A side effect of generating an immune system for defense against invading pathogens is the potential to develop destructive cells that recognize self-tissues. Typically, through the "education" of developing immune cells, the organism inactivates potentially self-destructive cells, resulting in what is called self-tolerance. I proposed to explore the molecular mechanisms responsible for the induction and maintenance of tolerance. Our lab has developed a model of induced immune tolerance to skin and islet allografts utilizing a donor-specific transfusion of spleen cells and a brief course of anti-CD40L antibody. Because the difficulty in isolation of tolerant T cells from this system is prohibitive to performing large screens on these cells directly, I have chosen to study an in vitro CD4+Th1 cell line, A.E7, which can be made anergic via stimulation through the T cell receptor in the absence of costimulation. I hypothesized that anergized T cells upregulate genes that are responsible for the induction and maintenance of anergy and therefore exhibit a unique RNA expression profile. I have screened anergic cells using Affymetrix GeneChips and identified a small number of genes that are differentially expressed long-term in the anergic population compared to mock-stimulated and productively activated controls. The results have been confirmed by quantitative RT-PCR for each of the candidates. One of the most promising, the zinc-finger transcription factor Egr-2, was verified to be expressed long-term by western blotting, demonstrating perfect correlation between Egr-2 protein expression and the anergic phenotype. Silencing Egr-2 gene expression by siRNA in A.E7 T cells prior to anergy induction rescues the cells from the inability to phosphorylate ERK-1 and ERK-2 and also results in increased proliferation in response to antigen rechallenge. In this study I report that Egr-2 is specifically expressed long-term in anergic cells, protein expression correlates inversely with responsiveness to antigen rechallenge, and that Egr-2 is required for the full induction of anergy in T cell clones.

Definitive evidence that natural killer (NK) cells mediate an antiviral effect in vivo was obtained using murine cytomegalovirus (MCMV) as a model system. Adoptive transfer studies using a variety of physical and immunochemical techniques to enrich and deplete NK cell activity showed that the cell population capable of mediating resistance (as assayed by enhanced survival and reduction in spleen virus titers) had the phenotype of an NK cell: a nylon wool nonadherent, asialo GM1+, NK 1.2+, ly 5+, Thy-1-, Ia-, low-density lymphocyte. Adoptive transfer of IL-2-dependent cloned NK cells (but not T cells) also provided resistance. NK cells did not provide resistance to lymphocytic choriomeningitis virus (LCMV). Selective depletion of NK cell activity by injection of mice with antibody to anti-asialo GM1 lowered resistance to MCMV, mouse hepatitis virus, and vaccinia virus but not to LCMV. NK cell depletion resulted in up to 1000-fold increases in spleen and liver virus titers, correlating with more severe pathology in these organs. NK cells were found to have antiviral effects early (0-3 days) but not late (6-9 days) postinfection. NK cell depletion resulted in markedly increased MCMV-induced suppression of T cell function, which is probably responsible for the delayed clearance of virus seen in these mice. NK cell depletion resulted in increased virus synthesis during persistent MCMV infection, but had no effect on the course of persistent LCMV infection, despite elevated NK cell and interferon (IFN) levels found in these LCMV-infected mice. The reason why NK cells play a role against MCMV but not LCMV infection was not due to differences in NK cells induced by these 2 viruses, but more likely due to target cell susceptibility. IFN pretreatment of MCMV-infected cells failed to protect them against NK cell-mediated lysis, whereas uninfected and LCMV-infected cells were almost totally protected. These IFN-pretreated, LCMV-infected cells were not resistant to cell-mediated lysis in general, as this treatment increased their sensitivity to virus-specific T cell-mediated lysis by 2- to 3-fold. This enhanced sensitivity to lysis correlated with increased surface expression of H-2 antigens, but not viral antigens. In summary, these studies provide compelling evidence that NK cells can mediate antiviral effects in vivo, and provide some insights into their mode of action and consequences of their disfunction.

