A RasGAP SH3 Peptide Aptamer Inhibits RasGAP-Aurora Interaction and Induces Caspase-Independent Tumor Cell Death

The Ras GTPase-activating protein RasGAP catalyzes the conversion of active GTP-bound Ras into inactive GDP-bound Ras. However, RasGAP also acts as a positive effector of Ras and exerts an anti-apoptotic activity that is independent of its GAP function and that involves its SH3 (Src homology) domain. We used a combinatorial peptide aptamer approach to select a collection of RasGAP SH3 specific ligands. We mapped the peptide aptamer binding sites by performing yeast two-hybrid mating assays against a panel of RasGAP SH3 mutants. We examined the biological activity of a peptide aptamer targeting a pocket delineated by residues D295/7, L313 and W317. This aptamer shows a caspase-independent cytotoxic activity on tumor cell lines. It disrupts the interaction between RasGAP and Aurora B kinase. This work identifies the above-mentioned pocket as an interesting therapeutic target to pursue and points its cognate peptide aptamer as a promising guide to discover RasGAP small-molecule drug candidates

The role of inducible costimulator-mediated phosphoinositide 3-kinase activation in the differentiation and function of follicular helper T cells

Antibodies are crucial components of the adaptive immune arsenal against invading pathogens. Production of high-affinity class-switched antibodies relies on follicular helper T (Tfh) cells, a distinct subset of CD4 helper T cells that migrate into B cell follicles and promote B cell differentiation into plasma cells during germinal center (GC) reactions. The CD28-like costimulatory receptor Inducible Costimulator (ICOS) is expressed on the surface of activated T cells and is crucial for the generation of Tfh cells in mice and humans, but the molecular mechanisms remained unknown. ICOS had been known as a potent activator of phosphoinositide 3-kinase (PI3K), but the role of ICOS-mediated PI3K activation in T cells has been poorly understood. The work presented here is a compilation of two studies that highlight the unique role of PI3K in ICOS-mediated Tfh cell differentiation and function. In the first study, presented in Chapter II, I analyzed a knock-in strain of mice possessing a point mutation in the cytoplasmic tail of ICOS that prevents binding of PI3K (ICOS-YF). I show that ICOS-mediated PI3K activation is crucial for the generation of Tfh cells, and in turn, GC formation, antibody class-switch, and affinity maturation. The ICOS-PI3K axis was crucial for the potentiation of T cell receptor (TCR)-mediated expression of IL-21 and IL-4, key cytokines involved in T cell-mediated B cell help. I also show data that strongly suggests that ICOS and CD28 have differential roles in the multistep process of Tfh cell differentiation, where CD28 is mainly involved in the early expansion of CD4 T cells through non-PI3K signaling mechanisms, while ICOS is involved in the later stages of Tfh cell differentiation in a PI3K-dependent manner. In the study presented in Chapter III, I show that ICOS costimulation enhances TCR-mediated activation of the key translation mediators 4E-BP1 and S6K, in a manner dependent on PI3K. Consistently, I show that the ICOS-PI3K axis enhances the formation of polysomes on IL-4 mRNA. Using an in vitro T-B cell co-culture system, I provide evidence that ICOS mutant CD4 T cells have impaired ability to induce B cell differentiation due to a limited production of IL-4. These findings suggest that ICOS-PI3K signaling facilitates targeted delivery of IL-4 from helper T cells to cognate B cells during T cell-B cell interactions in the GC. Thus, I demonstrate that PI3K is a key downstream signaling component in ICOS signaling during Tfh cell generation. I also show that ICOS-PI3K signaling can alter translational efficiency of pre-existing mRNAs suggesting ICOS' potential role in regulating the function of Tfh cells.Les anticorps sont des composantes cruciales de l'arsenal que le système immunitaire adaptatif utilise contre les pathogènes invasifs. La production d'anticorps de haute-affinité réarrangés par commutation isotypique nécessite l'apport des lymphocytes T auxiliaires folliculaires (Tfh), un sous-type de lymphocytes T auxiliaires CD4+ qui migrent dans les follicules nodules lymphatiques et y promeuvent la différentiation des cellules B en cellules plasmatiques, le tout durant les réactions du centre germinatif (GC). Le récepteur de costimulation de type CD28 Costimulateur Inductible (ICOS) est exprimé sur la surface des cellules T activées et joue un rôle critique dans la génération de cellules Tfh autant chez la souris que l'humain, cependant les mécanismes moléculaires demeurent inconnus. Jusqu'à présent, ICOS était reconnu comme un puissant activateur de la phosphatidyl inositol-3 kinase (PI3K), mais le rôle de l'activation de PI3K médiée par ICOS dans les cellules T demeure mal compris. Le travail présenté dans cette thèse retrace deux études qui décrivent le rôle unique de PI3K dans la fonction et la différentiation des cellules Tfh médiées par ICOS. Dans la première étude, présenté dans le Chapitre II, j'ai analysé une ligné de souris 'knock-in' possédant une mutation ponctuelle dans la région cytoplasmique de ICOS empêchant ainsi la liaison de PI3K (ICOS-YF). Je démontre que l'activation de PI3K médiée par ICOS demeure cruciale pour la génération de cellules Tfh, ainsi qu'en conséquence la formation des GC, la commutation isotypique d'anticorps et la maturation d'affinité. L'axe ICOS-PI3K s'avère critique pour la potentialisation de l'expression médiée par le récepteur de cellules T (TCR) de IL-21 et IL-4, des cytokines clés impliquées dans l'aide aux cellules B médiée par les cellules T. J'illustre également des résultats qui prouvent que ICOS et CD28 exercent des rôles distinct dans le processus complexe de la différentiation des cellules Tfh, où CD28 est principalement impliqué dans l'expansion précoce des cellules T CD4+ par l'entremise de mécanismes de signalisation indépendants de PI3K, tandis que ICOS s'engage plutôt de façon PI3K-dépendante dans les étapes tardives de la différentiation des cellules Tfh. Dans la seconde étude au Chapitre III, je révèle que la costimulation par ICOS augmente l'activation médiée par le TCR de médiateurs clés de la traduction de 4E-BP1 ainsi que S6K de façon dépendante à PI3K. De façon cohérente, je démontre que l'axe ICOS-PI3K augmente la formation de polysomes sur l'ARN messager d'IL-4. En utilisant un système in-vitro de co-culture de cellules T et B, je fourni des preuves que les cellules T CD4+ mutantes pour ICOS ont une capacité détérioré d'induire la différentiation de cellules B due à une production limitée d'IL-4. Ces découvertes suggèrent que la signalisation par ICOS-PI3K facilite l'acheminement dirigé d'IL-4 d'une cellule T auxiliaire à une cellule B apparentée durant une interaction entre les deux types cellulaires dans le GC. En conclusion, je démontre que PI3K est une composante de signalisation clé en aval de la signalisation par ICOS durant la génération de Tfh. De plus, je prouve que la voix ICOS-PI3K peut modifier l'efficacité de traduction d'ARN messager préexistant suggérant un rôle potentiel d'ICOS dans la régulation de la fonction des cellules Tfh

