Successful T cell activation depends on the recognition of antigenic peptides in the context of a Major Histocompatibility Complex molecule (pMHC) by the T cell antigen receptor (TCR), together with additional signals from co-stimulatory receptors such as CD28. Despite their importance, a thorough understanding of how TCR-pMHC binding properties relate to T cell functional responses remains unclear. In addition, there are no consensuses to the exact mechanism leading to CD28 receptor triggering. Activation of CD28 is dependent on the phosphorylation of key tyrosine residues within its cytoplasmic domain by Src family kinases. Just like the TCRs, CD28 receptors are susceptible to perturbations of the local kinase: phosphatase ratio. The K-S model postulates that upon ligand engagement, large RPTPs such as CD45 are segregated from the area of close contact, resulting in increased relative kinase concentration and CD28 receptor triggering. This hypothesis was tested in chapter 3, where elongated forms of CD80 were examined for their ability to costimulate primary T cells. CD28 costimulation was indeed diminished and there was reduced CD45 segregation from the elongated CD80 molecules. Additionally, CD28 habouring key Y170F tyrosine mutations were less susceptible to CD28 signal abrogation by elongated CD80 molecules. Interestingly, elongated CD80 molecules remained much less effective in mediating costimulation even when pMHC molecules were also elongated, suggesting that TCR-pMHC and CD28-CD80 size matching is not critical for costimulation. Despite the well-documented MHC-restriction requirement for TCR recognition, the relative energetic contributions of peptide versus MHC in TCR-pMHC interactions remain elusive. To address this question, the energetic footprints of four TCRs (1G4, JM22, A6 and G10) to HLA-A2 were determined via systematic alanine scanning mutagenesis on the HLA-A2 heavy chain in chapter 4. By targeting exclusive TCR contacting residues on the MHC, we conservatively estimate the contribution of MHCs for the four TCRs to range from 15% to over 70%. Several models have been formulated in an attempt to relate TCR-pMHC binding properties to T cell activation. Validity of the models was tested in chapter 5 using a supra-physiological TCR. By mutating key residues within the cognate pMHC, we generated a panel of TCR-pMHC with affinities that varies up to 105-fold. These reagents were used to stimulate Jurkat and primary T cells transduced with the supra-physiological TCR. Results in the Jurkat T cell system demonstrated the presence of an optimal off-rate (k_off) for TCR-pMHC interaction at low concentrations of pMHC concentration. The results argue against affinity models and the basic kinetic proofreading model for T cell activation.

The immune system is a complex network of tissues, cells and proteins which protects us against infections and invading pathogens we encounter every day. Immunosenescence refers to age-related impairments in immune function which may contribute to increased prevalence and severity of infectious disease in the elderly. How and why ageing affects the immune system is not fully understood. Using a naturally aged mouse model, work in this thesis shows that the abundance of a rare type of lymphocyte, known as NKT cells, increased across multiple immune organs. Additionally, macrophage abundance was also altered in the lymph nodes of aged mice. Invariant NKT (iNKT) cells express an invariant T cell receptor (TCR) which recognises lipids presented on the CD1d molecule. iNKT cells can be activated and respond to invading pathogens either by recognition of antigens through TCR-CD1d interactions or cytokine-dependent means. Less is known about NKT-like cells, which also express NK cell-associated surface markers, such as CD49b, but lack an invariant TCR. Data within this thesis show that both iNKT and NKT-like cell populations are abundant in the spleen and liver of aged mice. iNKT and NKT-like cells can be divided into subpopulations based on their expression of surface markers or transcription factors, and data suggests that not all subpopulations of these cells are affected by age equally. For instance, flow cytometry showed that while spleen-derived iNKT cells are significantly increased in aged mice, within the iNKT cell population the percentage representation of CD4+ cells are significantly reduced with age. Additionally, data indicates that both iNKT and NKT-like cells from aged mice show compromised responses to in vitro stimulation compared to young controls. Using bone marrow chimeras, where either young cells are reconstituted within an aged mouse or old cells are reconstituted within a young mouse, provided the opportunity to determine whether the aged environment contributes to this diminished response. Data demonstrates that the aged environment plays at least a partial role in these age-related changes to response to stimulation, however the young environment seems unable to reverse these changes. Macrophages are phagocytes which are found within all organs of the body. Studies in this thesis show that CD169+ macrophages have diminished numbers in the lymph nodes of aged mice, but this did not seem to affect the capture of the model antigen, dextran. Further studies revealed ageing affects macrophage populations differently in the different tissues within the body. For example, macrophage numbers remain constant in the spleen with ageing, but appear to increase in density in the lungs. To conclude, ageing can cause dramatic changes to the numbers and function of different cells of the immune system across multiple organs. Furthering our understanding of the ageing immune system and the underlying mechanisms which cause age-related decline in immune function is important to design strategies to improve the quality of the lives of the elderly.

