Immunosuppressive CD29+ Treg accumulation in the liver in mice on checkpoint inhibitor therapy

Abstract

Objective: Liver metastases are often resistant to immune checkpoint inhibitor therapy (ICI) and portend a worse prognosis compared with metastases to other locations. Regulatory T cells (Tregs) are one of several immunosuppressive cells implicated in ICI resistance of liver tumours, but the role played by Tregs residing within the liver surrounding a tumour is unknown.

Design: Flow cytometry and single-cell RNA sequencing were used to characterise hepatic Tregs before and after ICI therapy.

Results: We found that the murine liver houses a Treg population that, unlike those found in other organs, is both highly proliferative and apoptotic at baseline. On administration of αPD-1, αPD-L1 or αCTLA4, the liver Treg population doubled regardless of the presence of an intrahepatic tumour. Remarkably, this change was not due to the preferential expansion of the subpopulation of Tregs that express PD-1. Instead, a subpopulation of CD29⁺ (Itgb1, integrin β1) Tregs, that were highly proliferative at baseline, doubled its size in response to αPD-1. Partial and full depletion of Tregs identified CD29⁺ Tregs as the prominent niche-filling subpopulation in the liver, and CD29⁺ Tregs demonstrated enhanced suppression in vitro when derived from the liver but not the spleen. We identified IL2 as a critical modulator of both CD29⁺ and CD29^- hepatic Tregs, but expansion of the liver Treg population with αPD-1 driven by CD29⁺ Tregs was in part IL2-independent.

Conclusion: We propose that CD29⁺ Tregs constitute a unique subpopulation of hepatic Tregs that are primed to respond to ICI agents and mediate resistance.

Keywords: cancer; immunotherapy; liver.

Figure 1:. Tregs restrain the efficacy of immune checkpoint inhibitor against liver tumors.

B6 mice subcutaneous (sc) (A-B, IgG n=4, αPD-1 n=5) and intrahepatic (ih) (C-F) MC38 tumors were treated with αPD-1 as indicated. (F) Treg frequency of CD4+ cells in non-tumoral tissue from liver. (G-K) Intrahepatic MC38 tumor weights from B6 mice treated with αPD-1 with or without αCD25, with (I) Treg frequency of CD4⁺ T cells from liver and spleen and (K) PD-1+CD8:Treg ratio from TIL. (L-M) Intrahepatic MC38 tumor weights from FoxP3-DTR mice treated with DT and IgG control or αPD-1, respectively. (H-K) One-way Analysis of variance (ANOVA) was used. (B, E, F, M) Unpaired Student’s t-test was used. Data are shown as mean ± s.d. For all statistical tests, ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Data are representative of (A-K) 2 experiments or (L-M) 1 experiment.

Figure 2:. Checkpoint blockade causes Treg accumulation and activation in liver.

(A-C) Flow cytometry staining of Tregs from of B6 mice 3 days after one treatment with either IgG or αPD-1, represented by (A) representative flow cytometry plot showing frequency and (B) cell number of Tregs in the liver. (C) Ratio of change in Treg frequency of CD4 in αPD-1-treated mice in liver compared with spleen. (D-I) Activation markers on hepatic Tregs following αPD-1 treatment. (K + L) Fold change of Tregs in the livers and spleens of B6 mice treated with either αPDL1 or αCTLA4. (M) Frequency and (N + O) activation markers on hepatic Tregs from the livers of B6 mice treated with either IgG or αPD-1 five days following establishment of intrahepatic MC38 tumors. (A-O) n=4 or 5 per group. Unpaired Student’s T test was used, data are shown as mean ± s.d., ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Data are representative of (A-C, F-H, M-O) 2 experiments or (D-E, H-L) 1 experiment.

Figure 3:. Tregs proliferate in response to IL2 in the liver.

(A) FTY720 (20μg) with or without αPD-1 (200 μg) were injected to B6 mice and lymphocytes from liver and spleen were analyzed with flow cytometry 3 days after injection. The number of hepatic Tregs from αPD-1-treated B6 mice relative to IgG are shown for each treatment group. (B-C) Percentage of (B) Ki67+ and (C) Annexin V+ hepatic or splenic lymphocytes. Percentage of (D and F) Ki67+ and (E and G) Annexin V+ Tregs from (D-E) tumor-free and (F-G) MC38-bearing livers with or without αPD-1. (H) Percentage of Ly6C+ Tregs from tumor-free mice treated with IgG or αPD-1. (I + K) Expression of (I) CD25 on tumor-free murine Tregs and (K) IL2RA (CD25) in HCC-bearing human T cell subsets from the indicated tissues. (L) The liver’s frequency of Tregs in CD4 and (M) ratio of Tregs from tumor-free B6 mice treated with daily doses of control antibody or recombinant IL2-antibody complexes for 3 days, then harvested 3 days following the final injection. (N) Number of hepatic Tregs from mice treated with αPD-1 or IgG, with or without αIL2. (A), (K), and (N) One-way ANOVA was used. (B-C) Two-way AnOVA was used. (D-I) and (M) Unpaired student’s t test was used. Data are shown as mean ± s.d., ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Data are representative of 1 experiment.

