Tumor-associated macrophages trigger MAIT cell dysfunction at the HCC invasive margin

Abstract

Mucosal-associated invariant T (MAIT) cells represent an abundant innate-like T cell subtype in the human liver. MAIT cells are assigned crucial roles in regulating immunity and inflammation, yet their role in liver cancer remains elusive. Here, we present a MAIT cell-centered profiling of hepatocellular carcinoma (HCC) using scRNA-seq, flow cytometry, and co-detection by indexing (CODEX) imaging of paired patient samples. These analyses highlight the heterogeneity and dysfunctionality of MAIT cells in HCC and their defective capacity to infiltrate liver tumors. Machine-learning tools were used to dissect the spatial cellular interaction network within the MAIT cell neighborhood. Co-localization in the adjacent liver and interaction between niche-occupying CSF1R⁺PD-L1⁺ tumor-associated macrophages (TAMs) and MAIT cells was identified as a key regulatory element of MAIT cell dysfunction. Perturbation of this cell-cell interaction in ex vivo co-culture studies using patient samples and murine models reinvigorated MAIT cell cytotoxicity. These studies suggest that aPD-1/aPD-L1 therapies target MAIT cells in HCC patients.

Keywords: CODEX; HCC; MAIT cells; S(3)-CIMA; aPD-1/aPD-L1; immunotherapy; innate-like T cells; mucosal-associated invariant T cells; tumor immune microenvironment; tumor-associated macrophages.

Figure 1. Single-cell RNA sequencing reveals MAIT cell heterogeneity within the HCC bearing liver

(A) Uniform Manifold Approximation and Projection (UMAP) plot, showing clustering for different CD45⁺ immune cell types. (B) & (C) UMAP plots showing origin of single cells by patients (B) and tissue location (C). (D) Heatmap projections showing expression of selected indicated marker genes (corresponding to A) (E) Boxplot displaying frequency of main MAIT cell cluster, c9 amongst T cells. (F&G) Overall survival (OS) risk probability based on high or low expression of MAIT cell gene signature in TCGA using the MAIT signature by Yao et al. (F) or the MAIT signature derived from cluster c9 (G). (H) UMAP plot showing different MAIT cell subclusters. (I) Violin plots showing expression of selected markers of dysfunction and cytotoxicity in MAIT cell subclusters (as defined in H).

Figure 2:. MAIT cells are dysregulated in human and mouse HCC.

(A) Patient cohort & sampling strategy. (B) Representative flow cytometry plots showing identification of human hepatic MAIT cells (see also Fig.S4A) (C) Boxplot displaying MAIT cell frequencies as determined by flow cytometry (n=37). (D) Boxplots showing mean fluorescence intensity (MFI) of PD-1 (left), frequency of CD25⁺ (middle) and frequency of CD56⁺ hepatic MAIT cells as determined by flow cytometry (n=37). (E) Mouse model of syngeneic, orthotopic liver cancer. Photo showing mouse liver with intrahepatic HCC tumor. (F) Boxplots showing numbers of hepatic and tumor-infiltrating MAIT cells/g tissue. Results for for different tumor cell lines RIL-175, Hep55.1c, MC38 and B16-Mr1^WT are shown. (G) Frequency of PD-1⁺ (left), TIM-3⁺ (middle) and CD69⁺ (right) hepatic MAIT. Representative flow cytometry dot plots are shown (H) Boxplots showing MFI of transcription factors RORγt (left) and T-bet (right) in hepatic MAIT cells at d28. Comparison between tumor-free (n=8) vs tumor-bearing (n=9) mice. (I) Boxplots showing frequency of cytokines IFNy (left), TNFα (middle) and granzyme B (right) in hepatic MAIT cells. Comparison between tumor-free (n=7) vs tumor-bearing (n=7) mice. Representative flow cytometry dot plots are shown. (J) Mouse model of syngeneic, orthotopic liver cancer comparing tumor growth in C56BL/6 (Wildtype) and Mr1^−/− mice. (K) Top: Photo showing murine liver with a single orthotopic HCC lesion. Bottom: excised intrahepatic tumors comparing WT and Mr1^−/− mice. (L) Box plot showing the weight of intrahepatic Hep55.1c tumors at d12 comparing wildtype (n=20) and Mr1^−/− mice (n=21). (M) Box plot showing the weight of intrahepatic MC38 tumors at d12 comparing WT (n=15) and Mr1^−/− mice (n=14).