In this PhD thesis the potential of T-cells as therapy for disease are explored. The applications of genetically modified T-cells for treatment of cancer and autoimmune disease; the functionality and optimal activation of T-cells are discussed. Successful treatment of cancer with T-cell receptor (TCR)-modified T-cells was first reported in 2006, and is based on recognition of a specific peptide by the TCR in the context of the MHC molecule. As antigen presentation in tumors is often defective and to avoid MHC-restriction, chimeric antigen receptors (CAR) molecules containing an antibody part for recognition of cell surface antigens and TCR and co-receptor signaling domains have been developed. Activated T-cells mount an efficient immune response resulting in the killing of the cancer cell and initiating T-cell proliferation. The rationale for using genetically modified T-cells instead of isolating tumor infiltrating lymphocytes from the tumor and expanding them (TIL therapy) is that it is often very difficult to obtain viable lymphocytes that are able to expand enough in order to use them for therapy. This thesis explores the possibility of using prostate-specific antigens to target T-cells towards prostate cancer. The prostate has many unique tissue antigens but most patients with metastatic prostate cancer have undergone prostatectomy and consequently have “prostate antigen” expression only in cancer cells. We targeted the prostate antigens TARP and PSCA with a HLA-A2 restricted TCR and a CAR respectively. In both cases the tumor-specific T-cells were able to generate potent proliferative and cytotoxic responses in vitro. The PSCA CAR-modified T-cells delayed subcutaneous tumor growth in vivo. It is evident from our in vivo experiments that the PSCA CAR T-cells were unable to completely cure the mice. Therefore, we aimed to improve the quality of the transferred T-cells and their resistance to the immunosuppressive tumor microenvironment. Stimulation with allogeneic lymphocyte-licensed DCs improved the resistance to oxidative stress and antitumor activity of the T-cells. We further investigated the potential of genetically modified regulatory T-cells (Tregs) to suppress effector cells in an antigen-specific manner. Using a strong TCR we hypothesize that the phenotype of the TCR-transduced Tregs may be affected by antigen activation of those cells. We found that the engineered Tregs produced cytokines consistent with Th1, Th2 and Treg phenotypes.

Thesis (Ph.D. in Immunology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 138-153). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Orientador: Jose Andres Yunes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-14T02:57:03Z (GMT). No. of bitstreams: 1 Ganazza_MonicaAparecida_M.pdf: 2462267 bytes, checksum: 471302ae0756c283d944a7545a0ff930 (MD5) Previous issue date: 2009 / Resumo: A leucemia linfóide aguda (LLA) é o câncer mais comum da infância. Os atuais protocolos de tratamento da LLA levam à cura em 70% dos casos e parte do sucesso se deve à aplicação de diferentes tratamentos para os pacientes estratificados em diferentes grupos de risco, segundo fatores prognóstico pré-tratamento (contagem leucocitária e idade ao diagnóstico). Contudo, pacientes considerados em remissão podem apresentar conteúdo substancial de células neoplásicas, chamada doença residual mínima (DRM), cuja proliferação está associada com a recaída da doença e que podem estar em níveis indetectáveis pelas técnicas convencionais de análise morfológica. Vários trabalhos têm mostrado que é possível prever a evolução clínica dos pacientes com base na DRM, porém, no protocolo do Grupo Brasileiro de Tratamento da Leucemia Infantil (GBTLI), a DRM não é utilizada como critério de realocação dos pacientes nos grupos de risco. Desta forma, o presente estudo objetivou (1) obter dados prospectivos de DRM com o uso de reação em cadeia da polimerase (PCR) para detecção de rearranjos de genes de imunoglobulina (Ig) e receptores de células T (TCR); (2) comparar resultados de DRM com fatores prognósticos ao diagnóstico e de resposta utilizados nos protocolos do GBTLI e (3) avaliar o valor preditivo da DRM em pacientes tratados pelo protocolo GBTLI-99. No total, foram estudadas 91 amostras de LLA