The aim of this survey is also to determine vaccination coverage in compulsory vaccination of kids in the first years of life, compliance of vaccine dose timing with vaccination schedule and reasons why some children were not vaccinated. Another goal is to analyse the reasons for some kids in the first years of life being vaccinated late and whether some kids in groups most at risk are vaccinated against tuberculosis. We used a quantitative research method in the form of a cross-sectional study comprised of data collection and analysis. Our research sample consisted of children born in the years 2009, 2010 and 2011 in České Budějovice district. The reason why we have chosen this particular group of children is that (if applicable vaccination schedule had been adhered to) they were supposed to be fully vaccinated against diphtheria, tetanus, whooping cough, haemophilus influenzae b, Hepatitis B virus, poliomyelitis (DTPHibHB). This group of 831 children (427 boys and 404 girls) have been chosen by random sampling from practising paediatricians. These selected physicians provided us with data about all children born in above mentioned years. The results showed that overall vaccination coverage for Infanrix Hexa vaccine (four doses) is 98.2% and 97.26% for Priorix vaccine (two doses). Vaccination coverage for tuberculosis in the years 2009 and 2010 was 90.05%. In the first years of life, up to 88.1% of newborns were vaccinated against tuberculosis ? at least, according to vaccination schedule (and applicable Order). In the first year of life, 99.9% of children have been given three doses of Infanrix Hexa vaccine against diphtheria, tetanus, whooping cough, poliomyelitis, Haemophilus influenzae b invasive disease and Hepatitis B virus. In the first eighteen months of life, 97.4% of children in analysed sample were given fourth dose of said vaccine. 68.6% of children were given the first dose of Priorix-Tetra vaccine in the first fifteen months of life. In the following six to ten months, 75.4% of children were given the second dose. Further analysis revealed that in 40.5% of analysed records some vaccine dose timings were not met. The most common reason for vaccination delay was the state of health of the child. Two instances of vaccination delay were also caused by the omissions of parents. In 2010, indication for vaccination against tuberculosis was recorded for one newborn. In 2011, for four children from the analysed sample vaccination against tuberculosis was recommended. In all cases, vaccination against tuberculosis has been carried out in calmetisation centre. Taking into account my study and its results I can safely assume that the goals of my diploma thesis have been met and the hypotheses set forth have been confirmed by the research.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective therapeutic option for many malignant diseases. However, the efficacy of allo-HSCT is limited by the occurrence of destructive graft-versus-host disease (GVHD). Since allogeneic T cells are the driving force in the development of GVHD, their activation, proliferation, and differentiation are key factors to understanding GVHD pathogenesis. On the other hand, antigen-presenting cells (APCs) are essential for allogeneic T-cell priming and the development of GVHD. The T-box transcription factor T-bet is a master regulator for IFN-γ production and Th1 differentiation. T-bet also regulates the functions of APCs including dendritic cells (DCs) and B cells. Therefore, we investigated the role of T-bet in T cell responses, as well as on APC functions, in acute GVHD (aGVHD) using murine models of allogenic bone marrow transplantation (allo-BMT). In Chapter 2, we evaluated the roles of T-bet and IFN-γ in T-cell responses. T-bet-/- T cells induced significantly less GVHD compared with either wild-type (WT) or IFN-γ-/- counterparts in CD4-driven major histocompatibility complex (MHC)- or minor histocompatibility antigen (miHA)-mismatched models. We defined several T-bet-dependent but IFN-γ-independent molecules that may account for this distinct outcome. Further study indicates that T-bet also controls the optimal activity of Th17 cells to induce GVHD. Moreover, the compromised graft-versus-leukemia (GVL) effect of T-bet-/- T cells