Identification of two new HLA-A*1101-restricted tax epitopes recognized by cytotoxic T lymphocytes in an adult T-cell leukemia patient after hematopoietic stem cell transplantation

Abstract

We previously reported that Tax-specific CD8(+) cytotoxic T lymphocytes (CTLs), directed to single epitopes restricted by HLA-A2 or A24, expanded in vitro and in vivo in peripheral blood mononuclear cells (PBMC) from some adult T-cell leukemia (ATL) patients after but not before allogeneic hematopoietic stem cell transplantation (HSCT). Here, we demonstrated similar Tax-specific CTL expansion in PBMC from another post-HSCT ATL patient without HLA-A2 or A24, whose CTLs equally recognized two newly identified epitopes, Tax88-96 and Tax272-280, restricted by HLA-A11, suggesting that these immunodominant Tax epitopes are present in the ATL patient in vivo.

FIG. 1.

Induction of CTLs in PBMCs from post-HSCT patient 156 by stimulation with ILT-156 cells. (A) PBMCs from patient 156 (147 days post-HSCT) were cultured with periodic stimulation with formalin-fixed ILT-156 cells, and their IFN-γ-producing ability was evaluated by enzyme-linked immunosorbent assay at 17 days after initiation of culture, following 18 h of incubation with ILT-156 (closed circles), LCL-156 (open circles), HLA-A11-matched TCL-Kan (closed triangles) (HLA-A2/A11, B7/Bw46, Cw1/Cw3/Cw7, and DR2/DR9) and LCL-Kan (open triangles) (same HLA type as TCL-Kan), HLA-A26-matched ILT-Nkz-2 (closed squares) (HLA-A2/A26, B51/B54, and Cw1/−) and LCL-Nkz (open squares) (same HLA type as ILT-Nkz-2), or no addition (crosses) at an effector cell/target cell ratio of 10. Closed symbols, HTLV-I-positive cells; open symbols, HTLV-I-negative cells. (B) The IFN-γ-producing ability of the CTLs induced from patient 156 at 25 days in culture was determined after 18 h of incubation with ILT-156 cells at an effector cell/target cell ratio of 10, following preincubation of effector cells with CD4 or CD8 MAbs or preincubation of target cells with HLA class I or class II MAbs for 1 h at 37°C (3). (C) STR polymorphism in DNA extracted from CTLs from post-HSCT patient 156, ILT-156 cells, and ILT-167 cells was analyzed by using an AmpFlSTER SGM Plus PCR Amplification Kit, GeneScan 3.1, and Genotyper 2.5 software (Applied Biosystems, Foster City, CA). Electropherograms of four representative STR loci (D21S11, D18S51, D3S1358, and vMA) are shown. Peak height is measured against an arbitrary scale displayed on the y axis. The numbers of STRs are indicated in squares on the x axis. (D) IFN-γ production by CTLs from patient 156 (at 41 days of culture) after 18 h of incubation with ILT-156 cells, donor-derived HTLV-I-infected ILT-167 cells, and recipient-derived HTLV-I-negative LCL-156 cells at an effector cell/target cell ratio of 10. (E) The cytotoxicity of the CTLs from patient 156 (at 41 days of culture) against radiolabeled target ILT-156 cells was evaluated by 6-h ⁵¹Cr release assay in the presence of the indicated unlabeled competitor cells. Both the effector cell/target cell and competitor cell/radiolabeled target cell ratios were 40:1. All IFN-γ enzyme-linked immunosorbent assay values represent the means and standard deviations from duplicates, and ⁵¹Cr release assay values represent the means and standard deviations from triplicate assays.

FIG. 1.

