The Mechanisms Of Hematopoietic Stem Cells Microchimerism And Engineered Dendritic Cells Promoting The Activation Of Receptor T Cells

Objective:Microchimerism refers to the phenomenon that a small part of cells or DNA in an individual comes from another individual.Microchimerism is associated with graft tolerance,tissue repair,chronic GVHD and autoimmune disease.It is of great significance to study the process of hematopoietic stem cell microchimerism and its effect on immune function of T cells.Dendritic cells?DCs?are one of the most important antigen presenting cells and are the bridge of T cell activation.The engineered dendritic cells can enhance their regulatory effect on T cells.Because of its excellent physical and chemical properties,Mo S₂nanosheet?MSNs?has become a new star of nanomaterials,which has been developed into biological imaging probes,biosensors,photothermal therapy agents,drug carriers and tissue engineering scaffolds.Using it as an adjuvant to modulate immune responses is also a fascinating research topic.The body's anti-tumor immune response kills tumor cells mainly by activating T cells.Primary cutaneous T cell lymphoma?CTCL?is the most common cutaneous lymphoma,and the 5-year survival rate for late cutaneous T cell lymphoma remains less than 25%.Blocking the progression of cutaneous T-cell lymphoma early will prevent a high mortality rate in the late stage of the disease.Methods:1.To study the microchimerism induced by peripheral hematopoietic stem cell infusion and its effect on immune function of the recipient,we injected C57/BL6 spleen cells mobilized by G-CSF into CB6F1 without pretreatment to form microchimerism.By using the polychromatic flow technique to mark the differences between different cell subsets of mice and the H-2 antigens of donor and recipient cells,we can compare the differences and similarities of microchimerism formed by different cell subsets.In bioluminescence imaging tracing system,C57BL/6-Tg?Fluc+/-?mice were used to observe the microchimerism of donor cells in vivo.Polychromatic flow allowed us to further detect the activation of donor T cells and the amplification of hematological progenitor cells during microchimerism.Through flow sorting and RNA-seq technology,we detected the expression of RNA in T cells before and after microchimerism.2.Two few-layered MSNs at a respective size of 100-250 nm?S-MSNs?and400-500 nm?L-MSNs?were used to engineer DC.Bone marrow derived DCs were exposed to both sized MSNs at different doses?0,8,16,32,64,128?g/ml?for 48 h and subjected to analysis of surface marker expression,cytokine secretion.In vivo small animal imaging system tracked the homing of DCs and its effect on T cells in local lymph nodes was detected by flow cytometry.3.EG7-ova mouse T lymphoma cells were inoculated in C57BL/6 mice on day-2,and spleen cells from Balb/c mice mobilized by G-CSF were infused through the tail vein on day 0.The co-incubated DCs was injected into the right foot pad of the mice on day 0 and day 7 respectively,and the tail venous blood was taken from the mice on day7 and day 14 to detect donor cells chimerism by flow cytometry.The proliferation and activation of T cells in peripheral blood and popliteal lymph nodes were detected on day14 by flow cytometry and cell counter.Tumor volume was measured with vernier calipers on day 7,10 and 14.Results:1.The chimerism of donor cells in the DCI-6E7 group on+1d after cell transfusion was 1.76±0.49%and on day+4 reached the highest level of 2±0.54%.Then the chimerism fluctuated between 1.2 and 1.9%on day 7-14,showing no significant decrease.However,the chimerism rapidly decreased to 0.18%after day 14-21,and then continued to decline.Body weight of mice in the DCI-6E7 group increased gradually after cell transfusion,and no obvious abnormalities were observed in diet,hair color and body posture of mice.Peripheral blood white blood cells only slightly decreased and then recovered,while platelets and hemoglobin showed no significant changes.In the DCI-6E7 group,T cells,B cells and other subgroups only formed low percentage of donor chimerism?1.1%-4.5%?on day 1-14.Chimerism of each cell subgroup began to decline rapidly on day+14,and only a small number of donor was detected by flow cytometry on day+28.The proportions of CD3+,CD11b+and B220+cells in the DCI-6E7 showed only slight changes,fluctuating from 31.1%to 47.8%,25.6%-36%and 8.6%-24.3%,respectively.CD4/CD8 fluctuates 1.5-3.2 and lasts for>1.The fluorescence intensity of the DCI-6E7 group fluctuated from 0 to 12 hours,decreased on day+1,but showed no significant change on day 1-5,increased on day 5-7 day,and then decreased continuously.The fluorescence signal of the donor cells in the group DCI-6E7 was mainly concentrated in the lymph nodes and spleen.Donor-derived activated T cells in the DCI-6E7 group were amplified to varying degrees on day 7 after infusion,then gradually decreased.In the recipients,the relevant markers of T cell activation were significantly amplified om day 7-21 after cell infusion,and then gradually decreased,especially CD8+GZMB+increased from 2.4%?0d?to 33%?