|
|
Blood, Vol. 113, Issue 8, 1651-1660, February 19, 2009
Peripheral blood lymphocytes genetically modified to express the self/tumor antigen MAGE-A3 induce antitumor immune responses in cancer patients
Blood Fontana et al.
113: 1651
Supplemental materials for: Fontana et al
Files in this Data Supplement:
- Table S1. Patient HLA typing (PDF, 491 KB)
- Figure S1. Imaging of patient CIP-26 before and after treatment (JPG, 41.5 KB)
-
Pt CIP-26 underwent PET scan imaging before and after the vaccination treatment. During the 2nd cycle of the treatment (7th infusion) the patient experienced a complete clinical response (PET scan, November 2004).
- Figure S2. Identification of the MAGE-A3.A26 and B44 epitopes recognized by MAGE-A3-specific effectors of patient CIP-5 (JPG, 72 KB)
-
(A) MAGE-A3 cDNA fragments were obtained by enzymatic digestions with the restriction enzymes BglII, and either EcoRI, BamHI or HindIII. (B, C) MAGE-A3 cDNA fragments obtained by the enzymatic digestions were transfected in Cos-7 cells together with either HLA-A*2603 or -B*4402 cDNA alleles (B, C). Effectors cells recognized the BglII/EcoRI fragment but not the BglII/BamHI, indicating that the candidate peptide was contained in the 886–979 cDNA region. MAGE-3.A26 and -B44 effectors recognized Cos-7 cells transfected with either allele and pulsed with MAGE-A3250–258 peptide predicted by the SYFPETHI software (B, C).
- Figure S3. Dynamics of antitumor T cells frequency in patient CIP-5 (JPG, 74.9 KB)
-
PBMCs collected at different time points were stimulated with irradiated autologous tumor cells in limiting dilution conditions and tested for IFN-γ release against autologous tumor, activated T cells, and the NK target K562, after three weekly stimulations. Spontaneous antitumor effectors turned out to be 1.0 × 10−4 before vaccination and varied between 3.8 × 10–5 and 1.0 × 10–4 T cells during the course of the treatment. Frequency of anti–MelanA/MART-1.A2 and anti–MAGE-1.A26 T cells was estimated by the stimulation of multiple groups (105) of PBMCs with either the peptide (i.e. MelanA/MART-1.A2 or MAGE-1.A26), followed after 2 weeks by tetramer staining and IFN-γ release assay. Since the MelanA/MART-1.A26 peptide is unknown, the frequency of anti–MelanA/MART-1.A26 T cells was estimated by PBMC stimulation with tumor cells, followed by IFN-γ release assay on Cos-7 cells transfected with HLA-A26 and MelanA/MART-1 cDNAs. Anti–MelanA/MART-1.A2 effectors in the pre-vaccination sample were found at frequencies of 1.4 × 10–5 whereas after the treatment they varied from 2.5 × 10–6 to 1.2 × 10–6 among CD3+ lymphocytes. The frequencies of the anti MAGE-1.A26 effectors were 3.4 × 10–6 in the pre-vaccination sample, whereas they varied from 4.0 × 10−6 to 3.8 × 10−7 after vaccination. Even if stimulation with tumor cells may underestimate T-cell frequency compared to peptide stimulation, anti–MelanA/MART-1.A26 effectors were found to be approximately 5.4 × 10−5 in the pre-vaccination sample. They decreased after 14 infusions to 8.9 × 10−6, and then strongly increased up to 1 × 10−4 one year after the 15th infusion. After that they remained around 5 × 10−5 during the entire follow up. P stands for post-infusion.
- Figure S4. Comparison of anti–MAGE-A3.A26 frequency among TILs collected pre and post-treatment (JPG, 128 KB)
-
(A) Detection of anti–MAGE-A3.A26 T cells in TILs collected from a pre-treatment tumor sample of patient CIP-5. TILs were stimulated with M3-GML at 104 cells/well in the presence of IL-2 and IL-7. The microcultures were stimulated on days 7 and 14 in the same conditions. On day 21 cells were stained with MAGE-A3.A26 tetramer-PE and anti–CD8-FITC mAb and analyzed by flow cytometry. (B) Analysis of anti–MAGE-3.A26 precursor cells in TILs collected from a post-treatment tumor sample of CIP-5 and analyzed as in A. (C) The specificity of the post-treatment microcultures described in B was assessed by IFN-γ release assay at day 21 on UT-GML, autologous tumor cells (CIP-5 mel), Cos-7 cells transfected with HLA-A26 alone or together with MAGE-A3.
- Figure S5. Molecular analysis of circulating and tumor-infiltrating anti–MAGE-A3.A26 T cells in patient CIP-5 (JPG, 87.5 KB)
-
(A) Anti–MAGE-A3.A26 effectors isolated by MAGE-3.A26 tetramer from peripheral blood and tumor infiltrating lymphocytes of patient CIP-5, were analyzed by FACS and by RT-PCR for TCR Vβ chains usage. Both populations turned out to be Vβ7.1. (B) PCR products containing CDR3 region from both populations were sequenced and resulted to be identical. Anti–MAGE-3.A26 effectors expressed the Vβ7-J2.1 chain.
|
|
