Rozprawy doktorskie na temat „Intratumoral CD8 T cells”

CD8+ T cells play a crucial role in controlling liver tumours, such as hepatocellular carcinoma (HCC) however we have only limited knowledge of the precise dynamics of their interactions with hepatic parenchymal and non-parenchymal cells at the single-cell level. Previous work from our laboratory, demonstrated that in the context of HBV-expressing hepatocytes circulating effector CD8+ T cells (Teff) perform their immune surveillance function recognizing the antigen and kill virus-expressing hepatocytes extending cytoplasmic protrusions through endothelial fenestrations while still within liver sinusoids. Here we dissected whether similar or different mechanisms govern the capacity of Teff to home, migrate, recognize the antigen, and exert effector function within HCC. The first effort to dissect the project was the establishment of a new murine model of spontaneous HCC in which just the transformed hepatocytes express a nominal antigen, the oncogene SV40 large T antigen (TAg) and a fluorescent protein. We were able to obtain mice that develop spontaneous HCC lesions, highly proliferating and spread in a normal liver parenchyma. After the adoptive transfer of in vitro differentiated TAg-specific Teff in tumor-bearing mice, we observed that just some mice respond to the cytotoxic activity of the transferred cells, eliminating partially or completely the tumor, while in other mice the adoptive transferred cells have no beneficial effect on the tumor elimination. Thus, using a mathematical approach, we managed to pick the lesion volume as the fundamental parameter to predict the fate of each single HCC lesion: we called “responders” (R) the HCC lesions that are responsive to the cytotoxic activity of the TAg-specific Teff, with a single lesion volume <10 mm3, while we called “non-responders” (NR) the lesions that are not responding and have a single HCC lesion volume > 100mm3. We then studied the determinants that confer the therapeutic activity to the TAg-specific Teff in R and the ones that dampen the therapeutic activity in the NR lesions. Our main finding was that the vessel phenotype was completely different in R and NR. Our data support the hypothesis that some anatomic, hemodynamic, and environmental features acquired by each individual HCC lesion, during their growth, can influence their responsiveness to the Teff killing. The innovative nature of our work will elucidate new mechanisms whereby Teff fail to exert their immune function and cytotoxic activity in tumorigenic liver.
Le cellule T CD8+ giocano un ruolo cruciale nel controllo dei tumori del fegato come l’epatocarcinoma, tuttavia, abbiamo una conoscenza limitata delle precise interazioni a livello di singola cellula tra le cellule T CD8+ e le cellule parenchimali e non parenchimali del fegato. Precedenti studi del nostro laboratorio hanno dimostrato che, nel contesto in cui gli epatociti esprimono HBV, le cellule circolanti T CD8+ effettrici (Teff) svolgono l’attività di immuno sorveglianza riconoscendo l’antigene ed esercitando un’attività citotossica verso gli epatociti che esprimono il virus. E’ stato infatti dimostrato che queste cellule sono capaci di estendere protrusioni citoplasmatiche attraverso le fenestrature delle cellule endoteliali rimanendo all’interno del lume sinusoidale. In questo progetto studiamo quindi se un meccanismo simile è coinvolto nella capacità delle Teff di migrare, riconoscere l’antigene e svolgere le proprie funzioni effettrici nel contesto di epatocarcinoma. Il primo obbiettivo di questo studio è stato quello di creare un nuovo modello di epatocarcinoma spontaneo in cui solo le cellule neoplastiche esprimessero sia dei marker fluorescenti (ZsGreen e TdTomato) che l’oncogene SV40 large T-antigen (TAg). Siamo riusciti ad ottenere dei topi che sviluppassero epatocarcinomi spontanei ed altamente proliferanti sparsi in tutto il parenchima sano del fegato. Dopo aver differenziato in vitro le Teff specifiche per l’antigene TAg (TCR-I Teff), abbiamo trasferito queste cellule in topi in cui era presente il tumore e abbiamo osservato che solo alcuni topi rispondevano totalmente o parzialmente all’attività citotossica delle cellule trasferite, le quali erano in grado di eliminare il tumore. Questo effetto era correlato alla dimensione di ogni singola lesione tumorale. Infatti, usando un approccio matematico multi-parametrico abbiamo scoperto che il trasferimento delle cellule TCR-I Teff era efficace solo in lesioni neoformate con un volume < 10 mm3 (lesioni responder [R]). Invece, quando il volume della lesione era > 100 mm3, il tumore non rispondeva alla terapia cellulare e continuava a crescere (lesioni non-responder [NR]). Abbiamo quindi deciso di studiare i fattori in grado di conferire alle nostre cellule effettrici un’attività terapeutica nei R e quelli in grado di frenare la loro efficacia nelle lesioni NR. Il principale risultato che abbiamo ottenuto riguarda la differenza del fenotipo della vascolatura tra lesioni R e NR. I nostri dati supportano infatti l’ipotesi che alcune caratteristiche anatomiche, emodinamiche e ambientali acquisite dai tumori durante la loro crescita possano influenzarne la responsività alla terapia delle Teff. La natura innovativa del nostro lavoro chiarirà nuovi meccanismi con cui le Teff effettuano immuno sorveglianza ed esercitano le loro funzioni effettrici nel contesto di tumore epatico.

La présence de lymphocytes T (LT) CD8 dans le microenvironnement tumoral (TME) corrèle avec un bon pronostic dans de nombreux types de cancers solides. En périphérie, les LT régulateurs (Treg) jouent un rôle majeur dans le maintien d’une homéostasie immunitaire et empêchent le développement de pathologies auto-immunes. Néanmoins, dans le TME, les Treg (TA-Treg) ont un impact pronostic défavorable en inhibant la réponse immunitaire antitumorale. Sur le plan thérapeutique, il est indispensable d’éliminer ces TA-Treg ou leur fonction pour restaurer une réponse immunitaire antitumorale efficace. Pour cela, il reste important d’identifier des molécules membranaires permettant le ciblage sélectif de ces TA-Treg sans affecter les Treg périphériques pour éviter toute réaction auto-immune. L’analyse de données publiques de scRNA-seq comparant les LT (Treg, CD8, CD4) de tumeur, tissu sain et sang, a permis d’identifier l’expression sélective de CD177 par une population de TA-Treg dans plusieurs tumeurs solides. Si cette glycoprotéine est impliquée dans l’extravasation et la survie des neutrophiles, son rôle sur les Treg n’a été que peu décrit hormis dans quelques études confirmant l’expression de CD177 sur les TA-Treg dans plusieurs types tumoraux et montrant un effet suppresseur de ces TA-Treg CD177+ dans des cocultures avec des LT CD4 naïfs. Néanmoins, la caractérisation phénotypique et fonctionnelle de ces Treg reste peu explorée. CD177 interagit avec PECAM-1 qui est impliqué dans la transmigration des LT par interaction homophilique des domaines extracellulaires distaux immunoglobuline-like (IgD1/D2) avec les cellules endothéliales. De plus, il a été décrit que l’interaction avec le IgD6 extracellulaire de PECAM-1, zone de liaison de CD177, transmet un signal négatif via les motifs intracellulaires inhibiteurs (ITIM) et le recrutement de SHP2 qui bloque la signalisation TCR et la prolifération des LT. La réanalyse de données publiques de scRNA-seq de LT intra-tumoraux montre la restriction de l’expression de PECAM1 à des clusters de LT CD8 effecteurs mémoires suggérant qu’ils pourraient être la cible de la fonction immunosuppressive des Treg CD177+ dans le TME. Ainsi, dans l’objectif d’identifier un mécanisme de suppression des Treg spécifique des LT CD8 effecteurs dans l’environnement tumoral il est important de caractériser de manière approfondie ces TA-Treg CD177+ et d’identifier leurs interactions avec les LT CD8 PECAM-1+ dans le TME et leur impact sur la fonction de ces LT CD8. Ce travail de thèse a permis de démontrer, dans plusieurs types tumoraux, que CD177 identifie une population de Treg spécifiques de la tumeur, avec un phénotype activé. PECAM-1, la cible de CD177, est exprimé dans le TME par des LT CD8 effecteurs polyfonctionnels (GzmK, IFNү, TNFα), à forte capacité de prolifération. In situ sur coupe de tumeurs, des analyses de multi-immunofluorescence ont montré la colocalisation des Treg CD177+ et des LT CD8 PECAM-1+ au niveau du stroma tumoral, suggérant un lien entre ces deux populations. Par ailleurs, l’engagement de PECAM-1 IgD6, domaine de liaison de CD177, réduit l’activation et les fonctions des LT CD8 PECAM-1+ induites par le signal TCR en diminuant pZAP-70 et la sécrétion d’IFNү. Enfin, des premiers résultats sur tumeur ont montré que la culture de LT CD8 avec des TA-Treg CD177+ diminue la prolifération et la sécrétion d’IFNγ par les LT CD8 PECAM-1+ et l’ajout d’un anti-CD177 permet de lever en partie cette inhibition, suggérant le rôle de l’axe [CD177/PECAM-1] dans l’inhibition des LT CD8 PECAM-1+ par les TA-Treg CD177+. L’interaction [CD177/PECAM-1] représente la première mise en évidence d’un couple récepteur/ligand membranaire impliqué dans l’inhibition sélective des LT CD8 effecteurs par les TA-Treg dans le TME et CD177 apparait comme une cible prometteuse pour lever spécifiquement la suppression médiée par les Treg dans le TME sans altérer ceux de la périphérie
The presence of CD8 T cells in the tumor microenvironment (TME) correlates with good prognosis in many types of solid cancers. In the periphery, regulatory T cells (Treg) play a major role in maintaining immune homeostasis and preventing the development of autoimmune pathologies. However, in the TME, Treg (TA-Treg) have an unfavorable prognostic impact by inhibiting the anti-tumor immune response. Therapeutically, it is essential to eliminate these TA-Treg or their function to restore an effective anti-tumor immune response. For this, it remains important to identify membrane molecules allowing the selective targeting of these TA-Treg without affecting the Treg present in the periphery to avoid any autoimmune reaction. The analysis of public scRNA-seq data comparing T cells (Treg, CD8, CD4) from tumor, healthy tissue and blood, made it possible to identify the selective expression of CD177 by a subpopulation of TA-Treg in different solid tumors. If this glycoprotein is known for its involvement in the extravasation and survival of neutrophils, its role on Treg has been little described except in a few studies confirming the expression of CD177 on TA-Treg of several types of tumors and showing a suppressive impact of CD177+ TA-Treg in cocultures with naïve CD4 T cells. However, the phenotypic and functional characterization of these Treg remains little explored. CD177 interacts with PECAM-1 which is involved in T cells transmigration through homophilic interaction of distal extracellular immunoglobulin-like domains (IgD1/D2) with endothelial cells. Furthermore, it has been described that interaction with extracellular PECAM-1 IgD6, CD177 binding site, transmits a negative signal via inhibitory intracellular motifs (ITIM) and recruitment of SHP2 which blocks TCR signaling and the proliferation of T cells. Reanalysis of public scRNA-seq data from intra-tumoral T cells shows the restriction of PECAM1 expression to clusters of memory effector CD8 T cells suggesting that they could be the target of the immunosuppressive function of CD177+ Treg in the TME. Thus, with the aim of identifying a Treg suppression mechanism specific to effector CD8 T cells in the TME, it is important to characterize in depth these CD177+ TA-Treg and to identify their interactions with PECAM-1+ CD8 T cells in the TME and their impact on the function of these CD8 T cells. This thesis work demonstrated, in several tumor types, that CD177 identifies a population of TA- Treg, with an activated phenotype. PECAM-1, the target of CD177, is expressed in the TME by polyfunctional effector CD8 T cells (GzmK, IFNγ, TNFα) with a high proliferation capacity. In situ on tumor sections, multi-immunofluorescence analyses showed the colocalization of CD177+ Treg and PECAM-1+ CD8 T cells in the tumor stroma, suggesting a link between these two populations. Furthermore, engagement of PECAM-1 IgD6, CD177 binding domain, reduces the activation and functions of PECAM-1+ CD8 T cells induced by the TCR signal by decreasing pZAP-70 and IFNү secretion. Finally, initial results on tumors have shown that the culture of CD8 T cells with CD177+ TA-Treg reduces the proliferation and secretion of IFNγ by PECAM-1+ CD8 T cells and the addition of an anti-CD177 makes it possible to partly rescue this inhibition, suggesting the role of the [CD177/PECAM-1] axis in the inhibition of PECAM-1+ CD8 T cells by CD177+ TA-Treg. The [CD177/PECAM-1] interaction represents the first demonstration of a membrane receptor/ligand pair involved in the selective inhibition of CD8 T cells effectors by TA-Treg in the TME and CD177 appears as a promising target to specifically raise suppression mediated by TA-Treg in the TME without altering those in the periphery

