Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 122-140.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 137-158.

CD4 T cells and B cells are cells associated with the adaptive immune system. The adaptive immune system is designed to mount a rapid antigen-specific response to pathogens by way of clonal expansions of T and B cells bearing discrete antigen-specific receptors. During viral infection, interactions between CD4 T cells and B cells occur in a dynamic process, where B cells that bind to the virus internalize and degrade virus particles. The B cells then present viral antigens to virus-specific CD4 T cells that activate the B cells and cause them to proliferate and differentiate into virus-specific antibody-secreting cells. Yet, non-specific hypergammaglobulinemia and the production of self-reactive antibodies occur during many viral infections, and studies have suggested that viral antigen-presenting B cells may become polyclonally activated by CD4 T cells in vivo in the absence of viral engagement of the B cell receptor. This presumed polyclonal B cell activation associated with virus infection is of great medical interest because it may be involved in the initiation of autoimmunity or contribute to the long-term maintenance of B cell memory. In order to directly examine the interactions that occur between T cells and B cells, I asked what would happen to a polyclonal population of B cells that are presenting viral antigens, if they were transferred into virus-infected hosts. I performed these studies in mice using the well-characterized lymphocytic choriomeningitis virus (LCMV) model of infection. I found that the transferred population of antigen-presenting B cells had two fates. Some antigen-expressing B cells were killed in vivo by CD4 T cells in the first day after transfer into LCMV-infected hosts. However, B cells that survived the cytotoxicity underwent a dynamic polyclonal activation manifested by proliferation, changes in phenotype, and antibody production. The specific elimination of antigen-presenting B cells following adoptive transfer into LCMV-infected hosts is the first evidence that MHC class II-restricted killing can occur in vivo during viral infection. This killing was specific, because only cells expressing specific viral peptides were eliminated, and they were only eliminated in LCMV-infected mice. In addition to peptide specificity, killing was restricted to MHC class II high cells that expressed the B cell markers B220 and CD19. Mice depleted of CD4 T cells prior to adoptive transfer did not eliminate virus-specific targets, suggesting that CD4 T cells are required for this killing. I found that CD4 T cell-dependent cytotoxicity cannot be solely explained by one mechanism, but Fas-FasL interactions and perforin are mechanisms used to induce lysis. Polyclonal B cell activation, hypothesized to be the cause of virus-induced hypergammaglobulinemia, has never been formally described in vivo. Based on previous studies of virus-induced hypergammaglobulinemia, which showed that CD4 T cells were required and that hypergammaglobulinemia was more likely to occur when virus grows to high titer in vivo, it was proposed that the B cells responsible for hypergammaglobulinemia may be expressing viral antigens to virus-specific CD4 T cells in vivo. CD4 T cells would then activate the B cells. However, because the antibodies produced during hypergammaglobulinemia are predominantly not virus-specific, nonvirus-specific B cells must be presenting viral antigens in vivo. In my studies, the adoptively transferred B cells that survived the MHC class II-restricted cytotoxicity became polyclonally activated in LCMV-infected mice. Most of the surviving naïve B cells presenting class II MHC peptides underwent an extensive differentiation process involving both proliferation and secretion of antibodies. Both events required CD4 cells and CD40/CD40L interactions to occur but B cell division did not require MyD88-dependent signaling, type I interferon signaling, or interferon γ signaling within B cells. No division or activation of B cells was detected at all in virus-infected hosts in the absence of cognate CD4 T cells and class II antigen. B cells taken from immunologically tolerant donor LCMV carrier mice with high LCMV antigen load became activated following adoptive transfer into LCMV-infected hosts, suggesting that B cells can present sufficient antigen for this process during a viral infection. A transgenic population of B cells presenting viral antigens was also stimulated to undergo polyclonal activation in LCMV-infected mice. Due to the high proportion of B cells stimulated by virus infection and the fact that transgenic B cells can be activated in this manner, I conclude that virus-induced polyclonal B cell activation is independent of B cell receptor specificity. This approach, therefore, formally demonstrates and quantifies a virus-induced polyclonal proliferation and differentiation of B cells which can occur in a B cell receptor-independent manner. By examining the fate of antigen-presenting B cells following adoptive transfer into LCMV-infected mice, I have been able to observe dynamic interactions between virus-specific CD4 T cells and B cells during viral infection. Adoptive transfer of antigen-presenting B cells results in CD4 T cell-mediated killing and polyclonal activation of B cells during LCMV infection. Studies showing requirements for CD4 T cells or MHC class II to control viral infections must now take MHC class II-restricted cytotoxicity into account. Polyclonal B cell activation after viral infection has the potential to enhance the maintenance of B cell memory or lead to the onset of autoimmune disease.

