PD-L1 blockade restores CAR T cell activity through IFN-γ-regulation of CD163+ M2 macrophages

Abstract

Background: The immune suppressive tumor microenvironment (TME) that inhibits T cell infiltration, survival, and antitumor activity has posed a major challenge for developing effective immunotherapies for solid tumors. Chimeric antigen receptor (CAR)-engineered T cell therapy has shown unprecedented clinical response in treating patients with hematological malignancies, and intense investigation is underway to achieve similar responses with solid tumors. Immunologically cold tumors, including prostate cancers, are often infiltrated with abundant tumor-associated macrophages (TAMs), and infiltration of CD163⁺ M2 macrophages correlates with tumor progression and poor responses to immunotherapy. However, the impact of TAMs on CAR T cell activity alone and in combination with TME immunomodulators is unclear.

Methods: To model this in vitro, we utilized a novel co-culture system with tumor cells, CAR T cells, and polarized M1 or M2 macrophages from CD14⁺ peripheral blood mononuclear cells collected from healthy human donors. Tumor cell killing, T cell activation and proliferation, and macrophage phenotypes were evaluated by flow cytometry, cytokine production, RNA sequencing, and functional blockade of signaling pathways using antibodies and small molecule inhibitors. We also evaluated the TME in humanized mice following CAR T cell therapy for validation of our in vitro findings.

Results: We observed inhibition of CAR T cell activity with the presence of M2 macrophages, but not M1 macrophages, coinciding with a robust induction of programmed death ligand-1 (PD-L1) in M2 macrophages. We observed similar PD-L1 expression in TAMs following CAR T cell therapy in the TME of humanized mice. PD-L1, but not programmed cell death protein-1, blockade in combination with CAR T cell therapy altered phenotypes to more M1-like subsets and led to loss of CD163⁺ M2 macrophages via interferon-γ signaling, resulting in improved antitumor activity of CAR T cells.

Conclusion: This study reveals an alternative mechanism by which the combination of CAR T cells and immune checkpoint blockade modulates the immune landscape of solid tumors to enhance therapeutic efficacy of CAR T cells.

Keywords: immunotherapy, adoptive; macrophages; tumor microenvironment.

Figure 1

M2 macrophages suppress CAR T cells. (A) Illustration of the immune-suppression assay. CD14⁺ peripheral blood mononuclear cells were differentiated and polarized to M1 or M2 macrophages in vitro, and macrophages, CAR T cells, and tumor cells were co-cultured and evaluated for functional activities by flow cytometry. (B) Flow cytometry plots indicating the number of viable DU145-PSCA tumor cells in each condition. (C, D) CAR T cell-mediated tumor cell killing of DU145-PSCA prostate cancer (C) and CD19⁺ Daudi lymphoma (D) cells in the presence or absence of M1 or M2 macrophages after 6 and 3 days, respectively. PSCA-CAR T cell-mediated tumor cell killing was normalized to untransduced (UTD) T cells. (E–H) Proliferation (10 days) (E), 4-1BB activation (6 days) (F, G), and IFN-γ secretion (3 days) (H) of T cells in the presence or absence of M1 or M2 macrophages in the prostate cancer model. Proliferation and activation of T cells was measured by flow cytometry. Secreted IFN-γ in supernatant was measured by ELISA. Data represent at least two independent experiments using at least two different donors, in duplicate. CAR, chimeric antigen receptor; IFN, interferon; IL, interleukin; PSCA, prostate stem cell antigen.

Figure 2

CAR T cells alter M2 macrophage phenotypes. (A) Illustration of the immune-suppression assay to evaluate M2 macrophage phenotype. (B, C) Cell surface expression of CD80 (B) and CD163 (C) in M2 macrophages in the prostate cancer immune-suppression assay evaluated by flow cytometry. Data represent two independent experiments using two different donors, in duplicate (D) Illustration of M2 macrophage stimulation with conditioned media (CM) derived from PSCA-CAR T cell:tumor cell co-cultures. (E, F) Cell surface expression of CD80 (E) and CD163 (F) in M2 macrophages evaluated by flow cytometry 48 hours after stimulating with CM collected from co-culture of DU145-PSCA tumor cells and PSCA-CAR T cells. Data represent three independent experiments using three different donors, in duplicate. (G) Transcriptional changes by bulk RNA sequencing induced in M2 macrophages on stimulation with PSCA-CAR T cell-derived CM. Expression of selected immune-related genes is shown relative to a control condition stimulated with UTD T cell-derived CM. (H) Gene ontology enrichment analysis highlighting activated immune-related biological pathways in M2 macrophages on stimulation with PSCA-CAR T cell-derived CM. CAR, chimeric antigen receptor; IL, interleukin; PSCA, prostate stem cell antigen; UTD, untransduced.