Induction of CTLs in PBMCs from post-HSCT patient 156 by stimulation with ILT-156 cells. (A) PBMCs from patient 156 (147 days post-HSCT) were cultured with periodic stimulation with formalin-fixed ILT-156 cells, and their IFN-γ-producing ability was evaluated by enzyme-linked immunosorbent assay at 17 days after initiation of culture, following 18 h of incubation with ILT-156 (closed circles), LCL-156 (open circles), HLA-A11-matched TCL-Kan (closed triangles) (HLA-A2/A11, B7/Bw46, Cw1/Cw3/Cw7, and DR2/DR9) and LCL-Kan (open triangles) (same HLA type as TCL-Kan), HLA-A26-matched ILT-Nkz-2 (closed squares) (HLA-A2/A26, B51/B54, and Cw1/−) and LCL-Nkz (open squares) (same HLA type as ILT-Nkz-2), or no addition (crosses) at an effector cell/target cell ratio of 10. Closed symbols, HTLV-I-positive cells; open symbols, HTLV-I-negative cells. (B) The IFN-γ-producing ability of the CTLs induced from patient 156 at 25 days in culture was determined after 18 h of incubation with ILT-156 cells at an effector cell/target cell ratio of 10, following preincubation of effector cells with CD4 or CD8 MAbs or preincubation of target cells with HLA class I or class II MAbs for 1 h at 37°C (3). (C) STR polymorphism in DNA extracted from CTLs from post-HSCT patient 156, ILT-156 cells, and ILT-167 cells was analyzed by using an AmpFlSTER SGM Plus PCR Amplification Kit, GeneScan 3.1, and Genotyper 2.5 software (Applied Biosystems, Foster City, CA). Electropherograms of four representative STR loci (D21S11, D18S51, D3S1358, and vMA) are shown. Peak height is measured against an arbitrary scale displayed on the y axis. The numbers of STRs are indicated in squares on the x axis. (D) IFN-γ production by CTLs from patient 156 (at 41 days of culture) after 18 h of incubation with ILT-156 cells, donor-derived HTLV-I-infected ILT-167 cells, and recipient-derived HTLV-I-negative LCL-156 cells at an effector cell/target cell ratio of 10. (E) The cytotoxicity of the CTLs from patient 156 (at 41 days of culture) against radiolabeled target ILT-156 cells was evaluated by 6-h ⁵¹Cr release assay in the presence of the indicated unlabeled competitor cells. Both the effector cell/target cell and competitor cell/radiolabeled target cell ratios were 40:1. All IFN-γ enzyme-linked immunosorbent assay values represent the means and standard deviations from duplicates, and ⁵¹Cr release assay values represent the means and standard deviations from triplicate assays.

FIG. 1.

Induction of CTLs in PBMCs from post-HSCT patient 156 by stimulation with ILT-156 cells. (A) PBMCs from patient 156 (147 days post-HSCT) were cultured with periodic stimulation with formalin-fixed ILT-156 cells, and their IFN-γ-producing ability was evaluated by enzyme-linked immunosorbent assay at 17 days after initiation of culture, following 18 h of incubation with ILT-156 (closed circles), LCL-156 (open circles), HLA-A11-matched TCL-Kan (closed triangles) (HLA-A2/A11, B7/Bw46, Cw1/Cw3/Cw7, and DR2/DR9) and LCL-Kan (open triangles) (same HLA type as TCL-Kan), HLA-A26-matched ILT-Nkz-2 (closed squares) (HLA-A2/A26, B51/B54, and Cw1/−) and LCL-Nkz (open squares) (same HLA type as ILT-Nkz-2), or no addition (crosses) at an effector cell/target cell ratio of 10. Closed symbols, HTLV-I-positive cells; open symbols, HTLV-I-negative cells. (B) The IFN-γ-producing ability of the CTLs induced from patient 156 at 25 days in culture was determined after 18 h of incubation with ILT-156 cells at an effector cell/target cell ratio of 10, following preincubation of effector cells with CD4 or CD8 MAbs or preincubation of target cells with HLA class I or class II MAbs for 1 h at 37°C (3). (C) STR polymorphism in DNA extracted from CTLs from post-HSCT patient 156, ILT-156 cells, and ILT-167 cells was analyzed by using an AmpFlSTER SGM Plus PCR Amplification Kit, GeneScan 3.1, and Genotyper 2.5 software (Applied Biosystems, Foster City, CA). Electropherograms of four representative STR loci (D21S11, D18S51, D3S1358, and vMA) are shown. Peak height is measured against an arbitrary scale displayed on the y axis. The numbers of STRs are indicated in squares on the x axis. (D) IFN-γ production by CTLs from patient 156 (at 41 days of culture) after 18 h of incubation with ILT-156 cells, donor-derived HTLV-I-infected ILT-167 cells, and recipient-derived HTLV-I-negative LCL-156 cells at an effector cell/target cell ratio of 10. (E) The cytotoxicity of the CTLs from patient 156 (at 41 days of culture) against radiolabeled target ILT-156 cells was evaluated by 6-h ⁵¹Cr release assay in the presence of the indicated unlabeled competitor cells. Both the effector cell/target cell and competitor cell/radiolabeled target cell ratios were 40:1. All IFN-γ enzyme-linked immunosorbent assay values represent the means and standard deviations from duplicates, and ⁵¹Cr release assay values represent the means and standard deviations from triplicate assays.

FIG. 1.

