In metozoans, most of the key events that lead to cell activation and inhibition are controlled by tyrosine phosphorylation. Extracellular signals are transmitted by membrane bound receptors, which have intrinsic kinase activity or themselves recruit intracellular kinases to specialised inhibitory or activating phosphorylation motifs. In this way, the pattern of kinase activation creates its own turnover and can rapidly generate amplified signals by positive feedback, or recruit inhibitory proteins to counteract the signals. This process of inhibition is also constitutive since it requires continuous counter-inhibition by phosphatases at the cell surface and intracellularly even in the absence of ligands. The absence of phosphatase activity results in unbridled protein phosphorylation and form this and other data it has been proposed that the triggering of the T cell receptor and other co-receptors may result simply by physical exclusion of the large phosphatases such as CD45 from the vicinity of the receptors. Superagonist monoclonal antibodies may work in a similar way, by binding receptors close to the plasma membrane and excluding extracellular phosphatases. The work described in this thesis seeks to discover if antibody superagonists can be generated against the T cell inhibitory cell surface receptor PD-1 and test if this approach can attenuate the immune response. Using in vitro assays of lymphocyte activation and a mouse model expressing human PD-1, this study characterises a series of anti-PD-1 antibodies and shows how patterns of inhibitory activity varying according to binding sites. The inhibitory effects of the anti-PD1 antibodies are seen in the humoral, cellular and transplant immune responses. Agonistic anti-PD1 antibodies induce regulatory T cells and may have role in suppression of autoimmune disease. The thesis suggests that superagonism may be harnessed clinically to dampen the immune response, through activation of inhibitory receptors.

