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Thesis advisor: Thomas Chiles / Thesis advisor: James Kim / The purpose of this thesis is to describe some of the research I conducted in Dr. Markmann’s laboratory at Massachusetts General Hospital in the field of transplantation immunology. The first portion provides background information on the immune system and its different components, eventually providing an in-depth look at regulatory T cells, and their role in transplant immunology. The second portion of the thesis is dedicated to my experiments. This part presents the materials and methods used, the previous findings that led to my experiments, the data analysis and results, and a conclusive discussion. The research I did that is included in this thesis pertains to regulatory T cells. It is believed that a potential pathway to prolonging allograft survival is to drive antigen-specific CD4+Foxp3- T cells to convert to CD4+Foxp3+ T cells. I studied the effects of retinoic acid (RA) and anti-CD45RB treatment on Foxp3 expression in CD4+ T cells. These studies were done using different lines of transgenic mice that provided models of antigen-specificity. My in vitro data demonstrated that RA is capable of boosting TGF-β-mediated Foxp3 upregulation in a synergistic manner and that anti-CD45RB is also able to increase Foxp3 expression. In vivo RA experiments were inconclusive, and due to some misfortune and time constraints in vivo anti-CD45RB experiments were not conducted. Research funded by the following sources: 2R56AI048820, 5R01AI057851, and 5K01DK079207. / Thesis (BS) — Boston College, 2011. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: College Honors Program. / Discipline: Biology.

Airway hyperresponsiveness (AHR) is one of the primary features of allergic airways disease. Despite continuous allergen exposure atopic asthmatics do not develop progressively worsening AHR. The mechanism(s) that limit AHR are unknown. Two valid candidates are regulatory T cells (Treg) and antigen presenting cells (APC). Dendritic cells (DC) are the main APC within the airways. Presentation of allergens to T cells can result in the differentiation and expansion of different subsets of T cells including effector Treg cells. The precise role of Treg and DC in the attenuation of allergen-induced AHR remains unknown. The general aim of this thesis is to investigate mechanisms to limit AHR in a murine model of atopic asthma. Specific aims are to: 1. develop a murine model of allergen-induced attenuation of AHR, 2. determine the potential role of regulatory T cells (Treg) in allergen-induced AHR attenuation, and 3. determine the potential role of airway dendritic cells (DC) in allergen-induced AHR attenuation. Balb/c mice were sensitised with intraperitoneal Ovalbumin (OVA) in aluminium hydroxide and challenged with a single, 3-weeks or 6-weeks of OVA aerosols. Aerosols were 1% OVA in sterile saline delivered for 30 minutes for three days per week. Animals were sacrificed 24 hours after the final aerosol for measurements of lung function and Methacholine (MCh) responsiveness (low-frequency forced oscillation technique), collection of bronchoalveolar lavage fluid (BALF) and serum. '...' In contrast, 6-weeks of OVA challenges decreased Treg numbers back to control levels. Adoptive transfer of 1x106 Treg taken from DLN of 3-week challenged mice attenuated AHR in single-OVA recipients (p<0.05). Furthermore, in vivo depletion of Treg in 3-week OVA challenged mice restored AHR (p<0.05 compared with control). Similar proportions of CD4+ T cells became activated following both aerosol regimes, however total numbers of airway CD4+ T cells were decreased (p<0.05), and OVA-specific CD4+ T cell proliferation in DLN was reduced (p<0.05) after 3-weeks versus one OVA aerosol. Analysis of antigen handling by airway APC populations showed antigen uptake (OVA-647) and processing (DQ-OVA) by macrophages and airway DC subsets to be down-regulated (p<0.05) after 3-weeks of OVA aerosols. In addition, adoptive transfer of Treg into single-OVA recipients did not affect antigen handling by airway APC populations. These data suggest that Treg are responsible for allergen-induced attenuation of AHR in vivo in established airways disease. AHR attenuation was associated with an altered function of airway DC, resulting in reduced antigen capture and processing, leading to limited clonal expansion of antigen-specific CD4+ T cells with limited production of Th2 cytokines. Furthermore, Treg were not directly responsible for the down-regulation of allergen capture in the airways. In conclusion, knowledge of the role of Treg and DC in attenuation of AHR could potentially result in improved and more directed therapies for the attenuation of AHR in atopic asthmatics.

