Eosinophilia Induced By Radiation Contribute To Cytotoxic T Lymphocytes Infiltration And Improves Immunotherapy

Part1:Eosinophilia induced by radiation contribute to cytotoxic T lymphocytes infiltration and improves immunotherapyBackground:Recent studies have strongly suggested that the prognosis of cancer immunotherapy is largely dictated by intratumoral CD8⁺T cell infiltration and the nature of the individual tumor microenvironment?TME?.By inflaming the TME to favor CD8⁺T cell immunity,radiation is now widely considered a neoadjuvant for immunomodulation.While the local role of radiation is attributed to the type I interferon-mediated TME switch,the systematic role of tumor-focused radiation in immune regulation remains to be explored.In various animal models,we observed that radiotherapy?RT?creates a niche in tumors to recruit eosinophils,and this recruitment is closely associated with enhanced CD8⁺T cell infiltration.Eosinophil depletion dampens CD8⁺T cell infiltration and diminishes the overall antitumor efficacy of radiation,which demonstrates the causative role of eosinophils in promoting T cell-based antitumor effects.This eosinophil-CD8⁺T cell association was also identified in our retrospective cohort studies of radiation-treated cancer patients.Furthermore,in animal models,we showed that radiation enhanced the intratumoral infiltration of transferred cytotoxic T lymphocytes?CTLs?and chimeric antigen receptor?CAR?T cells,and the pattern of enhanced CAR-T cell infiltration was also validated in a CD19-CAR-T cell-infused lymphoma patient.Taken together,our data suggest that eosinophil mobilization can be considered a mechanistically relevant biomarker for predicting radiotherapy prognosis as well as a new strategy to enhance T cell-mediated immunotherapies for cancers.Methods:1.Radiotherapy promoted eosinophils infiltrate into tumor microenvironment.?1?The melanoma B16-F10 tumor-bearing model of C57BL/6 mice was established,and the tumor was locally irradiated with a single dose of 20Gy.Then the tumor tissue was taken7 days after radiation for RNA-seq detection and GSEA analysis,including inflammatory signals,granulocyte activity,and granulocyte immune signals.?2?Immunocyte subsets in tumor tissues were detected by flow cytometry 7 days after radiation.The subtypes included CD45⁺lymphocytes,DC cells,NK cells,eosinophils,CD4⁺T cells,CD8⁺T cells,monocytes,neutrophils,and Ly6C^hi cells.?3?Based on mouse melanoma B16-F10 model with bilateral tumor-bearing,only one side of the tumor received local 20Gy radiation,while the other side was not treated as control.Eosinophils activated in vitro were labeled with CFSE and transferred back into the irradiated mice,and 48 hours later tumor tissues were harvested for flow cytometry detection.?4?The chemotaxis effect of irradiated tumor tissues on eosinophils was detected by Transwell assay.?5?The mRNA expression of genes related to chemotaxis and survival of eosinophils in irradiated tumor tissues was detected by PCR.2.The recruitment of cytotoxic T lymphocytes?CTLs?was closely correlated with eosinophils-infiltration.?1?Based on mouse B16-F10 melanoma model,the mRNA expression of genes related to T cells'chemotaxis and function was detected by PCR assay.The percentage of eosinophil and CD8⁺T cells was detected by flow cytometry,and analyzed the correlation between them.?2?Mouse 4T1 breast cancer model was established and we detect the percentage of eosinophils and CD8⁺T cells in tumor tissues by flow cytometry.Further we detect the expression of genes related to T cells'chemotaxis and function,and eosinophils'chemotaxis and survival.?3?Mouse MC38 colon cancer model was established and we detect the percentage of eosinophils and CD8⁺T cells in tumor tissues by flow cytometry.Further we detect the expression of genes related to T cells'chemotaxis and function,and eosinophils'chemotaxis and survival.3.Eosinophil may act as prognostic marker of patients who had received radiotherapy.?1?The infiltration degree of eosinophils was predicted by observing the number of eosinophils in peripheral blood.?2?A total of 234 patients with non-small lung cancer?NSCLC?who had received radiotherapy were collected.We recorded the changes of the number of eosinophils in the patients'peripheral blood before and after radiotherapy,and analyzed the increase