Liver transplantation is the best therapy for patients with end-stage liver diseases and unresectable early hepatocellular carcinoma (HCC). Living donor liver transplantation (LDLT) has been successfully implemented as an alternative to deceased donor liver transplantation (DDLT) and likewise offers comparable excellent survival rate. However, the inferior post-transplant oncological outcomes are found in LDLT recipients with HCC. The liver grafts used in LDLT are usually small-for-size and less effective in coping with shear stress from transient portal hypertension, which results in small-for-size liver graft injury. Acute phase small-for-size liver graft injury may promote late phase tumor recurrence, whereas the underlying mechanism remains unclear. CXCL10, an inflammatory chemokine, initiates liver inflammatory response during hepatic ischemia-reperfusion (IR) injury and may link acute phase small-for-size liver graft injury and late phase tumor recurrence, yet the precise mechanisms remain elusive. Endothelial progenitor cells (EPCs) participate in tissue repair for graft recovery and also provide an angiogenic environment for tumor growth. Myeloid derived suppressor cells (MDSCs) and regulatory T cells (Tregs) can suppress the activation of the immune system and play a critical role in graft rejection and cancer development. We here established the rat orthotopic liver transplantation with whole graft or small-for-size graft model to study the impact of acute phase small-for-size liver graft injury on the mobilization of EPCs, MDSCs and Tregs, and intragraft CXCL10 and its receptor, CXCR3,gene expressions. We further subjected CXCL10-/-mice and CXCR3-/-mice to hepatic IR injury and major hepatectomy to study the role of CXCL10/CXCR3 signaling on the mobilization of EPCs, MDSCs and Tregs. We also investigated the effect of CXCL10 on EPC migration and tube formation in vitroas well as intratumoral microvessel density (MVD) in the rat liver transplantation with tumor growth model and EPCs on tumor growth in nude mice. Key findings: 1. Liver transplantation with small-for-size graft resulted in severe intragraft vascular injury and higher CXCL10 andCXCR3 gene expressions as well as more EPC, MDSC and Treg cell mobilizationin circulation than whole graft. 2. CXCL10-/-mice and CXCR3-/-mice had less circulating EPCs, MDSCs and Tregs than WT mice after hepatic IR injury and major hepatectomy. 3. CXCL10 recruited EPCs in dose-dependent and CXCR3-dependent manners and promoted EPC tube formation in vitro. 4. Higher intratumoral MVD was observed in small-for-size graft than in whole graft in liver transplantation with tumor growth model. 5. Tumor grew more quickly by combining EPC infusionin nude mouse orthotopic liver tumor model. In conclusion, acute phase small-for-size liver graft injury significantly mobilizes EPCs, MDSCs and Tregs after transplantation through CXCL10/CXCR3 signaling. More EPC mobilization and intragraft differentiation after transplantation with small-for-size liver graft may be related to higher intratumoral MVD in small-for-size liver graft after transplantation with tumor development. Therefore, targeting at post-transplant CXCL10/CXCR3 signaling may not only attenuate early phase liver graft injury but also prevent late phase tumor recurrence. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

Thesis (M. Clin. Sc.)--University of Adelaide, 1985. / Includes bibliographical references (leaves 99-106).

Tumor-infiltrating myeloid-derived suppressor cells (MDSCs), are important mediators of a tumor-permissive microenvironment that contributes to tumor growth and could account for the limited success of immunotherapeutic strategies. MDSCs suppress adaptive immunity by blocking T cell activation, inducing Treg accumulation, and inhibiting natural killer cell cytotoxicity against tumor cells. We investigated the roles of MDSCs in the regeneration of the exocrine pancreas associated with acute pancreatitis and the progression of acinar to ductal metaplasia. Acute pancreatitis was induced in wild type and P48+/Cre;LSL-KRASG12D mice using caerulein and an early influx of MDSCs into the pancreas was observed flow cytometry and immunocytochemistry. Numbers of Gr1(+)CD11b(+) MDSCs increased over 20-fold in pancreata of mice with acute pancreatitis to account for nearly 15% of intrapancreatic leukocytes and have T cell suppressive properties. This marked accumulation of MDSCs returned to normal values within 24 hours of the insult in wild type mice; however, in the oncogenic KRAS mice, MDSCs levels remained elevated. When intrapancreatic MDSCs were depleted by administration of a CXCR2 antagonist (SB265610) in wild type mice the severity of acinar damage was increased. This was also accompanied by a delayed regeneration determined morphologically and with the mitotic immunomarker phospho-histone H3. Isolated intrapancreatic MDSCs from treated mice induce naïve acinar cells to undergo acinar ductal metaplasia when co-cultured in collagen 3D cultures. Purified splenic MDSCs failed to induce the phenotypic transdifferentiation. We conclude that MDSCs are required for adequate pancreatic regeneration in wild type mice with acute pancreatitis and their persistent elevation in oncogenic KRAS mice is not only associated with immune-evasion, but may also function as direct enhancer of malignant proliferation.

