Arginase-1-specific T cells target and modulate tumor-associated macrophages

Abstract

Background: Arginase-1 (Arg1) expressing tumor-associated macrophages (TAMs) may create an immune-suppressive tumor microenvironment (TME), which is a significant challenge for cancer immunotherapy. We previously reported the existence of Arg1-specific memory T cells among peripheral blood mononuclear cells (PBMCs) and described that Arg-1-based immune modulatory vaccines (IMVs) control tumor growth and alter the M1/M2 macrophage ratio in murine models of cancer. In the present study, we investigated how Arg1-specific T cells can directly target TAMs and influence their polarization.

Methods: Murine Arg1-specific CD4+T cells isolated from splenocytes of animals vaccinated with an Arg1-derived peptide in the adjuvant montanide were co-cultured with either in vitro M2-differentiated bone marrow-derived macrophages or ex vivo isolated F4/80+TAMs. Human Arg1-specific CD4+T cell clones were co-cultured with Arg1-expressing TAMs generated in vitro from either PBMC-derived CD14+cells or the myeloid cell lines MonoMac1 and THP-1. MHC class II-restricted Arg-1 peptide presentation by macrophages was confirmed by immunopeptidomics. T-cell-mediated changes in the macrophage immune phenotype and cytokine microenvironment were examined using flow cytometry, RT-qPCR and multiplex immunoassay. The effect of Arg1-derived peptide IMV on TAMs in vivo was assessed by multiplex gene analysis of F4/80+cells.

Results: We show that Arg1-based IMV-mediated tumor control was linked to a decrease in multiple immunosuppressive pathways in the TAM population of the treated animals. Tumor-conditioned media (TCM) derived from Arg1-vaccinated mice induced significantly higher upregulation of MHC-II on exposed myeloid cells compared with controls. Furthermore, murine CD4+Arg1-specific T cells were able to target TAMs and effectively reprogram their phenotype ex vivo by secreting IL2 and IFNγ. Next, we established that human Arg1+TAMs present Arg1-derived peptides and are directly recognized by proinflammatory CD4+Arg1-specific T cell clones. These CD4+Arg1-specific T cells were able to reprogram TCM-conditioned macrophages as observed by increased expression of CD80 and HLA-DR.

Conclusions: TAMs may be directly targeted and modulated by Arg1-specific CD4+T cells. These findings provide a strong rationale for future clinical development of Arg1-based IMVs to alter the immune-suppressive TME by reprogramming TAMs and promoting a proinflammatory TME.

Keywords: Immune modulatory; Macrophage; T cell; Vaccine.

Figure 1. Arg1 IMV treatment-induced MC38 tumor growth delay is associated with changes in TAM phenotype in vivo. (A) MC38 tumor growth curves of mice treated with Arg1 IMV (n=12), OVA (n=9) peptide emulsion in montanide or peptide-free montanide emulsion (n=12). 100 µg of each peptide was injected on days 0 and 7 post-tumor inoculation. Statistical differences were identified by applying a mixed model. **p≤0.01; ***p≤0.001. (B) Arg1 IMV and Ova _323-339 peptide specific responses quantified by IFNγ ELISpot in CD4+T cells isolated from splenocytes of treated tumor bearing animals. (C) Heat map of differentially expressed genes in TAMs purified from Arg1 IMV-treated mice, in comparison with gene signature of OVA _323–339-treated animals. F4/80+macrophages were sorted from MC38 tumors 13 days after tumor inoculation. Gene expression is plotted as difference with mean log2 expression. (D–I) Difference individual gene expression for TAMs sorted from peptide-free montanide control, Ova or Arg1 IMV-vaccinated animals. Differences in Arg1 (D), Nos2 (E), Trem2 (F), Mrc1 (G), Marco (H) and Ccl24 (I) were evaluated by applying an unpaired T test. *p≤0.05, **p≤0.01. (J) Expression of MHC-II in bone marrow-derived macrophages cultured overnight with TCM derived Montanide, OVA _323-339 or Arg1 IMV treated MC38-bearing animals. All bars represent mean values±SEM. Statistical differences in flow data were analyzed by applying an unpaired student t-test. *p≤0.05. IMV, immune modulatory vaccine; ns, not significant; TAM, tumor-associated macrophage.

Figure 2. Murine Arg1-specific T cells reprogram Arg1-expressing macrophages ex vivo. (A) Arg1 expression determined through RT-qPCR analysis of murine bone marrow derived macrophages (BMDM) undifferentiated (M0, n=4) or differentiated into M2 (n=3). (B) Diagram representing the experimental set-up: (1) CD4+T cells were isolated from the spleens of mice vaccinated with either the peptide-free control or Arg1 IMV and co-cultured ON with (2) M2 differentiated BMDM. Created with BioRender.com. (C) Arg1 IMV peptide-specific T-cell responses quantified by IFNγ ELISpot in CD4+T cells isolated from splenocytes of vaccinated mice. (D) Phenotype changes in the macrophage population as determined by changes in the percentage of CD80^Hi, CD86^Hi, MHC-II^Hi, CD206^Hi and PD-L1^Hi macrophages in co-cultures with CD4+T cells isolated from control (gray) or Arg1 IMV (blue) treated animals as determined by flow cytometry. n=3 per group. (E) IL2 and IFNγ concentrations in co-culture supernatants from M2 macrophages alone (dark gray) or after ON co-culture with CD4+T cells from control (light gray) (n=2) or Arg1 IMV-treated mice (blue) (n=2) as measured by Luminex. (F) BMDM-M2-specific responses quantified by IFNƴ ELISpot using sorted CD4+T cells from OVA_323–339 (n=3) and Arg1 IMV (n=3) vaccinated mice. BMDM-M2 was co-incubated with CD4+T cells at a ratio of 1:5. (G) Arg1 expression in unsorted MC38 tumor bulk samples (n=9) and isolated F4/80+tumor-infiltrating macrophages (n=3) as measured by RT-qPCR. The expression of Arg1 was evaluated relative to hypoxanthine phosphoribosyltransferase (HPRT) expression. (H) Flow cytometry analysis of CD206 and PD-L1 expression on TAMs alone or TAMs after co-culture with CD4+T cells isolated from either control animals (light gray) (n=3), OVA-vaccinated animals (purple) (n=1) or Arg1 IMV vaccinated mice (blue) (n=2). TAMs were isolated from MC38 tumors. (I) representative contour plot showing PD-L1 and CD206 coexpression on TAMs after co-culture with CD4+T cells from control (gray) or Arg1 IMV (blue) treated mice. TAMs were isolated from MC38 tumors. Data are displayed as average±SEM. Statistical differences were analyzed by applying an unpaired Student’s t-test. *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001. E:T ratio of 5:1 was used. IMV, immune modulatory vaccines; ns, not significant; TAMs, tumor-associated macrophages.

