reduced the variability observed with other fOPA assays. The obtained data indicate the proposed fOPA as a reliable and useful tool for functional antibody assessment.
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http://dx.doi.org/10.1016/j.jim.2012.01.011DOI Listing
April 2012

Flow cytometry: an alternative method for direct quantification of antigens adsorbed to aluminum hydroxide adjuvant.

Anal Biochem 2011 Nov 22;418(2):224-30. Epub 2011 Jul 22.

Novartis Vaccines and Diagnostics, Cambridge, MA 02139, USA.

Flow cytometry (FC) has been widely used in biological research; however, its use for vaccine characterization has been very limited. Here we describe the development of an FC method for the direct quantification of two Neisseria meningitidis vaccine antigens, in mono- and multivalent formulations, while still adsorbed on aluminum hydroxide (AH) suspension. The antibody-based method is specific and sensitive. Because FC allows microscopic particle examination, the entire aluminum suspension carrying adsorbed antigen(s) can be analyzed directly. In addition to determining antigen concentration and identity, the assay is able to determine the distribution of the antigens on AH. High correlation coefficients (r(2)) were routinely achieved for a broad range of antigen doses from 0 to 150 μg/dose. Traditional assays for quantitative and qualitative antigen characterization on AH particles involve either complete aluminum dissolution or antigen desorption from the adjuvant. Because our direct method uses the whole AH suspension, the cumbersome steps used by traditional methods are not required. Those steps are often inefficient in desorbing the antigens and in some cases can lead to protein denaturation. We believe that this novel FC-based assay could circumvent some of the complex and tedious antigen-adjuvant desorption methods.
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http://dx.doi.org/10.1016/j.ab.2011.07.012DOI Listing
November 2011

Small molecule Toll-like receptor 7 agonists localize to the MHC class II loading compartment of human plasmacytoid dendritic cells.

Blood 2011 May 12;117(21):5683-91. Epub 2011 Apr 12.

Novartis Vaccines & Diagnostics, Siena, Italy.

TLR7 and TLR8 are intracellular sensors activated by single-stranded RNA species generated during viral infections. Various synthetic small molecules can also activate TLR7 or TLR8 or both through an unknown mechanism. Notably, direct interaction between small molecules and TLR7 or TLR8 has never been shown. To shed light on how small molecule agonists target TLRs, we labeled 2 imidazoquinolines, resiquimod and imiquimod, and one adenine-based compound, SM360320, with 2 different fluorophores [5(6) carboxytetramethylrhodamine and Alexa Fluor 488] and monitored their intracellular localization in human plasmacytoid dendritic cells (pDCs). All fluorescent compounds induced the production of IFN-α, TNF-α, and IL-6 and the up-regulation of CD80 and CD86 by pDCs showing they retained TLR7-stimulating activity. Confocal imaging of pDCs showed that, similar to CpG-B, all compounds concentrated in the MHC class II loading compartment (MIIC), identified as lysosome-associated membrane protein 1(+), CD63, and HLA-DR(+) endosomes. Treatment of pDCs with bafilomycin A, an antagonist of the vacuolar-type proton ATPase controlling endosomal acidification, prevented the accumulation of small molecule TLR7 agonists, but not of CpG-B, in the MIIC. These results indicate that a pH-driven concentration of small molecule TLR7 agonists in the MIIC is required for pDC activation.
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http://dx.doi.org/10.1182/blood-2010-12-328138DOI Listing
May 2011

Lack of allelic exclusion by secondary rearrangements of tumour B-cell receptor light chains in hairy cell leukaemia.

Hematol Oncol 2011 Mar;29(1):31-7

Sezione and Unità di Ematologia, Università di Siena & AOUS, Italy.