Anti-chlamydial Th17 responses are controlled by the inducible costimulator partially through phosphoinositide 3-kinase signaling.

We previously showed that mice deficient in the Inducible Costimulator ligand (ICOSL-KO) develop more severe disease and lung pathology with delayed bacterial clearance upon respiratory infection of Chlamydia muridarum. Importantly, the exacerbation of disease in ICOSL-KO mice was seen despite heightened IFN-γ/Th1 responses, the major defense mechanisms against Chlamydia. To gain insight into the mechanism of ICOS function in this model, we presently analyzed anti-Chlamydia immune responses in mice lacking the entire ICOS (ICOS-KO) versus knock-in mice expressing a mutant ICOS (ICOS-Y181F) that has selectively lost the ability to activate phosphoinositide 3-kinase (PI3K). Like ICOSL-KO mice, ICOS-KO mice showed worse disease with elevated IFN-γ/Th1 responses compared to wild-type (WT) mice. ICOS-Y181F mice developed much milder disease compared to ICOS-KO mice, yet they were still not fully protected to the WT level. This partial protection in ICOS-Y181F mice could not be explained by the magnitude of IFN-γ/Th1 responses since these mice developed a similar level of IFN-γ response compared to WT mice. It was rather IL-17/Th17 responses that reflected disease severity: IL-17/Th17 response was partially impaired in ICOS-Y181F mice compared to WT, but was substantially stronger than that of ICOS-KO mice. Consistently, we found that both polarization and expansion of Th17 cells were partially impaired in ICOS-Y181F CD4 T cells, and was further reduced in ICOS-KO CD4 T cells in vitro. Our results indicate that once the IFN-γ/Th1 response is above a threshold level, the IL-17/Th17 response becomes a limiting factor in controlling Chlamydia lung infection, and that ICOS plays an important role in promoting Th17 responses in part through the activation of PI3K

ICOS-KO cells but not ICOS-YF cells show an elevated IFN-γ production compared to WT cells upon restimulation.

