Image data acquisition and processing of FL tissues using IMC
In total, 39 lymph node samples from FL patients, including 26 FL samples at diagnosis and 13 paired samples at POD24, were subjected to IMC analysis after staining with 36 meta-tagged antibodies (Fig. 1A). As shown in Figure 1, the overlay of selected markers enabled the visualization of immune components, such as FL cell staining for CD19 and Bcl-2 in follicles (normal germinal center B cells do not express Bcl-2; Fig. 1B–D), CD4+ and CD8+ T cells (Fig. 1E, F), macrophages (CD68, Fig.1G), and structural cells such as FDCs (CD21, Fig. 1I) and FRCs (SMA and vimentin, Fig. 1I). In addition, the interfollicular region and follicles could be precisely gated using SMA, vimentin, and CD21 (Fig. 1I). After cell segmentation (Fig. 1J), spatial and phenotypic data were extracted and subjected to analysis of cell categories (Fig. 1K), marker expression (Fig. 1L), neighborhood relationships, and cell–cell interactions (Fig. 1M).
Landscape of the TIME in FL as revealed by IMC
To further illustrate the landscape of the TIME, the cell components were classified by their canonical marker expression. In total, 12 clusters in the tumor environment were displayed, including four FL subsets (clusters 1, 8, 10, and 11); two subsets of CD4+ helper T cells (Th) distinguished by inducible T-cell costimulator (ICOS) expression including cluster 2 (ICOS-Th) and cluster 12 (ICOS+Th); two types of tumor-associated Mφs (TAMs), including cluster 4 (CD163+Mφs) and cluster 6 (CD163−Mφs); CD8+ T cells (cluster 5); normal B cells (cluster 9); and Tregs (cluster 7) in the TIME (Fig. 2A). We further quantified clusters in the follicular and interfollicular regions. As a result, TAMs, ICOS-Th (cluster 2) and CD8+T cells as well as FRCs were significantly increased in the interfollicular regions, whereas four FL subsets, normal B cells and ICOS+Th (cluster 12) were more abundant in follicles (Fig. 2B, C). Consequently, cellular compositions in immune microenvironments of FL showed heterogeneities in spatial distribution that could be comprehensively revealed by IMC.
Evolution of immune and tumor compositions in the follicular and interfollicular area during POD24 in FL
Although immune compositions in the TIME at diagnosis were reported to be associated with POD24 in FL, the evolution of immune cells and its mechanisms in the TIME during POD24 are unknown. As cellular compositions were highly heterogeneous in the intra- and inter-follicle regions, we separately evaluated the dynamic alterations in inter- and intra-follicular region immune components during POD24 in FL. In the follicles, we found that the frequency of CD163−Mφs (cluster 6) was significantly higher at POD24 (Fig. 3A, C). However, unlike that in the follicular regions, the percentage of CD163+Mφs, rather than CD163−Mφs, in the interfollicular regions was markedly elevated at POD24 (Fig. 3B). In addition to that of CD163+ Mφs, the frequency of interfollicular ICOS-Th was significantly decreased after POD24 (Fig. 3B). Additionally, regarding FL cells, only the percentages of cluster 8 (CD27+CD45RO−HLA-DR+) in the interfollicular regions were significantly decreased at POD24, whereas FL subsets in the follicles were not significantly altered (Fig. 3B). Further phenotypic analysis showed higher expression of programmed cell death protein ligand 1(PD-L1) rather than PD-L2 in FL cells (cluster 8) in the interfollicular area after POD24 (Fig. 3D). Thus, TAMs evolved distinctly in the follicular and interfollicular areas along with a few other subsets, including Tcons and cluster 8.