L'expression de récepteurs inhibiteurs tels PD-1, TIM-3, LAG-3, TIGIT sur les lymphocytes T participe à l'immunosuppression dans l'environnement tumoral. Le ciblage de PD-1 par les immunothérapies anti-PD-1/PD-L1 notamment révolutionne depuis peu la prise en charge de nombreux types de cancers en particulier dans le mélanome, cancer du poumon et aussi du rein. Dans la plupart des cancers comme dans celui du poumon et le mélanome, l'infiltrat CD8 et la réponse Th-1/IFN-gamma sont associés à un meilleur pronostic, contrairement aux tumeurs du rein et aux hémopathies. Les travaux de ma thèse s'intéressent à la caractérisation de l'expression des récepteurs inhibiteurs des lymphocytes, notamment PD-1 et TIM-3 dans le carcinome rénal et dans le lymphome. Mes travaux de thèse ont été réalisés avec des outils d'exploration du microenvironnement tumoral permettant une analyse multiplexe de nombreux paramètres in situ. En théorie jusqu'à 7 protéines peuvent être mises en évidence à la fois, j'ai pu mettre au point des comarquages de 4 protéines membranaires et/ ou nucléaires + Dapi ainsi que la détection d'ARN in situ dans les tumeurs. L'utilisation d'un outil technologique de microscopie à fluorescence multispectrale a permis une étude fine de la coexpression de PD-1 et Tim-3 sur les lymphocytes T CD8 (LT-CD8) grâce à la visualisation aisée de la colocalisation de ces 3 marqueurs. De la même façon, j'ai mis en évidence de l'expression de leurs ligands PD-L1 et Galectin-9 (Gal-9) dans l'environnement des tumeurs du rein. J'ai démontré que la co-expression de Tim-3 et PD-1 sur les CD8 dans le carcinome rénal avait un rôle délétère aussi bien sur le plan (i) fonctionnel puisque les LT-CD8 sécrétaient moins d'IFN-gamma, (ii) et clinique puisque les patients présentant un infiltrat de LT-CD8 double positifs pour PD-1 et TIM-3 récidivaient plus fréquemment. La présence des ligands PD-L1 et Gal-9 a été mise en évidence dans l'environnement tumoral laissant suggérer des interactions possibles avec les récepteurs inhibiteurs exprimés par les LT. J'ai également caractérisé les LT-CD8 PD-1+ TIM-3+ dans les lymphomes en combinant un marquage CD20 (quadruple + Dapi). Selon le type de lymphome, TIM-3 était coexprimé avec PD-1 à la surface des CD8 et plus ou moins au contact avec les cellules lymphomateuses CD20+. D'autre part, lorsque TIM-3 était coexprimé, les LT-CD8 étaient plus volontiers proliférant (comarquage Ki-67) en comparaison aux PD-1+ TIM-3-. Afin de poursuivre dans la caractérisation Th-1/IFN-gamma, j'ai élaboré la mise au point de la détection d'ARN dans les lymphocytes T in situ dans la tumeur, permettant de faire des liens avec leur fonctionnalité. Au total mes travaux de thèse ont permis de mettre en évidence des biomarqueurs immunologiques composites en lien avec l'expression des récepteurs inhibiteurs PD-1 et/ou TIM-3 et la perte de fonctionnalité (IFN-gamma) des lymphocytes T intratumoraux
It has been mainly described that the inhibitory receptors coexpression (PD-1, TIM-3, LAG-3, TIGIT) by lymphocytes in the tumor microenvironment (TME) induces a local immunosuppression. Targeting these receptors particularly PD-1 and its ligand PD-L1 is of great clinical benefit in cancer many types treatment (melanoma, renal and lung cancer in particular). In the most cases of cancer, like melanoma and lung cancer, a CD8-T cell and Th-1/IFN-gamma response is of good prognosis. But this is not the case in renal cancer and in hemopathies. My PhD work attempts to characterize clinical and biological implication of PD-1 and TIM-3 expression by intra-tumor lymphocytes in the setting of renal cancer and lymphoma. My PhD work has been conducted thanks to new methods of multiplexed characterization of the TME. Multispectral immunofluorescence lead to identify 7 parameters at the same time, and in this study I elaborated the identifications of lymphocytes markers in situ within the tumor: 4 membrane and/or nuclear proteins + nuclei (Dapi counterstain) and also coupled with the RNA detection. This tool allows me to accurately study the coexpression of PD-1 and TIM-3 at the CD8-T cell surface thanks to colocalisation identification and counting of these 3 markers. With the same method, I found that PD-L1 and Gal-9, which are PD-1 and TIM-3 ligands, were also expressed in the TME of renal carcinoma. I found that the coexpression of TIM-3 together with PD-1 in the CD8-T cells had a double relevance (i) at functional level, CD8-T cells were less able to secrete gamma-IFN (ii) at clinical level, patients harboring a higher infiltrate were more likely to relapse. The presence of PD-L1 and Gal-9 suggested interactions with inhibitory receptors of T cells. I also characterized CD8-T cells expressing PD-1 and TIM-3 in lymphomas, combining a CD20 staining (quadruple staining + Dapi). TIM-3 was more or less expressed depending of the lymphoma type near to CD20+ cells. TIM-3 PD-1 CD8-T cells were more likely Ki-67+ compared to TIM-3- cells, suggesting a more proliferative capacity. In order to continue the characterization of the Th-1/gamma-IFN-gamma immune response, I elaborate a technic to detect the gamma-IFN RNA in situ, together with lymphocytes staining, allowing the exploration of functionality within the tumor. To summarize, during my PhD work I could characterize composite immune biomarkers linked to the functionality of CD8-T cell and gamma-IFN Th-1 response

