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. 2009 Aug 1;15(15):4944-53.
doi: 10.1158/1078-0432.CCR-08-3332. Epub 2009 Jul 14.

Functional unresponsiveness and replicative senescence of myeloid leukemia antigen-specific CD8+ T cells after allogeneic stem cell transplantation

Gregory L Beatty  1 , Jasmine S Smith, Ran Reshef, Kunal P Patel, Theresa A Colligon, Barbara A Vance, Noelle V Frey, F Brad Johnson, David L Porter, Robert H Vonderheide
Affiliations

Functional unresponsiveness and replicative senescence of myeloid leukemia antigen-specific CD8+ T cells after allogeneic stem cell transplantation

Gregory L Beatty et al. Clin Cancer Res. .

Abstract

Purpose: The therapeutic effect of allogeneic hematopoietic stem cell transplantation (HSCT) for patients with myeloid malignancies has been attributed in part to a graft-versus-leukemia effect that is dependent on donor T lymphocytes. CD8(+) T-cell responses to MHC class I-restricted tumor epitopes, not just allogeneic antigens, may help mediate antileukemia effects after HSCT, but the specificity and function of such cells are not completely understood.

Experimental design: We examined the diversity, phenotype, and functional potential of leukemia-associated antigen-specific CD8(+) T cells in patients with myeloid leukemia following allogeneic HSCT. Screening for antigen-specific T cells was accomplished with a peptide/MHC tetramer library.

Results: Patients with acute myelogenous leukemia or chronic myelogenous leukemia in remission following HSCT exhibited significant numbers of peripheral blood CD8(+) T cells that recognized varying combinations of epitopes derived from leukemia-associated antigens. However, these cells failed to proliferate, release cytokines, or degranulate in response to antigen-specific stimuli. As early as 2 months after HSCT, CD8(+) T cells from patients were predominantly CD28(-) CD57(+) and had relatively short telomeres, consistent with cellular senescence.

