Liver myeloid-derived suppressor cells expand in response to liver metastases in mice and inhibit the anti-tumor efficacy of anti-CEA CAR-T

Abstract

Chimeric antigen receptor-modified T cell (CAR-T) technology, a promising immunotherapeutic tool, has not been applied specifically to treat liver metastases (LM). While CAR-T delivery to LM can be optimized by regional intrahepatic infusion, we propose that liver CD11b+Gr-1+ myeloid-derived suppressor cells (L-MDSC) will inhibit the efficacy of CAR-T in the intrahepatic space. We studied anti-CEA CAR-T in a murine model of CEA+ LM and identified mechanisms through which L-MDSC expand and inhibit CAR-T function. We established CEA+ LM in mice and studied purified L-MDSC and responses to treatment with intrahepatic anti-CEA CAR-T infusions. L-MDSC expanded threefold in response to LM, and their expansion was dependent on GM-CSF, which was produced by tumor cells. L-MDSC utilized PD-L1 to suppress anti-tumor responses through engagement of PD-1 on CAR-T. GM-CSF, in cooperation with STAT3, promoted L-MDSC PD-L1 expression. CAR-T efficacy was rescued when mice received CAR-T in combination with MDSC depletion, GM-CSF neutralization to prevent MDSC expansion, or PD-L1 blockade. As L-MDSC suppressed anti-CEA CAR-T, infusion of anti-CEA CAR-T in tandem with agents targeting L-MDSC is a rational strategy for future clinical trials.

Fig. 1

L-MDSC expand in response to LM and suppress CAR-T. LM were established by splenic injections of either MC38 or MC38CEA tumor cells, and livers were harvested 2 weeks of tumor growth. Flow cytometry was used to evaluate the expansion of L-MDSC in response to LM. a Sequential gating of L-MDSC as live, CD11b+, and Gr-1+ liver leukocytes from normal or tumor-bearing livers. Cells were enriched by immunomagnetic beading for CD45+ NPC prior to staining. b Dot plot confirming high level of Gr-1 and CD11b co-expression among liver leukocytes. c L-MDSC were co-cultured with CFSE-labeled anti-CEA CAR-T stimulated by irradiated MC38CEA cells, and pooled results from three independent experiments are graphed. The percentages of cells having undergone division (CFSE-low) were normalized to the unstimulated group. Bar graphs represent mean ± SEM; dot plots and histograms are representative of ≥3 mice per group and have been confirmed with at least two separate experiments (*p ≤ 0.05, **p ≤ 0.01)

Fig. 2

L-MDSC depletion improves CAR-T efficacy. LM were established by splenic injections of MC38CEA cells, and saline or anti-Gr-1 antibody was injected intraperitoneally on days 7 and 11 post-tumor establishment. Livers were harvested for NPC isolation, staining, and analysis after 14 days. a Flow cytometry dot plots of the L-MDSC (live, CD11c−CD11b+Gr-1+) population with or without anti-Gr-1 treatment. Cells were enriched by immunomagnetic beading for CD45+ NPC prior to staining. b Absolute L-MDSC numbers per 100,000 cells in liver isolate were calculated according to cell counts. c Mice with established LM received infusions of UT or CAR-T after 7 days, in addition to anti-Gr-1 (aGr-1) treatment in designated groups on days 7 and 11. Flow cytometry was used to determine the percentages of viable CEA+ tumor cells among all liver NPC after killing at day 26. d Survival curves for in vivo examination of ≥12 animals with LM treated with UT or CAR-T with and without Gr-1-antibody. Bar graphs represent mean ± SEM; dot plots are representative samples, survival significance displayed as relative to CAR-T+aGr-1 group (**p ≤ 0.01)