could be essentially reversed by IL-17 neutralization. Thus, targeting T-bet or regulating its downstream effectors independent of IFN-γ may be a promising strategy to control GVHD in the clinic. In Chapter 3, we evaluated the role of T-bet on APCs and found that T-bet-/- recipients developed much milder GVHD than their WT counterparts in MHC-mismatched or CD4-depedent miHA-mismatched models. As the functional readout of APCs, allogeneic donor T cells, particular CD4 subpopulation, significantly reduced IFN-γ production, proliferation and migration, and caused less damage in liver and gut in T-bet-/- recipients. We further observed that T-bet on recipient hematopoietic APCs, particular DCs, was primarily responsible for donor T-cell response and pathogenicity in GVHD. In fact, Trail/DR5 interaction served as a major signaling pathway responsible for donor T-cell apoptosis and impaired GVHD development in T-bet-/- recipients. Furthermore, T-bet expression on the host is largely dispensable for the GVL effect. Taken together, we propose that T-bet is a potential therapeutic target for the control of GVHD through regulating T cells as well as APCs. We believe further exploration and understanding of the immunobiology of T-bet in controlling the activities of T cells and APCs in GVHD will expand therapeutic options for the continuing success of allo-HSCT.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / O estudo teve como objetivo construir e validar quanto ao conteÃdo e aparÃncia uma cartilha educativa para a prevenÃÃo da transmissÃo vertical do HIV. Tratou-se de uma pesquisa metodolÃgica. Para realizaÃÃo do estudo, seguiram-se as etapas: submissÃo do projeto ao comità de Ãtica em pesquisa; levantamento bibliogrÃfico; elaboraÃÃo da cartilha e validaÃÃo do material por juÃzes especialistas e representantes do pÃblico-alvo. Implementou-se, inicialmente, o levantamento bibliogrÃfico a partir de 15 publicaÃÃes do MinistÃrio da SaÃde do Brasil acerca dos cuidados que as mÃes devem ter para a prevenÃÃo da transmissÃo materno-infantil do HIV. A cartilha construÃda foi intitulada âComo prevenir a transmissÃo do HIV de mÃe para filho? Fique por dentro!â, abordando os cuidados para a prevenÃÃo da transmissÃo vertical desde o prÃ-natal, perpassando pelo parto e puerpÃrio. Na etapa de elaboraÃÃo da cartilha, foram elaborados os textos a partir das informaÃÃes levantadas na pesquisa bibliogrÃfica e consultada uma especialista em desenho para confeccionar as figuras. Para a validaÃÃo de aparÃncia e conteÃdo, foram selecionados nove juÃzes conforme critÃrios prÃ-estabelecidos e para a validaÃÃo de aparÃncia pelo pÃblico-alvo, foram selecionadas 30 mulheres, sendo 23 gestantes e 7 puÃrperas HIV positivas, captadas em duas maternidades pÃblicas da cidade de Fortaleza-Ce. O perÃodo de coleta de dados com juÃzes e mulheres se deu entre agosto e dezembro de 2013. Para a coleta de dados, foram utilizados dois instrumentos, um direcionado aos juÃzes e outro para o pÃblico-alvo. Quanto à validade de conteÃdo da cartilha, foi utilizado o Ãndice de Validade de ConteÃdo (IVC), com ponto de corte de 0,78. Quanto à validade de aparÃncia pelos juÃzes e pÃblico-alvo, foram considerados validados os itens que obtivessem nÃvel de concordÃncia mÃnimo de 75% nas respostas positivas. As sugestÃes e opiniÃes foram compiladas e apresentadas em quadros. A cartilha educativa em estudo foi a primeira a ser desenvolvida dentro da temÃtica e mostrou-se como um material validado, visto que apresentou bom IVC global de 0,87 e nÃvel de concordÃncia excelente entre juÃzes, variando de 91,1% a 100% e o pÃblico-alvo que oscilou de 93,3% a 100%. Diante das sugestÃes e contribuiÃÃes durante o processo de validaÃÃo, a cartilha passou por modificaÃÃes, ajustes e acrÃscimos a fim de tornÃ-la mais eficaz, de forma que a cartilha passou de 20 pÃginas para 28 em sua versÃo final. Acredita-se que o uso deste material com mulheres HIV positivas, desde o perÃodo prÃ-concepcional atà o pÃs-parto, facilitarà a prÃtica da enfermagem, tendo em vista que se constitui em uma tecnologia ilustrada capaz de favorecer o diÃlogo entre profissionais e mulheres, facilitar a aquisiÃÃo de conhecimentos e proporcionar o empoderamento das mulheres, gestantes e puÃrperas HIV positivas. / The