Epidermal Resident And Monocyte-derived Langerhans Cells Regulate Melanoma Progression In Bidirectional Way By Affecting The Immune Microenvironment

Part ?.The number and distribution of epidermal resident Langerhans cells and monocyte-derived Langerhans cells in different tumor stages of primary cutaneous melanomaObjective:Melanoma immunosuppressive tumor microenvironment could promote tumor invasion and metastasis.Epidermal Langerhans cells(LCs)could present antigens and induce T cell responses.Recently,it has been found that during epidermal inflammation,monocytes were recruited into the epidermis and differentiate into LCs,which are monocyte-derived LCs(moLCs).moLCs could promote the progression of psoriasis through pro-inflammatory function.Studies have shown that LCs above melanoma were significantly reduced,and its presence was related to the better prognosis of melanoma patients.This suggests that LCs may be important for the formation of anti-melanoma immunity.However,some studies have found that there were more LCs in primary melanoma than nevus or normal skin.The current research has not distinguished the different LC subpopulations in melanoma.Therefore,further analysis of the changes in the number and distribution of different LC subpopulations in melanoma with different tumor stage will provide directions for studying the role of LCs in melanoma.Methods:LCs in normal skin of mice were compared with the LCs in melanoma from C57 mice challenged with B16 melanoma cells.The LC subpopulations in the tumors were determined by flow cytometry.Changes in the percentage of resident Langerhans cells(rLC s)and moLCs in melanoma were detected on day 11,14,and 18 after tumor challenge.Changes in the percentage of rLCs and moLCs in tumor-draining lymph nodes were detected on day 0,3,6,10,14,and 18 after tumor challenge.The distributions of rLCs and moLCs in melanoma of mice were detected by immunofluorescence.The number of rLCs and moLCs in different T stages of melanoma and their changes in intratumoral and peritumoral distribution were detected by immunohistochemistry.Results:(1)Percentage and distribution of LC subpopulations in different stages of mouse melanoma model?There were rLCs(CD45+Langerin+CD11b+XCR1-Ly6C-)and moLCs(CD45+Langerin+CD11b+XCR1-Ly6C+)in melanoma of mice.?rLCs in mice were mainly located in the epidermis overlying the melanoma.moLCs were mainly located at the junction of the epidermis and dermis.? As the tumor progressed,the proportion of rLCs gradually decreased,and the proportion of moLCs gradually increased.The number of LCs in TDLNs increased firstly and then decreased with tumor progression.The proportion of rLCs was about 90%.(2)Number and distribution of LC subpopulations in different stages of human cutaneous melanoma specimens? Both moLCs and rLCs existed in human cutaneous melanoma.moLCs and rLCs were mainly distributed in the skin surrounding the tumor,but rarely in the center of the tumor.rLCs were mainly distributed in the epidermis,while moLCs were mainly distributed at the junction of the epidermis and dermis.? As the tumor progressed,the rLCs in the epidermis overlying melanoma decreased,and the rLCs in the epidermis adjacent to melanoma increased and then decreased.The number of rLCs in the epidermis overlying melanoma was significantly more than that in the epidermis adjacent to melanoma in T1 stage.The number of rLCs in the epidermis overlying melanoma was significantly less than that in the epidermis adjacent to melanoma in the T2-T4 stage.The rLCs in melanoma with ulceration were mainly distributed in the epidermal adjacent to melanoma.?The number of moLCs in melanoma was relatively small.As the tumor progressed,both intratumoral and peritumoral moLCs gradually increased.However,moLCs were mainly distributed at the junction of the epidermis and dermis adjacent to melanoma.Conclusions:(1)There were two subgroups of LCs in human and mouse melanomas-namely rLCs and moLCs.(2)As the tumor progressed,the proportion of rLCs decreased and the proportion of moLCs increased.(3)rLCs were mainly distributed in the epidermis overlying the tumor in thin melanoma.rLCs were mainly distributed in the epidermis adjacent to tumor in thicker melanoma.(4)Intratumoral and peritumoral moLCs significantly increased.The changes in the number and distribution of rLCs and moLCs indicated that they may have different functions in melanoma.Part ?.Eliminating mouse epidermal resident Langerhans cells or monocyte-derived Langerhans cells regulated the growth and immune microenvironment of cutaneous melanoma bidirectionallyObjective:The existence of LCs in melanoma was related to the better prognosis of patients.However,current research indicates that LCs in melanoma and sentinel lymph nodes may had defects in phenotype and function,thereby triggering and maintaining highly immunosuppressive tumor microenvironment.And there were no research reports on the role of moLCs in melanoma.LCs are resident macrophages from embryonic yolk sac and fetal liver.Tissue-resident macrophages and monocyte-derived macrophages could promote tumor progression of pancreatic cancer through different functions.Combining the number and distribution characteristics of rLCs and moLCs in melanoma in our previous results,we speculated that rLCs and moLCs in melanoma may also play pro-tumor or anti-tumor role in the progression of