Imbalanced evolution in TAMs and CD8+ T cells surrounding FL cells results in a more immunosuppressive TIME in follicles after POD24 in FL
Although immune and most tumor cells reside in the follicles, they do not accurately represent the neighbors of FL-cells owing to the existence of other components in the follicles, such as normal B cells and FRCs or FDCs. Therefore, we evaluated the evolution of the immune cells neighboring FL-cells at POD24 according to their location information. Consistent with the total follicle data, the percentage of CD163−Mφs in the vicinity of FL-cells was significantly increased after POD24, whereas CD8+ T cells around FL cells showed a significant decrease (Fig. 4A). Further phenotypic analysis showed that CD163−Mφs around FL cells displayed higher expression of PD-L1 and PD-L2 after POD24 (Fig. 4C). Regarding CD8+T cells neighboring FL cells, higher expression of lymphocyte-activation gene 3 (LAG3), but not programmed cell death protein 1(PD-1), was observed in the POD24 group (Fig. 4B). These data suggest that immune cells in follicles are involved in the POD24 of FL; notably, an increase of CD163−Mφs with upregulation of PD-1 ligands, and a decrease in cytotoxic CD8+T cells, but with upregulated expression of immune checkpoints, might facilitate the formation of an immunosuppressive microenvironment during POD24 in FL.
Dynamic changes in interactions between tumor and immune cells during POD24 in FL
Microenvironments presented at POD24 in FL were found to have evolved to be more immunosuppressive, as mentioned previously herein. Cytotoxic T cells were considered the main effector cells that kill FL cells, whereas Treg and TAMs were the main immunosuppressive cells restricting CD8+T cell functions in the TIME. Tumor cells that interact with different immune cells could represent different immune response statuses. We classified FL cells according to the co-localization patterns with Mφs, Tregs, and CD8+T cells, as shown in Fig. 5A. Eight interaction patterns between FL cells and the three types of immune cells were described, and the percentages of FL cells with different interaction patterns accounting for total FL cells were calculated (Fig. 5A). As a result, we found that the proportion of FL cells interacting with CD8+T cells alone (pattern 2) was significantly reduced in follicles after POD24 (Fig. 5B). However, FL cells interacting with Mφs alone (patterns 4) and simultaneously with and Tregs (Pattern 7) were robustly increased in follicles after POD24 (Fig. 5C, D). Similarly, a significant decrease in FL cells interacting with CD8+T cells alone (pattern 2) and an increase in FL cells interacting with Mφs alone (pattern 4) were observed in the interfollicular regions after POD24 (Fig. 5E, F). These data suggest that more FL cells are protected from immune attack via spatial distancing from cytotoxic T cells and interacting immunosuppressive cells, and especially Mφs.
More frequent cooperation between TAMs and Tregs in hijacking cytotoxic T cells contributes to TIME evolution into enhanced immune-escaping potential during POD24 in FL
Although CD8+T cells contributed to the predominant cytotoxic effects on tumor cells, their cytotoxic function was generally suppressed by regulatory cells such as Tregs and TAMs. However, whether Tregs and TAMs suppressed CD8+T cells separately or cooperatively in the TIME of FL and how the interaction pattern evolves during POD24 remained unknown. To explore the immunosuppressive pattern of TAMs and Tregs, we analyzed the fractions of Tregs around TAMs, accounting for total Tregs (Fig. 6A). As a result, a larger fraction of Tregs around Mφs was observed after POD24 (Fig. 6B). An analysis of the interaction patterns among Tregs revealed that more Tregs cooperated with Mφs after POD24 in a manner similar to that of Patterns 4 and 7 (Fig. 6C, D), but the frequency of Tregs inhibiting CD8+T cells near FL cells alone (Pattern 5) was significantly decreased (Fig. 6E), indicating that Tregs evolved to serve their regulatory function by cooperating more frequently with Mφs, rather than by themselves, after POD24. CD8+T cells are the main targets of Tregs and Mφs. Therefore, we similarly classified CD8+T cells according to their interaction patterns, as mentioned previously herein (Fig. 7A). Consistently, more CD8+T cells were hijacked simultaneously by Tregs and Mφs (Pattern 6) after POD24 (Fig. 7B, C). However, more CD8+T cells near FL cells were inhibited by Mφs but not Tregs (Pattern 7, 5 Fig. 7C,D). Taken together, these data suggest that Mφs not only serve an immunosuppressive function by themselves but also cooperate with Tregs more frequently to simultaneously inhibit cytotoxic T cells, to rationally yield enhanced immune-evading potentials of the TIME after POD24.