10 | Nature | www.nature.com Article d 5 10 15 20 *** * **** ** 30 40 50 60 Ki-67 + CD4 + T H (%) 10 13 17 21 PE–Tetramer BV421–PD-1 0% 83.0% 9.19% 7.85% 37.8% 41.7% 0 –10 3 10 3 10 4 10 5 0 –10 3 10 3 10 4 10 5 0.2% 20.3% 0 –10 3 10 3 10 4 10 5 e 10 15 0 1,000 2,000 Isotype 44.H1 Anti-PD-1 44.H1 + anti-PD-1 10 15 0 100 200 300 400 500 ** *** **** **** **** **** 0 5 10 15 20 0 50 100 Survival Isotype 44.H1 44.H1 + anti-PD-1 Anti-PD-1 Isotype 8 8 8 8 0 0 44.H1 9 9 9 9 0 0 Anti-PD-1 9 9 9 8 2 2 Combo 9 9 9 9 7 7 0 5 10 15 20 23 4 7 4 9 17 *** g h i j 0 10 20 30 40 20 0 20 40 60 80 100 P = 0.07 Time after inoculation (d) Untreated Anti-PLA2G2D Ki-67 + T reg (%) 10 15 20 25 30 0 500 1,000 1,500 Pla2g2d –/– + anti-PD-1 Pla2g2d –/– Pla2g2d +/+ + anti-PD-1 Pla2g2d +/+ **** * f Tumour volume (mm 3 ) Time after inoculation (d) Time after inoculation (d) Time (d) Time after inoculation (d) Time after inoculation (d) Tet + (% of CD8 + ) * Isotype 44.H1 Anti-PD-1 44.H1 + anti-PD-1 IL-2 + (% of CD8 + ) * Isotype 44.H1 Anti-PD-1 44.H1 + anti-PD-1 **** a * 0 10 20 30 40 50 0 1 2 3 4 TDLNs Tet + (% of CD8 + ) 0 10 20 30 40 Ki-67 + (% of CD8 + Tet + ) Isotype Anti-PLA2G2D * *** 0 1 2 3 4 Blood P = 0.07 P = 0.06 0 20 40 60 80 ** 0 80 85 90 95 100 Tumour Ki-67 + (% of CD8 + ) 10 13 17 21 10 13 17 21 10 13 17 21 Time after inoculation (d) Time after inoculation (d) Time after inoculation (d) 0 20 40 60 80 0 5 10 15 20 0 5 10 15 20 25 * ** *** ** P = 0.06 Isotype Anti-PLA2G2D c b BB515–Ki-67 ** **** Naive CM EM 0 20 40 60 80 100 Ki-67 + cells (%) BV650–CD44 Isotype Anti-PLA2G2D Isotype Anti-PLA2G2D 4.34% 4.79% 10.2% 80.7% 6.01% 7.86% 12.2% 73.9% 0 10 4 10 5 0 10 4 10 5 0 –10 2 10 2 10 3 10 4 0 –10 3 10 3 10 4 10 5 Tumour volume (mm 3 ) Tumour volume (mm 3 ) Fig. 5 | Dual targeting of PLA2G2D and PD-1 checkpoints improves anti- tumour immunity. a , Immune monitoring in isotype- or anti-PLA2G2D antibody-treated mice bearing LN6-987AL tumours (see Fig. 4a). Graphs show percentages of TRP2 tetramer-positive (Tet + ) or Ki-67 + (Tet + or total) CD8 + T cells across the indicated tissues ( n = 6 per group). b , Representative flow-cytometry analysis showing Ki-67 versus CD44 signals on TDLN CD8 + T cells from isotype- or anti-PLA2G2D antibody-treated mice (day 10). c , Percentages of Ki-67 + naive, central memory (CM) and effector memory (EM) CD8 + T cells in blood from isotype- or anti-PLA2G2D antibody-treated mice (day 13; n = 6 per group). d , Percentages of Ki-67 + CD4 + T helper (T H ) cells (top) and T reg cells (bottom) in blood from isotype- or anti-PLA2G2D antibody-treated mice ( n = 6 per group). e , Representative flow-cytometry analysis showing TRP2 tetramer versus PD-1 signals on tumour CD8 + T cells from isotype- or anti-PLA2G2D antibody- treated mice (day 13). f , Tumour volumes in Pla2g2d +/+ and Pla2g2d −/− mice after inoculation with MC38 tumour cells and treatment with isotype control ( n = 9) or anti-PD-1 ( n = 10) antibodies. g , Kaplan–Meier survival curve of humanized PLA2G2D mice inoculated with B16F10 melanoma cells and treated with isotype control (grey; n = 8), anti-PLA2G2D (44.H1; purple; n = 9), anti-PD-1 (blue; n = 9) or anti-PLA2G2D (44.H1) and anti-PD-1 (combo; red; n = 9) antibodies. h , i , Tumour volumes in humanized PLA2G2D mice after inoculation with B16F10 ( h ; isotype, n = 13; other, n = 16) or LN6-987AL ( i ; isotype, n = 9; other, n = 10) melanoma cells. j , Percentages of tumour-specific (Tet + ) CD8 + T cells (left) and IL-2 + CD8 + T cells (right) in tumours from mice treated with isotype control ( n = 9), anti-PLA2G2D (44.H1), anti-PD-1 or anti-PLA2G2D (44.H1) and anti-PD-1 antibodies ( n = 10 per group). Line and bar graphs denote mean ± s.e.m. P values by two-way ANOVA with Fisher’s LSD ( a , d , f , i ) or Tukey’s multiple comparisons test ( h ), two-sided unpaired t -test ( c ), one-way ANOVA with Tukey’s multiple comparisons test ( j ) or log-rank test ( g ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (exact P values in Supplementary Data).