Interleukin (IL)-7 is an essential non-redundant cytokine and throughout the life-span of a T cell signaling via the IL-7 receptor influences cell survival, proliferation and function. It is therefore no surprise that expression of the IL-7 receptor alpha-chain (CD127) is tightly regulated. In this study I establish IL-7 down regulates CD127 gene transcription and surface protein expression in primary human CD8 T cells through two mechanisms. Upon binding IL-7, surface CD127 is rapidly internalized and phosphorylated at the critical tyrosine residue Y449. Concurrent activation of the JAK/STAT5 pathway stimulates expression of CIS, a member of the SOCS family of proteins. CIS protein already expressed at basal levels and induced by IL-7 bind directly to CD127 as demonstrated by Coimmunoprecipitation assays and colocalize with both CD127 and the early endosomal marker EEA1. Subsequent proteasomal degradation of CD127 and CIS is dependent on an E3 ligase. Through siRNA-mediated knockdowns I confirm CIS plays a predominant role in the IL-7 mediated degradation of CD127. The mechanism by which IL-7 suppresses CD127 transcripts in primary human CD8 T cells was also examined. Through qPCR and nuclear run-on assays I illustrate that IL-7 suppresses CD127 gene transcription in a time- and dose-dependent manner. The IL-7 mediated suppression of CD127 transcripts is dependent on JAK/STAT5 signaling. Notably, cycloheximide blocked IL-7’s ability to down-regulate CD127 transcripts suggesting IL-7 stimulates the de novo synthesis of a transcriptional repressor of the CD127 gene. Through PCR arrays, qPCR and Western blot analysis the IL-7 inducible transcription factor c-Myb was identified as a candidate repressor. The region within the CD127 gene promoter required for IL-7 mediated transcriptional suppression was identified through progressive truncations using firefly luciferase as a reporter gene and is located from -1760 to -2406 bp upstream of the TATA box and contains three putative c-Myb binding sites. Using siRNA-mediated knockdown and transient over-expression, I illustrate c-Myb suppresses CD127 gene transcription in primary human CD8 T cells. A thorough understanding of the mechanisms by which IL-7 regulates CD127 expression is imperative and may reveal novel insights into the contribution of abnormal IL-7 signaling to diseases affecting immune function.

Cytotoxic T-lymphocytes are important for the control of viral replication during HIV infection, however the magnitude and breadth of HIV-specific CD8+ T-cell response does not correlate well. The purpose for this study was the examination of the HLA-B*2705-specific CD8+ T-cell response to the KRWIILGLNK (KK10) epitope as a definitive model of immune control over HIV replication. The breadth of the T-cell receptor (TCR) repertoire was determined for an association between the qualitative nature of this response and immune escape and therefore, disease progression. Methodology was developed and validated for TCR repertoire analysis in formaldehyde fixed antigen-specific CD8+ T-cells. The TCR repertoire for the KK10-specific CD8+ T-cell response was defined in cross-section and longitudinally for 6 HLA-B*2705+ patients. Comparison was made to cognate HLA-A*0201 CMV NV9 and HLA-B*2705 EBV RL9-specific CD8+ T-cell populations using the Simpson??s diversity index and the Morisita-Horn similarity index for standardized repertoire analysis. HLA-B*2705 KK10-specific TCR repertoire was not found to be a determinant of control. Greater clonotype variation was found within CMV-specific CD8+ T-cell populations, suggesting an association with reactivation of CMV and disease state. An association was found between KK10-specific population diversity and the prevalence of cognate KK10 epitope in vivo. Cross-reactivity observed for dominant KK10-specific clonotypes suggested that avidity of CD8+ T-cells was important for in vivo survival. Phenotype and function was tested through multiparameter analysis of HIV and CMV-specific CD8+ T-cells. Increased frequency of CD127 (IL-7R) and Bcl-2 expression within dominant populations was suggestive of selective advantage. Division of dominant and sub-dominant CMV-specific CD8+ T-cell populations into ??early?? and ??late?? differentiation phenotypes indicated virus-specific mechanisms of clonotype turn over. No simple association of TCR expression was found for HIV and CMV-specific CD8+ T-cells with published examples of definitive TCR bias. Over-represented TCR ??-chain families of patients were found in association with public clonotypes. Convergent recombination of TCR genes was demonstrated as a mechanism for the prevalence of shared clonotypes. Standardized assessment of T-cell repertoire successfully identified mechanisms of antigen-specific CD8+ T-cell recruitment. A substantial increase in sample numbers is required before this methodology can be used to accurately demonstrate the importance of TCR repertoire usage in the control of human viral infection.

