Establishment of the reversible peptide-major histocompatibility complex (pMHC) class I Histamer technology: tool for visualization and selection of functionally active antigen-specific CD8+ T lymphocytes

Sabine Tischer 1 2 Till Kaireit 1 2 Constana Figueiredo 1 2 Oliver Hiller 1 2 Britta Maecker-Kolhoff 0 1 Ren Geyeregger 3 Stephan Immenschuh 2 Rainer Blasczyk 1 2 Britta Eiz-Vesper 1 2 0 Department of Pediatric Hematology and Oncology, Hannover Medical School , Hannover, Germany 1 Integrated Research and Treatment Center (IFB-Tx), Hannover Medical School , Hannover, Germany 2 Institute for Transfusion Medicine, Hannover Medical School , Hannover, Germany 3 Children's Cancer Research Institute , St. Anna Kinderkrebsforschung, Department Pediatrics, Medical University Vienna , Vienna, Austria Multimers of soluble peptide-major histocompatibilty complex (pMHC) molecules are used in both basic and clinical immunology. They allow the specific visualization and isolation of antigenspecific T cells from ex vivo samples. Adoptive transfer of antigen-specific T cells sorted by pMHC multimers is an effective strategy for treatment of patients with malignancies or infectious diseases after transplantation. We developed a new reversible pMHC multimer called 'Histamer' to enable the specific detection and isolation of antiviral T cells from peripheral blood. HLA-A*02:01/CMVpp65495-503 Histamer (A02/CMV Histamer) was generated by coupling 6xHis-tagged pMHC molecules onto cobaltbased magnetic beads. The specificity of the Histamer was evaluated by flow cytometry. Sorting of antiviral CD8+ cytotoxic T lymphocytes (CTLs) was performed by magnetic cell separation, followed by the monomerization of the Histamer after addition of the competitor l -histidine. Sorted T cells were analyzed for phenotype and function. The reversible pMHC Histamer proved to be highly specific and sensitive. CMV-specific T cells of up to 99.6% purity were isolated using the Histamer technology. Rapid and complete disassembly of the T-cell surface-bound A02/CMV Histamer followed by the subsequent dissociation of the pMHC monomers from CD8+ CTL receptors was achieved using 100 mM l -histidine. The function of CMV-specific T cells enriched by Histamer staining did not differ from CTLs induced by standard T-cell assays. This reversible T-cell staining procedure preserves the functionality of antigenspecific T cells and can be adapted to good manufacturing practice conditions. The pMHC Histamer technology offers full flexibility and fulfills all requirements to generate clinical-grade T lymphocytes. Introduction Cellular immunodeficiency after allogeneic hematopoietic stem cell transplantation (HSCT) or solid organ transplantation (SOT) leaves the patient susceptible to a wide range of infections (1, 2). Viral and fungal infections are serious complications, which are difficult to control with drugs and are associated with high morbidity and mortality. Adoptive immunotherapy using antigen-specific T cells is an efficient tool to control viral infections after allogeneic HSCT or SOT, virusinduced malignancies and metastatic melanomas (37). The rationale behind T-cell transfer for infections is the hypothesis that a quantitative deficiency of antigen-specific T cells confers patient susceptibility to viral reactivation and primary infection of a variety of infectious agents, including EpsteinBarr virus (EBV), adenovirus (ADV) and cytomegalovirus (CMV) (8). Accumulating evidence indicates that effective antiviral drug therapy relies on specific immune reconstitution. Therefore, synergism between donor lymphocyte infusion (DLI) and antiviral drug therapy will have to be addressed by 562 Reversible pMHC class I Histamer technology further studies (911). However, the application for DLI in the treatment of infections is limited and associated with a significant risk of graft-versus-host disease (GvHD). Therefore, adoptive immunotherapy should be performed using antigenspecific T-cell infusions. In this context, peptide-major histocompatibility complex (pMHC) class I multimer technology is an aspiring powerful tool for obtaining antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) for adoptive T-cell transfer in cancer or infectious disease patients after HSCT or SOT (12, 13). In recent years, the direct visualization, quantification, phenotypical characterization and isolation of antigenspecific T cells by multimeric pMHC complex has attracted major attention (14, 15). The analysis of T-cell responses to autoantigens, infectious diseases and tumor cells using pMHC multimer staining reagents has been described previously (16). Altman et al. (1996) were the first to use the avidity effect of pMHC multimerization to stain T cells (17). Structurally, the heterotrimeric pMHC complex is composed of an MHC heavy chain (hc), the 2 microglobulin (B2M) light chain and an antigen-specific peptide, which is presented in a groove formed between the hc 1 and 2 domains (18). The pMHC staining technology exploits fluorescently tagged backbones to create pMHC multimers for visualization of antigen-specific T cells, whereas fluorophores are not necessary for separation of antigen-specific T cells. Several pMHC multimer techniques have been developed using dimers (19, 20), tetramers (17, 21), pentamers (13, 2224), dextramers (25), octamers (26), streptamers (8, 27) and clinimers (28, 29) for visualization, characterization and sorting of antigen-specific T cells. All of these pMHC multimers use the natural T-cell receptor (TCR) ligand (the peptideMHC complex) as the staining probe. Due to the low avidity of TCR/pMHC interactions, pMHC monomers have to be multimerized in order to raise the complex stability to a detectable level. The various means of pMHC multimerization, such as avidin/streptavidin-based tetramers, are reviewed elsewhere (12, 21). As summarized by Constantin et al. (30) and Einsele et al. (8, 31), other techniques for T-cell isolation have various limitations by altering phenotype and function. Therefore, application of pMHC multimers seems to be a safe and promising tool for isolation of antigen-specific T cells without affecting their naivety. pMHC multimers proved useful in initial clinical trials for isolation and expansion of specific T cells for adoptive therapy after HSCT, especially in CMV infection (11, 13, 32, 33). Even though these studies are promising, T cells are manipulated during the enrichment process, and TCR/pMHC interactions could alter the functional status of enriched T cells. Such manipulation could reduce T-cell viability during isolation and expansion and even in vivo if the pMHC multimers remain on the T-cell surface (27). Moreover, the administration of pMHC multimer-enriched antigen-specific T cells into patients requires more complex protocols for production according to good manufacturing practice (GMP) guidelines. This poses financial and regulatory barriers to such cell treatments (11). The reversible conventional pMHC streptamer technique was devised in order to solve these problems (11, 27, 28). This novel reagent allows isolation of T cells without altering their original phenotype a (...truncated)