T-Cell Immunity to Two HLA-A2-Restricted Self-Determinants of Cyclin E May Contribute to Remission After Stem Cell Transplantation.

Abstract 686

Cyclin E1 (CCNE1) and cyclin E2 (CCNE2) are tightly regulated cell cycle genes in normal cells but are over-expressed and constitutively active in breast cancer and in the majority of hematological malignances. To validate CCNE as a potential target antigen for T-cells in leukemia, we first confirmed aberrant CCNE1 and CCNE2 protein in PBMC from 26 (93%) of 28 patients (CML = 16; AML = 7; ALL =2; NHL = 3) by Western Blot compared to 4 (33%) of 12 healthy controls (p < 0.0005). Next, we screened the sequences of CCNE1 and CCNE2 for HLA-A*0201 binding motifs and identified a pair of homologous nonameric peptides with highest predicted binding to HLA-A*0201 using an NCBI algorithm. The peptides, denoted CCNE1_M (¹⁴⁴ILLDWLMEV¹⁵²) and CCNE2L (¹⁴⁴ILLDWLLEV¹⁵²), differed at P7 (M or L), and both differed from mouse sequence at P1 (V). Synthetic mouse and human peptides were used to confirm high affinity HLA-A2 binding on T2 cells by FACS analysis and peptide-pulsed T2 were used to elicit peptide-specific CTLs from healthy HLA-A2⁺ PBMC in vitro. CCNE1_M-CTL lines specifically lysed both CCNE1_M-loaded and CCNE2_L-loaded T2 cells, while no CTL could be elicited with mouse peptide. Similarly, CCNE2_L-stimulated CTL lines killed CCNE1_M-loaded and CCNE2_L-loaded T2 cells but not non-loaded T2 cells. Using CCNE1_M and CCNE2_L HLA-A2 tetramers, we found that either tetramer could bind equally to either the CCNE1_M- or CCE2_L-derived CTL lines, suggesting that both peptides could be cross-recognized by CTL lines elicited with either peptide. To further study the cross-recognition and potential immune dominance of both peptides and to determine their potential anti-leukemia activity, CCNE1_M- and CCNE2_L-CTL clones were derived by limiting dilution assay. Two peptide-specific CTL clones from each of the lines showed 25% and 26% specific lysis, respectively, of leukemia cells at E:T 10:1. Neither CCNE-specific CTLs showed lysis of BM cells that were obtained from the same patient during remission, nor HLA-A2⁺ BM cells from a healthy donor. Next, we compared the T-cell antigen receptor (TCR) avidity of these CCNE1_M- and the CCNE2_L-CTL clones by measuring tetramer dissociation half-times (t_1/2) at 25°C using CCNE1_M/HLA-A2 and CCNE2_L/HLA-A2 (and control pp65/HLA-A2) tetramers analyzed by flow cytometry. The decay of normalized (to time = 0) tetramer-bound fluorescence versus time was linear for each clone with either tetramer (R² = 0.85 to 0.91), showing that tetramer binding avidity could be used to proportionally determine TCR affinity. Furthermore, first order kinetics could be used to determine the t_1/2 of each of the clones. The t_1/2 of CCNE1_M/HLA-A2 tetramer was 85 min and 25 min, respectively, while the t_1/2of CCNE1_L/HLA-A2 was 30 min and 11 min, respectively, for the CCNE1_M-CTL and the CCNE2_L-CTL. This suggests that while both peptides were cross recognized by unique T-cell clones (with unique TCR, determined by TCR-Vβ sequence comparisons), CCNE1_M appeared to be immunodominant. To determine whether immune response (IR) to either peptide occurred in leukemia patients, we studied PBMC from 18 patients (10 CML; 8 ALL) before and 3–6 mo after SCT with CCNE1_M/HLA-A2- and CCNE2_L/HLA-A2-tetramer assay. The mean number of CCNE1_M-CTL and CCNE2_L-CTL cells increased after SCT (p< 0.002 in CCNE1_M-CTL and CCNE2_L-CTL) compared to no change in mean number of pp65-CTL before/after SCT. IR (defined as ≥ 20% increase of specific CTL after SCT) to either CCNE1_M or CCNE2_L did not correlate with type of leukemia, donor-recipient HLA disparity (matched or mismatched), or disease status prior to SCT by Fisher's exact test. However, in 8 CML patients not in remission prior to SCT, IR to either CCNE1 or CCNE2 occurred more frequently in patients who achieved CR compared to those that did not achieve CR after SCT (100% vs. 33%, respectively; p < 0.04). These findings were confirmed in an additional 25 AML patients with active disease at SCT. To study whether the peptide-specific CTL were functional, we measured IFN-γ and TNF-αa production after peptide stimulation by Luminex bead assay and by intracellular cytokine flow cytometry (CFC). The assays showed production of IFN-γ and TNF-αa cytokines by T-cells after stimulation with CCNE1_M or CCNE2_Lpeptides. Taken together, these results show that CCNE1_M and CCNE2_Lself-peptides from constitutively active cell cycle proteins are novel leukemia-associated antigens that could be studied in immunotherapy strategies.