Erik Manting, Stefan Kostense, Debbie van Baarle, and Frank Miedema
Department of Clinical Viro-Immunology, CLB Amsterdam, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 3261, Fax: +31 20 512 3310
Tetrameric complexes of HLA molecules can be used to stain antigen-specific T cells in FACS analysis. The enumeration and phenotypical analysis of antigen-specific cellular immune responses against viral, tumour or transplantation antigens has applications in various experimental and clinical settings. At the Department of Clinical Virology, tetramers are synthesised for the analysis of cellular immunity against viral infections in HIV infected individuals.
Cells present part of their proteinaceous content to the immune system via the proteolytic generation of peptides which are transported to the lumen of the endoplasmic reticulum, where they meet human leukocyte antigen (HLA) class I molecules. Peptides only bind to HLA molecules with a sufficient binding affinity and the HLA-peptide complexes are then transported to the cell surface. Here, they can be recognised by cytotoxic CD8+ T cells that are specific for both the type of HLA molecule and the peptide. This non-self recognition takes place when an epitope is derived from an abnormal cellular protein, derived from a virus or a tumour, or when the type of HLA molecule is not matched with the immune system, because it is present on a donor-organ. Non-self recognition leads to activation and proliferation of the responsive T cells and results in the lysis and removal of cells presenting the specific HLA-peptide complexes.
The cellular immune response consists of two components, namely the cytolytic CD8+ T cell response and a CD4+ T helper response. The helper response supports the proliferation and activity of CD8+ T cells by producing the nescessary growth factors and cytokines and therfore plays a pivotal role in the immune system. CD4+ T cells recognise antigen in the context of a different class of HLA molecules. These HLA class II molecules are only present on antigen-presenting cells and bind to peptides derived from extracellular proteins. Hereto, the molecules are loaded in the ER with an invariant polypeptide, which is proteolysed and replaced by peptides from endocytosed extracellular proteins before the HLA molecules reach the cell surface.
To optimise antigen presentation and T cell recognition, each individual possesses several types of HLA class I and II molecules, which are distinguished by subunit composition. HLA class I molecules consist of an invariant small beta-2-microglobulin subunit and a variable larger alpha subunit that contains the antigen binding site. The overall structure of HLA class I and II molecules is similar, but class II molecules are composed of two equally sized alpha and beta subunits, that both contribute to the antigen binding site and HLA type. In vitro synthesised soluble HLA-peptide complexes are used as tetrameric complexes to stain antigen specific T cells in FACS analysis. These reagents are termed tetramers (Altman et al., Science 274: 94-96, 1996).
Soluble HLA class I-peptide complexes can be synthesised in vitro by separately expressing alpha chain and beta-2-microglobulin in the bacterium Escherichia coli (Garboczi et al., Proc. Natl. Acad. Sci. USA 89: 3429-3433, 1992). To make it a soluble protein, the alpha chain is expressed without its carboxy-terminal transmembrane segment, which is replaced by a biotinylation domain (Schatz, Biotechnology 13: 1138-1143, 1993). After their purification as inclusion bodies (high density protein aggregates formed in E. coli), the HLA subunits are denatured in 8 M urea, mixed and refolded by dilution in buffer containing excess peptide (Figure 1A-C). The refolded HLA-peptide complexes are then enzymatically biotinylated by the E. coli enzyme BirA and purified by gel filtration chromatography. Because HLA-peptide complexes bind to T cell receptors via low-affinity interactions, the molecules are oligomerised to increase their binding avidity for T cells. A fluorescently labelled streptavidin molecule is used as a scaffold to bind four biotinylated HLA molecules. After mixing streptavidin and biotinylated HLA molecules in an approximately 1:4 ratio, the tetramers are purified in a second gel filtration chromatography step and are ready for use (Figure 1D).
HLA class II molecules poorly refold in vitro and so far no publications have described HLA class II tetramers that were synthesised similar to class I tetramers. Tetramers have been succesfully produced from HLA molecules obtained from eukaryotic expression systems. In this case, the antigenic peptide was covalently attached to one of the HLA subunits via a flexible peptide linker (Kozono et al., Nature 369: 151-154, 1994). As an alternative means to produce HLA-peptide oligomers, fusion proteins consisting of two HLA molecules linked to an IgG scaffold have been succesfully used for antigen-specific T cell staining with both HLA class I and II molecules (Dal Porto, Proc. Natl. Acad. Sci. USA 90: 6671-6675, 1993). Studies with different oligomers of streptavidin-bound HLA class I-peptide complexes have however demonstrated that optimal binding requires an assembly of at least three HLA-peptide complexes (Boniface et al., Immunity 9: 459-466, 1998).