Myeloid precursor cell reprogramming into a myeloid-derived suppressor cell (MDSC) contributes to high mortality rates in mouse and human sepsis. S100A9 mRNA and intracellular protein levels increase during early sepsis and remain elevated in Gr1+CD11b+ MDSCs after pro-inflammatory sepsis transitions to the later chronic anti-inflammatory and immunosuppressive phenotype. The purpose of this study was to determine whether intracellular S100A9 protein might sustain Gr1+CD11b+ MDSC repressor cell reprogramming during sepsis. We used a chronic model of sepsis in mice to show that S100A9 release from MDSCs and circulating phagocytes decreases after early sepsis and that targeting the S100a9 gene improves survival. Surprisingly, we find that intracellular S100A9 protein translocates from the cytosol to nucleus in Gr1+CD11b+ MDSCs during late sepsis and promotes expression of miR-21 and miR-181b immune repressor mediators. We further provide support of this immunosuppression pathway in human sepsis. This study may inform a new therapeutic target for improving sepsis outcome.

OBJECTIVE. To determine if the levels of circulating myeloid-derived suppressor cells increase with progression of prostate cancer (PCa); to determine if such cells could contribute to the relative inefficiency of PCa immunotherapy. MATERIALS AND METHODS. We analyzed peripheral blood mononuclear cells isolated from untreated PCa patients (uPCa; N=18; mean age±SD: 72.1± 6.9 years), tPCa (N = 22; 72.8 ± 9.8 years) and age matched controls (AMC; N = 12; 68.8 ± 7.5 years). We quantified surface marker phenotype, differentiation potential, effects on T cell proliferation and intracellular cytokines. RESULTS. We observed an unexpectedly high percentage of a type of myeloid-derived suppressor cells, CD14+HLA-DR low/- monocytes, in tPCa (30.7±15.0% of CD14+ cells) relative to AMC (4.1+6.5%, P<0.0001) and uPCa (10.6 ± 14.3%, P = 0.0001). The levels of CD14+ HLA-DR low/- cells were significantly correlated with circulating PSA levels and treatment with LHRH-agonist leuprolide in combination with either an antiandrogen or dexamethasone. Monocytes from tPCa inhibited autologous T cell proliferation statistically significantly more effectively than AMC monocytes and were defective in their ability to differentiate into phenotypically mature dendritic cells. Isolated CD14+HLA-DRlow/- cells expressed higher levels of intracellular interleukin-10 and suppressed T cell proliferation more effectively than isolated CD14+HLA-DR+ cells. CONCLUSIONS. This is the first report of CD14+ cells exhibiting reduced expression of HLADRmolecules in PCa patients. These cells suppress immune cell function in vitro and, plausibly, in vivo, a finding that must be factored into the design of immunotherapy protocols for PCa patients.