Analyses of the tumour immunoglobulin (Ig) gene (IG) heavy (H) and light chains show heterogeneity of mutational status, but reveal common features of ongoing IGH isotype-switching with multiple IGH isotype expression and preference of IG lambda (IGL) light chain with selective use of IGLJ3. Phenotypic and immunogenetic analyses were performed in a series of 105 HCL patients to estimate prevalence of multiple IG light chain expression by the tumour cells. By phenotype, 3/105 HCL (2.9%) expressed double tumour-related Ig kappa (K) and L light chain proteins. By immunogenetic analysis, functional mutated double IGK(I) /IGK(II) , IGK(I) /IGL(I) and IGL(I) /IGL(II) transcripts were cloned and sequenced in 3/71 (4.2%) HCL. These latter three HCL expressed multiple IGH isotypes with mutated IGHVDJ rearrangements at the time of AID transcript expression. Most interestingly, the three cases had reinduced RAG1 transcript. In the double IGL expresser, single-cell analysis documented co-expression of the tumour-related IGLs in 5/6 cells (83%). In the IGK/IGL co-expresser, evidence of surface IgK/IgL isotype proteins confirmed functionality of the tumour-derived transcripts. The evidence of double light chain expression in single HCs and the new observation of RAG re-induction suggest ongoing selective influences on the BCR that may promote or maintain the HCL clone in the periphery.
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http://dx.doi.org/10.1002/hon.952DOI Listing
March 2011

Intranasal administration of CpG induces a rapid and transient cytokine response followed by dendritic and natural killer cell activation and recruitment in the mouse lung.

J Innate Immun 2010 30;2(2):144-59. Epub 2009 Oct 30.

Department of Immunology, Novartis Vaccines Research Center, Siena, Italy.

CpG-containing oligodeoxynucleotides are potent mucosal adjuvants and effective as stand-alone treatment of respiratory infections in mice. Although CpG is also used as a type 1 helper immunomodulator in the treatment of asthma and allergic disease, immune modulation following intranasal application has not been fully characterized yet. Using a B-type CpG, we monitored RNA expression profiles, cytokine production and cellular activation in lung tissue and bronchoalveolar lavages ex vivo and cytokine production of purified cell populations in vitro. CpG triggered the upregulation of many transcripts, including interferon response genes and proinflammatory cytokine genes, between 3 h and 4 days. Overlapping subsets of these cytokine proteins were induced in vitro in purified CD11c+ cells, B cells and alveolar macrophages from the lung, thus identifying these cells as direct targets of CpG. While lung B cells strongly respond to CpG in vitro, less activation is found ex vivo, suggesting efficient CpG sequestering or rapid B cell migration after activation. In contrast, a type II alveolar epithelial cell line did not respond to CpG in vitro. We noted selective recruitment of plasmacytoid dendritic cells (DCs) into the lung tissue, and of conventional DCs and natural killer (NK) cells into the lung tissue and bronchoalveolar space. Furthermore, CpG induced activation of intrapulmonary DCs, NK and T cells. We hypothesize that CpG-linked adjuvanticity and clearance of respiratory pathogens are mediated by two major mechanisms: transient induction of the interferon pathway limiting microbial survival and selective recruitment of DCs and NK cells, which allows for better adaptive responses.
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http://dx.doi.org/10.1159/000254948DOI Listing
July 2010

Adjuvanted H5N1 vaccine induces early CD4+ T cell response that predicts long-term persistence of protective antibody levels.

Proc Natl Acad Sci U S A 2009 Mar 23;106(10):3877-82. Epub 2009 Feb 23.

Research Center, Novartis Vaccines and Diagnostics, Via Fiorentina 1, Siena 53100, Italy.

Immune responses to vaccination are tested in clinical trials. This process usually requires years especially when immune memory and persistence are analyzed. Markers able to quickly predict the immune response would be very useful, particularly when dealing with emerging diseases that require a rapid response, such as avian influenza. To address this question we vaccinated healthy adults at days 1, 22, and 202 with plain or MF59-adjuvanted H5N1 subunit vaccines and tested both cell-mediated and antibody responses up to day 382. Only the MF59-H5N1 vaccine induced high titers of neutralizing antibodies, a large pool of memory H5N1-specific B lymphocytes, and H5-CD4(+) T cells broadly reactive with drifted H5. The CD4(+) response was dominated by IL-2(+) IFN-gamma(-) IL-13(-) T cells. Remarkably, a 3-fold increase in the frequency of virus-specific total CD4(+) T cells, measurable after 1 dose, accurately predicted the rise of neutralizing antibodies after booster immunization and their maintenance 6 months later. We suggest that CD4(+) T cell priming might be used as an early predictor of the immunogenicity of prepandemic vaccines.
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http://dx.doi.org/10.1073/pnas.0813390106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2646626PMC
March 2009

Human plasmacytoid dendritic cells are unresponsive to bacterial stimulation and require a novel type of cooperation with myeloid dendritic cells for maturation.