HLA class I tetramers have been used in a wide array of experimental and clinical settings, to detect T cells directed against viral, tumor and transplantation antigens. At the Department of Clinical Viro-Immunology, the role of antigen-specific CD8+ T cells in the immune defense against human immunodeficiency virus (HIV) and other virusses is studied using frozen peripheral blood mononuclear cells (PBMC) samples from HIV+ subjects participating in the Amsterdam Cohort for AIDS Studies. Figure 2A demonstrates the presence of T cells specific for an HIV epitope in an HIV infected individual. Longitudinal analysis of tetramer staining reveals the HIV-specific T cells dynamics during the course of infection (Figure 2B). In the example shown, a loss of HIV-specific T cells occurs concomitantly with a drop in CD4+ T cells counts and an increased viral load, indicative of a loss of immune control. Circulating antigen-specific T cells can be enumerated by tetramer staining, but may not nescessarily reflect the functional cellular immune response. We therefore compared tetramer staining with interferone (IFN)-gamma Elispot, a method that is based on the antigen-induced IFN-gamma production by activated T cells (Figure 3). In some human immunodeficiency virus (HIV)-infected patients we have found a complete lack of T cells responsive to Epstein-Barr virus (EBV) antigens while EBV-specific cells were still detected by tetramer staining. This absence of functional CD8+ T cells may be related to an inadequate T helper response, which is diminished during the course of HIV infection. These experiments demonstrate a complementary role of tetramer staining and functional analysis of the T cell response. Whereas tetramer staining has a high sensitivity and detects all epitope-specific T cells, the functionality of T cells is an independent indicator of disease progression, especially in immunodeficiency-related diseases.
In order to use tetramers, several requirements have to be met. Epitopes that play a role in the immune response against the antigen of interest must have been defined and mapped to a certain HLA allele. Obviously, the tetramers containing this HLA-peptide epitope complex can only be used in HLA-matched samples. Secondly, the immunodominance of the epitope may differ between individuals and appears to depend on the HLA background. It is therefore important to define the recognition of an epitope in both positive and negative controls. Negative controls are individuals that have not been in contact with the antigen, or that are not HLA-matched. Positive controls are T cell lines specific for the antigen or, preferably, the exact epitope in the context of the matched HLA molecule. Samples from individuals that are positive in IFN-gamma Elispot should also contain T cells that can be stained by tetramers containing the same peptide. So far, tetramers have been mainly used to stain T cells obtained from PBMC or cell cultures. Tetramer staining of T cell infiltrates in tissue may be problematic, since the clustering of T cell receptors required for interaction with the oligomeric HLA complexes will not take place after freezing or fixation.
The CLB tetramer facility
To synthesize tetramers, we have collected a plasmid library containing several HLA class I alpha chain alleles. These plasmids, and the plasmid encoding the beta-2-microglobulin allow for the high-yield overexpression of the separate subunits as inclusion bodies in Escherichia coli. After chemical unfolding of the proteins and the subsequent refolding by dilution or dialysis of the HLA molecules from 8 M urea solution in the presence of the appropriate peptide, refolded monomers are biotinylated and purified (Figure 1). We use a Superdex 200 16/60 column (Pharmacia) for separation of the refolded monomers from the BirA enzym, unassociated HLA subunits, loose peptide and biotin by fast performance liquid chromatography (FPLC). After a slow and stepwise mixing of a four-fold excess of the biotinylated monomers with fluorescently labelled streptavidin, the tetramers are purified in a second FPLC gel filtration chromatography step. After concentration of the purified tetramers, they are ready for use. When possible, we test our tetramers on cell-lines isolted from patients with proven responses to the specific epitope before every experiment. Due to the dissociation and diffusion of the peptides from the HLA molecules, tetramers are intrinsically unstable. Depending on the affinity of peptide binding to the HLA molecule, the lifetime of tetramers may vary between several weeks to several months when stored at 4 0C in the dark. In our laboratory, tetramer staining is combined with various functional assays, including intracellular cytokine staining and IFN-gamma Elispot after peptide stimulation. We are currently developing technology to produce HLA class II tetramers.
For inquiries on the synthesis of HLA class I tetramers, please contact
Erik Manting (Ph.D.)
Department of Clinical Viro-Immunology
1066 CX Amsterdam
Tel: +31 20 512 3261 / 3310 (Fax)
Fig. 1. Synthesis of HLA class I tetramers. A) HLA subunits are expressed in E. coli as inclusion bodies. B) Purified inclusion bodies are dissolved in 8 M urea. C) Subunits are mixed and refolded in buffer containing excess peptide. D) Refolded HLA-peptide complexes are biotinylated and bound to fluorescently labelled streptavidin.
Fig. 2. Tetramer staining of HIV-specific CD8+ cells in an HIV-infected individual.
A) Phycoerythrin (PE)-labelled tetramers of HLA-A*0201 and an HLA-A*0201 restricted peptide (SLYNTVATL, Gag 77-85) were used to stain lymphocytes present in PBMC of the HLA-A2-matched donor. Note that tetramer staining occurs only with CD8+ cells, stained with a tricolor-labelled monoclonal antibody against CD8.
B) Longitudinal analysis of the tetramer staining () in relation to viral load () and CD4+ T cell counts () (adapted from Ogg et al., J. Virol. 73: 9153-9160).
Fig. 3. Complementary information is obtained by combining tetramer staining with functional assays like IFN-gamma Elispot. Especially in HIV+ patients, circulating antigen-specific T cells may be dysfunctional, and in HIV infected individuals suffering from EBV-related non-Hodgkin lymphoma we found declining functional EBV-specific CD8+ T cells, rather than lower numbers of T cells stained with tetramers containing EBV peptides.