Promiscuity of the AlloHLA-A2 Restricted T Cell Repertoire Hampers the Generation of Minor Histocompatibility Antigen-specific Cytotoxic T Cells across HLA Barriers

Stimulation of miHA-specific T cells with HLA-A2/miHA complex-coated DCs. A, HLA-A2 expression by HLA-A2^pos DCs (left) and HLA-A2/miHA complex-coated HLA-A2^neg DCs (right) before (filled histogram) and after (open histogram) complex coating. B, Cytolytic activity by an HA-1-specific T cell clone (filled diamonds) and an HA-2 specific T cell clone (filled squares) in response to DCs coated with various amounts of HLA-A2/HA-1 or HLA-A2/HA-2 complexes (micrograms per 10⁶ DCs) or to HLA-A2^pos DCs pulsed with HA-1 or HA-2 peptides (open symbols). C, A polyclonal HA-1-specific T cell line generated from an HLA-A2^pos HA-1^neg donor was restimulated 2 times with autologous HLA-A2/HA-1 complex-coated DCs. HA-1^A2 tetramer binding was determined on days 0, 7, and 14. The cytolytic activity directed against HLA-A2^neg EBV-LCLs (open bars) or HLA-A2^pos EBV-LCLs naturally expressing HA-1 (filled bars) was tested in parallel on days 0 and 14.

Generation of polyclonal alloHLA-A2/HA-1 specific T cells from HLA-A2^neg donors. CD8⁺ T cells from HLA-A2^neg donors M#1 (A, C) and M#2 (B, D) were stimulated with autologous HLA-A2/HA-1 complex-coated DCs (A, B) or with alloHLA-A2^pos HA-1 peptide-pulsed DCs (C, D). HA-1^A2 tetramer binding and cytolytic activity (effector:target ratio, 80:1) directed against TAP-deficient T2 cells (open bars) or HA-1 peptide-pulsed T2 cells (filled bars) were determined after 5 rounds of stimulation. A-D show preferential recognition of HA-1^A2 by T cells stimulated with HLA-A2/HA-1 complex-coated DCs but not by T cells stimulated with HLA-A2^pos HA-1 peptide-pulsed DCs.

Specificity of polyclonal HA-1^A2 tetramer binding T cells generated from donor M#1. A, tetramer binding profiles of HA-1^A2 tetramer binding CD8⁺ T cells isolated from donor M#1 before and after sorting. B, Left, Cytolytic activity of tetramer binding (filled bars) and tetramer-nonbinding (open bars) CD8⁺ T cells isolated from donor M#1 directed against HLA-A2^pos HA-1^neg EBV-LCLs, the same EBV-LCLs exogenously pulsed with HA-1 peptide, HLA-A2^pos EBV-LCLs naturally expressing HA-1 or HLA-A2^neg EBV-LCL. Right, Cold target inhibition of the cytolytic activity of the HA-1^A2 tetramer binding CD8⁺ T cells from donor M#1 in response to T2 cells pulsed with HA-1 peptide (filled bars). Unlabeled T2 pulsed with HA-2 or HA-1 peptide, or HLA-A2^pos HA-1^neg EBV-LCLs were used as cold targets at a 10:1 cold:hot ratio. C, IFN-γ production by HA-1^A2 tetramer binding CD8⁺ T cells from donor M#1 or a HA-1-specific T cell clone in response to aAPCs presenting a single set of HLA-A2/HA-1 or HLA-A2/HA-1 complexes.

Left, Specificity of polyclonal HA-1^A2 tetramer binding T cells generated from donor M#2. tetramer binding profile of HA-1^A2 tetramer binding CD8⁺ T cells isolated from donor M#2 after sorting. Right, Cytolytic activity of tetramer binding (filled bars) and tetramer-nonbinding (open bars) CD8⁺ T cells isolated from donor M#2 directed against HLA-A2^pos HA-1^neg EBV-LCLs, the same EBV-LCLs exogenously pulsed with HA-1 peptide, HLA-A2^pos EBV-LCLs naturally expressing HA-1 or HLA-A2^neg EBV-LCL.

Direct isolation of alloHLA-A2/HA-2-specific T cells from HLA-A2^neg parous female donors F#1 and F#5. A, B, Left, Analysis of CD8⁺ cells obtained from the peripheral blood of HLA-A2^neg parous female donor F#1 or F#5 after a first enrichment sort for CD8⁺ and HA-2^A2 tetramer binding cells. The rectangles represent the gate set for the second sort. Center, tetramer binding profiles (HA-2^A2, HA-1^A2, CMV^A2, HY^A2) of CD8⁺ T cells derived from donors F#1 or F#5 after 28 d of nonspecific expansion. Right, Cytolytic activity of these CD8⁺ T cells directed against HLA-A2^pos HA-1^neg EBV-LCLs, the same EBV-LCLs exogenously pulsed with HA-1 peptide or HLA-A2^neg EBV-LCL. C, Left, Staining of CD8+ T cells from donor F#5 with pooled tetramers. Center and right, Staining of the CD8+ T cell population from donor F#5 with HA-1^A2 tetramer-APC and HA-2^A2 tetramer-PE (center) or with HA-1^A2 tetramer-APC and CMV^A2 tetramer-PE (right). A single subset of the donor F#5-derived T cell population binds the HA-2^A2, HA-1^A2, and CMV^A2 tetramers with varying affinities.

Specificity of HA-2^A2 tetramer binding T cells derived from donor F#1. A, HA-2^A2 and HA-1^A2 tetramer binding by T cell clone I-4, derived from donor F#1 using a single-cell-per-well sorting procedure. B, IFN-γ production of T cell clone I-4 in response to aAPCs presenting a single set of HLA-A2/HA-2 (left) or HLA-A2/HA-1 (right) complexes. C, Cytolytic activity of T cell clone I-4 in response to varying EBV-LCLs displaying HA-2-independent recognition of HLA-A2^pos target cells despite HA-1-specific tetramer binding and IFN-γ production shown in A and B.