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Background: Life-prolonging tumour removal surgery is associated with increased metastasis and disease recurrence. Natural Killer (NK) cells are critical for the anti-tumour immune response. Postoperatively, NK cell cytotoxicity and interferon-gamma (IFNγ) production are profoundly suppressed and this dysfunction has been linked to increased metastases/poor patient outcomes. NK cell activity depends on the integration of signals through receptors and can be modulated by soluble factors, including transforming growth factor- beta (TGFβ). The postoperative period is characterized by the expansion of myeloid-derived suppressor cells (sxMDSCs), which inhibit NK cell effector functions. I hypothesize that impaired NK cell IFNγ production is due to altered signaling pathways caused by sxMDSC-derived TGFβ. Methods: Postoperative changes in NK cell receptor expression, receptor-dependent phosphorylation of downstream targets, and rIL-2/12-stimulated IFNγ production were assessed using newly developed whole blood assays utilizing peripheral blood samples from cancer surgery patients. Isolated healthy NK cells were incubated in the presence of healthy/baseline/postoperative day (POD) 1 plasma or isolated sxMDSCs and NK cell phenotype and function were assessed. NK cells were also cultured with plasma in the presence/absence of a TGFβ blocking monoclonal antibody (mAb) or a TGFβ RI small molecule inhibitor (smi). Single-cell RNA-sequencing was performed on six colorectal cancer surgery patients at baseline and on POD1. S6 phosphorylation was used as a proxy for mammalian target of rapamycin complex (mTORC) 1 activity to investigate the mechanism of TGFβ-mediated NK cell dysfunction. Results: Intracellular NK cell IFNγ, activating receptors CD132 (IL-2R), CD212 (IL-12R), NKG2D, and DNAM-1, and the phosphorylation of downstream targets STAT5, STAT4, p38 MAPK, and S6 were significantly reduced on POD1. TGFβ was increased in patient plasma on POD1. The dysfunctional phenotype could be phenocopied in healthy NK cells through the addition of rTGFβ1 or by incubation with POD1 plasma. This dysfunctional phenotype could be prevented with the addition of an anti-TGFβ mAb or a TGFβ RI smi in culture. RNA-sequencing revealed a reduction in transcripts associated with mTOR effector functions, suggesting an impairment in mTOR. S6 phosphorylation was maintained with the addition of TGFβ-specific therapies. The hyporesponsive NK cell phenotype was reproduced upon culture of healthy NK cells with sxMDSCs and sxMDSCs were shown to produce soluble TGFβ in culture. Conclusion: Surgically stressed NK cells display a dysfunctional phenotype, which could be prevented in vitro through the addition of TGFβ-specific blocking therapies. sxMDSCs produced TGFβ and co- incubation induced dysfunction in healthy NK cells. The recovery of impaired S6 phosphorylation with TGFβ-specific therapies suggests that TGFβ is inducing NK cell dysfunction via inhibition of mTORC1 activity. The perioperative period of immunosuppression presents a window of opportunity for novel therapeutics to prevent metastases and cancer recurrence among cancer surgery patients.

Natural killer (NK) cell deficiency (NKD) occurs when an individual’s major clinical immunodeficiency derives from abnormal NK cells and is associated with several genetic etiologies. Three categories of β actin-related diseases with over 60 ACTB variants have previously been identified, none with a distinct NK cell phenotype. An individual with mild developmental delay, macrothrombocytopenia, susceptibility to infections, molluscum, and EBV-associated lymphoma had functional NK cell deficiency for over a decade. A de novo ACTB variant encoding G342D β actin was identified and was consistent with the individual’s developmental and platelet phenotype. This novel variant also was found to have a direct impact in NK cells, as its expression in YTS (YTS-NKD) cells caused increased cell spreading in lytic immune synapses created on activating surfaces. YTS-NKD cells were able to degranulate and perform cytotoxicity, but demonstrated defective serial killing owing to prolonged conjugation to the killed target cell and thus were effectively unable to terminate lytic synapses. G342D β actin results in a novel mechanism of functional NKD via increased synaptic spreading and defective lytic synapse termination with resulting impaired serial killing leading to overall reductions in NK cell cytotoxicity.