ZTLs vermitteln die Eliminierung von infizierten und entarteten Zellen durch Apoptose. Neuste Erkenntnisse unserer Gruppe haben gezeigt, dass eine Subpopulation der CD8+ T-Zellen, anstelle der zytotoxischen Marker das Oberflächenmolekül CD40L exprimiert. Die Expression von CD40L ist bislang als Schlüsselmolekül für die CD4+ T-Zell vermittelte Hilfe bekannt, welche durch Bindung an den CD40 Rezeptor auf anderen Immunzellen induziert wird. Das von den CD4+ T–Zellen ausgehende CD40L Signal ist besonders für die T-Zell abhängige B-Zell Aktivierung und die Bildung von Keimzentren essentiell, in denen B-Zellen heranreifen und hochaffine Antikörper produzieren um den Organismus vor eindringenden Erregern zu schützen. Aufgrund der CD40L-assoziierten Helferfunktion sollte in dieser Arbeit untersucht werden, welche Auswirkungen die Interaktion von CD8+CD40L+ T-Zellen mit B Zellen hat. In in vitro Studien konnte gezeigt werden, dass 50% der antigen-spezifischen CD8+ T-Zellen nach Aktivierung durch B-Zellen CD40L hochregulieren. Sowohl auf RNA- als auch auf Proteinebene induzierten CD8+CD40L+ T-Zellen einen B-Zell Phänotyp, der stark dem von CD4+ T-Zellen stimulierten B-Zellen ähnelte. In Infektionsversuchen mit dem B-Zell-trophen Virus MHV-68 konnte gezeigt werden, dass transgene Mäuse mit CD40L defizienten CD8+ T-Zellen im Vergleich zu Kontrolltieren eine signifikante Reduktion der Keimzentrums-B-Zellen in den Lymphknoten der oberen Halsregion aufweisen. Eine genauere Betrachtung des B-Zell Repertoires von IgG Gedächtniszellen ergab jedoch, dass die Sequenzen der IGHJ3 Genfamilie bevorzugt für die Modifikation der CDR3 Region in Mäusen mit CD40L defizienten CD8+ T-Zellen verwendet wird, die eine entscheidende Rolle bei der Antigenerkennung spielt. Zusammengefasst kann mit dieser Arbeit zum ersten Mal gezeigt werden, dass CD8+CD40L+ T-Zellen Helferfunktionen durch Unterstützung der B-Zell Aktivierung und Bildung von Keimzentren übernehmen können.
CTLs are important for the elimination of infected and degenerated cells by inducing apoptosis of the target cells. Recently our group identified a sub-population of CD8+ T cells expressing CD40L instead of common CTL markers. To that date, transient CD40L expression on T cells has been only described as a function of activated CD4+ T cells, which displays this key molecule for CD4+ T cell mediated help by binding to the CD40 receptor on other immune cells. Particularly, CD40L signaling provided by CD4+ T cells is indispensable for T cell dependent B cell activation and GC responses, which generate B cells secreting high affinity antibodies that protect the host from invading pathogens. Due to its associated helper functions, this thesis aimed to dissect whether CD40L positive CD8+ T cells are restricted to cytotoxic killing or if this sub-population possesses similar properties as CD4+ T cells when interacting with B cells. In vitro co-culture experiments showed that 50% of murine antigen specific CD8+ T cells up-regulated CD40L upon activation by antigen presenting B cells. When compared to CD40L deficient CD8+ T cells, the interaction of CD8+ CD40L+ T cells induced remarkable changes in B cells on the RNA and protein level and triggered a B cell phenotype resembling that of B cells primed by CD4+ T cells. By the infection of mice with the B cell trophic virus MHV-68, it was found that E8IcrexCD40Lflox transgenic mice lacking CD40L only on matured CD8+ T cells, exhibited a significant decrease of GC B cells in superficial cervical lymph nodes at the acute state of infection compared to WT mice. A closer look into the memory B cell repertoire revealed a preferred usage of the murine IGHJ3 gene family that modifies the CDR3 and thus the recognition groove of the B cell antibody in E8IcrexCD40Lflox mice. In summary, this work provides sufficient evidence that CD8+ CD40L+ T cells adopt helper-like functions by supporting B cell activation and subsequent GC formation.

There have been substantial advances in our understanding of the CD4+CD25+ regulatory T cell subset, but the possibility of an equivalent regulatory subset within the CD8+ T cell population has received less attention. In the course of studies investigating immunological abnormalities in patients with the inflammatory arthritis Ankylosing Spondylitis (AS), novel human CD8+ T cells that have a regulatory phenotype and function have been identified. CD8+/TCRαβ+ T cells were isolated from the peripheral blood of both AS patients and healthy donors and subsequently expanded as T cell lines using antilogous LPS activated dendritic cells. These lines contained IL-4 producing CD8+ T cells which were then cloned non-specifically by limiting dilution. Conventional CD8+ T cells are cytotoxic but these clones were not. They proliferated and produced cytokines in an autoreactive HLA class I restricted fashion; the cytokines produced included IL-4, IL-5 and TGFβ1, but not IFN-γ or IL-10. The clones expressed markers commonly associated with CD4+CD25+ regulatory T cells, including high levels of CTLA-4 and Foxp3. They suggested IFN-γ production and proliferation by CD4+ T cells in vitro in a cell contact dependent manner, which appeared to involve surface CTLA-4 since suppression could be blocked using an anti-CTLA-4 mAb. Increased numbers of these IL-4+ CD8+ T cells were found in AS patients’ PBMC stimulated directly ex vivo compared to healthy donors, and high numbers correlated with decreased numbers of pro-inflammatory IFN-γ producing T cells, suggesting they may actively regulate inflammation in vivo. Thus, human CD8+ regulatory T cells may be up-regulated in the peripheral blood under certain pathological conditions such as in response to chronic inflammation. AS is a remitting and relapsing inflammatory disease; therefore it is possible that these cells contribute to periods of remission by actively regulating inflammation in vivo.

Dengue virus, which has four serotypes, has several clinical manifestations including asymptomatic infection, a self-limiting febrile illness termed dengue fever (DF) and a severe form characterized by plasma leakage termed dengue hemorrhagic fever (DHF). The pathogenesis of DHF is not fully understood and many studies have shown that it is more prevalent during secondary infection. In addition to a mechanism termed antibody dependent enhancement (ADE), the role of T-cells in the pathogenesis of dengue also has been investigated. It has been hypothesized that upon secondary infection dengue-specific memory T-cells generated during a previous infection, which are cross-reactive and have low avidity for the current serotype dominate the T-cell response. This phenomenon is called 'Original antigenic sin' and the consequence of this low avidity T-cell response may be ineffective viral elimination leading to increased production of inflammatory cytokines which could cause plasma leakage. To study CD8+ T-cell responses to dengue-peptide variants, HLA-A11-restricted NS3 133-142-specific T-cell clones were generated and their cytotoxicity, proliferation and cytokine production in response to variant epitopes was tested. The results support that the magnitude of T-cell responses is related to the strength of the TCR-peptide-MHC interaction. AG129 mice, which lack both IFN (symbol missing) and IFN (symbol missing) receptors, show susceptibility to dengue virus infection and develop symptoms seen during human infection such as vascular leakage. This allows for investigation of the role of T-cell responses generated towards sequential infections with well defined serotypes. Experiments that would be very difficult to carry out in human dengue patients. Splenocytes from sequentially infected AG129 mice were assayed for their response to whole dengue proteins from serotype 1 and 2 virus. New epitopes were identified and CD8+ T-cell lines and clones were generated and their functions studied using peptide variants. The results showed that dengue-specific cross-reactive memory CTLs displayed better recognition of epitopes encountered during primary immunisa tion as compare to those recognised during secondary immunisation, which supports the idea of original antigenic sin in dengue-specific CD8+ T -cells. In conclusion, this study focuses on cross-reactive dengue-specific CD8+ T-cells and their functions when recognizing heterologous dengue peptides to clarify their role in pathogenesis.

CD8+ T cells are essential for host protection against intracellular pathogens and tumors. During antigen-driven responses, CD8+ T cell fate is governed by transcriptional and epigenetic processes that allow naïve CD8+ T cells to develop into a wide range of effector and conventional memory cell subsets. Over the last decades, novel techniques and major efforts led to a better understanding of the origin, nature, and short- and long-term effects of these processes on individual CD8+ T cells. Under certain conditions, naïve CD8+ T cells can acquire memory phenotype and functions in an antigen-independent manner. Although homeostatic cytokines and initial activation pathways that drive the development of these unconventional memory cells had been identified, the ensuing transcriptional profile of these cells and their degree of similarity with conventional memory cells remained ill-defined. The epigenetic events that accompany unconventional memory formation were also not known.Here, we show that innate memory cells, a type of thymic unconventional memory cells, are transcriptionally close to conventional memory cells but only partially epigenetically programmed toward the full memory fate. We also show that the sole overexpression of the transcription factor Eomesodermin (EOMES), a master regulator of effector and conventional memory cells, is able to drive many of the phenotypical, functional, transcriptional, and epigenetic features of innate memory cells, and to induce the recruitment of BRG1, a member of chromatin remodeling complexes, to innate memory gene regulatory regions. We further show that the in vivo interleukine-4-dependent development of innate memory cells is largely dependent on BRG1. We bring to light that, in innate memory cells, EOMES is recruited in many instances to genomic regions previously bound by the transcription factor RUNX3. Overall, we provide insights into the mechanisms that allow memory cell formation and T cell receptor stimulation to be uncoupled.
Doctorat en Sciences médicales (Médecine)
info:eu-repo/semantics/nonPublished

