Resident Kupffer cells and neutrophils drive liver toxicity in cancer immunotherapy

Abstract

Immunotherapy is revolutionizing cancer treatment but is often restricted by toxicities. What distinguishes adverse events from concomitant antitumor reactions is poorly understood. Here, using anti-CD40 treatment in mice as a model of T_H1-promoting immunotherapy, we showed that liver macrophages promoted local immune-related adverse events. Mechanistically, tissue-resident Kupffer cells mediated liver toxicity by sensing lymphocyte-derived IFN-γ and subsequently producing IL-12. Conversely, dendritic cells were dispensable for toxicity but drove tumor control. IL-12 and IFN-γ were not toxic themselves but prompted a neutrophil response that determined the severity of tissue damage. We observed activation of similar inflammatory pathways after anti-PD-1 and anti-CTLA-4 immunotherapies in mice and humans. These findings implicated macrophages and neutrophils as mediators and effectors of aberrant inflammation in T_H1-promoting immunotherapy, suggesting distinct mechanisms of toxicity and antitumor immunity.

Fig. 1.. aCD40 triggered canonical anti-tumor Th1 cytokines throughout a tumor-bearing host.

(A) Rationale and workflow to study of irAEs using IL-12 and IFN-𝛾 reporter mice. (B) Flow cytometry plots exemplifying IL-12p40-EYFP induction in liver on day two following aCD40. Y-axis = viability dye (Zombie Aqua). (C) IL-12p40-EYFP induction across tissues on day two following aCD40. Values calculated based on % of CD45⁺ events that are EYFP⁺ (n = 3–16 mice/group). (D) Flow cytometry plots exemplifying IFN-𝛾-EYFP induction in liver on day two following aCD40 treatment. Y-axis as in (B). (E) IFN-𝛾-EYFP induction across tissues on day two following aCD40. Values calculated as in (C) (n = 5–14 mice/group). (F) MC38 tumor volumes and changes in body weight for mice treated or not with aCD40 with or without IL-12 or IFN-𝛾 neutralization (n = 5–7 mice/group). (G) TC-1 tumor volumes and changes in body weight for mice treated or not with aCD40 with or without IL-12 neutralization (n = 7–9 mice/group). Data are represented as mean ± SEM. For comparisons between two groups, Student’s two-tailed t test was used. For comparisons between multiple groups, one-way ANOVA was used. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 2.. IL-12 and IFN-𝛾 crosstalk after immunotherapy was causative of inflammatory pathology.

(A) Hematoxylin and eosin (H&E) staining of fixed liver tissue from mice treated with aCD40, with or without IL-12 or IFN-𝛾 neutralization, two days after aCD40. Necrotic lesions (dashed yellow lines). (B) Quantification of necrotic lesion area as a percent of total liver area in H&E section (n = 3–7 mice/group). (C) Diagram depicting generation of bone marrow chimeras to study the requirement for IFNgR1 signaling on hematopoietic vs. radio-resistant cells. (D) Changes in body weight from mice as depicted in 2C, two days following aCD40 (n = 4–6 mice/group). (E) H&E staining of fixed liver tissue from mice sufficient for IFNgR1 only in hematopoietic cells (left) or only in radio-resistant cells (right). Necrotic lesions (dashed yellow lines). (F) Flow cytometry quantification of IL12-EYFP⁺ cells in livers of mice treated or not with aCD40, with or without IFN-𝛾 neutralization (n = 3–4 mice/group). (G) Flow cytometry data as in (F), but from IFN-𝛾-EYFP mice with or without IL-12 neutralization (n = 5–6 mice/group). Data are represented as mean ± SEM. For comparisons between two groups, Student’s two-tailed t test was used. For comparisons between multiple groups, one-way ANOVA was used. ***p < 0.001, ****p < 0.0001.

Fig. 3.. DC-independent sources of IL-12 were sufficient to drive toxicity.