Thesis (MScMedSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Background Individuals who are latently infected with Mycobacterium tuberculosis (M.tb) are able to quell the infection by balancing the innate and adaptive immune responses. Glucocorticoids (GCs) can affect this balance and can increase the risk of reactivation of TB. The three month injectable contraceptive medroxyprogesterone acetate (MPA) is widely used by women in developing countries, where TB is rife. MPA, unlike the two monthly contraceptive norethisterone enanthate (NET), possesses selective glucocorticoid activity, and could therefore alter immune responses to TB. Aims The aim of my investigation was to elucidate the immune modulatory effects of the synthetic progestins, MPA and NET, compared to the endogenous hormones, cortisol and progesterone, in Mycobacterium bovis Bacillus Calmette–Guérin (BCG) or anti-CD3 stimulated peripheral blood mononuclear cells (PBMC). I aim to determine the effects of MPA, NET, cortisol and progesterone on the receptor expression of glucocorticoid and various progesterone receptors. I investigate the effect of the above mentioned hormones on the downstream signalling cascades in the presence or absence of either BCG or anti-CD3. The overall immune modulation will be determined with regard to the cytokine production in PBMCs. Methods The presence of receptors for these steroid hormones in PBMCs was verified and BCG, anti-CD3 and hormone induced changes in receptor expression determined through RT-PCR. The impact of cortisol, MPA, NET and progesterone on BCG or anti-CD3 mediated activation of downstream signalling molecules were determined by Western blot as well as Luminex analysis. Results and Conclusion My results show that BCG and anti-CD3 mediated activation of the T cell receptor associated signalling molecules, Lck, ZAP-70, LAT was inhibited by the steroid hormones. Similarly several kinases including JNK, ERK and p38 and transcription factors including STAT3, STAT5 and CREB were differentially affected by the hormones. The inhibition of phosphorylation seen in the different signalling molecules indicated an inhibition of activation of downstream signalling cascades. To investigate the impact of the hormone induced changes in the signalling cascades on the expression of inflammatory and anti-inflammatory cytokines Luminex analysis was performed on the supernatant of the BCG and anti-CD3 stimulated PBMC cultures. Cortisol and MPA, but not NET and progesterone, significantly inhibited the secretion of IL-1α, IL-1β, IL-6, IL-10, TNF-α, IL-12 and IL-13. These results suggest that the immune suppressive effects of MPA are likely mediated through a combination of direct genomic GR action as well as through direct or indirect inhibition of several signalling molecules. The inhibition of the IFN-γ, IL-12, IL-1and IL-6 secretion by MPA could potentially increase the risk of susceptibility to TB in women using this contraceptive. Therefore the absence of glucocorticoid activity seen with NET could make this contraceptive a better choice for women in TB endemic areas. / AFRIKAANSE OPSOMMING: Agtergrond Individue wat latent met Mikobakterium tuberkulose (M.tb) geïnfekteer is, is in staat om die infeksie te onderdruk deur die ingebore en aanpasbare immuunrespons te balanseer. Glukokortikoïede (GCs) kan hierdie balans beïnvloed en kan die risiko van heraktivering van tuberkulose (TB) verhoog. Die drie maande inspuitbare voorbehoedmiddel medroksiprogestroon-asetaat (MPA) word algemeen gebruik deur vroue in ontwikkelende lande, waar TB volop is. MPA, in teenstelling met die twee maandelikse voorbehoedmiddel noretisteroon enantaat (NET), beskik selektiewe glukokortikoïed aktiwiteit, en kan dus die immuunrespons teenoor TB verander. Doelwitte Die doel van my studie was om die immuunregulerende effekte van die sintetiese progestiene, MPA en NET, toe te lig , in vergelyking met die endogene hormone, kortisol en progesteroon, in Mycobacterium bovis Bacillus Calmette - Guerin (BCG) of anti- CD3 gestimuleerde perifere bloed mononukleêre selle (PBMSe). Ek het beoog om die gevolge van MPA, NET, kortisol en progesteroon op die reseptor uitdrukking van glukokortikoïede en verskeie progesteroon reseptore te bepaal. Ek het ondersoek ingestel op die effek van die bogenoemde hormone op die sein transduksie in die teenwoordigheid of afwesigheid van óf BCG of anti-CD3. Die algehele immuun -modulasie sal bepaal word met betrekking tot die produksie van sitokiene in PBMSe . Metodes Die teenwoordigheid van reseptore vir die steroïedhormone in PBMSe is geverifieer en BCG en anti-CD3 en die veranderinge deurdie hormone in verband met die reseptor uitdrukking bepaal deur RT -PCR. Die impak van kortisol, MPA, NET en progesteroon op BCG of anti- CD3 aktivering van sein transduksie molekules is bepaal deur ‘Western blot’ asook Luminex analise. Resultate en gevolgtrekking My resultate toon dat BCG en anti-CD3 die aktivering van die T-sel reseptor wat verband hou met sein molekules , LCK , ZAP -70 , en LAT word geïnhibeer deur die steroïedhormone . Van die kinases insluitend JNK , ERK en p38 en transkripsie faktore, insluitend STAT3 , STAT5 en CREB is beïnvloed deur die hormone. Die inhibisie van fosforilering gesien in die verskillende sein molekules dui daarop aan dat 'n inhibisie van aktivering van sein transduksie. Die impak van die hormoon veroorsaak veranderinge in die sein transduksie met betrekking tot die uitdrukking van inflammatoriese en anti -inflammatoriese sitokiene Luminex analise is uitgevoer op die supernatant van die BCG en anti-CD3 gestimuleerde PBMS kulture. Kortisol en MPA, maar nie NET en progesteroon , het aansienlik die produksie van IL-1α , IL-1β , IL-6 , IL-10 , TNF-α , IL-12 en IL-13 geïnhibeer. Hierdie resultate dui daarop dat die immuunstelsel se onderdrukkende effekte van MPA is waarskynlik bemiddel deur 'n kombinasie van direkte genomiese GR interaksie sowel as deur direkte of indirekte inhibisie van verskeie sein molekules . Die inhibisie van die IFN-γ, IL-12, IL-1 en IL-6 sekresie deur MPA kan potensieel die risiko verhoog van vatbaarheid vir TB in vroue wat hierdie voorbehoedmiddel gebruik. Daarom oor die afwesigheid van glukokortikoïede aktiwiteit wat gesien is met NET, kan maak laat hierdie voorbehoedmiddel 'n beter keuse vir vroue in TB endemiese gebiede.