Diferents estudis han assenyalat la importància del sistema immunitari adaptatiu en la etiopatogènia de la malaltia de Parkinson (PD). La infiltració de limfòcits T s’ha descrit tant en models animals com en teixit postmortem humà. Alguns autors han proposat les modificacions post-traduccionals de l’α-sinucleïna com a possible antigen que indueix la resposta immunitària adaptativa. Per tant, l’objectiu principal d’aquesta tesi va ser determinar si els limfòcits T participen en l’inici i la progressió de la PD. A més, volíem saber si l’α-sinucleïna es comportava com un neoantigen. Vam analitzar i caracteritzar fenotípicament els limfòcits T que infiltren la substantia nigra pars compacta (SNpc) en teixit postmortem humà en diferents etapes de la malaltia. Teixit de PD i casos incidental de cossos de Lewy (iLBD), els quals són considerats un estadi inicial pre-motor de la malaltia, van ser analitzats. Vam estudiar la relació entre la infiltració de limfòcits T amb la mort de neurones dopaminèrgiques i la sinucleinopatia, dos pedres angulars de la malaltia. Vam detectar una infiltració bifàsica de limfòcits T citotòxics CD8+ (CTL) a la SNpc. Inesperadament, el primer i més important pic es produeix quan la sinucleinopatia i la mort dopaminèrgica no estan establerts. La infiltració de CTL es redueix un cop la sinucleinopatia i la mort dopaminèrgica comencen. La infiltració de CTL torna a augmentar en casos de PD on la densitat de limfòcits T CD8+ correlaciona amb la mort neuronal. Resultats semblants van ser obtinguts en un altra àrea cerebral afectada en la PD com és ara el locus coeruleus (LC). El fet que els CTLs fessin contacte amb neurones dopaminèrgiques i correlacionessin amb la seva pèrdua, suggereix un possible rol en la mort dopaminèrgica. Per anar més enllà, vam detectar que els CTLs expressaven maquinària citotòxica com ara granzims i interferó-g. La infiltració de CTLs granzims+ a la SNpc estava augmentada en casos iLBD indicant una resposta immunitària adaptativa aguda en estadis inicials de la malaltia. Un alt percentatge de CTLs eren limfòcits T memòria residents de teixit identificats per CD103. La presentació antigènica via micròglia MHC-II+ estava reduïda en estadis inicials de la malaltia. Baixes densitats de micròglia MHC-II+ tipus ameboide/activada correlacionaven amb més pèrdua dopaminèrgica, suggerint un rol positiu de la micròglia MHC-II+. Per tal de determinar el millor model rosegador per analitzar el rol de limfòcits T, vam caracteritzar la resposta immunitària en ratolins injectats amb MPTP i en rates que sobre-expressaven α-sinucleïna. Vam trobar una infiltració transitòria de limfòcits T CD4+ i CD8+ que precedien la mort dopaminèrgica en el model MPTP subagut i que correlacionaven amb una afectació estriatal. Tot i així, l’eliminació de la tolerància immunitària a través de la depleció dels Tregs no va augmentar el dany nigroestriatal. Finalment, també vam observar la infiltració de limfòcits T CD4+ i CD8+ a la SNpc en rates sobre-expressant α-sinucleïna. No obstant, aquestes rates no mostraven ni canvis motors ni dany nigroestriatal. En conclusió, la resposta immunitària adaptativa en cervells de casos amb la PD és diferent a l’observada en models animals de la malaltia. A la SNpc, els limfòcits T CD4+ no estaven augmentats i la infiltració de CTL precedia la sinucleinopatia. Aquests resultats assenyalen el fet que l’α-sinucleïna no sembla ser l’antigen que provoca un atac citotòxic. En general, aquesta tesi ha demostrat que la infiltració de CTL és un esdeveniment inicial en la malaltia precedint tant la mort dopaminèrgica com la sinculeinopatia. Per tant, el sistema immunitari adaptatiu pot ser una bona diana terapèutica tant en estadis inicials com finals de la malaltia. Emperò, urgeix la necessitat d’establir nous models animals que recapitulin la resposta humana immunitària adaptativa.
Diferentes estudios han señalado la importancia del sistema inmune adaptativo en la etiopatogenia de la enfermedad de Parkinson (PD). La infiltración de linfocitos T se ha descrito tanto en modelos animales como en tejido postmortem humano. Algunos autores han propuesta las modificaciones post-traduccionales de la α-sinucleína como posible antígeno que induzca la respuesta inmune adaptativa. Por lo tanto, el objetivo principal de esta tesis fue determinar si los linfocitos T participan en el inicio y la progresión de la PD. Además, queríamos saber si la α-sinucleína se comportaba como un neoantígeno. Analizamos y caracterizamos fenotípicamente los linfocitos T que infiltran la substantia nigra pars compacta (SNpc) en tejido postmortem humano en diferentes etapas de la enfermedad. Tejido de PD y casos incidentales de cuerpos de Lewy (iLBD), los cuales son considerados un estadio inicial pre-motor de la enfermedad, fueron analizados. Estudiamos la relación entre la infiltración de linfocitos T con la muerte de neuronas dopaminérgicas y la sinucleinopatía, dos piedras angulares de la enfermedad. Detectamos una infiltración bifásica de linfocitos T citotóxicos CD8+ (CTL) en la SNpc. Inesperadamente, el primer y más importante pico se produce cuando la sinucleinopatía y la muerte dopaminérgica aún no están establecidas. La infiltración de CTL se reduce cuando la sinucleinopatía y la muerte dopaminérgica empiezan. La infiltración de CTL vuelve a aumentar en los casos de PD donde la densidad de linfocitos T CD8+ correlaciona con la pérdida neuronal. Resultados parecidos fueron obtenidos en otra área cerebral afectada en la PD como es el locus coeruleus (LC). El hecho que los CTLs contactasen con neuronas dopaminérgicas y correlacionasen con su pérdida, sugiere un posible rol en la muerte dopaminérgica. Más específicamente, detectamos que los CTLs expresaban maquinaria citotóxica como granzimas e interferón-g. La infiltración de CTLs granzimas+ en la SNpc estaba augmentada en casos iLBD indicando una respuesta inmune adaptativa aguda en estadios iniciales de la enfermedad. Un elevado porcentaje de CTLs eran linfocitos T memoria residentes de tejido identificados por CD103. La presentación antigénica vía microglía MHC-II+ estaba reducida en estadios iniciales de la enfermedad. Bajas densidades de microglía MHC-II+ tipo ameboide/activada correlacionaban con más pérdida dopaminérgica, sugiriendo un rol positivo de la microglía MHC-II+. Para determinar el mejor modelo roedor con la intención de analizar el rol de los linfocitos T, caracterizamos la respuesta inmune adaptativa en ratones inyectados con MPTP y en ratas que sobre-expresaban α-sinucleína. Encontramos una infiltración transitoria de linfocitos T CD4+ y CD8+ que precedían la muerte dopaminérgica en el modelo MPTP subaguda y que correlacionaban con una afectación estriatal. Aún así, la eliminación de la tolerancia inmunitaria vía la depleción de los Tregs no augmentó el daño nigroestriatal. Finalmente, también observamos la infiltración de linfocitos T CD4 y CD8+ en la SNpc en ratas sobre-expresando α-sinucleína. No obstante, estas ratas no mostraban ni cambios motores ni daño nigroestriatal. En conclusión, la respuesta inmune adaptativa en cerebros de casos con la PD es diferente a la observada en modelos animales de la enfermedad. En la SNpc, los linfocitos T CD4+ no están augmentados y la infiltración de CTL precede la sinucleinopatía. Estos resultados señalan el hecho que la α-sinucleína no parace ser el antígeno que provoca un ataque citotóxico. En general, esta tesis ha demostrado que la infiltración de CTL es un evento inicial en la enfermedad precediendo tanto la muerte dopaminérgica como la sinucleinopatía. Por lo tanto, el sistema inmune adaptativo puede ser una buena diana terapéutica tanto en estados iniciales como finales de la enfermedad. Aún así, urge la necesidad de establecer nuevos modelos animales que recapitulen la respuesta humana inmune adaptativa.
Mounting evidence has pointed out that the adaptive immune system has an important role in Parkinson’s disease (PD) etiopathogenesis. T cell infiltration has been described in both PD experimental animal models and post-mortem human tissue. Some authors have proposed α-synuclein posttranslational modifications as the antigen eliciting this adaptive immune response. Thus, the main goal of this thesis was to determine whether T cells participate in the onset and progression of the disease. Moreover, we wanted to know whether α-synuclein behaved as a neoantigen. In order to overcome this, we analyzed and phenotypically characterized substantia nigra pars compacta (SNpc) infiltrating T cells in post-mortem human tissue at distinct disease stages. PD and incidental Lewy Body disease (iLBD) cases, which are considered to be an early pre-motor stage of the disorder, were analyzed. We studied the relationship between T cell infiltration with dopaminergic cell loss and synucleinopathy, two hallmarks of the disorder. We found a biphasic SNpc CD8+ cytotoxic T lymphocyte (CTL) infiltration. Strikingly, the first and highest peak was found when synucleinopathy and dopaminergic cell loss were not established. SNpc CTL infiltration subsided when synucleinopathy and dopaminergic cell loss started. SNpc CTL infiltration again increased in PD cases where CD8+ T cell densities correlated with neuronal death. Similar results were also obtained in another PD brain affected area such as locus coeruleus (LC). The fact that SNpc CTLs made contact with dopaminergic neurons and correlated with dopaminergic cell loss, suggests a likely role in dopaminergic cell death. To delve further into this concept, we found that SNpc CTLs expressed cytotoxic machinery i.e. granzymes and interferon-g. Infiltrating SNpc granzyme+ CTLs were found increased in iLBD cases indicating an acute adaptive immune response in early stages of the disease. A high percentage of SNpc CTLs were tissue resident memory T cells identified by CD103 expression. Antigen presentation by means of MHC class-II+ microglia was reduced in early stages of the disease. Low densities of ameboid/activated MHC class-II+ microglial cells correlated with higher dopaminergic cell loss, suggesting a positive role of MHC class-II+ microglia in the disease. To determine the best rodent model to assess the T cell role in PD, we characterized the immune response in MPTP injected mice and rats overexpressing α-synuclein. We found a transient CD4+ and CD8+ T cell infiltration preceding dopaminergic cell death in the subacute MPTP injected mice which correlated with striatal damage. Nonetheless, breaking immune tolerance through systemic Treg depletion did not increase nigrostriatal damage. Finally, we also observed CD4+ and CD8+ T cell SNpc infiltration in rats overexpressing α-synuclein. However, these rats did show neither behavioural motor changes nor nigrostriatal damage. To conclude, human PD-specific brain adaptive immune response reported in our study is different to the one observed in PD experimental animal models. In SNpc human tissue CD4+ T cells were not elevated, and CTL infiltration preceded synucleinopathy. These results point out the fact that α-synuclein seems not to be the antigen for the cytotoxic attack elicited by CD8+ T cells. Overall, this thesis demonstrated that CTL infiltration is an early event of the disease preceding both α-synuclein deposition and dopaminergic cell loss. Thus, targeting the adaptive immune response in both early and late stages of the disease may have beneficial effects. Nevertheless, there is a need to establish new PD experimental animal models which recapitulate the human adaptive immune response.
Universitat Autònoma de Barcelona. Programa de Doctorat en Neurociències

