MHC class I-associated phosphopeptides are the targets of memory-like immunity in leukemia

Abstract

Deregulation of signaling pathways is a hallmark of malignant transformation. Signaling-associated phosphoproteins can be degraded to generate cancer-specific phosphopeptides that are presented by major histocompatibility complex (MHC) class I and II molecules and recognized by T cells; however, the contribution of these phosphoprotein-specific T cells to immune surveillance is unclear. We identified 95 phosphopeptides presented on the surface of primary hematological tumors and normal tissues, including 61 that were tumor-specific. Phosphopeptides were more prevalent on more aggressive and malignant samples. CD8(+) T cell lines specific for these phosphopeptides recognized and killed both leukemia cell lines and human leukocyte antigen-matched primary leukemia cells ex vivo. Notably, healthy individuals showed robust CD8(+) T cell responses against many of these phosphopeptides within the circulating memory compartment. This immunity was significantly reduced or absent in some leukemia patients. This reduction correlated with clinical outcome; however, immunity was restored after allogeneic stem cell transplantation. These results suggest that phosphopeptides may be targets of cancer immune surveillance in humans, and point to their importance for development of vaccine-based and T cell adoptive transfer immunotherapies.

Figure 1

Characterizing HLA-bound phosphopeptides displayed on tumor and matched healthy tissue. (a) Phosphopeptide display isolated from HLA-A2 and HLA-B7 molecules from 11 primary tumor samples (ALL1, AML1, CLL1-4, HCL1, MCL), EBV-transformed B-cells (B-LCL) and HLA-matched healthy tissue (T-cells, B-cells and bone marrow). Red dots and green dots indicate phosphopeptide antigens selected for further study in patients with CLL and AML respectively. (b) Comparison of the number of individual phosphopeptides identified between HLA-A2 and HLA-B7 in both normal and malignant tissue. (c) Comparison of the number of unique phosphopeptides identified between normal, indolent malignant and aggressive malignant tissue.

Figure 2

The distribution and characteristics of leukemia-associated phosphopeptides. (a, b) Euler diagrams depicting the distribution of HLA-B7-restricted phosphopeptides among different leukemias normal tissues (a) and within different B-cell malignancies (b). (c,d,f) Logoplots of residue frequency at each position of all 9mer HLA-B7 phosphopeptides (c); 9mer non-phosphorylated HLA-B7 peptides from the ImmuneEpitope database (d); and predicted HLA-B7 phosphopeptide binders with a pSer at position 4 (f). The position of phosphoserine for all B7-predicted binders (e).

Figure 3

Generation of phosphopeptide-specific T-cells, from healthy donors, which recognize and kill leukemic targets. (a) Expansion of LSP-1 (RQA(pS)IELPSMAV) specific T-cells from healthy donor PBMCs using DCs and tetramer selection. (b,c) Peptide antigen specificity (b) and recognition (c) of leukemia cell lines by LSP-1-specific T-cells. (d,e) T-cell mediated killing of leukemia cell lines (d) or primary leukemia samples (e) by phosphopeptide-specific T-cell lines but not an HLA-mismatched primary tumor. (f) NCOA1 (RPT(pS)RLNRL)-specific T-cell mediated killing of three HLA-matched CLL tumor samples was inhibited by adding autologous NCOA1 loaded cold targets.

Figure 4

Phosphopeptide-specific immunity is present in healthy individuals within the circulating memory T-cell compartment. (a) Marked heterogeneity of phosphopeptide-specific immunity in 10 healthy donors against 86 antigens revealed by 7-day ELISpot assays. (b) ELISpot results for immunodominant phosphopeptides directly ex vivo and following 14-days in vitro expansion when tetramer binding is evident. (c) Expanded T-cells do not recognize the unphosphorylated counterpart peptides. (d) The relative magnitude of phosphopeptide-specific T-cells compared against common viral epitopes following overnight culture (n=3). (e) Analysis of memory-flow-sorted CD8+ enriched T-cells by 7-day ELISpot reveal phosphopeptide-specific responses reside in the memory compartment.

Figure 5

Leukemia-associated phosphopeptide-specific immunity is lacking in CLL patients. (a) Comparative ELISpot analysis of enriched CD8+ T-cells from patients with CLL and healthy donors against 12 CLL-associated antigens reveals two distinct groupings. (b,c) Analysis of the average patient response against each phosphopeptide between the groups and healthy donors (b) reveals both Group1 (n=12) and Group2 (n=12) have suppressed phosphopeptide-immunity yet responses to mitogens are intact (c). (d,e,f) Kaplan-Meier analysis of overall survival (OS, d), progression free survival (PFS, e) and time to first treatment (TTFT, f) is reduced in Group1 patients, but not statistically significant. **P < 0.01 by Student’s t test comparing average responses for each phosphopeptide between HD and either Group 1 or Group 2.

Figure 6

Phosphopeptide-specific immunity is lacking in patients with AML and restored following stem cell transplantation. (a) AML-associated phosphopeptide-specific immunity is lacking in AML patients in remission compared against healthy individuals using a panel of 12 phosphopeptide antigens. (b) Analysis of the average patient response against each phosphopeptide (n=12) between the groups and healthy donors reveals patients have suppressed immunity. (c) Analysis of total immunity against all 12 antigens between healthy donors (n=12) and each patient pre- and post-transplant (n=12) reveal the recovery of AML-associated phosphopeptide immunity following SCT. (d,e) Immune reconstitution of donor anti-phosphopeptide immunity in two AML patients demonstrating large expansions of phosphopeptide-specific T-cells. (f) T-cell line specific to MLL(EPR) generated from patient AML4 is able to kill the HLA-B7 transfected KG-1a leukemia cell line. ***P < 0.001, **P<0.01 by Student’s t test.