CD45 high “Infiltrating Leukocytes”

1. Supplementary Figures * * * * * * * * CD45lowCD11b+ “Microglia” CD45highCD11b- “Lymphocytes” CD45high “Infiltrating Leukocytes” CD45highCD11b+ “GIMs” CD45high “Infiltrating Leukocytes” CD45highCD11b+ “GIMs” CD45highCD11b- “Lymphocytes” CD45lowCD11b+ “Microglia” Suppl. Figure S1: Immunophenotyping of the K1492 glioma microenvironment in response to sterile injury. C57Bl/6 mice implanted with K1492 cells and analyzed by flow cytometry at 15 days-post implantation, 3 days post-treatment with intracranial PBS (PBS, n=4) or untreated (NoTx, n=4). Non-tumour bearing mice were used as a control (NT, n=4). Quantified numbers of each cell type isolated from the tumour-bearing hemisphere (top). Error bars represent standard error, and asterisks indicate statistical difference (p<0.05). Scatter graph of isolated immunocytes as a percent of the total CD45 population present in the tumour-bearing hemisphere compared to PBS treatment (bottom).

2. K1492 + 7dpTx MYXV Suppl. Figure S2:Immunophenotyping of the K1492 glioma microenvironment after Myxoma virus treatment.A – 14 day K1492 tumour in wildtype mice with Iba1 staining demonstrating the ‘gradient of activation.’ Series of three photos stitched together from 10X objective. White line denotes approximate tumour border. B - C57Bl/6 mice were implanted with K1492 cells and treated with MYXV 14 days post-implantation. Formalin-fixed paraffin sections were stained with H&E, Myxoma virus protein M-T7 or for the microglial/macrophage marker Iba1 (First row 25X; Second row 200X). K1492 NoTx Iba1 200x panels represent within tumour (left) and adjacent to tumour (right) without MYXV treatment. Representative pictures of 2-3 animals/group. White arrows show areas of polymorphonuclear cell infiltration while black arrow show areas of focal necrosis. K1492 + 3dpTx MYXV K1492 14days post-implantation, Iba1 stain K1492 + 1dpTx MYXV A M-T7 Iba1 H&E B

3. 1dpTx 1dpTx H&E Magnification H&E M-T7 Suppl. Figure S3: Virus infected areas of K1492 tumours are accompanied by polymorphonuclear cell infiltration. Formalin-fixed paraffin sections were stained using Hematoxylin and Eosin (H&E) or immunohistochemical for MYXV early protein M-T7. White arrows show areas of intense infiltration of polymorphonuclear cells. Far right picture is 400X displaying polymorphonuclear phenotype.

4. A K1861 B H&E IbaI High Grade Mid-High Grade Suppl. Figure S4:K1861 and Spontaneous NPcis gliomas in C57Bl/6 mice have significant microglia and/or GIM recruitment. A –NPcis cell line K1861 was orthotpically grafted into C57Bl/6 mice and tumours were stained for Iba1 14 days post-implantation.B -C57Bl/6 mice heterozygous for Nf1 and Tp53 inactivating mutations spontaneously develop astrocytomas. Two such spontaneous astrocytomas, a high grade (top) and mid-high grade (bottom) were stained with haematoxylin and eosin (H&E) or with the microglia/macrophage marker Iba1.

5. A B Suppl. Figure S5: Non-tumour bearing CCR2-deficient animals are only slightly impaired in clearing MYXV. Naïve CCR2-null mice (n=5) or WT (n=5) were infected with 5x106 FFUs of vMyx-FLuc and followed for bioluminescence (A) and survival (B). Experiment was terminated after 100 days.