During priming, CD8+ T cells integrate a plethora of signals that affect their differentiation into subsets of CD8+ T cells with distinct migratory properties and functions. Given that CD8+ T cells exert their protective function via cell-cell contacts, the migratory patterns and spatial distribution of CD8+ T cell subsets induced by primary challenge are of critical importance to the host. Dendritic cells (DCs), as the primary initiators of these responses, play a pivotal role in shaping the size and differentiation status of CD8+ T cells that emerge. However, inadequate markers for CD8+ T cell subsets have hindered study of their lineage relationships, as well as their migratory behaviors. Here, we use a novel marker for identification of CD8+ T cell subsets to interrogate whether subsets of DCs skew CD8+ T cell fate decisions, the differentiation pattern of CD8+ T cell subsets, and the migratory behavior of these CD8+ T cell subsets. Within secondary lymphoid organs (SLOs), CD8+ T cells encounter subsets of DCs that may differentially impact subsequent T cell fate decisions. While distinct DC subsets were found to influence CD8+ T cell priming and subsequent differentiation in vitro, these differences were masked when priming occurred in vivo. This prompted us to delve deeper into how CD8+ T cell subsets are defined in vivo. Classically, memory CD8+ T cells are divided into two subsets: central memory T cells (TCM) and effector memory T cells (TEM). Based on the variable expression of the chemokine receptor, CX3CR1, we define TCM as CX3CR1- and TEM as CX3CR1high. Additionally, a previously undefined subset of T cells was identified that express intermediate levels of CX3CR1. Flow cytometric analysis of the subsets migrating through murine peripheral tissues in the memory phase established CX3CR1int cells as the dominant subset, thus these cells have been termed peripheral memory T cells (TPM). Lineage tracing of these three subsets established a uni-directional relationship where increasing levels of CX3CR1 marked further terminal differentiation: TCM (CX3CR1-) to TPM (CX3CR1int) to TEM (CX3CR1hi). The finding that TPM, and not TEM, migrated through peripheral tissues was intriguing because it contradicted previous studies suggesting that TEM had this migratory pattern. To resolve this contradiction, we visualized the migration of TEM precursors, CX3CR1hi effector T cells (TEff), by intravital multi-photon microscopy (IV-MPM) in real-time. Surprisingly, CX3CR1hi TEff adhered to and patrolled along the dermal endothelium of mice. Specifically, migration was enriched along arteriolar endothelium and tended to be against blood flow. Patrolling occurred for both CX3CR1hi TEff and TEM and was limited to CX3CR1hi CD8+ T cells and CX3CR1hi monocytes, but was not dependent on functional CX3CR1. Moreover, addition of cognate antigen (Ag) resulted in rapid stopping of Ag-specific T cells, suggesting that patrolling T cells scan arteriolar endothelium for cognate Ag. Together, these results challenge the paradigm that TEM function by migration through peripheral tissues and establish a new migratory behavior by TEM and their effector precursors that promotes intravascular scanning of arteriolar endothelium.
Medical Sciences

Professional antigen presenting cells (APCs) represent an important link between the innate and adaptive immune system. Macrophages (MΦs) and dendritic cells (DCs) serve as sentinels in the periphery collecting samples from their environment and processing this information. These cells then present antigenic fragments to T cells in the context of self-MHC molecules. Although a clear role for both of these APCs in the stimulation of already activated or memory T cells has been established, the ability of MΦs to activate naive T cells is still unknown. In this thesis the ability of bone marrow-derived MΦs and DCs to prime naive CD8+ and CD4+ T cells was investigated. Using adoptively transferred transgenic CFSE-Iabeled P-14 T cells, specific for gp33 from lymphocytic choriomeningitis virus in the context of Db, we were able to demonstrate the ability of both MΦs and DCs to induce naive CD8+ T cells proliferation. Once primed by MΦs these T cells gained effector function as shown by interferon- γ (IFN-γ) production and in vivo cytolysis. In addition, immunization of wild type animals with gp33-pulsed MΦs, as well as DCs, led to greater than a 95% reduction in lymphocytic choriomeningitis virus titers. To rule out the role of cross-presentation in the observed priming, two models were used. In the first model, lethally irradiated F1 bxs chimeras reconstituted with either H-2s or H-2b bone marrow were used as host for the adoptive transfer experiments. Since the gp33 peptide binds to Db, the H-2s reconstituted animals should be unable to cross-present the peptide to the P-14 T cells. Using this model, we were able to clearly demonstrate the ability of MΦs to activate naive P-14 T cells to undergo division. Additional experiments, demonstrated that these MΦ primed T cells went on to develop into effector cells. Finally, the ability of the MΦ primed T cells to develop into functional memory cells was demonstrated. To confirm the chimera results, these experiments were repeated using β2 microglobulin deficient animals (whose cells don't express MHC I) as host in adoptive experiments. MΦs were able to stimulate the naive P-14 T cells to divide and gain effector function as demonstrated by the ability to produce IFN-γ. In contrast to the CD8 system, MΦ were poor stimulators of D011.10 CD4+ T cell proliferation. Additionally, D011.10 T cells stimulated by DCs were able to produce interleukin-2 (IL-2), IL-4, tumor necrosis factor and granulocyte-macrophage colony stimulating factor where as MΦ stimulated D011.10 T cells were only able to produce IL-2. In conclusion this body of work clearly demonstrates the in vivo ability of MΦ to stimulate CD8+ T cell proliferation, effector function, as well as the formation of functional CD8+ T cell memory. Whether or not the nature of the memory pools stimulated by the two APCs is exactly the same is still unknown and needs further investigation. The ability of APCs other than DCs to stimulate functional protective memory needs to be considered in the quest to design vaccines that offer broad-spectrum protection.

T Zellen können in zwei Hauptpopulationen mit unterschiedlichen Aufgaben unterschieden werden. CD4+ T Zellen exprimieren im Zuge ihrer Aktivierung CD40L, welches ein zentraler kostimulatorischer Rezeptor zur Induktion von B-Zell basierter humoraler Immunität, APC Aktivierung und einer effizienten Effektor CD8+ T Zell Entwicklung ist („Helfer-Funktion“). Im Gegensatz dazu sind die zytotoxischen CD8+ T Zellen dazu vorbestimmt, infizierte oder maligne Zellen direkt abzutöten. Jedoch wurde eine Fraktion von CD8+ T Zellen identifiziert, die nach Aktivierung CD40L exprimiert. Bisher ist nicht verstanden, wie in solchen CD8+ T Zellen a) die CD40L Expression reguliert ist, b) wann und wie die Fähigkeit CD40L zu exprimieren implementiert wird und c) was die Folgen für das Immunsystem sind. In dieser Arbeit konnten wir zeigen, dass sowohl in CD4+ als auch in CD8+ T Zellen die CD40L Expression durch DNA-Methylierung regulatorischer Regionen des CD40LG Lokus reguliert wird. Die Demethylierung zentraler Elemente wird im Thymus implementiert, manifestiert sich mit der T-Zell Reifung und geht mit einer zunehmenden Stabilität der CD40L Expression einher. Erhöhte Expression von CD5 und NUR77 in CD40L+ CD8+ SP Thymozyten weisen auf eine positive Selektion mit hoher Affinität gegen Selbst-peptide während der Reifung im Thymus hin, welche das weitere Schicksal der CD40L exprimierenden CD8+ T Zellen beeinflusst. Naive CD40L+ CD8+ T Zellen besitzen ein anderes TCR Repertoire als CD40L- CD8+ T Zellen und reifen im Zuge ihrer Aktivierung bevorzugt zu Gedächtniszellen mit Zytokin- und Chemokinrezeptorprofilen von Tc2, Tc17 und Tc22 Zellen heran. Mit ihrem nicht-zytotoxischen Phänotyp und ihrer Genexpressionsignatur ähneln diese Zellen stark Helfer-CD4+ T Zellen und können von den klassisch zytotoxischen Tc1 und Tc17+1 Zellen durch ihre IL-6R und fehlende SLAMF7 Expression sowie der Expression von Markern die auf eine Fähigkeit in die Haut zu wandern schließen lassen, unterschieden werden. Zusammenfassend zeigen wir hier, dass naive CD8+ T Zellen von den frühsten Entwicklungsstadien im Thymus an nicht homogen sind und die Fähigkeiten über CD40L Expression eine Helferfunktion auszuüben beziehungsweise über die Sekretion zytolytischer Moleküle Zielzellen abzutöten unabhängig vom CD4+ or CD8+ T-Zell Status sind. Zellen mit Zytokin- und Genexpressionsignaturen, die mit denen der CD8+ Helfer-T Zellen übereinstimmen, wurden von uns und anderen in Geweben (Haut, Lunge) identifiziert und tragen zu den verschiedensten autoinflammatorischen Erkrankungen bei. Diese Arbeit insinuiert daher die Notwendigkeit einer grundlegenen Neubewertung der CD8+ T Zell Fähigkeiten und Funktionen in Immunantworten.
The T cell compartment consists of two major subsets with diverse assignments. CD4+ T cells express CD40L upon activation, a central co-stimulatory receptor to induce B cell mediated humoral immunity, activate APCs and prime efficient effector CD8+ T cell development (“helper function”). In contrast, cytotoxic CD8+ T cells are predetermined to kill infected or malignant cells directly. However, a fraction of CD8+ T cells expressing CD40L upon activation was identified. So far, it is not understood in CD8+ T cells a) how CD40L expression is regulated, b) when and how the ability of CD40L expression is implemented and c) what are the implications for the immune system. In this thesis, we found that CD40L expression is regulated by DNA-methylation of regulatory regions of the CD40LG locus in CD4+ as well as CD8+ T cells. The de-methylation of central elements is implemented in the thymus and increases with T cell maturation reflected by enhanced stability of CD40L expression. Elevated CD5 and NUR77 expression of CD40L+ CD8+ SP thymocytes suggests that high affine detection of self-peptides during positive selection in the thymus implements CD40L expression ability and predetermines the fate of the CD40L imprinted CD8+ T cells. CD40L+ naïve CD8+ T cells differ in their TCR repertoire from their CD40L- counterparts and preferentially mature into memory cell subsets with cytokine and chemokine receptor profiles of Tc2, Tc17 and Tc22 cells. With their non-cytotoxic phenotype and gene expression signatures, the CD40L+ memory CD8+ T cell subsets Tc2, Tc17 and Tc22 widely resemble helper CD4+ T cells and can be distinguished from classical cytotoxic Tc1 and Tc17+1 cells by their IL-6R and absent SLAMF7 expression and their skin migratory phenotype. Altogether, we demonstrate that from the earliest developmental stages in thymus onwards naive CD8+ T cells are not homogenous and the abilites to provide “CD40L based help” or “cytotoxicity mediated killing” are independent of the CD4+ or CD8+ T cell status. Cells with helper-type CD8+ T cell cytokine and gene-expression signatures were found at barrier sites (skin, lung) by us and others where they contribute to multiple autoinflammatory diseases. Therefore, this work insinuates the need to revisite CD8+ T cell capablities and function in immune responses.

