The Role Of Alarmin HMGN1 In The Induction Of Anti-tumor Immune Responses In Murine Models

Alarmins are structurally distinct endogenous mediators which are capable of enhancing the induction of innate and adaptive immune responses by promoting the recruitment and maturation of antigen-presenting cells(APCs),including dendritic cells(DCs).High-mobility group nucleosome-binding protein 1(HMGN1)is a novel alarmin reported by Professor Yang et al in 2012.It has been elucidated that extracellular HMGN1 was able to locally recruit DC,induce DC maturation through Toll-like receptor 4(TLR4)and promote antigen-specific immune responses when antigen was co-administrated.Previous studies found that alarmin HMGN1 acted as immunoadjuvant and contributed to the induction of tumor-associated antigen(TAA)specific anti-tumor immune responses.However,the mechanisms of HMGN1-induced TAA specific anti-tumor immune responses and its role in tumor therapeutic vaccine have not yet been fully elucidated.This project mainly focused on the mechanisms of the alarmin HMGN1-induced anti-tumor immune responses as well as the role of HMGN1 acted as adjuvant in the preventive and adoptive immunotherapy models.This research was divided into the following two chapters:Chapter 1,B16F1 murine melanoma cell lines stably expressing HMGN1 as well as corresponding control cell line were successfully established.B16-s N1 cell line secretory expressed protein HMGN1,while B16-m N1 cell line expressed membraneanchoring HMGN1.Wild-type B16 cell line transfected with empty lentivirus was also established as control,named after B16-PCDH.The secretory expression of HMGN1 by B16-s N1 cell line and the membrane-anchoring HMGN1 by B16-m N1 cell line were validated by western blot in vitro.B16-s N1,B16-m N1 tumors grew slower than control B16-PCDH tumors in vivo,albeit all cell lines proliferated equally in vitro,suggesting the generation of resistance to HMGN1-expressing tumors.Chapter 2,the three established stable B16F1 cell lines were irradiated and prepared as tumor cell vaccines.In murine models,the three cellular vaccines with PBS as blank control,were inoculated subcutaneously into C57BL/6 mice for three times,respectively.Fluorescence Activated Cell Sorter(FACS)showed that splenocytes from both B16-s N1(IR)and B16-m N1(IR)immunized mice had significantly higher proportion of Interferon-?(IFN-?)+CD8+ T cells as well as T-bet+CD8+ T cells than the PBS control group.In contrast,the proportion of myeloid-derived suppressor cells(MDSC)or regulatory T(Treg)cells was not significantly different among all groups.The cytotoxicity assays furtherly confirmed that splenocytes from B16-m N1(IR)immunized mice had better capacity of specificly-killing B16F1 cells.ELISA demonstrated that IFN-? had a relatively higher level in the serum of mice immunized with B16-m N1(IR)when compared with that in the control group.These results indicated that B16-m N1(IR)cellular vaccine can significantly enhance anti-tumor immune responses in vivo by promoting tumor specific Tc1 immune responses.In tumor prophylatic model,our results found that the tumor formation in groups immunized with B16-s N1(IR)and B16-m N1(IR),respectively,grew obviously slower than the control group.Meanwhile,tumor weights were lighter in the former two groups as well.The difference in the group immunized with B16-m N1(IR)and the control group was statistically significant(P<0.05).These results suggested that membrane-expression HMGN1 can prevent the occurrence of melanoma in vivo.In adoptive immunotherpy model,it has been observed that the melanoma mice treated with splenocytes from B16-m N1(IR)immunized donors had significantly slower rate of tumor growth than the control group,moreover,tumors immunized with B16-s N1(IR)and B16-m N1(IR)showed more tumor infiltrating CD8+T and CD4+T cells,whereas there was no difference of tumor infiltrating Treg among all the groups.It indicated that membrane-anchored HMGN1 acted as adjuvant may help to enhance the effect of adoptive immunotherapy.In summary,we can conclude that: alarmin HMGN1 acted as immunoadjuvant can contribute to the induction of anti-tumor immune responses in vivo by promoting tumor specific Tc1 immune responses.Alarmin HMGN1 may potentially be a promising adjuvant in the development of prophylactic and therapeutic tumor vaccines.