Figure 4:. Single cell RNA profiling of hepatic Tregs from MC38 tumor-bearing mice following αPD-1 treatment.

MC38 tumors were established for 7 days in FoxP3-GFP mice, then treated with either IgG or αPD-1 for 3 days. Mice were sacrificed on day 10. Tregs were FACS-purified and pooled based on group: Liver IgG (n=3), Liver αPD-1 (n=4), Spleen IgG (n=5), Spleen αPD-1 (n=5). (A) FACS-purified hepatic and splenic Tregs depicted by tSNE plot, resolving 8 clusters of Treg subsets. (B) Top differentially expressed genes defining each cluster. (C) Relative percentages of each Treg cluster derived from IgG or αPD-1-treated liver or spleen. (D) MA plots comparing expression of select genes from C2 with eTreg or rTreg clusters. (E-F) GSEA plots showing relative enrichment of C2 cells for the indicated gene sets. (G-M) Violin plots of relative expression of select genes within the rTreg, C2, and eTreg clusters. (N-O) Trajectory analysis using RNA velocity of Tregs from the (N) liver and (O) spleen. rTreg: resting Treg, Tfr: follicular Treg, eTreg: effector Treg, fTreg: fragile Treg. (G-M) One-way ANOVA was used. ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. (E-F) FWER p values are displayed. Data are representative of 1 experiment.

Figure 5:. CD29 expression on Tregs predicts response to αPD-1.

(A) Frequency of CD29⁻ positive T lymphocytes from B6 livers. (B) Expression level of Ki67 in CD29⁺ and CD29^neg hepatic T lymphocyte subsets. (C-D) Percentage of Annexin V⁺ cells within CD29⁺ and CD29^neg hepatic T lymphocyte populations. (E) Representative flow cytometry plot and (F) quantification of percentage of Tregs and (G) Ki67 levels from liver and spleen Tregs expressing CD29, Ly6C, or double-negative (DN). (H) Percentage of PD-1+ Tregs within the subsets CD29⁺, Ly6C, or DN. (I-M) Number of Treg, Ki67 percentage and levels, and ICOS expression on the above subsets of hepatic Tregs from B6 mice treated with either IgG or αPD-1. (A-D) and (H) One-way ANOVA was used. (F-G) and (I-M) Two-way ANOVA was used. Data are shown as mean ± s.d., ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Data are representative of 2 experiments.

Figure 6:. CD29+ Tregs are suppressive in the liver and dependent on IL2.

(A) In vitro suppression assay. FoxP3-GFP Tregs were purified using FACS from the livers or spleens of mice bearing MC38 liver tumors treated for 3 days with either IgG or αPD-1, then co-cultured with VPD450-labeled CD4⁺GFP^neg− Responder T Cells (Tres) in the presence of irradiated APCs and αCD3 for 5 days. Suppression was calculated by gating the VPD450-low Tres cells compared with unstimulated. (B) Treg quantification following rIL2c treatment from experiment from Figure 3L, stratified by CD29 expression. (C) Quantification of hepatic Tregs from mice 3 days after treatment with combination αIL2 and αPD-1 as in Figure 3N. (A) and (C) Two-way ANOVA was used. (B) One-way ANOVA was used. Data are shown as mean ± s.d., ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Data are representative of (A) and (C) 2 experiments or (B) 1 experiment.

Figure 7:. ITGB1-containing Treg subset in human liver cancer.

(A) Re-clustering of Tregs from liver samples derived from patients bearing primary liver cancer (GSE151530). (B) Expression of 6-gene signature in human Treg clusters based on top 6 DEGs from mouse C2. (C) Comparisons of 6-gene signature in (B) between C1 and all other clusters. (D) Expression of CD25 on matched human Tregs from the indicated tissues. Tregs were defined as CD45⁺CD3⁺CD4⁺FOXP3⁺. (E) Gating of CD29^high and CD29^low Treg subsets on a representative tumor sample, gated off FMO. (F) Ki67+ percentage of Tregs from the indicated tissues in either CD29^high or CD29^low subsets. (C) Unpaired Student’s t-test was used. (D) Unpaired one-way ANOVA was used. (F) Paired two-way ANOVA was used. Data are shown as mean ± s.d., ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.