1 |
Using the CRISPR/Cas9 system to understand the biology of natural killer cells and unleash their function in the tumour microenvironmentRojas, Eduardo January 2021 (has links)
NK cell based anti-tumour therapies demonstrate high efficacy in targeting hematological malignancies, however, treatments for advanced solid tumours face challenges. The immunosuppressive environment produced by tumours prevents NK cells from maintaining cytotoxic activity and reducing tumour burden. Enhancing NK cell activation is essential to improve their function against solid tumours. Genetic manipulation of primary NK cells with viral and non-viral methods has seen a drastic improvement in recent years. Lentiviral vectors are being used to generate CAR-NK cells ex vivo, while refinement of electroporation protocols has allowed for the generation of stable gene knockouts in primary NK cells. To establish and validate the generation of a stable knockout in primary human NK cells we focused on targeting the NCAM-1 (CD56) surface adhesion molecule. The high surface expression of CD56 in NK cells makes it a suitable target to establish the knockout protocol. Furthermore, despite its levels of expression being correlated to different functional phenotypes, the role of CD56 in NK cell function is not understood.
Here we have shown that current lentiviral transduction protocols are not viable methods to deliver the sgRNA/Cas9 system into primary NK cells. However, we found that nucleofection of the sgRNA/Cas9 complex into NK cells is an efficient method to generate gene knockouts. Using newly generated CD56KO NK cells we have shown that the expression of CD56 has no effect on NK cell cytotoxicity, cytokine production, proliferation, and in vivo tissue trafficking.
In parallel, we have also identified an intracellular pathway that is active in the tumour microenvironment and could inhibit NK cell function. Recent studies on the intracellular signaling of the E3 ubiquitin-protein ligase Cbl-b have highlighted its role in inhibiting NK cell tumour lytic and anti-metastatic activity. Immunosuppressive factors produced by tumours activate the Cbl-b pathway, leading to the targeted degradation of signaling proteins required for NK cell activation. We have shown that Cbl-b is upregulated in ex vivo expanded NK cells cultured with GAS6 or ovarian cancer ascites. Therefore, the generation of human primary Cbl-bKO NK cells could be a beneficial asset to enhance NK cell cancer immunotherapy. / Thesis / Master of Science (MSc)
|
2 |
Investigation into the Role of CBL-B in Leukemogenesis and MigrationBadger-Brown, Karla Michelle 15 September 2011 (has links)
CBL proteins are E3 ubiquitin ligases and adaptor proteins. The mammalian homologs – CBL, CBL-B and CBL-3 show broad tissue expression; accordingly, the CBL proteins play roles in multiple cell types. We have investigated the function of the CBL-B protein in hematopoietic cells and
fibroblasts.
The causative agent of chronic myeloid leukemia (CML) is BCR-ABL. This oncogenic fusion down-modulates CBL-B protein levels, suggesting that CBL-B regulates either the development or progression of CML. To assess the involvement of CBL-B in CML, bone marrow transduction and transplantation (BMT) studies were performed. Recipients of BCR-ABL-infected CBL-B(-/-) cells succumbed to a CML-like myeloproliferative disease with a longer latency than the wild-type recipients.
Peripheral blood white blood cell numbers were reduced, as were splenic weights. Yet despite the reduced leukemic burden, granulocyte numbers were amplified throughout the animals. As well, CBLB(-/-)
bone marrow (BM) cells possessed defective BM homing capabilities. From these results we
concluded that CBL-B negatively regulates granulopoiesis and that prolonged latency in our CBL-B(-/-) BMT animals was a function of perturbed homing.To develop an in vitro model to study CBL-B function we established mouse embryonic fibroblasts (MEFs) deficient in CBL-B expression. Transduction of the wild-type and CBL-B-deficient MEFs with BCR-ABL did not confer transformation; nevertheless, the role of CBL-B in fibroblasts was evaluated. The CBL-B(-/-) MEFs showed enhanced chemotactic migration toward serum in both Transwell migration and time-lapse video microscopy studies. The biochemical response to serum was
extensively evaluated leading to the development of a model. We predict that CBL-B deficiency either: (a) augments GRB2-associated binding protein 2 (GAB2) phosphorylation leading to enhanced
extracellular signal-regulated kinase (ERK) and protein kinase B (PKB / Akt) signaling, or (b) alleviates negative control of Vav3 resulting in stimulation of Rho effectors. In either case, our results reveal a
negative regulatory role for CBL-B in fibroblast migration.