Figure 3:. Generation of a spatially resolved immune cell atlas of human liver cancer by CODEX technology

(A) Antibody panel design and CODEX workflow (B) Seven-color overview of a human HCC sample LHCC35, stained with the 37-plex CODEX panel (see A). Representative annotation of gross tissue regions is shown. (C) Middle: Seven-color overview of a whole tissue section derived from LHCC41. ROI are labeled 1–4 and correspond to higher-magnification multi-color images. ① liver parenchyma in the adjacent liver: T cells (CD3⁺), granulocytes (CD15⁺) and macrophages (CD68⁺) patrol inside the liver sinusoids (LYVE-1⁺). ② Periportal region showing branches of the portal vein (PV), arteria hepatic (A) and bile ducts (EPCAM⁺panCK⁺, B). ③ Tertiary lymphoid structure in the rim regions with aggregates of B cells (CD19⁺), helper T cells (CD4⁺), regulatory T cells (FOXP3⁺) and cytotoxic T cells (CD8⁺) within an aSMA-rich environment. ④ ROI in the tumor core shows proliferating (Ki-67⁺) tumor cells (HNF4a⁺) in an endothelial cell-rich (CD34⁺) environment. T cells (CD3⁺) and dendritic cells (CD11c⁺) are also shown.

Figure 4:. Identification and analysis of spatial distribution of immune cell phenotypes using CODEX imaging.

(A) UMAP plot, color coded for different immune cell types identified by unsupervised clustering of CD45⁺cells in the CODEX dataset. (B) & (C) UMAP plots showing origin of single cells color coded by tissue locations (B, see also Fig. 3B) and patients (n=15, C). (D) UMAP projections showing expression of indicated marker proteins per immune cell cluster. (E) Heatmap showing expression of indicated marker proteins per immune cell cluster (corresponding to A). (F) Comparison of total CD45⁺ leukocyte density in the three tissue regions. (G) Correlation of MAIT, T_reg and NK cell frequencies as determined by flow cytometry (top panel, n=37) and CODEX (middle panel, n=15). The bottom panel shows correlation of frequencies derived from paired patient samples (n=12) and measured by either flow cytometry or CODEX. Each datapoint corresponds to a paired measurement in either adjacent liver or tumor core. Pearson r and p values as indicated. (H) Sankey flow diagram of HCC samples representing indicated immune cell populations sorted on the y-axis from highest (top) to lowest (bottom) cell density in the specified histopathological compartments.The line width is scaled to cell density across the three regions. Pie charts at the bottom represent frequency of different immune cells within each spatial category.

Figure 5:. MAIT cell neighborhood analysis identifies the cellular interaction network underlying the immunosuppressive MAIT cell niche

(A) Schematic of S³-CIMA (Supervised Spatial Single Cell Imaging Analysis) algorithm used on CODEX single-cell data to investigate the MAIT cell neighborhoods in different tissue regions. (B) Boxplots showing frequencies of selected cells from different tissue regions compared with randomly selected cells in the background. (C) Bubble plot displaying S³-CIMA classification of cell types in whole tissue sections with neighborhood size k=40 nearest neighboring cells. Y-Axis shows the ratio of the number of all selected cell type CT in nearest neighbor of the anchor cell $\left(𝒦n{n}_{CT}^S:\right)$ to the number of all cell type CT in nearest neighbor of the anchor cell$\left(𝒦n{n}_{CT}\right)$. X-axis shows the ratio of the Number of all selected cell type CT $\left({𝒦}_{CT}^S:\right)$ to the number of all cells of cell type CT ${𝒦}_{CT}$. Color-coding corresponds to the enrichment score (ES) as displayed in (C). Size of the bubble displays the ratio: number of all selected cell type CT in nearest neighbor of the anchor cell $\left(𝒦n{n}_{CT}^S\right)$ to the number of all selected cells in nearest neighbor of the anchor cell $𝒦n{n}^S$ or $\frac{𝒦n{n}_{CT}^S}{𝒦n{n}^S}$ (D) Waterfall plot displaying enrichment score of indicated cell populations in the MAIT cell neighborhood (data for adjacent liver is shown) as selected by S³-CIMA. Values >1 indicate specific enrichment in the MAIT cell niche. (E) Five-color overlay images of a CODEX datasets displaying interactions between PD-L1⁺ (turquoise) CD163⁺ (pink) macrophages and PD-1 (yellow) on MAIT cells (TCRVa7.2^+, red) in the adjacent liver. Examples for 4 different patient samples (are shown. (F) Mean expression of PD-L1 on selected cell populations in the normal liver tissue region as determined by CODEX imaging. (G) Representative histograms showing M2 polarization of TAMs (defined as CD45⁺CD68⁺) as determined by expression of CD163 (left) and PD-L1 (right) on macrophages from healthy PBMCs (grey), HCC patient PBMCs (black), and adjacent, non-tumor tissue (red).