study aimed to construct and validate the contents and appearance of an educational booklet for the prevention of vertical transmission of HIV. This was a methodological research. To conduct the study, we followed the steps: submission of the project to the research ethics committee; literature survey; development and validation of material by expert judges and representatives of the target audience. Initially it was implemented the bibliographic from 15 papers of the Ministry of Health of Brazil about the care that mothers should have for the prevention of mother to child transmission of HIV. The booklet developed was titled "How to prevent transmission of HIV from mother to child? Get inside!", addressing care for the prevention of vertical transmission in pre-natal, childbirth and postpartum. In the preparation of the primer stage, the texts were compiled from the information gathered in the literature and consulted one expert in design for making the figures. Nine judges were selected to validate appearance and content, according to pre-established criteria and to validate appearance by the target audience, 30 women were selected, with 23 pregnant and 7 postpartum HIV positive captured in two public hospitals in the city of Fortaleza - Ce. The period of data collection with judges and women occurred between August and December 2013. To collect data, two instruments, one directed to the judges and one for the target audience, were used. For the validity of the content of the booklet, we used the Content Validity Index ( CVI ), with a cutoff of 0.78. For the validity of appearance by the judges and audience were considered validated items that obtain a minimum level of agreement of 75 % for positive responses. The suggestions and opinions were collated and presented in tables. The educational booklet in the study was the first to be developed within the theme and showed up as a validated material, since it showed good overall CVI of 0.87 and excellent level of agreement between judges, ranging from 91.1% to 100% and between the audience that ranged from 93.3% to 100% . Given the suggestions and contributions during the validation process, the booklet has undergone modifications, adjustments and additions to make it more effective, so that the playbook went from 20 pages to 28 in the final version. It is believed that the use of this material with HIV positive women from pre-conception period to the postpartum facilitate nursing practice, because that is illustrated in a technology capable of promoting dialogue between professionals and women, facilitate the acquisition of knowledge and provide the empowerment of women, HIV positive pregnant women and postpartum women.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2014-12-18T12:34:27Z No. of bitstreams: 1 Dissertação - Davi Mello Cunha Crescente Alves - 2013.pdf: 853772 bytes, checksum: 72911b50f56ac854e4084c11c9191154 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2014-12-18T14:28:20Z (GMT) No. of bitstreams: 1 Dissertação - Davi Mello Cunha Crescente Alves - 2013.pdf: 853772 bytes, checksum: 72911b50f56ac854e4084c11c9191154 (MD5) / Made available in DSpace on 2014-12-18T14:28:20Z (GMT). No. of bitstreams: 1 Dissertação - Davi Mello Cunha Crescente Alves - 2013.pdf: 853772 bytes, checksum: 72911b50f56ac854e4084c11c9191154 (MD5) Previous issue date: 2013-12-18 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The White-Nose syndrome is an emergent infectious disease that had already killed almost six millions North American bats and spread more than two thousand kilometers. Even so, studies about their possible impacts upon hosts are still lacking, principally upon all the susceptible North American bats. We predicted the consequences of the WNS spread in the North American hosts by generating an environmental suitability map for the disease, and then, we overlaid with the extension of occurrence of all hibernating bats in North America. We assumed that all intersection localities will somehow negatively affect bat’s local populations, and we reassessed their conservation status based on their potential population reduction. 16% of the North American hibernating bat fauna were considered threatened under this WNS potential spread. We believe our results could contribute with governments conservation actions. / (Sem resumo)