Orientador: Maria Terezinha Serrão Peraçoli / Resumo: Introdução: A pré-eclâmpsia (PE) é uma patologia obstétrica e uma das principais causas de morbimortalidade materna e fetal. Na PE ocorre um estado de má adaptação da tolerância imunológica, caracterizada por ativação anormal do sistema imune inato e adaptativo. As células T reguladoras (Treg) representam uma população de linfócitos T responsáveis pela manutenção da tolerância e controle da inflamação, enquanto células Th17 medeiam diferentes tipos de reações inflamatórias. Portanto, o balanço entre células Treg e Th17 pode ser crítico para a tolerância ao feto e prevenção da PE. Objetivo: Avaliar as subpopulações de células T CD4+ (Th1, Th2, Th17 e Treg) e o perfil de citocinas produzido por essas células, em gestantes portadoras de pré-eclâmpsia, classificadas em PE precoce e PE tardia. Métodos: Foram estudadas 60 gestantes, sendo 20 normotensas e 40 portadoras de PE, pareadas pela idade gestacional. As gestantes com PE foram classificadas de acordo com o aparecimento das manifestações clínicas em PE precoce (< 34 semanas de gestação; n=20) e PE tardia (≥ 34 semanas de gestação; n=20). Células mononucleares do sangue periférico (PBMCs), obtidas das gestantes foram avaliadas quanto à produção de citocinas pró e anti-inflamatórias e à expressão de fatores de transcrição envolvidos na caracterização das subpopulações de células T CD4+. A expressão dos fatores de transcrição intracitoplasmáticos de células Th1 (T-bet), Th2 (GATA-3), Th17 (RORc) e Treg (FoxP3) foi avaliada por c... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Un vérificateur de modèle peut produire une trace de contreexemple, pour un programme erroné, qui est souvent difficile à exploiter pour localiser les erreurs dans le code source. Dans ma thèse, nous avons proposé un algorithme de localisation d'erreurs à partir de contreexemples, nommé LocFaults, combinant les approches de Bounded Model Checking (BMC) avec un problème de satisfaction de contraintes (CSP). Cet algorithme analyse les chemins du CFG (Control Flow Graph) du programme erroné pour calculer les sous-ensembles d'instructions suspectes permettant de corriger le programme. En effet, nous générons un système de contraintes pour les chemins du graphe de flot de contrôle pour lesquels au plus k instructions conditionnelles peuvent être erronées. Ensuite, nous calculons les MCSs (Minimal Correction Sets) de taille limitée sur chacun de ces chemins. La suppression de l'un de ces ensembles de contraintes donne un sous-ensemble satisfiable maximal, en d'autres termes, un sous-ensemble maximal de contraintes satisfaisant la postcondition. Pour calculer les MCSs, nous étendons l'algorithme générique proposé par Liffiton et Sakallah dans le but de traiter des programmes avec des instructions numériques plus efficacement. Cette approche a été évaluée expérimentalement sur des programmes académiques et réalistes. / A model checker can produce a trace of counter-example for erroneous program, which is often difficult to exploit to locate errors in source code. In my thesis, we proposed an error localization algorithm from counter-examples, named LocFaults, combining approaches of Bounded Model-Checking (BMC) with constraint satisfaction problem (CSP). This algorithm analyzes the paths of CFG (Control Flow Graph) of the erroneous program to calculate the subsets of suspicious instructions to correct the program. Indeed, we generate a system of constraints for paths of control flow graph for which at most k conditional statements can be wrong. Then we calculate the MCSs (Minimal Correction Sets) of limited size on each of these paths. Removal of one of these sets of constraints gives a maximal satisfiable subset, in other words, a maximal subset of constraints satisfying the postcondition. To calculate the MCSs, we extend the generic algorithm proposed by Liffiton and Sakallah in order to deal with programs with numerical instructions more efficiently. This approach has been experimentally evaluated on a set of academic and realistic programs.