pediátrica classificadas e tratadas pelo protocolo GBTLI-99. Duas metodologias foram empregadas, metodologia do grupo brasileiro (91 casos) e metodologia Biomed-1 (78 casos), as quais são baseadas em PCR com primers consenso para as regiões VDJ das imunoglobulinas e TCR seguido por análise de formação de homo/heteroduplex. Ambos métodos mostraram-se tecnicamente factíveis, reprodutíveis e capazes de detectar marcadores moleculares para a análise da DRM na maioria dos casos, a saber, 2 ou mais marcadores de DRM em 85,7 e 76,9% do total das amostras analisadas, respectivamente. As mesmas metodologias foram aplicadas para a análise da DRM nos dias 14 e 28 da terapia de indução e demonstraram concordância de 96% na detecção da DRM. Gênero masculino, idade <1 e _ 9 anos, imunofenótipo LLA-T, classificação em alto risco, ausência de CD10, presença de blastos no dia 14, presença de mais de 5% de blastos leucêmicos na medula óssea (MO) do D14 e resposta ao tratamento associaram-se com DRM positiva no D14. Já DRM positiva no D28 mostrou estar associada com imunofenótipo LLA-T, ausência de calla (CD10), presença de mais de 5% de blastos leucêmicos na MO no D28 e resposta ao tratamento. O valor preditivo da DRM, nas condições testadas, mostrou-se pouco útil para casos classificados como sendo de risco baixo pelo GBTLI/99. Para casos de alto risco, entretanto, a ausência de DRM no D14 caracteriza um grupo de pacientes com sobrevida similar a pacientes de risco básico. Novos protocolos para análise da DRM foram propostos baseados nas metodologias aplicadas no presente estudo visando otimizar o tempo e minimizar os custos / Abstract: Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer. The recent ALL treatment protocols can achieve the complete remission in 70% of cases and this success is due to different treatments for patients stratified into different risk groups , according to pre-treatment prognostic factors (white count cells and diagnosis age). Therefore, patients considered in remission may have substantial contents of neoplasic cells, the minimal residual disease (MRD). The proliferation of such neoplasic cells is associated with disease relapse and they can be undeteced by conventional methods. It has been demonstrated that the clinical evolution of patients based on MRD can be forecasted. Therefore, in the Brazilian Group for Childhood Leukemia Treatment (GBTLI), MRD is not used as a reallocation criterion of patients in risk groups. The present study aimed to (1) obtain MRD data through the use of polymerase chain reaction (PCR) for the detection of immunoglobulin (Ig) and T cell receptor genes rearrangements (TCR); (2) compare the results of MRD with prognostic factors at diagnosis and response factors used in the GBTLI protocols and (3) estimate the predictive value of MRD in patients treated by the GBTLI-99 protocol. In total, 91 samples of pediatric ALL classified and treated with the GBTLI-99 protocol were studied using two methodologies: Brazilian group methodology (91 cases) and Biomed-1 methodology (78 cases), both using the consensus PCR primers for the immunoglobulin and TCR VDJ regions followed by homo/heteroduplex formation. These methods were feasible, reproducible and able to detect molecular markers for MRD analysis in most cases, since they provided the detection of two or more markers in 85,7% and 76,9% of the samples analyzed, respectively. The same methodologies were applied in the MRD analysis on days 14 and 28 of induction therapy and the results are in agreement in 96% of the MRD detection. Male, age (<1 e _ 9 years), immunophenotyping (T-ALL), high risk classification, calla (CD10) absence, presence of blasts in peripheral blood on day 14, presence of more than 5% of leukemic blasts in the bone marrow (BM) on day 14 of treatment response were associated with positive MRD on D14. Already, positive MRD on day 28 of treatment were associated with immunophenotyping (T-ALL), CD10 absence, presence of more than 5% of leukemic blasts in the BM on D28 and treatment response. MRD predictive value on the conditions tested proved to be useful for the patients classified by GBTLI/99 as basic risk. However, for high risk cases, the absence of MRD on D14 defines a new group of patients with similar survival to basic risk patients. New protocols to the MRD analysis were proposed based on methodologies applied in this study to optimize the time and minimize the costs / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