HIV-2 differs from HIV-1 in that many infected people experience normal survival, whilst only 20% progress rapidly to AIDS. Understanding mechanisms of delayed HIV-2 disease progression could provide new insights into HIV control. The Caio Community Cohort was established in Guinea-Bissau in the setting of high HIV-2 prevalence. This thesis investigates the role of polymorphic host restriction factors of the TRIM family in HIV-2 outcome. TRIM proteins are a family of E3 ubiquitin-ligases, where closely-related TRIM5α and TRIM22 are thought to inhibit HIV-1 transcription, uncoating and budding. There was an association between TRIM5α amino acid substitution R136Q and reduced HIV-2 viral load/prolonged survival. Conversely, P479L was enriched among HIV-2 infected participants and progressors with CD4+ T cell decline. TRIM22 was highly polymorphic in this cohort, revealing three novel coding variants. Although most substitutions were located in the putative virus-interacting PRYSPRY domain, two in the coiled-coil, D155N and R242T, showed significant and divergent associations with survival. R242T was enriched in HIV-2 infected participants, who progressed to death at twice the rate of wild-type controls. In silico studies predicted D282, D360, and R321 of TRIM22 to be highly conserved, exposed residues, for which polymorphisms would be deleterious. When aligned with sequences from the potent HIV-1 restriction factor, rhesus macaque TRIM5α, TRIM22 substitutions R321K, T415I, and D360Y were spatially relevant to residues involved in HIV-1 restriction. The role of TRIM22 in HIV restriction was supported by in vitro pilot studies showing that TRIM22 was upregulated by HIV-1 infection in a lymphoid cell line and co-localised with the HIV-1 capsid protein p24. Overexpression of TRIM22 resulted in the restriction of VSV-G pseudotyped HIV-1 and SIVmac. The R242T substitution diminished TRIM22's restriction of HIV-1 and SIVmac: protein analysis suggested that this may be due to the inability of the R242T mutant to fully dimerise.

Les peptides antimicrobiens sont considérés comme une nouvelle classe prometteuse d'agents anti-infectieux. Afin de développer de nouveaux agents efficaces et non toxiques, des stratégies de conception rationnelle peuvent être utilisées. Dans cette perspective, nous avons utilisé une approche computationnelle pour concevoir trois peptides synthétiques ([I5, R8] MP, EcDBS1R6 et PaDBS1R1). En déterminant la concentration minimale inhibitrice, nous avons montré que tous les peptides sont actifs contre les bactéries Gram-négatif et -positif. Seul [I5, R8] MP a montré une activité antifongique. La mesure de la concentration de peptide provoquant 50 % de mortalité cellulaire a permis de montrer que les peptides étaient faiblement ou non hémolytiques, sans toxicité vis-à-vis des cellules embryonnaires rénales humaines HEK-293. La cinétique bactéricide a révélé que PaDBS1R1 et [I5, R8] MP tuent rapidement E. coli en comparaison à S. aureus et que EcDBS1R6 élimine rapidement les deux souches. Des études de perméabilisation et de dépolarisation combinées à de la microscopie électronique à haute résolution (FEG-SEM) ont montré un mécanisme membranolytique des peptides. L'analyse de la structure des peptides par spectroscopie de dichroïsme circulaire et résonance magnétique nucléaire, ainsi que par modélisation moléculaire lors de leur interaction avec une membrane modèle, révèle une conformation en hélice alpha amphipatique. En conclusion, notre étude indique que l'évaluation structurale et fonctionnelle de peptides antimicrobiens synthétiques conçus de manière rationnelle représente une stratégie prometteuse pour le développement de nouveaux agents antimicrobiens. / Antimicrobial peptides (AMPs) have been considered as a potential novel class of antimicrobial compounds. In order to generate new potent and non-toxic antimicrobial agents, rational design strategies may be employed. In this view, we used a computational method to design three synthetic AMPs ([I5, R8] MP, EcDBS1R6 and PaDBS1R1). By determining the minimum inhibitory concentration, we found that all the peptides were active against Gram-negative and -positive