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. 2022 Oct;10(10):e005326.
doi: 10.1136/jitc-2022-005326.

Arginase-2-specific cytotoxic T cells specifically recognize functional regulatory T cells

Stine Emilie Weis-Banke #  1 , Thomas Landkildehus Lisle #  1 , Maria Perez-Penco  1 , Aimilia Schina  1 , Mie Linder Hübbe  1 , Majken Siersbæk  2 , Morten Orebo Holmström  1   3 , Mia Aaboe Jørgensen  1 , Inge Marie Svane  1 , Özcan Met  1 , Niels Ødum  3 , Daniel Hargbøl Madsen  1 , Marco Donia  1 , Lars Grøntved  2 , Mads Hald Andersen  4   3
Affiliations

Arginase-2-specific cytotoxic T cells specifically recognize functional regulatory T cells

Stine Emilie Weis-Banke et al. J Immunother Cancer. 2022 Oct.

Abstract

Background: High expression of the metabolic enzyme arginase-2 (ARG2) by cancer cells, regulatory immune cells, or cells of the tumor stroma can reduce the availability of arginine (L-Arg) in the tumor microenvironment (TME). Depletion of L-Arg has detrimental consequences for T cells and leads to T-cell dysfunction and suppression of anticancer immune responses. Previous work from our group has demonstrated the presence of proinflammatory ARG2-specific CD4 T cells that inhibited tumor growth in murine models on activation with ARG2-derived peptides. In this study, we investigated the natural occurrence of ARG2-specific CD8 T cells in both healthy donors (HDs) and patients with cancer, along with their immunomodulatory capabilities in the context of the TME.

Materials and methods: A library of 15 major histocompatibility complex (MHC) class I-restricted ARG2-derived peptides were screened in HD peripheral blood mononuclear cells using interferon gamma (IFN-γ) ELISPOT. ARG2-specific CD8 T-cell responses were identified using intracellular cytokine staining and ARG2-specific CD8 T-cell cultures were established by enrichment and rapid expansion following in vitro peptide stimulation. The reactivity of the cultures toward ARG2-expressing cells, including cancer cell lines and activated regulatory T cells (Tregs), was assessed using IFN-γ ELISPOT and a chromium release assay. The Treg signature was validated based on proliferation suppression assays, flow cytometry and quantitative reverse transcription PCR (RT-qPCR). In addition, vaccinations with ARG2-derived epitopes were performed in the murine Pan02 tumor model, and induction of ARG2-specific T-cell responses was evaluated with IFN-γ ELISPOT. RNAseq and subsequent GO-term and ImmuCC analysis was performed on the tumor tissue.

Results: We describe the existence of ARG2-specific CD8+ T cells and demonstrate these CD8+ T-cell responses in both HDs and patients with cancer. ARG2-specific T cells recognize and react to an ARG2-derived peptide presented in the context of HLA-B8 and exert their cytotoxic function against cancer cells with endogenous ARG2 expression. We demonstrate that ARG2-specific T cells can specifically recognize and react to activated Tregs with high ARG2 expression. Finally, we observe tumor growth suppression and antitumorigenic immunomodulation following ARG2 vaccination in an in vivo setting.

Conclusion: These findings highlight the ability of ARG2-specific T cells to modulate the immunosuppressive TME and suggest that ARG2-based immunomodulatory vaccines may be an interesting option for cancer immunotherapy.

Keywords: Arginase; Immunomodulation; Tumor Microenvironment; Vaccination.

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Conflict of interest statement

