| Objective Cancer immunotherapy has achieved great success in hematological cancers.However,immune cells are highly heterogeneous population and can vary highly on clonal expansion,migration and function status,making it difficult to evaluate and predict patient response to immune therapy.Conventional technologies only yield information on the average population information of the treatment,masking the heterogeneity of individual T cell activation status,formation of immune synapse as well as efficacy of tumor cell killing at single cell level.To fully interrogate these single cell events in detail,here,we present a microfluidic microwell array device that enables massive parallel analysis of immunocyte's heterogeneity upon its interaction pairs with tumor cell at single-cell level.By precisely controlling the number and ratio of tumor cells and T cells,our technique can interrogate the dynamics of CD8+T cell and leukemia cell interaction inside 6,400 microfluidic wells simultaneously.We demonstrated that by investigating interactions of T-cell and tumor cell pairs at single cell level using our microfluidic chip,details hidden in bulk investigations,such as heterogeneity in T cell killing capacity,time-dependent killing dynamics as well as drug treatment induced dynamics shift can be revealed.This method opens avenue to investigate cancer immunotherapy efficacy and resistance at single cell level and can explore our understanding of fundamental cancer immunity as well as interrogate cancer immunotherapy efficacy for personalized therapy.Methods1.We developed a microfluidic well array device integrated with the situ incubation system and time-lapse imaging of the microscope to trace dynamic immune and leukemia cell's interaction at the single-cell level.2.By adjusting the initial concentration of the input cell,from 1?10~6/m L to20?10~6/m L,and different effector-target cell ratios,the optimal T cell-leukemia cell pair can be obtained in a single microfluidic chip well.3.We selected OT-I?OVA system to validate our system.3 groups were loaded as following:OT-I T cells with OVA+as positive control;2:na?ve T cells isolated from wild type C57BL6 mice,with OVA+as negative control;3:OT-I T cells,with OVA-C1498 cells as negative control.4.We preloaded OT-I T cells with Fluo-4 dye to study immune synapse formation heterogeneity.5.We added anti-mouse PD-1 antibody to the T cell medium whereas control group is set up without addition of anti-PD-1.We then monitored T cell killing against leukemia cells.Results1.There is significant higher cytotoxicity in the positive control OT-I-OVA+group as compared with the other two groups.Furthermore,The total dead cell numbers increased continuously in the OT-I-OVA+group over time,which was significantly higher than that of the na?ve T and OTI-OVA-group(32.89%,16.09%and 14.75%,respectively).We calculated that 63.28%OT-I cells killed one or more OVA coated C1498 cells within 5 hours.When incubated with non-coated C1498 cells,the baseline killing was minimal(9.51%).Also,with na?ve CD8 T cells,the killing of C1498 cells was also minimal(8.30%).2.A strong heterogeneity in time required for T cell to effectively kill a tumor cell since TCR engagement and T cell activation.Most(51%)T cells were able to kill tumor cells within 50 minutes.Only 16.3%of T cells can kill tumor cells with 20minutes,and 14.5%T cell can kill tumor cells between 20-30 minutes.18.2%T cell would require greater than or equal to 60 minutes to kill tumor cell since initial activation.3.We qualitatively analyzed how initial distances affect T cells'killing preformation.Our results showed that the initial distances between T cell and leukemia cells varied from 0?m to 18?m.85%of the killing occurred when OT-I T cells and leukemia cells are in direct contact,whereas 15%of the killing occurred between cells with initial distance between 1 and 15?m.4.T cells of the same origin show heterogeneity in killing capacity:54.5%of T cells killed 1 cell within 5 hours whereas 4.9%killed 2 target cells and only 0.78%T cells killed 3 or more target cells,the rest 39.8%T cells did not exert any killing.5.Total of 522 killing events were analyzed for time since initial tumor cell-T cell contact to tumor cell death.The killing time required varied from 20 to 300minutes.Importantly,95%of the killing activities occurred later than for 90 minutes since T cells'initial contact with target cells.The most killing occurred between 3.5hours to 4 hours,which accounts for 22.4%of the total killing.6.We analyzed the killing efficiency of 1024 OT-I cells in the control group and777 in the anti-PD-1 antibody treatment group,and found that the overall killing efficiencies of OT-I in control and the treatment group were respectively 37.52%±5.33 vs 61.48%±7.86(and 63.28%±2.93 vs 73.04%±3.71 at 3.5 and 5hours,the difference was statistically significant(p<0.05).7.The killing capacity of single OT-I cells was significantly enhanced after immune checkpoint blockade.In the control group,Inert T cells which did not kill any tumor cells consist of 39.8%of total T cells.With anti-PD-1 treatment,inert T cell percentage was reduced to 27.3%.Moreover,OT-I cells that killed 1,2 and 3 target cells in the control group accounted for 54.47%,4.92%and 0.77%of total population,respectively,all were significantly lower than 60.77%,10.53%and1.43%in experimental group with ant-PD-1 treatment.8.In the control group without PD-1 antibody,the number of target cells that killed by T cells within 2 hours accounted for 12.06%of total tumor cells,while in the experimental group,30.85%killing occurred within 2 hours.The time window where most killing occurred was 3-3.5 hours in the treatment group,which was half an hour sooner as compared to 3.5-4 hours in control group.Conclusion Using our microfluidic chip and OT-I?OVA system can well observe T cell antigen recognition and killing in vitro,and it is feasible to study the heterogeneity of T cell-target cell interaction at the single cell level.This study successfully described the heterogeneity of individual immune cells and target cells,and the impact of immune checkpoint suppression(PD-1/PD-L1)on cellular heterogeneity.In addition,from a methodological perspective,this research provides a new technical platform for exploring intercellular gap junctions,intracellular lipid changes,paracrine and cell drug resistance.This platform has the advantages of convenience,speed,high throughput and easy operation.It can provide help for the early diagnosis of diseases and the interaction between cells.This technology platform opens a new way to study the efficacy and drug resistance of cancer immunotherapy at the single cell level,enhances our exploration to the basic theory of cancer immunotherapy,and provides theoretical support for the design of personalized cancer immunotherapy. |
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