of eosinophils in peripheral blood and progression-free survival?PFS?.?3?A total of 249 patients with nasopharynx cancer?NPC?who had received radiotherapy were collected.We recorded the changes of the number of eosinophils in the patients'peripheral blood before and after radiotherapy,and analyzed the increase of eosinophils in peripheral blood and PFS.4.Eosinophils were essential for CD8⁺T cell-mediated anti-tumor immunity.?1?The eosinophils were depleted by anti-Siglec-F neutralizing antibody in B16-F10tumor-bearing mice.?2?The proportion of CD8⁺T cells and effector CD8⁺T cells were detected by flow cytometry,and the correlation was analyzed with eosinophils.?3?IFN-gamma?IFNG?-related mRNA signaling in mouse tumor tissues was detected by PCR.?4?Mouse tumor volume was measured after eosinophils deleption.5.Eosinophils promoted the efficacy of adoptive cell transfer?ACT?immunotherapy.?1?Activated CD8⁺T cells from Pmel mice were transferred intravenously?i.v.?into the irradiated C57BL/6 mice.?2?CAR-T cells were transferred i.v.into the irradiated NSG mice.6.Patients receiving radiotherapy would benefit more from CAR-T therapy.?1?To monitor the disease progression of patients enrolled in clinical trials.?2?The count of blood cells?CBC?of patients was processed.?3?To detect the local puncture samples.Results:1.Radiation promoted eosinophil infiltrate into tumor microenvironment.?1?Radiation induced the inflammatory response within the tumor niche,gene enrichment related to granulocyte activation,migration and chemotaxis was increased,and the immune signals of granulocyte were enhanced.?2?The proportion of immune cells in the irradiated tumor tissues was increased,and we observed that the percentage of CD8⁺T cells and eosinophils was increased significantly.?3?More CFSE positive eosinophils were detected in the irradiated side of the tumors.?4?The ability to recruit eosinophils in irradiated tumor tissue was stronger than in nonirradiated ones.?5?Factors related to eosinophilic chemotaxis and survival expressed higher in irradiated tumor tissues than in nonirradiated ones.2.Eosinophil-infiltration was closely related to the recruitment of cytotoxic T lymphocytes.?1?PCR detection revealed that mRNA expression of T cell chemotactic and functional related molecules in irradiated tumor tissues was significantly increased.Flow cytometry revealed that the proportion of eosinophils in mouse irradiated tumor tissues was highly correlated with the proportion of CD8⁺T cells as well as effector CD8⁺T cells.?2?Flow cytometry revealed that in the mouse 4T1 breast cancer model,the proportion of infiltrated CD45⁺immune cells,eosinophils and CD8⁺T cells in irradiated tumor tissues was significantly increased,and the proportion of eosinophils was highly correlated with the proportion of CD8⁺T cells.PCR detection revealed that mRNA expression of molecules related to the chemotaxis and function of T cells,the chemotaxis and survival of eosinophils was significantly elevated.?3?Flow cytometry revealed that in the mouse MC38 colon cancer model,the proportion of infiltrated CD45⁺immune cells,eosinophils and CD8⁺T cells in irradiated tumor tissues was significantly increased,and the proportion of eosinophils was highly correlated with the proportion of CD8⁺T cells.PCR detection revealed that mRNA expression of molecules related to the chemotaxis and function of T cells,the chemotaxis and survival of eosinophils was significantly elevated.3.Eosinophils may act as prognostic molecules in patients who had received radiotherapy.?1?A feasible method was established to predict the infiltration degree of eosinophils in tumor tissues through counting the CBC of eosinophils in peripheral blood of patients with cancer.?2?The number of eosinophils in peripheral blood of patients with NSCLC increased significantly after radiotherapy.Patients with an eosinophilic hyperplasia of 1.67 times or more had a longer PFS.?3?The number of eosinophils in peripheral blood of patients with NPC increased significantly after radiotherapy.Patients with an eosinophilic hyperplasia of 2.0 times or more had a longer PFS.4.Eosinophils were essential for CD8⁺T cells-mediated anti-tumor immunity.?1?Flow cytometry showed that the antibody effectively cleared eosinophils in mice.?2?The proportion of CD8⁺T cells and effector CD8⁺T cells was