Many virus infections induce a transient state of immune suppression in the infected host. Virus-induced T cell suppression can be caused by T cell activation-induced cell death (AICD), dendritic cell (DC) apoptosis, DC dysfunction, and/or the enhanced expression of immune-suppressive cytokines. It has been previously demonstrated that naïve bystander CD8 T cells derived from hosts experiencing an acute virus-specific T cell response underwent AICD when polyclonally activated by anti-CD3 in vitro (Zarozinski et al., 2000). Susceptibility of naïve bystander T cells to AICD could prevent the development of a new T cell response during an ongoing immune response, and thus render infected hosts immune suppressed. Although immune suppression could result in an enhanced susceptibility to superinfections, virus-infected individuals are more commonly resistant to superinfecting pathogens. Because of these seemingly contradictory conditions, we sought to investigate how acute viral infections impact naïve bystander CD8 T cells in vivo. More specifically, we asked whether bystander CD8 T cells are susceptible to immune suppression or whether they can contribute to the resistance to superinfections. In order to address this, we examined the responses of bystander CD8 T cells activated with cognate antigen during acute viral infections in vivo. We generated several in vivomodels using P14 (LCMV glycoprotein-specific), HY (male antigen-specific), and OT-I (ovalbumin-specific) transgenic CD8 T cells, which we defined as bystander during acute infections with lymphocytic choriomeningitis virus (LCMV), Pichinde virus (PV), vaccinia virus (VV), and murine cytomegalovirus (MCMV). Consistent with the enhanced susceptibility to cell death noted in vitro, we found that bystander CD8 T cells activated with cognate antigen in vivo during acute viral infections underwent markedly reduced proliferation. Virus-induced transient T cell suppression in vivo was not exclusively mediated by Fas-FasL- or TNF-induced AICD or due to an enhanced susceptibility to apoptosis. Instead, immune suppression in vivowas associated with a delayed onset of division, which we found not to be due to a defect in antigen presentation, but rather due to a T cell intrinsic defect. Despite the suppressed proliferation of TCR-stimulated bystander CD8 T cells in vivo, we found an enhancement of the effector functions exerted by bystander CD8 T cells activated during acute viral infections. During acute viral infections or after stimulation with type 1 IFN (IFN-αβ) inducers, some bystander CD8 T cells were sensitized to immediately exert effector functions such as IFN-γ production and degranulation upon stimulation with high affinity cognate antigen. Sensitization of naïve CD8 T cells required self-MHC I and indirect effects of IFN-αβ, while IL-12, IL-18, and IFN-γ were not individually required. IL-15 was not required for the rapid expression of IFN-γ, but was required for up-regulation of granzyme B (GrzB). P14 and OT-I CD8 T cells, which are capable of homeostatic proliferation, could be sensitized by poly(I:C), but HY CD8 T cells, which are poor at homeostatic proliferation, could not, suggesting that the requirement for MHC I may be to present low affinity cryptically cross-reactive self antigens. Sensitized naive CD8 T cells up-regulated the t-box transcription factor Eomesodermin (Eomes), which can regulate these rapid effector functions. In conclusion, we demonstrate in this thesis that acute viral infections impact naïve bystander CD8 T cells such that their response to cognate antigen is altered. Prior to cognate antigen engagement, bystander CD8 T cells up-regulated Eomes, CD122, and GrzB. Following cognate antigen engagement, bystander CD8 T cells rapidly degranulated and expressed the effector cytokine IFN-γ. The ability of bystander CD8 T cells to rapidly exert effector functions may contribute to the resistance of virus-infected individuals to superinfections. Despite these rapid effector functions, the proliferation of TCR-stimulated bystander CD8 T cells was markedly inhibited. This reduced proliferation was found not to be a defect in antigen presentation, but was a T cell intrinsic defect in initiating division. Thus, bystander CD8 T cells were also susceptible to virus-induced immune suppression. It is also likely that virus-specific CD8 T cells that are not activated until later in the response, so-called latecomer CD8 T cells, may also be susceptible to immune enhancement and suppression. Thus, latecomer CD8 T cells would be able to rapidly exert effector functions at the expense of proliferation. Taken together, we propose that during an immune response, due to spatial and temporal gradients of antigen and inflammation, it is likely that a combination of heterogeneous T cells with different signal strengths and sequences of exposure from cytokines and peptide-MHC constitute the total T cell response to pathogens.

Chronic inflammation is associated with carcinoma development in several clinical settings, and we sought to investigate the role of T cells in this phenomenon using the DMBA/TPA two-stage chemical carcinogenesis protocol. We demonstrate that, paradoxical to models of immunosurveillance, wild-type (WT) mice have a markedly higher rate of tumor formation relative to strains lacking CD8+ T cells. Adoptive transfers of antibody-coated magnetic bead-enriched peripheral CD8+ T cells into TCRáâ-/- mice confirmed that the increased mean tumor area and progression to carcinoma was attributable to the presence of CD8+ T cells. All analyzed strains of mice in which the CD8 compartment was intact (WT, CD4-/-) showed significant increases in tumor susceptibility. Putative tumor-promoting (T-pro) cells (TCRáâ+CD8+CD44+CD62L- tumor infiltrating lymphocytes, TILs) were directly compared to their phenotypic equivalents in peripheral blood lymphocytes (PBLs). In WT and CD4-deficient mice, CD8+ TILs consistently revealed a markedly higher relative expression, by RT-PCR, of IFNã, TNFá and COX-2, and a striking decrease in expression of perforin. Cytokine-bead analysis (CBA) comparison of CD8+ and CD4+ TIL in tumors from WT mice confirmed the increased expression by the CD8+ TIL of IFNã and TNFá. To our knowledge, this is the first demonstration of increased carcinogenesis attributable to CD8+ TILs, characterized by their high IFNã, TNFá, and COX-2 production and defective perforin production relative to phenotypically equivalent PBLs. These studies may have mechanistic implications for the role of T cells in inflammation-associated carcinogenesis.

Proinsulin is an auto-antigen in type 1 diabetes in the Non-obese Diabetic (NOD) mouse. The CD8+ T cell clone, G9C8, which utilises a T cell receptor (TCR) comprising TRAV8-1*01TRAJ9 and TRBV19*01TRBD1TRBJ2-3, recognises insulin B15-23 presented by H-2Kd, escapes negative selection and rapidly causes diabetes upon adoptive transfer into immunocompromised NODscid mice. To understand how these insulin B15-23 reactive CD8+ T cells develop, G9C8-derived single TCR TRAV8-1*01TRAJ9 chain transgenic NOD mice were generated. These mice were bred with different proinsulin-expressing NOD mice to generate proinsulin1 deficient, proinsulin2 deficient, proinsulin2 over-expressing and proinsulin1 and 2 deficient mice with a mutated proinsulin transgene, preventing G9C8 antigen recognition. Although proinsulin-specific CD8+ TCR repertoire changes in TRBV19 were seen in these mice, the proportion of insulin B15-23 reactive CD8+ T cells was unaffected by proinsulin expression. Interestingly, TCR clonotyping of these insulin B15-23 reactive T cells revealed minimal shared sequences between the strains, with mice exhibiting individual clonal expansions. However, by isolating TRBV19-expressing insulin B15-23-responsive T cells, shared sequences across the different proinsulin-expressing mice were identified, with a requirement of TRBJ2-3 for insulin recognition (used by the original G9C8 T cell clone). Furthermore, male proinsulin2 deficient TRAV8-1*01TRAJ9 mice developed diabetes, with a higher incidence seen upon antibiotic administration. Although the TCR repertoire was unaffected by antibiotics, the gut microbial diversity was greatly reduced in all the mice with age, independent of antibiotic use, with firmicutes bacteria comprising 90-97% of the microbiota. In summary, proinsulin expression and antibiotics modify diabetes susceptibility; however, insulin B15-23 reactive T cells develop independently of antigen expression and are not directly affected by antibiotics. This data suggests that iii non-antigen dependent mechanisms exist in controlling the development of auto-reactive T cells, but T cell activation and pathogenicity is influenced indirectly by a combination of antigen expression and gut microbiota.

Tc17 cells and the semi-invariant human mucosal associated invariant T (MAIT) cells are important CD8+ tissue-homing cell populations. Both are characterized by high expression of CD161 (++) and type-17 differentiation, yet their origins and relationships remain poorly defined. By transcriptional and functional analyses it is demonstrated that a pool of polyclonal, pre-committed type-17 CD161++CD8αβ+ T cells exists in cord blood, from which a prominent MAIT cell (TCR Vα7.2+/Vβ2 or 13.2) population emerges post-natally. During this expansion, CD8αα T-cells appear exclusively within CD161++CD8+/MAIT subset, sharing cytokine production (IL17, IL-22 and IFN-γ), chemokine-receptor expression (CCR2, CCR6 and CXCR6), TCR-usage and transcriptional profiles with their CD161++CD8αβ+ counterparts. These data demonstrate the origin and differentiation pathway of MAIT cells from a naïve type-17 pre-committed CD161++CD8+ T cell pool and the distinct phenotype and function of CD8αα cells in man. The CD161++CD8αβ and CD8αα T cell subsets are reduced in the peripheral circulation in chronic hepatitis B and C and are enriched in the liver in chronic hepatitis C. Their potential role in immunity to chronic viral hepatitis B and C is demonstrated by their expression of activation/exhaustion markers CD69, CD25, HLA-DR and PD-1. In addition a substantial distinct CD161-CD8β^low population is demonstrated in chronic hepatitis B, co-characterised by a CD28^low, HLA-DR^high phenotype and high expression of IFN-γ, with important implications for the development of immunotherapy and vaccination.