La primo-infection est un moment critique de l’établissement du réservoir justifiant de l’initiation d’un traitement précoce. Nous avons initié un essai randomisé évaluant l’impact de deux ans d’un traitement antirétroviral intense (essai ANRS147 OPTIPRIM, trithérapie versus pentathérapie) sur le réservoir et avons initié des études physiopathologiques au cours de cet essai. Nous montrons ainsi la faible diversité génétique des virus en primo-infection dans les compartiments sanguins et rectaux. Le réservoir s’établit dès le premier mois de l’infection par diffusion d’un cluster viral homogène au sein des lymphocytaires T CD4 naïfs (TN) et mémoires centrales (TCM), transitionnelles (TTM), effectrices (TEM) quiescents. Il en résulte une perturbation de l’homéostasie lymphocytaire associée à une faible contribution au réservoir des cellules peu différenciées à longue demi-vie, TN et TCM. Par ailleurs nous montrons que la majorité des patients au moment de leur primo-infection n’ont pas la capacité de développer des réponses T CD8 à même de supprimer la réplication virale comme chez les patients HIV Controllers. Après deux ans de traitement, nous observons que la diversité virale n’a pas évolué, par contre la taille du réservoir est fortement réduite. Les anomalies de l’homéostasie lymphocytaire T CD4 persistent, par contre le traitement très précoce a permis de protéger les TN et TCM. Il n’y a pas de bénéfice additionnel d’une pentathérapie mais nous avons validé le concept qu’un traitement précoce permet d’induire un contrôle virologique au long cours après arrêt de traitement. Nos résultats indiquent qu’un traitement plus long que deux ans permettrait de renforcer la diminution du réservoir. Ces résultats seront à prendre en compte pour l’élaboration de futurs essais en primo-infection visant à réduire le réservoir pour une rémission au long cours. / HIV primary infection is a critical period in the establishment of the reservoirs that justifies the initiation of an early treatment. We started a randomised trial to assess the impact of a two-year intense HAART (ANRS147 OPTIPRIM trial: five-drug therapy versus. three-drug therapy) on the blood reservoir; within this this trial, we included some pathophysiological studies. Thus, we show that during the primary infection, viruses have a low genetic diversity in blood and rectal compartments. The reservoir establishes itself as early as the first month of the infection by spreading a homogeneous viral cluster in CD4 T cells subsets, naive T cells (TN), central memories (TCM), transitional memories (TTM), effector memories (TEM), and resting T cells. This results in a disruption of the lymphocyte homeostasis, linked to the low contribution to the reservoir of TN and TCM, which are little differentiated cells with long half-lives. Moreover, we show that, at the time of the primary infection, the majority of patients do not have the ability to develop CD8 T cells responses that could suppress the viral replication, as HIV Controllers patients do. After two years of treatment, we observe that there is no evolution of the viral diversity, but the size of the reservoir is significantly reduced. The abnormalities of the CD4 T cells lymphocyte homeostasis remain, but the very early treatment was able to protect the TN and TCM. The five-drug therapy does not have any additional benefit, but we confirm the idea that early treatment can induce long-term virological control after the discontinuation of the treatment. Our results show that a treatment lasting more than two years would be able to reinforce the reduction of the reservoir. These results should be taken into account in the development of future trials aiming to reduce the reservoir in patients treated at the time of primary infection for a sustainable remission.