Embryonic stem cells (ESC) possess immune privileged properties and have the capacity to modulate immune activation. ESCs can persist across allogeneic immunological barriers, prevent lymphocyte proliferation in mixed lymphocyte reaction (MLR) assays and can promote graft acceptance. However, clinical application of live ESC to treat immunological disorders is not feasible as live ESC can form teratoma in-vivo. In order to harness these properties of ESCs without adverse risk to patients, we hypothesized that ESC derived extracts may retain immune modulatory properties of whole cells and therefore could be used to abrogate allo-immune responses. We found addition of ESC-extracts from human lines H1 and H9, significantly prevented T cell proliferation in allogeneic MLRs. These results were confirmed using murine J1 ESC line. In-vitro studies showed human ESC EXT were able to modulate maturation of human monocyte derived dendritic cells (DC) by suppressing up-regulation of important co-stimulatory and maturation markers CD80, HLA-DR and CD83. In addition, DCs educated in the presence of human ESC extracts significantly lost their ability to stimulate purified allogeneic T cells compared to control extract treated DCs. We also determined that ESC extracts have an independent effect on T cells. ESC extracts prevented T cell proliferation in response to anti CD3/CD28 stimulation. In MLRs, ESC derived factors significantly down-regulated IL-2 and IFN-γ expression, while up-regulating TGF-β and Foxp3 expression. Furthermore, lymphocytes and purified T cells activated with anti-CD3/CD28, ConA and PMA proliferated poorly in the presence of ESC derived factors, while proliferation in response to ionomycin was not affected. Western blot analysis indicated that ESC derived factors prevented PKC-θ phosphorylation without influencing total PKC-θ levels. Moreover, IκB-α degradation was abrogated, confirming absence of PKC-θ activity. Therefore, ESC extracts have potent immune suppressive properties and may have clinical applications in ameliorating transplant rejection and autoimmune conditions.

Embryonic stem cells (ESCs) possess hypo-immunogenic properties and have the capacity to modulate allogeneic immune response. ESCs have been shown to reduce immune activation in response to third party antigen presenting cells (APCs) in vitro and have the capacity to promote allograft survival in vivo. Clinical use of live ESCs to treat immunological disorders, however, risks teratoma or ectopic tissue formation. Accordingly, the way lab is studying the immune modulatory potentials of ESC-derived factors and recently, found that dendritic cells (DCs) treated with human ESC extracts are poor stimulators of purified allogeneic T cells compared to those DCs treated with vehicle or fibroblast extracts. In the present study, I found that ESC-derived extracts directly inhibit T cell proliferation and suppress their activation without inducing cell death. Furthermore, ESC extracts are able to suppress Th1 polarization while increasing the numbers of Foxp3+ CD4+ CD25+ regulatory T cells. Moreover, I found that a protein called Milk fat globule-EGF factor 8 (MFG-E8) appears to be highly expressed in ESCs. Importantly, neutralizing MFG-E8 substantially abrogated the immune suppressive effects of ESC extracts on T cell activation. These findings lead to future studies to further define specific immunomodulatory factors derived from ESCs for potential applications.

Embryonic stem cells (ESC) possess immune privileged properties and have the capacity to modulate immune activation. ESCs can persist across allogeneic immunological barriers, prevent lymphocyte proliferation in mixed lymphocyte reaction (MLR) assays and can promote graft acceptance. However, clinical application of live ESC to treat immunological disorders is not feasible as live ESC can form teratoma in-vivo. In order to harness these properties of ESCs without adverse risk to patients, we hypothesized that ESC derived extracts may retain immune modulatory properties of whole cells and therefore could be used to abrogate allo-immune responses. We found addition of ESC-extracts from human lines H1 and H9, significantly prevented T cell proliferation in allogeneic MLRs. These results were confirmed using murine J1 ESC line. In-vitro studies showed human ESC EXT were able to modulate maturation of human monocyte derived dendritic cells (DC) by suppressing up-regulation of important co-stimulatory and maturation markers CD80, HLA-DR and CD83. In addition, DCs educated in the presence of human ESC extracts significantly lost their ability to stimulate purified allogeneic T cells compared to control extract treated DCs. We also determined that ESC extracts have an independent effect on T cells. ESC extracts prevented T cell proliferation in response to anti CD3/CD28 stimulation. In MLRs, ESC derived factors significantly down-regulated IL-2 and IFN-γ expression, while up-regulating TGF-β and Foxp3 expression. Furthermore, lymphocytes and purified T cells activated with anti-CD3/CD28, ConA and PMA proliferated poorly in the presence of ESC derived factors, while proliferation in response to ionomycin was not affected. Western blot analysis indicated that ESC derived factors prevented PKC-θ phosphorylation without influencing total PKC-θ levels. Moreover, IκB-α degradation was abrogated, confirming absence of PKC-θ activity. Therefore, ESC extracts have potent immune suppressive properties and may have clinical applications in ameliorating transplant rejection and autoimmune conditions.

