Inhibition of the BTK-IDO-mTOR axis promotes differentiation of monocyte-lineage dendritic cells and enhances anti-tumor T cell immunity

Abstract

Monocytic-lineage inflammatory Ly6c+CD103+ dendritic cells (DCs) promote antitumor immunity, but these DCs are infrequent in tumors, even upon chemotherapy. Here, we examined how targeting pathways that inhibit the differentiation of inflammatory myeloid cells affect antitumor immunity. Pharmacologic inhibition of Bruton's tyrosine kinase (BTK) and the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) or deletion of Btk or Ido1 allowed robust differentiation of inflammatory Ly6c+CD103+ DCs during chemotherapy, promoting antitumor T cell responses and inhibiting tumor growth. Immature Ly6c+c-kit+ precursor cells had epigenetic profiles similar to conventional DC precursors; deletion of Btk or Ido1 promoted differentiation of these cells. Mechanistically, a BTK-IDO axis inhibited a tryptophan-sensitive differentiation pathway driven by GATOR2 and mTORC1, and disruption of the GATOR2 in monocyte-lineage precursors prevented differentiation into inflammatory DCs in vivo. IDO-expressing DCs and monocytic cells were present across a range of human tumors. Thus, a BTK-IDO axis represses differentiation of inflammatory DCs during chemotherapy, with implications for targeted therapies.

Keywords: BTK; Bruton's tyrosine kinase; IDO; antigen-presenting cells; chemotherapy; dendritic cells; immunotherapy; indoleamine 2,3-dioxygenase; tumors.

Conflict of interest statement

Declaration of interests Y.Z. has received clinical trial support from NewLink Genetics (now Lumos Pharma), which holds the rights to indoximod. S.E. was an employee of ArQule (now a wholly owned subsidiary of Merck & Co., Kenilworth, NJ, USA), which holds the rights to ArQ531. E.K. was an employee of NewLink Genetics (now Lumos Pharma). T.L.M. receives consulting income from FLX Therapeutics. B.R.B. holds intellectual property interests in the therapeutic use of IDO inhibitors; receives remuneration as an advisor to Magenta Therapeutics and BlueRock Therapeutics; and receives research funding from BlueRock Therapeutics, Rheos Medicines, and Equilibre Biopharmaceuticals. T.S.J. has received clinical trial funding from NewLink Genetics (now Lumos Pharma). D.H.M. holds patents and intellectual property interests in the therapeutic use of IDO inhibitors and has received consulting income and research support from NewLink Genetics (now Lumos Pharma). The other authors declare no competing interests.

Copyright © 2021 Elsevier Inc. All rights reserved.

Figures

Figure 1.. Pharmacological inhibition of BTK and IDO promotes differentiation of tumor-associated monocyte-lineage DCs

(A-B) Adoptive transfer of monocyte-lineage (cMoP) or dendritic (CDP) precursors into Batf3-deficient hosts, followed by injection of OT-I T cells i.v., and vaccination with ovalbumin protein (OVA) in CpG+incomplete Freund’s adjuvant (IFA), panel A. Flow-cytometry analysis of OT-I proliferation, and phenotype of the transferred precursor cells (B). Representative of 3 independent experiments. (C) Flow cytometry analysis of endogenous Ly6c+CD103+ DCs (gated CD11c+ cells) in vaccine-draining lymph nodes (VDLNs) following vaccination of WT mice versus Batf3−/− (OVA+CpG+IFA vaccine, no adoptive-transfer). * p<0.001 by t-test. (D) Flow cytometry phenotyping of immature Ly6c+c-kit+ precursor population in normal resting LNs in C57BL/6 mice. (E) Flow cytometry analysis of BTK and IDO expression in gated Ly6c+c-kit+ cells from normal LNs, or disaggregated B16F10 melanoma tumors. *pSee also Figure S1

Figure 2.. Pharmacological inhibition of BTK and IDO promotes anti-tumor T cell responses after chemotherapy and reduces tumor growth

