Soluble Recombinant CMVpp65 Spanning Multiple HLA Alleles for Reconstitution of Antiviral CD4+ and CD8+ T-Cell Responses After Allogeneic Stem Cell Transplantation

Institute for Transfusion Medicine, Hannover Medical School, D-30625 Hannover, Germany.
Journal of immunotherapy (Hagerstown, Md.: 1997) (Impact Factor: 4.01). 11/2009; 33(1):60-72. DOI: 10.1097/CJI.0b013e3181b56dcc
Source: PubMed


The reactivation of the human cytomegalovirus (CMV) can be prevented or controlled by the adoptive transfer of ex vivo expanded donor-derived CMV-specific T lymphocytes. Several methods for expansion and adoptive transfer of CMV-specific T cells have been developed using either defined CMV peptides or peptide pools for antigen-specific T-cell stimulation. The majority of studies have focused on the lower matrix protein (pp65) and the immediate-early protein-1 (IE-1) of CMV as immunodominant targets. We investigated the behavior of secretory CMVpp65 (sCMVpp65) with respect to its capacity to stimulate pp65-specific T cells independently of human lymphocyte antigen (HLA) type and even in donors unresponsive to the immunodominant HLA-A*0201-restricted CMVpp65495-503 peptide. To facilitate the eukaryotic expression and isolation procedures, we constructed an HLA-A*0201/CMVpp65 fusion protein that is secreted into the supernatant of human embryonic kidney 293 (HEK293) cells. CMV-specific CD4 and CD8 T cells generated by culturing unfractionated peripheral blood mononuclear cells in the presence of recombinant sCMVpp65 did not differ in function with regard to cytotoxicity and interferon-gamma (IFN-gamma) production compared with cytotoxic T cells induced using the well-studied HLA-A*0201-restricted CMVpp65495-503 peptide. We demonstrated that polyclonal CMV-specific T cells could be generated from CMV-seropositive individuals expressing HLA alleles for which no immunogenic epitopes have been identified so far. The production of recombinant sCMVpp65 can easily be adapted to good manufacturing practice conditions and can be used to generate large numbers of immunogenic pathogen-derived proteins for therapeutic applications.

