|
|
Blood, Vol. 112, Issue 5, 1844-1852, September 1, 2008
Targeting a single mismatched minor histocompatibility antigen with tumor-restricted expression eradicates human solid tumors
Blood Hambach et al.
112: 1844
Supplemental materials for: Hambach et al
Files in this Data Supplement:
- Table S1. Characteristics of the tumor cell lines used in this study (PDF, 15.9 KB) -
The tumor cell lines show a differential pattern of the immunogeneic HA-1H allele, the non-immunogeneic HA-1R allele and the mHag H-Y on genomic level and of HA-1 mRNA expression. The tumor cell lines are derived from solid tumors of different entities.
- Figure S1. Expression of HA-1 mRNA and cell surface molecules in the tumor cell lines (JPG, 76.4 KB)
-
(A) HA-1 mRNA expression levels in different tumorcell lines. Depicted are the relative levels of HA-1 mRNA expression in different tumor cell lines using EBV LCLsas reference determined by quantitative PCR. Results were normalized with 18S mRNA. (B) Expression of cell surface molecules in different tumor cell lines and EBV LCLs determined by flow cytometry. X-axis: HLA-A2, the co-stimulatory molecules CD80 and CD86 and the cell adhesion molecules ICAM and VCAM, L-, E and P-Selectin, VLA-4, Mac-1 and LFA-3. Y-axis: mean fluorescence intensity (MFI) in relation to the isotype control.
- Figure S2. Detailed characterization of the human HA-1 CTL clones (JPG, 90.1 KB)
-
(A) Response of HA-1 specific CTL clones to serial dilutions of HA-1 peptide loaded on EBV LCLs in a Cr51 release assay. (B) HA-1 CTL clones were analyzed for the markers CD45RA and CD62L in order to categorize them as naïve (N), central memory (CM), effector memory (EM) or EM-RA cells. Subsequently, HA-1 CTL clones were stained for CD27 and CD28 to determine the differentiation stage of the CTLs. PBMCs are shown as controls.
- Figure S3. Infiltration of microtumors by different CTL clones (JPG, 66 KB)
-
Microtumors were co-incubated with different HA-1 CTLs and H-Y CTLs for 3 days in an effector-target ration of 15:1 and tumor infiltration by CTLs was determined by immunohistochemistryfor CD8. X-axis: the HA-1 CTL clones 1.7, 2.12 and 3HA15 and the H-Y CTL clone 21-17; Y-axis: number of CD8 + staining cells/mm2 on median 7 parallel sections (3-13) per CTL clone.
- Figure S4. Long-term monitoring of microtumor growth after CTL treatment (JPG, 77.6 KB)
-
Microtumors were generated from (A) tumor cell lines from in vitro culture or (B) MDA-MB 231 cells explanted from s.c. tumors grown in NOD/scid mice for 4 weeks. Each line represents mean microtumor area +/− SEM (median 3 (range 3-5) microtumors per condition). X-axis: day of culture; y-axis: microtumor area (mm2). Treatment: HA-1 CTLs (E:T ratio 15:1) ♦; H-Y CTLs (E:T ratio 15:1) ◇; allo-HLA-A2 CTLs (E:T ration 15:1) ●; medium ■. Tumor growth in the medium control wells was terminated on day 6 due to beginning overgrowth of the culture well.
- Figure S5. In vivo distribution of HA-1 CTLs (JPG, 46 KB)
-
NOD/scid mice with s.c. MDA-MB 231 macrotumors received i.v. a single dose of 30 × 106 HA-1 CTLs (clone 1.7). HA-1 CTLs were quantified in lung, liver, spleen, bone marrow, peripheral blood and the solid tumor at 1, 3, 7, 14 or 21 days after CTL transfer. Depicted are the absolute numbers of viable human CTLs in the individual tissues. Bars correspond to mean +/− S.E.M. (4 mice per measurement time point).
- Figure S6. Kinetic of CTLs in the peripheral blood in the pulmonary microtumor model (JPG, 37.6 KB)
-
On the X-axis is depicted the time after HA-1 or H-Y CTL inoculation. On the Y-axis is depicted the mean CD8 cell counts +/− SEM in the peripheral blood of mice (6 mice per group). Treatment: closed squares: HA-1 CTL clone; triangles: H-Y CTLs.
- Figure S7. Connective tissue in MDA MB-231 tumors grown in vitro and in vivo (JPG, 185 KB)
-
In vitro microtumors, pulmonary metastases and s.c. tumors were stained with H&E (A) and with the connective tissue stains Masson’s Trichrome(B) and Resorcin-Fuchsin counterstained with van Gieson’s (C). (B) Masson’s Trichrome: collagen – green, cytoplasm – red, nuclei – dark brown; arrowheads: collagen (green). (C) Resorcin-Fuchsin counterstained with van Gieson’s: elastic fibres - black, collagen – red, cytoplasm – yellow/brown, nuclei – brown; arrowheads: micro-and macrotumor: collagen (red), pulmonary metastases: elastic fibres(black); bar,100 µm.
|
|
