WT1-specific T-cell responses in high-risk multiple myeloma patients undergoing allogeneic T cell–depleted hematopoietic stem cell transplantation and donor lymphocyte infusions by Eleanor M. Tyler, Achim A. Jungbluth, Richard J. O'Reilly, and Guenther Koehne Blood Volume 121(2):308-317 January 10, 2013 ©2013 by American Society of Hematology

WT1-CTL frequencies increase after DLI WT1-CTL frequencies increase after DLI. Ten patients received 2 or more DLI after transplantation, after which WT1-CTL responses continued to be monitored. 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. Eleanor M. Tyler et al. Blood 2013;121:308-317 ©2013 by American Society of Hematology

WT1-CTL emergence is associated with disease reduction and stabilization. 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. Eleanor M. Tyler et al. Blood 2013;121:308-317 ©2013 by American Society of Hematology

Kinetics and characterization of WT1-CTL. 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. Eleanor M. Tyler et al. Blood 2013;121:308-317 ©2013 by American Society of Hematology

WT1 epitope spreading occurs after DLI 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. 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. Eleanor M. Tyler et al. Blood 2013;121:308-317 ©2013 by American Society of Hematology

WT1 is expressed in CD138+ plasma cells in the BM of MM patients. 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×). Eleanor M. Tyler et al. Blood 2013;121:308-317 ©2013 by American Society of Hematology

WT1-CTL are detected in MM patients pretransplantation and are negatively associated with disease severity. 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. Eleanor M. Tyler et al. Blood 2013;121:308-317 ©2013 by American Society of Hematology