Fig. 3

L-MDSC expansion is driven by tumor-associated GM-CSF. LM were established by splenic injections of MC38CEA cells, and isotype control (IgG) or anti-GM-CSF antibody (aGM-CSF) was injected intraperitoneally on days 4, 6, and 8 post-tumor establishment. a Flow cytometry was used following harvest after 9 days to evaluate MDSC frequency (live, CD11b+Gr-1+ cells) (right, representative of three independent experiments). For this experiment, MDSC percentage values reflect analysis of bulk NPC not subjected to fractionation based on CD45 expression. b Serum GM-CSF and L-MDSC absolute numbers as determined by flow cytometry were quantified from mice at various time points post-LM establishment or CTRL (non-tumor bearing). c Whole liver lysate GM-CSF was measured from mice following LM establishment or CTRL animals. d The percentage of cells acquiring an MDSC phenotype (CD11b+Gr-1+) was determined by flow cytometry from bone marrow cells cultured for 3–4 days with exposure to recombinant GM-CSF, GM-CSF-producing MC38CEA tumor, or tumor-conditioned medium. Bar graphs represent mean ± SEM; dots plots are representative of ≥3 mice per group and have been confirmed with ≥2 separate experiments (*p ≤ 0.05, ***p ≤ 0.001)

Fig. 4

L-MDSC suppression through PD-1/PD-L1 is modulated by GM-CSF. a Flow cytometry was used to determine PD-1 expression on fresh CAR-T (CAR-T gated as CD3+Wi2+ cells) relative to FMO controls. b Flow cytometry was used to determine PD-L1 expression on L-MDSC (gated CD11c−CD11b+Gr-1+) isolated from tumor-bearing mice after 2 weeks of tumor growth relative to FMO controls. c Representative dot plots indicating PD-L1 expression as determined from BM-derived MDSC (gated as live, CD45+CD11c−CD11b+Gr-1+ cells) following exposure to recombinant GM-CSF or GM-CSF-producing tumor ex vivo. d Pooled results from two independent experiments are graphed. Bar graphs represent mean ± SEM; dot plots and histograms are representative of ≥3 mice per group and have been confirmed with ≥2 separate experiments (*p ≤ 0.05)

Fig. 5

L-MDSC suppress CAR-T through STAT3-dependent PD-L1 expression. a Representative CFSE histograms that demonstrate CAR-T proliferation (second panel) were suppressed by L-MDSC (third panel) and rescued by the addition of anti-PD-L1 antibody (aPD-L1). b Pooled results from three independent experiments are graphed, with proliferation normalized to that of the minimal proliferation value. The percentage of proliferating CAR-T was calculated based upon CFSE-low cells. c Phosphorylated STAT3 (pSTAT3) and total STAT3 (TSTAT3) expression levels in MDSC (CD11b+NPC) and non-MDSC (CD11b-fraction) from tumor-bearing livers were assayed by Western blot. d pSTAT3 to TSTAT3 ratio obtained from the Western blot analysis was calculated for MDSC and non-MDSC. e Flow cytometry was used to determine PD-L1 expression on L-MDSC following ex vivo culture with the STAT3 inhibitors JSI-124 and celastrol. f Pooled results from four independent experiments are graphed. Bar graphs represent mean ± SEM; dot plots and histograms are representative of ≥3 mice per group and have been confirmed with ≥3 separate experiments. Normalized proliferation = experimental %proliferation/%proliferation of CAR-T+MDSC (**p ≤ 0.01, ***p ≤ 0.001)

Fig. 6

Neutralization of GM-CSF and PD-L1 improves regional anti-CEA CAR-T efficacy. a Mice were injected with luciferase-expressing MC38CEA cells to establish LM and received treatments as indicated in addition to daily systemic IL2 support. b Mice were injected with luciferin and imaged for 15 days to evaluate tumor progression expressed as bioluminescence (photon/s). c Representative images of tumor burden at indicated time points. d Bar graphs indicate quantified tumor bioluminescence presented at day 15 following LM establishment. Groups were untreated (n = 3), untransduced (UT, n = 5), CAR-T (n = 11), CAR-T+aGr-1 (n = 10), CAR-T+aPD-L1 (n = 5), CAR-T+aGM-CSF (n = 10), CAR-T+aPD-L1+aGM-CSF (n = 11). e Survival of the animals treated with antibodies was tracked for up to 45 days after tumor injections. Kaplan–Meier curves were constructed and groups compared with the log-rank test. CAR-T+anti-GM-CSF treatment was significantly higher than CAR-T alone (*p = 0.03)