Blood 2009 Apr 27;113(18):4232-9. Epub 2009 Jan 27.

Research Center, Novartis Vaccines and Diagnostics, Siena, Italy.

Dendritic cell (DC) populations play unique and essential roles in the detection of pathogens, but information on how different DC types work together is limited. In this study, 2 major DC populations of human blood, myeloid (mDCs) and plasmacytoid (pDCs), were cultured alone or together in the presence of pathogens or their products. We show that pDCs do not respond to whole bacteria when cultured alone, but mature in the presence of mDCs. Using purified stimuli, we dissect this cross-talk and demonstrate that mDCs and pDCs activate each other in response to specific induction of only one of the cell types. When stimuli for one or both populations are limited, they synergize to reach optimal activation. The cross-talk is limited to enhanced antigen presentation by the nonresponsive population with no detectable changes in the quantity and range of cytokines produced. We propose that each population can be a follower or leader in immune responses against pathogen infections, depending on their ability to respond to infectious agents. In addition, our results indicate that pDCs play a secondary role to induce immunity against human bacterial infections, which has implications for more efficient targeting of DC populations with improved vaccines and therapeutics.
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http://dx.doi.org/10.1182/blood-2008-10-186890DOI Listing
April 2009

Role of intracellular calcium and S-glutathionylation in cell death induced by a mixture of isothiazolinones in HL60 cells.

Biochim Biophys Acta 2009 Mar 11;1793(3):572-83. Epub 2008 Dec 11.

Department of Molecular Biology, University of Siena, via Fiorentina 1, 53100 Siena, Italy.

Previously we reported that brief exposure of HL60 cells to a mixture of 5-chloro-2-methyl-4-isothiazolin-3-one (CMI) and 2-methyl-4-isothiazolin-3-one (MI) shifts the cells into a state of oxidative stress that induces apoptosis and necrosis. In this study, flow cytometric analysis showed that CMI/MI induces early perturbation of calcium homeostasis, increasing cytosolic and mitochondrial calcium and depleting the intracellular endoplasmic reticulum (ER) stores. The calcium chelator BAPTA-AM reduced necrosis and secondary necrosis, the loss of DeltaPsim and S-glutathionylation induced by necrotic doses of CMI/MI, but did not protect against CMI/MI-induced apoptosis, mitochondrial calcium uptake and mitochondrial hyperpolarization. This indicates that increased cytoplasmic calcium does not have a causal role in the induction of apoptosis, while cross-talk between the ER and mitochondria could be responsible for the induction of apoptosis. GSH-OEt pretreatment, which enhances cellular GSH content, reduced S-glutathionylation and cytosolic and mitochondrial calcium levels, thus protecting against both apoptosis and necrosis shifting to apoptosis. Therefore, the degree of GSH depletion, paralleled by the levels of protein S-glutathionylation, may have a causal role in increasing calcium levels. The mitochondrial calcium increase could be responsible for apoptosis, while necrosis is associated with cytoplasmic calcium overload. These findings suggest that S-glutathionylation of specific proteins acts as a molecular linker between calcium and redox signalling.
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http://dx.doi.org/10.1016/j.bbamcr.2008.11.018DOI Listing
March 2009

Coligation of the hepatitis C virus receptor CD81 with CD28 primes naive T lymphocytes to acquire type 2 effector function.

J Immunol 2008 Jul;181(1):174-85

Novartis Vaccines, Siena, Italy.