Made available in DSpace on 2015-12-06T23:47:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2008 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Embora esteja bem estabelecido, há mais de quinze anos, que linfócitos T CD8+ restritos por moléculas de MHC-Ia são importantes no controle da parasitemia e sobrevivência de camundongos infectados com Trypanosoma cruzi, pouco se sabia da especificidade desses linfócitos. A ausência de epítopos bem definidos impedia o estudo detalhado da cinética da resposta imune e dos mecanismos de controle da imunodominância. Assim, o objetivo inicial desta tese foi a identificação de epítopos reconhecidos pelos linfócitos T CD8+ ativados durante a infecção pelo T. cruzi. Uma vez definidos esses epítopos, estudamos a cinética de ativação dessas células e alguns dos parâmetros que a controlavam. Por fim, estudamos os possíveis mecanismos de imunodominância durante a resposta imune. Durante a infecção experimental com a cepa Y de T. cruzi, nós observamos que os epítopos VNHRFTLV, IYNVGQVSI ou TEWETGQI foram reconhecidos por camundongos infectados C57BL/6, BALB/c ou B10.A, respectivamente. Esses epítopos, expressos por membros da família das trans-sialidases, geraram forte resposta imune medida pela citotoxicidade in vivo ou pela produção de IFN-γ ex vivo (Elispot). A cinética da ativação desses linfócitos T CD8+ se iniciou no pico da parasitemia e dependeu da carga parasitária. Ou seja, quanto mais tempo a parasitemia demorou para atingir o pico, mais lenta foi a aparição das células específicas. Uma vez que a resposta chegou ao máximo, essa se manteve alta por meses e só então começou a declinar lentamente. O fenótipo dos linfócitos T CD8+ específicos de memória é CD62Llow, característico de células de memória efetoras. Tanto a cinética, quanto o fenótipo dessas células diferiram dos achados observados para vírus, bactérias e outros protozoários intracelulares. Em animais previamente vacinados com DNA plasmidial ou proteínas recombinantes, uma significativa aceleração da resposta imune específica foi observada, a qual correlacionou com a imunidade protetora. A fim de estudarmos os fatores que contribuíram para ativação dessas células, nós utilizamos camundongos geneticamente deficientes. Observamos que a deficiência para a produção de IL-12 e Interferon tipo I não causou nenhuma redução na geração das células citotóxicas específicas. O mesmo foi observado em animais deficientes para os receptores do tipo Toll 2, 4 ou 9. Por outro lado, animais geneticamente deficientes que não expressavam as moléculas de MHC-II ou CD4 apresentaram significativa redução na resposta específica de linfócitos T citotóxicos. O estudo dos mecanismos que controlam a imundominância da resposta imune mediada por linfócitos T CD8+ específicos foi feito, inicialmente, pela comparação da resposta imune em camundongos C57BL/6. Observamos que a resposta imune para o epítopo VNHRFTLV foi imunodominante sobre os demais epítopos restritos pelo MHC-Ia H-2Kb . A fim de determinar se essa imunodominância poderia ser exercida sobre epítopos restritos por MHC-Ia H-2Kk (TEWETGQI) ou H- 2Kd (IYNVGQVSI), comparamos as respostas imunes em camundongos infectados homozigotos e heterozigotos. No caso do epítopo VNHRFTLV, nós observamos que a resposta imune se manteve alta em ambas as linhagens de camundongo. Já no caso dos dois outros epítopos restritos por MHC-Ia, H-2Kk ou H-2Kd , observamos uma significativa redução na resposta imune dos animais heterozigotos em relação aos homozigotos. Essa competição não foi dependente do tempo ou da dose de parasitas inoculados. Também não foi observada quando os animais foram imunizados com adenovírus recombinantes contendo esses mesmos epítopos. O mais importante foi observar que a imunodominância pode ser evitada quando duas cepas de parasitas, contendo epítopos imundominantes distintos, foram utilizadas simultaneamente para infecção, sugerindo que há uma competição pelas células apresentadoras de antígenos durante o “priming”. Esse mecanismo de imundominância reduz a magnitude e o repertório da resposta imune e pode ser um mecanismo sofisticado de escape do parasita para evitar a eliminação completa pelos mecanismos efetores do hospedeiro. / Although it is well established for more than 15 years that CD8+ lymphocytes restricted by MHC-Ia molecules are important for the control of the parasitemia and survival during rodent infection with Trypanosoma cruzi, little was known about the specificity of these lymphocytes. The lack of well defined epitopes hindered the detailed study of the kinetic of the immune response and the mechanisms of control of the immunodominance. Therefore, the initial objective of this thesis, was to identify epitopes recognized by CD8+ T