Figure 6:. CSF1R⁺CD163⁺PD-L1⁺ TAMs impair human MAIT cell function ex vivo in a contact and PD-L1 dependent manner.

(A) Experimental setup (B) Frequency of IFNγ⁺ MAITs after co-culture with CD163⁺ Mφ or CD163⁻ hepatic MNCs at indicated ratios. (C) Representative flow cytometry plots. Data include n=7 independent patient samples. (D) Scheme of the transwell experiment. Box plots shows frequency of IFNγ⁺ MAITs after co-culture with CD163⁺ Mφ at 1:6 ratio either in contact dependent or independent manner. Data include n=6 independent patient samples. (E) Representative histogram showing PD-L1 expression on sorted CD163⁺Mφ cells compared to CD163⁻ cells (F) Box plots shows frequency of IFNγ⁺ MAITs after co-culture with CD163⁺ Mφ at 1:6 ratio in presence or absence of aPD-L1 (20ng/mL). Data include n=7 independent patient samples, n=6 control group samples as in (D). (G) Hepatic CD45⁺ sorted myeloid cell clusters from Fig. 1A were selected and reclustered. UMAP projection showing subtypes of myeloid-derived cells and each cluster is color-coded according to the annotations indicated in the figure. (H) Heatmap projections showing expression of selected indicated marker genes among myeloid cell clusters (corresponding to clusters in G) (I) Experimental setup: C57BL/6 mice were injected orthotopically with syngeneic HCC cell line RIL-175 or left tumor-free. At d28 mice were sacrificed and liver and tumor-infiltrating MNCs were isolated. CD45⁺ sorted cells were subsequently subjected to single-cell RNA-sequencing. (J) UMAP projection showing the landscape of murine CD45⁺ cells pooled from tumor-free and tumor-bearing animals. (K) Heatmap projections showing expression of selected indicated marker genes among murine immune cell clusters (corresponding to clusters in J)

Figure 7:. MAIT dysfunction in murine HCC is reversed by PD-L1 blockade in vivo and by depletion of CSF1R⁺ TAMs.

(A) MC38 tumor-bearing, transgenic MM^DTR or wildtype mice received DT (250ng/mouse) s.c. every other day starting d1. (B) Representative flow plot showing frequency of hepatic F4/80⁺/CD11b^int macrophages (gated on CD45⁺CD3⁻CD19⁻NK1.1⁻) or CD11c⁺MHC-II⁺ cDC (gated on CD45⁺CD3⁻CD19⁻NK1.1⁻F4/80⁻) for MM^DTR or WT mice after injection of DT. (C) On d11, tumors were harvested, and the cell number (left) and frequency (right) of tumor infiltrating MAIT cells was determined. Comparison between Wildtype (n=10) vs MM^DTR mice (n=6). (D) Experimental setup as in (A). Boxplots summarizing cytokine expression of intrahepatic MAIT cells for IFN γ, granzyme B, and TNFα after 4h of ex vivo stimulation.. Representative flow cytometry dot plots are shown. Comparison between Wildtype (n=10) vs MM^DTR mice (n=6) is shown. (E) RIL-175 tumor-bearing C57BL/6 mice received aPD-L1 or IgG control i.p. at d5, d10, d15 & d20. (F&G) On d23, tumors were harvested, and the cell number (F) and frequency (G) of tumor infiltrating MAIT cells was determined by flow cytometry. Comparison between IgG Control (n=10) vs aPD-L1 (n=9) is shown. (H) Experimental setup as in (E) granzyme B expression of intrahepatic MAIT cells after 4h of ex vivo stimulation. Comparison between IgG Control and aPD-L1 i.p. treatment. (J) Cd274^fl/fl (Control), Lyz2^CRECd274^fl/fl and Csf1r^CRECd274^fl/fl lines received RIL=175 intrahepatic and livers and tumors were harvested on d28. (K) Tumors were harvested, and the number of tumor- infiltrating MAIT cells was determined by flow cytometry. n=11 for Cd274^fl/fl, n=10 for Lyz2^CRECd274^fl/fl and n=7 Csf1rCRECd274fl/fl (L) Boxplots showing MFI of granzyme B (K) in hepatic MAIT cells after 4h of ex vivo stimulation. Comparisons as in (L). (M) C57BL/6 or Mr1^−/− mice received intrahepatic injection of syngeneic HCC cell line Hep55.1c. 5-OP-RU/CpG treatment and/or aPD-L1 was administered intraperitoneally at indicated time points. (N&O) Bar graphs showing fold change of Hep55.1c tumor weights at d17 in comparison to the mean tumor weight of the control treated C57BL/6 (Wildtype) (N) or Mr1^−/− mice (O).