+14d?,and then gradually decreased to 2.5%?+28d?.The number of genes that significantly up-regulated in CD4+cells was more than the number of genes that down-regulated on10d after cell transplantation.However,the number of genes that significantly down-regulated in CD8+cells was more than the number of genes that were up-regulated.They are involved in various biological processes,cellular components and molecular functions,and are involved in regulating a variety of signaling pathways?including immune and inflammatory responses,autoimmune diseases,infectious diseases,tumors,etc.?.Many of these genes are involved in immune and inflammatoryVresponses,and they regulate inflammatory responses by regulating the differentiation of TH1/TH2 and TH17 cells or directly through the adhesion pathway between cells.2.Different-sized MSNs of all doses?0-128?g/ml?did not affect the viability of DCs.A significantly elevated expression of CD40,CD80,CD86 and CCR7 could be measured on both S-MSNs and L-MSNs treated DCs at dose of 128?g/ml.The secretion of IL-12p70 remained unchanged while a decreased secretion of IL-1?and prompted TNF-?was observed to an extent correlated with the dose of MSNs treatment.A significantly increase of IL-6 could be only observed in 128?g/m L L-MSNs treated DCs.MSNs treatment dramatically improve the ex vivo movement and in vivo homing ability of both the local resident and blood circulating DCs.The rearrangement of cytoskeleton regulated by ROS elevation was responsible for enhanced DCs homing ability.A more robust CD4+and CD8+T cell proliferation and activation?characterized by high expression of CD107a,CD69 and ICOS?was observed in mice vaccinated by MSNs treated DCs.The exposure of MSNs did not interrupt LPS induced DC activation,homing and T cell priming.3.The cytotoxicity of OVA and MSNs was low.OVA alone could not promote DCs maturation,while co-culture with MSNs could promote DCs maturation,which characterized by increased CD40,CD80 and CD86,and increased IL-6 secretion.Peripheral hematopoietic stem cell infusion can produce microchimerism of donor cells in tumor mice,which tends to decrease with the extension of time.Peripheral hematopoietic stem cell infusion can stimulate the expansion of peripheral blood B and T cells in tumor mice.In particular,CD4+and CD8+cells were stimulated,and the cells expressed activated markers of CD107a,CD69 and ICOS expanded in large numbers.DCs immunity can stimulate the expansion of B cells and T cells in local lymph nodes of mice,and Mo S₂modified can further amplify this effect.In particular,it stimulated the proliferation of CD4+cells and CD8+cells expressed CD107a,CD69and ICOS.OVA-DCs immunotherapy can inhibit tumor growth in mice,and Mo S₂engineered DCs can further inhibit tumor growth in mice.Peripheral blood hematopoietic stem cells alone can also inhibit tumor growth.The combination of peripheral blood hematopoietic stem cell infusion and Mo S₂-OVA-DCs immunity had the best inhibitory effect on tumor cells.Conclusions:1.By in vivo imaging,we observed the microchimerism of donor cells in the recipient.Compared with traditional macrochimerism,infusion associated microchimerism only slightly reduced blood cells without GVHD,activated T cells of the recipient,stimulated the amplification of bone marrow hematopoietic stem progenitor cells of the recipient,and regulated the immune function and inflammatory response of the recipient.These results provide a basis for studying the formation of microchimerism and its role in anti-tumor,autoimmune diseases and hematopoiesis.2.In the engineered DC,at a relatively high dose?128 g/ml?,MSNs can improve the in vitro maturation and lymph node homing of DCs,significantly improve the activation ability of DCs,and cause stronger CD4+and CD8+T cell immune response.In addition,ROS-induced cytoskeletal rearrangement were involved in the improvement of the motor capacity of MSNs modified DCs.As the first systematic study on the modification of DC by MSNs,our findings provide a new method for the modification of DC and provide supporting evidence for the use of MSNs as an immunomodulation adjuvant.3.The infusion of blood stem cells and Mo S₂nanosheets can inhibit tumor growth by regulating DCs,and the combination of the both can play a stronger role in inhibiting tumor growth.The pathway to play this role may be associated with the activation and amplification of T cells in peripheral blood and local lymph nodes,but further research and exploration are needed.