Development and dynamics of robust T-cell responses to CML under imatinib treatment

Abstract

Novel molecular targeted therapies, such as imatinib for chronic myelogenous leukemia (CML), represent the first agents that inhibit cancer cells more than other dividing cells, such as immune cells. We hypothesize that imatinib may create a window in which the immune response is partially restored while apoptotic leukemic cells are present, thus rendering leukemic cells immunogenic as patients enter remission. To detect and quantify antileukemia immune responses in an antigen-unbiased way, we used cryopreserved autologous pretreatment blood samples (representing predominantly leukemic cells) as stimulators to detect antileukemia T-cell responses in CML patients in remission on imatinib. We studied patients over time to address the dynamics of such responses. Our data show that antileukemia T-cell responses develop in the majority of CML patients (9 of 14) in remission and that CD4(+) T cells producing tumor necrosis factor-alpha (median 17.6%) represent the major response over interferon-gamma. This confirms the immune system's ability to respond to leukemia under certain conditions. Such responses may be further amplified as a potential therapy that synergizes with imatinib for improved control of CML.

Figure 1

Antileukemia T-cell responses develop in remission after imatinib. (A) IFN-γ ELISPOT data (triplicate) depicted from P12: leukemic cells alone (first row), remission sample alone (second row), and leukemic and remission samples (last row). (B) Detection of antileukemia immune responses in 9 of 14 patients via IFN-γ ELISPOT. Leukemic cells alone (□), remission sample alone (■), and leukemic and remission samples (). The following stimulated remission samples from the patients are shown and depicted as median and range: P1, 5 months; P2, 12 months; P3, 6 months; P4, 18 months; P6, 14 months; P8, 15 months; P11, 4 months; P12, 5 months; P13, 12 months. All responses were statistically significant (P < .05) compared with leukemic or remission samples alone. (C) TNF-α and IFN-γ production (CFC) by CD4⁺ T and CD8⁺ T cells in stimulated remission samples (■) from 6 patients. Leukemic cells alone (□) and remission samples alone (). The same remission samples shown in panel B were analyzed (*P < .05, statistically significant responses over background).

Figure 2

CD4⁺ and CD8⁺ T-cell responses via flow cytometry. TNF-α (A,C) and IFN-γ responses (B,D) (first column, leukemic cells alone; second column, remission sample alone; third column, leukemic and remission samples) in CD4⁺ and CD8⁺ T cells of P11, 4 months (A,B) and P8, 15 months (C,D). (E) CD8⁺ CD107⁺ responses (third column, leukemic and remission samples) in patient P11, 4 months compared with leukemic cells alone (first column) and remission sample alone (second column).

Figure 3

Dynamics of antileukemia immune responses. (A) Immune responses in 4 patients via IFN-γ ELISPOT analysis. Stimulated remission samples (■), leukemic cells alone (□), and remission samples alone (). Background levels are connected by lines (*P < .05, statistically significant responses over background). Ratio of the tumor load measured by molecular responses (bcr-abl transcript/bcr-abl control gene, left panel, ○) and levels of Ph⁺ cells in chromosome analysis (right panel, ▵) at the corresponding time points. (B) TNF-α (■) and IFN-γ(▾) responses by CD4⁺ and CD8⁺ T cells (CFC) from patients P4 and P13 over time. Leukemic cells alone (TNF-α (■), IFN-γ(▾)) and remission samples alone (TNF-α (), IFN-γ(). Background levels are connected by lines (*P < .05, statistically significant responses over background). (C) Tumor load over time from patients P5(▴), P6(▾), P7 (♦), P8 (●), connected by solid lines, and patients P9 (□), P11 (▿), and P14 (○), connected by dotted lines. Error bars indicate ranges of triplicate measurements. Results are representative of 2 or 3 independent experiments.

Figure 4

Responses of T-cell clones to tumor lysate, but not to TAAs. (A) TNF-α and IFN-γ responses of 2 CD4⁺ and 2 CD8⁺ T-cell clones stimulated with autologous leukemic cells (■) via multiplex cytokine array compared with unstimulated T-cell clones () and T-cell clones stimulated with remission sample (). (B,C) Responses in T-cell clones stimulated with remission sample pulsed with lysate from autologous leukemic cells (■) and remission sample pulsed with lysate from the AML cell line THP1 () using CFC for TNF-α and IFN-γ (B) and blocked with anti-CD4 and anti-MHC II antibodies or anti-CD8 and anti-MHC II antibodies, respectively (C). Results were compared with PHA and ionomycin-stimulated T-cell clones. (D) T-cell responses in CD8⁺ T-cell clones pulsed with HLA-A0201 restricted peptides from LAAs and TAAs using ELISPOT assay compared with PHA stimulated CD8⁺ T-cell clones, and CD8⁺ T-cell clones stimulated with a peptide from the melanoma-associated antigen gp100. Results are shown as median and range.

Figure 5

Immune responses in remission after imatinib after prestimulation. (A) Immune responses in prestimulated remission samples using IFN-γ ELISPOT. Remission samples from P10 stimulated with autologous leukemic cells (■, third column), with lysate from autologous leukemic cells (■), unstimulated remission samples (), and leukemic cells alone (□). Results are shown as median and range. Immune responses in prestimulated remission samples using CFC: (B) 28 months; (C) 32 months. Remission samples from P10 stimulated with autologous leukemic cells (third column), unstimulated remission samples (second column), and leukemic cells alone (first columns). (D) Tumor load from patient P10 over time (○).