Competing interests: MHA is named as an inventor on various patent applications relating to the therapeutic use of arginase peptides, including ARG2 peptides, for vaccination. These patent applications have been transferred to the company IO Biotech ApS whose purpose is to develop immunomodulatory vaccines for cancer treatment. MHA is cofounder, shareholder and scientific advisor of IO Biotech ApS. IMS is cofounder, shareholder and clinical advisor of IO Biotech ApS. The remaining authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Identification of an ARG2 peptide that elicits CD8+ T-cell responses. (A, left) IFN-γ ELISPOT screening of responses to 15 different HLA-A2 predicted ARG2-derived peptides in five healthy HLA-A2+ donors. 3×105 cells were plated per well. Control and peptide stimulations were done in triplicates. Peptide-specific IFN-γ secreting cells are given as the difference of spots between cells in the peptide-stimulated wells and control wells. (A, right) Representative examples of the ELISPOT responses (shown in A). (B, left) IFN-γ ELISPOT responses of three HDs to A2S05. 3×105 cells were plated per well. Control and peptide stimulations were done in triplicates. Bars represent the mean IFN-γ spot count±SD. (B, right) Representative examples of the ELISPOT responses (shown in B). (C) Representative intracellular cytokine staining for IFN-γ and TNF-α secreting in samples from three HDs stimulated with control or A2S05 gating strategy is available in online supplemental figure S6. DP: TNF-α+ IFN-γ+. DP, double positive; HD, healthy donor; IFN-γ, interferon gamma; PBMC, peripheral blood mononuclear cell; TNF-α, tumor necrosis factor alpha.
Figure 2
Figure 2
A2S05 elicits responses in both HLA-A2+ and HLA-A2− donors and responses are also detectable ex vivo. (A) IFN-γ ELISPOT responses to A2L2 and A2S05 in 17 HDs. 3.5×105 cells were plated per well. Control and peptide stimulations were done in triplicates. Peptide-specific IFN-γ secreting cells are given as the difference of spots between cells in the peptide-stimulated wells and control wells. Each dot represents one donor and bars represent the mean. (B) IFN-γ ELISPOT responses to A2S05 in 30 HDs and 13 patients with cancer (2 patients with prostate cancer and 11 patients with melanoma) with known HLA types. Each dot represents one donor and bars represent the mean. (C, left) Ex vivo ELISPOT responses to A2S05. 9×105 cells were plated per well and control and peptide stimulations were done in three to six replicates. Bars represent the mean IFN-γ spot count±SD. *P≤0.05 (according to the DFR rule) (C, right) Representative examples of the ex vivo ELISPOT responses (shown in C). (D) HLA-typing data from the three HDs and one patient with cancer (AA01, melanoma) with strong responses to A2S05. HLA types shared between donors are highlighted in yellow. DFR, distribution free resampling rule; HD, healthy donor; IFN-γ, interferon gamma; PBMC, peripheral blood mononuclear cell.
Figure 3
Figure 3
ARG2-specific CD8+ T cells recognize A2S05 in the context of HLA-B8. (A) Generation of A2S05-specific T-cell cultures. ARG2-specific CD8+ T cells were expanded from three HDs and one patient with cancer (AA01, melanoma). The specificity of the T-cell cultures against A2S05 was assessed by intracellular cytokine staining for TNF-α and IFN-γ. CD107a was included as a marker of cytotoxicity. Bars show the percentage of CD8+ T cells expressing CD107a and secreting IFN-γ, TNF-α or both (DP) in response to Ctrl stimulation or A2S05 stimulation (pep). (B–D) ARG2-specific T cells from two donors (HD78 and HD93) were evaluated in IFN-γ ELISPOT with cancer cells lines prepulsed with A2S05 peptide. The same cell lines without peptide stimulation were included as Ctrls. 3×104 ARG2-specific T cells were plated with 1×104 cancer cells (E:T of 3:1). The cell lines were either HLA-A1+ (B), HLA-C7+, (C) or HLA-B8+ (D). T cells plated alone (−) or T cells plated with A2S05 (+pep) served as negative and positive Ctrls, respectively. Bars represent the mean IFN-γ spot count±SD of technical triplicates. (E) 51Cr-release assay of FM6 and FM6 stimulated with IFN-γ (100 U/mL) for 24 hours prior to the assay with A2S05-specific T cells from HD78. Error bars represent mean±SD of technical duplicates. Ctrl, control; DP, double positive; E:T, effector-totarget ratio; -HD, healthy donor; IFN-γ, interferon gamma; TNF-α, tumor necrosis factor alpha.
Figure 4
Figure 4
ARG2-specific T cells recognize and react to stimulated Tregs (Tregs). (A–C) IFN-γ ELISPOT responses of ARG2-specific CD8+ T cells to in vitro activated and expanded Tregs (Tregs) or resting CD4+ T cells (Trest). 5×104 ARG2-specific CD8+ T cells were plated per well with 5×103 target cells (E:T of 10:1). Bars show the mean IFN-γ spot count±SD. Background from Tregs and Trest have been corrected in the bars. All conditions were plated in six replicates. *P≤0.05 (according to the DFR). (D) Flow cytometry assessment of the Treg population after coculture of activated PBMCs with autologous CD8+ T cells (Ctrl T cells) or ARG2-specific T cells for 6 hours. Cocultures were set up with a 3:2 ratio of activated PBMCs to T cells. Activated PBMCs alone were used to set the gates for Tregs, which are similar to the gates used for sorting of Tregs for the ELISPOT experiment in (C). Bars show the mean decrease in Treg population when cocultured with Ctrl T cells or ARG2-specific T cells compared with PBMCs plated alone±SD of three technical replicates (depicted as dots). *P=0.0194 (unpaired t test). (E) RT-qPCR analysis of ARG2 expression in the Tregs and Trest used for the ELISPOT (in A–C). The bulk culture of activated PBMCs from which the Treg and Trest were isolated was also included. ARG2 expression was normalized to the housekeeping gene POL2RA and presented as fold change versus Trest. Bars show the mean±SD of technical triplicates. (F) Western Blot analysis assessing ARG2 expression in sorted Tregs and Trest from HD93. Cell lines Set2 and UKE-1 are included as ARG2 positive and negative Ctrls, respectively. (G, H) IFN-γ ELISPOT responses of ARG2-specific CD4+ T cells to in vitro activated and expanded Tregs or Trest. 