Activation of immunity to self-antigens is the goal in cancer immunotherapy, whereas blocking such responses is the goal in autoimmune disease. Thus, it is not surprising that investigation into cancer immunotherapy might also produce insights for the treatment of autoimmune disease. Heat shock protein, gp96, based therapies lead to the robust activation of CD8+ cytotoxic T cells that can slow tumor growth in 60-70% of mice, but only lead to the elimination of tumors in 30-40% of animals. The primary goal of the current studies was to understand why vaccination with a secreted gp96 vaccine was not efficacious in a larger proportion of animals, and identify combination therapies that enhanced the anti-tumor activity of gp96-Ig vaccination. It was found that in mice bearing established tumors, some mice responded well to vaccination with gp96-Ig, and that the induction of CD8+ T cells was found to correlate with tumor rejection; indicating that the proportion of mice that failed to reject tumors had established mechanisms of tumor-mediated suppression of anti-tumor immunity. The mechanism of this suppression was found to differ between various tumor models, so combination therapy sought to amplify CD8+ T cell responses directly, rather than by indirectly inhibiting suppressive factors induced by established tumors. It was found that antibody-based therapies leading to the stimulation of TNFRSF25, a powerful T cell co-stimulatory receptor, caused synergistic expansion of tumor-specific T cells when given in combination with gp96-Ig vaccination and led to enhanced rejection of multiple tumor types. Interestingly, TNFRSF25 agonistic antibodies were also found to directly stimulate the proliferation of natural CD4+FoxP3+ regulatory T cells. This activity was found to be beneficial in the prevention of allergic lung inflammation when administered prior to antigen challenge. These studies have therefore identified the conditions required for successful tumor elimination following gp96-Ig vaccination, and discovered that a TNFRSF25 agonistic antibody may be used to enhance anti-tumor immunity induced by gp96-Ig. These studies have also identified TNFRSF25 stimulation as the most powerful, and physiologically relevant, method to selectively induce Treg proliferation in vivo ever discovered, with important consequences for the treatment of autoimmune inflammation.

Solid organ transplantation has evolved from being an experimental procedure to a life-saving treatment for patients with end-stage organ failure. The risk of losing a transplant due to acute rejection is very low with the use of modern immunosuppressive protocols and the short-term results are impressive. However, long-term outcomes are suboptimal and transplant recipients are at increased risks for severe complications such as cancers, opportunistic infections and cardiovascular events. The previous struggle to achieve short-term survival has turned into a search for new strategies to improve patient and transplant longevity. Regulatory T cells (TRegs), a subset of T cells, occur naturally in the immune system and have the capacity to down regulate immune responses. Under normal conditions they maintain self-tolerance and prevent excessive immune activation. Functional TReg defects lead to a massive autoimmune response and are not compatible with life. Preclinical data support that TRegs can be used as a cell therapy to prevent transplant rejection, with the potential to minimize the need for traditional immunosuppression and improve the long-term outcome. This thesis aims to enhance the translation of TReg cell therapy to clinical organ transplantation. In particular, strategies for isolation and expansion of TRegs from uremic patients awaiting kidney transplantation have been assessed. A non-invasive imaging technique to study T cell products after intravenous administration was developed, for use in future clinical trials. The performance of a novel cell purification technique was investigated to potentially improve the clinical production of TRegs. The thesis demonstrates that TRegs can be isolated and expanded from uremic patients to display potent suppressive properties in vitro. The mode of isolation and expansion affect the functional characteristics, where cells purified with cytometry based techniques and expanded with mature dendritic cells were the most advantageous. T cells can be labeled using the radioactive tracer [111In]oxine with preserved viability and subsequently followed in vivo with SPECT/CT for more than 1 week after intravenous administration. The use of microfluidic switch technology offers a novel way of purifying TRegs at high speed, purity and viability, under conditions compatible with clinical use.