Conclusions: Circulating leukemia-specific CD8(+) T cells are prominent in myeloid leukemia patients after HSCT, but such cells are largely functionally unresponsive, most likely due to replicative senescence. These findings carry important implications for the understanding of the graft-versus-leukemia effect and for the rational design of immunotherapeutic strategies for patients with myeloid leukemias.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
Viral and leukemia-associated antigen-specific CD8+ T cells in myeloid leukemia patients following allogeneic HSCT. PBMC from myeloid leukemia patients following allogeneic HSCT were labeled with anti-CD8, anti-CD14, anti-CD4, and peptide/HLA-A2 tetramers and analyzed. Cells were gated on CD8+ CD4neg CD14neg lymphocytes. Representative examples of CD8+ T cells recognizing various viral and leukemia-associated antigenic epitopes are shown for 6 patients compared to the HTLV-1 negative control tetramer. Patient identification numbers are shown to the left of each plot. The percentage of tetramer-specific CD8+ cells out of CD8+ CD4neg CD14neg lymphocytes is shown on each plot.
Fig. 2
Fig. 2
Leukemia-associated antigen-specific CD8+ T cells lack proliferative capacity. CD8+ CD4neg CD14neg cells were analyzed from uncultured (fresh) PBMC for binding to HLA-A2-specific tetramers. PBMC were then subjected to in vitro stimulation with peptide and CD8+ CD4neg CD14neg cells were analyzed for binding to tetramer after 7 days. (A) Viral specific CD8+ T cells specific for either Flu or CMV show response to peptide stimulation. (B) Leukemia-associated antigen-specific CD8+ T cells show lack of proliferative capacity in the majority of cases. (C) Summary of results for 8 patients in whom PBMC were analyzed for in vitro stimulated (IVS) proliferation of viral and leukemia-associated antigen-specific CD8+ T cells. Evidence for proliferation was determined by observing a greater than two fold increase in the percentage of antigen-specific CD8+ T cells at analysis following IVS. ✕, no proliferation. ●, proliferation. No symbol, not done.
Fig. 2
Fig. 2
Leukemia-associated antigen-specific CD8+ T cells lack proliferative capacity. CD8+ CD4neg CD14neg cells were analyzed from uncultured (fresh) PBMC for binding to HLA-A2-specific tetramers. PBMC were then subjected to in vitro stimulation with peptide and CD8+ CD4neg CD14neg cells were analyzed for binding to tetramer after 7 days. (A) Viral specific CD8+ T cells specific for either Flu or CMV show response to peptide stimulation. (B) Leukemia-associated antigen-specific CD8+ T cells show lack of proliferative capacity in the majority of cases. (C) Summary of results for 8 patients in whom PBMC were analyzed for in vitro stimulated (IVS) proliferation of viral and leukemia-associated antigen-specific CD8+ T cells. Evidence for proliferation was determined by observing a greater than two fold increase in the percentage of antigen-specific CD8+ T cells at analysis following IVS. ✕, no proliferation. ●, proliferation. No symbol, not done.
Fig. 2
Fig. 2
Leukemia-associated antigen-specific CD8+ T cells lack proliferative capacity. CD8+ CD4neg CD14neg cells were analyzed from uncultured (fresh) PBMC for binding to HLA-A2-specific tetramers. PBMC were then subjected to in vitro stimulation with peptide and CD8+ CD4neg CD14neg cells were analyzed for binding to tetramer after 7 days. (A) Viral specific CD8+ T cells specific for either Flu or CMV show response to peptide stimulation. (B) Leukemia-associated antigen-specific CD8+ T cells show lack of proliferative capacity in the majority of cases. (C) Summary of results for 8 patients in whom PBMC were analyzed for in vitro stimulated (IVS) proliferation of viral and leukemia-associated antigen-specific CD8+ T cells. Evidence for proliferation was determined by observing a greater than two fold increase in the percentage of antigen-specific CD8+ T cells at analysis following IVS. ✕, no proliferation. ●, proliferation. No symbol, not done.
Fig. 3
Fig. 3
Leukemia-associated antigen-specific CD8+ T cells do not degranulate or release cytokines following cognate-peptide interaction. PBMC were stimulated in vitro with peptide for 4 hours. CD8+ CD4neg CD14neg T cells were gated on tetramer-positive cells and analyzed by flow cytometry for the ability of antigen-specific CD8+ T cells to mobilize CD107a and for cytoplasmic expression of IFN-γ following antigenic stimulation. (A) Flu-specific CD8+ T cells from normal donors (ND) stimulated with either negative control peptide or Flu peptide. (B) PR1-specific and PRA142-specific CD8+ T cells from three representative patients.
Fig. 3
Fig. 3
Leukemia-associated antigen-specific CD8+ T cells do not degranulate or release cytokines following cognate-peptide interaction. PBMC were stimulated in vitro with peptide for 4 hours. CD8+ CD4neg CD14neg T cells were gated on tetramer-positive cells and analyzed by flow cytometry for the ability of antigen-specific CD8+ T cells to mobilize CD107a and for cytoplasmic expression of IFN-γ following antigenic stimulation. (A) Flu-specific CD8+ T cells from normal donors (ND) stimulated with either negative control peptide or Flu peptide. (B) PR1-specific and PRA142-specific CD8+ T cells from three representative patients.
Fig. 4
Fig. 4
Phenotype characterization of leukemia-associated antigen-specific CD8+ T cells. PBMC were labeled with anti-CD8, anti-CD14, anti-CD4, and HLA-A2 tetramers and analyzed for expression of CD28 and CD57. (A) CD28 and CD57 expression on bulk CD8+ T cells is shown separately for each of the 13 patients (one symbol per patient) and plotted versus the duration since HSCT. Mean frequency and standard deviation of CD28 and CD57 expression on bulk CD8+ T cells from 20 normal donors is presented on the right for comparison (filled square shown as mean +/− 1 SD). (B) Representative examples of the expression of CD28 and CD57 on leukemia-associated antigen-specific CD8+ T cells from two patients following allogeneic HSCT, shown in comparison to viral-specific CD8+ T cells from 3 normal donors. Plots are shown after gating on tetramer-positive CD8+ CD4neg CD14neg lymphocytes.
Fig. 4
Fig. 4
Phenotype characterization of leukemia-associated antigen-specific CD8+ T cells. PBMC were labeled with anti-CD8, anti-CD14, anti-CD4, and HLA-A2 tetramers and analyzed for expression of CD28 and CD57. (A) CD28 and CD57 expression on bulk CD8+ T cells is shown separately for each of the 13 patients (one symbol per patient) and plotted versus the duration since HSCT. Mean frequency and standard deviation of CD28 and CD57 expression on bulk CD8+ T cells from 20 normal donors is presented on the right for comparison (filled square shown as mean +/− 1 SD). (B) Representative examples of the expression of CD28 and CD57 on leukemia-associated antigen-specific CD8+ T cells from two patients following allogeneic HSCT, shown in comparison to viral-specific CD8+ T cells from 3 normal donors. Plots are shown after gating on tetramer-positive CD8+ CD4neg CD14neg lymphocytes.
Fig. 5
Fig. 5
Telomere shortening in CD8+ T cells following allogeneic HSCT. Whole genomic DNA from purified CD8+ T cells obtained from the peripheral blood of patients (□; 002, 004, 005, 012) and normal donors (○; 20–40 yo and 40–60 yo) was analyzed by southern blot for mean telomere length. The donor age for each patient was as follows: 002 (65 yo), 004 (51 yo), 005 (32 yo), and 012 (62 yo). Each symbol indicates an individual patient or normal donor. The filled circle(●) indicates the normal donor (62 yo, female) for patient 012, filled square (■). Solid lines indicate the mean for each group. p-values for the comparisons indicated were calculated using Student’s t test.
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