Virtual memory (VM) CD8+ T cells represent a population of antigen-inexperienced T cells with an apparent memory phenotype. In lymphoreplete germ-free mice VM CD8+ T cells represent 10-20% of all peripheral CD8+ T cells. Their origin correlates with the levels of self-reactivity where the main factor that determinates the T-cell fate decision is the strength of homeostatic signals. In the first part of this thesis, we demonstrated that VM CD8+ T cells and naïve CD8+ T cells had distinct TCR repertoire and T-cell subsets contained different clonotypes. Moreover, 'VM clones' were enriched among VM T cells and were also present in naïve T cells. In contrast, 'naïve clones' were almost exclusively detected in naïve T cells. Next, we characterized the signaling of particular OVA-reactive TCRs from both naïve and VM subsets. We confirmed that 6 out of 8 tested TCRs were responsive to Kb-OVA. In the last part of the thesis, we developed and optimized a qPCR-based method for the relative quantification of specific T-cell clonotypes prior to and during the immune response. This method will serve as a tool for studying the biology of particular VM and naïve T-cell subsets and their role during the immune response. Keywords: T-cell receptor, homeostatic signaling, self-reactivity, virtual memory cells, T cells

The immune system generates discrete lineages of cells that are designed to respond optimally to environmental cues and infectious agents. Two distinct lineages of T cells, distinguished by expression of either an αβ or γδ T cell receptor (TCR), arise from a common progenitor in the thymus. The type of pathogen and the cytokine milieu directs effector differentiation of αβ T cells in the periphery through the induction of specific transcriptional networks. γδ T cell development is distinct from that of αβ T cells in its ordered rearrangement of TCR genes and the pairing of Vγ and Vδ chains to generate γδ T cell subsets that home to specific tissues. Unlike conventional αβ T cells, γδ T cells express a preactivated or memory phenotype prior to pathogen encounter, and recent evidence indicates that effector functions may be programmed during thymic development. To better understand the development and function of γδ T cells, we analyzed the gene expression profiles of subsets of γδ T cells segregated by TCR repertoire and maturation state in the thymus. We also determined the impact of TCR signaling and trans-conditioning on γδ T cell subset-specific gene signatures by analysis of Itk-/- and Tcrb-/- γδ T cell subsets. Our analysis has defined three stages of γδ T cell subset-specific differentiation, and indicates that γδ T cells may consist of at least two separate lineages, distinguished by the expression of a Vγ2 or Vγ1.1 TCR, that arise from different precursors during thymic development. Key transcriptional networks are established in immature γδ T cells during the first phase of development, independent of TCR signaling and trans-conditioning, with Vγ2+ cells expressing modulators of WNT signaling, and Vγ1.1+ cells expressing high levels of inhibitor of DNA binding 3 (ID3), which regulates E2A/HEB proteins. The second stage involves the further specification of the Vγ2+ subset specific gene signature, which is dependent upon ITK-mediated signals. In the third stage, terminal maturation of γδ T cell subsets occurs, dependent on both TCR and trans-conditioning signals. The expression patterns of Vγ1.1+ subsets that differ in Vδ usage diverge, and all subsets further elaborate and reinforce their effector programming by the distinct expression of chemokine and cytokine receptors. Alteration of WNT signaling or E2A/HEB activity results in subset specific defects in effector programming, indicating that the transcriptional networks established at the immature stage are crucial for the functional maturation of γδ T cells. These data provide a new picture of γδ T cell development, regulated by multiple checkpoints that shape the acquisition of subset-specific molecular signatures and effector functions.

