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Blood, Vol. 109, Issue 12, 5346-5354, June 15, 2007
CD4 cells can be more efficient at tumor rejection than CD8 cells
Blood Perez-Diez et al.
109: 5346
Supplemental materials for: Perez-Diez et al, Vol 109, Issue 12, 5346-5354
Supplemental Methods Evaluation of siRNA sequences against MHC class II molecule by transient transfection experiments
We designed eight targeting sequences (sequences no. 1 to 8, Figures S1 and S2) of siRNA against MHC class II molecule (H-2A b) based on suggestions from Dr. Tuschl’s lab1 and onalgorithms from Dharmacon (Lafayette, CO), and purchased synthetic siRNA oligonucleotides from Dharmacon. To screen for the best sequence, we transiently transfected three different cell lines: the Ab-expressing P12 cell line, 293T cells simultaneously co-transfected with the and the  chains of Ab, and MB49. Down-regulation of Ab, for P12 (Figure S1) inhibition of its de novo expression, for 293T, (Figure S1); or inhibition of its IFN -induced expression, for MB49 (Figure S2), was evaluated by flow cytometry. Generation of control viruses and virus titration
Control viruses were generated using empty pLL3.7 plasmids and shRNA plasmids against the bacterial galactosidase gene (GTGACCAGCGAATACCTGT, 1915-1933 of coding region 2). Viruses were titrated on the MB49 cell line, and the titer assessed on the basis of GFP expression of the target cells. Sorting MB49-Ab(i)
MB49-Ab(i) was stimulated with IFN- and FACS sorted two times to select for cells showing the most down-regulation of Ab expression. We sorted MB49Ø and MB49-LacZ(i) in the same way, gating on cells with same range of GFP levels as the sorted MB49-Ab(i) population. REFERENCES
1. Elbashir SM, Harborth J, Weber K, Tuschl T. Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods. 2002;26:199-213.
2. Qin XF, An DS, Chen IS, Baltimore D. Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5. Proc Natl Acad Sci U S A. 2003;100:183-188.
Files in this Data Supplement:
- Figure S1. Down-regulation of MHC Class II (Ab) and suppression of its de novo expression by different siRNA oligonucleotides (PDF, 28.9 KB) -
P12 cells, which express Class II Ab molecules, were transfected (105) in 24 well plates with nothing (mock), with siRNA Ab sequences (numbers 1 to 4), or with a control siRNA sequence for luciferase, and tested four days later for Ab expression by flow cytometry. Simultaneously, 293T cells (4 × 105) were co-transfected with the and chains of Ab and with the different siRNA oligonucleotides (as for P12), and tested two days later for Ab expression. Mock transfected cells lines were stained with isotype control antibody as well. Empty line is the Ab expression by mock transfected cells. Numbers represent the mean and median fluorescence of the grey histograms.
- Figure S2. Oligonucleotides numbers 2 and 3 compared against four new siRNA sequences (numbers 5 to 8) on IFN-γ stimulated MB49 (PDF, 29.4 KB) -
MB49 cells (2 × 105) were transfected in 24 well plates with nothing (mock), or with siRNA Ab sequences (numbers 2, 3 and 5 to 8, Figure S1). Twenty-four hours later, transfectants were stimulated with 500 IU/mL rmIFN- and tested two days later for Ab expression by flow cytometry. Mock tranfected MB49 was stained with isotype control antibody as well. Empty line is the Ab expression by mock transfected MB49. Gating on viable cells was by 7AAD exclusion. Numbers represent the mean and median fluorescence intensity of the grey histograms. Sequence number 2 was selected for cloning into the lentiviral vector.
- Figure S3. Lentivirus constructs used to generate MB49 stable transfectants (PDF, 20.7 KB)
- Figure S4. MB49 Ab(i) expresses low levels of Class II molecules after in vitro stimulation with IFN-γ (PDF, 23.3 KB) -
The three stable transfectants of MB49 and the parental line (mock) were stimulated with 500 IU/mL rmIFN- for two days and tested for eGFP and Ab expression by flow cytometry. Mock tranfected MB49 was stained with isotype control antibody as well (left panel). Numbers represent the percentage of Ab positive cells. Gating on viable cells was by 7AAD exclusion.
- Figure S5. B16 upregulates Class I and Class II molecules in vitro after IFN-γ stimulation (PDF, 43.6 KB) -
Expression of MHC Class I (Db) and Class II (Ab) by in vitro-grown B16 tumor cells that were either untreated (-) or incubated for two days with 500 IU/mL of IFN- (+IFN-). Unshaded area is staining seen with an isotype control.
- Figure S6. Lower levels of Class II expression by the tumor do not affect Marilyn’s antitumor activity (PDF, 15.3 KB) -
RagKO mice were challenged with either 105 MB49mock cells (no symbol, dashed lines), MB49-Ø (circles), MB49-LacZ(i) (squares), or MB49-Ab(i) (diamonds) cells. One day later, half of the mice in each group received 106 Marilyn cells (closed symbols). One representative experiment out of three is shown. Data in Figure 4B showed that lower expression of Class II by MB49 did not inhibit the anti-tumor effect of CD4 T cells. Here we show that the lower expression of Class II by MB49 also does not improve the anti-tumor effect, showing that, in this model, Class II expression by the tumor does not have a tolerogenic effect on CD4 cells.
- Figure S7. H-Y expression on tumor cell lines from male mice (PDF, 44.1 KB) -
RT-PCR of TC-tet, WR21, TRAMP-C2, IP2-E4, 3B-11 and 2F-2B tumor cell lines, done as in Fig 1B. Spleen cells from female B6 mice, and the MB49 tumor cell line, were used as negative and positive controls respectively.
- Figure S8. Marilyn CD4 T cells are primed in vivo after immunization of CD3KO-γcKO H-2k mice with male splenocytes (PDF, 14 KB) -
CD3KO-cKO H-2k mice were challenged with 105 MB49 cells subcutaneously (two middle panels) or not (left panel) and received 106 Marilyn cells one day later. Mice receiving Marilyn cells were immunized at days 2, 5, 7, 9, 13, and 17 after tumor challenge with 3 × 106 CD3KO male splenocytes. Twenty-three days after the first immunization, spleen cells were harvested and stained for TCR-, CD4 and CD44. Staining of spleen cells from an immunized female B6 mouse served as a positive control (right panel). Cells were gated on TCR-+ cells. Numbers represent the percentage of CD4 cells that are CD44 high for each mouse.
- Figure S9. Rachel CD4 TCR transgenic mice delayed the growth of an H-2k tumor (PDF, 15.7 KB) -
Female Marilyn (squares), female Rachel (diamonds) and male Marilyn (no symbol) mice were challenged with 3 × 105 3B-11 H-2k tumor cells. Tumor size was measured every 3-4 days. Each line represents a different mouse. Male Marilyn mice are a surrogate for RagKO mice, as they have no T cells and will not respond to the tumor: the tumor grows with similar kinetics as in the RagKO mice (Figure 7C). Rachel mice showed a stronger antitumor effect than MataHari mice even against this tumor that they cannot directly recognize.
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