Protein kinase inhibitors substantially improve the physical detection of T-cells with peptide-MHC tetramers

Abstract

Flow cytometry with fluorochrome-conjugated peptide-major histocompatibility complex (pMHC) tetramers has transformed the study of antigen-specific T-cells by enabling their visualization, enumeration, phenotypic characterization and isolation from ex vivo samples. Here, we demonstrate that the reversible protein kinase inhibitor (PKI) dasatinib improves the staining intensity of human (CD8+ and CD4+) and murine T-cells without concomitant increases in background staining. Dasatinib enhances the capture of cognate pMHC tetramers from solution and produces higher intensity staining at lower pMHC concentrations. Furthermore, dasatinib reduces pMHC tetramer-induced cell death and substantially lowers the T-cell receptor (TCR)/pMHC interaction affinity threshold required for cell staining. Accordingly, dasatinib permits the identification of T-cells with very low affinity TCR/pMHC interactions, such as those that typically predominate in tumour-specific responses and autoimmune conditions that are not amenable to detection by current technology.

Expansion of highly differentiated CD8+ T-cells or NK-cells in patients treated with dasatinib is associated with cytomegalovirus reactivation

“…Furthermore, dasatinib effectively abrogated downregulation of both TCR and CD8 in the CD8 high subpopulation in vitro, which could be a contributing factor in the generation and maintenance of persistent LGL expansions (data not shown). 26 Importantly, the dominant TCR clonotypes and phenotypic features of the CD8 high and CD8 low NLVPMVATVspecific subsets were similar (Figure 3), thereby indicating that these cells were derived from the same precursors and that the observed CD8 downregulation likely reflected a state of recent activation. One minor deviation from this general observation occurred in patient 13, in whom the NLVPMVATV-specific CD8 low population appeared more differentiated (CD45RO dim CD57 þ ) and expressed greater levels of programmed death-1 (PD-1); this is consistent with antigen-driven effects in the context of combined T-cell and NK-cell LGL expansions.…”

“…In addition, high CD8 expression may reflect the inhibition of co-receptor downregulation by dasatinib. 7,26 CD8 low T-cells have been observed primarily in chronic infections with persistent antigenic stimula- …”

Modification of MHC Anchor Residues Generates Heteroclitic Peptides That Alter TCR Binding and T Cell Recognition

“…The HLA A p 0201-restricted Melan-A/MART-1 [26][27][28][29][30][31][32][33][34][35] ELAGIGILTV-specific MEL5 clone and the HLA A p 0201-restricted preproinsulin [15][16][17][18][19][20][21][22][23][24] ALWGPDPAAAspecific 1E6 clone were described previously (22)(23)(24). PBMCs from an HLA A p 0201 + donor were primed with either ELAGIGILTVor EAAGIGILTV as described previously (25).…”

“…For the recognition screen, 2 3 10 3 1E6 CD8 + T cells were cultured in triplicate for 16 h with position 2 (p2) variants of the wild type preproinsulin [15][16][17][18][19][20][21][22][23][24] peptide (ALWGPDPAAA) at a concentration of 1 mg/ml. Supernatant was harvested and analyzed for TNF-a production by ELISA according to the manufacturer's instructions (Life Technologies, Paisley, U.K. + T cells were washed and rested overnight in RPMI 1640 containing 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, and 2% heatinactivated FCS (R2 medium; Life Technologies).…”

“…Autoimmune disease provides such a context, and we therefore focused on the 1E6 CD8 + T cell clone, which is specific for the HLA A p 0201-restricted human preproinsulin [15][16][17][18][19][20][21][22][23][24] peptide ALWGPD-PAAA. This clone was recently derived from the peripheral blood of a patient with T1D; a clone with an identical TCR was subsequently isolated from the same patient 1 y later, thereby demonstrating the long-term persistence in vivo of T cells with this specificity (22).…”