Adaptive immune response plays a key role in maintaining homeostasis of the organism. T cells use an immense repertoire of T-cell receptors (TCRs) to discriminate between self and foreign antigens with very high sensitivity. Although we have many clues outlining how an ideal TCR repertoire is selected, and a good understanding of the TCR signaling machinery, there are still some key aspects of these processes that remain controversial. The objective of this thesis is to extend our knowledge of the very proximal events of TCR signaling, with special focus on interaction of TCR coreceptors with lymphocyte-specific kinase LCK. Coreceptor-LCK interaction has been described to regulate several aspects of T- cell development and response. We observed dynamic change of this interaction in course of T-cell development. Interestingly, CD4 and CD8 coreceptors displayed differential dynamics of interaction with LCK. Our data suggest that such disparity in coreceptor- LCK interaction leads to selection of more self-reactive TCR repertoire in CD8+ T cells. Moreover, when the highly self-reactive CD8+ T cells get to the periphery, the homeostatic signals drive their differentiation towards a more tolerogenic memory-like phenotype. To finally resolve the role of coreceptor-LCK interaction in the T-cell...

Early in the course of human immunodeficiency virus (HIV) infection a population of latently infected cells is established which persists despite long-term anti-retroviral treatment. This latent reservoir of HIV-infected cells, which reflects mechanisms of transcriptional repression, is the major barrier to cure. Efforts to target the latent reservoir have been inefficient, indicating a need for a more complete understanding of how HIV transcription is regulated. The molecular networks involved in the regulation of HIV transcription remain incompletely defined. I hypothesized that utilization of a high throughput enhanced yeast one-hybrid assay would reveal novel host transcription factor-long terminal repeat (LTR) interactions and transcriptional networks that regulate HIV. The screen identified 42 human transcription factors and 85 total protein-DNA interactions with HIV LTRs. I investigated a subset of these factors for transcriptional activity in cell-based models of infection. Krüppel-like factors 2 and 3 (KLF2 and KLF3) are repressors of HIV-1 and HIV-2 transcription whereas PLAG1-like zinc finger 1 (PLAGL1) is an activator of HIV-2 transcription. These factors regulate HIV expression through direct protein-DNA interactions and correlate with epigenetic modifications of the HIV LTR. Multiple signals converging from the cellular environment and cell-cell interactions converge at the HIV LTR to determine HIV replication and transcription. Previous work in our lab has shown that strong signaling through the T cell receptor (TCR) was required to support HIV expression and the establishment of an inducible latent infection, whereas weak TCR signaling was insufficient for these outcomes. I hypothesized that dendritic cells-CD4+ T cell interactions provide signals that compensate for weak TCR signaling, supporting HIV-1 expression and generation of inducible latent infection. I used CD4+ T cells that express chimeric antigen receptors in a dendritic cell coculture model to deliver differential signals to CD4+ T cells during cell-to-cell transmission of HIV. I found that signals from dendritic cells compensate for weak TCR signaling, facilitating cell activation, HIV expression and establishment of an inducible infection.

Suite à la rencontre d’un antigène (Ag) présenté à la surface des cellules présentatrice de l’Ag (CPA), les lymphocytes T naïfs, ayant un récepteur des cellules T (RCT) spécifique de l’Ag, vont proliférer et se différencier en LT effecteurs (1). Suite à l’élimination de l’Ag la majorité des LTe vont mourir par apoptose alors que les restants vont se différencier en LT mémoire (LTm) protégeant l’organisme à long terme. Les mécanismes qui permettent la différenciation des LTe en LTm sont encore inconnus. Pour comprendre comment les LTm CD8+ sont générés à partir des LTe, nous avons émis l’hypothèse que la densité de l’Ag présenté par les CPA peut avoir un impact sur la sélection des LT CD8+ répondant l’Ag à se différencier en LTm. De manière intéressante, nos résultats montrent qu’une immunisation avec des cellules dendritiques (DCs) exprimant un haut niveau de complexe CMH/peptide à sa surface permet le développement de LTm. À l’inverse, le développement des LTm est fortement réduit (10-20X) lorsque les souris sont immunisées avec des DCs exprimant un niveau faible de complexes CMH/peptide à leur surface. De plus, la quantité d’Ag n’a aucune influence ni sur l’expansion des LT CD8+ ni sur