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Tumor-associated macrophages in PTL
AD
B
C
Figure 1. Characterization of cell immunophenotypes with mIHC. (A-C) Representative images from 4-plex mIHC stainings. Panels (low, intermediate, and high) show representative images from the corresponding tertiles, based on the content of different immune cell subtypes. The insets highlight cells with higher magnification. PD-L1=blue, PD-L2=red, CD68=white, c-Maf=green (A); PD-L1=blue, CD163=red, CD20=white, PD1=green (B); CD3=blue, CD8=red, CD4=white, PD1=green (C). Scale bar 40 mm. (D) Proportions of distinct immune cell subpopulations from all cells. PD-L1+CD68+ indicating the content of PD-L1+ TAMs, PD-L1+CD163+ and PD- L1+CD68+c-Maf+ the content of PD-L1+ M2-polarized TAMs, PD-1+CD3+CD4+ and PD-1+CD3+CD8+ the content of PD-1+ TILs, and PD-L1+CD20+ the content of PD-L1+ lymphoma cells.
range 0.1-99%) expressed PD-L1. Due to a low proportion of PD-L2+ cells (0.06%) (data not shown), PD-L2 was excluded from further analyses.
We further observed that a high number of PD-L1+ cells, high proportion of PD-L1+CD68+ macrophages from all cells, as well as a high proportion of PD-L1+CD68+ macrophages from all CD68+ macrophages (PD- L1+CD68+/CD68+), associated with favorable OS when analyzed as continuous variables (Table 3). In order to use an objective cutoff, we stratified the patients into three equal subgroups based on tertiles of the PD-L1+CD68+ macrophage counts (high, intermediate, low). The 5-year OS and DSS rates were clearly worse for the patients with a low number of PD-L1+CD68+ macrophages (≤4.75% corresponding to the lowest tertile of the patients) in com- parison to the patients with intermediate or high numbers (>4.75%, 5-y OS, 39% vs. 66%, P=0.014; 5-y DSS, 53% vs. 76%, P=0.056; Figure 2A). When PD-L1+CD68+ macrophage count was included in a multivariate analysis with IPI, both factors had independent prognostic value for OS (Table 4). In contrast, neither PD-L1+ lymphoma cells, PD-L1+CD68– cells nor any other TAM phenotypes were significantly associated with survival (Table 3). When comparing the three PD-L1+CD68+ TAM subgroups (high, intermediate and low), no significant differences in age, molecular subtype, IPI score or treatments were observed (Table 1). However, high PD-L1+CD68+ macrophage count was associated with limited disease stage. When the patients treated in the pre-rituximab era were removed from the analyses, a trend towards worse survival was maintained for the patients with low number of PD-L1+CD68+ macrophages (≤5.97%, the lowest tertile; OS, P=0.093, Online Supplementary Figure S2A). These
Table 3. Cox regression analysis at the univariate level showing asso- ciation of cell immunophenotypes with overall survival.
Cell immunophenotype
PD-L1+
HRa 95% CI
0.983 0.967-0.999
1.009 0.995-1.023
0.993 0.978-1.008
0.981 0.955-1.007
0.986 0.964-1.008
0.965 0.933-0.999
0.987 0.975-0.998
1.012 0.982-1.043
0.835 0.668-1.044
0.734 0.518-1.041
0.996 0.978-1.014
0.989 0.969-1.010
0.194 0.053-0.712
0.089 0.008-0.999
0.042 0.003-0.537
P
0.038
0.209
0.376
0.146
0.196
0.042
0.027
0.437
0.113
0.083
0.666
0.298
0.013
0.050
0.015
CD20+ PD-L1+CD20+ PD-L1+CD68- CD68+
PD-L1 CD68
+++ PD-L1+CD68+/CD68
–+ PD-L1 CD68
CD68+c-Maf+ PD-L1+CD68+c-Maf+ CD163+ PD-L1+CD163+
+ CD3
PD-1+CD3+CD4+
PD-1+CD3+CD8+
aHR: hazard ratio; CI: confidence interval; PD-L1+CD68+ implies the number of PD-
L1+CD68+ TAMs from all cells; PD-L1+CD68+/CD68+ implies the number of PD-L1+CD68+ +
TAMs from all CD68 TAMs. Boldface font indicates statistical significance (P<0.05).
results highlight the clinical relevance and possible func- tional connection of PD-L1+ TAMs for PTL progression.
Association of PD1+ TILs with survival
Given the prognostic value of PD-L1+ TAMs, we then determined their association with T cells by mIHC. The marker CD3 was used to identify all T cells. Subpopulations of T cells were then defined by the pres-
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