Extended Data Fig. 4: Anti-human-CD25NIB (RG6292) depletes Treg and drives T cell activation in tumor-bearing humanized mice.
Stem cell humanized female NOG mice bearing an established s.c. BxPC-3 tumor were injected i.p. with vehicle, RG6292 [4 mg/kg] or Ipilimumab [10 mg/kg]. After 72 hrs, splenocytes, blood lymphocytes and tumor infiltrating lymphocytes were isolated and evaluated for counts of activated CD8+ T cells (huCD45+, huCD3+, huCD8+ huCTLA-4+) and Tregs (huCD45+, huCD3+, huCD4+, huFoxP3+) as well as for markers of recent T cell activation. (a) Ipilimumab as well as RG6292 decreased the intratumoral Treg counts. An increase of intratumoral activated CD8+ T cell count was only evident after administration of RG6292. Normalized counts were plotted for the respective treatment groups. Each symbol represents one animal (n=5 mice), CD8 and Treg cells are connected for the same animals (b) Intratumoral CD8+ T cells after RG6292 treatment were highly activated and had increased levels of HLA-DR, PD-1 and CTLA-4 (MFI as well as % of positive cells). Each symbol represents one animal (n=5 mice). The box and whiskers plots show minima and maxima and the median. Statistical analysis of RG6292 and Ipilimumab treated groups against vehicle group is indicated. Data was analyzed using 2-way ANOVA, Dunnet’s multiple comparisons test (ns=p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001) (p-value between RG6292 and Ipilimumab was 0.0001 for CTLA4 MFI on CD8 T cells and 0.0008 for HLA-DR on CD8 T cells. (c) Representative FACS plots showing CD25 expression versus FoxP3 expression in CD4+ T cells and PD-1 expression versus CTLA-4 expression in CD8+ T cells for vehicle, RG6292 and Ipilimumab treated animals.
