Tetramer staining of antigen-specific T cells
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.
Background
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).
Tetramers
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 Ipeptide 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).
Applications
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 IFNgamma 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.)
CLB
Department of Clinical Viro-Immunology
Plesmanlaan 125
1066 CX Amsterdam
The Netherlands
Tel: +31 20 512 3261 / 3310 (Fax)
Email: e_manting@clb.nl
alpha-chain
beta-2- microglobulin
A
B
C
D
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.
A
2000
0%
0
10000
1000
viral load (copies/ml)
1000000
100000
CD4+ cells/ml
2%
tetramer staining
(% CD8+ cells)
B
100
0
time (months)
150
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).
HLA-EBV-peptide tetrameric complexes
PBMC
EBV-specific INF  producing T cells
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.