BACKGROUND: Natural killer (NK) cells are traditionally considered part of the innate immune system but have recently been shown to possess adaptive qualities similar to T cells in response to an infection with a pathogen. In addition to possessing adaptive features, NK cells have also been found to reside in different organs such as the liver, spleen, and lymph nodes and differ based on phenotypic markers and their responses to different cytokines. Utilizing these findings, several groups have isolated and identified CD49a as a marker for tissue-resident NK cells. In the liver, CD49a has also been shown to be a positive indicator for NK cell memory-like responses in murine models. Building off work that demonstrated antigen-specific responses in rhesus macaques, this project focuses on characterizing the phenotypic markers and functional profile of CD49a+ NK cells in non-human primates. To better understand the role of CD49a in memory-like NK cells outside of the liver, this project utilized spleen samples from rhesus macaques infected with SIV/SHIV. This work aims to help us better understand the dysfunction NK cells experience as a result of HIV-1 infection in humans and also to demonstrate the changes NK cells experience as the disease progresses. A thorough understanding of the adaptive capabilities of NK cells can pave the way for targeted therapies to increase NK cell antiviral activity in HIV and other infections. METHODS: To characterize the functional and phenotypic profiles of CD49a+ NK cells by multiparameter flow cytometry, thirteen samples of spleen from rhesus macaques were thawed and then stained with two different protocols. A phenotyping protocol involved staining with antibodies against surface markers as well as intracellular markers T-Bet and Eomes. For the functional characterization protocol, the same thirteen samples were stained intracellularly after being stimulated with a cocktail of PMA and ionomyocin. The antibodies used in this were for functional markers. Of the thirteen samples used, six were infected with SHIVSF162P3, two were infected with SIVmac239X, and the remaining five were uninfected. After staining, these samples were analyzed on an BD LSRII from BD Biosciences. The data obtained were then analyzed using FlowJo software to study NK cells, which were characterized as CD45+CD14-CD20-CD3-CD159+. RESULTS: The analysis compared NK cells with T cells, B cells, and NKB cells. Some increases were seen among CD49a+ NK cells in the frequency of CD336+ (NCR2/NKp44), CD337+ (NKp30), and CD366+ (Tim-3) after infection. Although there were some mild increases in CD107a and TNF- in infected samples compared to uninfected, a significant increase was observed in the frequency of IFN-ɣ among infected CD49a+ NK cells compared to uninfected. CONCLUSION: When comparing samples that were infected vs uninfected, it appears there were some mild decreases after infection in the ratio of NK cells to other lymphocytes. In addition, there did not appear to be a significant increase in the frequency of CD49a+ among these NK cells as a result of the infection. However, among the CD49a+ subpopulation, there were some observed non-significant decreases in CD56-CD16+ cells. Furthermore, there was found to be an almost significant increase in TNF- (p = 0.06) among CD49a+ cells after infection. These findings demonstrate an increase in cytotoxic activity in splenic NK cells associated with an adaptation to the virus. Although there does not appear to be significant changes in the ratio of NK cell populations in the spleen, the changes observed in phenotypic and functional markers associated with CD49a+ demonstrate an increase in the cytotoxic activity of NK cells as a result of infection with SIV/SHIV. However, it remains to be seen if CD49a is a direct indicator of this type of infection. Future work geared toward memory-like NK cells in non-human primate splenic tissue could look at the contrast in CD49a+ NK cells from different states of infection with HIV-1 and/or SIV (acute vs chronic) to better understand the integrin’s role in adaptation.

Since their discovery, natural killer cells (NK) cells have been implicated as important players in cancer immunosurveillance. In recent years, researchers have taken advantage of this role by developing NK cell-based immunotherapies in the fight against cancer. While these treatments have been moderately successful against hematological malignancy, they are less effective against solid cancers. This lack of success partially results from the immunosuppressive effects of the tumor microenvironment (TME). While tumors use myriad processes to evade the immune system, the avid consumption of nutrients common to NK and cancer cell metabolism and the production of toxic waste products can have significant deleterious effects on NK cell anti-tumor function. However, it may be possible to avoid some of this tumor-induced inhibition of NK cell anti-tumor function by manipulating NK cell metabolism and/or environmental conditions. Recent studies have revealed that different activation regimens can affect the metabolic dependencies of different NK cell subsets. Furthermore, studies have identified potential targets in the TME that can make the environment less hostile for infiltrating NK cells. By considering the interrelationship of NK cell metabolism and function—especially in the TME—this thesis illuminates potential strategies to modulate immunometabolic suppression. Despite the promising work already done, many gaps in the knowledge of NK cell metabolism remain. Future work will need to investigate the specific molecular mechanisms linking metabolism and function, the role of tissue-resident NK cells in cancer immunosurveillance, and the influences of chronic disease and altered systemic metabolism on NK cell anti-tumor activity.

