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Copenhagen, Denmark

Scholler J.,Immudex | Singh M.,Lionex GmbH and Helmholtx Center for Infection Research | Bergmeier L.,Kings College London | Brunstedt K.,Immudex | And 5 more authors.
Journal of Immunological Methods

The objective of this study was to produce and evaluate the immunogenic potential of a recombinant HLA-class I antigen linked to dextran. The HLA-A*0201 heavy chain and β2 microglobulin were cloned by PCR amplification of overlapping oligonucleotides and produced in E. coli. These were assembled with a CMV binding peptide motif, the HLA complex was biotinylated and bound by streptavidin coated dextran at a ratio of 24 HLA to 1 dextran molecule (termed Dextramer). Allostimulation of human PBMC in vitro and in vivo immunization of Balb c mice with the HLA-A*0201 construct elicited CD4+ and CD8+ T cell proliferative responses, IgG specific antibodies in mice and in human T cell proliferation and APOBEC3G mRNA. These adaptive and innate immune responses induced by a novel recombinant HLA construct in human cells and mice suggest their application as a potential vaccine candidate against HIV infection. © 2010. Source

Wang Y.,Kings College London | Whittall T.,Kings College London | Rahman D.,Kings College London | Bunnik E.M.,AMC Medical Research | And 7 more authors.

The AID/APOBEC family (activation induced deaminase/apolipoprotein B mRNA editing cytokine deaminase) in B cells play important roles in adaptive and innate immunity. Whereas APOBEC3G has been studied in CD4+ T cells and myeloid cells its functional potential in B cells has received little attention. AID combines two critical functions of antibodies, class switching and affinity maturation and may serve as a functional surrogate of protection. These functions were studied following systemic immunization of rhesus macaques with recombinant HLA constructs, linked with HIV and SIV antigens and HSP70 to dextran. The results showed significant upregulation of AID in CD20+ B cells, APOBEC 3G in CD27+ memory B cells and CD4+ effector memory T cells. After immunization the upregulated APOBEC 3G and AID were directly correlated in B cells (p<0.0001). Following challenge with SHIV SF162.P4 the viral load was inversely correlated with AID in B cells and APOBEC 3G in B and T cells, suggesting that both deaminases may have protective functions. Investigation of major interactions between DC, T cells and B cells showed significant increase in membrane associated IL-15 in DC and CD40L in CD4+ T cells. IL-15 binds the IL-15 receptor complex in CD4+ T and B cells, which may reactivate the DC, T and B cell interactions. The overall results are consistent with AID inhibiting pre-entry SHIV by eliciting IgG and IgA antibodies, whereas APOBEC 3G may contribute to the post-entry control of SHIV replication and cellular spread. © 2012 Wang et al. Source

Kasmar A.G.,Harvard University | Van Rhijn I.,Harvard University | Van Rhijn I.,University Utrecht | Magalhaes K.G.,Harvard University | And 16 more authors.
Journal of Immunology

Human CD1a mediates foreign Ag recognition by a T cell clone, but the nature of possible TCR interactions with CD1a/lipid are unknown. After incubating CD1a with a mycobacterial lipopeptide Ag, dideoxymycobactin (DDM), we identified and measured binding to a recombinant TCR (TRAV3/TRBV3-1, K D of ≈100 μM). Detection of ternary CD1a/lipid/TCR interactions enabled development of CD1a tetramers and CD1a multimers with carbohydrate backbones (dextramers), which specifically stained T cells using a mechanism that was dependent on the precise stereochemistry of the peptide backbone and was blocked with a soluble TCR. Furthermore, sorting of human T cells from unrelated tuberculosis patients for bright DDM-dextramer staining allowed recovery of T cells that were activated by CD1a and DDM. These studies demonstrate that the mechanism of T cell activation by lipopeptides occurs via ternary interactions of CD1a/Ag/TCR. Furthermore, these studies demonstrate the existence of lipopeptide-specific T cells in humans ex vivo. Copyright © 2013 by The American Association of Immunologists, Inc. Source

Tario J.D.,Roswell Park Cancer Institute | Chen G.L.,Roswell Park Cancer Institute | Hahn T.E.,Roswell Park Cancer Institute | Pan D.,Roswell Park Cancer Institute | And 7 more authors.
Cytometry Part B - Clinical Cytometry

The enumeration of antigen-specific T cells is increasingly relevant in clinical and research settings. This information is useful for evaluating immune responses to treatment, monitoring the efficacy of anticancer vaccines, and for detecting self-reactive T cells in autoimmune disorders. Quantifying antigen-specific T cells can be accomplished via IFNγ ELISpot assay, the measurement of intracellular cytokine production by flow cytometry, or by lymphocyte proliferation assays in response to antigen. While robust, these technologies are labor-intensive and can take several days to obtain results. New technology has led to more powerful tools for quickly and accurately measuring antigen-specific T cells by flow cytometry via fluorescently-labeled TCR-specific multimers. In this study, we evaluated the use of an assay based on Dextramer reagents for enumerating cytomegalovirus (CMV) antigen-specific T cells (CASTs). Assay performance characteristics were assessed by establishing Dextramers' sensitivity (median=0.4; range=0.1-1.4 CASTs μl-1), determining their specificity (100%), evaluating assay robustness with different leukocyte sources and assay reproducibility via interlaboratory and interinstrument investigations. Furthermore, the levels of CASTs in 95 peripheral blood samples from 62 unique blood and marrow transplants recipients correlated well between Dextramers and Tetramers (R2=0.9042). © 2014 International Clinical Cytometry Society. Source

Kim Y.-H.,Leiden University | Faaij C.M.J.M.,Leiden University | van Halteren A.G.S.,Leiden University | Schrama E.,Leiden University | And 9 more authors.
Biology of Blood and Marrow Transplantation

HY-specific T cells are presumed to play a role in acute graft-versus-host disease (aGVHD) after female-to-male stem cell transplantation (SCT). However, infiltrates of these T cells in aGVHD-affected tissues have not yet been reported. We evaluated the application of HLA-A2/HY dextramers for the in situ detection of HY-specific T cells in cryopreserved skin biopsy specimens. We applied the HLA-A2/HY dextramers on cryopreserved skin biopsy specimens from seven male HLA-A2+ pediatric patients who underwent stem cell transplantation with confirmed aGVHD involving the skin. The dextramers demonstrated the presence of HY-specific T cells. In skin biopsy specimens of three male recipients of female grafts, 68% to 78% of all skin-infiltrating CD8+ T cells were HY-specific, whereas these cells were absent in biopsy specimens collected from sex-matched patient-donor pairs. Although this study involved a small and heterogeneous patient group, our results strongly support the hypothesis that HY-specific T cells are actively involved in the pathophysiology of aGVHD after sex-mismatched stem cell transplantation. © 2012 American Society for Blood and Marrow Transplantation. Source

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