(A) Intracellular IL-12p40 staining from tumors (left) or livers (right) of Batf3^+/+ or Batf3^–/– mice two days after aCD40 (n = 5–8 mice/group). (B) Whole mount imaging of livers from Batf3^+/+ Il12-EYFP or Batf3^–/– Il12-EYFP mice, given or not aCD40, two days after treatment. Lectin-rhodamine (blue); IL12-EYFP (green). Tissue lesions (dashed yellow lines). (C) Quantification of IL12-EYFP⁺ cells from livers of mice as in (B). (D) H&E staining of livers from Batf3^+/+ or Batf3^–/– mice treated with aCD40, with or without IL-12 neutralization, two days after aCD40, quantified as in Fig. 1B (n = 3–10 mice/group). Necrotic lesions (dashed yellow lines). (E) H&E staining of liver tissue taken from WT mice or Zbtb46-Dtr bone marrow chimeras treated with aCD40. Necrotic lesions (dashed yellow lines). Data are represented as mean ± SEM. For comparisons between two groups, Student’s two-tailed t test was used. For comparisons between multiple groups, one-way ANOVA was used. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 4.. Resident Kupffer cells were a source of IL-12.

(A) scRNAseq pipeline (left) and UMAP representation (right) comparing tumor and liver IL-12-EYFP⁺ cells from aCD40-treated reporter mice (light blue, tumor, n = 3 mice; dark blue, liver, n = 2 mice). (B) UMAP of EYFP⁺ cells in tumor (n = 2295 cells) and liver (n = 5157 cells) colored by cell state annotation. (C) Il12b expression and dendritic cell, macrophage, and Kupffer cell markers in EYFP⁺ cells from (A). Colorbar saturated at the 99.5^th expression percentile measured across all EYFP⁺ cells in tumor or liver. (D) Quantification of transcripts depicted in (C) across Il12b⁺ cell states. Mean of each biological replicate is shown (dots) with standard error of each replicate-specific mean; count per 10,000, CP10K. (E) Flow cytometry of liver IL12-EYFP⁺ cells from aCD40-treated reporter (representative example of n = 4 mice). (F) Contribution of IL12-EYFP⁺ cells with different myeloid cell phenotypes as defined in (E) (average from n = 4 mice). (G) Schematic for parabiosis study to analyze chimerism of liver EYFP⁺ cells after aCD40. (H) Flow cytometry data gating IL-12-EYFP⁺ CD11b^–/lo F4/80⁺ KCs in IL-12 reporter (top) and non-reporter (bottom) livers. (I) Proportions of EYFP⁺ cells with a KC (CD11b^–/lo F4/80⁺) or migratory macrophage (CD11b⁺ F4/80⁺) phenotype in each parabiont (n = 3 mice/group). Data are represented as mean ± SEM. For comparisons between two groups, Student’s two-tailed t test was used. *p < 0.05.

Fig. 5.. IFN-𝛾-sensing Kupffer Cells drove liver toxicity.

