Extended Data Fig. 6: Loss of NRP1 upregulated Id3 and CXCR5 expression on pT_EX infiltrating primary B16-gp100 tumors.

The E8I^CreNrp1^L/L and E8I^Cre strains on pMel-1 background were further crossed with an Id3-GFP reporter mouse strain, resulting in one mutant allele carrying the modified Id3 locus with insertion of sequence encoding GFP into the ATG initiation codon (Id3^gfp/+). a. Scheme for pMel-T cell adoptive transfer. Briefly, congenically-mismatched bulk CD8⁺ T cells were purified from the following 3 groups of naive mice (pMel-1×E8I^CrexId3^+/+(CD45.2⁺Thy1.1⁺), pMel-1×E8I^CrexId3^gfp/+ (CD45.2⁺Thy1.1⁺Thy1.2⁺) and pMel-1×E8I^CreNrp1^L/LxId3^gfp/+ (CD45.2⁺Thy1.2⁺)), co-transferred at 1:1:1 ratio into CD45.1 recipients, followed by B16-gp100 tumor implantation one day later. Tumor-infiltrating CD8⁺ T cells were analyzed on D12 or D18. b, Frequency of Id3-GFP⁺ within Nrp1^+/+Id3^gfp/+ and Nrp1^–/–Id3^gfp/+ donor cells; c, Representative flow cytometry plots (left) depicting the phenotype of intratumoral Id3-GFP⁺ vs. Id3-GFP^– cells. Bar plot (right) tabulating the composition of Id3-GFP⁺ and Id3-GFP^– fractions, from Nrp1^+/+ or Nrp1^–/–-derived donors, respectively (n = 6 per group). d, Expression of CXCR5 and Ki67 on B16-gp100 tumor-infiltrating pMel-T cells, recovered on D21 post implantation. Cells shown in the representative plot were gated on Nrp1^+/+ and Nrp1^–/– donors by the congenic markers. Bar graphs tabulating the genomic mean fluorescence intensity (gMFI) and the percentage of CXCR5⁺ cells within Ki67⁺ and Ki67^– fraction within each genotype. Data were pooled from 2 independent experiments, with 9 recipient mice per experiment in a-c, 4 replicates per group in d. Error bars, mean±s.e.m; Statistical significance was determined by two-tailed paired Student’s t test (b, d).

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