Induction of CTLs in PBMCs from post-HSCT patient 156 by stimulation with ILT-156 cells. (A) PBMCs from patient 156 (147 days post-HSCT) were cultured with periodic stimulation with formalin-fixed ILT-156 cells, and their IFN-γ-producing ability was evaluated by enzyme-linked immunosorbent assay at 17 days after initiation of culture, following 18 h of incubation with ILT-156 (closed circles), LCL-156 (open circles), HLA-A11-matched TCL-Kan (closed triangles) (HLA-A2/A11, B7/Bw46, Cw1/Cw3/Cw7, and DR2/DR9) and LCL-Kan (open triangles) (same HLA type as TCL-Kan), HLA-A26-matched ILT-Nkz-2 (closed squares) (HLA-A2/A26, B51/B54, and Cw1/−) and LCL-Nkz (open squares) (same HLA type as ILT-Nkz-2), or no addition (crosses) at an effector cell/target cell ratio of 10. Closed symbols, HTLV-I-positive cells; open symbols, HTLV-I-negative cells. (B) The IFN-γ-producing ability of the CTLs induced from patient 156 at 25 days in culture was determined after 18 h of incubation with ILT-156 cells at an effector cell/target cell ratio of 10, following preincubation of effector cells with CD4 or CD8 MAbs or preincubation of target cells with HLA class I or class II MAbs for 1 h at 37°C (3). (C) STR polymorphism in DNA extracted from CTLs from post-HSCT patient 156, ILT-156 cells, and ILT-167 cells was analyzed by using an AmpFlSTER SGM Plus PCR Amplification Kit, GeneScan 3.1, and Genotyper 2.5 software (Applied Biosystems, Foster City, CA). Electropherograms of four representative STR loci (D21S11, D18S51, D3S1358, and vMA) are shown. Peak height is measured against an arbitrary scale displayed on the y axis. The numbers of STRs are indicated in squares on the x axis. (D) IFN-γ production by CTLs from patient 156 (at 41 days of culture) after 18 h of incubation with ILT-156 cells, donor-derived HTLV-I-infected ILT-167 cells, and recipient-derived HTLV-I-negative LCL-156 cells at an effector cell/target cell ratio of 10. (E) The cytotoxicity of the CTLs from patient 156 (at 41 days of culture) against radiolabeled target ILT-156 cells was evaluated by 6-h ⁵¹Cr release assay in the presence of the indicated unlabeled competitor cells. Both the effector cell/target cell and competitor cell/radiolabeled target cell ratios were 40:1. All IFN-γ enzyme-linked immunosorbent assay values represent the means and standard deviations from duplicates, and ⁵¹Cr release assay values represent the means and standard deviations from triplicate assays.

FIG. 2.

Mapping of HTLV-I Tax epitopes recognized by CTLs from post-HSCT patient 156. LCL-156 cells were pulsed with 10 μM of a series of 24-mer synthetic oligopeptides covering the N-terminal half (A) and a series of 15-mer oligopeptides covering the C-terminal half (B) of the Tax amino acid sequence, and their susceptibility to CTLs of post-HSCT patient 156 was measured by IFN-γ enzyme-linked immunosorbent assay following 18 h of incubation at an effector cell/target cell ratio of 10. Values represent the means and standard deviations from duplicate assays.

FIG. 2.

Mapping of HTLV-I Tax epitopes recognized by CTLs from post-HSCT patient 156. LCL-156 cells were pulsed with 10 μM of a series of 24-mer synthetic oligopeptides covering the N-terminal half (A) and a series of 15-mer oligopeptides covering the C-terminal half (B) of the Tax amino acid sequence, and their susceptibility to CTLs of post-HSCT patient 156 was measured by IFN-γ enzyme-linked immunosorbent assay following 18 h of incubation at an effector cell/target cell ratio of 10. Values represent the means and standard deviations from duplicate assays.

FIG. 3.

Detection of Tax88-96 and Tax272-280-specific CTLs by tetramers in PBMCs from post-HSCT patient 156. CTLs from post-HSCT patient 156 at 41 days after initiation of culture (A), uncultured PBMCs from post-HSCT patient 156 (B), and uncultured PBMCs from donor 167 (C) were stained with PE-Cy5-labeled CD8 MAbs (HIT8a; BD PharMingen) together with PE-conjugated HLA-A*1101/Tax88-96 (left), HLA-A*1101/Tax272-280 (center), or a mixture of both tetramers (right). Both tetramers were provided by the National Institute of Allergy and Infectious Diseases Tetramer Facility, Emory University, and were used at a dilution of 1:800. Numbers in the upper right corners indicate percentages of CD8-positive cells bound to the tetramer as analyzed on a flow cytometer (1). A total of 100,000 events were collected in each case.