(A) Schematic for bone marrow chimeras sufficient or deficient for IL-12-producing cDCs. (B) Quantification of IL-12-producing DCs (left) or KCs (right) from livers of mice shown in (A) two days after aCD40. (C) H&E of livers from mice as depicted in (A). Necrotic lesions (dashed yellow lines). (D) Flow cytometry quantification of IL-12-producing DCs (F4/80^– CD11c⁺ MHCII⁺) or KCs (CD11b^–/lo F4/80⁺), two days after aCD40, given Control or Clodronate Liposomes (n = 4–5 mice per group). (E) H&E of livers from aCD40-treated mice given Control or Clodronate Liposomes. Necrotic lesions (dashed yellow lines). (F) Quantification of lesions as shown in (E) (n = 5 mice/group). (G) H&E with quantification from livers of control (left) or Clec4f-Dtr (right) mice 2 days following aCD40 (n = 3–4 mice/group). Necrotic lesions (dashed yellow lines). (H) Flow cytometry quantification of liver IFN-𝛾-eEYFP⁺ cells from mice given Control or Clodronate Liposomes, two days following aCD40 (n = 4 mice/group). (I) Flow cytometry quantification of liver IL-12-producing CD11b^–/lo F4/80⁺ from mice treated or not with aCD40, with or without IL-12 or IFN-𝛾 neutralization (n = 3–5 mice/group). (J) Diagram of mice containing both WT and Ifngr1^–/– hematopoietic cells (left). Flow cytometry data comparing IL-12 production in Ifngr1^+/+ vs. Ifngr1^–/– CD11b^–/lo F4/80⁺ KCs from livers of bone marrow chimeras two days after aCD40 (right). (K) Flow cytometry data comparing IL-12 production in CD11b^–/lo F4/80⁺ KCs from livers of Clec4f-cre^o/o Ifngr1^fl/fl vs. Clec4f-cre^+/o Ifngr1^fl/fl mice two days after aCD40. (L) H&E of livers from Clec4f-cre^o/o Ifngr1^fl/fl (left) or Clec4f-cre^+/o Ifngr1^fl/fl (right) mice 2 days following aCD40. Necrotic lesions (dashed yellow lines). Data are represented as mean ± SEM. For comparisons between two groups, Student’s two-tailed t test was used. For comparisons between multiple groups, one-way ANOVA was used. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 6.. IFN-𝛾, IL-12 and macrophages, but not DCs, induced a pathogenic neutrophil response.

(A) UMAP of CD45⁺ cells from livers of untreated (n = 5,879 cells) or aCD40-treated (n = 12,892 cells) mice, colored by major cell-type (n = 2 mice per condition). (B) Fold change in the relative abundance of major cell types in sequenced CD45⁺ cells from livers of aCD40-treated vs. untreated mice. (C) Flow cytometry data of neutrophils (CD11b⁺ Ly-6G⁺) in livers of untreated or aCD40-treated mice. (D) Quantification of flow cytometry data as shown in (C) (n = 5 mice/group). (E) Flow cytometry quantification of liver neutrophils from mice treated or not with aCD40, with or without IL-12 or IFN-𝛾 neutralization (n = 3–5 mice/group). (F) Flow cytometry quantification of liver neutrophils from WT or Zbtb46-Dtr mice following aCD40 (n = 4–6 mice/group). (G) Flow cytometry quantification of liver neutrophils from mice treated with aCD40 and control or clodronate liposomes (n = 5 mice/group). (H) MPO staining of livers from untreated and aCD40-treated mice. ROI, region of interest (n = 5 mice/group). Necrotic lesions (dashed red lines). (I) H&E staining of livers from aCD40-treated mice, given (or not) anti-Gr-1 or anti-Ly-6G mAbs with or without a CXCR2 inhibitor. Necrotic lesions (dashed yellow lines). (J) Quantification of liver lesions from aCD40-treated mice, given (or not) anti-Gr-1 mAbs (n = 6–7 mice/group). (K) Quantification of liver lesions from aCD40-treated mice, given (or not) anti-Ly-6G mAbs with or without a CXCR2 inhibitor (n = 3–5 mice/group). (L) Quantification of liver lesions from aCD40-treated mice sufficient or deficient for CSF3R (n = 4–5 mice/group). (M) MC38 tumor volumes for mice treated (or not) with aCD40, with or without neutrophil targeting. (n = 6–8 mice/group) (N) Flow cytometry quantification of IL-12-EYFP⁺ cells in livers of mice treated (or not) with aCD40, given or not anti-Ly6G mAbs (n = 4–5 mice/group). (O) Flow cytometry quantification of IFN-𝛾-EYFP⁺ cells in livers of mice treated (or not) with aCD40, given or not anti-Ly6G mAbs (n = 4–5 mice/group). Data are represented as mean ± SEM. For comparisons between two groups, Student’s two-tailed t test was used. For comparisons between multiple groups, one-way ANOVA was used. *p < 0.05, **p < 0.01, ****p < 0.0001.