lymphocytes activated during the T. cruzi infection. Once we defined these epitopes, we studied the kinetics of activation of these cells and some of the parameters that controlled it. Finally, it was possible to study the mechanisms of immunodominance during the immune response. During the experimental infection with Y strain of T. cruzi, we observed that the epitopes VNHRFTLV, IYNVGQVSI or TEWETGQI were recognized by C57BL/6, BALB/c or B10.A infected mice, respectively. These epitopes, expressed by members of the trans-sialidase family of surface proteins, generated strong immune response as measured by the in vivo cytotoxicity or by the production of interferon-γ (Elispot). The kinetics of the activation of these CD8+ T cells initiated on the peak of parasitemia and depended on the parasite load. The longer delayed the parasitemia to reach its peak, slower was the appearance of these specific T cells. When the immune response reached the maximum, it was kept high for months and then, it declined slowly. The phenotype of memory CD8+ T lymphocytes was CD62LLow characteristic of effector memory cells. The kinetic one and phenotype of these cells had differed from the findings observed for other intracellular viruses, bacteria and protozoan parasites. In animals previously vaccinated with plasmidial DNA or recombinant proteins, a significant acceleration of the specific immune response was observed that correlated with the protective immunity. To study the factors that contributed for the activation of these cells, we used genetically deficient mice. We observed that deficiency for production of IL-12 and Interferon type I did not cause any reduction in the specific cytotoxic response. The same was observed in animals deficient for the Toll-like receptors 2, 4 or 9. On the other hand, genetically deficient animals that did not express MHC-II or CD4 molecules had significant reduction in the cytotoxic response mediated by CD8+ T cells. Finally, we described that following infection of mice with T. cruzi, an immunodominant CD8+ T cell immune response was developed directed to the epitope VNHRFTLV. To determine whether this immunodominance was exerted over other non H-2Kb -restricted epitopes, we measured during infection of heterozygote mice, immune responses to three distinct epitopes, all expressed by members of the trans-sialidase family, recognized by H-2Kb , H-2Kk (TEWETGQI), or H-2Kd (IYNVGQVSI)- restricted CD8+ T cells. Infected heterozygote or homozygote mice displayed comparably strong immune responses to the H-2Kb -restricted immunodominant epitope. In contrast, H-2Kk or H-2Kd -restricted immune responses were significantly impaired in heterozygote infected mice when compared to homozygote ones. This interference was not dependent on the dose of parasite or the timing of infection. Also, it was not seen in heterozygote mice immunized with recombinant adenoviruses expressing T. cruzi antigens. Finally, we observed that the immunodominance was circumvented by concomitant infection with two T. cruzi strains containing distinct immunodominant epitopes, suggesting that the operating mechanism most likely involves competition of T cells for limiting APCs. This type of interference never described during infection with a human parasite may represent a sophisticated strategy to restrict priming of CD8+ T cells of distinct specificities, avoiding complete pathogen elimination by host effector cells, and thus favoring host parasitism. / BV UNIFESP: Teses e dissertações

Cytotoxic CD8 T lymphocytes kill virus-infected cells and are critical for viral clearance from the body. Cytokines, particularly those sharing the common gamma receptor chain (gamma c), play a key role in this cytotoxic function as well as in the growth, differentiation and homeostasis of CD8 T lymphocytes. In order to exert these biological effects, cytokine-dependent signal transduction via the Janus kinase (Jak) / Signal Transducers and Activators of Transcription (STAT) pathway, the phosphoinositide 3-kinase (PI3-K) and mitogen-activated protein kinase (MAPK) pathways is required. In HIV infection however, the CD8 T lymphocytes become defective and are characterized by impaired cytotoxicity, altered differentiation patterns, and increased susceptibility to apoptosis. I hypothesized that impaired cytokine responsiveness resulting from defects in cytokine-dependent signal transduction contributes to the CD8 T cell impairment observed in HIV+ patients. I investigated the activation of the Jak/STAT signaling pathway to cytokines in CD8 T cells from HIV+ patients. Interestingly, these cells were responsive to IL-2, IL-4, IL-10, IL-15, and IL-21 at the level of their respective STAT activation. However, impairment of the IL-7 / IL-7Ralpha