Natürlich vorkommende regulatorische T-Zellen spielen eine entscheidende Rolle für die intestinale Immunhomöostase und Limitierung von (Auto)-Immunität. Sie exprimieren den Transkriptionsfaktor Foxp3 und an der Oberfläche die α-Kette des IL-2 Rezeptors (CD25). Im Tiermodell verhindern regulatorische T-Zellen Autoimmunopathien, Transplantatabstoßungen und entzündliche Darmerkrankungen. Da Foxp3+ regulatorische T-Zellen nur äußerst geringe Mengen an Interleukin-2 synthetisieren, sind sie auf eine adäquate Versorgung angewiesen. Konventionelle T-Zellen werden als bedeutende IL-2 Quelle für Treg-Zellen vermutet, doch über die Mechanismen und räumlich-zeitliche Dynamik der Treg-Effektor-Zellinteraktion ist bisher nur wenig bekannt. In dieser Arbeit wurden Foxp3+ regulatorische T-Zellen in Mausgeweben analysiert und Zellinteraktionen mit Effektorzellen im Darm charakterisiert. Es wurde ein theoretisches Modell zur Evaluierung von Zell-Zellkontakten erarbeitet und experimentell überprüft. Es konnte gezeigt werden, dass in der Akutphase einer T-Zell-induzierten Kolitis und im Kolon von gesunden Wildtyp-Mäusen Foxp3+ regulatorische T-Zellen an Ki-67+ proliferierenden T-Zellen akkumulieren. Diese Zellinteraktionen sind abhängig von Interleukin-2, da IL-2 defiziente Mäuse keine signifikanten Treg-Effektor-Zellakkumulationen aufweisen. Die Analyse der Genexpression konnte zeigen, dass Ki-67+ Zellen Interleukin-2 produzieren. Lokal sezerniertes Interleukin-2 könnte als Sensor für Entzündungsprozesse chemotaktisch auf Foxp3+ regulatorische T-Zellen wirken und die Akkumulation an proliferierenden, IL-2 produzierenden Effektorzellen bedingen. Dieser Mechanismus könnte einerseits zur lokalen Versorgung mit IL-2 dienen und gleichzeitig regulierend auf Effektorzellen in unmittelbarer Umgebung wirken. Dieser Prozess würde zur Erhaltung von regulatorischen T-Zellen in der Peripherie und zur Sicherung der intestinalen Immunbalance beitragen.

Au cours de ces 20 dernières années, les inhibiteurs de "checkpoint blockade" sont une des stratégies les plus prometteuses pour l’immunothérapie du cancer. une partie de l'effet anti-tumoral de ces inhibiteurs reposerait sur la déplétion des lymphocytes T régulateurs (TREG). ici, nous avons examine si la forte expression d'ICOS sur ces cellules pourrait être utilisée comme marqueur pour cibler les TREG et améliorer le rejet tumoral. nous rapportons qu'un nouvel acm anti-ICOS humain deplete préférentiellement les TREG dans un modèle de souris nsg humanisées (cd34+) menant a une augmentation du rapport cd8+/treg. toutefois, cela ne suffisait pas a influer sur la croissance de la lignée de cancer du sein mda-mb-231. nous avons administre du cyclophosphamide (CTX) a faible dose pour induire de la mort cellulaire immunogène et stimuler la réponse anti-tumorale. le traitement des animaux avec une combinaison CTX + anti-ICOS conduit 0 une réduction drastique de la croissance tumorale alors que les traitements individuels ont des effets modérés. Grâce a la cytométrie de masse (cytof), nous avons observe une plus forte expression de cd45ro, hla-dr et ki-67 sur les ltcd8+ du groupe traite par la combinaison. de plus, d'autres analyses suggèrent que les monocytes et PDC humains et les cellules myéloïdes murines pourraient être impliqués dans cet effet. au final, nos résultats sont la première démonstration que les souris humanisées peuvent être utilisées pour développer de nouvelles immunothérapies anti-cancer et indiquent que le ciblage TREG avec une combinaison d'ACM anti-ICOS et la chimiothérapie est une strat2gie pertinente pour renforcer la réponse anti-tumorale. / Checkpoint blockade inhibitors are the most promising and effective strategy for t-cell mediated cancer immunotherapy of the past 20 years. Part of the anti-tumoral effect of these checkpoint inhibitors might be due to regulatory T cell (treg) depletion. Here, we investigated whether the reported high expression of icos on treg might be used as a flag to target treg and improve tumor rejection. We report that a novel anti-human icos mab preferentially depleted treg in immunodeficient nsg mice reconstituted with cd34+ progenitors, leading to an increased cd8+/treg ratio. However, this was insufficent to affect growth of the breast cancer cell line mda-mb-231. We thus administered low dose cyclophosphamide (ctx) to induce immunogenic cell