6. A * * * * NK1.1-CD3+ “T cells” NK1.1+CD3- “NK cells” B WT-K1492 + MYXV CCR2- K1492 WT- K1492 CCR2-K1492 + MYXV CD45 CD11b + * C * Suppl. Figure S6:CCR2-deficient mice have an exaggerated recruitment of NK and T cells to the K1492 glioma in response to Myxoma treatment. Wildtype (WT) or CCR2-deficient (CCR2) C57Bl/6 mice implanted with K1492 cells and analyzed by flow cytometry at 15 days-post implantation, 3 days post-treatment with Myxoma virus (MYXV) or untreated (NoTx). Initial live gating was around small lymphocyte population followed by interrogation of the CD45high population within this gate. Quantified numbers of each cell type isolated from the tumour-bearing hemisphere demonstrating: A - NK cells as measured by NK1.1+CD3- and T cells as NK1.1-CD3+; B – Representative scatter plots from the small gated lymphocytes on the CD45/CD11b scatter plot. NK1.1 staining is grey population. C - Subsequent quantification of flow cytometry experiment using only the NK1.1 and DX5 antibodies to look at NK cell populations recruited to K1492 gliomas 3 dpTx. Error bars represent standard error and asterisks represent significant differences within mouse strain but between treatment groups. Plus signs represent significant differences between mouse strains within treatment groups. (n=3, p<0.05).

7. A * B Suppl Figure S7: Minocycline administration mimics CCR2-null viral clearance kinetics but does not result in a survival advantage. Minocycline hydrochloride (Sigma) was prepared fresh in DMSO and heated to 37oC immediately before every treatment. Wildtype C57Bl/6 mice implanted with K1492 cells were treated with Minocycline (Mino) or 50 mg/Kg twice a day starting at 10-11dpi, 50 mg/Kg once a day from 12-16dpi, and then 25 mg/Kg from 17-20dpi or with DMSO (Vehicle). Viral administration was given at 14dpi. A - Real-time monitoring of viral infection with bioluminescence using vMyx-FLuc. Error bars represent standard error and asterisks represent significant differences (Mann-Whitney, p<0.05; MYXV n=5, MYXV+Mino n=7). B – Survival of animals treaded with this regimen (Log-rank, Mantel-Cox, p=0.8630).

8. A * * + + + + NK1.1-CD3+ “T cells” NK1.1+CD3- “NK cells” B K1492 K1492 + MYXV CD45 K1492-IL2Rγ K1492-IL2Rγ MYXV CD11b Suppl. Figure S8: K1492 tumours in IL2Rγ mice are significantly depleted of NK and T cell populations.Wildtype (WT) or IL2Rγ-deficient (IL2Rγ) C57Bl/6 mice implanted with K1492 cells and analyzed by flow cytometry at 15 days-post implantation, 3 days post-treatment with Myxoma virus (5x106 FFUs vMyx-GFP; MYXV) or untreated. Initial live gating was around small lymphocyte population followed by interrogation of the CD45high population within this gate. Quantified numbers of each cell type isolated from the tumour-bearing hemisphere demonstrating: A - NK cells as measured by NK1.1+CD3- and T cells as NK1.1-CD3+; Error bars represent standard error and asterisks represent significant differences within mouse strain but between treatment groups. Plus signs represent significant differences between mouse strains within treatment groups (n=3, p<0.05). B – Representative scatter plots from the small gated lymphocytes on the CD45/CD11b scatter plot. NK1.1 staining is grey population.

9. A * * B Suppl. Figure S9: Non-tumour bearing IL2Rγ-deficient animals are impaired in clearing MYXV. Naïve IL2Rγ-null mice (n=5) or WT (n=5) were infected with 5x106 FFUs of vMyx-FLuc and followed for bioluminescence (A) and survival (B). Experiment was terminated after 100 days.

10. A NoTx vs. CPA, p=0.0771 CPA vs. MYXV + CPA, p=0.1191 C CD45 CD11b Suppl Figure S10: Single low-dose cyclophosphamide combined with Myxoma treatment did result in improved treatment efficacy (A) or viral infection (B). However, repeated low-dose treatments resulted in severe lymphoablation of tumour-resident as well as treatment recruited leukocytes (C).