Figure 3

CAR T cells induce PD-L1 expression in M2 macrophages. (A–C) PD-L1 expression in macrophages and DU145-PSCA tumor cells in the immune-suppression assay. Data represent three independent experiments using three different donors, in duplicate. (D) Immunostaining of CD163, CD68, PD-L1 and CD3 in a humanized MISTRG mouse model following CAR T cell therapy against intratibial LAPC9 prostate xenografts. (E, F) PD-L1 induction at the protein (E) and messenger RNA (F) levels following inhibition of IFN-γ signaling. Anti-IFN-γR1 antibody was used to block IFN-γ signaling in the presence of recombinant IFN-γ or PSCA-CAR T cell-derived CM collected from the DU145-PSCA tumor cell killing assay. (G) PD-L1 induction following inhibition of various signaling pathways. PSCA-CAR T cell-derived CM was applied to M2 macrophages in the presence of various small molecule inhibitors: fludarabine (STAT1 i), C188-9 (STAT3 i), itacitinib (JAK1 i), AG490 (JAK2 i), AZD1480 (JAK1/2 i), Bay11-7082 (NFκB i), Akti VIII (AKT i), CZC24832 (PI3K i), rapamycin (mTOR i). PD-L1 induction was evaluated by flow cytometry 48 hours after CM stimulation. Data represent at least two independent experiments using at least two different donors, in duplicate. CAR, chimeric antigen receptor; CM, conditioned media; IFN, interferon; PD-L1, programmed death ligand-1; PSCA, prostate stem cell antigen; UTD, untransduced.

Figure 4

PD-L1 blockade restores CAR T cell function in the presence of suppressive M2 macrophages. CAR T cell function was evaluated in the prostate cancer immune-suppression assay in the presence of PD-L1 blockade. (A) Flow cytometry plots indicating the number of viable DU145-PSCA tumor cells in each condition in the presence or absence of anti-PD-L1 antibody, atezolizumab (Atezo). (B–D) Quantification of PSCA-CAR T cell-mediated killing of DU145-PSCA tumor cells (B), T cell activation (C), and IFN-γ secretion (D). (E) DU145-PSCA tumor cell killing of CAR T cells in the presence or absence of two clinically approved anti-PD-L1 antibodies, Atezo and avelumab (Ave), and Ave that lacks CH₂ domain (Ave (ΔCH₂)). Tumor killing and T cell activation were evaluated by flow cytometry, and IFN-γ secretion was evaluated by ELISA. Data represent at least two independent experiments using at least two different donors, in duplicate. CAR, chimeric antigen receptor; IFN, interferon; PD-L1, programmed death ligand-1; PSCA, prostate stem cell antigen; UTD, untransduced.

Figure 5

Combination of PD-L1 blockade and CAR T cell therapy depletes M2 macrophages via IFN-γ signaling. (A, B) Analysis of M2 macrophages in the prostate cancer immune-suppression assay in the presence or absence of PD-L1 blockade. (C, D) Analysis of M2 macrophages stimulated with PSCA-CAR T cell-derived CM in the presence or absence of PD-1 or PD-L1 blockade. (E–G) Representative brightfield images and analysis of M2 macrophage stimulated with PSCA-CAR T cell-derived CM in the presence or absence of PD-L1 and/or IFN-γR1 blockade. The number of total viable M2 macrophages (A, C, F) and the frequency and number of CD163⁺ M2 macrophages (B, D, G) were evaluated by flow cytometry. Data represent at least two independent experiments using at least two different donors, in duplicate. CAR, chimeric antigen receptor; CM, conditioned media; IFN, interferon; PD-L1, programmed death ligand-1; PD-1, programmed cell death protein-1; PSCA, prostate stem cell antigen; UTD, untransduced.