Previously, our lab demonstrated that heterologous immunity could result in either gain or loss of protective immunity and alteration in immune pathology following infection by a second un-related pathogen. One of the prototypical models to study T cell-mediated heterologous immunity involves two distantly related arenaviruses, namely lymphocytic choriomeningitis virus (LCMV) and Pichinde virus (PV). Each virus encodes a cross-reactive CD8 epitope that has six out of eight in amino acid (aa) similarity with respect to its counterpart at the position 205-212 of the nucleoprotein (NP205). Heterologous challenge between LCMV and PV results in 1) expansion of the cross-reactive NP205-specific CD8 T cell responses and alteration of the immunodominance hierarchy and 2) partial protective immunity (heterologous immunity). Our lab showed that cross-reactive NP205-specific CD8 T cell receptor (TCR) repertoires become extremely narrowed following a heterologous challenge between LCMV and PV. Therefore, I questioned if LCMV NP205 epitope escape variants could be isolated during a dominant but narrowed crossVI reactive NP205-specific CTL response. In the first part of my thesis, I describe the isolation of a LCMV NP-V207A CTL escape variant in vivo using PV-immune animals challenged with LCMV clone 13. The LCMV NP-V207A variant contains a point mutation, which results in the switching of valine to alanine at the third non-anchoring residue of the LCMV NP205 CD8 epitope. Immunization of mice with the LCMV NP-V207A variant results in a significantly diminished cross-reactive NP205-specific CD8 T cell response. This suggests that the point mutation is responsible for the loss in the immunogenicity of the LCMV NP205 CD8 epitope. In addition, an in vitrorescued(r) recombinant LCMV variant (r/V207A) that encodes the original mutation also induces a highly diminished cross-reactive NP205-specific CD8 T cell response in mice. In agreement with the result obtained from the intracellular cytokine assays (ICS), MHC-Ig dimers loaded with the LCMV NP205 (V-A) peptide could only detect a minute population of cross-reactive NP205-specific CD8 T cells in mice infected with r/V207A variant virus. All the data indicate that the point mutation results in a significant loss in immunogenicity of the LCMV NP205 CD8 epitope. So far, no direct link between the cross-reactive NP205-specific CD8 T cells and heterologous immunity had been established in this system. Therefore, we immunized mice with either LCMV WT or the LCMV NP-V207A variant virus and showed that a significant loss of heterologous immunity is associated with the group immunized with LCMV NP-V207A variant virus. Again, r/V207Aimmune animals also displayed a significant loss in heterologous immunity following PV challenge. This suggests that the cross-reactive NP205-specific CD8 T cells mediate the majority of heterologous immunity between LCMV and PV in vivo. In comparison to the PV-immune control group, PV clearance kinetics mediated by the cross-reactive NP205-specific CD8 T cells were significantly delayed. Finally, these data also suggest that bystander activation plays very little role in heterologous immunity between LCMV and PV. Many studies in murine systems and humans suggest that cross-reactive T cells are often associated with immune pathology. We showed that in mice that were sequentially immunized with PV and LCMV (PV+LCMV WT double immune mice), there was a development of a high incidence and high level of immune pathology known as acute fatty necrosis (AFN) following a final PV challenge. The data suggest that these cross-reactive NP205-specific CD8 T cells might play an important role in immune pathogenesis. Therefore, we asked if the cross-reactive NP205-specific CD8 T cells play a role in immune pathogenesis by comparing the incidence of AFN between the (PV+LCMV WT) and the (PV+LCMV NP-V207A) double immune mice following a final PV challenge. In agreement with our hypothesis, the result showed the (PV+LCMV NP-V207A) double immune mice developed a significantly lower incidence of AFN compared to the (PV+LCMV WT) double immune mice. However, linear correlation studies comparing the frequency of different antigen-specific CD8 T cell populations within the (PV+LCMV WT) double immune mice before challenge and the severity of AFN following the PV challenge suggest that two opposing antigen-specific CD8 T cell populations are involved in determining the final outcome of the immune pathology. The PV NP38-45-specific CD8 T cell response (PV NP38) appears to be more protective than the cross-reactive NP205-specific CD8 T cell response. In addition, a positive linear correlation between the ratio of cross-reactive NP205 to PV NP38 and the severity of AFN seem to suggest that these cross-reactive populations are important contributors to immune pathogenesis. Peptide titration studies examining the functional avidities to different antigenic specificities suggest that both populations consist of high avidity TCR and peptide MHC (TCR:pMHC) interactions. However, skewing within the cross-reactive NP205 specific CD8 T cell response towards the LCMV NP205 epitope response in one of the (PV+LCMV WT) double immune mice suggests that cross-reactive NP205 specific CD8 T cells could constitute a sub-optimal response to a PV challenge. In summary, I questioned what might be some of the immunological consequences of heterologous immunity in this model. First of all, we have established a direct link between the cross-reactive NP205-specific CD8 T cell response and heterologous immunity in LCMV and PV. Second of all, I demonstrated that a LCMV NP205 epitope escape variant could be selected in vivo under the conditions of heterologous immunity. In addition, I showed that PV clearance kinetic was significantly delayed in cross-reactive NP205-mediated heterologous immunity as compared to homologous challenge. Finally, we demonstrated that cross-reactive NP205-specific CD8 T cells could play an important role in immune pathogenesis in this model. However, correlation data indicate that two opposing antigen-specific CD8 T cell populations could ultimately decide the outcome and magnitude of immune pathology in each individual mouse. All the data presented above strongly suggest that the cross-reactive NP205 CD8 T cells play a crucial role in immune pathology in this model system by 1) interfering with the regular establishment of immunodominance hierarchy orders, or 2) exhibiting a sub-optimal protective immunity due to the nature of the cross-reactive epitope.