bacteria. Only [I5, R8] MP was found to display antifungal activity. The determination of the peptide concentration producing 50% of cell lysis revealed low or no hemolytic activity, with no cytotoxicity towards human embryonic kidney cells HEK-293. During time-kill assays more rapid bactericidal effects were observed for PaDBS1R1 and [I5, R8] MP against E. coli compared to S. aureus. For the peptide EcDBS1R6, identical killing curves were obtained for both bacterial strains. Membrane permeabilization and depolarization studies combined with field emission gun scanning electron microscopy (SEM-FEG) revealed that a membranolytic mechanism occurs for these peptides. When analyzed by circular dichroism and nuclear magnetic resonance microscopy or by molecular dynamics simulations during interaction with a membrane model, peptides were shown to adopt an amphipathic alpha-helical conformation. In conclusion, our results indicate that the structural and functional evaluation of rationally designed synthetic AMPs represents a promising strategy for the development of potent novel antimicrobial agents.

Respiratory syncytial virus (RSV) is the most common cause of acute lower respiratory tract infection in young children worldwide, accounting for an estimated 33.8 million cases of respiratory disease, over 3 million of which require hospitalization, and between 66,000 and 199,000 deaths in this susceptible population. Additionally, severe RSV infection early in life is associated with an increased risk of wheeze and other airway disorders later in life. Despite this, there is currently no vaccine or economically reasonable prophylactic regimen to prevent infection. While disease is typically more severe in infancy RSV can infect throughout the lifespan repeatedly as the body does not develop protective immunity during primary or subsequent infection. The mechanisms behind this incomplete immunity are unclear. RSV has been reported to infect numerous extra-epithelial cell types. Interestingly, viral infection in human mesenchymal stem cells (MSCs) has been reported, but the consequences are poorly understood. MSCs are an immune regulatory cell population present in nearly every organ including the nasal mucosa and the lung. They play a role in regulating immune responses and mediating tissue repair. In the following studies we sought to determine whether RSV infection of MSCs enhances their immune regulatory functions and contributes to RSV-associated lung disease. RSV was shown to replicate in human MSCs by fluorescence microscopy, plaque assay, and expression of RSV transcripts. RSV-infected MSCs showed differentially altered expression of cytokines and chemokines such as IL-1β, IL-6, IL-8 and SDF-1 compared to normal human bronchial epithelial cells. Notably, RSV-infected MSCs exhibited significantly increased expression of IFN-β (~100-fold) and indoleamine-2,3-dioxygenase (IDO) (~70-fold) compared with mock-infected MSCs. IDO was identified in cytosolic protein of infected cells by Western blots and enzymatic activity was detected by tryptophan catabolism assay. Treatment of PBMCs with culture supernatants from RSV-infected MSCs reduced their proliferation in a dose dependent manner. This effect on PBMC activation was reversed by treatment of MSCs with the IDO inhibitors 1-methyltryptophan and vitamin K3 during RSV infection. We also demonstrated the pathway leading to IDO expression in RSV infected MSCs. Neutralizing IFN-β prevented IDO expression and activity indicating its role as a viral response factor perhaps “high jacked” by the virus in immune escape. Treatment of MSCs with an endosomal TLR, but not a RIG-I, inhibitor prevented IFN-β and IDO expression. Additionally, TLR3/dsRNA complex inhibitor was able to block IFN-β stimulation, while a TLR7/8/9 inhibitory ODN did not, suggesting that endosomal TLR3 detection of RSV dsRNA was leading to IFN-β and IDO expression. Together, these findings indicate that RSV infection of MSCs triggers their immune regulatory function via TLR3 recognition of dsRNA, upregulating IFN-β expression and IDO activity, ultimately affecting immune cell proliferation. This finding may account for the lack of protective RSV immunity and consequent repeated infections throughout one's lifetime.