A growing body of evidence has demonstrated that soy isoflavone consumption may protect against the development of various chronic diseases. This protection could be linked to isoflavone-induced alterations in immune function. However, recent in vitro and animal studies suggest that soy isoflavones may either enhance or suppress immunocompetence, depending upon the isoflavone concentration, target tissue, and a number of other factors. To date, no study has investigated the effect of dietary soy isoflavone supplementation on immune parameters in humans. Therefore, the purpose of this double-blind, placebo-controlled, 4 wk intervention trial was to investigate whether supplementation with soy isoflavones alters indices of immune function in postmenopausal women. Twenty healthy women (50-69 yr), who were not on hormone replacement therapy, were randomly divided into 2 treatment groups. The supplemented group (n=10) consumed soy isoflavone tablets (100 mg/d) for 4 wk, while the control group (n=10) received placebo tablets. Fasting blood samples were drawn at baseline and on d 28 to assess specific immune parameters. In addition, plasma concentrations of genistein and daidzein were quantified at baseline and at the end of the intervention period. Despite high individual variability among subjects, there was a significant increase (p<0.005) in plasma isoflavone concentration in the supplemented group. However, all assessed immune parameters remained unchanged after supplementation and did not differ between the 2 treatment groups. In conclusion, this study suggests that short-term soy isoflavone supplementation at physiologically attainable concentrations does not alter the aforementioned immune parameters in healthy postmenopausal women. Due to the conflicting data concerning the effect of dietary soy isoflavones on immune function, further research in this area is warranted. / Master of Science

A growing body of evidence has demonstrated that soy isoflavone consumption may protect against the development of various chronic diseases. This defense could be linked to isoflavone-induced alterations in immune function. However, to date, no study has examined the effect of soy isoflavone supplementation on human immunity in vivo. Establishing whether isoflavones affect immunity in aging adults is particularly relevant since compromised immune function has been observed in this population. Therefore, the purpose of this double-blind, placebo-controlled, 4-wk intervention trial was to investigate whether supplementation with soy isoflavones influenced the distribution and/or function of specific lymphocytes in postmenopausal women. Healthy postmenopausal women (50-69 y), who were not using hormone replacement therapy, were randomly divided into 2 treatment groups. The experimental group (n=9) consumed two-50 mg soy isoflavone tablets/d for 4 wk, while the control group (n=9) received placebo tablets. Fasting blood samples were drawn at baseline and on d 28 to assess distribution of T-helper cells (CD3+CD4+), T-cytotoxic cells (CD3+CD8+), total T lymphocytes (CD3+), B lymphocytes (CD19+) and natural killer (NK) cells (CD16+CD56+) via flow cytometry. Cytotoxicity of NK cells was quantified based on lactate dehydrogenase release of lysed K562 cancer cells following co-culture with NK cells from subjects. Analysis of plasma isoflavone concentrations by HPLC demonstrated a significant increase (p<0.005) in plasma genistein concentration in the experimental group after 4 wk of supplementation. However, there was no alteration in lymphocyte distribution or NK cell activity in response to isoflavone supplementation, suggesting that short-term soy isoflavone supplementation does not alter these parameters of immunity in healthy postmenopausal women. / Master of Science