5×104 ARG2-specific CD4+ T cells were plated per well with 5×103 target cells (E:T of 10:1). Bars show the mean IFN-γ spot count±SD of six technical replicates. Background from Tregs and Trest have been corrected in the bars. All conditions were plated in six replicates. *P≤0.05 (according to the DFR). Ctrl, control; DFR, distribution free resampling rule; HD, healthy donor; IFN-γ, interferon gamma; PBMC, peripheral blood mononuclear cell; Treg, regulatory T cell; Trest, resting T cell.
Figure 5
Figure 5
Characterization of sort-purified Tregs. (A) MFI of FOXP3-PE in sorted Tregs and Trest from the three donors following intracellular staining. (B–F) RT-qPCR analysis of the expression of IL2RA (B), TNFRSF18 (C), IKZF2 (D), CTLA4 (E), or IL10 (F) in the Tregs and Trest. The expression of each Treg signature gene was normalized to the housekeeping gene POL2RA and presented as fold change versus Trest with bars showing the mean±SD of technical triplicates. (G) RT-qPCR analysis of PDCD1 expression in Tregs and Trest. Data is represented as relative expression to the housekeeping gene POL2RA (arbitrary units) with bars showing the mean±SD of technical triplicates. (H) In vitro Treg suppression assay. (Left) Percentage of undivided CD8+ T cells after 5 days of co-culture with sort-purified Tregs (+Treg) or Trest (+Trest). Bars indicate the mean of three technical triplicates (depicted as dots). **P=0.0069 (unpaired t-test). (Middle) Proliferation index of CD8 T cells after 5 days of coculture with Tregs or Trest. Bars indicate the mean of three technical triplicates (depicted as dots). **P=0.0022 (unpaired t-test). (Right) CFSE staining of CD8+ T cells cocultured with Tregs or Trest for 5 days. The gray bar depicts the undivided population. CFSE, carboxyfluorescein succinimidyl ester; IL, interleukin; MFI, mean fluorescence intensity; Treg, regulatory T cell; Trest, resting T cell.
Figure 6
Figure 6
An ARG2-based immunomodulatory vaccine reduces tumor growth, induces activation of ARG2-specific CD4+ and CD8+ T cells and promotes a proinflammatory tumor microenvironment. (A) Overview of the experimental timeline. (B) Average Pan02 tumor growth for mice receiving a Ctrl or ARG2-peptide immunomodulatory vaccine. Mice (n=10 per group) were inoculated and vaccinated according to the experimental overview (in A). Data are represented as mean±SEM. ****P<0.0001 (two-way analysis of variance test). (C) IFN-γ ELISPOT on splenocytes isolated from mice receiving the Ctrl (n=3) or ARG2-based (n=6) vaccine from the study in shown in B. Briefly, 8×105 splenocytes were plated per well. Ctrl and peptide stimulations were performed in triplicates. Peptide-specific IFN-γ secreting cells were quantified as the difference in the number of spots counted between the peptide-stimulated and Ctrl wells. Each dot represents one mouse and bars represent the mean±SEM. *P=0.0238 (Mann-Whitney test). (D) ARG1 and ARG2-specific IFN-γ-secreting cells among splenocytes from Pan02 tumor-bearing mice receiving the ARG2-based peptide vaccine were assayed with an IFN-γ ELISPOT. In this assay, 8×105 splenocytes were plated per well with either ARG1 or ARG2 peptide. Each dot represents one mouse, and bars represent the mean±SEM. *P=0.0022 (Mann-Whitney test). (E) ARG2-specific IFN-γ-secreting cells present in CD4+ and CD8+-sorted T cells isolated from splenocytes of Pan02 tumor-bearing mice treated with the ARG2-based peptide vaccine were assayed with an IFN-γ ELISPOT assay. In this assay, 2.8×105 T cells were plated together with 6×105 antigen-presenting cells (splenocytes from a naïve mouse) with or without ARG2 peptide. Each dot represents one sample (pooled from two mice), and bars represent the mean±SEM. (F) Randomly selected tumors from Pan02 tumor-bearing mice from the experiment (in B) were harvested (n=4–6 per group) on day 31. Tumor RNA was extracted, and bulk RNAseq was performed. Differentially expressed genes (false discovery rate (FDR) <0.05 and absolute log2 fold change >0) in Pan02 tumors from ARG2-vaccinated mice compared with Ctrl vaccinated mice were identified and presented in a volcano plot, n=282 upregulated and n=33 downregulated genes. (G) Immune-related biological processes (Gene Ontology analysis) associated with significantly upregulated genes in RNAseq data (described in F). (H–J) Bar plots showing the total absolute immune infiltration score (arbitrary unit) of all immune populations (H) or the immune score ratio of M1:M2 macrophages (I) and CD8 T cells to Tregs (J). Immune population scores were generated with ImmuCC algorithm, using bulk tumor RNAseq (as described in F). Each dot represents one mouse, and bars represent the mean±SEM. Ctrl, control; IFN-γ, interferon gamma; Treg, regulatory T cell.
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