death and stimulate anti-tumor response. Treatment of humanized mice with a combination of ctx+ anti-icos mab led to a drastic reduction in tumor growth whereas single treatments had only moderated effect. Using mass cytometry (cytof), we observed higher expression of cd45ro, hla-dr and ki67 on tcd8+ of the combined-treatment group. Accordingly, depletion of cd8+ t cells partly abolished the therapeutic effect of the combination. Moreover, additional analyses suggest that human monocytes and pdc and murine myeloid cells are involved in this effect. Altogether, our results represent the first demonstration that humanized mice can be used to develop novel therapeutic strategies for cancer immunotherapy and indicate that targeting treg with a combination of anti-icos mab and chemotherapy is a relevant strategy to release the immune response to the tumor.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第22122号 / 医科博第107号 / 新制||医科||7(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 生田 宏一, 教授 濵﨑 洋子, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Chronic inflammation is associated with the pathophysiology of obstetrical disorders (e.g. preterm birth [PTB]) and gynecological diseases (e.g. endometriosis); however, the exact mechanism(s) for these conditions are unknown. Numerous immunological conditions and disease states (e.g. inflammatory bowel disease, Crohn’s disease, systemic lupus erythematomus) also disrupt the microbiome homeostasis by inducing a number of changes in the microbial flora when compared to that of healthy individuals. Furthermore, the gastrointestinal (GI) microbiome is one of the principal regulators of circulating estrogens which are known to directly impact the female reproductive disorders endometriosis and PTB. Thus, an alteration of microbial species could indicate a shift in immune balance from homeostatic to pro-inflammatory, and an aberrant estrogen metabolism that precipitates the development of disease stages in endometriosis and/or PTB. The Braundmeier-Fleming lab has developed a systems biology model that investigates the interactions between the immune system, microbial dynamics (in the GI and reproductive system) and estrogen metabolism, in women, as a potential diagnostic tool for endometriosis and PTB. This dissertation, therefore, examined how inflammation triggered by female reproductive disorders (endometriosis or PTB) alter the systemic and localization immune responses, the microbial communities in the urogenital (UG), peritoneal and GI mucosal epithelium, as well as levels of excreted conjugated estrogen. The first specific hypothesis is that inflammation associated with endometriosis alters microbial dynamics and functions that are distinct from those of non-diseased patients. Preliminary data indicated that reproductive tract microbial communities from patients with endometriosis are unique when compared to non-disease patients. Therefore, the central aims of this study are to identify the immune and microbial profiles of patients diagnosed with endometriosis and determine if an alteration of these profiles impact estrogen signaling, thus driving disease pathogenesis. Additionally, I hypothesized that surgery or hormonal therapy will temporarily restore the microbiome and estrogen levels of patients with endometriosis. Differences in systemic (blood) regulatory T cell (Treg) and T-helper 17 (Th17) cell populations (tolerant and inflammatory, respectively) were measured by flow cytometry, and the immune mediators was measured by serum cytokine levels via 10-plex-ELISA kits. Immunohistochemistry was used to identify resident Th17/Treg immune cell distribution within the endometrium and ectopic endometriotic lesions, and RORγt+/FOXP3+ transcripts within these same tissues were analyzed by real-time-qPCR. We implemented high-throughput non genomic sequencing targeting bacterial-V4 16S rRNA and robust bioinformatics analyses to characterize microbial composition/diversity within the GI (fecal swab), vaginal (vaginal swab), and UG (urine) cavities. Alterations in estrogen metabolism, parent estrogens and metabolites, in urine were analyzed via LC-MS/MS. Patients with endometriosis exhibit 1) systemic and localized inflammation within ectopic and endometrial tissues, 2) altered GI/UG microbial dynamics, 3) aberrant levels of endogenous estrogen and estrogen