Identification of a novel PD-L1 positive solid tumor transplantable in HLA-A*0201/DRB1*0101 transgenic mice

Abstract

HLA-A*0201/DRB1*0101 transgenic mice (A2/DR1 mice) have been developed to study the immunogenicity of tumor antigen-derived T cell epitopes. To extend the use and application of this mouse model in the field of antitumor immunotherapy, we described a tumor cell line generated from a naturally occurring tumor in A2/DR1 mouse named SARC-L1. Histological and genes signature analysis supported the sarcoma origin of this cell line. While SARC-L1 tumor cells lack HLA-DRB1*0101 expression, a very low expression of HLA-A*0201 molecules was found on these cells. Furthermore they also weakly but constitutively expressed the programmed death-ligand 1 (PD-L1). Interestingly both HLA-A*0201 and PD-L1 expressions can be increased on SARC-L1 after IFN-γ exposure in vitro. We also obtained two genetically modified cell lines highly expressing either HLA-A*0201 or both HLA-A*0201/ HLA-DRB1*0101 molecules referred as SARC-A2 and SARC-A2DR1 respectively. All the SARC-L1-derived cell lines induced aggressive subcutaneous tumors in A2DR1 mice in vivo. The analysis of SARC-L1 tumor microenvironment revealed a strong infiltration by T cells expressing inhibitory receptors such as PD-1 and TIM-3. Finally, we found that SARC-L1 is sensitive to several drugs commonly used to treat sarcoma and also susceptible to anti-PD-L1 monoclonal antibody therapy in vivo. Collectively, we described a novel syngeneic tumor model A2/DR1 mice that could be used as preclinical tool for the evaluation of antitumor immunotherapies.

Keywords: HLA transgenic mouse; PD-L1; T cells; cancer immunotherapy; sarcoma.

Figure 1. Novel transplantable sarcoma cell line in A2/DR1 mice

(A) Generation of the mouse sarcoma cell line derived from a naturally occurring tumor in a 23- months-old female A2/DR1 mouse. (B) contrast microscopy aspects are shown. (C) Sry male-specific amplification from SARC-L1 cell line, and from female and male tissues by PCR. lane 1: 100 bp marker, lane 2: SARC-L1 DNA, lane 3: female DNA, lane 4: male DNA. (D) CD45 and EpCAM surface expression (solid line), and isotype (dotted line). (E) E-cadherin and vimentin expression by western blot analysis from SARC-L1, HT-29 (epithelial control) and CT26 (mesenchymal control). (F) Morphological analysis (hemalun and eosin staining) of SARC-L1 tumor. (G) Immunohistochemistry on SARC-L1 tumor with a positive staining for anti-alpha-smooth muscle actin antibody (left) and a negative staining for anti-pankeratin (middle left), anti-PS100 (middle right), anti-desmin (right).

Figure 2. Genes expression profiling of SARC-L1

(A) Representative cluster of significant enrichment of biological processes obtained with Enrichr website. MAPK pathway, cell cycle processes and TGFβ response are highly activated in this cell line. B, C 3 related sarcoma genes (NBEA, ARSG, MYLK) were quantified by real-time quantitative PCR (B) Electrophoresis was performed with the products from the RTqPCR in a 2% agarose gel. (C) Relative expression of NBEA, ARSG and MYLK genes of SARC-L1 cell using WEHI-164 cells as gene of reference. The mRNA transcripts were calculated using the 2^ΔΔCt method. Amplification of the samples was performed in duplicate and the G6PDH mRNA transcript was used as housekeeping gene of reference (three independent experiments).

Figure 3. Effect of human sarcoma-related cytotoxic drugs on SARC-L1 cells

(A) cells were cultured in presence of increasing doses of chemotherapies during 48 h and the percentage of apoptotic cells was measured using Annexin-V/7-AAD staining. Dot plots of SARC-L1 cells and percentage of early (Annexin-V+/7−AAD−) and late (Annexin-V+/7−AAD+) apoptotic cells. Dot plots are representative of three independent experiments. Bars represent means + SEM from the three independent experiments. (B) Average tumor size in the groups of A2/DR1-mice treated by i.p injection of gemcitabine 120 mg/kg, or cisplatin 7.5 mg/kg or doxorubicin 5 mg/kg, or saline (group control). Mean +/− SEM (n = 4–5 mice/group) *P < 0.05; ***P < 0.001 (Student test). (C) Kaplan–Meier curves. Log-rank (Mantel-Cox) tests are shown: *P < 0.05; **P < 0.01 ***P < 0.001.

Figure 4. Analysis of MHC molecules expression by SARC-L1 cell line

(A–D) Representative MHC molecules expression by SARC-L1. Human MHC (HLA-A2, and HLA-DR) are shown by flow cytometry (A) and confocal microscopy (B). Expression of HLA-A2 and HLA-DR on SARC-L1 cell line cultured in vitro in presence of murine recombinant IFN-γ (100 ng/ml) (D) Representative mouse murine MHC molecules expression by SARC-L1 (H2 Kb, H2 Kd, IA/IE). Specific markers (black line), isotype control (dotted line). (E, F) Tumor microenvironment of SARC-L1 in A2/DR1 mice were analyzed among CD45 positive tumor infiltrating cells. Gating strategy (left), histogram (right) represents mean of percentage +/− SEM (bar) ofimmune cells subpopulation (n = 10 mice). Effector cells (E) and immunosuppressive cells (F) Results are representative of three experiments analysed independently.

Figure 5

Presence of PD-1/PD-L1 axis in SARC-L1 tumor microenvironment A-B PD-L1 expression on SARC-L1 by flow cytometry (A) and confocal microscopy (B, C) PD-L1 expression on SARC-L1 by flow cytometry. SARC-L1 cells were cultured in presence of recombinant mIFN-γ or (D) indicated chemotherapies during 24 h (E) PD-L1 expression on ex vivo freshly isolated tumor cells from A2/DR1 mouse (F) PD-1 and TIM-3 expression. Representative histogram (right) in percentages +/− SEM (bar) of PD-1+ or TIM-3+ among CD4+ and CD8+ TILs cells (n = 10 mice) (G) Individual tumor size in SARC-L1 tumor bearing mice (n = 8 mice/group) treated with anti-PD-L1 or isotype control (untreated). Mice were treated or not by 4 × 200 μg anti-PD-L1 (n = 4–5 mice). (H) Percentage of CD8+ CD3+ TILs (left), CD8+ TIL/Treg ratio (right) in each group. Histogram represent mean + SEM.

Figure 6. Transduction of SARC-L1 with syngeneic MHC-Class I and II molecules as potential tools to improve tumor-specific immunity model

(A) MHC-Class I (HLA-A2.1) and MHC-Class II (HLA-DR.1) expression on SARC-A2 and SARC-A2DR1 tumor cells cultured overnight in the presence or not of recombinant mouse IFN-γ. (B) Tumor size measurement in A2/DR1 mice engrafted with 2.105 SARC-L1, SARC-A2 or SARC-A2DR1 cells (n = 10–17 mice).