Approximately one-third of the world’s population is currently infected with Mycobacterium tuberculosis (Mtb), the bacillus that causes tuberculosis. Globally, it is the second leading cause of death by a single infectious agent. An effective vaccine is needed to stop this ongoing pandemic, but efforts to design one are hampered by our limited understanding of host immunity to this pathogen. CD8+ T cells are elicited during tuberculosis and are required for optimum host resistance. They produce cytokines such as IFN-γ and can directly lyse infected cells. During infection, the expansion and differentiation of effector CD8+ T cells is a dynamically regulated process that is influenced by the inflammatory milieu of the infected host. Currently, the signals governing CD8+ T cell responses during tuberculosis are not well characterized. Utilizing a mouse model of disease, we address the effects of key cytokines on CD8+ T cells, beginning with IL-12, type 1 interferons (IFN), and IL-27. All three of these cytokines are produced by innate immune cells during tuberculosis and have profound effects on host resistance. IL-12 proves most essential for robust CD8+ T cell expansion and IFN-γ production and also drives the terminal differentiation of short-lived effector cells. However, IL-12 is not acting alone, and type 1 IFN and IL-27 each have non-redundant roles supporting expansion in infected lungs. Thus, CD8+ T cells reflect the inflammatory environment of the host, responding in different degrees to each cytokine present. We next examine the role of IL-21, a cytokine produced by activated CD4+ T cells. In the absence of IL-21 signaling, CD8+ T cell expansion and effector functions are severely compromised. IL-21 is also essential to prevent CD8+ T cell exhaustion at later time points during disease. These observations are the first to describe an essential role for IL-21 in the host immune response to Mtb. Together, these studies establish IL-12 and IL-21 as essential regulators of CD8+ T cells during tuberculosis, and indicate type 1 IFN and IL-27 support expansion in the lungs. We believe these observations have implications for future immunotherapies and rational vaccine design.
Medical Sciences

Axial spondyloarthritis (AxSpA) is an inflammatory disease affecting the axial skeleton. The infiltrate of T-cells in the structural lesions has been found to contribute to bone remodeling, but consensus relating the functional contribution of different T-cell subsets to pathogenesis has not been reached yet. Aim of the project was to characterize circulating T-cells and their homing markers from axSpA patients in order to identify cellular populations that could migrate to inflamed tissues and be implicated in axSpA. We found an altered proportion of circulating naïve and memory T-cells in axSpA patients, and a skew in favor of CD8+ T-cells expressing the chemokine receptor CCR4. Since CCL17 and CCL22, the two ligands for CCR4, are found to be elevated in the sera of axSpA patients, we investigated in details the role of CD8+CCR4+ T cells in axSpA. Our data showed that circulating CD8+CCR4+ T-cells display an effector memory phenotype and express homing markers for tissues that are target of the disease. Noteworthy, CD8+CCR4+ T cells from axSpA patients were activated, expressed markers of proliferation and acquired a cytotoxic phenotype, as demonstrated by the increased production of granzyme and perforin. CD8+CCR4+ T cells from axSpA patients upregulate the transcription of genes involved in bone mineralization and downregulate genes involved in osteoclast differentiation, indicating their possible involvement in bone remodeling. Furthermore, CD8+CCR4+ T cells stimulated with PMA and ionomycin were able to produce and release TNF and IL-8, two cytokines involved in osteoclastogenesis, indicating that CD8+CCR4+ T-cells after stimulation would be able to promote osteoclasts differentiation and neutrophils recruitment. Taken together our data suggest that CD8+CCR4+ T cells might exert a pathogenic role in axSpA, by releasing mediators of tissue damage, bone remodeling and recruitment of other pro inflammatory cells.

Melanoma is the third most commonly diagnosed form of cancer amongst Australians and Australia has the highest incidence of melanoma in the world. At present, there are no vaccines that are approved for the treatment of melanoma. Previous research performed in the laboratory resulted in the development of a syngeneic, whole cell melanoma vaccine engineered to express high levels of the B7.1 T cell co-stimulatory molecule, in addition to using high doses of interferons to upregulate MHC Class I molecules on the surface of the vaccine cells. Results from extensive mouse trials determined that the incorporation of B7.1 into the vaccine cell model directly stimulated CD8+ T cells to effectively kill melanoma cells in CTL assays and protect mice from developing tumours following injection with live tumour cells expressing intermediate levels of B7.1, thereby replacing the requirement for CD4+ T cells. Subsequently, the vaccine technology was translated into a human allogeneic, whole cell vaccine that completed testing in 2011 on stage IV melanoma patients in a Phase I clinical trial at the Princess Alexandra Hospital, Brisbane. The purpose of this thesis was to extend this anticancer vaccine technology by investigating whether the addition of a second co-stimulatory molecule, 4-1BBL, onto the surface of the vaccine cells could replace or reduce the requirement for DCs in the stimulation and activation of CD8+ T cells targeting the cancer cells.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Science, Environment, Engineering and Technology
Full Text

The aim of this project was to explore the role of the tyrosine kinases JAK1 and JAK3 in cytokine signalling, focusing on interleukin-2 signalling in CD8⁺ effector T lymphocytes. Initial experiments compared the effects of the pan JAK1/JAK3 inhibitor tofacitinib, the selective JAK1 inhibitor GSK186, and the selective JAK3 inhibitor GSK192 on IL-2 control of effector CD8+ cytotoxic T cells (CTL). On the basis of these preliminary data, a detailed analysis of the effect of tofacitinib on effector CD8⁺ T lymphocytes was performed. Phosphorylation events regulated by tofacitinib were identified using mass spectrometry analysis of SILAC (stable isotope labelling with amino acids in cell culture) labelled CTL. Tofacitinib regulated a selective number of phosphorylation sites, with less than 1.2% of the CTL phosphoproteome significantly regulated by tofacitinib treatment following 4hrs tofacitinib treatment. Proteins with downregulated phosphorylation sites were enriched in functions related to the Jak-STAT signalling, regulation of gene expression, and MAPK signalling cascades. Proteins with upregulated phosphorylations were also enriched in functions related to regulation of gene transcription. The proteome of tofacitinib treated CTL was defined by label free mass spectrometry. Approximately 4.5% of the CTL proteome was significantly regulated following 24 hours tofacitinib treatment, suggesting tofacitinib regulates the expression of a selective subset of proteins. Tofacitinib treatment resulted in the downregulation of proteins involved in ribosome biosynthesis, steroid biosynthesis, regulation of transcription and the cell cycle; and the upregulation of proteins with hydrolase activity, and with roles in the lysosome and extracellular exosomes. The phosphoproteomic and proteomic data demonstrates that JAK kinase dependent IL-2 signalling regulates essential processes in CTL by controlling a selective number of phosphorylation events and proteins. Validation of proteins identified as regulated following tofacitinib treatment identified new targets of IL-2 signalling in CTL, including the transcription factor NFIL3. NFIL3 was shown to be upregulated in CD8⁺ T lymphocytes following stimulation with IL-2 and regulated perforin and CD62L expression, suggesting a role in the regulation of CTL effector function.

Infection with HIV is a continuing global health problem. Antiretroviral therapy effectively suppresses viraemia, but the viral reservoir in latently infected CD4+ T-cells persists for decades and is the main obstacle to HIV eradication. The central role of CD8+ T-cellmediated immune control of natural adult HIV infection has long been established and recent studies suggest that CD8+ T-cells are likely to be crucial in eliminating reactivated reservoirs. However, the questions of what constitutes an effective anti-HIV CD8+ T-cell response and how to induce it therapeutically are outstanding. Moreover, even less is known about the antiviral function of CD8+ T-cells in paediatric HIV infection. Although outnumbered by adult infections, paediatric infection may offer opportunities to achieve HIV cure that could be more widely applicable to cure strategies in adults. Here, I explore the specific aspects of the interplay between host and virus in order to gain a better understanding of the optimal CD8+ T-cell-mediated immune responses. I demonstrate that in contrast to the Gag-specific response that mediates viral control in the context of HLA-B*57, among HLA-B*14-restricted responses, Env-specific cells are more potent than those targeting Gag. This is associated with higher antigen sensitivity and stronger selection pressure in the Env-specific population. I define two broadly distinct phenotypes of HIV non-progression in children. These are characterised by differential protein specificity of the antiviral CD8+ T-cell response but children of both phenotypes demonstrate strong selection pressure exerted on the virus by CD8+ T-cells in the first weeks of life. Next, in the context of the HIV vaccine tested in the Phambili trial, I report an HLA-specific effect of vaccine on disease progression, as a result of altering the natural CD8+ T-cell immunodominance hierarchy. Finally, I demonstrate a new method which allows to selectively deplete human HIV-specific CD8+ T-cell in vitro, and thereby to evaluate the contribution and efficacy of particular CD8+ T-cell specificities to viral inhibition - a critical question to HIV vaccine design and reservoir eradication studies. The work described here adds important insights to our understanding of the HIV-specific CD8+ T-cell responses that are generated from birth through childhood and into adulthood and is relevant to the future studies directed at harnessing the most effective CD8+ T-cell response to achieve HIV cure.

Although chronic lymphocytic leukaemia (CLL) is a B-cell malignancy, T-cells from CLL patients often display abnormal characteristics when compared to age-matched healthy donors. One example is the preferential expansion of CD8+ T-cells within a subgroup of CLL patients resulting in an inverted CD4:CD8 ratio (CLLIR). This thesis describes a detailed analysis of the T-cell compartment with an emphasis on CD8+ T-cells. It confirms that CLL patients have abnormal memory T-cell subset distributions, with skewing to more differentiated memory subsets (EM/EMRA CD8+ T-cells) and an increase in a replicative senescent phenotype (CD57+). Furthermore, CLLIR patients demonstrated significantly inferior prognosis compared to their normal ratio counterparts (CLLNR). The aim of this thesis was to further characterise the expanded CD8+ T-cells within the CLLIR subgroup to define more precise prognostic markers and to understand the role of these T-cells in CLL disease. Polychromatic phenotyping of CLL patients (n = 99) was carried out using 8-colour flow cytometry, testing for markers associated with senescence (CD57/KLRG-1), exhaustion (PD-1) and activation (HLA-DR/CD38). This allowed the identification of multiple discrete subsets of T-cells, with a greater complexity of subsets seen in CLL patients compared to healthy donors. There was also an increase in senescent phenotypes within CLLIR patients, confirming previous results. However, the use of additional markers identified increased frequencies of an ‘activated senescent’ phenotype (CD8+CD57+HLA-DR+) within CLLIR patients. This phenotype was shown to have a prognostic effect on progression-free survival in both multivariate and univariate analysis, with higher frequencies conferring inferior prognosis. Multivariate analyses also revealed CD4+ subsets, including those with a CD4+HLA-DR+PD-1+ phenotype, to be of prognostic value. This prompted a phenotypic assessment of the CD4+ T-cell compartment. Like the CD8+ population, CD4+ T-cells showed increased frequencies of senescent phenotypes within CLLIR patients. There was also an exacerbation of CD57+HLA-DR+ and HLA-DR+PD-1+ phenotypes within the CLLIR subgroup, the latter being the strongest prognostic phenotype identified by multivariate analysis within the entire study. To further evaluate the prognostic potential and stability of the CD4:CD8 ratio in CLL, preliminary phenotypic analysis was performed on a small cohort of treated and untreated patients. These results showed a disproportionate number of patients with inverted ratio in the treated patient group. Furthermore, the prognostic phenotypes CD8+CD57+HLA-DR+ and CD4+HLA-DR+PD-1+ were over represented within the treated subgroup. Finally, single telomere length analysis (STELA) of CD8+ T-cells in CLL was performed to explore potential changes in the replicative history between CLLNR and CLLIR patients. Overall, the mean telomere lengths of T-cells from CLLIR patients was similar to that of CLLNR patients, suggesting that the expanded CD8+ compartment does not necessarily arise from a long-lived population that has undergone multiple rounds of division. The STELA did reveal two additional subgroups of CLL patients that had either “short” or “long” telomeres within their T-cell memory subsets. This observation may provide an additional molecular marker that could be investigated either in combination or alone for prognostic relevance. Overall the results from the phenotypic analysis and STELA suggest that there is an active ongoing T-cell response in CLL, but the nature of the stimulus is unclear. The expanded T-cells in CLL are unusual and do not follow the patterns of T-cell exhaustion and senescence seen in chronic viral diseases and other cancers. Importantly, this study has defined two phenotypes (CD8+CD57+HLA-DR+ and CD4+HLA-DR+PD-1+) that have greater prognostic power than the inverted CD4:CD8 ratio. These may be useful in identifying patients who are likely to develop more aggressive clinical disease.

