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

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Les dernières avancées dans les traitements des hémopathies aboutissent à un meilleurs taux de rémission complète ainsi qu' à de meilleurs taux de survie après traitement. Cependant les risques de rechutes restent élevés. Notre projet s'inscrit dans la compréhension du rôle des cellules NK dans l'évolution de ce type de pathologies. Dans une première partie nous nous sommes intéressés à la polyglobulie de Vaquez. Cette pathologie présente une évolution lente et progressive, et elle est caractérisée par une mutation de JAK2 présente dans la lignée myéloïde chez plus de 95% des patients. Nous avons cherché à détecter la mutation dans les cellules NK de patients, puis, pour savoir si la mutation avait un effet sur les NK, nous avons exploré leurs fonctions in vitro. Nos résultats ont montré que, bien que la mutation soit présente dans les cellules NK, elle ne semble pas avoir d'impact sur les fonctions des cellules NK que nous avons pu tester. Nous en avons conclu que l'évolution de la polyglobulie de Vaquez en leucémie n'était peut-être pas due à une perte de fonction des NK mais plutôt à leur inhibition par l'environnement cellulaire.Dans une deuxième partie nous avons étudié la régulation des natural cytotoxicity receptors dans la leucémie aiguë myéloïde. D'apres des travaux antérieurs nous avons émis l'hypothèse que l'expression des trois NCR aurait une régulation commune s'effectuant au niveau de transcription de leurs gènes. Nos recherches bio-informatiques ainsi que notre expérimentation d'immunoprécipitation de la chromatine (Chip) montrent que le facteur de transcription ETS-1 semble être impliqué dans la régulation commune aux trois NCR. / The latest advances in blood disorders treatments lead to a better complete remission rate and a better survival rate after treatment. However, the risk of relapse remains high. Our project is included in the understanding of NK cells role in the development of these diseases.In a first part, we focused on polycythemia Vera for several reasons: the pathology has a slowly progressive disease, and it is characterized by the presence of JAK2 mutation for > 95% patients. We wanted to know if this mutation was found in NK cells from PV patients and what effects the mutation had on NK cells functions. Our results have shown that although the mutation was found in NK cells, it appears to have no impact on NK cells functions. We conclude that the evolution of PV to leukemia is not due to a loss of NK cell functions but to their inhibition by cellular environment.In a second part, we investigated the regulation of natural cytotoxicity receptors in acute myeloid leukemia because previous works have shown that NCR are weakly expressed in AML patients, that this down-regulation is acquired during evolution of AML and reversible after complete remission, ant that NCR weak expression is related to poor prognosis. We supposed that the expression of the three NCR has a common regulation at genes transcription level. Our bioinformatic researches and our experiment of chromatin immunoprecipitation show that ETS-1 transcription factor is a good candidate involved in the common regulation of the three NCR.

published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Early pregnancy is characterized by complex interactions between blood vessels, leukocytes, and conceptus-derived trophoblasts within the gestational uterus. Uterine Natural Killer (uNK) cells become the most abundant leukocyte during decidualization and produce a wide array of angiogenic factors, yet little is known regarding their early pregnancy functions. To characterize the role(s) of uNK cells, whole mount in situ immunohistochemistry of live early implant sites was performed. A timecourse examination of murine early pregnancy (virgin, and gd4.5-9.5) implantation sites was performed. Comparison of Gd6.5, 8.5 and 9.5 implant sites from BALB/c+/+ controls (BALB/c) and BALB/c-Rag2-/-Il2rg-/- (alymphoid) identified anomalies that result from the absence of lymphocytes. In alymphoid decidua basalis, mesometrial angiogenesis was widespread but pruning of nascent vessels within alymphoid decidua basalis was deficient. As early gestation progressed, vessels of alymphoid decidua basalis showed no evidence for remodeling. Alymphoid implantation sites showed ~24h delay in uterine lumen closure and embryonic development. To determine if uNK cells would normalize the anomalies observed in alymphoid implantation sites, adoptive cell transfer of NK+ B- T- marrow to alymphoid mice was performed. All of the above anomalies were reversed by adoptive transfer of NK+B-T- marrow. My results suggest that uNK cells support vascular growth and development which ensures the decidua can support the growing conceptus early in pregnancy prior to formation and function of the placenta. Human decidual NK cells may fill similar roles and be important targets for strategies designed to correct intra-uterine growth restriction. / Thesis (Master, Anatomy & Cell Biology) -- Queen's University, 2013-08-02 08:42:06.487

Department of Biochemistry, Faculty of Science, Charles University in Prague 2010 Structure and function of C-type lectin NK cell receptors studied by recombinant expression and protein crystallography Abstract of Ph.D. thesis Ondřej Vaněk Supervisor: Prof. RNDr. Karel Bezouška, DSc. Natural killer cells (NK cells) were found out for their ability to spontaneously kill certain allogeneic tumour cell lines, without any previous sensitization. NK cells are part of non- adaptive immune response with very short reaction time against pathogens such as viruses, intracellular bacteria, parasites, and they are responsible for elimination of certain tumour cells and thus they are able to fight against malignancy and formation of metastasis. Activity of NK cells is regulated by the balance between activation and inhibitory signals mediated by the NK cell surface receptors. From the structural point of view, the majority of NK cell surface receptors could be classified as the C-type lectin or immunoglobulin-like receptors. One of many C-type lectin subgroups are type II lymphocyte receptors that are expressed on the NK cell surface. This study had two main aims. The first one was to find suitable expression and purification systems for selected C-type lectin receptors of NK cells and the other one was to perform their...