Review
Multimer monitoring of CMV-specific T cells in research and in clinical applications,☆☆,

https://doi.org/10.1016/j.diagmicrobio.2013.11.007Get rights and content

Abstract

Multimer monitoring has become a standard technique for detection of antigen-specific T cells. The term “multimer” refers to a group of reagents based on the multimerisation of molecules in order to raise avidity and thus stabilize binding to their ligand. Multimers for detection of antigen-specific T-cell responses are based on major histocompatibility complex class I peptide complexes. Multimer staining enables fast and direct visualization of antigen-specific T cells; thus, it is widely applied to assess antiviral immunity, e.g., monitor patients in vaccination trials or confirm purity of cell products for adoptive transfer. Assessment of T-cell immunity against persistent pathogens like cytomegalovirus (CMV) is of major importance in immunosuppressed patients. Recent advancements of multimers facilitate reversible labeling and allow isolation of epitope-specific T cells for adoptive transfer. Here, we give an overview on the different multimers and their applications, with an emphasis on CMV-specific T-cell responses.

Introduction

The specific cellular immune response can be detected by indirect or direct staining methods (Lacey et al., 2004) using flow cytometry (fluorescence-activated cell sorting [FACS]) for the analysis or enzyme-linked immunosorbent assay (ELISA)–based detection. Indirect detection depends on restimulation of specific cells and has been long established in form of ELISA, enzyme-linked immunospot assay (ELISpot), intra- or extra-cellular staining of cytokines, and effector molecules (e.g., interferon γ [IFN-γ], tumor necrosis factor α, interleukin 2, Granzyme B, Perforin) and activation markers (e.g., CD69, CD25, CD154). Direct detection of virus-specific cells was established about 20 years ago and relies on the combination of major histocompatibility complex (MHC) alleles and specific peptide epitopes. Multimer staining allows direct labeling and thus visualization of epitope-specific T cells by binding of the T-cell receptor (TCR) to multimeric complexes of MHC-molecules and specific epitopes. Since Altman et al. (1996) first described the use of tetramers, many different variants of the idea to multimerise MHC-epitope complexes in order to raise avidity and create stable bonding of the TCR to the reagent have been introduced.

In the context of hematopoietic stem cell transplantation (HSCT), reactivation of persistent viruses like adenovirus, cytomegalovirus (CMV), or Epstein-Barr virus (EBV) contributes significantly to morbidity and mortality. Reconstitution of the antiviral immune response and control of viral reactivation is mainly achieved by reconstitution of CD3+CD8+ antiviral T lymphocytes (CTLs). Apart from routine screening for viral reactivation and treatment with antiviral drugs (Biron, 2006), adoptive transfer of antiviral immunity either with the graft or after HSCT with donor leukocyte infusion (DLI) or selected virus-specific T cells has been investigated and successfully applied over the last years (Einsele et al., 2008, Gerdemann et al., 2012, Sili et al., 2012). For investigation of antiviral T-cell reconstitution and follow-up analysis of adoptively transferred T cells for persistence and expansion, in vivo multimer staining became a valuable tool over the last years.

Here, we give an overview on the different multimer techniques, compare multimer staining to functional assays, and summarize clinical studies employing the multimer technology in order to investigate antiviral immunity, with an emphasis on CMV.

Section snippets

Overview of different multimers

Specific T cells recognize their target antigen by binding of the TCR to an MHC-epitope complex on the surface of antigen-presenting cells (APCs). In vivo, this interaction is not limited to a single TCR-MHC interaction, but several of these molecules are present on the cell surface. In addition, costimulatory molecules enhance the interaction. While the affinity to a specific TCR can be mimicked in vitro by loading recombinant or purified MHC molecules with an epitope, the avidity of a single

General considerations for multimer staining

Depending on the expected frequency of the target cell population, an appropriate number of T cells have to be acquired to ensure reliable detection of multimer-positive cells. Addition of fluorescent beads (e.g., flow count, Beckman Coulter) enables 1-platform quantification of the number of specific T cells. Dead cells should be excluded to avoid unspecific background. Therefore, multimer staining requires the use of additional antibodies like αCD3 and αCD8 antibodies to enable gating on CD3+

MHC-II multimers to analyze CD4 responses

MHC class II multimers were developed shortly after the MHC class I–based multimers (Crawford et al., 1998, Novak et al., 1999). They were subsequently used in mouse and human studies to analyze autoreactivity (Falta et al., 2005, Massilamany et al., 2011), to detect and prime tumor-specific CD4 T cells (Poli et al., 2013), and to analyze pathogen specific CD4 T cells (Lacey et al., 2004, Meyer et al., 2000). Yang et al. (2006) presented a multiplexing approach combining multiple HLA class II

Method comparison

Several assays to detect antigen-specific T-cell responses have been developed in the past years and compared to each other concerning their applicability in research and clinical monitoring (Lacey et al., 2004, Hobeika et al., 2005). The benefits and limitations of flow cytometry-based multimer or intracellular cytokine staining (ICS) and ELISpot with an emphasis on possible clinical application are summarized in Table 2.

Multimer monitoring and multimer-based enrichment of CMV-CTL

Multimers were used to enumerate CMV-CTL in order to a) investigate kinetics of immune reconstitution, b) identify high-risk patients, c) identify suitable donors and isolate CMV-CTL for adoptive transfer, or d) follow adoptively transferred CTL in patient samples. An (incomplete) chronological overview with focus on detection of CMV-CTL by multimers as well as ICS and ELISpot and interpretation of CMV-CTL levels is provided in Table 3.

Conclusions and future perspectives

Multimer monitoring of antiviral T cells has become a valuable tool in assessing the immune reconstitution and response in immune compromised patients after SOT or HSCT. Due to the low amount of blood needed, fast and easy assay conduction, and high reproducibility and reliability of the results, multimer monitoring is suitable to serve as quality measure for purified cells for adoptive transfer, for monitoring of expansion of specific T cells in vivo, also after adoptive transfer of T cells or

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    Disclosures: The authors have nothing to disclose.

    ☆☆

    Funding: The authors and their studies referenced here were/are in part funded by: Celleurope; IFB-Tx, reference number: 01EO0802 German Ministry of Education and Research; DZIF, Partnerside Hannover-Braunschweig; Hilfe für Krebskranke Kinder Frankfurt e.V.; Alfred and Angelika Gutermuth-Stiftung; Dieter-Schlag-Stiftung; LOEWE Center for Cell and Gene Therapy Frankfurt (Hessisches Ministerium für Wissenschaft und Kunst, ref. number: III L4-518/17.004, 2010); and HiLF.

    Author contributions: SB wrote the manuscript, designed the figure and the tables, and provided data on multimer staining in clinical routine. JO wrote the manuscript, performed literature searches, and designed the tables. PV provided data on multimer monitoring in stem cell donors and stability tests and proofread the manuscript. ST provided data on histamers and critical discussion of the manuscript. MB provided data on multimer staining in clinical routine and critical discussion of the manuscript. BEV provided data on multimer monitoring in stem cell donors and on histamers and critical discussion of the manuscript. UK provided data on multimer staining in clinical routine and critical discussion of the manuscript. EMW provided data on multimer staining in clinical routine and critical discussion of the manuscript.

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