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    • "Typically, they represent 0.5% to 4% of the CD8+ T-cell pool and 0.05% to 1.6% of the CD4+ T helper (Th) cell pool (Rentenaar et al., 2000; Cwynarski et al., 2001). Most protocols for the generation of virus-specific T cells use peptide-loaded monocyte-derived dendritic cells (DCs), artificial antigen-presenting cells (aAPCs), or CMV-infected immature dendritic cells as stimulator cells (Sun et al., 1999; Peggs et al., 2001; Carlsson et al., 2003; Oelke et al., 2003; Lozza et al., 2005; Paine et al., 2007, 2010; Lilleri et al., 2008). However, these protocols are difficult to standardize and often laborious to adapt to GMP conditions. "
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    ABSTRACT: Infection with and reactivation of human cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus (ADV) are frequent and severe complications in immunocompromised recipients after hematopoietic stem cell transplantation (HSCT) or solid organ transplantation (SOT). These serious adverse events are associated with significant morbidity and mortality. Donor lymphocyte infusions (DLIs) are often used to treat both viral infections and leukemia relapses after transplantation but are associated with potentially life-threatening graft-versus-host disease (GvHD). Adoptive immunotherapy with virus-specific cytotoxic effector T cells (CTLs) derived from seropositive donors can rapidly reconstitute antiviral immunity after HSCT and organ transplantation. Therefore, it can effectively prevent the clinical manifestation of these viruses with no significant acute toxicity or increased risk of GvHD. In conditions, where patients receiving an allogeneic cord blood transplant or a transplant from a virus-seronegative donor and since donor blood is generally not available for solid organ recipients, allogeneic third party T-cell donors would offer an alternative option. Recent studies showed that during granulocyte colony-stimulating factor (G-CSF) mobilization, the functional activity of antiviral memory T cells is impaired for a long period. This finding suggests that even stem cell donors may not be the best source of T cells. Under these circumstances, partially human leukocyte antigen (HLA)-matched virus-specific CTLs from healthy seropositive individuals may be a promising option. Therefore frequency assessments of virus-specific memory T cells in HLA-typed healthy donors as well as in HSCT/SOT donors using a high throughput T-cell assay were performed over a period of 4 years at Hannover Medical School. This chapter will address the relevance and potential of a third-party T-cell donor registry and will discuss its clinical implication for adoptive T-cell immunotherapy
    Frontiers in Immunology 01/2012; 3:410. DOI:10.3389/fimmu.2012.00410
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    • "Cytolytic activity of induced antiviral T cells was determined once weekly after the first (day 14) and second restimulation cycle (day 21) in a non-radioactive flow cytometric assay using autologous CFSE [5- or 6-(N-succinimidyloxicarbonyl)-3',6'-O,O'-diacetylfluorescein)]-labeled CMVpp65495-503 peptide-loaded autologous PBMCs [38] as target cells. In order to exclude alloreactivity the generated T cells were also tested against unloaded CFSE-labeled PBMCs. "
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    ABSTRACT: Heat shock protein 70 (HSP70) has gained major attention as an adjuvant capable of inducing antigen-specific CD8(+) and CD4(+) T-cell responses. The ability of HSP70/peptide complexes to elicit cytotoxic T-cell (CTL) responses by cross-presentation of exogenous antigens via HLA class I molecules is of central interest in immunotherapy. We examined the role of HSP70/CMVpp65(495-503)-peptide complex (HSP70/CMV-PC) in HLA class I-restricted cross-presentation for ex vivo expansion of CMV-specific CTLs. CMV-specific T cells generated from PBMCs of HLA-A*02:01/CMV-seropositive donors were stimulated for 21 days with HSP70/CMV-PC and analyzed in functional assays. As a control PBMCs were cultured in the presence of CMVpp65(495-503) peptide or HSP70. Increase of CMV-specific CTLs was visualized by pentameric HLA-A*02:01/CMVpp65(495-503) complex. About 90% of HSP70/CMV-PC generated T cells were CMV-specific and exhibited significantly higher IFN-γ secretion, cytotoxic activity, and an increased heme oxygenase 1 (HO-1) gene expression as compared to about 69% of those stimulated with CMVpp65(495-503) peptide. We decided to classify the HLA-A*02:01/CMV-seropositive donors as weak, medium, and strong responder according to the frequency of generated A2/CMV-pentamer-positive CD8(+) T cells. HSP70/CMV-PC significantly induces strong antiviral T-cell responses especially in those donors with low memory precursor frequencies. Blockage of CD91 with α2-macroglobulin markedly reduced proliferation of antiviral T cells suggesting a major role of this receptor in the uptake of HSP70/CMV-PC. This study clearly demonstrates that HSP70/CMV-PC is a potent mediator to induce stronger T-cell responses compared to antiviral peptides. This simple and efficient technique may help to generate significant quantities of antiviral CTLs by cross-presentation. Thus, we propose HSP70 for chaperoning peptides to reach an efficient level of cross-presentation. HSP70/peptide complexes may be particularly useful to generate stronger T-cell responses in cases of low precursor frequencies and may help to improve the efficiency of antigen-specific T-cell therapy for minor antigens.
    Journal of Translational Medicine 10/2011; 9(1):175. DOI:10.1186/1479-5876-9-175 · 3.93 Impact Factor
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    ABSTRACT: The glycoprotein CD86 is an important costimulatory molecule that has been shown to be predominantly expressed on APCs, such as dendritic cells, macrophages, and B cells. More recently, CD86 was also detected on T cells in specific pathological conditions. The mechanisms of how CD86 might be induced and its functional role in T cells are not well understood. In the present study, we showed that treatment with IL-2 markedly upregulated CD86, but not CD80, in human CD4(+) and CD8(+) T cells. This upregulation occurred in the absence of bystander cells, and isolated naive CD4(+) or CD8(+) T cells exhibited different time-dependent CD86-expression patterns in response to IL-2. Upregulation of CD86 on activated T cells was reduced by Abs that block IL-2 and IL-2Rα (CD25), indicating a receptor-mediated mechanism. IL-2-dependent CD86 upregulation was blocked by pharmacological inhibitors of the NFAT and mammalian target of rapamycin pathways and was largely reduced by simultaneous exposure to IFN-α. Importantly, a marked increase in CD86 on T cells was also observed in vivo in IL-2-treated patients. In conclusion, IL-2 upregulates CD86 expression on human CD4(+) and CD8(+) T cells via a receptor-dependent mechanism that involves the NFAT and mammalian target of rapamycin pathways.
    The Journal of Immunology 02/2012; 188(4):1620-9. DOI:10.4049/jimmunol.1100181 · 4.92 Impact Factor
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