Thesis (M.S.)--University of California, San Diego, 2009.
Title from first page of PDF file (viewed June 22, 2009). Available via ProQuest Digital Dissertations. Includes bibliographical references (p. 43-45).

Previous research has provided evidence that CD8 T cells mediate immunity against infection with Theileria parva. However, the immunity induced by one parasite strain doesn‟t give complete protection against other strains and this is associated with parasite strain specificity of the CD8 T cell responses. There is evidence that such strain specificity is a consequence of the CD8 T cell responses of individual animals being focused on a limited number of immunodominant polymorphic peptide-MHC determinants. Dominant responses to the Tp2 antigen have been demonstrated in animals homozygous for the A10 MHC haplotype. Three Tp2 epitopes recognised by A10+ animals (Tp249-59, Tp250-59 and Tp298-106) have been defined. This project set out to investigate the dominance of these epitopes and to examine the T cell receptor (TCR) repertoires of the responding T cells. The specific objectives were to: (i) Determine the dominance hierarchies of the three defined Tp2 epitopes in both A10-homozygous and -heterozygous cattle. (ii) Examine the clonal repertoires of epitope-specific responses by analysis of TCR gene expression. (iii) Isolate full-length cDNAs encoding TCR α and β chain pairs from T cell clones of defined epitope specificity and use them to generate cells expressing the functional TCRs. Using MHC class I tetramers the relative dominance of CD8 T cell responses were found to differ between A10-homozygous and heterozygous cattle. All A10-homozygous cattle examined had detectable responses to all 3 Tp2 epitopes, the Tp249-59 epitope consistently being the most dominant. By contrast, only some A10-heterozygous cattle had detectable responses to Tp2 and when present the response was specific only for the Tp298-106 epitope. Analyses of the sequences of expressed TCR β chains showed that the responses in individual animals were clonotypically diverse, but often contained a few large expanded clonotypes. The TCRs of Tp298-106–specific T cells showed preferential usage of the Vβ13.5 gene and the frequent presence of a “LGG” motif within the CDR3 of the B chain. A conserved (public) TCRβ clonotype shared by the Tp250-59-specific CD8 T cells from all A10-homozygous cattle was identified. The TCRα chains co-expressed with this public TCRβ clonotype were identified for a number of T cell clones. Lentivirus transduction of Jurkat cells with three full-length TCR α and β chain pairs resulted in successful expression of one of the α/β chain pairs as a functional TCR, thus providing the basis for future work to generate bovine T cells expressing defined TCRs in vitro.

Infectious Disease & Immunity
Ph.D.
CAR-T cell immunotherapy is a highly efficacious treatment for CD19-positive hematological malignancies, however, some patients are non-responsive for reasons that are not well understood. Clinical efficacy has been correlated with long-term persistence, a propensity that can be predicted by the differentiation state of transplanted cells. Despite this, decades-old methods for expanding T cells have not been updated to prevent the deleterious effects of excessive differentiation in CAR-T cells. Uncoupling proliferation and differentiation is a long-held goal in the field of immunotherapy with both cytokines and pharmacological approaches being implemented to dissociate these parallel processes. Histone methyltransferases rewire transcriptional programs in T cells and simultaneously regulate multitudes of genes, making them attractive targets for modifying the proliferation-differentiation axis. Despite this, only a handful of studies have examined their role in regulating the transcriptional programs of human CD8+ T cells. MLL4 (encoded by KMT2B) belongs to the six-member group of MLL histone methyltransferases. MLL1, a paralog of MLL4, has been implicated in regulating the maintenance of IL-4 and GATA-3 expression in TH2 CD4 memory T cell populations, however the function of MLL4 in human CD8+ T cells is unknown. We report a critical role for MLL4 in the proliferation and differentiation of CD8+ T cells. CRISPR-Cas9-editing of MLL4 uncoupled the processes of proliferation and differentiation, increasing proliferation but maintaining central memory T cell (TCM)-like populations, allowing for the production of increased numbers of TCM-like CD62L+CD45RO+ cells. Pharmacologically inhibiting the MLL4-Menin complex with MI-2 during T cell expansion enriched the frequency of minimally differentiated TCM-like CD8+ T cells. TCM-associated CD62L, CCR7, CD122 and CD127 surface markers were upregulated and early memory-associated transcription factor TCF7, LEF1, EOMES, and FOXP1 transcripts were increased. CD8+ CAR-T cells expanded in the presence of MI-2 responded earlier, while improving both tumor burden and survival in a NSG xenograft model of human leukemia. This finding has important translational impact in improving the persistence and proliferative capacity of CD8+ CAR-T cells.
Temple University--Theses

CD8 T cells are essential for the elimination of intracellular pathogens and tumor cells. Understanding how naïve CD8 T cells differentiate into effector cells capable of eliminating pathogens and to generate adequate memory cells during immune responses is fundamental for optimal T cell vaccine design. In this PhD thesis work we addressed two central questions: 1) What are the mechanisms by which early effector T cells could act as pro-inflammatory effectors? And what is their role in the immune response? 2) How heterogeneous are CD8 responses? Could different pathogens modulate CD8 T cell differentiation programs and be responsible for CD8 cell-to-cell heterogeneity? Could they also generate memory cells with different protection capacities? To address these questions related to the diversity of CD8 T cell differentiation during immune responses, we used the single cell RT-PCR technique to detect ex vivo expression of mRNA in each individual cell, and Brefeldin A injected mice to detect ex vivo intracellular proteins. As experimental system to evaluate in vivo cell activation we used T cell receptor transgenic (TCR-Tg) CD8 T cells. Since the use of TCR-Tg cells to study immune responses has been subjected to criticism (due to high frequency of naïve-precursor transfers), in a first Ms. we compared the behavior of TCR-Tg and endogenous (non-transgenic and present at low frequency) cells in the same mouse. We found fully overlapping behavior between these two cell populations, which reinforced the advantage of using TCR-Tg cells to study CD8 immune responses. In addition, we concluded that the frequency of naïve-precursors do not induce diversity on CD8 T cell differentiation patterns. In a second Ms. we evaluated the impact of different pathogens in the diversity of CD8 T cell properties during two different immune responses: OT1 TCR-Tg cells (specific for OVA antigen) in the response to LM-OVA (Listeria Monocytogenes expressing OVA) infection; and P14 TCR-Tg cells (specific for GP33 epitope) in the response to Lymphocytic choriomeningitis vírus (LCMV) infection. We found that OT1 and P14 cells had different properties. As this difference could also be attributed to the different TCR avidity between OT1 and P14 cells, we then compared the behavior of P14 and OT-1 cells in the same mouse, co-injected with LM-OVA and LM-GP33. Since no differences were then detected, these results demonstrated that priming with different pathogens generates CD8 T cells with different characteristics that are not determined by TCR usage, but rather by the infection context. In addition, when looking for the protection capacity of endogenous CD8 memory cells generated in bacterial or viral context, we found that memory cells generated after LCMV priming were more efficient in responding to a second challenge, than memory cells generated after LM-GP33 priming. We also found that this better protection is associated with a T cell effector memory (TEM) phenotype associated with the LCMV infection, in contrast with a T cell central memory (TCM) phenotype generated after LM-OVA infection. These results demonstrate that different pathogens are responsible for diversity of CD8 T cell differentiation patterns and that even when distinct pathogens are efficiently eliminated during the primary immune response the quality of the memory generated may differ. In a third Ms. we studied the mechanisms by which effector CD8 T cells attracted other cell types in the early days of an immune response. We used two experimental systems: the response of OT1 TCR-Tg cells to LM-OVA infection; and the response of anti-HY TCR-Tg cells to male cells ("sterile"-non infectious context). In both cases we found that immediately after activation, CD8 T cells expressed high levels of pro-inflammatory cytokines and chemokines (such as TNFα, XCL1, CCL3 and CCL4). (...)

Thesis (Ph. D.)--University of California, San Diego, 2009.
Title from first page of PDF file (viewed Jan, 14, 2010). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 108-125).

Factors regulating expression of the IL-7 receptor throughout the CD8 T-cell life-span have not been fully characterized but IL-7 has been shown to down regulate the IL-7 receptor alpha-chain (CD127) at the cell surface. Here we show that IL-7 induced a decline in the level of total CD127 transcripts in CD8 T-cells. Also, IL-7 had no effect on CD127 mRNA stability, or alternative splicing. We also find that down regulation of CD127 by IL-7 requires de novo transcript and protein synthesis. Reporter assay analysis of the CD127 promoter showed that sequences up to 3kb upstream of the TATA box are required for basal CD127 gene expression. Within this region a Glucocorticoid Response Element near -2.2kb was identified as an important regulator of CD127 transcription. Conversely, IL-7 did not down regulate the ∼3kb promoter construct suggesting that the IL-7 responsive elements may lie outside this region.