signaling axis was identified and characterized by a defect in STAT5 signaling. The impaired STAT5 activation correlated with a low IL-7Ralpha surface expression. The expanded population of IL- 7Ralphalow-expressing CD8 T cells, found particularly in viremic HIV+ patients, expressed higher levels of the transcriptional repressor Growth Factor Independent-1 (GFI1) compared to their IL-7Ralphahigh counterparts. This prompted further investigations into the role of GFI1 in IL-7Ralpha regulation in primary human CD8 T cells as a model. Though silencing of GFI1 did not modulate basal IL-7Ralpha expression, exogenous overexpression negatively regulated IL-7Ra surface levels. The gc cytokines, IL-2, IL-4, IL-7, and IL-15, but not IL-21, were found to efficiently suppress IL-7Ralpha expression however, only IL-4 simultaneously upregulated GFI1 expression. RNA interference studies targeting GFI1 in IL-4 stimulated CD8 T cells established a specific role for GFI1 in sustaining the suppression of IL-7Ralpha expression. Furthermore, transient downregulation of GFI1 in CD8 T cells subjected to IL- 4-dependent proliferation reduced their proliferative capacity. Other functions identified for GFI1 were in the suppression of CXCR4 and Bax expression in CD8 T cells. Studies aimed at identifying the signal transduction pathways responsible for regulating GFI1 and IL-7Ralpha expression revealed that IL-4-mediated downregulation of IL-7Ralpha expression required activation of the Jak/STAT and the PI3K pathways. On the other hand, IL-4-induced upregulation of GFI1 expression was mediated via the PI3K pathway. The JNK and P38 MAPK pathways appeared to be important as regulators of basal IL-7Ralpha expression levels, but had no statistically significant effects on GFI1 expression. To conclude, these studies have clarified the important biological effects of GFI1 in mature human CD8 T lymphocytes. Furthermore, exposure to IL-4 may generate CD8 T cell populations with an exhausted phenotype similar to those found in chronically-infected HIV+ patients, characterized by reduced cytotoxic activity and increased IL-4 production. Thus, the IL-4 study model may prove valuable for investigating the activity of human CD8 T cells in such chronic diseases and those characterized by a type 2 cytokine profile.

The innate immune response mediates immediate control of the pathogen and is followed by the acquired immune response which is slower but ensures comprehensive elimination of the pathogen. Dendritic cells are unique innate immune cells that can phagocytose the pathogen and generate pathogen-associated antigenic peptides for presentation to T cells in order to initiate the acquired immune response. Dendritic cells also express cytokines which facilitate pathogen control and development of acquired immune responses, thus acting as a bridge between innate and acquired immune responses. CD8+ T cells are important cells of the adaptive immune system that play a key role in mediating clearance and protection against intracellular pathogens. Upon engagement by antigen-presenting cells, CD8+ T cells undergo massive expansion followed by a swift, extensive contraction to restore homeostasis. The mechanisms behind the expansion and contraction of CD8+ T cells are yet to be completely elucidated. FoxO3a is a transcription factor that is involved in the regulation of various vital cellular processes ranging from cell proliferation and cell metabolism to stress resistance and cell death. I have, therefore, investigated the role of FoxO3a signaling in the activation of dendritic cells and CD8+ T cells. My initial experiments indicated that FoxO3a regulates the homeostasis of various immune cells including CD8+ T cells and dendritic cells. CD8+ T cells lacking FoxO3a displayed enhanced proliferation, as evaluated by cell imaging, CFSE dilution and Ki67 staining, upon polyclonal stimulation in vitro. The modulation of cell proliferation by FoxO3a seemed to be p27kip-independent, as evaluated by western blotting. At later stages of stimulation, FoxO3a-deficient CD8+ T cells underwent reduced cell death, as assessed by cell counting and 7-AAD staining, and this seemed to be independent of Bim, Caspase 8 or Caspase 3 activation. In addition, FoxO3a regulated cytokine expression by CD8+ T cells while displaying similar NFκB activation in comparison to WT CD8+ T cells. Similar results were observed in dendritic cells upon LPS stimulation in vitro, wherein cytokine expression was higher in the FoxO3a-deficient dendritic cells and they also displayed enhanced antigen presentation to CD8+ T cells, as evaluated by CFSE dilution. Taken together, these results indicate that FoxO3a acts as a negative regulator of CD8+ T cell and dendritic cell activation.