The two studies detailed herein expand our knowledge of CBL-B function. They strongly
suggest that CBL-B can modulate granulocyte proliferation and point toward a role for CBL-B in the motility of numerous cell types.
|
3 |
Investigation into the Role of CBL-B in Leukemogenesis and MigrationBadger-Brown, Karla Michelle 15 September 2011 (has links)
CBL proteins are E3 ubiquitin ligases and adaptor proteins. The mammalian homologs – CBL, CBL-B and CBL-3 show broad tissue expression; accordingly, the CBL proteins play roles in multiple cell types. We have investigated the function of the CBL-B protein in hematopoietic cells and
fibroblasts.
The causative agent of chronic myeloid leukemia (CML) is BCR-ABL. This oncogenic fusion down-modulates CBL-B protein levels, suggesting that CBL-B regulates either the development or progression of CML. To assess the involvement of CBL-B in CML, bone marrow transduction and transplantation (BMT) studies were performed. Recipients of BCR-ABL-infected CBL-B(-/-) cells succumbed to a CML-like myeloproliferative disease with a longer latency than the wild-type recipients.
Peripheral blood white blood cell numbers were reduced, as were splenic weights. Yet despite the reduced leukemic burden, granulocyte numbers were amplified throughout the animals. As well, CBLB(-/-)
bone marrow (BM) cells possessed defective BM homing capabilities. From these results we
concluded that CBL-B negatively regulates granulopoiesis and that prolonged latency in our CBL-B(-/-) BMT animals was a function of perturbed homing.To develop an in vitro model to study CBL-B function we established mouse embryonic fibroblasts (MEFs) deficient in CBL-B expression. Transduction of the wild-type and CBL-B-deficient MEFs with BCR-ABL did not confer transformation; nevertheless, the role of CBL-B in fibroblasts was evaluated. The CBL-B(-/-) MEFs showed enhanced chemotactic migration toward serum in both Transwell migration and time-lapse video microscopy studies. The biochemical response to serum was
extensively evaluated leading to the development of a model. We predict that CBL-B deficiency either: (a) augments GRB2-associated binding protein 2 (GAB2) phosphorylation leading to enhanced
extracellular signal-regulated kinase (ERK) and protein kinase B (PKB / Akt) signaling, or (b) alleviates negative control of Vav3 resulting in stimulation of Rho effectors. In either case, our results reveal a
negative regulatory role for CBL-B in fibroblast migration.
The two studies detailed herein expand our knowledge of CBL-B function. They strongly
suggest that CBL-B can modulate granulocyte proliferation and point toward a role for CBL-B in the motility of numerous cell types.
|
4 |
The Role of CBL Family Proteins in Dendritic Cell Development, Homeostasis, and Functional QuiescenceTong, Haijun 03 1900 (has links)
Les cellules dendritiques sont des cellules du système immunitaire inné qui jouent un rôle
important dans la reconnaissance immunitaire contre les agents pathogènes étrangers. Elles
peuvent également prévenir les maladies auto-immunes à l'état basal. En raison de l'importance
des cellules dendritiques dans la régulation immunitaire, il est important de comprendre comment
le développement, l'état d'homéostasie et de quiescence des ces cellules sont contrôlées dans des
conditions physiologiques et pathologiques. Cette étude permettra non seulement de mieux
comprendre le contrôle de la régulation immunitaire, mais aussi de contribuer au développement
de nouvelles approches pour traiter les maladies infectieuses et auto-immunes, ainsi que les
cancers.