melanoma through different mechanisms.Methods:The muLangerin-DTR-EGFP mouse and anti-CSF1R monoclonal antibody model were used to remove mouse epidermal rLCs to observe melanoma growth and immune microenvironment changes.Langerin+rLCs in the epidermis and Langerin+dendritic Cells(DCs)in the dermis would be eliminated in muLangerin-DTR-EGFP mice received intraperitoneal injection of diphtheria toxin.Langerin+DCs could be replenished from DCs precursors in peripheral blood after one week,while LCs could not.We challenged LangerinDTR-EGFP mice with B16 melanoma cells in this window period to explore the effects of rLCs clearance on the growth and immune microenvironment of melanoma.Intraperitoneal injection of anti-CSF1R monoclonal antibody could remove monocytes,macrophages and rLCs.One week later,monocytes and macrophages could recover.We challenged C57 mice with B16 melanoma cells at that time to explore the effects of rLCs clearance on melanoma growth and immune microenvironment.C57 mice challenged with B16 melanoma cells were injected intraperitoneally with Ly6C monoclonal antibody(once every other day for a total of 5 times)on day 7 after tumor challenge to remove monocytes and monocyte-derived cells,including moLCs.The effects on melanoma growth and immune microenvironment were examined.Results:(1)The growth of melanoma accelerated after the rLCs were removed in the muLangerin-DTR-EGFP mice.The proportion of CD8+T cells secreting IFN-? reduced in rLCs removement group.The proportion of Tregs in tumor-draining lymph nodes increased in rLCs removement group.(2)The growth of melanoma accelerated and the proportion of IFN-?+CD4+T cells reduced after rLCs were removed with anti-CSF 1R monoclonal antibody.(3)Melanoma grewth more slowly after the use of Ly6C monoclonal antibody.However,the proportions of tumor Tregs,IFN-?+CD4+T cells,CD8+T cells and IFN-?+CD8+T cells did not change significantly.Conclusion:rLCs could inhibit the growth of melanoma by promoting anti-tumor immune response.Monocytes and monocyte-derived cells,including moLCs,could promote tumor growth.Part ?.Tolerogenic phenotype and adaptive immune response were different between human CD34-derived Langerhans cells and monocytederived Langerhans cellsObjective:Melanoma could induce the tolerance of DCs by secreting multiple inhibitory factors that impair DCs activation and antigen presentation.TGF-? is the most important immunosuppressive factor in the tumor microenvironment.TGF-? is also a key cytokine to maintain the steady state and tolerant state of LCs.Therefore,TGF-? may also become an important cytokine for melanoma-induced LCs immune tolerance.CD34+hematopoietic stem cells and monocytes could be induced into CD34-LCs or moLCs under the stimulation of cytokines,respectively.The transcriptome of CD34-LCs was similar to human epidermal LCs.CD34-LCs showed the strong ability to stimulate the proliferation and activation of allogeneic T cells.moLCs could also stimulate allogeneic T cell proliferation,but their cytokine secretion levels were low.In this part,we aimed to study the phenotype and function of TGF-?1-induced tolerogenic CD34-LCs and moLCs.Methods:Human umbilical cord blood CD34+stem cells were induced into CD34-LCs by cytokines FLT3L,TPO,SCF,GM-CSF,TGF-?1 and TNF-?.Human peripheral blood monocytes were induced into moLCs by the cytokines GM-CSF,TGF-?1,IL-4,TNF-? and dexamethasone.CD34-LCs and moLCs were induced by 0,5,or 20 ng/mL of TGF-?1.when indicated,CD34-LCs and moLCs were continuously cultured with inflammatory cytokines mixtures.CD80,CD86,HLA-DR,and PD-L1 on CD34-LCs and moLCs were detected by flow cytometry.Transcriptomes of CD34-LCs and moLCs were tested by RNA-sequencing.The abilities of CD34-LCs and moLCs to stimulate naive CD4+T cells,memory CD4+T cells,and memory CD8+T cells to proliferate and differentiate were detected by flow cytometry.Results:(1)moLCs expressed lower CD80,CD86,and HLA-DR,and higher PD-L1 compared with that of CD34-LCs in the basal state.As the concentration of TGF-?1 increased,the expressions of CD80 and HLA-DR on the surface of CD34-LCs decreased gradually,while the expression of PD-L1 on the surface of moLCs gradually increased.(2)RNAsequencing results showed that there were significant differences in gene expression profiles between moLCs and CD34-LCs.moLCs expressed more chemokines that attracted monocytes and neutrophils,adhesion/matrix-related proteins,TGF-?1,etc.CD34-LCs expressed more molecules similar to rLCs,such as Epcam,E-cadherin,and migration molecules(CXCR4 and CCR7).(3)The ability of TGF-?1-induced tolerogenic moLCs to induce Tregs form memory CD4+T cells was equivalent to that of CD34-LCs and was dependent on TGF-?1 concentration and moLCs number.The ability of TGF-?1-induced tolerogenic moLCs to induce Tregs from naive CD4+T cells was better than that of CD34LCs and was dependent on TGF-?1 concentration and moLCs number.The ability of TGF?1-induced tolerogenic moLCs to induce the proliferation and TNF-? secretion of memory CD8+T cells was generally weaker than that of CD34-LCs.Conclusion:moLCs expressed lower costimulatory molecules and higher PD-L1 than that of rLCs.moLCs were more likely to be induced immune tolerance than that of CD34-LCs.moLCs may accelerate melanoma immune microenvironment formation by inducing more Tregs and inhibiting the proliferation and activation of memory CD8+T cells.