Costimuli provide supplementary signals required by naive T cells to become fully activated upon Ag encounter. Tetraspanins are a large family of transmembrane proteins that can costimulate T cells when engaged in vitro. In this study, we describe for the first time that coligation of the tetraspanins CD81, CD82, or CD9 with the costimulatory molecule CD28 in vitro leads to proliferation of naive T cells. When activated through this pathway, both CD4+ and CD8+ naive T cells differentiate into type 2 effector cells, which produce IL-4, IL-5, IL-13, and IL-10, together with IL-2 and TNF-alpha, but little to no IFN-gamma. These effector cells descend from precursors that display early and strong production of IL-4, STAT6 phosphorylation, and up-regulation of the transcription factor GATA-3, suggesting a direct skewing toward Th2 differentiation without a Th0 intermediate. The hepatitis C virus envelope protein E2 is the only ligand known for CD81. Therefore, we propose that this new type of Ag-independent T cell activation may occur in hepatitis C virus-infected individuals, contributing to liver inflammation, impaired type 1 immune responses, and recurrent flares of type 2 immunity associated with chronic infection.
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http://dx.doi.org/10.4049/jimmunol.181.1.174DOI Listing
July 2008

Combination adjuvants for the induction of potent, long-lasting antibody and T-cell responses to influenza vaccine in mice.

Vaccine 2008 Jan 7;26(4):552-61. Epub 2007 Dec 7.

Novartis Vaccines, Via Fiorentina 1, Siena, Italy.

Influenza is controlled by protective titres of neutralizing antibodies, induced with the help of CD4 T-cells, and by antiviral T-cell effector function. Adjuvants are essential for the efficient vaccination of a naïve population against avian influenza. We evaluated a range of adjuvants for their ability to enhance, in naïve mice, protective hemagglutination inhibition (HI) titres, which represent the generally accepted correlate of protection, virus-neutralizing titres and T-cell responses to a new generation influenza vaccine produced in cell culture. The selected adjuvants include alum, calcium phosphate (CAP), MF59, the delivery system poly-(lactide co-glycolide) (PLG) and the immune potentiator CpG. MF59 was clearly the most potent single adjuvant and induced significantly enhanced, long-lasting HI and neutralizing titres and T-cell responses in comparison to all alternatives. The combination of alum, MF59, CAP or PLG with CpG generally induced slightly more potent titres. The addition of CpG to MF59 also induced a more potent Th1 cellular immune response, represented by higher IgG2a titres and the induction of a strongly enhanced IFN-gamma response in splenocytes from immunized mice. These observations have significant implications for the development of new and improved flu vaccines against pandemic and inter-pandemic influenza virus strains.
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http://dx.doi.org/10.1016/j.vaccine.2007.11.054DOI Listing
January 2008

The acquired immune response to the mucosal adjuvant LTK63 imprints the mouse lung with a protective signature.

J Immunol 2007 Oct;179(8):5346-57

Novartis Vaccines and Diagnostics, Siena, Italy.

LTK63, a nontoxic mutant of Escherichia coli heat labile enterotoxin (LT), is a potent and safe mucosal adjuvant that has also been shown to confer generic protection to several respiratory pathogens. To understand the mechanisms of action underlying the LTK63 protective effect, we analyzed the molecular and cellular events triggered by its administration in vivo. We show here that LTK63 intrapulmonary administration induced in the mouse lung a specific gene expression signature characterized by the up-regulation of cell cycle genes, several host defense genes, chemokines, chemokine receptors, and immune cell-associated genes. Such a transcriptional profile reflected the activation of alveolar macrophages and the recruitment to the lung of T and B cells and innate immune cells such as granulocytes, NK, and dendritic cells. All of these events were T cell dependent and specific for LTK63 because they were absent in SCID and nude mice. Additionally, we showed that LTK63 induces a potent adaptive immune response against itself directed to the lung. We propose that acquired response to LTK63 is the driving force for the local recruitment of both adaptive and innate immune cells. Our data suggest that LTK63 acts as an airway infection mimic that establishes a generic protective environment limiting respiratory infection by innate immune mechanisms and by improving adaptive responses to invading pathogens.
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http://dx.doi.org/10.4049/jimmunol.179.8.5346DOI Listing
October 2007

Invariant NKT cells sustain specific B cell responses and memory.

Proc Natl Acad Sci U S A 2007 Mar 27;104(10):3984-9. Epub 2007 Feb 27.

Novartis Vaccines, Via Fiorentina 1, 53100 Siena, Italy.