significantly reduced after the removal of eosinophils,and was highly correlated with the proportion of eosinophils.?3?PCR detection showed that the IFNG-related mRNA signals were decreased significantly after the removal of eosinophils by anti-Siglec-F neutralizing antibody.?4?Eosinophil depletion weakened the control of radiotherapy on tumor-bearing mouse.5.Eosinophils promoted the efficacy of ACT immunotherapy.?1?Radiotherapy promoted the recruitment of activated specific CD8⁺T cells to the locally irradiated tumor in C57BL/6 mice.?2?Radiotherapy promoted the recruitment of activated CAR-T cells to the locally irradiated tumor in NPG mice.6.Patients receiving radiotherapy would benefit more from CAR-T therapy.?1?Combined radiotherapy with CAR-T cell therapy would control tumor progression better.?2?The number of eosinophils in the patient's CBC increased after radiotherapy.?3?PCR detection of tumor puncture showed that more transferred CAR-T cells were infiltrated into the irradiated tumor.Conclusions:Radiation reprogrammed the tumor immune microenvironment.Radiation induced eosinophilia was closely related to the recruitment of cytotoxic T lymphocytes.Eosinophils may act as prognostic molecules in patients who had received radiotherapy.Eosinophils were essential for CD8⁺T cells-mediated anti-tumor immunity.Eosinophils promoted the efficacy of ACT immunotherapy.Radiotherapy enhanced the efficacy of CAR-T immunotherapy as adjuvant therapy.Part 2: Local mutational diversity drives intratumoral immune heterogeneity in non-small cell lung cancerBackground:Cancer is a genetic disease,with the growth of tumour cells initiated by mutations that activate oncogenic drivers.This process is combined with the genetic or non-genetic activation or inactivation of genes that promote or suppress the proliferation of tumours.In many cancers,oncogenesis is accompanied by the accumulation of mutations,which can provide a selective advantage to populations of cancer cells by increasing their degree of genetic diversity,accelerating their evolutionary fitness.Yet this diversity comes at a cost: the further a cancer cell diverges from a normal cell,the more likely it is to be recognized as foreign by the immune system.Although long considered a possibility,it has been demonstrated only in the past several years that the total mutation burden?TMB?of tumours contributes to immune recognition of cancer and that it may,at least partly,determine individual response to cancer immunotherapy.Combining whole exome sequencing,transcriptome profiling,and T cell repertoire analysis,we investigate the spatial features of surgically-removed biopsies from multiple loci in tumor masses of 15 patients with non-small cell lung cancer?NSCLC?.This revealed that the immune microenvironment has high spatial heterogeneity such that intratumoral regional variation is as large as inter-personal variation.While the local total mutational burden is associated with local T-cell clonal expansion,local anti-tumor cytotoxicity does not directly correlate with neoantigen abundance.Together,these findings caution against that immunological signatures can be predicted solely from TMB or microenvironmental analysis from a single locus biopsy.Methods:We recruited 15 patients with newly diagnosed NSCLC and performed a variety of multiple genomic and immunogenic genome analyses for multiple biopsy samples from each primary tumor mass,including whole-exome sequencing?WES?,transcriptome profiling?RNA-Seq?,and T-cell repertoire sequencing.In order to further improve the accuracy of tumor immune microenvironment assessment,we developed a machine learning method to integrate multi-dimensional immune-related variables for data mining.Using a random forest algorithm,278 variables were input to classify the immune microenvironment of tumor sites into "hot" and "cold" tumor regions.Results:1.Mutational burdens correlate with local T-cell expansion.2.Mutational burden is loosely related to cytolytic activity.3.Spatially heterogeneous tumor immune microenvironments.4.Mutational heterogeneity is related to immune heterogeneity.Conclusions:The immune microenvironment has high spatial heterogeneity among intratumoral regions,and a combinational omics strategy about immunological signatures is needed to comprehensively evaluate the TME immunophenotype.