Differential effects of PD-L1 versus PD-1 blockade on myeloid inflammation in human cancer

Abstract

BACKGROUNDPD-1 and PD-L1 have been studied interchangeably in the clinic as checkpoints to reinvigorate T cells in diverse tumor types. Data for biologic effects of checkpoint blockade in human premalignancy are limited.METHODSWe analyzed the immunologic effects of PD-L1 blockade in a clinical trial of atezolizumab in patients with asymptomatic multiple myeloma (AMM), a precursor to clinical malignancy. Genomic signatures of PD-L1 blockade in purified monocytes and T cells in vivo were also compared with those following PD-1 blockade in lung cancer patients. Effects of PD-L1 blockade on monocyte-derived DCs were analyzed to better understand its effects on myeloid antigen-presenting cells.RESULTSIn contrast to anti-PD-1 therapy, anti-PD-L1 therapy led to a distinct inflammatory signature in CD14+ monocytes and increase in myeloid-derived cytokines (e.g., IL-18) in vivo. Treatment of AMM patients with atezolizumab led to rapid activation and expansion of circulating myeloid cells, which persisted in the BM. Blockade of PD-L1 on purified monocyte-derived DCs led to rapid inflammasome activation and synergized with CD40L-driven DC maturation, leading to greater antigen-specific T cell expansion.CONCLUSIONThese data show that PD-L1 blockade leads to distinct systemic immunologic effects compared with PD-1 blockade in vivo in humans, particularly manifest as rapid myeloid activation. These findings also suggest an additional role for PD-L1 as a checkpoint for regulating inflammatory phenotype of myeloid cells and antigen presentation in DCs, which may be harnessed to improve PD-L1-based combination therapies.TRIAL REGISTRATIONNCT02784483.FUNDINGThis work is supported, in part, by funds from NIH/NCI (NCI CA197603, CA238471, and CA208328).

Keywords: Cancer immunotherapy; Hematology; Immunology.

Conflict of interest statement

Conflict of interest: AR is an employee of Genentech.

Figures

Figure 1. CONSORT flow diagram for non–small cell lung cancer and asymptomatic myeloma clinical trials.

(A) Diagram reporting the distribution of αPD-L1 and αPD-1 treatment in patients with non–small cell lung cancer and subsequent data analysis. (B) Flow diagram reporting the process of screening, enrollment, allocation, follow-up, and assessment through the phases of the clinical trial of single-agent atezolizumab in patients with asymptomatic myeloma.

Figure 2. PD-L1 blockade leads to distinct transcriptomic changes in circulating monocytes and T cells.

RNA was extracted from magnetic bead isolated CD14+ monocytes and CD3+ T cells from patients with lung cancer before and after therapy with either anti–PD-L1 (atezolizumab; n = 5) or anti–PD-1 (nivolumab; n = 6 previously published; ref. 10) and analyzed using affymetrix human transcriptome array 2.0. (A) Distribution of differentially regulated genes upregulated and downregulated in monocytes and T cells following therapy with anti–PD-L1 or anti–PD-1. (B) Differentially regulated genes in monocytes following therapy with anti–PD-L1 (selected from top 50 differentially regulated genes). (C) Single cell RNA sequencing was performed before and after therapy with either anti–PD-L1 (n = 3) or anti–PD-1 (n = 4). Figure shows the number of shared differentially expressed (Wilcoxon rank-sum with Bonferroni’s correction, P < 0.05) genes after versus before treatment between all anti–PD-L1 treated monocytes and all anti–PD-1–treated monocytes. (D) Uniform manifold approximation and projection (UMAP) plots of monocytes from single cell RNA sequencing of anti–PD-L1 monocytes before and after treatment (left panel: blue, after treatment; red, before treatment) and monocyte groups identified by unsupervised clustering (right panel). Cluster 1 represents CD16+ monocytes; clusters 2, 3, and 4 represent CD16– monocytes.

Figure 3. PD-L1 blockade leads to distinct plasma cytokine profiles.

Plasma collected before and after therapy with anti–PD-L1 (n = 10) or anti–PD-1 (n = 20, as previously published; ref. 10) was analyzed using Luminex multiplex/ELISA. Figure shows changes in plasma IP-10 (A), IL-18 (B), GRO-α/CXCL1 (C), IFN-α2 (D), and sCD40L (E) following therapy with anti–PD-L1 or anti–PD-1. (*P < 0.05, **P < 0.01, ***P < 0.001, §P = 0.06 by Mann-Whitney U test).

Figure 4. Changes in circulating immune cells following therapy with anti–PD-L1 in asymptomatic myeloma (AMM).

PBMCs isolated from blood of AMM patients before therapy (PreC1) and following therapy with atezolizumab on day 15 (C1D15), as well as before cycles 2–7 (C2–C7) were analyzed using single cell mass cytometry or CyTOF. (A) Changes in circulating CD3+, CD4+, CD8+ T cells, monocytes, and B cells. Data are shown as fold change compared with pretherapy (PreC1) levels. (B) Expression of CD40, HLA-DR, and CD16 on circulating monocytes before therapy (Pre), on day 15 following first dose (C1D15), and before second dose of atezolizumab (C1D21) in 2 different patients (PT1 and PT2). (C) Changes in circulating monocytes in MM patients receiving atezolizumab in another clinical trial (NCT02431208). Each line represents an individual patient. EOT, end of therapy.

Figure 5. Changes in circulating T cells following therapy with anti–PD-L1 in asymptomatic myeloma (AMM).

