Abstract
Invariant natural killer T (iNKT) cells are innate-like T cells that are abundant in liver sinusoids and play a critical role in tumor immunity. However, the role of iNKT cells in pancreatic cancer liver metastasis (PCLM) has not been fully explored. In this study, we employed a hemi-spleen pancreatic tumor cell injection mouse model of PCLM, a model that closely mimics clinical conditions in humans, to explore the role of iNKT cells in PCLM. Activation of iNKT cells with α-galactosylceramide (αGC) markedly increased immune cell infiltration and suppressed PCLM progression. Via single cell RNA sequencing (scRNA-seq) we profiled over 30,000 immune cells from normal liver and PCLM with or without αGC treatment and were able to characterize the global changes of the immune cells in the tumor microenvironment upon αGC treatment, identifying a total of 12 subpopulations. Upon treatment with αGC, scRNA-Seq and flow cytometry analyses revealed increased cytotoxic activity of iNKT/NK cells and skewing CD4 T cells towards a cytotoxic Th1 profile and CD8 T cells towards a cytotoxic profile, characterized by higher proliferation and reduced exhaustion marker PD1 expression. Moreover, αGC treatment excluded tumor associated macrophages. Lastly, imaging mass cytometry analysis uncovered the reduced epithelial to mesenchymal transition related markers and increased active CD4 and CD8 T cells in PCLM with αGC treatment. Overall, our findings uncover the protective function of activated iNKT cells in pancreatic cancer liver metastasis through increased NK and T cell immunity and decreased tumor associated macrophages.
| Original language | English (US) |
|---|---|
| Article number | 216149 |
| Journal | Cancer Letters |
| Volume | 561 |
| DOIs | |
| State | Published - May 1 2023 |
Funding
This study was supported by Henry Ford Immunology Program grants (T71016 and T71017) to QSM and LZ, by the National Institutes of Health ( NIH ) grants 1R01AI119041 to QSM , RO1AR072046 to LZ , R33AR076803 to IA and QSM, and R01 CA232347 and CA257520 to DF. Our scRNA-seq data revealed complex infiltrating immune profiles in livers of control, LM, and αGC-treated LM mice. T cells (e.g., CD4 T and CD8 T) failed to infiltrate the liver within the context of pancreatic cancer, as the fractions of CD4 T cells and CD8 T cells were markedly reduced in LM mice compared to control mice, supporting the notion that pancreatic cancer cells exclude T cell liver infiltration, resulting in immunological silencing during progression of liver metastasis. Encouragingly, we found that T lymphoid cells, including CD4 T (Cd3e, Cd3d, and Cd4), CD8 T (Cd3e, Cd3d, and Cd8a), iNKT(Cd3e, Cd3d, Ncr1, and Nkg7), NK (Ncr1, Nkg7, Gzma, and Prf1), and B cells (Cd79a, Ms4a1, and Cd19) were markedly more abundant in αGC treated LM mice than in LM mice, and were also more enriched than in control mice. In addition, macrophages (Csf1r, Itgam), neutrophils (Ly6g, S100a9, and S100a8), plasmacytoid dendritic cells (pDC) (Tlr7 and Bst2), and mast cells (Cpa3, Cd34, and Kit) were also increased in αGC-treated LM mice relative to LM mice. Furthermore, the fractions of macrophages (Csf1r, Itgam, Arg1, and Cd68), myeloid-derived suppressor cells (MDSC) (Cxcl2, Cd14, S100a9, and S100a8), and conventional dendritic cells (cDC) (Btla, Cd24, and Itgax) were reduced in αGC treated LM mice relative to LM mice. (Fig. 2B and C). These data indicate that activated iNKT cells enhance T lymphoid cell liver infiltration and markedly alter the immune profile in PCLM.Recognition of tumor cells by cytotoxic CD8 T cells requires binding with antigen presented on MHC I molecules, which is expressed on all nucleated cells. However, pancreatic cell lines, including FC1242 used in our study, as well as cells from many human pancreatic tumors have lower MHC I levels [59–61], which could be a reason for limited T cell infiltration in the context of pancreatic tumor. In the present study, increased IFN-γ from Th1 CD4 T cells could increase MHC I levels, which might support CD8 T cell function. Moreover, iNKT cells activated with αGC significantly increased infiltration of CD8 T cells with improved cytotoxic properties and higher proliferation. T cell exhaustion is a factor that limits T cell function in cancer, and the blockade of molecules such as PD-1 and CTLA4 can rescue exhausted cells. Our study found that iNKT cells activated with αGC protected cytotoxic CD8 T cells from exhaustion, which restored their antitumor function. In addition, we also observed a novel B-CD8 T cell subpopulation in liver immune cells, which comprised up to 10% of the healthy liver CD8 T cells; However, B-CD8 T cells were markedly reduced in LM mice, and the role of this subpopulation in the progression of tumor metastasis is still unclear.This study was supported by Henry Ford Immunology Program grants (T71016 and T71017) to QSM and LZ, by the National Institutes of Health (NIH) grants 1R01AI119041 to QSM, RO1AR072046 to LZ, R33AR076803 to IA and QSM, and R01 CA232347 and CA257520 to DF.
Keywords
- CD4 T
- CD8 T cell
- Invariant natural killer T(iNKT) cells
- Pancreatic cancer liver metastasis
- Tumor immunity
- scRNA-seq
- α-galactosylceramide (αGC)
ASJC Scopus subject areas
- Oncology
- Cancer Research
