Preview

PDF, English - main document Download (37MB) | Terms of use

Abstract

Assessment of T cell responses towards disease-associated antigens is of key interest in order to develop and monitor immunotherapeutic interventions. Patient sample material, however, is usually highly limited, which hinders comprehensive screening of putative novel antigens by conventional methods such as the enzyme-linked immunospot (ELISPOT) or peptide major histocompatibility complex (pMHC) multimer staining. To overcome this issue, several alternative assays have recently been proposed that allow simultaneous measurement (i.e. multiplex) of a larger pool of T cell specificities within a single assay reaction. However, these technological approaches are currently not well suited for a broad and routine laboratory usage. Therefore, we have developed a novel sensitive method called T-Plex Assay for the multiplex detection of antigen-specific T cell responses using flow cytometry, which can be implemented easily into routine laboratory practice. The elaborated T-Plex Assay concept is based on cell-sized, fluorescently color-coded magnetic polystyrene beads such as the Luminex xMAP MagPlex® microspheres, which we have conjugated to defined recombinant pMHC-I or pMHC-II molecules. This pMHC conjugation in turn converts the beads into barcoded, artificial antigen-presenting cells (aAPCs) with the capacity to drive antigen-specific T cell activation. In addition, on the same pMHC-coated bead surface we co-immobilized monoclonal antibodies specific for various T cell effector cytokines such as interferon-γ that are released upon T cell activation. These assembled barcoded aAPCs with cytokine capture capacity serve ultimately as cell contact-dependent biosensors for cognate T cell populations. Executing the T-Plex Assay concept, we developed an easy-to-use and profoundly optimized T-Plex Assay protocol facilitating highly reproducible as well as simultaneous measurement of theoretically up to 80 antigen-specific T cell responses within a single reaction, while allowing for complete recovery of the viable T cell sample post analysis. As a proof of concept, we demonstrated the T-Plex Assay-based detection of various HLA-A*02:01-restricted viral antigen-specific T cell populations within healthy donor peripheral bloodderived T cells. Here, the T-Plex Assay displayed a similar sensitivity compared to conventional standard assays such as the pMHC multimer staining. Moreover, we used the T-Plex Assay in a small pilot study of four cancer patients to validate the immunogenicity of several in silico predicted HLA-A* 02:01-restricted tumor neoantigens. Unfortunately, analysis of cancer patient-derived peripheral blood by the T-Plex Assay or corresponding pMHC multimer staining did not reveal any T cell population specifically responding to the tested putative neoantigens. In an interlinked study aspect, we established a novel manufacturing strategy for efficient and easily parallelized small-scale production of correctly folded and ready-to-use soluble pMHC-I and pMHC-II molecules based on various mammalian cell transient gene expression systems. Here, we elaborated several expression constructs encoding for covalently assembled pMHC-I as well as pMHC-II Fc-fusion proteins. These pMHC-Fc proteins were successfully produced and notably bound in an antigenspecific manner to their cognate T cell populations. Although the true potential of the T-Plex Assay remains to be further analyzed, we think that the T-Plex Assay including our soluble pMHC production strategy holds great promise for clinical applications aiming for the routine discovery and validation of novel disease-associated T cell antigens particularly in cases where cell material is limited.