Embryonic stem cells (ESC) possess immune privileged properties and have the capacity to modulate immune activation. ESCs can persist across allogeneic immunological barriers, prevent lymphocyte proliferation in mixed lymphocyte reaction (MLR) assays and can promote graft acceptance. However, clinical application of live ESC to treat immunological disorders is not feasible as live ESC can form teratoma in-vivo. In order to harness these properties of ESCs without adverse risk to patients, we hypothesized that ESC derived extracts may retain immune modulatory properties of whole cells and therefore could be used to abrogate allo-immune responses. We found addition of ESC-extracts from human lines H1 and H9, significantly prevented T cell proliferation in allogeneic MLRs. These results were confirmed using murine J1 ESC line. In-vitro studies showed human ESC EXT were able to modulate maturation of human monocyte derived dendritic cells (DC) by suppressing up-regulation of important co-stimulatory and maturation markers CD80, HLA-DR and CD83. In addition, DCs educated in the presence of human ESC extracts significantly lost their ability to stimulate purified allogeneic T cells compared to control extract treated DCs. We also determined that ESC extracts have an independent effect on T cells. ESC extracts prevented T cell proliferation in response to anti CD3/CD28 stimulation. In MLRs, ESC derived factors significantly down-regulated IL-2 and IFN-γ expression, while up-regulating TGF-β and Foxp3 expression. Furthermore, lymphocytes and purified T cells activated with anti-CD3/CD28, ConA and PMA proliferated poorly in the presence of ESC derived factors, while proliferation in response to ionomycin was not affected. Western blot analysis indicated that ESC derived factors prevented PKC-θ phosphorylation without influencing total PKC-θ levels. Moreover, IκB-α degradation was abrogated, confirming absence of PKC-θ activity. Therefore, ESC extracts have potent immune suppressive properties and may have clinical applications in ameliorating transplant rejection and autoimmune conditions.

Embryonic stem cells (ESCs) possess hypo-immunogenic properties and have the capacity to modulate allogeneic immune response. ESCs have been shown to reduce immune activation in response to third party antigen presenting cells (APCs) in vitro and have the capacity to promote allograft survival in vivo. Clinical use of live ESCs to treat immunological disorders, however, risks teratoma or ectopic tissue formation. Accordingly, the way lab is studying the immune modulatory potentials of ESC-derived factors and recently, found that dendritic cells (DCs) treated with human ESC extracts are poor stimulators of purified allogeneic T cells compared to those DCs treated with vehicle or fibroblast extracts. In the present study, I found that ESC-derived extracts directly inhibit T cell proliferation and suppress their activation without inducing cell death. Furthermore, ESC extracts are able to suppress Th1 polarization while increasing the numbers of Foxp3+ CD4+ CD25+ regulatory T cells. Moreover, I found that a protein called Milk fat globule-EGF factor 8 (MFG-E8) appears to be highly expressed in ESCs. Importantly, neutralizing MFG-E8 substantially abrogated the immune suppressive effects of ESC extracts on T cell activation. These findings lead to future studies to further define specific immunomodulatory factors derived from ESCs for potential applications.