Notre laboratoire a montré que C-CBL et CBL-B, deux membres de la famille CBL des
ubiquitine ligases E3, jouent un rôle redondant dans la régulation négative du développement et
de l'activation des cellules T et B. En l'absence de CBL dans les cellules T ou B, les souris
développent des maladies auto-immunes sévères, indiquant que C-CBL et CBL-B jouent un rôle
dans le système auto-immun. Partant de ces observations, nous proposons que CBL-B et C-CBL
peuvent également jouer un rôle similaire dans le développement et la fonction des cellules
dendritiques. Pour étudier cette possibilité, nous avons généré une souris knockout de Cbl
spécifiques aux cellules dendritiques (dKO). Nous avons trouvé que cette mutation provoque une
modification de l'homéostasie d'un sous-ensemble des cellules dendritiques (DC), y compris une
augmentation marquée des CD8a+ cDCs et une réduction des pDC dans la rate. Cette modification
est causée par la prolifération accrue des CD8a+ cDCs. Dans les CD8a+ cDCs mutantes, les voies
de signalisation PKB et ERK sont constitutivement activées. Blocage de la signalisation de MTOR
par la rapamycine atténue de manière significative l'hyperprolifération des CD8a+ cDCs in vitro
et in vivo, indiquant que l'hyperactivation de MTOR est en partie responsable de l'augmentation
CD8a+ cDCs. Les protéines CBL contrôlent l'ubiquitination et la dégradation du récepteur FLT3,
suggérant que les protéines CBL contrôlent ainsi l'homéostasie de CD8a+ cDCs.
Outre ces effets sur le développement des cellules dendritiques, nous avons trouvé que les souris
Cbl dKO développent des inflammations sévères du foie et d'autres organes, caractérisées par une
infiltration massive de leucocytes et une activation importante des cellules lymphocytes T
périphériques. Les souris mutantes produisent des niveaux élevés de cytokines inflammatoires et de chimiokines, telles que le TNF-α, l'IL-6 et le CCL2. Les souris mutantes développent une
maladie inflammatoire du foie. L'ensemble de ces observations montrent que les protéines CBL
jouent un rôle essentiel dans le maintien de la quiescence immunitaire chez la souris. Puisque les
souris dKO Cbl développent principalement une inflammation sévère du foie, il serait intéressant
d'étudier si les voies contrôlées par les protéines CBL contribuent également au développement
d'une inflammation du foie chez l'homme. / Dendritic cells (DCs) are innate immune cells that play an important role in immune recognition
against foreign pathogens. They may also sense self-cues and prevent autoimmune diseases under
the steady-state. Given the importance of DCs in immune regulation, it is conceivable that
understanding how DCs development, homeostasis and functional quiescence are regulated under
physiological and pathological conditions will not only bring insight into our knowledge how
immune regulation is controlled but also some new approaches to treat infectious and autoimmune
diseases and even cancers.
Dr. Gu’s lab previously has shown that C-CBL and CBL-B, two members of the CBL family of
E3 ubiquitin ligases, play a redundant negative regulatory role in both T cells and B cells
development and activation. In the absence of CBL family of proteins in either T or B cells, mice
develop severe autoimmune diseases, indicating that C-CBL and CBL-B restrain immune system
against self. Based on these discoveries, we propose that C-CBL and CBL-B may also have a
similar regulatory role in DC development and function. To study this possibility, we have
generated DC-specific Cbl dKO mice. We have found that the Cbl dKO mutation results in an
altered homeostasis of DC subsets, including a marked increase of CD8a+ cDCs and reduction of
pDCs in the spleen (SP). This alteration is due to the enhanced proliferation of CD8a+ cDCs rather
than the preferential lineage commitment to CD8a+ cDCs. In the mutant CD8a+ cDCs, both the
PKB signaling pathway and ERK signaling pathways are constitutively activated. Blockage of
MTOR signaling by Rapamycin significantly attenuates the hyperproliferation of CD8a+ cDCs
both in vitro and in vivo, indicating that hyperactivation of MTOR is at least one of the reasons
leading to CD8a+ cDC expansion. CBL proteins regulate ubiquitination and degradation of FLT3.
Based on these results, we conclude that CBL proteins control CD8a+ cDC homeostasis through
promoting FLT3 ubiquitination and degradation.
In addition to the altered DC development, we have found that Cbl dKO mice develop severe
liver and other organ inflammation characterized by massive leukocytes infiltration and profound
peripheral T cell activation. Mutant mice produce high levels of inflammatory cytokines and
chemokines including TNF-a, IL-6, CCL2, etc. Most strikingly, the mutant mice develop a similar
liver inflammatory disease even in the absence of T and B cells. These findings together indicate
that CBL proteins play an essential role in the maintenance of immune quiescence in mice. Since
Cbl dKO mice mainly develop severe liver inflammation, it will be interesting to study whether
the pathways controlled by CBL proteins also contribute to the development of liver inflammation
in humans.
|
Page generated in 0.0158 seconds