Figure 1.
Mice with the citation (cit) allele of H2-Aa strongly inhibited melanoma growth. (A) Left panel: B16F10 melanoma volume on day 20 post-injection s.c. of B16F10 cells into the flank of third generation (G3) descendants of a single G1 male mouse, with REF (+/+), HET (+/mutant), or VAR (mutant/mutant) genotypes for H2-Aa (n = 7 B6, 5 REF, 9 HET, 10 VAR). Right panel: Manhattan plot showing −log10(P values) (Y axis) plotted versus chromosomal positions of mutations (X axis) identified in the G1 founder of the affected pedigree using a recessive model of inheritance. Horizontal red or orange lines represent thresholds of P = 0.05 with or without Bonferroni correction, respectively. The strongest mutation–phenotype association is for a mutation in H2-Aa. (B)H2-Aa transcript diagram showing the location of the citation mutation (red asterisk), a G to A transition of the fifth nucleotide of intron 1. Corresponds to 1,128-bp NCBI reference sequence NM_010378.3. (C) RT-PCR analysis of H2-Aa using primers complementary to sequences in exons 1 and 4. No H2-Aa cDNA could be detected in H2-Aacit/cit splenocytes. (D) WT or H2-Aacit/cit splenic B cell (CD19+) MHC-II surface expression detected by flow cytometry. (E–G) Tumor growth curves of B16F10 melanoma (E) (n = 7 +/+, 22 +/cit, 9 cit/cit), YUMM1.G1 melanoma (F) (n = 11 +/+, 14 cit/cit), and MC38 colon carcinoma (G) (n = 10 +/+, 8 cit/cit) after s.c. inoculation on day 0 into the flank of mice. No PD1 antibody was administered. (H) Survival curves of mice after i.v. inoculation with B16F10 melanoma on day 0. Mice were intraperitoneally (i.p.) injected with anti-PD1 or vehicle (PBS) twice per week till the end of the experiment (death or euthanasia) (n = 16–24 per group). (I) Tumor growth curve of B16F10 melanoma in which H2-Aa was knocked out (KO) after s.c. inoculation on day 0 into the flank of mice (n = 7 +/+, 9 cit/cit). (J) Tumor growth curve in the presence of cell depleting antibodies. B16F10 cells were injected s.c. on day 0 into the flank of mice. Anti-CD4, anti-CD8, anti-NK1.1, or control IgG, were injected i.p. into H2-Aacit/cit (cit) mice on days 0, 3, 6, 9, 12, and 15 after tumor inoculation to deplete the corresponding cells (n = 4 per group). (K–M) Frequency of tumor infiltrating lymphocytes (K), CD8 T cells, Treg, tTreg, and pTreg (L), and the phenotype of CD8 T cells (M) in B16F10 tumors collected on day 13 after B16F10 inoculation (n = 4 per group). Data points represent individual mice (A and K–M). Data are representative of one experiment (A) or two independent experiments (C–M). WT littermates (C–M) and WT C57BL/6J mice from JAX (A) were used as controls. Error bars indicate SD (A left panel, K–M) or SEM (E–G, I, and J). P values were determined by Student’s t test (A left panel, K–M), two-way ANOVA with post-hoc Tukey test (E–G, I, and J), or log-rank test (H). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Mice with the citation (cit) allele of H2-Aa strongly inhibited melanoma growth. (A) Left panel: B16F10 melanoma volume on day 20 post-injection s.c. of B16F10 cells into the flank of third generation (G3) descendants of a single G1 male mouse, with REF (+/+), HET (+/mutant), or VAR (mutant/mutant) genotypes for H2-Aa (n = 7 B6, 5 REF, 9 HET, 10 VAR). Right panel: Manhattan plot showing −log10(P values) (Y axis) plotted versus chromosomal positions of mutations (X axis) identified in the G1 founder of the affected pedigree using a recessive model of inheritance. Horizontal red or orange lines represent thresholds of P = 0.05 with or without Bonferroni correction, respectively. The strongest mutation–phenotype association is for a mutation in H2-Aa. (B)H2-Aa transcript diagram showing the location of the citation mutation (red asterisk), a G to A transition of the fifth nucleotide of intron 1. Corresponds to 1,128-bp NCBI reference sequence NM_010378.3. (C) RT-PCR analysis of H2-Aa using primers complementary to sequences in exons 1 and 4. No H2-Aa cDNA could be detected in H2-Aacit/cit splenocytes. (D) WT or H2-Aacit/cit splenic B cell (CD19+) MHC-II surface expression detected by flow cytometry. (E–G) Tumor growth curves of B16F10 melanoma (E) (n = 7 +/+, 22 +/cit, 9 cit/cit), YUMM1.G1 melanoma (F) (n = 11 +/+, 14 cit/cit), and MC38 colon carcinoma (G) (n = 10 +/+, 8 cit/cit) after s.c. inoculation on day 0 into the flank of mice. No PD1 antibody was administered. (H) Survival curves of mice after i.v. inoculation with B16F10 melanoma on day 0. Mice were intraperitoneally (i.p.) injected with anti-PD1 or vehicle (PBS) twice per week till the end of the experiment (death or euthanasia) (n = 16–24 per group). (I) Tumor growth curve of B16F10 melanoma in which H2-Aa was knocked out (KO) after s.c. inoculation on day 0 into the flank of mice (n = 7 +/+, 9 cit/cit). (J) Tumor growth curve in the presence of cell depleting antibodies. B16F10 cells were injected s.c. on day 0 into the flank of mice. Anti-CD4, anti-CD8, anti-NK1.1, or control IgG, were injected i.p. into H2-Aacit/cit (cit) mice on days 0, 3, 6, 9, 12, and 15 after tumor inoculation to deplete the corresponding cells (n = 4 per group). (K–M) Frequency of tumor infiltrating lymphocytes (K), CD8 T cells, Treg, tTreg, and pTreg (L), and the phenotype of CD8 T cells (M) in B16F10 tumors collected on day 13 after B16F10 inoculation (n = 4 per group). Data points represent individual mice (A and K–M). Data are representative of one experiment (A) or two independent experiments (C–M). WT littermates (C–M) and WT C57BL/6J mice from JAX (A) were used as controls. Error bars indicate SD (A left panel, K–M) or SEM (E–G, I, and J). P values were determined by Student’s t test (A left panel, K–M), two-way ANOVA with post-hoc Tukey test (E–G, I, and J), or log-rank test (H). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

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