Two important mechanisms of lymphocyte-mediated cytotoxicity, one perforin based and the other Fas ligand (FasL) based, have been characterized recently. It has also been shown that CD44, an adhesion molecule, can participate in signaling cytotoxic activity of cytotoxic T lymphocytes (CTLs). In the current study we tested the hypothesis that activation of natural killer (NK) or lymphokine activated killer (LAK) cells induces the expression of FasL, perforin, and CD44 which together contribute towards increased cytolytic activity. To this effect, we used wild-type mice, perforin-knockout mice, and mice lacking a functional FasL. We observed that both interleukin-2 (IL-2) and Poly I:C triggered NK/LAK cells to lyse targets through the perforin- and FasL- pathways. In addition, Fas+ tumor targets were more susceptible to lysis by poly I:C and IL-2 activated NK/LAK cells when compared to Fas- targets. Furthermore, Fas- tumor cells injected subcutaneously into syngeneic mice could grow and induce tumors, whereas, Fas+ tumors were rejected. IL-2 treatment increased the CD44 expression on NK cells, which was responsible for the lysis of endothelial cells through its ligand, hyaluronate. Upregulation of perforin and FasL in activated NK/LAK cells may explain why such cells can kill a wide variety of tumor cells efficiently. On the other hand, activated NK/LAK cells express increase increased levels of CD44 and use this molecule to mediate cytotoxicity of endothelial cells, which may account for the vascular leak seen during IL-2 therapy. / Master of Science

Les virus du Chikungunya (CHIKV) et de la dengue (DENV) sont deux virus émergents qui sévissent dans les régions tropicales et subtropicales du monde entier et qui sont transmis par les moustiques du genre Aedes. Ces dernières années, leur transmission a surtout progressé dans les zones urbaines et périurbaines touchant des millions d’individus et faisant de ces deux pathogènes des sujets majeurs de préoccupation pour la santé publique. Le Chikungunya et la Dengue sont des infections dites aiguës entrainant une mise en place rapide de la réponse immunitaire innée qui joue un rôle majeur dans le contrôle et l’évolution de la maladie. Les cellules Natural Killer (NK) représentent une population cellulaire clé de la réponse innée et jouent un rôle crucial dans les mécanismes de défense mis en place. A travers une étude ex vivo et in vitro, nous nous sommes intéressées à la caractérisation des cellules NK à travers (i) une étude phénotypique et fonctionnelle des cellules NK chez des patients infectés en phase aiguë par le CHIKV, DENV-2 ou par les deux virus et (ii) à la caractérisation des interactions entre les cellules NK et les cellules cibles infectées par le virus. L’ensemble de ces données contribue à mieux identifier l’implication des cellules NK dans le contrôle des infections par le CHIKV et DENV-2 permettant ainsi de mieux comprendre les mécanismes à l’origine des dérèglements de la réponse immunitaire. Au cours des dernières épidémies, plusieurs cas de patients coinfectés par les deux virus ont été répertoriés. De plus, l’expansion géographique des moustiques Aedes pourrait amener à une augmentation du nombre de cas de coinfections sans que les mécanismes sous jacents aux coinfections ne soient étudiés. Afin de pouvoir réponse à certaines questions concernant ce phénomène, nous avons mis en place un modèle expérimental de coinfection par CHIKV et DENV-2 chez le macaque Rhésus. / Chikungunya (CHIKV) and Dengue (DENV) virus are both re-emerging viruses transmitted by Aedes mosquitoes and responsible of widespread outbreaks in tropical and subtropical country. Recently, transmission of both viruses had emerged in urban and peri-urban area infecting millions of persons. Chikungunya and Dengue are both acute infections where innate immunity rapidly takes place and play a crucial role in the control and in the evolution of the disease. Natural Killer cells (NK) represent one of the major cellular population of innate immunity and play a crucial role in defense mechanism. By way of ex vivo and in vitro studies, we characterized NK cells by (i) a phenotypic and functional study of NK cells in CHIKV, DENV-2 infected patients or CHIKV/DENV-2 co-infected patients and (ii) characterization of NK cells interactions with infected target cells. During last outbreaks, several cases of co-infected patients were reported. Moreover, geographic spread of Aedes mosquitoes could increase number of coinfection cases without underlying mechanisms being explored. In order to respond to certain questions regarding coinfections, we realized a co-infected CHIKV and DENV-2 experimental model in Rhesus macaques.Together, these data will contribute to better identify NK cells implication in the control of CHIKV and DENV-2 infections allowing a better comprehension of mechanisms that causes immune system disorder.