<p>Single cell suspensions of splenocytes or lung mononuclear cells were prepared at day 14 post-infection and were restimulated <i>in vitro</i> as described in <i>Materials and Methods</i>. The amounts of IFN-γ in culture supernatants were measured by ELISA. Data are presented as mean ± SD (7 ICOS-WT, 4 ICOS-KO, and 7 ICOS-YF mice) from one representative experiment out of three independent experiments with consistent results. * <i>p</i><0.05, *** <i>p</i><0.001.</p

Reduced levels of IL-17 production in ICOS-KO and ICOS-YF cells upon restimulation correspond to disease severity.

<p>Single cell suspensions of splenocytes or lung mononuclear cells were prepared at day 14 post-infection and were restimulated <i>in vitro</i> as described in <i>Materials and Methods</i>. The amounts of IL-17 in culture supernatants were measured by ELISA. Data are presented as mean ± SD (7 ICOS-WT, 4 ICOS-KO, and 7 ICOS-YF mice) from one representative experiment out of three independent experiments with consistent results. * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001.</p

Severity of lung inflammation corresponds to the level of bacterial load.

<p>Lungs were isolated at day 14 post-infection and processed to stain with hematoxylin and eosin as described in <i>Materials and Methods</i>. <b>A</b> Representative images of the lung tissues were shown at low (20X) and high (100X) magnification. <b>B</b> and <b>C</b> Semi-quantitative analysis of lung inflammation. Slides were examined by a blinded pathologist and the inflammatory grades (<b>B</b>) and the composition of immune cell infiltrates (<b>C</b>) were analyzed as described in <i>Materials and Methods</i>. Data represent mean ± SD of 4 slides from 4 mice per group. * <i>p</i><0.05, *** <i>p</i><0.001.</p

Reduced Th17 responses in ICOS-KO and ICOS-YF mice correspond to disease severity.

<p>Single cell suspensions of spleen and lung mononuclear cells of WT, ICOS-KO and ICOS-YF mice were prepared at day 14 post-infection and the IL-17 expressing CD4 T cells were detected by intracellular cytokine staining as described in <i>Materials and Methods</i>. Representative flow cytometric plots (<b>A</b>) and a summary of the results (<b>B</b>) are shown. Data are presented as mean ± SD (7 ICOS-WT, 4 ICOS-KO, and 7 ICOS-YF mice) from one representative experiment out of three independent experiments with consistent results. * <i>p</i><0.05, ** <i>p</i><0.01.</p

ICOS-YF mice display partially impaired ability to control <i>Chlamydia</i> infection.

<p>Three groups of mice, ICOS-WT, KO, and YF were intranasally infected with 1000 IFUs of <i>Chlamydia</i> and monitored daily for 14 days. <b>A</b> Time course of body weight change. The original body weights of the three groups were similar and the body weight changes were plotted as percentage of the original weight. Asterisks (*) on KO line indicate statistical significance of KO verse WT, while asterisks on YF line indicate statistical significance of YF vs WT. Pounds (#) on YF line indicate statistical significance of KO vs YF. <b>B</b> Bacterial burdens in the lung. Mice were euthanized at day 14 post-infection and lungs were aseptically collected and processed to assess bacterial growth <i>in vivo</i> as described in <i>Materials and Methods</i>. Data are presented as mean ± SD (7 ICOS-WT, 4 ICOS-KO, and 7 ICOS-YF mice) from one representative experiment out of three independent experiments with consistent results. * or # <i>p</i><0.05, ** or ## <i>p</i><0.01, *** or ### <i>p</i><0.001.</p

IFN-γ producing T cells in the spleen increased in ICOS-KO mice but marginally decreased in ICOS-YF mice.

<p>Splenocytes were prepared at day 14 post-infection and the IFNγ producing cells were quantified by intracellular cytokine staining as described in <i>Materials and Methods</i>. Representative flow cytometric plots (<b>A</b>) and a summary of the results (<b>B</b>) are shown. Data are presented as mean ± SD (7 ICOS-WT, 4 ICOS-KO, and 7 ICOS-YF mice) from one representative experiment out of three independent experiments with consistent results. * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001.</p