fcγr murine mastocytoma cell line p815  (ATCC)

Journal: The Journal of Clinical Investigation

Article Title: Mitochondrial complex II orchestrates divergent effects in CD4 + and CD8 + T cells

doi: 10.1172/JCI194134

Figure Lengend Snippet: ( A ) B6 and BALB/c mice were transplanted with WT or SDHA-KO T cells. Survival rate and clinical GVHD score are shown (syngeneic n = 8/group, allogeneic n = 10/group). ( B – D ) BALB/c mice received HCT with P815 cells (MHC class I + class II – ). ( B ) BALB/c mice received syngeneic or allogeneic T cells from WT or SDHA-KO mice and were infused concurrently with 1.0 × 10 3 luciferase + P815 cells. ( C ) Tumor-related mortality (syngeneic n = 5, allogeneic n = 11/group) and clinical GVHD score ( n = 5/group). ( D ) Representative bioluminescence images and tumor burden on day 14 after HCT ( n = 3–10/group). ( E ) BALB/c mice received HCT (WT or SDHA-KO B6 → BALB/c). Representative flow cytometry images and Annexin V + 7-AAD + cells in donor-derived (H-2Kb + ) CD4 + and CD8 + T cells day 7 after HCT ( n = 6/group). ( F ) Representative flow cytometry measuring proliferation of donor-derived (H-2Kb+CD45.2 + ) CD4 + and CD8 + T cells day 7 after HCT ( n = 3–6/group). ( G ) Representative flow cytometry images and granzyme A and perforin levels in donor-derived (H-2Kb + CD45.2 + ) CD8 + T cells day 7 after HCT ( n = 3–10/group). ( H ) Representative flow cytometry images and granzyme A, granzyme B, and perforin levels in donor-derived (H-2Kb + CD45.2 + ) CD4 + T cells day 7 after HCT ( n = 3–10/group). ( I ) WT and SDHA-KO mice were inoculated with B16-F10 melanoma cells on day 0 and injected with anti–PD-1 or isotype control antibody on days 7, 10, 13, and 16. Tumor growth was measured ( n = 6–9/group). Two-tailed Mann-Whitney U test for GVHD score and Annexin V + 7-AAD + , Mantel-Cox log rank test for survival, 1-way ANOVA with Tukey’s post hoc test ( D and F – H ), and 2-way ANOVA with Tukey’s post hoc test ( I ) were used (mean ± SEM). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: P815 leukemia cells (catalog TIB-64) were purchased from ATCC and were transduced with luciferase ( ).

Techniques: Luciferase, Flow Cytometry, Derivative Assay, Injection, Control, Two Tailed Test, MANN-WHITNEY

Journal: Cancer Discovery

Article Title: Intrinsic Properties of the Lymph Node Render It Immunologically Susceptible to Metastasis

doi: 10.1158/2159-8290.CD-24-1847

Figure Lengend Snippet: Sequestration of IL2 by Tregs permits survival of tumor cells in the LN. A, Experimental design to isolate OT-I from the tdLN of 6694c2-OVA tumor–bearing Foxp3 DTR mice treated with PBS, DT, or DT + anti-IL2 and coculture them with M167M1-OVA tumor cells. B and C , Representative images ( B ) and normalized killing ( C ) of M167M1-OVA-mCherry cells cocultured 1:1 with OT-I cells isolated from PBS, DT, and DT + anti-IL2 groups. Scale bar, 400 μm. D, Experimental setup: tdLNs were harvested from a patient with PDAC at rapid autopsy, sliced into 2-mm cubes, and cultured for 24 hours in media ± rIL2. CD8 + T cells were isolated from tdLN samples and incubated with αCD3 pulsed P815 cells. E, F, and G, Quantification of annexin V staining in P815 target cells after incubation with CD8 + T cells derived from control tdLNs or tdLNs exposed to rIL2 ( E ), representative plot ( F ) and quantification ( G ) of GZMB substrate (delivered GZMB, GranToxiLux Kit) within target cells that were incubated with CD8 + T cells from control or rIL2-exposed tdLNs. H, Representative images of LLC-OVA tumor growth in LNs from Foxp3 DTR mice treated, from left to right, with vehicle control, DT, DT + anti-CD8, DT + anti-IL2, rIL2, and rIL2 with anti-CD8. Scale bar, 3 mm. I and J, Quantification of OVA-mCherry MFI ( I ) and YFP + /mCherry + tumor area per LN ( J ) across groups. K, Barplots representing the frequency of LLC-OVA tumor growth in LNs across groups ( n = 10 per group). L and M, Limiting dilution plots comparing the frequency of 6694c2-OVA ( L ) and LLC-OVA ( M ) tumor formation in the LN after intra-LN injection (black line) or in the lungs after intravenous injection (red line). Shaded area represents the 95% confidence interval. Statistical differences between tumor formation efficiency after LN injection vs. intravenous injection are listed for each cell line [color of the P value indicates which injection method was more efficient at forming tumors (red = intravenous, black = intra-LN)]. Statistical significance was assessed using generalized linear models for multiple regressions followed by Bonferroni multiple comparisons correction. Total n = 80 mice, 20 per condition. Barplots represent the fold difference between the efficiency of tumor formation after intra-LN injection compared with intravenous injection using parental cells (left), OVA + cells (middle), and OVA + cells in Treg-depleted mice (right). Dotted line represents no fold difference. Statistical differences determined with unpaired two-tailed Student t test ( E and G ), one-way ANOVA with Tukey HSD correction for multiple comparisons ( C , I , and J ), Fisher exact test corrected for multiple comparisons ( K ) or as described above. Ctrl, control; MFI, mean fluorescence intensity; n.s. not significant. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. ( A and D, Created with BioRender.com .)

Article Snippet: P815 mastocytoma cells were generously provided by Michael Betts, originally from the ATCC.

Techniques: Isolation, Cell Culture, Incubation, Staining, Derivative Assay, Control, Injection, Two Tailed Test, Fluorescence