Non-invasive Tracking And Monitoring Of γδ T Cell Tumor Immunotherapy With PET Imaging

Research background:γδT cell-based immunotherapy has been rolled out as a promising treatment strategy for malignant tumors due to their potent anti-tumor cytotoxicity,ease of expansion and unrestricted major histocompatibility complex（MHC）feature.However,the application ofγδT cell therapy still has a major problem to be solved:the dynamics and outcomes ofγδT cells in tumor sites are poorly understood.The dynamic behavior ofγδT cells limits the application of traditional blood pharmacokinetic analyses or tissue biopsies,which may only partially capture indirectlyγδT cell temporal and spatial distributions relative to the tumor,and cannot satisfy the dynamic monitoring of overall distribution ofγδT“living”cells in vivo.Therefore,developing strategies for in vivo tracking and quality assessment ofγδT cells will help to understand the reasons behind the success of immunotherapy and,more importantly,the reasons for treatment failure,which will be important to advance the development,clinical application and industrial development ofγδT cellular immunotherapy.This study aims to develop non-invasiveγδT cell tracking strategies,including theγδT cell bioorthogonal labeling based on metabolic glycoengineering and click chemistry forγδT cell NIRF/PET multimodal imaging;endogenous PET imaging ofγδT cells with VLA-4（very late antigen-4,biomarker for activatedγδT cells）targeted ⁶⁸Ga-LLP2A;and dual-PET imaging ofγδT cells combing granzyme B（downstream effector ofγδT cells）targeted ⁶⁸Ga-GZP with VLA-4 targeted⁶⁸Ga-LLP2A,to establish a multi-modal and multi-level molecular imaging platform for in vivoγδT cell tracking and therapeutic monitoring,so as to dynamically monitor and evaluate the systemic distribution,tumor infiltration and retention,and cell functional activity ofγδT cells.Ⅰ NIRF/PET imaging ofγδT cells via bioorthogonal labelingPurpose:The aim was to develop an efficientγδT cell NIRF/PET imaging strategy by combining metabolic glycoengineering and bioorthogonal click chemistry.Methods:γδT cells were incubated with N-azidoacetyl-D-mannosamine-tetraacylated（Ac₄Man NAz）,and azide（N₃）groups were then incorporated onto the surface ofγδT cells via metabolic glycoengineering to obtain N₃-γδT cells.Subsequently,a NIRF dye,Cy5.5dibenzylcyclooctyne（DBCO-Cy5.5）were conjugated with N₃-γδT cells via bioorthogonal copper-free click chemistry,resulting in Cy5.5-γδT cells.To investigate the in vivo biodistribution profiles of adoptiveγδT cells,NIRF imaging was performed after intravenous injection of Cy5.5-γδT cells into Burkitt’s lymphoma cell Daudi tumor-bearing mice.To radiolabel N₃-γδT with ⁶⁸Ga,a clickable tracer ⁶⁸Ga-NETA-DBCO was prepared.Its bioorthogonal binding ability with N₃-γδT cells was examined using in vitro cell uptake assay with unmodifiedγδT cells as a control.To track the adoptive N₃-γδT cells by PET imaging with short half-life radiometal ⁶⁸Ga,a pre-targeted technique was applied by administrating the N₃-γδT cells into mice 3 days in advance,and then performing ⁶⁸Ga-NETA-DBCO PET/CT imaging and biodistribution assays.The infiltration ofγδT cells on tumor tissues was identified by flow cytometry and tissue immunofluorescence.Results:γδT cells could be conveniently labeled with Cy5.5 with a minimal effect on cell viability and function.In Daudi tumor-bearing mice,NIRF images of Cy5.5-γδT cell-injected Daudi tumor-bearing mice showed a strong NIRF signal throughout the whole body,including the tumor site,on day 1,and NIRF signal intensity reached a maximum two days post-injection,and the tumor-to-muscle ratio peaked on day 3.The adopted cells could be clearly observed via non-invasive NIRF imaging over 10 days.In contrast,a weak NIRF signal was observed in the saline-injected mice.N₃-γδT cells showed higher uptake of ⁶⁸Ga-NETA-DBCO as compared to that ofγδT cells（p<0.001）,validating the specific binding of ⁶⁸Ga-NETA-DBCO to the N₃-γδT cells.In the pre-targeted PET images,significant high tumor uptake of ⁶⁸Ga-NETA-DBCO was observed in mice 3 days post injection of N₃-γδT cells,which was 1.5-fold higher than that of control mice（1.295±0.097 vs.0.839±0.068%ID/g,p<0.05）.The infiltration ofγδT cells was noticeably confirmed via flow cytometry and immunofluorescence studies.Conclusion:These findings demonstrated that a bioorthogonal direct cell labeling method followed by NIRF/PET imaging can provide crucial insights into the biodistribution,migration,and tumor-homing efficiency of adoptiveγδT cells in vivo,representing a new potentialγδT cell imaging technology.