Fig. 7.. TNF-a-expressing, IFN-𝛾-responsive neutrophils determined toxicity but not tumor control.

(A) Gene Ontology (GO) results based on scRNAseq transcripts significantly enriched in liver neutrophils from aCD40-treated compared to untreated mice. (B) Single-cell expression of Tnf in liver neutrophils from mice treated or not with aCD40. Colorbar saturated at 99.5^th expression percentile measured across all CD45⁺ immune cells. (C) Relative contributions by different cell types to Tnf transcription based on transcript counts and relative representation of each cell type. (D) H&E staining, with quantification, of livers from aCD40-treated mice, with or without TNF-a neutralization (n = 5 mice/group). Necrotic lesions (dashed yellow lines). (E) Diagram of mice containing both WT and Ifngr1^–/– neutrophils. Both populations were sorted from livers two days after aCD40 and taken for RNAseq (left). Quantification of Tnf transcripts (gene of interest) in these cells (right). (F) Single-cell expression of Cd274 in liver neutrophils from mice treated (or not) with aCD40. Colorbar as in (B). (G) Quantification of Cd274 transcripts (gene of interest) from WT and Ifngr1^–/– neutrophils from livers of mice in (E). (H) H&E staining, with quantification, of livers from aCD40-treated mice, given (or not) anti-PD-L1 followed by anti-Rat IgG2b depleting mAbs (n = 5 mice/group). Necrotic lesions (dashed yellow lines). (I) Flow cytometry quantification of liver neutrophils from mice treated (or not) with aCD40, given (or not) anti-PD-L1 followed by anti-Rat IgG2b depleting mAbs (n = 6 mice/group). (J) Flow cytometry quantification of liver IL-12⁺ cells from mice treated as in (I) (n = 6 mice/group). (K) Flow cytometry quantification of tumor IL-12⁺ cells from mice treated as in (I) (n = 6 mice/group). (L) MC38 tumor volumes for mice treated as in (I) (n = 7 mice/group). Data are represented as mean ± SEM. For comparisons between two groups, Student’s two-tailed t test was used. For comparisons between multiple groups, one-way ANOVA was used. *p < 0.05, ***p < 0.001, ****p < 0.0001.

Fig. 8.. IFN-𝛾, IL-12 and neutrophil responses in human irAEs.

(A) Comparison of fold changes in single-cell gene expression for key cytokines and receptors in immune cells from human colon (immunotherapy-induced colitis vs patients not receiving immunotherapy) and mouse liver (aCD40-treated vs untreated). Red, yellow quadrants show conserved responses to therapy. See also Table S5. (B) Comparison of gene expression changes in selected immune cell types from mouse livers and human colons from immunotherapy conditions as in (A). Pearson correlation (R) for genes changing in mice and human homologs, calculated based on direction of change. The 100 genes with greatest fold change in mice were used for the analysis. See also Table S5. (C) Scatterplot comparing changes in gene expression for monocytes/macrophages in mouse livers and human colons from immunotherapy conditions as in (A). Up to 100 genes were selected based on: (1) FDR<0.05 and magnitude of change >2-fold in mouse; (2) existence of a 1:1 human homolog. Red, yellow quadrants show conserved responses to therapy; genes with conserved responses listed. See also Table S5. (D) Cancer diagnosis, treatment, toxicity score, and granulocyte inflammation scores from livers of 24 cancer patients who developed irAEs. Additional information is available in Table S6. n.a. = not assessed. (E) MPO staining of liver tissue from four patients diagnosed with cancer, treated with ICBs, and who developed hepatitis. Dashed red lines indicate lobular hepatitis. Additional information available in Table S6. (F) Example of CD15 staining in liver tissue from patient as in (E). Additional information is available in Table S6. (G) Quantification of neutrophil score (left) and CD15⁺/MPO⁺ granulocyte score (right) in irAE livers from cancer patients, treated with ICB who developed mild, moderate, or severe liver toxicities as assessed by histological analysis of liver biopsies.