Supplemental materials 1
Supplemental Fig. 1 Immunogram This immunogram summarizes patient clinical data and immune parameters at corresponding time points for Patient IMF-32. The top panel illustrates the patient s treatment history with ipilimumab and prior chemotherapy with a platinum analog/vinblastine/temozolomide. Panel A shows percent changes in target lesions by maximum diameter. Surgical resection of left inguinal lymph nodes is represented by X. Panel B illustrates changes in white blood cell (WBC, 10 3 /mm 3 ) and absolute lymphocyte count (ALC, 10 3 /mm 3 ). Panel C shows changes in CD4 + and CD8 + T- cells characterized by flow cytometry of PBMCs. Panel D shows changes in CD4 + ICOS hi and CD4 + FoxP3 + expression as assessed by multiparameter flow cytometry. Panel E depicts changes in gp100- and tyrosinase-specific CD8 + IFN-γ + T-cells and HLA-A*0201 gp100 209-217 and HLA-A*0201 tyrosinase 369-377 tetramer-reactive CD8 + T- cells as determined by multiparameter flow cytometry. 2
Supplemental Fig. 2 Immunohistochemical staining of metastatic tumor tissue for gp100 and tyrosinase tumor antigens from Patient IMF-32 Paraffin-embedded tissue from a metastatic melanoma tumor resected from the left lung in April of 2007 was analyzed for expression of gp100 and tyrosinase antigens using HMB45 and T311 antibodies, respectively. Representative images demonstrate no staining for gp100 but staining for tyrosinase (Panels A and B, respectively). Higher magnification images confirm this staining pattern (Panels C and D, respectively). Similar staining patterns were noted in other metastatic lesions. 3
Supplemental Fig. 3 Immunohistochemical staining of metastatic tumor tissue for class I and II MHC expression in Patient IMF-32 Parafin-embedded tissue from a metastatic melanoma tumor resected from the left inguinal region in February of 2006 was analyzed for expression of MHC class I/II molecules. Representative images demonstrate no staining for class I and II MHC molecules within the tumor tissue but staining at the periphery of the tumor and surrounding soft tissue (Panels A and B, respectively). Higher magnification images confirm staining for class I and II MHC molecules only in cells surrounding the metastatic melanoma tumor but not the melanoma cells (Panels C and D, respectively). Similar staining patterns were noted in other metastatic lesions. 4
Supplemental Fig. 4 Changes in gp100- and tyrosinase-specific tetramer-reactive CD8 + T-cells following ipilimumab therapy in Patient IMF-24 Tetramer staining was accomplished in the same manner as Patient IMF-32. Panel A: representative dot plots reveal a significant increase in HLA-A*0201 gp100 209-217 but not HLA-A*0201 tyrosinase 369-377 tetramer-reactive CD8 + T- cells with ipilimumab therapy. Panel B: further characterization of the gp100-specific tetramer-reactive CD8 + T- cells reveals that most of these are CCR7 - CD45RA -, consistent with an effector-memory phenotype. 5
Supplemental Fig. 5 Changes in gp100- and tyrosinase-specific CD8 + IFN-γ + T-cells and evidence of polyfunctional responses in Patient IMF-24 ICS staining was performed as in Patient IMF-24. Representative dot plots show an increase in gp100-specific but not tyrosinase-specific CD8 + IFN-γ + T-cells following ipilimumab therapy. Control data (no peptide added) are provided in the top row. The bottom two rows show evidence of polyfunctionality, wherein the CD8 + IFN-γ + T-cells express MIP-1β and CD107a. 6
Supplemental Fig. 6 Hematoxylin & eosin and immune-specific staining cutaneous tumor site following ipilimumab therapy in Patient IMF-11 The site of prior cutaneous metastasis and clear clinical response was biopsied for evaluation for microenvironmental responses following ipilimumab therapy. Panel A: H&E staining showed a tumorous nodule with melanin pigment, infiltrated with macrophages and lymphocytes and without evidence of tumor cells or melanocytes. Panel B: Immunostaining demonstrated that these lymphocytes were predominantly of the CD8 + T-cell subtype. 7