Studies of Receptor Interactions and Effects of Alarmins

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

Alarmins are characterized by having in vitro chemotactic and or in vivo recruitment activity for cells expressing GiPCR, together with the capacity to interact with other receptors resulting in the activation of immature dendritic cells (iDC) to develop into mature antigen-presenting cells capable of interacting with T lymphocytes. This enables the DC to present antigens they have phagocytized and processed to T lymphocytes and results in adaptive immune responses. Consequently, the in vitro stimulant effects on DC is used by us to predict the capacity of an alarmin to promote immune responses in vivo. These alarmins, if administered together with an antigen result in considerable augmentation of both in vivo cellular and humoral immune responses. We previously showed that HMGN1 knockout mice exhibit reduced resistance to tumor (EG-7 or EL-4) challenge. Conversely, tumor cells (EG-7 or EL-4) when transfected to overexpress HMGN1 showed a marked reduction in the rate of growth in normal mice. These observations indicated that HMGN1 is capable of augmenting antitumor immunity. We therefore injected a recombinant HMGN1 protein directly intratumorally into CT26 colon tumors in mice to proximate the adjuvant and CT26-associated antigen(s). This therapeutic vaccine trial did slow the tumor growth and prolonged the survival of mice but did not cure any of the mice. We therefore improved the potency of the tumor vaccine by employing combinations of immunotherapeutic antitumor reagents to cure mice with larger tumors. We screened all the TLR ligands to identify which cooperated best with HMGN1 on the activation of DCs. This led to the identification of R848, a TLR7/8 ligand as capable of maximally synergizing with HMGN1 in stimulating DC maturation to markedly increase their production of IL-12 and TNF. We have been able to show that intratumoral injections of the combination of immunotherapeutic agents consisting of HMGN1, R848 (Resiquimod), a checkpoint inhibitor such as anti PDL-1 or anti CTLA4 antibody or a low dose of cytoxan successfully cured large (1cm diam.) tumors of the colon (CT26), kidney (RENCA), thymoma (EG7) lung (Lewis Lung) and liver (Hepa1-6) in mice, resulting in the generation of immune resistance to re-challenge with the same tumor. We have termed this combinational regimen as 'therapeutic vaccination', shortened "TheraVac". We have also treated mice bearing tumors spontaneously arising from genetically modifications with TheraVac and succeeded in slowing their growth and prolonging their life span significantly. In addition, we have been able to cure 80% of mice bearing resistant B16/F10 melanoma tumors with a combination of TheraVac and cGAMP, a natural agonist of the STING pathway. We also investigated the mechanisms accounting for the synergistic stimulatory effects of HMGN1, R848, and cGAMP. Trigger of various signaling pathways in DCs by HMGN1, R848, and cGAMP results in synergistic activation of various transcription factors (e.g. NF-kB, MAPK, IRF3 and IRF7), which, in turn, accounts for synergistic induction of proinflammatory cytokines (e.g. IL-12, TNF, and type I IFNs) and marked upregulation of the phenotypic markers indicative of DC maturation. Synergistic production of IL-12 enhanced Th1 polarization favoring cellular antitumor immunity. We have previously reported that we can cleave the HMGN1 molecule and show that the N terminal domain preserve the immunostimulating effects. In collaboration with a group led by Prof. Kouji Matsushima, we recently identified a smaller peptide component of the N terminus of HMGN1 that is biologically active. This so-called "MinP" peptide in conjunction with anti-PD-L1 (an immune checkpoint blockade antibody) has demonstrated immune mediated antitumor effect in mice. To overcome the limitation of administration of TheraVac using intratumoral route, we have been trying to develop a TheraVa-functionalized nanoparticle to carry TheraVac preferentially to tumors upon intravenous administration. We have recently succeeded in doing so after we successfully designed and synthesized a ROS-sensitive mesoporous silica nanoparticle (MSN) functionalized with TheraVac (MSN@TheraVac), which when injected intravenously selectively accumulated in the tumors and more importantly cured 100% of the CT26-bearing mice. Another achivement we made in the laboratory is that we have developed more potent TheraVac versions by replacing R848 with FSL-1 or by adding cGAMP into the original TheraVac regimen. In addition to our previous publication showing that alpha-synuclein (aS) is a potent chemotactic protein for phagocytes, we have recently demonstrated that aS is also a potent activator of dendritic cells and therefore functions as an alarmin that is critical for host immunity. This is of particular interest since mutations of aS are casually associated with the development of familiar Parkinson's Disease. Mice with deletion of the aS genes have deficient cellular and humoral immune responses, indicative of the important immunological function of aS. Thus, our findings show aS, in addition to its involvement in the development of neurodegenerative diseases including Parkinson's disease, has potent proinflammatory/immunological effects.