Ⅱ Visualizing γδ T cells by very late antigen-4-targeted positron emission tomographyPurpose: Direct cell labeling is the difficulty in differentiating between live and dead cells and tracing proliferated cells,which is of limited use in preclinical and clinical study of γδ T cell immunotherapy.The aim was to investigate whether VLA-4（very late antigen-4）,a crucial component in the effective trafficking of lymphocytes,could serve as a biomarker to non-invasively visualize γδ T cells and monitor the γδ T cell therapy.Methods: VLA-4 expression and binding specificity were examined in γδ T cells,peripheral blood mononuclear cells（PBMCs）,and breast cancer cell MDA-MB-231 and lung cancer cell A549 using Western blot and cell binding assays.To evaluate the detection sensitivity of VLA-4-targeted tracer,⁶⁸Ga-LLP2 A,to γδ T cells,we prepared different amounts of γδ T cells incubated with ⁶⁸Ga-LLP2 A in 96-well PCR plates for in vitro PET phantom study,and subcutaneously transplanted varying numbers of γδ T cells mixed with 50 % Matrigel in shoulders of nude mice for in vivo PET phantom study.Longitudinal PET imaging of MDA-MB-231-and A549-bearing mice with or without adoptive transfer of γδ T cells were performed at indicated time points to map the migration and localization of γδ T cells in vivo.Imaging data were verified by ex vivo biodistribution studies,and the infiltration of γδ T cells and the co-localization of γδ T cells and VLA-4 was validated by immunohistochemistry studies.Results: VLA-4 expression was highest in γδ T cells,while PBMCs and tumor cells（MDAMB-231,A549）displayed significantly lower expression.Higher uptake of ⁶⁸Ga-LLP2 A was observed in γδ T cells,whereas PBMCs and γδ T cells pretreated with unlabeled LLP2 A showed negligible accumulation,revealing the high specificity of ⁶⁸Ga-LLP2 A to γδ T cells.In phantom studies,the PET signal was positively correlated with the amounts of γδ T cells,and the detection limitation were 2,000 cells in vitro and 4,000 cells in vivo.For longitudinal PET imaging studies,tumor accumulation was similar between mice with adoptive transfer of γδ T cells（γδ T cells-mice）and control mice initially;however,γδ T cells-mice showed increasing PET signal intensity in tumors and displayed higher uptake than control mice starting at day 3 after cell injection and onward（p<0.05）,indicating the tumor-specific homing of γδ T cells to tumor.The presence of VLA-4-expressing γδ T cells in tumors was confirmed via histological analysis.Adoptive transfer γδ T cells slowed down the growth of MDA-MB-231 tumor and prolonged the survival of tumor-bearing mice.⁶⁸Ga-LLP2 A PET imaging showed the gradually increased PET signal with time and the number of treatments,further demonstrating the feasibility of ⁶⁸Ga-LLP2 A in the therapeutic monitoring of γδ T cells.Conclusion: Herein,we reported the first molecular probe,⁶⁸Ga-LLP2 A,for in vivo endogenous imaging of γδ T cells in live tumors,which advances PET imaging of γδ T cells and supports the translation of imaging agents for immunotherapeutic monitoring.