Figure 3. Human Arg1-specific CD4+ T cells but not tumor antigen-specific T cells impact Arg-1 expressing myeloid cells and promote a proinflammatory microenvironment. (A) ARG1 expression in human THP-1 cells as determined by RT-qPCR. THP-1 cells were either undifferentiated (M0), treated with 200 U/mL IFNγ (M1-like), IL-13 20 U/mL (M2-like), or tumor-conditioned media (TCM-THP1) for 48 hours prior to expression analysis. All bars represent average values ±SEM. (B) Intracellular cytokine staining of IFNγ and TNFα production by Arg1-specific CD4+ T cell clone reacting against HLA-matched myeloid cancer cell line THP-1. Myeloid cells were either used undifferentiated (M0) or differentiated with TCM derived from ovarian cancer cells (TCM-THP-1) for 48 hours. E:T ratio of 4:1 was used. (C) Arg1-derived peptide sequences presented by the THP-1 cells differentiated in vitro into a TAM-like phenotype using TCM (blue) aligned with the ArgLong2 peptide (black) as identified by mass spectrometry-based immunopeptidome profiling. Illustration of the experimental workflow created with BioRender.com. (D) representative histograms (left) and bar plots (right) representing changes in % of HLA-DR+ cells and PD-L1 MFI on TCM-THP-1 after co-culture with an HLA-matched Arg1-specific CD4+ T cell clone as determined by flow cytometry. (E) IL2, IFNγ, TNFα, IL6 and IL8 cytokine concentrations in culture supernatants of either an Arg1-specific T cell clone co-cultured with HLA-matched TCM-THP1, TCM-THP1 alone, or the Arg1-specific CD4+ T cell clone alone. (F) IL2, IFNγ, TNFα, IL6 and IL8 cytokine concentrations in culture supernatants of either an Arg1-specific T cell clone co-cultured with TCM-MonoMac1 cells with or without HLA-DR blockade, TCM-MonoMac1 cells alone, or an Arg1-specific CD4+ T cell clone alone. (G) Representative histograms (left) and bar plot representation (right) of changes in MFI of HLA-DR and PD-L1 on TCM-MonoMac1 cells after co-culture with HLA-matched Arg1-specific CD4+ T cell clone as determined by flow cytometry. TCM-MonoMac1 cells were co-cultured with the Arg1-specific clone directly or after pre-treatment with HLA-DR blocking antibody. All T-cell and myeloid co-cultures were set up at a E:T ratio of 1:10. Error bars represent SEM of two technical replicates. (H) Representative histograms (left) and bar plot representation (right) of changes in MFI of HLA-DR and PD-L1 on TCM-MonoMac1 cells after co-culture with HLA-matched Mart1-specific CD4+T cell clone as determined by flow cytometry. Mart1-peptide loaded TCM-MonoMac1 cells (orange histograms) were used as positive control. All T-cell and myeloid co-cultures were set up at a E:T ratio of 1:10.

Figure 4. Human Arg1-specific CD4+ T cell clones recognize and polarize CD14+ monocyte derived Arg-1 expressing myeloid cells to promote a proinflammatory microenvironment. (A) ARG1 expression as determined by RT-qPCR in undifferentiated human CD14+ cells or CD14+ cells differentiated to a TAM-like phenotype by in vitro treatment with ovarian cancer cell-derived tumor-conditioned media (TCM-CD14) for 48 hours. All bars represent average values ±SEM. (B) Intracellular cytokine staining of IFNγ and TNFα production by Arg1-specific CD4+ T cell clone reacting against the Arg1-derived epitope (ArgLong2) (left) or autologous PBMC derived myeloid cell (right). Myeloid cells were either used undifferentiated (M0) or differentiated with TCM derived from breast cancer cells (TCM-CD14) for 48 hours. E:T ratio of 4:1 was used. (C) Representative histograms and (D) bar plot representation of changes in MFI of HLA-DR, CD80 and PD-L1 on TCM-CD14 cells after co-culture with autologous Arg1-specific CD4+ T cell clone as determined by flow cytometry. (E) IL2, IFNγ, TNFα, IL6 and IL8 cytokine concentrations in culture supernatants of either an Arg1-specific T cell clone co-cultured with autologous TCM-CD14, TCM-CD14 alone or the Arg1-specific CD4+ T cell clone alone. PBMC, peripheral blood mononuclear cell.