PBMCs isolated from blood of AMM patients before therapy (PreC1) and following therapy with atezolizumab on day 15 (C1D15), as well as before cycles 2–7 (C2–C7), were analyzed using single cell mass cytometry or CyTOF. (A) Changes in CD4 and CD8 naive and memory T cells during therapy with atezolizumab. Data are shown as fold change compared with levels before starting therapy (PreC1). (B) Ki-67+ proliferating naive (CCR7+RO–), central memory (Tcm; CCR7+RO+), and effector memory (Tem; CCR7–RO+) T cells before (Pre), 15 days following start of therapy (C1D15), and before cycle 2 (C1D21) of therapy with atezolizumab. Figure shows data from 2 separate patients. (C) PBMCs obtained pre therapy (PreC1), 15 days after starting therapy (C1D15), or before cycles 2–7 (C2-C7) were evaluated for the presence of SOX2-specific T cell reactivity using overlapping peptides encompassing the entire SOX2 antigen as previously described (10). Figure shows SOX2 T cell reactivity in the 2 patients. Data reported as fold change compared with before therapy (PreC1) for SOX2 reactive submix versus nonreactive mix as control.

Figure 6. Changes in monocytes and effector T cells in BM following therapy with anti–PD-L1 in AMM by mass cytometry.

BM was collected from AMM patient before and after 2 cycles (6 weeks) of therapy with atezolizumab. Mononuclear cells were isolated and analyzed using single cell mass cytometry, as well as single cell RNA sequencing. (A) Bar graph shows changes in CD3, CD4, CD8 T cells, CD14+ myeloid cells and B cells at 6 weeks following therapy with atezolizumab. (B) Changes in CD14+ myeloid cells. (C) Histogram showing changes in HLA-DR expression in CD14+ myeloid cells following therapy with atezolizumab. (D) Proportions of granzyme and T-bet positive naive, Tcm, and Tem cells in the marrow before start of therapy, as well as 6 weeks following therapy with atezolizumab.

Figure 7. Changes in monocytes and effector T cells in BM following therapy with anti–PD-L1 in AMM by single cell RNA sequencing.

BM was collected from AMM patient before and after 2 cycles (6 weeks) of therapy with atezolizumab. CD138– BM cells obtained before therapy and 6 weeks after therapy were characterized using sc-RNA–Seq. (A) t-SNE plot with 9 distinct populations determined by unsupervised clustering. Figure also shows distribution of the immune cells from before therapy BM (Pre) and after therapy BM (Post). (B) Percent of immune cells from before therapy (Pre) and after atezolizumab therapy (Post) within the clusters shown in A.

Figure 8. PD-L1 blockade leads to functional changes in DCs.

Immature Mo-DCs generated from healthy blood donors were either left untreated (control, Cntr) or treated with either anti–PD-L1 antibody (200 μg/mL), anti–PD-1 antibody (200 μg/mL), or their respective isotype control antibodies (Ig-G2b and Ig-G1) at 200 μg/mL or CD40L (250 ng/mL). Culture supernatants were analyzed for changes in cytokines using Luminex assay. Representative data from 7 healthy donors (HDs). (A) DC maturation following treatment with either anti–PD-L1, anti–PD-1, or isotype control. Figure shows fold change in CD83 and CD80 double-positive DCs compared with untreated cells. (B) Changes in secreted IL-8, IL-6, TNF-α, and IL-1β following treatment with anti–PD-L1 or anti–PD-1. (C) Treatment with anti–PD-L1 leads to early activation of caspase-1. Fold change of activated caspase-1 in immature Mo-DCs following treatment with anti–PD-L1 or anti–PD-1 for 4 hours. Figure shows fold change compared with untreated cells (Cntr). (D) Changes in respiratory capacity of DCs following treatment with anti–PD-L1. Immature Mo-DCs (n = 3 HDs) were either left untreated (control; Cntr) or were treated with anti–PD-L1 (200 μg/mL for 3 hours), and their spare respiratory capacity was analyzed using Seahorse XFe96 analyzer. Basal, coupled, maximal, and spare respiratory capacities were analyzed. Line graph shows data from a representative patient. Bar graph on the right shows data from all 3 different donors (mean ± SEM). (*P < 0.05, **P < 0.01, ***P < 0.001; A, C, D used Mann-Whitney U test, and B used Kruskal Wallis test).

Figure 9. PD-L1 blockade synergizes with CD40L to improve antigen-specific T cell expansion.

Immature Mo-DCs generated from healthy blood donors were either left untreated (control, Cntr) or treated with CD40L (250 ng/mL) alone (–) or with anti–PD-L1 antibody (200 μg/mL), anti–PD-1 antibody (200 μg/mL), or their respective isotype control antibodies (Ig-G2b and Ig-G1) at 200 μg/mL. (A) Anti–PD-L1 treatment synergizes with CD40L to improve DC maturation. Figure shows fold change in DC maturation (assessed by increase in CD83 and CD80 double-positive cells) compared with control cells. (B) Representative data from one donor showing increased DC maturation with concurrent treatment with CD40L and PD-L1. (C) Immature Mo-DCs (HLA-A2.1+) were stimulated with CD40L alone or CD40L plus anti–PD-L1 antibody. After overnight culture, DCs were loaded with HLA-A2.1-specific influenza matrix peptide (FMP) at 0.1 μg/mL and used to stimulate autologous T cells. After 10–12 days of DC–T cell coculture, expansion of influenza-specific T cells was analyzed using FMP-specific tetramer. (*P < 0.05, Kruskal Wallis test).