The Streptamer technology allows the reversible isolation and staining of antigen-specific T cells. This technology combines a current T cell isolation method with the Strep-tag technology. In principle, the T cells are separated by establishing a specific interaction between the T cell of interest and a molecule that is conjugated to a marker, which enables the isolation. The reversibility of this interaction and the fact that it is performed at low temperatures is the reason for the successful isolation and characterization of functional T cells. Because T cells remain phenotypically and functionally indistinguishable from untreated cells, this method offers new strategies in clinical and basic T cell research.
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Classic methods in T cell research
T cells play an important role in the adaptive immune system. They are capable of orchestrating, regulating and coordinating complex immune responses. A wide array of clinically relevant aspects are associated with the function or malfunction of T-cells: Autoimmune diseases, control of viral or bacterial pathogens, development of cancer or graft versus host responses. Over the past years, various methods (ELISPOT assay, intracellular cytokine staining, secretion assay) have been developed for the identification of T cells, but only major histocompatibility complex (MHC) procedures allow identification and purification of antigen-specific T cells independent of their functional status.
In principle, MHC procedures are using the T cell receptor (TCR) ligand, which is the MHC-peptide complex, as a staining probe. The MHC interacts with the TCR, which in turn is expressed on the T cells. Because TCR-MHC interactions have only a very weak affinity towards each other, monomeric MHC-epitope complexes cannot provide stable binding. This problem can be solved by using multimerized MHC-epitopes, which increases the binding avidity and therefore allows stable binding. Fluorochromes conjugated to the MHC-multimers then can be used for identification of T cells by flow cytometry. Nowadays, MHC molecules can be produced recombinantly together with the antigenic peptides which are known for a fast-growing number of diseases.
The Streptamer backbone
The Streptamer staining principle combines the classic method of T cell isolation by MHC-multimers with the Strep-tag/Strep-Tactin technology. The Strep-tag is a short peptide sequence that displays moderate binding affinity for the biotin-binding site of a mutated streptavidin molecule, called Strep-Tactin. For the Streptamer technology, the Strep-Tactin molecules are multimerized and form the "backbone", thus creating a platform for binding to strep-tagged proteins. Further, the Strep-Tactin backbone has a fluorescent label to allow flow cytometry analysis. Incubation of MHC-Strep-tag fusion proteins with the Strep-Tactin backbone results in the formation of a MHC-multimer, which is capable for antigen-specific staining of T cells.
Reversible staining
Because the molecule d-biotin has a much higher affinity to Strep-Tactin than Strep-tag, it can effectively compete for the binding site. Therefore, a MHC multimer based on the interaction of Strep-tag with Strep-Tactin is easily disrupted in the presence of relatively low concentrations of d-biotin. Without the Strep-Tactin backbone, the single MHC-Strep-tag fusion proteins spontaneously distatch from the TCR of the T cell, because of weak binding affinities (monomeric MHC-epitope complexes cannot provide stable binding, see above).
Advantages and applications
The advantages of the Streptamer technology become clear in comparison with other methods that aim to identify and purfify T cells. The main drawback using MHC multimers is that these multimers represent the natural ligand bound to the TCR. Therefore, placement of MHC multimer-stained cells into in vitro cell culture is expected to suffer from functional alterations of purified T cell populations like TCR internalization, activation, overstimulation and cell death. A strong negative influence on the function and phenotype in term of function and survical of MHC multimer-stained T cells was also found in vivo. This intrinsic problem of MHC multimer staining substantially limits the applications of the technology for basic T cell research as well as in clinical medicine. Only if the multimer staining is performed at low temperatures (4 °C), the T-cell phenotype is not altered because TCR-MHC-mediated signaling events do not become activated.
The Streptamer technique enables a reversible identification and purification of antigen specific T cell population at 4 °C, thus it does not affect the functional status of the cells. The addition of a Strep-tag competitor, d-biotin, results in the rapid disassembly of MHC multimers. Subsequently, the dissociation of monomeric MHC-peptide molecules from the T cell surface takes place. Therefore, this method comprises the advantages of conventional MHC multimer staining procedures, but it further allows removal of the staining reagents before negative effects of ligand-binding can begin to take place. The Streptamer technology is not only especially suited for clinical applications because of the reversibility of the TCR-MHC interaction, furthermore two other requirements are fulfilled. First, d-biotin (also known as vitamin H) is non-toxic for T-cells in low concentrations, which are used for Streptamer disassembly. Second, the amount of d-biotin that might be transferred with Streptamer isolated T-cells are far lower than d-biotin concentrations found in conventional vitamin supplementations, thus, unlikely to be harmful.
This method gains clinical relevance by now allowing direct ex vivo isolation (by fluorescence-activated cell sorting (FACS) or magnetically activated cell separation) and adoptive transfer of defined antigen specific T cell populations. This may now be realized as very effective therapeutic strategy. Another application is the improvement of T cell cloning strategies: expanded T cell lines or clones can be easily purified from contaminating cells or cell debris before transfer to clinical applications. Thus, Streptamer-sorted T cells comprise a highly defined population reducing the probability of unwanted side effects.