Over-expression of PD-1 Does Not Predict Leukemic Relapse after Allogeneic Stem Cell Transplantation

Abstract

Blockade of the T-cell exhaustion marker PD-1 to re-energize the immune response is emerging as a promising cancer treatment. Relapse of hematologic malignancy after allogeneic stem cell transplantation limits the success of this approach, and PD-1 blockade may hold therapeutic promise. However, PD-1 expression and its relationship with post-transplant relapse is poorly described. Because the donor immunity is activated by alloresponses, PD-1 expression may differ from nontransplanted individuals, and PD-1 blockade could risk graft-versus-host disease. Here we analyzed T-cell exhaustion marker kinetics and their relationship with leukemia relapse in 85 patients undergoing myeloablative T-cell-depleted HLA-matched stem cell transplantation. At a median follow-up of 3.5 years, 35 (44%) patients relapsed. PD-1 expression in CD4 and CD8 T cells was comparably elevated in relapsed and nonrelapsed cohorts. Helios⁺ regulatory T cells and CD8 effector memory cells at day 30 emerged as independent predictors of relapse. Although leukemia antigen-specific T cells did not overexpress PD-1, single-cell analysis revealed LAG3 and TIM3 overexpression at relapse. These findings indicate that PD-1 is an unreliable marker for leukemia-specific T-cell exhaustion in relapsing patients but implies other exhaustion markers and suppressor cells as relapse biomarkers.

Keywords: Biomarker; Graft-versus-leukemia effect; Post-transplant relapse.

Figure 1.

(A) Cumulative incidence of relapse. (B) Kaplan-Meier estimate of post-relapse survival in subjects with the time to relapse >180 vs. ≤ 180 days.

Figure 2.

Comparison of cellular immune subsets between the relapsed and non-relapsed cohorts. (A) Percentages of PD-1⁺ in CD4 cells. (B) Percentages of PD-1⁺ in CD8 cells. (C) Percentages of Helios⁺FoxP3⁺ in CD4 cells. (D) Absolute numbers of PD-1⁺CD4 cells. (E) Absolute numbers of PD-1⁺CD8 cells. (F) Absolute numbers of effector memory CD8 cells.

Figure 3.

Cumulative incidence of relapse in (A) subjects with %Helios⁺T_regs CD4 cells at day 30 <10% vs. ≥ 10%. (B) subjects with absolute number of CD8 effector memory cells at day 30 <150 /uL vs. ≥150 /uL.

Figure 4.

Comparison of exhaustion marker expressions in T cell memory subsets between the relapsed cohort and paired donor cohorts. (A) Percentages of PD-1⁺ in CD4 cells. (B) Percentages of LAG3⁺ in CD4 cells. (C) Percentages of TIM-3⁺ in CD4 cells. (D) Percentages of PDL-1⁺ in CD4 cells. (E) Percentages of PD-1⁺ in CD8 cells. (F) Percentages of LAG3⁺ in CD8 cells. (G) Percentages of TIM-3⁺ in CD8 cells. (H) Percentages of PDL-11⁺ in CD8 cells. Abbreviations: CM, central memory; EM, effector memory; SCM, stem cell memory; TEMRA, effector memory RA.

Figure 5.

(A) Correlation between leukemia associated antigen (LAA) and antigen specific T cells in six subjects. Five LAAs (AURAK, NY-ESO,1, MAGEA3, PRAME, WT1) were quantified in peripheral blood by reverse transcription polymerase chain reaction (RT-PCR) and leukemia specific T cells were simultaneously measured by enzyme-linked immunosorbent spot-forming cell assay (Elispot). (B) Representative flow data of antigen specific T cells in PD-1 negative or positive fractions (UPN1 and UPN3). HIV was used as negative control and OKT3 as positive control. Abbreviation: 2^nd R, second relapse; PR, post-relapse; R, relapse; SFU; spot forming units; R, relapse.

Figure 6.

Single cell RNA-seq analysis of PRAME and CMVpp65 specific T cells at relapse in UPN1. (A) Cluster analysis of the four pooled samples. Clusters were roughly classified into three categories: CMVpp65 and PRAME positive T cells, clusters 2, 6, 9, 10, 11 shown in red boxes; CMVpp65 and PRAME negative T cells, clusters 1, 3, 7, 8, 13 shown in blue boxes; and non-specific T cells, cluster 4, 5, 12 shown in orange boxes. (B) Cluster analysis of each four samples: CMVpp65 positive or negative cells and PRAME positive or negative cells. (C) Comparison of expression profiles of exhaustion markers in CMVpp65 and PRAME positive T cells at single cell level. PDCD1 (PD-1) in left, LAG3 in middle, and HAVCR2 (TIM-3) in right.