l’acquisition de leurs fonctions effectrices, mais affecte de manière critique la génération des LTm. Nos résultats suggèrent que le nombre de RCT engagé lors de la reconnaissance de l’Ag est important pour la formation des LTm. Pour cela nous avons observé par vidéo-microscopie le temps d’interaction entre des LTn et des DCs. Nos résultats montrent que le temps et la qualité de l’interaction sont dépendants de la densité d’Ag présenté par les DCs. Effectivement, nous observons une diminution dans le pourcentage de LT faisant une interaction prolongée avec les DCs quand le niveau d’Ag est faible. De plus, nous observons des variations de l’expression des facteurs de transcription clefs impliqués dans la différenciation des LTm tels qu’Eomes, Bcl-6 et Blimp-1. Par ailleurs, la densité d’Ag fait varier l’expression du Neuron-derived orphan nuclear receptor 1 (Nor-1). Nor-1 est impliqué dans la conversion de Bcl-2 en molécule pro-apoptotique et contribue à la mort par apoptose des LTe pendant la phase de contraction. Notre modèle propose que la densité de l’épitope contrôle la génération des CD8+ LTm. Une meilleure compréhension des mécanismes impliqués dans la génération des LTm permettra le développement de meilleures stratégies pour la génération de vaccin. Dans un second temps, nous avons évalué le rôle du signal RCT dans l’homéostasie des LTm. Pour ce faire, nous avons utilisé un modèle de souris transgénique pour le RCT dont son expression peut être modulée par un traitement à la tétracycline. Ce système nous a permis d’abolir l’expression du RCT à la surface des LTm. De manière intéressante, en absence de RCT exprimé, les LTm CD8+ peuvent survivre à long terme dans l’organisme et rester fonctionnels. De plus, une sous population des LTm CD4+ a la capacité de survivre sans RCT exprimé dans un hôte lymphopénique alors que l’autre sous population nécessite l’expression du RCT. / Following antigen (Ag) encounter presented at surface of antigen presenting cell (APC), naïve T lymphocytes, which express a T cell receptor (TCR) specific for Ag, undergo massive proliferation and differentiate into effector T cells (1). After elimination of the pathogen, most effector T cells die, while the remaining differenciates into memory T cells (LTm) which are responsible for long-term protection of the organism. The mechanism that promotes the differentiation of effectors T cells into memory T cells is still largely unknown. To understand how Tm cells are generated from effectors, we hypothesized that the density of antigen on the APC could have an impact on the selection of CD8+ T cell responders differentiating into memory. Very interestingly, our results show that immunization of mice with dendritic cells (DCs) expressing high levels of peptide-MHC complexes on their surface allow a strong development of LTm. In contrast, the development of memory T cells was strongly reduced (10-20X) when mice were immunized with DCs expressing two-fold less level of peptide-MHC complexes. In agreement with the results described above, the amount of Ag does not have any influence on T cell expansion and acquisition of effector functions, but critically affects memory T cell generation. Our data suggest that the numbers of TCR engaged in MHC/peptide recognition are important for the formation of memory T cells. To do that, we evaluated by time-lapse videomicroscopy the time of interaction between LTn and DCs. Effectively, we observed a significant reduction in the percentage of cells making prolonged interaction with DCs when the level of Ag is decreased. Moreover, we observed a modification in the expression of key transcription factors involved in the differentiation of Tm cells, such as Eomes, Bcl6 and Blimp-1. Further analysis reveals that the Ag density influences the expression of Neuron-derived orphan nuclear receptor 1 (Nor1). Nor-1 is involved in the conversion of Bcl-2 into a pro-apoptotic molecule and contributes to effector death by apoptosis during contraction phase. Our model proposes that density of Ag controls the generation of LTm. A better understanding of the role of TCR signals in the generation of LTm will help to develop better vaccination strategies. Second time, we have evaluated the role of TCR signals in Tm cell homeostasis. To do that, we have used a tetracycline-inducible expression system of the TCR in mice. This system allows us to abolish TCR expression on Tm cells. Interestingly, we show that the ablation of TCR expression did not influence the survival and functionnality of Ag-specific CD8+ LTm cells. Furthermore, our results show that a subset of CD4 Tm cells can survive in the absence of TCR expression in nonlymphopenic hosts while another subset requires the TCR expression to survive.