A study on the immunostimulatory effects of alarmin HMGN1 in cancer immunity

概要

Introduction:
The aim of this thesis is to elucidate the immunostimulatory effects of an alarmin high-mobility nucleosome binding protein 1 (HMGN1) in cancer immunity. HMGN1 has been considered as an immune stimulator to trigger the activation and migration of dendritic cell (DCs) and has also been reported as a potential adjuvant for T cell-mediated therapy. T cell-mediated therapies, such as immune checkpoint inhibitors or T-cell transfer, provide remarkable therapeutic benefit for cancer patients, but not all patients response equally well to those therapies. The failure of T cellmediated therapies may result from the problems of insufficient antigen presentation, coinhibition, immunosuppression, and T cell exhaustion happened within the tumor microenvironment. To tackle these problems, the researchers have attempted to combine various types of immune stimulators with T cell-mediated therapy to strengthen the antigen presenting ability of tumor-infiltrating DCs, facilitate the generation of tumor-reactive CD8+ T cells, and reinvigorate the effector functions of those CD8+ T cells from exhaustion. Here I use HMGN1 as adjuvant in combination with T cell-mediated therapies, such as anti-CD4 depleting antibody or anti-PD-L1 antibody.

Methods:
Recombinant HMGN1 proteins were produced in E. coli and purified using sequential fractionation by heparin affinity column, ion exchange column and reverse-phase column. The purity of and the endotoxin level of HMGN1 were confirmed before in vivo treatment. The HMGN1 peptides were synthesized by Eurofins Genomics, Japan. The anti-tumors effects of HMGN1, anti-CD4 depleting antibody, anti-PD-L1 antibody, and their combined treatment were monitored in Colon26, B16F10, LLC, and EO771 subcutaneous murine models. The tumorinfiltrating CD8+ T cell population, proliferation, differentiation, exhaustion, and gene expression profile were determined by high-dimensional flow cytometric analysis, transcriptome analysis, and quantitative real-time PCR.

Results:
This thesis started by studying synergistic antitumor effects brought by HMGN1, in combination with either anti-CD4 depleting antibody or anti-PD-L1 antibody. First, I found that combination therapy of HMGN1 and anti-CD4 depleting antibody are able to expand intratumoral CD8+ T cells and prolong their antitumor activities by rescuing them from T cell exhaustion. The additional HMGN1 treatment further reinvigorated the effector functions of exhausted CD8+ T cells, which addresses the problem of T cell exhaustion during T cell-mediated therapy. Motivated by the synergistic antitumor effects brought by HMGN1, I began to identify the immunostimulatory domain on HMGN1 and synthesized the minimized HMGN1 immunostimulatory peptide (minP1). For the development of clinical agent, synthetic peptides have advantage over recombinant proteins, as they are quickly and easily synthesized without endotoxin contamination, and are relatively inexpensive. Finally, I combined the minP1 with anti-PD-L1 antibody to evaluate whether minP1 could improve the therapeutic efficacy of antiPD-L1 treatment. The minP1 synergized with anti-PD-L1 antibody to enhance antigen presenting function of CCR7+ mDCs, facilitated the generation of tumor-reactive CD8+ T cells, expanded Ly108+ TIM-3- stem-like CD8+ T cells in the tumor, and thereby leading to tumor regression and even tumor clearance in some mice.

Discussion:
In chapter III, I observed that those mice receiving intraperitoneal administration of low-dose HMGN1 with anti-CD4 depleting antibody had an increasing number of tumor-infiltrating CD8+ T cells with the less exhausted phenotypes and the capacity to produce multiple cytokines. The cellular and molecular mechanisms underlying the increase in the number of functional CD8+ T cells after combination treatment remain elusive. It is likely that the depletion of the Foxp3+ CD4+ regulatory T cells, which suppresses CD8+ T cell expansion in the draining lymph node through the cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) medicated downregulation of B7 co-stimulatory molecules in DCs, may contribute to the increased number of tumorinfiltrating CD8+ T cells. In chapter IV, I identified the domain of immunostimulatory functions on HMGN1. Previous studies have found that there are nucleosome binding and chromatin unfolding domains on HMGN1 based on their distinct intracellular functions. In terms of extracellular functions, my previous works have demonstrated that HMGN1 induces immunostimulatory responses that brings synergistic antitumor effects; however, the domain of immunostimulatory functions on HMGN1 is not well understood. It is worth noting that I have identified a 23 amino acid residues EPKRRSARLS AKPPAKVEAK PKK within HMGN1 nucleosome binding region, which retains the immunostimulatory responses and the synergistic antitumor effects of HMGN1. This finding helps resolve the location of immunostimulatory domain on HMGN1 and prompt me and other researchers to look deeper at the immunostimulatory domain for understanding how HMGN1 binds and interacts with its candidate receptors, such as MD2 or Gαi protein coupled receptor (GiPCR). Like high mobility group box-1 protein (HMGB1), HMGN1 may elicit different immunostimulatory responses by activating multiple signaling pathways, such as TLR2, TLR4 or receptor for advanced glycation endoproducts (RAGE) pathway. The receptors of HMGN1 and their related downstream signaling pathways are under investigation.

Conclusion:
This thesis describes the molecular and cellular mechanism of HMGN1 while it used as an adjuvant for cancer immunotherapy, identified the immunostimulatory domain from HMGN1, and highlights the possible approach to combining HMGN1 (or minP1) with anti-CD4 depleting antibody or anti-PD-L1 antibody to strengthen the efficacy of antitumor responses, which may address the current problems happened during T cell-mediated therapy of cancer.