Invariant natural killer T (iNKT) cells are innate-like lymphocytes recognizing CD1d-restricted glycolipid antigens, such as alpha-galactosylceramide (alphaGC). We assessed whether iNKT cells help B lymphocyte responses and found that mice immunized with proteins and alphaGC develop antibody titers 1-2 logs higher than those induced by proteins alone. Activation of iNKT cells enhances protection against infections such as influenza and elicits higher frequencies of memory B cells and higher antibody responses to booster immunizations. Protein vaccination with alphaGC, but not with conventional adjuvants, elicits IgG responses in mice lacking MHC class II molecules, demonstrating that iNKT cells can substitute for CD4(+) T cell help to B cells. Interestingly, the decay of circulating antibodies is faster in mice lacking iNKT cells. These findings point to a homeostatic role for iNKT cells on critical features of the antibody response such as immunity and B cell memory.
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http://dx.doi.org/10.1073/pnas.0700191104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1805488PMC
March 2007

Functional specialization of human circulating CD16 and CD1c myeloid dendritic-cell subsets.

Blood 2007 Jun 1;109(12):5371-9. Epub 2007 Mar 1.

Molecular Immunology Department, Research Center, Novartis Vaccines, Siena, Italy.

Human blood contains 2 populations of dendritic cells (DCs): plasmacytoid and myeloid (mDC). mDCs are subdivided into 3 subsets using the surface markers CD16, CD1c, and BDCA-3. Their role as pathogen sentinels and adjuvant targets was tested by phenotypic and functional analysis. We show that mDC subsets are immature and express mRNA for most toll-like receptors (TLRs), except for TLR3 in CD16-mDCs. The most represented subsets, CD16- and CD1c-mDCs, are similarly responsive to all TLR agonists. Among 31 cytokines tested, both subsets produce CXCL8 (IL-8)/tumor necrosis factor-alpha (TNF-alpha)/IL-6/CCL3 (MIP-1 alpha)/CCL4 (MIP-1beta)/IL-1 beta. CXCL8 (IL-8) is the predominant cytokine produced by CD1c-mDCs on TLR engagement, whereas all other cytokines, particularly TNF-alpha, are secreted in 10-fold to 100-fold higher amounts by CD16-mDCs. CD16-mDCs cocultured with human umbilical vein endothelial cells induce a significantly higher production of CXCL10 (IP-10), granulocyte-macrophage colony-stimulating factor, and granulocyte colony-stimulating factor than CD1c-mDCs. In addition, interleukin-3 and type I interferons are stimuli specifically for DC maturation rather than cytokine secretion, whereas TNF-alpha is almost ineffective in inducing either function, suggesting a mechanism of T-cell-DC crosstalk and of rapid induction of antigen-presenting cell function during viral infection rather than inflammation. In conclusion, CD16-mDCs show strong proinflammatory activity, whereas CD1c-mDCs appear to be mainly inducers of chemotaxis.
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http://dx.doi.org/10.1182/blood-2006-08-038422DOI Listing
June 2007

Activation of naïve B lymphocytes via CD81, a pathogenetic mechanism for hepatitis C virus-associated B lymphocyte disorders.

Proc Natl Acad Sci U S A 2005 Dec 9;102(51):18544-9. Epub 2005 Dec 9.

Chiron Vaccines, 53100 Siena, Italy.

Infection with hepatitis C virus (HCV), a leading cause of chronic liver diseases, can associate with B lymphocyte proliferative disorders, such as mixed cryoglobulinemia and non-Hodgkin lymphoma. The major envelope protein of HCV (HCV-E2) binds, with high affinity CD81, a tetraspanin expressed on several cell types. Here, we show that engagement of CD81 on human B cells by a combination of HCV-E2 and an anti-CD81 mAb triggers the JNK pathway and leads to the preferential proliferation of the naïve (CD27-) B cell subset. In parallel, we have found that B lymphocytes from the great majority of chronic hepatitis C patients are activated and that naïve cells display a higher level of activation markers than memory (CD27+) B lymphocytes. Moreover, eradication of HCV infection by IFN therapy is associated with normalization of the activation-markers expression. We propose that CD81-mediated activation of B cells in vitro recapitulates the effects of HCV binding to B cell CD81 in vivo and that polyclonal proliferation of naïve B lymphocytes is a key initiating factor for the development of the HCV-associated B lymphocyte disorders.
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http://dx.doi.org/10.1073/pnas.0509402102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1310512PMC
December 2005

Chlamydia pneumoniae genome sequence analysis and identification of HLA-A2-restricted CD8+ T cell epitopes recognized by infection-primed T cells.