L’existence d’un partage des tâches a été démontrée au sein de nombreux systèmes biologiques et ce notamment en immunologie où il a été décrit dans le contexte de différentes sous-populations d’un même type cellulaire. Les cellules dendritiques plasmacytoïdes (pDC) jouent un rôle clé lors des infections virales. Les pDCs ont la capacité de sécréter de grandes quantités d’interférons de type I et de se différencier en cellules dendritiques matures capables d’activer une réponse immunitaire adaptative. Il a été proposé que ces fonctions innées et adaptatives soient séquentiellement induites après activation virale. Au cours de ma thèse, je me suis intéressée à ces deux fonctions principales des pDC et je suis arrivée à la description de différentes sous-populations de pDC activées : PD-L1+CD80- (P1), PD-L1+CD80+ (P2) and PD-L1-CD80+ (P3), démontrant qu’il existe un partage des tâches entre ces sous-types. P1 produit spécifiquement de l’IFN-α, indiquant une spécialisation en immunité innée, et promeut une réponse tolérogénique des cellules T CD4. Inversement, P3 induit une forte activation des cellules T CD4 naïves et une polarisation de type Th2, démontrant une spécialisation fonctionnelle dans l’immunité adaptative. P2 possède un profil fonctionnel intermédiaire. Plutôt qu’un lien séquentiel, nos résultats indiquent une exclusion réciproque des fonctions innées et adaptatives entre ces différents sous-types de pDC / Under microbial stimulation plasmacytoid pre-dendritic cells (pDC) secrete large amounts of type I interferon (IFN) and differentiate into mature dendritic cells capable of activating T cells. These innate and adaptive functions are thought to be induced sequentially in pDC through triggering of the IRF-7 and NFkB pathways, respectively. We found that viral activation of pDC induced their differentiation into three phenotypically distinct subsets: PD-L1+CD80- (P1), PD-L1+CD80+ (P2) and PD-L1-CD80+ (P3). P1 specifically produced IFN-α, indicating a specialization in innate immunity, while promoting weak activation and high IL-10 expression in CD4 T cells. Conversely, P3 showed increased expression of surface costimulatory molecules, improved migratory capacity, strong naïve CD4 T cell activation, and induction of Th2 differentiation. P2 had an intermediate functional profile. No conversion could be induced between subsets. We identified P1 in psoriatic skin, and blood from active lupus patients. Our results indicate reciprocal exclusion, rather than sequential link, of innate and adaptive pDC functions, with important implications in immune regulation and immunopathology.

This is a semantic study, dealing with style and technique in the short stories of D. B. Z. Ntuli. The study as a whole analyses Ntuli' s first six volumes of short stories. The first chapter is an introduction, dealing with the aim of the study. The second sub-section after aim is Ntuli's biographical notes. Full details of this author from high school attendance to his contribution during his working experience are given. Ntuli's biography is followed by the scope of study. Under this sub-heading, short story volumes to be analysed are clearly stated. The fourth sub-heading is the method of approach and a conclusion. Chapter two deals with various types of repetition, a literary technique. It analyses Ntuli's use of language, and repetition of sentences approaching it from different angles. Chapter three and four deal with choice of words. The former chapter handles the various types of language elements semantically and the latter deals specifically with the ideophone. The ideophone is sub-divided into two sub-sections: classification and usage. Chapter five deals with proverbial expressions and these are sub-divided into two sections: idioms and proverbs. The usage of idiomatic expressions is discussed under: verbs, nouns and qualificatives, while the proverbs are analysed under classification and syntax. Imagery is dealt with in chapter six. Imagery is further sub-divided into four categories: metaphor, simile, personification and symbolism. Style and structure are discussed in chapter seven. In this chapter various elements of language forms are handled: types of sentenceidiophonic; negative forms of the ideophone, with conjunctives; sentences with adverbs; the demonstratives; titles of short story volumes and naming of characters. Chapter eight is the general conclusion, reflecting on Ntuli's style and technique with special emphasis on his unique use of the language. Reference is made to discoveries regarding the author's use of vocabulary, and his techniques in using repetition as well as avoiding it, which is part of his style. His choice of words and how he arranges them on paper is also discussed. Ntuli's choice of titles in naming his short story volumes is summed up showing that these have been influenced by his background. The study concludes by suggesting areas that still require further analysis in Ntuli 's short stories. / African Languages / D. Litt. et Phil. (African Languages)

