Figure 2. Uptake of cellular debris induces the TREM2 gene program in mo-macs.

a) (top) Confocal immunofluorescence IF) imaging of murine KP-GFP tumor-bearing lungs depicting GFP⁺ tumor cells (green) and TREM2⁺ cells (red) at different timepoints during tumor progression (day 7, 14, 21, and 28 post-inoculation). (bottom, left) Confocal IF imaging of KP-GFP tumor-bearing lungs after 28 days of engraftment and growth showing intracellular staining of nuclei (DAPI) and GFP (green) in TREM2⁺ cells (red). Middle and right images represent magnification of the region of interest (ROI, white outline); middle panel depicts cells staining for both DAPI and GFP (white arrow), and the right panel depicts cells staining for both GFP and TREM2 (white arrow). Scale bar, 50 μm. (bottom, right) Pictorial diagram showing relationship between efferocytosis and TREM2.

b) Volcano plot of differentially expressed genes between GFP^lo and GFP^hi mo-macs. Statistical significance defined based on an adjusted P < 0.01. Significant genes are highlighted in either red (up-regulated upon tumor antigen capture) or blue (down-regulated upon tumor antigen capture). Data representative of two independent experiments.

c) Expression of Trem2 signature genes and other select genes encoding machinery involved in phagocytosed cargo transport, antigen processing, and lipid metabolism by GFP^lo and GFP^hi mo-macs. Values shown as the logarithm of fold change of absolute number of transcripts relative to the mean value across replicates.

d) Absolute expression (transcripts per million) of genes encoding LDL receptor, NPC1, and PLIN2 in GFP^lo and GFP^hi mo-macs. Paired values shown for paired GFP^lo and GFP^hi mo-macs that were purified from the same biological replicate. Data from two independent experiments. (mean ± standard error of mean (S.E.M.); unpaired two-tailed t-test at a 95% confidence interval)

e) Absolute expression (transcripts per million) of genes encoding GPR183 and MARCKSL1 in GFP^lo and GFP^hi mo-macs. Paired values shown for paired GFP^lo and GFP^hi mo-macs that were purified from the same biological replicate. Data from two independent experiments. (mean ± standard error of mean (S.E.M.); unpaired two-tailed t-test at a 95% confidence interval)

f) Relative qPCR quantification of Trem2, Gpnmb, and Apoe transcripts from in vitro differentiated bone marrow-derived macrophages (BMDMs) with or without (control) exposure to apoptotic KP-GFP cells. Briefly, BMDMs were differentiated and polarized with IL-4 and GM-CSF. KP-GFP cells were exposed to ultraviolet (UV) light for 30 min, left to undergo apoptosis overnight, and then added to BMDMs. After a four hour co-culture period, CD45⁺ CD64⁺ MerTK⁺ GFP⁺ cells were sorted by flow cytometry for PCR analysis. BMDMs cultured in the absence of apoptotic tumor cells were used as a negative control. Transcripts are shown as the two-fold difference in gene expression (ΔCt) over Ptprc expression. Data representative of two independent experiments. Values are shown as mean transcripts per million (TPM) ± standard error of mean (S.E.M.). (Unpaired two-tailed t-test at a 95% confidence interval)