High-Plex Imaging and Cellular Neighborhood Spatial Analysis Reveals Multiple Immune Escape and Suppression Patterns in DLBCL

Background: DLBCL is a heterogeneous disease of malignant B cells and tumor microenvironment (TME). As a malignancy arising from the immune system, DLBCL tumor cells are known to deploy immune escape and/or suppression strategies to evade immunosurveillance. A deep understanding of the interactions between tumor and its TME is critical to any enduring treatment outcome. Here, we systematically characterize the spatial patterns of major tumor infiltrating immune cells in DLBCL using high-plex imaging data, and characterize them in known high-risk patient groups such as EFS24 failures, ABC subtype, double-hit, and a high risk transcriptional subtype A7 (Stokes submitted).

Methods: We generated multiplexed ion beam imaging (MIBI) data for two cohorts (cohort 1 and cohort 2) of newly-diagnosed DLBCL cases (N=55 and N=30) with 17 and 33 markers, respectively. Both cohorts had associated RNAseq and FISH data, enabling determination of molecular high-risk patients. The first cohort also contained MIBI imaging for N=5 normal lymph node (NLN) samples, while the second cohort had associated progression-free survival (PFS) data with R-CHOP treatment. The imaging analysis identified major immune cell phenotypes and their relative abundances. Largely following the methods of Bhate 2022, we calculated cellular neighborhoods (CNs) consisting of each cell and its 20 nearest neighbors for all 532,755 and 643,114 cells in the two cohorts, respectively.

We clustered the cellular composition of the CNs to identify characteristic CN clusters for each cohort, and investigated the spatial arrangement, or context (SC) of these CNs, and the CN cluster-specific expression of certain markers and their association with ABC subtype, DHIT, and A7. For cohort 2, we also performed univariate Cox proportional hazard modeling with PFS data.

Results: Seven unique CN clusters were identified, each enriched with different types of infiltrating immune cells, which we named with ascending tumor content as: CD4-rich (6.3% tumor), CD8-rich (21%), M1/DC-rich (33%), Tumor/DC mix (46%), M2-rich (56%), Tumor/T mix (59%), and Tumor-rich (87%). These clusters, initially identified in cohort 1, were largely replicated in cohort 2.

When contrasting ABC vs. GCB subtypes, we find that ABC has fewer Tumor-rich (p = 0.01) but higher abundance of M2-rich CNs (p=0.043). These M2-rich CNs, which are enriched with immune suppressive CD163+ macrophages, are present only in DLBCL and not in NLN samples, indicating a role of suppressive macrophages in ABC that is previously under-appreciated. CD163+ macrophages are associated with poor outcome in the 2^ndcohort (HR=4.4 +/- 0.61, p = 0.015), confirming similar findings in literature (Bohn 2012, Nam 2014, Reiss 2019).

As expected, tumor-rich CNs are associated with PFS24 failures (p = 0.026), and in DHIT (p = 0.0023), and A7 (p < 0.001) samples, in both cohorts. A7 samples lacked the two T-cell rich CNs, consistent with its low immune infiltration characteristic (Stokes submitted). Interestingly, the abundance of tumor/DC-mix CNs is also associated with A7 high-risk patients in both cohorts (p < 0.01).

The two T cell-rich CNs were least enriched for tumor cells, indicating a structure of T cell exclusion from tumor-rich regions in DLBCL. We identified a SC motif of "T-cell islands" where CD4-rich CNs are enclosed by CD8-rich CNs, which are adjacent to a layer of tumor/T mix CNs and/or M2-rich CNs, followed by tumor-rich CNs (example in Figure 1B). We believe that this layered structure serves as an immune escape strategy, whereby CD4+ T helper cells are excluded from direct contact with tumor cells, which may reduce their antigen presentation capacity; while CD8-rich CN adjacency with M2-rich CNs could result in immunosuppression of cytotoxic CD8+ T cells.

Among our other findings in cohort 2, infiltrating CXCR5+ CD8+ T cells are prognostic (PFS HR=0.23; p=0.021), and are highly enriched among the three highest tumor-rich CNs (p < 10^-6).

Conclusions: Using high-plex imaging and cellular neighborhood spatial analysis we have uncovered multiple spatial patterns between DLBCL tumor cells and their immune cell neighbors, which indicate that tumors exploit multiple mechanisms of immune escape or suppression. Detailed understanding of these mechanisms may provide new insights into tumor-TME interactions that will be useful for improving immune-oncology and combination strategies in DLBCL.

Reiss:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Huang:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Nakayama:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Stokes:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Gandhi:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company.