metabolites, 4) dampened inflammation (caused by disease) due to hormonal therapy, 5) altered bacteria populations in the gut and vaginal canal of patients with endometriosis due to hormonal therapy treatment, and 6) increased post-surgical variability in microbial community dynamics. The second specific aims examined the hypothesis that induction of endometriosis in baboons (P. Anubis) results in chronic systemic and tissue specific inflammation through regulation of Th17 and Treg populations. Further, the induction of endometriosis altered GI/UG/peritoneal cavity microbial communities that are distinct from non-diseased animals. Utilizing a non-human primate animal model of induced endometriosis allowed us to characterize factors involved at the early onset of endometriosis and throughout the disease progression. We collected samples from 8 baboons at pre-inoculation (no evidence of disease) and at 3, 6, 9, and 15 months post-induction of the disease. We found that the induction of endometriosis decreased peripheral Tregs cells while Th17 cells increased at all post-induction collections with reduced ratio of total Tregs to Th17 cells indicating systemic inflammation. Microbial community diversities as well as abundances at each sample site (GI, UG [vagina, urine] tracts and peritoneal cavity) were also altered at post-induction. These results therefore suggest that induction of endometriosis in non-human primates caused an inflammatory shift. Disease induction also resulted in altered vaginal, urinary and fecal microbial profiles, which may drive inflammation through the production of inflammatory mediators. The last specific aims studied the hypothesis that patients who deliver preterm have a systemic and placental inflammatory phenotype and abnormal estrogen levels during pregnancy that are distinct from those of patients with term delivery. Biological samples were collected at 8-12 weeks, 20-24 weeks, 32-36 weeks, at delivery and 6 weeks postpartum. Subjects with PTB showed signs of systemic inflammation with an elevation in Th17:Treg ratio, greater Th17 and lower levels of natural Tregs during the 2nd trimester, and lower inducible Tregs during the 3rd trimester and at delivery. Placental tissues from subjects with PTB also had an inflammatory immune phenotype (higher Th17) within the decidua basalis and maternal-fetal interface. Immunological shifts from tolerant to inflammatory were observed in both patient groups, but these shifts occurred early in gestation for subjects with PTB and at a later gestational age for subjects delivering at term. Levels of conjugated parent estrogens and estrogen metabolites were reduced in subjects with PTB, indicative of an abnormal production of estrogen. These analyses gave us a better understanding of the inflammatory cascade with estrogen metabolism associated with pregnancy, and how these effects are correlated with premature labor. The data from this study suggest that the levels of endogenous estrogen and estrogen metabolites of estrogen metabolism were abnormal in PTB and endometriosis disease models of inflammation compared to their respective controls. In the human and non-human primate model of endometriosis studies, we observed that both patients and baboons with endometriosis had systemic and resident inflammatory phenotypes and an alteration in mucosal microbial community dynamics compared to their respective controls. All together, our long-term goal is to identify factors from the microbiome and/or the immune system that would allow us to have early non-invasive diagnostics for endometriosis or to predict which mothers are most at risk to encounter PTB. Furthermore, it would allow us to determine whether the mucosal microbiome may be a good indicator of immune stress, and if alternative therapies can alter microbial community dynamics—thereby eliminating immune stress associated with female reproductive diseases. These findings may have a substantial impact on the obstetrical care and management of patients with endometriosis and women at risk for PTB, as well as provide evidence to support the development of novel therapeutics to treat these diseases.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第17951号 / 医博第3835号 / 新制||医||1000(附属図書館) / 30781 / 京都大学大学院医学研究科医学専攻 / (主査)教授 河本 宏, 教授 生田 宏一, 教授 斎藤 通紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM