Chronic myelogenous leukemia shapes host immunity by selective deletion of high-avidity leukemia-specific T cells

Abstract

We have shown that cytotoxic T lymphocytes specific for PR1, an HLA-A2-restricted nonopeptide derived from proteinase 3, kill leukemia cells and may contribute to the elimination of chronic myelogenous leukemia (CML) after treatment with IFN or allogeneic bone marrow transplant. Some patients with persistent disease also have circulating PR1-specific T cells, however, suggesting the likelihood of immune tolerance. Here we show that both high- and low-avidity PR1-specific T cells from the peripheral blood of healthy donors can be identified and selectively expanded in vitro. Although high-avidity PR1-specific T cells killed CML more effectively than low-avidity T cells, only high-avidity T cells underwent apoptosis when stimulated with high PR1 peptide concentration or when exposed to leukemia that overexpressed proteinase 3. No high-avidity PR1-specific T cells could be identified or expanded from newly diagnosed leukemia patients, whereas low-avidity T cells were readily expanded. Circulating high-avidity PR1-specific T cells were identified in IFN-sensitive patients in cytogenetic remission, however. These results provide evidence that CML shapes the host immune response and that leukemia outgrowth may result in part from leukemia-induced selective deletion of high-avidity PR1-specific T cells.

Figure 1

Lower doses of PR1 peptide induce CTLs with higher intensity PR1/HLA-A2 tetramer staining that correlates with TCR avidity and inversely with effector function threshold. (a) PBMCs collected from healthy HLA-A2.1⁺ donors were stimulated weekly with PR1 peptide-pulsed T2 cells at the peptide concentrations indicated above each FACS plot. After 4 weeks, resulting cultures were stained with CD8 (FITC) Ab and PR1/HLA-A2 tetramer, and the percentage of CD8⁺ cells that stain with tetramer are noted within each FACS plot. (b) Surface HLA-A2 expression on T2 cells increases linearly with increasing concentration of PR1 peptide from 2 μM and 200 μM. T2 cells were incubated with PR1 peptide at the concentrations shown and surface HLA-A2 expression was measured by flow cytometry. (c) CTLs elicited with PR1 at 0.2 μM (open circles) or 20 μM (filled squares) PR1 were incubated for 4 hours at 37°C with PR1 peptide-pulsed T2 cells at the indicated peptide concentrations at an effector/target (E/T) ratio of 10:1 (adjusted based on the number of tetramer-positive CTLs), and percentage of specific lysis was determined. (d) Tetramer decay (t_1/2) was determined to be 58 minutes and 19 minutes by plotting normalized antigen-specific fluorescence at the indicated time points for 28-day-old PR1/HLA-A2 tetramer-stained CTLs elicited with 0.2 μM (open circles) or 20 μM PR1 (filled squares), respectively. Dissociation kinetics of PR1/HLA-A2 tetramer staining were determined at 4°C in the presence of saturating concentrations of BB7.2 Ab to prevent rebinding of tetramer and in the presence of PI (1 μg/ml) to eliminate dead cells from the FACS gate.

Figure 2

High-avidity PR1-specific CTLs cause more specific lysis of CML BM cells than low-avidity PR1-specific CTLs. After 4 weeks in culture, PR1-stimulated CTLs were coincubated in a 4-hour microcytotoxicity assay with bone marrow cells, and specific lysis was determined. Six replicate wells were used for each dilution of effector cells. Specific lysis is plotted versus E/T ratio, and effector number was normalized for the number of PR1/HLA-A2 tetramer-staining cells in the bulk culture. (a) High-avidity PR1-specific CTLs from a healthy donor showed greater specific lysis of CML target cells than low-avidity PR1-specific CTLs. (b) PR1-specific CTL line from a CML patient 3 months after IFN treatment preferentially lyse autologous BM target cells taken at time of diagnosis over healthy HLA-A2⁺ BM cells from a third party, and the amount of CML target cell lysis is similar to that produced by healthy donor-derived low-avidity PR1-specific CTLs.