Allogeneic hematopoietic stem cell transplantation (allo-HCT) has been a successful cellular therapy for patients suffering from hematological malignancies for many decades; however, the beneficial effects of graft-versus-leukemia (GVL) are classically offset by graft-versus-host disease (GVHD). GVHD occurs when major and/or minor human leukocyte antigen (HLA) mismatches between donor and recipient cause rapid expansion and activation of donor effector T cells (Teffs) resulting in end organ damage to the recipient’s epithelial tissues. Given the lymphoproliferative nature of this disease, the standard treatment option is broad immunosuppression, which can result in primary disease relapse, steroid refractory GVHD, and/or opportunistic infection. A more targeted therapy that can selectively suppress GVH responses with maintained GVL responses would achieve the optimal goal of allo-HCT. Regulatory T cells (Tregs) both natural (nTregs) or induced (iTregs) could be potential cellular therapies for the treatment of GVHD, given their innate suppressive function. Initial clinical trials using nTregs have yielded positive results; however, nTreg cellular therapy has been cumbersome due to the necessity for large scale ex vivo expansion given their low yield within an apheresis product and non-specific suppression. Conversely, iTregs can be generated from naïve T cells thus decreasing ex vivo culture times and can be educated with specific antigen thus providing targeted suppression, but a consensus on their efficacy for GVHD therapy has not been reached. Therefore, we investigated the efficacy of antigen specific iTreg therapy for the prevention of GVHD while maintaining GVL responses. In Chapter 2, we evaluated the effectiveness of monoclonal HY-specific iTregs in GVHD attenuation. We chose HY as a target antigen because it is a naturally processed, ubiquitously expressed minor mismatch antigen carried by only male donors/recipients cited to increase GVHD prevalence when donor and recipient are sex-mismatched. Utilizing HY-transgenic mice in which all T cells recognize HY antigen exclusively, we generated HY specific iTregs which effectively attenuating GVHD in male, but not female recipients in three murine bone marrow transplantation (BMT) models (major mismatch, parent to F1, and miHAg mismatch). We found HY specific iTregs lost stability in female recipients but remained stable and suppressive in male recipients suggesting expression of HY antigen was required for their suppressive function and stability. GVL responses were not compromised with the addition of HY specific iTregs in recipient mice using a pre-established tumor model. Thus, HY-specific iTregs can be generated and suppress GVHD in an antigen-dependent manner while sparing the GVL effect. In Chapter 3, we extend our findings in Chapter 2, which provided proof of principle that antigen specific iTregs effectively control GVHD; however, this therapy has a limited translational potential. Therefore, we generated alloreactive CD4 and CD8 iTregs and evaluated GVHD attenuation and GVL preservation in either full or haplo-MHC mismatched BMT models. We found alloreactive CD4 iTregs significantly suppress lethal GVHD, but completely abrogated the GVL effect against aggressive tumors. Conversely, alloreactive CD8 iTregs moderately attenuated GVHD and possessed direct cytotoxicity against tumor cells. Therefore, to rescue the impaired GVL effect mediated by CD4 iTregs, we established a combinational therapy with CD8 iTregs. Indeed we found combination CD4 and CD8 iTreg therapy significantly suppressed GVHD while sparing GVL responses compared to either CD4 or CD8 singular therapy. Mechanistically, this was achieved by potent suppression of both CD4 and CD8 Teffs coupled with preserved cytolytic molecule expression by both CD8 iTregs and Teffs. Taken together, we propose antigen specific iTreg therapy can effectively attenuate GVHD while preserving GVL responses. We further uncovered unique characteristics of CD4 and CD8 iTregs that can be exploited to achieve the optimal cellular therapy following allo-HCT.