abstract: Intracerebral hemorrhage (ICH) is a devastating type of acute brain injury with high mortality and disability. Acute brain injury swiftly alters the immune reactivity within and outside the brain; however, the mechanisms and influence on neurological outcome remains largely unknown. My dissertation investigated how ICH triggers focal and systemic immune responses and their impact hemorrhagic brain injury. At the focal level, a significant upregulation of interleukin (IL)-15 was identified in astrocytes of brain sections from ICH patients. A transgenic mouse line where the astrocytic IL-15 expression is controlled by a glial fibrillary acidic protein promoter (GFAP-IL-15tg) was generated to investigate its role in ICH. Astrocyte-targeted expression of IL-15 exacerbated brain edema and neurological deficits following ICH. Aggravated ICH injury was accompanied by an accumulation of pro-inflammatory microglia proximal to astrocytes in perihematomal tissues, microglial depletion attenuated the augmented ICH injury in GFAP-IL-15tg mice. These findings suggest that IL-15 mediates the crosstalk between astrocytes and microglia, which worsens ICH injury.Systemic immune response was investigated by leveraging the novel method of obtaining and analyzing bone marrow cells from the cranial bone flaps of ICH patients. A swift increase of hematopoietic stem cell (HSCs) population in the bone marrow was identified, along with a shift towards the myeloid cell lineage. Human findings were mirrored in an ICH mouse model. Fate mapping these HSCs revealed increased genesis of Ly6Clow monocytes in the bone marrow, which transmigrate into the hemorrhagic brain and give rise to alternative activation marker bearing macrophage. Blockade of the β3-adrenergic receptor or inhibition of Cdc42 abolished ICH-induced myeloid bias of HSCs. Importantly, mirabegron, a Food and Drug Administration-approved β3 adrenergic receptor agonist, and a Cdc42 activator, IL-3, enhanced bone marrow generation of Ly6Clow monocytes and improved recovery. These results suggest that brain injury modulates HSC lineage destination to curb distal brain inflammation, implicating the bone marrow as a unique niche for self-protective neuroimmune interactions. Together, these results demonstrate how acute brain injury exerts a profound yet distinct effect on immune responses within and outside the brain and sheds new light on neuroimmune interactions with potential clinical implications. / Dissertation/Thesis / Doctoral Dissertation Neuroscience 2020

HCV is remarkable at disrupting human immunity to establish chronic infection. The accumulation of Treg cells at the site of infection and upregulation of inhibitory signaling pathways (such as T-cell Ig and mucin domain protein-3 (Tim-3) and galectin-9 (Gal-9)) play pivotal roles in suppressing antiviral effector T (Teff) cells that are essential for viral clearance. While Tim-3/Gal-9 interactions have been shown to negatively regulate Teff cells, their role in regulating Treg cells is poorly understood. To explore how Tim-3/Gal-9 interactions regulate HCV-mediated Treg-cell development, here we provide pilot data showing that HCV-infected human hepatocytes express higher levels of Gal-9 and TGF-β, and upregulate Tim-3 expression and regulatory cytokines TGF-β/IL-10 in co-cultured human CD4+ T cells, driving conventional CD4+ T cells into CD25+Foxp3+ Treg cells. Additionally, recombinant Gal-9 protein can transform TCR-activated CD4+ T cells into Foxp3+ Treg cells in a dose-dependent manner. Importantly, blocking Tim-3/Gal-9 ligations abrogates HCV-mediated Treg-cell induction by HCV-infected hepatocytes, suggesting that Tim-3/Gal-9 interactions may regulate human Foxp3+ Treg-cell development and function during HCV infection.

HCV is remarkable at disrupting human immunity to establish chronic infection. The accumulation of Treg cells at the site of infection and upregulation of inhibitory signaling pathways (such as T-cell Ig and mucin domain protein-3 (Tim-3) and galectin-9 (Gal-9)) play pivotal roles in suppressing antiviral effector T (Teff) cells that are essential for viral clearance. While Tim-3/Gal-9 interactions have been shown to negatively regulate Teff cells, their role in regulating Treg cells is poorly understood. To explore how Tim-3/Gal-9 interactions regulate HCV-mediated Treg-cell development, here we provide pilot data showing that HCV-infected human hepatocytes express higher levels of Gal-9 and TGF-β, and upregulate Tim-3 expression and regulatory cytokines TGF-β/IL-10 in co-cultured human CD4+ T cells, driving conventional CD4+ T cells into CD25+Foxp3+ Treg cells. Additionally, recombinant Gal-9 protein can transform TCR-activated CD4+ T cells into Foxp3+ Treg cells in a dose-dependent manner. Importantly, blocking Tim-3/Gal-9 ligations abrogates HCV-mediated Treg-cell induction by HCV-infected hepatocytes, suggesting that Tim-3/Gal-9 interactions may regulate human Foxp3+ Treg-cell development and function during HCV infection.