Invasive aspergillosis (IA) is a major opportunistic fungal infection in patients with haematological malignancies. Morbidity and mortality rates are high despite anti-fungal treatment, as the compromised status of immune system prevents the host from responding optimally to conventional therapy. This raises the consideration for immunotherapy as an adjunctive treatment. In this study, we evaluated the utility of expanded human NK cells as treatment against Aspergillus fumigatus infection in vitro and in vivo. The NK cells were expanded and activated by K562 cells genetically modified to express 4-1BB ligand and membrane-bound interleukin-15 (K562-41BBL-mbIL-15) as feeders. The efficacy of these cells was investigated in A. fumigatus killing assays in vitro and as adoptive cellular therapy in vivo. The expanded NK cells possessed potent killing activity at low effector-to-target ratio of 2:1. Fungicidal activity was morphotypal-dependent and most efficacious against A. fumigatus conidia. Fungicidal activity was mediated by dectin-1 receptors on the expanded NK cells leading to augmented release of perforin, resulting in enhanced direct cytolysis. In an immunocompromised mice pulmonary aspergillosis model, we showed that NK cell treatment significantly reduced fungal burden, hence demonstrating the translational potential of expanded NK cells as adjunctive therapy against IA in immunocompromised patients.

Background: CD100, also known as Sema4D, is an immune semaphorin constitutively expressed on natural killer (NK) cells and T cells. As an immune activation molecule, CD100 has important immunoregulatory effects on NK functions by enhancing the interactions between NK cells and target cells. The aim of this study was to investigate whether hepatitis C virus (HCV) infection affects CD100 expression, and whether interferon-α treatment enhances NK killing activity to facilitate HCV clearance via CD100. Methods: Expression of CD100 on NK cells was evaluated by flow cytometry in patients with chronic HCV infection, with or without pegylated interferon-α-based therapy. NK cell cytotoxicity and interferon (IFN)-γ production were measured by flow cytometry upon culturing the NK cells with K562 and Huh7.5 or HCV JFH-1-infected Huh7.5 cells. Results: The frequency of CD100+ NK cells in HCV-infected individuals was slightly suppressed compared to healthy subjects. IFN-α treatment could significantly upregulate CD100 expression, which was confirmed by in vitro studies using peripheral blood mononuclear cells cocultured with HCV-expressing Huh7.5 cells or IFN-α. Importantly, the expression of CD100 on NK cells from HCV patients was inversely associated with the HCV-RNA levels in the early phase of IFN-α therapy, and the IFN-α upregulated CD100 led to an enhanced NK killing activity through ligations with its receptors plexin-B1/B2 on target cells. Conclusion: These results implied a novel mechanism by which IFN-α enhanced CD100/Plexin-B1/B2 interaction plays an important role in promoting NK functions in patients with chronic hepatitis C.

Marathon runners (N = 22) who had completed at least seven marathons (X ± SEM = 23.6 ± 5.7) and had been training for marathon race events for at least 4 yr (12.3 ± 1.3) were compared with sedentary controls (N = 18). Although the two groups were of similar age (38.7 ± 1.5 and 43.9 ± 2.2 yr, respectively) and height, the marathon runners were significantly leaner and possessed a VO2max 60% higher than that of the controls. Neutrophil counts tended to be lower in the group of marathoners, while other leukocyte and lymphocyte subsets were similar to controls. Mitogen-induced lymphocyte proliferation did not differ between groups. Natural killer cell cyto-toxic activity (NKCA) was significantly higher in the marathoners versus controls (373 ± 38 vs 237 ± 41 total lytic units, respectively, a 57% difference, P = 0.02). For all subjects combined (N = 40) and within the group of marathon runners (N — 22), percent body fat was negatively correlated with NKCA (r = -0.48, P = 0.002; r = -0.49, P = 0.019, respectively), and age was negatively correlated with Con A-induccd lymphocyte proliferation (r = -0.41, P = 0.009; r = -0.53, P = 0.011, respectively). These data indicate that NKCA but not mitogen-induced lymphocyte proliferation is higher in marathon runners relative to sedentary controls.

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.

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