In the aftermath of cancer surgery, Natural killer (NK) cells are severely suppressed. NK cells are critical for anti-tumour surveillance and their postoperative dysfunction creates an opportunity for metastases. I hypothesized that NK cell suppression is mediated by multiple suppressive mechanisms of surgery-induced Myeloid Derived Suppressor Cells (Sx-MDSCs). In this thesis, I first show that NK cell dysfunction is far worse than previously described. In a cohort of colorectal cancer (CRC) surgery patients (n=42), the ability of NK cells to secrete IFN-gamma in response to stimulation was suppressed for up to 2 months after surgery. Secondly, since Sx-MDSCs have been poorly characterized in humans, I thoroughly phenotyped Sx-MDSCs from cancer surgery patients using flow cytometry (n=32 patient samples) and single-cell RNA sequencing (n=6 patient samples). Additionally, upon screening a library of 150 compounds, I showed that Sx-MDSC rely on PI3K signaling for their suppression of NK cells in ex vivo NK cell suppression assays. The third part of this thesis explores the contribution of Sx-MDSCs to the rapid reduction in postoperative arginine, the perioperative importance of arginine for NK cells, and the therapeutic effects of a perioperative arginine enriched supplement (AES) on metastases in murine models of surgical stress. Here, I showed that perioperative AES attenuates postoperative metastases by accelerating NK cell recovery after surgery. These promising preclinical data combined with evidence from the scientific literature led us to initiate a Phase II randomized-controlled clinical trial assessing the ability of perioperative AES to improve NK cell function after surgery in CRC patients (n=12/arm). In the last part of this thesis, I present the results from our clinical trial, which showed only a transient and, at best, modest improvement in NK cell function. Importantly, this may have been heavily influenced by poor postoperative patient compliance in taking the AES. In conclusion, this body of work describes the multifactorial role that Sx-MDSCs play in mediating postoperative NK cell suppression, and that safe, effective, and targeted perioperative interventions should be further investigated as a strategy to attenuate metastatic disease recurrence after surgery.

Pancreatic ductal adenocarcinoma (PDAC) is a highly fatal cancer, accounting for 3.2% of new cancer cases yearly but nearly 8% of all yearly cancer mortalities. Over the past twenty years, our understanding of cancer biology has greatly improved which has resulted in vastly improved prognoses for many cancers. However, the prognosis of pancreatic ductal adenocarcinoma has not improved despite the advance in cancer treatments. This is especially apparent with cancer immunotherapies, a newer therapeutic strategy that utilizes the innate defense mechanism of the body to target malignancies. Immune checkpoint inhibitors are a type of cancer immunotherapy that act by inhibiting the PD-1/PD-L1 and CTLA-4 immune checkpoint pathways and allowing T lymphocytes to proliferate and generate an antitumor response. They have greatly improved the prognosis for many types of malignancies, but clinical studies show that immune checkpoint inhibition has had a limited effect on the prognosis of PDAC. Recent studies have demonstrated that the immune microenvironment of PDAC is highly immunosuppressive, which is a probable factor in limiting the therapeutic efficacy of immune checkpoint inhibitors. Myeloid derived suppressor cells (MDSCs) are a main component of the immune microenvironment in PDAC. They are immature cells of myeloid origin that express CD11b+Gr-1+ on their surface, making them phenotypically distinct from mature dendritic cells. Their infiltration of the PDAC microenvironment early on in the course of the disease is promoted in a large part by the cytokine GM-CSF. MDSCs are believed to contribute to the limited efficacy of immune checkpoint inhibitor therapy both directly and indirectly. Indirect mechanisms are mediated by promoting the activity of other immunosuppressive cells in the PDAC microenvironment such as tumor associated macrophages and regulatory T lymphocytes. MDSCs induce the transformation of naïve CD4+ T lymphocytes into protumorigenic regulatory T lymphocytes. They also promote the polarization of macrophages to the tumor associated macrophage phenotype (IL-10high IL-12low) by secreting IL-10, which decreases IL-12 synthesis by macrophages present in the tumor microenvironment. On top of mediating immunosuppression through other cell types, MDSCs directly mediate immunosuppression by decreasing the amounts of amino acids necessary for anti-tumor immunity in the tumor microenvironment and disrupting the activity of antigen presenting cells and the signaling needed to initiate a cytotoxic T lymphocyte response. The decreased amount of arginine limits the ability of T cells to proliferate, resulting in a weaker cytotoxic response. These mechanisms limit the antitumor response against pancreatic ductal adenocarcinoma, resulting in the decreased response to immune checkpoint inhibitor therapy observed in clinical trials. Future attempts to strengthen the anti-tumor immune response must be combinatorial therapies that incorporate therapeutic strategies that seek to alleviate MDSC-mediated immunosuppression of T lymphocytes from the tumor microenvironment in addition to the more widely available immune checkpoint inhibitor therapy. Such therapeutics are currently being studied in murine models and have shown promising preliminary results but have yet to have been examined in clinical trials. These therapies are an ideal avenue to explore in a search for more effective therapy for this highly lethal disease.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19560号 / 医博第4067号 / 新制||医||1013(附属図書館) / 32596 / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 小川 修, 教授 長澤 丘司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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