(A-C) Measurement of Ly6c+CD103+ DCs as a pharmacodynamic marker. B16F10 tumors were treated with combinations of CTX, different BTK-inhibitors and different IDO-inhibitors to produce a varying range of antitumor efficacy. Response was measured as final tumor size, and Ly6c+CD103+ DCs were quantitated in tumors by flow cytometry. (A) Synergy between CTX, indoximod and graded doses of ibrutinib, by tumor size and Ly6c+CD103+ DCs, both measured on day 15. (B) Linear regression analysis of Ly6c+CD103+ DCs versus tumor response across graded doses of the IDO-inhibitor linrodostat (10-5-2-0 mg/kg/day) plus CTX (given day 10) and ibrutinib (16 mg/kg/day), on a schedule similar to panel A. Regression analysis shows pooled data from all groups, measured on day 15. (C) Ly6c+CD103+ DCs versus final tumor size (day 18) across different combinations of BTK-inhibitor ArQ531 (50 mg/kg/day i.p.) with CTX (day 10) and indoximod. Regression analysis shows pooled data from all groups, measured on day 18. (Based on preclinical pharmacology, the ArQ531 was started on day 6 in these experiments.) (D) C57Bl/6 mice were pre-loaded with resting (naïve) TCR-transgenic pmel-1 T cells, then implanted with B16F10 tumors and treated with CTX + ibrutinib + indoximod, or no treatment. Flow cytometry analysis of Thy1.1+ pmel-1 cells in tumors on day 15. Representative of 3 independent experiments. (E) Flow cytometry analysis showing destabilization of tumor-associated Treg cells (gated CD4+Foxp3+). B16F10 tumors treated with the three regimens as shown, or no treatment. *pSee also Figure S2

Figure 3.. Deletion of Btk or Ido1 replaces pharmacologic inhibition and allows differentiation of inflammatory monocyte-lineage DCs within tumors following chemotherapy

(A) B16F10 tumors implanted in Batf3−/− or WT mice, then treated with CTX + ibrutinib + indoximod, as in Figure 1F. In all panels in the figure, *p<0.001 by ANOVA. (B) Batf3−/− mice bearing B16F10 tumors received adoptive-transfer of sorted cMoPs or CDPs from CD45.1+ WT bone marrow. Control mice received no transfer. All mice were then treated with CTX + ibrutinib + indoximod. Bar graphs show flow cytometry analysis of homing to tumors (left), and phenotype of transferred cells and endogenous host T cells (right) in tumors at the end of therapy. (C) Ido1−/− mice or WT C57BL/6 controls were implanted with B16F10 tumors and treated with CTX alone or CTX + ibrutinib doublet. Pooled data from 4 independent experiments. (D) Batf3−/− mice with established B16F10 tumors received adoptive-transfer of cMoPs from either Btk−/− donors or WT controls. All mice were then treated with CTX + indoximod doublet alone, or CTX + indoximod + ibrutinib. Pooled data from 4 independent experiments. Flow cytometry was performed to measure the ability of each cMoP population to generate Batf3+CD11c+ DCs in tumors (using the CTX + ibrutinib + indoximod regimen), and the phenotype of CD8+ T cells in tumors on day 15. See alsoFigure S3

Figure 4.. Immature Ly6c+c-kit+ precursor cells and inflammatory monocytic DCs in tumor-draining lymph nodes share genetic similarities with pre-cDC1 and cDC1

(A) Ly6c+c-kit+ precursor cells (CD11cNEG) were sorted from tumor-draining LNs (TDLNs) of untreated B16F10 tumors (day 14). Chromatin accessibility was assessed by ATAC-seq (A). Tracks are compared against published datasets for mature cDC1 (Brown et al., 2019) and pre-cDC1 (Durai et al., 2019). Three independent experiments were performed with similar results; a representative track is shown. (B-E) scRNA-seq was performed on total cells from TDLNs of B16F10 tumors, 48 hrs after treatment with CTX + ibrutinib + indoximod. Analysis was performed using Loupe browser. Unsupervised UMAP clustering (B), with heat-map (C) of genes used to assign cluster descriptions. (D) Clusters 2 and 3 were pooled, and individual cells classified as falling in either “monocytic” or “non-monocytic” sub-population based on expression of at least one monocytic-signature gene (Ly6c1, Ly6c2, Csf1r or Lyz2). (E) Expression of selected individual genes by cells classified as either “monocytic-signature” or non-monocytic in pooled Clusters 2+3. See alsoFigure S4

Figure 5.. BTK and IDO in human monocyte-derived DCs inhibit an inflammatory differentiation pathway driven by GATOR2 and mTORC1

Human monocytes were cultured with growth factors and cytokine cocktail as described in Methods, followed by activation with IFNγ to drive terminal differentiation, with or without inhibitory TGFβ. (A) In vitro culture schema, and flow cytometry analysis of gated CD83+ cells on day 8 for each treatment group. Representative of at lease 5 independent experiments. *pIDO1, BTK or components of the GATOR1-GATOR2 complex using siRNA (or scrambled siRNA control). siRNA was added day 5 along with FITC-labeled tracer oligos to identify transfected cells. Cells were analyzed by flow cytometry on day 8, and transfected cells gated as CD83+ FITC+ . (D) Effect of silencing of the DEPDC5 subunit of GATOR1. At least 3-5 experiments per group, *p<0.001 vs both other groups. (E) Effect of silencing IDO1. At least 3-5 experiments per group, *p<0.001 vs both other groups. (F) Effect of silencing BTK on the stability of IDO. At least 5 experiments per group, *p<0.001. (G) Effect of disrupting the WDR24 subunit of GATOR2 on the ability of indoximod (200 uM) to restore mTORC1 activity (p-S6) in vitro. At least 4 experiments per group, *p<0.001 vs both other groups. See alsoFigure S5