Chronic hepatitis C virus (HCV) infection has global effects on the immune system. CD8+ T-cells, responsible for viral clearance and control, are dysfunctional for as yet unknown reasons. It is hypothesized that IL-7 signaling pathway deficiencies contribute to this impairment. Blood-derived CD8+ T-cells in chronic HCV mono- and HIV-HCV co-infection had lower IL-7-induced activation of STAT5 and production of Bcl-2, and lower proliferation in co-infection, compared to controls. Lower Bcl-2 production was also associated with increased fibrosis. These changes were independent of the IL-7 receptor α expression and suppressor of cytokine signaling 1 or 3 expression. Intrahepatic CD8+ T-cells in HCV-infection did not activate STAT5 above basal levels with cytokine stimulation and had lower Bcl-2 expression than blood-derived cells. In conclusion, bulk CD8+ T-cells were impaired in response to IL-7 and the IL-7 signaling pathway may be one mechanism by which CD8+ T-cells are impaired in chronic HCV infection.

Interleukin (IL)-7 is an essential non-redundant cytokine and throughout the life-span of a T cell signaling via the IL-7 receptor influences cell survival, proliferation and function. It is therefore no surprise that expression of the IL-7 receptor alpha-chain (CD127) is tightly regulated. In this study I establish IL-7 down regulates CD127 gene transcription and surface protein expression in primary human CD8 T cells through two mechanisms. Upon binding IL-7, surface CD127 is rapidly internalized and phosphorylated at the critical tyrosine residue Y449. Concurrent activation of the JAK/STAT5 pathway stimulates expression of CIS, a member of the SOCS family of proteins. CIS protein already expressed at basal levels and induced by IL-7 bind directly to CD127 as demonstrated by Coimmunoprecipitation assays and colocalize with both CD127 and the early endosomal marker EEA1. Subsequent proteasomal degradation of CD127 and CIS is dependent on an E3 ligase. Through siRNA-mediated knockdowns I confirm CIS plays a predominant role in the IL-7 mediated degradation of CD127. The mechanism by which IL-7 suppresses CD127 transcripts in primary human CD8 T cells was also examined. Through qPCR and nuclear run-on assays I illustrate that IL-7 suppresses CD127 gene transcription in a time- and dose-dependent manner. The IL-7 mediated suppression of CD127 transcripts is dependent on JAK/STAT5 signaling. Notably, cycloheximide blocked IL-7’s ability to down-regulate CD127 transcripts suggesting IL-7 stimulates the de novo synthesis of a transcriptional repressor of the CD127 gene. Through PCR arrays, qPCR and Western blot analysis the IL-7 inducible transcription factor c-Myb was identified as a candidate repressor. The region within the CD127 gene promoter required for IL-7 mediated transcriptional suppression was identified through progressive truncations using firefly luciferase as a reporter gene and is located from -1760 to -2406 bp upstream of the TATA box and contains three putative c-Myb binding sites. Using siRNA-mediated knockdown and transient over-expression, I illustrate c-Myb suppresses CD127 gene transcription in primary human CD8 T cells. A thorough understanding of the mechanisms by which IL-7 regulates CD127 expression is imperative and may reveal novel insights into the contribution of abnormal IL-7 signaling to diseases affecting immune function.

