Tumor-derived Immunoglobulin Like Transcript 5 And 4 Induce Suppressive Immunocyte Infiltration In Colorectal Cancer

Purpose:Infiltration of immunosuppressive cells in the tumor microenvironment(TME)induced colorectal cancer(CRC)progression and their resistance to immunotherapy.Identification of tumor specific factors to modulate inhibitory immunocyte infiltration would provide alternative and novel targets for CRC immunotherapy.Immunoglobulin like transcripts(ILT)5 and Immunoglobulin like transcripts 4 are negative regulators of myeloid cell activation.Studies have shown that ILT5 is mainly expressed on myeloid cells and can negatively regulate immune functions of various cells.However,the expression and functional role of ILT5 in solid tumors is still unknown.ILT4 is enriched in solid tumor cells,facilitating their proliferation,invasion,and metastasis.But its role of immune regulation in colorectal cancer is still unclear.Here we aimed to analyze the expression of ILT5 and ILT4 in CRC,and delineate how tumor-derived ILT5/ILT4 regulates immune cell infiltration and distribution in CRC.Method:1.The expression of ILT5 in tumor cell lines and epithelial cell lines was evaluated by real-time PCR and western blot.2.Immunohistochemical(IHC)staining,immunofluorescence(IF)staining and public database analysis were used to detect the expression of ILT5 in CRC tissues.The characteristics of immunocyte infiltration was determined and analyzed by IHC in CRC tissues.3.Gather statistical analysis of the correlation of ILT5 expression,immune cell infiltration and clinicopathological parameters.4.The correlation between ILT5 expression,immune cell infiltration and patient survival status was statistically analyzed.5.The MC38 mouse colorectal cancer cell line with overexpressed/control,knockdown/control of PIR-B(orthologs of ILT5 in mouse)was subcutaneously injected in C57BL/6 mice,forming a tumor transplantation model,to explore the regulation function of ILT5 of CRC.Immune cell infiltration characteristics in mouse peripheral blood were detected by flow cytometry,and PIR-B expression and its correlation with immune cell infiltration were analyzed by IHC in paraffin embedded sections of mouse tumor tissues.6.IHC staining and public database analyses determined the correlation of ILT4 expression with different T cell subset densities,IFN-y levels,and patient outcomes.7.The correlation between ILT4 expression,immune cell infiltration and survival status was statistically analyzed.Result:ILT5 was highly expressed in CRC cells compared with normal colorectal epithelial cells.Enriched ILT5 in tumor cells was correlated with advanced tumor stages and poor patient survival.Tumor-derived ILT5 inhibited the infiltration of T cells especially that of CD3+and CD8+T cells in the TME,creating suppressive T cell contexture.Furthermore,ILT5 directed M2-like polarization of tumor-associated macrophages(TAMs).Inhibition of tumor-derived ILT5 restored the immunosuppressive T cell and TAM contexture and restricted CRC progression.High ILT4 expression in CRC cells was associated with decreased T cell infiltration,disease progression,and poor patient survival.T cell subset analyses indicated that ILT4-high patients showed reduced CD8+T cell but elevated FOXP3+regulatory T(Treg)cell frequencies in the TME.High ILT4 levels predicted lower IFN-y production by tumor-infiltrating lymphocytes(TILs),especially by CD8+T cells in human CRC tissues.Conclusion:ILT5 is enriched in CRC cells,functioning as a negative prognostic biomarker.ILT5 inhibits the infiltration of CD3+and CD8+T cells and induces M2-like polarization of TAMs,creating immunosuppressive TME for CRC progression.ILT4 in CRC cells induced immunosuppressive T cell subset infiltration and impaired IFN-y production in TILs,suggesting that ILT4 might be a potential immunotherapeutic target and prognostic biomarker.