Vaccine 2005 Oct;23(42):5028-37

Chiron Vaccines, Via Fiorentina 1, 53100 Siena, Italy.

In the present study, we performed in silico analysis of Chlamydia pneumoniae genome sequence to identify human HLA-A2-restricted T cell epitopes. Thirty-one Chlamydia-specific protein antigens were selected and peptides were derived thereof using an HLA-A2 epitope predictive algorithm. Firstly, we tested binding of 55 selected 9mer peptides to HLA-A2 in vitro. Next, infection of HLA-A2 transgenic mice with C. pneumoniae elementary bodies and assessment of effector CD8+ T cells allowed us to identify which of the epitopes binding to HLA-A2 in vitro were recognized by C. pneumoniae infection-primed CD8+ T cells. Finally, we could confirm that CD8+ T cells in association with HLA-A2 recognized the most reactive peptides when the corresponding full-length genes were used to DNA-immunize HLA-A2 transgenic mice. By using this approach, a novel HLA-A2-restricted epitope in the outer membrane protein A (OmpA) of C. pneumoniae was identified, which proved to mediate specific lysis of peptide-loaded target cells.
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http://dx.doi.org/10.1016/j.vaccine.2005.04.048DOI Listing
October 2005

Comparable functions of plasmacytoid and monocyte-derived dendritic cells in chronic hepatitis C patients and healthy donors.

J Hepatol 2005 Jan;42(1):61-7

Immunology and Virology Department, Chiron Vaccines Research Center, via Fiorentina 1, 53100 Siena, Italy.

Background/aims: Dendritic cells (DCs) play a key role in immune responses through antigen presentation and cytokine secretion. Hepatitis C virus (HCV) is able to escape elimination by the immune system and often establishes a chronic infection. To investigate whether DC dysfunction is involved in this process, we have studied monoycte-derived DCs (Mo-DCs) and plasmacytoid DCs (pDCs), which produce large amounts of IFN-alpha, from chronic HCV patients and healthy donors.

Methods: We have assessed TNF-alpha and IFN-alpha production by pDCs using intracellular staining after total PBMCs stimulation with unmethylated CG dinucleotides (CpGs). The induction of allogeneic T cell proliferation by immature Mo-DCs was measured using the MLR assay. The up-regulation of maturation markers and the production of TNF-alpha in response to LPS were analyzed using flow cytometry and ELISA, respectively.

Results: We have detected comparable frequencies of pDCs producing TNF-alpha and IFN-alpha in both chronic HCV patients and healthy donors and we have found that immature Mo-DCs from both patients and donors similarly induce allogeneic T cell proliferation and mature and secrete TNF-alpha in response to LPS.

Conclusions: Our results demonstrate that both pDC and Mo-DCs are not impaired in HCV infected patients.
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http://dx.doi.org/10.1016/j.jhep.2004.09.014DOI Listing
January 2005

Production of profibrotic cytokines by invariant NKT cells characterizes cirrhosis progression in chronic viral hepatitis.

J Immunol 2004 Jul;173(2):1417-25

Experimental Immunology Unit, Cancer Immunotherapy and Gene Therapy Program, DIBIT, H. San Raffaele Scientific Institute, Milano, Italy.

