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. 2007 Sep 15;110(6):1924-32.
doi: 10.1182/blood-2007-03-076844. Epub 2007 May 15.

Graft-versus-leukemia effects associated with detectable Wilms tumor-1 specific T lymphocytes after allogeneic stem-cell transplantation for acute lymphoblastic leukemia

Katayoun Rezvani  1 , Agnes S M Yong, Bipin N Savani, Stephan Mielke, Keyvan Keyvanfar, Emma Gostick, David A Price, Daniel C Douek, A John Barrett
Affiliations

Graft-versus-leukemia effects associated with detectable Wilms tumor-1 specific T lymphocytes after allogeneic stem-cell transplantation for acute lymphoblastic leukemia

Katayoun Rezvani et al. Blood. .

Abstract

To determine whether the leukemia-associated Wilms tumor antigen (WT1) contributes to a graft-versus-leukemia (GVL) effect after allogeneic stem-cell transplantation (SCT) for acute lymphoblastic leukemia (ALL), we studied CD8(+) T-cell responses to WT1 in 10 human lymphocyte antigen (HLA)-A*0201-positive ALL patients during the early phase of immune recovery after SCT (days 30-120). Seven of 10 patients had detectable WT1 expression in their peripheral blood (PB) before SCT by quantitative reverse-transcription polymerase chain reaction. Using WT1/HLA-A*0201 tetramers and intracellular interferon-gamma (IFN-gamma) staining, WT1(+) CD8(+) T-cell responses after SCT were found only in patients with detectable WT1 expression before SCT (5 of 7 vs. 0 of 3; P < .05). To monitor the kinetics of WT1(+) CD8(+) T-cell responses and disease regression after SCT, absolute WT1(+) CD8(+) T-cell numbers and WT1 expression were studied for each time point. The emergence of WT1(+) CD8(+) T cells was associated with a decrease in WT1 expression, suggesting a WT1-driven GVL effect. Loss of WT1(+) CD8(+) T-cell responses was associated with reappearance of WT1 transcripts, consistent with a molecular relapse (P < .001). WT1(+) CD8(+) T cells had a predominantly effector-memory phenotype (CD45RO(+) CD27(-)CD57(+)) and produced IFN-gamma. Our results support the immunogenicity of WT1 after SCT for ALL and highlight the potential for WT1 vaccines to boost GVL after SCT for ALL.

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Figures

Figure 1
Figure 1
CD8+ T-cell responses to WT1 in donors and patients with ALL after SCT. (A) Tetramer analysis of PBMCs was performed by 6-color flow cytometry. Longitudinal data from patient 1 are presented. WT1/HLA-A*0201+ CD8+ T cells were gated on CD3+ events after passing through a small lymphocyte gate; HIV/HLA-A*0201 tetramer was used similarly as a negative control. (B) Comparison of frequency of WT1/HLA-A*0201+ CD8+ T cells in samples before SCT and after SCT. The values represent the maximal WT1/HLA-A*0201+ CD8+ T-cell response for each patient before and after SCT. Bars represent means. (C) IFN-γ production by CD8+ T cells in PBMC samples from patient 1, cultured for 6 hours with (top panel) or without (negative control; bottom panel) WT1 peptide. Results are expressed as percentages of CD8+ T cells. (D) Frequencies of WT1-specific CD8+ T cells by tetramer analysis (formula image) and WT1-specific IFN-γ producing CD8+ T cells (formula image).
Figure 2
Figure 2
Relationship between WT1-specific CD8+ T-cell responses and WT1 gene expression in peripheral blood of patients with ALL and donors. (A) WT1 gene expression in peripheral blood samples from healthy donors, patients with ALL before SCT and after SCT. (B) Correlation between WT1 and BCR-ABL expression in PB samples from patient 2. (C) Relationship between WT1-specific CD8+ T-cell frequencies in PB samples after SCT and WT1 gene expression in PB samples obtained before SCT. Bars represent medians.
Figure 3
Figure 3
Longitudinal phenotypic characterization of tetramer-positive CD3+ CD8+ T cells. Analysis of PBMCs was performed by 6-color flow cytometry in 2 ALL patients at defined time points after SCT. CD45RO, CD27, and CD57 phenotype of CD3+ CD8+ T-cell gated tetramer positive lymphocytes on samples from patient 1 is presented here. (A) CMVpp65495/HLA-A*0201+ CD8+ T cells. (B) WT1/HLA-A*0201+ CD8+ T cells
Figure 4
Figure 4
WT1-specific CD8+ T-cell responses in peripheral blood in relation to disease response as measured by WT1/ABL gene expression. Results in 7 individual patients with detectable WT1 gene expression before SCT are shown (A, patient 1; B, patient 2; C, patient 3; D, patient 4; E, patient 5; F, patient 6; G, patient 7). The number of days after transplantation is shown on the x-axis. WT1/HLA-A*0201+ CD8+ T cells are expressed as absolute numbers/mL of peripheral blood (left, y-axis; dark blue); the shaded area represents absolute numbers of CMVpp65495/HLA-A*0201+ CD8+ T cells. WT1 gene expression in peripheral blood is expressed as the ratio of WT1/ABL (right, y-axis; red). Times of DLI (black arrow), GVHD (med blue), and relapse (green) are depicted on each graph. G indicates GVHD grade; S, skin; P, prednisone; m, months; CyA, cyclosporine A; D, dacluzimab; I, infliximab.
Figure 5
Figure 5
CMV-specific CD8+ T cells in patients and donors. (A) After SCT, patients have significantly greater frequencies of CMVpp65495/HLA-A*0201+ CD8+ T cells than donors and patients before SCT. Data from multiple time points after SCT are presented. (B) Longitudinal analysis of CMVpp65495/HLA-A*0201+ CD8+ T cells after SCT in patient 4. HIV/HLA-A*0201 was used as a negative control for tetramer staining. Results are expressed as percentages of CD8+ T cells. (C) Longitudinal analysis of IFN-γ production by CD8+ T cells in post-SCT PB samples from patient 4 cultured for 6 hours with CMV peptide (top panel) or without peptide (negative control, bottom panel). Results are expressed as percentages of CD8+ T cells.
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