Figure 6.. Disrupting the GATOR2-mediated sufficiency signal in monocyte-lineage precursors prevents differentiation into inflammatory DCs in vivo

(A-D) In vitro transduction of wild-type CD45.1+ mouse bulk bone marrow cells, using siRNA against the Wdr24 subunit of GATOR2 (or scrambled control siRNA), plus a FITC-oligo tracer. FITC+ (transduced) cMoPs cells (Ly6c+ c-fms+ c-kit+) were then sorted and transferred into Batf3−/− recipients for vaccination or tumor studies. (A) Rescue of vaccine response by WT cMoPs with either control siRNA or Wdr24 silencing. (B-D) Rescue of anti-tumor response to CTX + ibrutinib + indoximod therapy in Batf3−/− mice by transfer of WT cMoPs, with or without siRNA silencing of Wdr24. All figures represent pooled data from 3-5 independent experiments. (B) Growth curves. *pBatf3−/− with no cMoP transfer; and WT cMoPs with no siRNA transduction) are included for comparison. (C) Wdr24 protein and markers of mTOR activity in tumors on day 15 (+5 days after CTX). (D) Homing of cMoPs to tumors (left, showing transferred cells as a fraction of total Ly6c+ cells in tumor) for un-transduced cMoPs vs Wdr24-silenced and control siRNA. Phenotypic markers (right) on gated CD45.1+ transferred cMoPs at the end of treatment (day 15). p<0.001 by ANOVA.

Figure 7.. Identification of IDO-expressing DCs and monocytic cells across a range of human tumors

(A-C) Multi-spectral immunofluorescence images of human melanoma biopsies stained for BTK (green pseudocolor), IDO (red) and CD33 (magenta). Dual-positive BTK+IDO+ cells show as yellow. Morphology of the BTK+IDO+ population is representative of over 20 melanoma biopsies. Scale bars are 50 μm. (D-G) UMAP analysis was performed on scRNA-seq data from ref. (Cillo et al., 2020), comprising pooled populations of tumor-infiltrating immune cells, control tonsil, and peripheral blood mononuclear cells (PBMC). (D) Outline shows the monocyte-Mϕ-DC (MoMacDC) region, with clusters arbitrarily numbered 1-6. (E) UMAP analysis of gated tumor-infiltrating immune cells, showing cluster distribution of IDO-expressing cells. BTK+IDO1+ dual-positive cells (red) and single-positive IDO1+BTK− cells (blue), with merged distribution (right). Bar graph compares total IDO1+ cells in the MoMacDC region from tumor versus tonsil and PBMC, as a percentage of total immune cells (T, B and all other cells). See also Figure S6B. (F) Heatmap analysis of the clusters in panel D, showing Z-score (normalized gene expression relative to the total of tumor infiltrating cells) for a curated list of genes used to assign identification of each cluster. (G) Percentage of total IDO1+ cells in Clusters 1-6 in tumor-infiltrating cells. Data-points represent values for individual patients (n=26), bars represent mean of pooled samples. Pie chart shows the contribution from each Cluster 1-6 to the total IDO1+ population (pooled samples). (H) UMAP analysis of sc-RNAseq data for 15 additional tumor types from ref. (Cheng et al., 2021) (see also Figure S7). Analysis of total IDO1+ cells in the myeloid population (which corresponds to the MoMacDC region in panel D). Cells were classed as either DC1, LAMP3+ cDC, or DC2, using the authors’ assignment in the original reference; then CD14+ cells were identified in the remaining cells. The corresponding data from panel G are included as the first tumor type (HNC), for comparison. The lower bar graph shows the contribution of each cluster to the total IDO1+ population (corresponding to the pie-chart of panel G). Abbreviations: HNC, head and neck; BRCA, breast; CRC, colorectal; ESCA, esophageal; HCC, hepatocellular; LYM, lymphoma; MEL, melanoma; MYE, myeloma; NPC, nasopharyngeal; OV-FTC, ovarian; PAAD, pancreatic; STAD, gastric; THCA, thyroid; UCEC, endometrial. See alsoFigure S6andS7.