Ⅲ Dual-PET tracers targeting very late antigen-4 and granzyme B for immune-monitoring of γδ T cellsPurpose: Non-invasive molecular imaging of γδ T cells by PET is a promising approach with the ability to provide spatial,temporal and functional information,which may help to predict the patient responsiveness and therapeutic outcome.Very late antigen-4（VLA-4）is a crucial component involved in the efficient trafficking of lymphocytes and is highly expressed on γδ T cells;Granzyme B is a downstream effector of cytotoxic T cells and natural killer cells to induce apoptosis in tumors providing information on antitumor activity.The aim was to assess the application value of a dual-PET combined imaging strategy targeting VLA-4(⁶⁸Ga-LLP2A)and granzyme B(⁶⁸Ga-GZP)for immune-monitoring of γδ T cell therapy.Methods: Granzyme B activity of γδ T cells was determined by enzymatic assay with granzyme B substrate Ac-IEPD-p NA.Varying numbers of γδ T cells mixed with 50 % Matrigel were subcutaneously transplanted in shoulders of nude mice for in vivo PET phantom study.To analyze the feasibility of ⁶⁸Ga-GZP as an imaging probe for detecting the functional distribution of γδ T cells in vivo,γδ T cells/PBS were injected into the A549 tumor-bearing mice,and then performed ⁶⁸Ga-GZP and ⁶⁸Ga-LLP2 A PET imaging on the 7th/8th day post cell-injection,respectively.⁶⁸Ga-GZP and ⁶⁸Ga-LLP2 A PET imaging of A549 tumor-bearing mice with adoptive transfer of γδ T cells were performed at early（D 1/2）and late（D 5/6）time points post cell administration.To evaluate the widespread application of ⁶⁸Ga-LLP2 A and ⁶⁸Ga-GZP combined PET imaging strategies,Burkitt’s lymphoma Daudi cells with high levels of VLA-4 were selected to construct the tumorbearing model.⁶⁸Ga-GZP and ⁶⁸Ga-LLP2 A PET imaging were performed on day 7/8 after γδ T cells or PBS injection.Delineate regions of interest（ROI）to semi-quantitative analyze the immune-monitoring ability of two probes.The infiltration of γδ T cells in tumor tissues were verified by immunohistochemistry.Results: High expression of granzyme B was detected in γδ T cells,while A549 and Daudi cells displayed significantly lower expression.In phantom studies,⁶⁸Ga-GZP could detect the subcutaneously planted γδ T cells and PET signal was positively correlated with the amounts of γδ T cells.In A549 tumor-bearing mice,both ⁶⁸Ga-GZP and ⁶⁸Ga-LLP2 A lightened the tumors with high contrast at day 7 and day 8 after cell injection,respectively,which were significantly higher than that of the control group.In addition,the longitudinal imaging results showed increasing tumor uptakes of ⁶⁸Ga-GZP and ⁶⁸Ga-LLP2 A over time,indicating the gradually tumor-specific homing of γδ T cells.In Daudi tumor-bearing mice,we found an unexpected mismatch between ⁶⁸Ga-LLP2 A and ⁶⁸Ga-GZP imaging results,with ⁶⁸Ga-LLP2 A imaging showing significantly higher tumor uptake in the treatment group than the control group（p<0.001）,while no significant difference was found in ⁶⁸Ga-GZP imaging（p=0.31）.The difference of ⁶⁸Ga-GZP detection results on infiltrating γδ T cells in different tumors may be correlated with the expression level of tumor protease inhibitor-9（direct inhibitor of granzyme B in humans）.In vitro analysis of granzyme B activity by enzymatic assay showed that the granzyme B activity of γδ T cells was significantly inhibited by the co-incubation with PI-9 positive Daudi cell extracts,while not affected by PI-9 negative A549,indicating the expression level of PI-9 in tumor cells affects the granzyme B activity,and thus affects the detection results of ⁶⁸Ga-GZP.Immunohistochemistry results showed obvious infiltration of γδ T cells in both Daudi and A549 tumors,and Daudi tumor tissues highly expressed PI-9 while no obvious expression of PI-9 was found in A549 tumor,which was consistent with the WB results.Conclusion: This study highlights the potential of dual-PET tracer imaging strategy targeting VLA-4(⁶⁸Ga-LLP2A)and granzyme B(⁶⁸Ga-GZP)for immune-monitoring of γδ T cell therapy.Additionally,the PI-9 expression in tumor affects the activity of granzyme B produced by γδ T cells,and thus affects the result evaluation of PET imaging targeting granzyme B.This emphasizes the importance of dual-PET combined imaging strategy,which validates and complements each other,and contribute to understand the relationship between the tumor environment and gamma-delta T cell immune responses.Meanwhile,this strategy may also provide valuable information for γδ T cells combined with PI-9 related immunotherapy.