WT1-specific T-cell responses in high-risk multiple myeloma patients undergoing allogeneic T cell-depleted hematopoietic stem cell transplantation and donor lymphocyte infusions

Abstract

While the emergence of WT1-specific cytotoxic T lymphocytes (WT1-CTL) has been correlated with better relapse-free survival after allogeneic stem cell transplantation in patients with myeloid leukemias, little is known about the role of these cells in multiple myeloma (MM). We examined the significance of WT1-CTL responses in patients with relapsed MM and high-risk cytogenetics who were undergoing allogeneic T cell-depleted hematopoietic stem cell transplantation (alloTCD-HSCT) followed by donor lymphocyte infusions. Of 24 patients evaluated, all exhibited WT1-CTL responses before allogeneic transplantation. These T-cell frequencies were universally correlated with pretransplantation disease load. Ten patients received low-dose donor lymphocyte infusions beginning 5 months after transplantation. All patients subsequently developed increments of WT1-CTL frequencies that were associated with reduction in specific myeloma markers, in the absence of graft-versus-host disease. Immunohistochemical analyses of WT1 and CD138 in bone marrow specimens demonstrated consistent coexpression within malignant plasma cells. WT1 expression in the bone marrow correlated with disease outcome. Our results suggest an association between the emergence of WT1-CTL and graft-versus-myeloma effect in patients treated for relapsed MM after alloTCD-HSCT and donor lymphocyte infusions, supporting the development of adoptive immunotherapeutic approaches using WT1-CTL in the treatment of MM.

Trial registration: ClinicalTrials.gov NCT01131169.

Figures

Figure 1

WT1-CTL frequencies increase after DLI. Ten patients received 2 or more DLI after transplantation, after which WT1-CTL responses continued to be monitored. (A) Percentages of CD8 and CD4 T cells producing IFN-γ in response to WT1 peptides were determined after transplantation, before administration of DLI (pre-DLI; □), and at multiple time points after DLI, by intracellular IFN-γ analyses. ■ indicates the maximum IFN-γ response observed post-DLI. (B) Percentages of CD8-positive WT1-CTL were also quantified in pre-DLI samples and post-DLI by MHC-tetramer analyses in HLA-A*0201/0301–expressing patients, by staining with the HLA-A2/A3-RMF tetramers. (C) Absolute numbers of WT1-CTL present pre-DLI and post-DLI were quantified by multiplying the percentages of WT1-CTL determined by MHC-tetramer or IFN-γ analyses (presented in panels A and B), by each patient's ALC. (D) Comparing the absolute number of WT1-CTL pre- and post-DLI reveals a 6.6-fold increase in WT1-CTL. (E) Comparison of each patient's ALC recorded pre-DLI and post-DLI reveals no significant difference between the 2 time points. *P < .04 by Wilcoxon matched-pairs signed rank test. Δ P = .0011 by 2-tailed Mann-Whitney test.

Figure 2

WT1-CTL emergence is associated with disease reduction and stabilization. WT1-CTL frequencies in the PB generally increase after DLI and fluctuate over the course of disease. WT1-CTL frequencies were quantified by intracellular IFN-γ assay in freshly isolated PBMCs. Absolute numbers of WT1-CTL were then computed and compared with the clinical marker of disease, M-spike gamma. (A) Representative FACS plots of intracellular IFN-γ production by CD8+ and CD4+ T cells against autologous nonpulsed and WT1 peptide-pulsed target PBMC (UPN1, time point indicated by “FACS” arrow in panel D). (B) UPN9; (C) UPN4; (D) UPN1; (E) UPN8; (F) UPN10.

Figure 3

Kinetics and characterization of WT1-CTL. Monitoring the emergence of WT1-CTL post-DLI in UPN7. (A) Absolute number of WT1-CTL determined by HLA-A*0201/RMF MHC-tetramer staining. (B) Frequency and phenotypes of PB and BM-resident WT1-CTL in UPN7 from time point indicated by “FACS” arrow in panel C. Effector memory T cells dominate the PB, whereas BM-resident T cells are predominantly central memory in phenotype. (C) Longitudinal phenotypic analysis of WT1-CTL over the course of the disease in UPN7. TN indicates naive T cells; TE, effector T cells; TEM, effector memory T cells; and TCM, central memory T cells.

Figure 4

WT1 epitope spreading occurs after DLI. Immunogenic epitopes were identified in 3 healthy donor lymphocyte products and in the 3 patients who received those DLI, at varying time points after T-cell infusion. Epitopes inducing IFN-γ responses were mapped using the grid shown, whereby subpools inducing dominant responses intersect to reveal the single common peptide containing the immunodominant epitope. (A) CD8+ T-cell production of IFN-γ in response to WT1 peptide subpools, in DLI products for, and PBMC isolated from UPN1, UPN7, and UPN9 post-DLI. (B) CD4+ T-cell production of IFN-γ in response to WT1 peptide subpools, in DLI products for, and PBMC isolated from UPN1, UPN7, and UPN9 post-DLI. (C) Epitope mapping grid outlining subpool peptide compositions. The mapping grid consisting of 24 subpools each containing up to 12 WT1-derived pentadecapeptides. Each peptide is uniquely contained within 2 intersecting subpools. For example, subpools 6 and 24, to which CD8+ and CD4+ T cells from the DLI product for UPN1 both react, uniquely share peptide 138.

Figure 5

WT1 is expressed in CD138+ plasma cells in the BM of MM patients. The expression of WT1 in MM cells was longitudinally assessed over the course of disease in MM patients before and after alloTCD-HSCT and DLI. Paraffin-fixed BM biopsies from MM patients were double stained with monoclonal antibodies to CD138 (MI15; DAB, brown) and WT1 (6F-H2; nFu, red). Immunohistochemical analysis of WT1 expression was performed and biopsies were graded negative, focal, +, ++, +++, or ++++ based on the percentage of CD138+ PC staining positive for WT1. Representative biopsy stains are shown. (A) ++++ > 75% of CD138+ PC stain positive for WT1; UPN5. (B) ++ > 25%-30% of CD138+ cells are WT1+; UPN10. (C) Focal < 5% of CD138+ cells are WT1+; UPN10. (D) Negative, no CD138+ are positive for WT1; UPN10 (20×).

Figure 6

WT1-CTL are detected in MM patients pretransplantation and are negatively associated with disease severity. Pretransplantation frequencies of WT1-CTL in the PB of MM patients were quantified by intracellular IFN-γ assay or MHC-tetramer analyses. (A) Percentage of CD3+ cells in healthy donors and MM patients producing IFN-γ in response to nonpulsed autologous PBMC, or PBMC pulsed with WT1 peptide pools. (B) Absolute numbers of CD3+ cells producing IFN-γ in response to nonpulsed or WT1 peptide pool–pulsed PBMC were quantified by multiplying the percentage of cells producing IFN-γ by the patient's absolute lymphocyte count. (C) Comparing disease severity in patients with greater or less than 10 WT1-CTL/μL of PB. Absolute numbers of WT1-CTL were determined by IFN-γ or MHC-tetramer analyses and compared with pretransplantation M-Spike gamma levels. *P ≤ .0003, 2-tailed Wilcoxon matched pairs signed rank test; Δ, P ≤ .0303, 2-tailed Mann-Whitney test.