Invariant (inv)NKT cells are a subset of autoreactive lymphocytes that recognize endogenous lipid ligands presented by CD1d, and are suspected to regulate the host response to cell stress and tissue damage via the prompt production of cytokines. We investigated invNKT cell response during the progression of chronic viral hepatitis caused by hepatitis B or C virus infection, a major human disease characterized by a diffused hepatic necroinflammation with scarring fibrotic reaction, which can progress toward cirrhosis and cancer. Ex vivo frequency and cytokine production were determined in circulating and intrahepatic invNKT cells from controls (healthy subjects or patients with nonviral benign or malignant focal liver damage and minimal inflammatory response) or chronic viral hepatitis patients without cirrhosis, with cirrhosis, or with cirrhosis and hepatocellular carcinoma. invNKT cells increase in chronically infected livers and undergo a substantial modification in their effector functions, consisting in the production of the type 2 profibrotic IL-4 and IL-13 cytokines, which characterizes the progression of hepatic fibrosis to cirrhosis. CD1d, nearly undetectable in noncirrhotic and control livers, is strongly expressed by APCs in cirrhotic ones. Furthermore, in vitro CD1d-dependent activation of invNKT cells from healthy donors elicits IL-4 and IL-13. Together, these findings show that invNKT cells respond to the progressive liver damage caused by chronic hepatitis virus infection, and suggest that these cells, possibly triggered by the recognition of CD1d associated with viral- or stress-induced lipid ligands, contribute to the pathogenesis of cirrhosis by expressing a set of cytokines involved in the progression of fibrosis.
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http://dx.doi.org/10.4049/jimmunol.173.2.1417DOI Listing
July 2004

Innate immune responses support adaptive immunity: NKT cells induce B cell activation.

Vaccine 2003 Jun;21 Suppl 2:S48-54

IRIS Research Center, Chiron Vaccines, Via Fiorentina 1, I-53100 Siena, Italy.

Invariant NKT cells are a peculiar subset of T lymphocytes whose features, highly conserved both in the mouse and the human system, strongly recall those of other "innate lymphocytes". Following recognition of CD1d-presented glycosphingolipid antigens invariant NKT promptly release high amount of diverse cytokines concurring to the activation of the actors of both innate and acquired immune responses. For this reason, in recent years NKT cells have been the object of intensive study, aimed to understand their role in diverse patho-physiological conditions and to exploit the possibility to take advantage of their "adjuvant-like" activity in the formulation of new vaccines. As antibodies are an essential part of many immune responses, we focused our attention on invariant NKT-B cell interactions analyzing their influences on B cell activation and effector functions. The results of this study demonstrate that human invariant NKT cells can provide direct help for B cell proliferation and antibody production through CD1d-restricted mechanisms. Remarkably, help to B lymphocytes by invariant NKT cells is delivered also in the absence of exogenous antigen, suggesting the existence of an endogenous ligand presented by CD1d on B cells.
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http://dx.doi.org/10.1016/s0264-410x(03)00200-7DOI Listing
June 2003

CD1d-restricted help to B cells by human invariant natural killer T lymphocytes.

J Exp Med 2003 Apr 14;197(8):1051-7. Epub 2003 Apr 14.

IRIS Research Center, Chiron Vaccines, 53100 Siena, Italy.

Invariant natural killer T (NKT) cells are a highly conserved subset of T lymphocytes expressing a semi-invariant T cell receptor (TCR), which is restricted to CD1d and specific for the glycosphingolipid antigen alpha-galactosylceramide. Their ability to secrete a variety of cytokines, which in turn modulate the activation of cells of both innate and acquired immune responses, suggests that invariant NKT cells exert a regulatory role mainly via indirect mechanisms. A relevant question is whether invariant NKT cells can directly help B cells. We document here that human invariant NKT cells are as efficient as conventional CD4+ Th0 lymphocytes in promoting proliferation of autologous memory and naive B lymphocytes in vitro, and in inducing immunoglobulin production. Help to B cells by invariant NKT cells is CD1d-dependent and delivered also in the absence of alpha-galactosylceramide, suggesting that NKT cells recognize an endogenous ligand presented by CD1d on B cells. The two major subsets of invariant NKT cells, CD4+ and double negative (CD4-CD8-), express comparable levels of CD40 ligand and cytokines, but differ in helper functions. Indeed, both subsets induce similar levels of B cell proliferation, whereas CD4+ NKT cells induce higher levels of immunoglobulin production. These results suggest a direct role for invariant NKT cells in regulating B lymphocyte proliferation and effector functions.
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http://dx.doi.org/10.1084/jem.20021616DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2193881PMC
April 2003