Figure 3

Only low-avidity PR1-specific CTLs are elicited from peripheral blood of CML patients. PBMCs from three different HLA-A2⁺ CML patients were stimulated weekly with PR1-pulsed T2 cells with PR1 ranging from 0.002 μM to 200 μM. After 4 weeks, resultant cultures were stained with CD8 Ab and PR1/HLA-A2 tetramer and analyzed by FACS. The percentage of CD8⁺ cells that stain with relevant tetramer is indicated within each FACS plot. (a) Cultures elicited with 0.2 μM, 0.02 μM, and 0.002 μM PR1 resulted in CTLs with lower-intensity tetramer staining than CTLs from healthy donors elicited with similar doses of PR1. (b) PBMCs from an untreated chronic phase CML patient (CML no. 4) were studied weekly prior to restimulation with PR1-pulsed T2 cells with PR1/HLA-A2 tetramer. Only PR1-specific CTLs with low-intensity tetramer staining emerge over the 4 weeks, and no relatively high tetramer intensity CTLs are present. (c) PBMCs from CML no. 2 stimulated weekly with 0.2 μM pp65 peptide elicited CTLs with high-intensity pp65/HLA-A2 tetramer staining after 4 weeks in culture.

Figure 4

High-avidity PR1-specific CTLs are identified in the peripheral blood of (a) IFN sensitive CML patients in cytogenetic remission, but not in (b) IFN-resistant or in (c) untreated newly diagnosed CML patients. PBMCs were stained with CD8, dump (CD14 + CD19), and PR1-HLA-A2 tetramer. Patients 5–8 were treated for a minimum of 9 months with IFN. The percentage of Ph⁺ chromosomes in a simultaneous BM specimen is indicated above each FACS plot, and the percentage of CD8⁺ cells with high-avidity PR1-specific CTLs is indicated within each FACS plot.

Figure 5

High-avidity PR1-specific CTLs undergo apoptosis 18 hours after stimulation with high-concentration PR1 peptide. PBMCs from a healthy donor 28 days after weekly restimulation with either 0.2 μM or 20 μM PR1-pulsed T2 cells established relatively high- and low-avidity PR1-CTL, respectively (far left panels). The resulting PR1-CTLs were washed and combined in a 1:1 ratio, based on the number of tetramer-positive cells, with T2 cells pulsed with either 0.2 μM or 20 μM PR1 peptide. After 16 to 18 hours, cells were stained with annexin V Ab, and live cells were analyzed based on PI staining. The percentage of CD8⁺ cells that are tetramer-positive is shown in the far left panels, and the percentage of tetramer-positive cells that stain with annexin V are shown in the remaining panels. (a) Annexin V expression increased on high-avidity PR1-CTLs exposed to high-concentration (20 μM) PR1, but not after exposure to low (0.2 μM) concentration PR1. Annexin V upregulation was blocked by pretreating peptide-pulsed T2 cells with anti–HLA-A2 (BB7.2) prior to coculture with PR1-CTL. (b) Annexin V was not upregulated 18 hours after coculture of low-avidity PR1-CTLs with either low-concentration (0.2 μM) or high concentration (20 μM) PR1 peptide.

Figure 6

High-avidity PR1-CTLs undergo apoptosis 18 hours after coincubation with HLA-A2⁺ CML cells that overexpress proteinase 3. High- and low-avidity PR1-CTLs were combined in a 1:1 ratio, based upon the number of tetramer-positive cells, with CML BM cells from untreated HLA-A2⁺ and HLA-A2^– patients. Annexin V staining was measured on live cells, based on PI staining, 18 hours after coincubation. The percentage of CD8⁺ cells that are tetramer-positive is shown in the left panels, and the percentage of tetramer-positive cells that stain with annexin V are shown in the remaining panels. (a) Annexin V was upregulated in the high-avidity PR1-CTLs after coincubation with HLA-A2⁺ cells, but not after coincubation with HLA-A2^– cells. Remaining low-avidity PR1-CTLs in the culture did not upregulate annexin V. (b) In contrast, low-avidity PR1-CTLs did not upregulate annexin V after coincubation with either HLA-A2⁺ or HLA-A2^– CML BM cells. (c) Overall MHC-I expression and proteinase 3 expression was similar in both CML BM target cells, as measured by surface staining with pan-HLA-A,B,C Ab. (d) Proteinase 3 expression was 2.8- and 3.3-fold higher in the HLA-A2⁺ and the HLA-A2^– patient BM, respectively, compared with healthy donor BM cells.