Regulatorische T-Zellen (Treg) eröffnen neue immuntherapeutische Wege zur Kontrolle unerwünschter Immunreaktionen, jedoch wirft die Heterogenität dieser Zellen die Frage auf, welche Treg-Population für die klinische Anwendung. Darauf basierend werden in dieser Arbeit drei Fragestellungen bearbeitet: i) Bestimmung der Häufigkeit von Tregs und deren Subpopulationen in verschiedenen Altersgruppen bei Empfängern einer Organtransplantation (Tx) und einer gesunden Kontrollgruppe; ii) Vergleich der Suppressorkapazität verschiedener Treg-Populationen und in vitro-Expansion der Zellen unter Erhaltung ihrer Funktionalität; iii) Klärung der Differenzierungsmerkmale von Tregs und deren Verknüpfung mit konventionellen T-Zellen (Tconv) mittels Analyse des T-Zell-Rezeptor- (TCR) Repertoires. Sowohl bei gesunden Probanden als auch bei Tx-Empfänger konnte eine altersabhängige Verschiebung von naiven (TregN) hin zu dominant zentralen Gedächtnis-Zellen (TregCM) beobachtet werden, Treg von Tx-Empfängern hatten mehr Effektor-Memory-Zellen (EM) und sie waren mehr aktiviert. In Bezug auf die Kontrolle der frühen Tconv zeigen TregCM eine erhöhte Suppressorkapazität im Vergleich zu TregN. Außerdem sind im Gegensatz zu TregN nur TregCM dazu in der Lage, Apoptose bei Responderzellen zu induzieren. Der Grund hierfür könnte in der stärkeren Expression von CTLA-4 auf TregM liegen. Die Expansionskultur führte zur phänotypischen Veränderung der TregN, deren Umwandlung in TregCM mit einer verbesserten Suppressoraktivität verbunden ist. Die Daten legen nahe, dass das Expandieren mit gesamt Treg für die Adoptive-Treg-Therapie optimal sind, da sie der größte Anteil von ihnen die hochpotenten TregCM sind. TCR-Studien mittels Next Generation Sequencing zeigen weiter, dass TregM aus TregN entstehen, anstatt aus Tconv, in einem Antigen-gesteuerten Prozess. Diese Daten belegen erstmalig neue Erkenntnisse hinsichtlich der Unterschiede der TCR-Repertoires von TregM und Tconv beim Menschen. / Regulatory T cells (Treg) offer new immunotherapeutic options to control undesired immune reactions, but the heterogeinetiy of Treg raises the question which Treg population should be used for clinical translation Thus, this project involves three main parts: i) investigating Treg frequency and subsets distribution with age in healthy donors and transplant (Tx) patients; ii) comparing the suppressive capacity of Treg subsets and expanding them in vitro without losing functionality; iii) clarifyjing the differiation relationship of Treg subsets and their relation to conventional T cells (Tconv) by T cell receptor (TCR) repertoire analysis. From both healthy donors and Tx patients, an age-dependent shift from naïve Treg (TregN) to the dominant central-memory Treg (TregCM) was observed,; However,Treg in Tx patients contained more effector-memory EM cells, , and they were pre-activated due to the exposure to allo antigens,. Regarding control of early Tconv activation, TregCM showed enhanced suppressive capacity compared to TregN; furthermore, only TregCM could induce apoptosis of responder cells while TregN could not, which may result from thehigherexpression of cytotoxic T-lymphocyte antigen 4 (CTLA-4) on TregM. Following in vitro expansion of the Treg subsets, however, TregN converted mainly into TregCM phenotype with enhanced suppression activity. The poor proliferation capacity of TregEM might indicate EM as the terminal differential stage. These data suggest that expansion with total Treg is optimal for adoptive Treg therapy as the majority of them are the highly potent TregCM. Lastly, TCR repertoire study by next generation sequencing (NGS) indicate that TregM derived from TregN rather than Tconv in an antigen-driven process. The highest similarity of the TCR repertoires was observed between TregCM and TregEM. These data reveal new insights for the first time into the distinct TCR repertoires of Treg subsets and Tconv in human by NGS technology.