Seminario de Título entregado a la Universidad de Chile en cumplimiento parcial de los requisitos para optar al Grado de Magister en Ciencias Biológicas. / El sistema nervioso y el sistema inmune se encuentran íntimamente relacionados comunicándose bidireccionalmente mediante citoquinas, neuropéptidos y neurotransmisores, como la dopamina. Este neurotransmisor ejerce su efecto a través de cinco receptores dopaminérgicos que poseen diferentes grados de afinidad, contando con la mayor afinidad el receptor 3 (D3R). Su presencia se ha reportado en diferentes células del sistema inmune. La estimulación del D3R promueve la producción de IFN-γ en linfocitos T CD4+, favoreciendo su diferenciación a un perfil Th1. Sin embargo, el rol de este receptor en los linfocitos T CD8+ aún no se ha estudiado a cabalidad. En esta tesis se estudió la incidencia del D3R en la formación del perfil citotóxico en linfocitos T CD8+, que poseen un TCR transgénico específico para el péptido OVA(257-264) alojado sobre el MHC de clase I H2-Kb (linfocitos OT-I). La diferenciación in vitro muestra que la carencia del D3R en linfocitos OT-I (OT-I D3RKO) resulta en una reducción significativa en la producción de IFN-γ, TNF-α, IL-2 y en la expresión de CD25 (cadena α del receptor de IL-2) en respuesta a la estimulación con péptido OVA(257-264). Estas deficiencias se revierten al añadir IL-2 exógena a los cultivos, sugiriendo que el mecanismo de acción por el cual el D3R favorece la diferenciación hacia un fenotipo efector productor de IFN-γ y TNF- α ocurre a través de la producción de IL-2. Concordante con lo anterior, se observó que la expresión del D3R en linfocitos OT-I promueve la expansión clonal y la protección antitumoral frente a melanoma. En conjunto los resultados obtenidos en linfocitos T CD8+ sugieren que el D3R promueve la formación de un perfil citotóxico, induciendo la producción de IFN-γ y favoreciendo una alta tasa de expansión clonal, lo que conlleva a una mayor sobrevida frente a un desafío tumoral. / The nervous system and the immune system are intimately related, communicating bi-directionally through cytokines, neuropeptides and neurotransmitters. Dopamine is a neurotransmitter that exerts its effect through five dopaminergic receptors which display different degrees of affinity. The D3R, which display the highest affinity for dopamine, has been found expressed in CD4+ T cells, where its stimulation promotes high production of IFN-γ, favoring the acquisition of the Th1 profile. However, the role of this receptor in CD8+ T lymphocytes has not yet been fully studied. In this thesis the role of D3R in the acquisition of the cytotoxic profile by CD8+ T lymphocytes was studied. For this purpose, in this thesis CD8+ T-cells bearing a transgenic TCR specific for the recognition of the OVA peptide(257-264) over the class I MHC H2-Kb (OT-I lymphocytes) were used. In vitro differentiation experiments show that lack of D3R in OT-I lymphocytes (OT-I D3RKO) results in a significant reduction in the production of IFN-γ, TNF-α and on the expression of CD25 (α chain of the IL-2 receptor) in response to the stimulation with OVA peptide(257-264). These effects were reversed by adding exogenous IL-2 into the cultures, suggesting a mechanism by which D3R favors the differentiation towards an effector phenotype through the production of IL-2. In vivo experiments show that D3R expressed on OT-I lymphocytes favors a higher clonal expansion and thereby an stronger protection against melanoma tumors. The results obtained suggest that D3R promotes the formation of a more efficient cytotoxic profile capable of producing higher percentages of IFN-γ and higher rates of clonal expansion, which leads to greater survival in the face of a tumor challenge. / Junio 2020

archives@tulane.edu / Immune signatures are patterns of gene and protein expression in immune cells that characterize states of activation and response. As such, signatures indicative of viral control during natural infection may guide vaccine development efforts to achieve similar patterns of protection. Here, we used nonhuman primate (NHP) models of Zika virus (ZIKV) and simian immunodeficiency virus (SIV, as a model for HIV) to explore outcomes of infection in these important human pathogens. We employed a multifaceted approach including high dimensional flow cytometry and RNA sequencing to understand cellular responses to ZIKV generally and during pregnancy, as well as to identify the impacts of infection in astrocytes, a neuroglial target of ZIKV thought to be important in the development of neurologic disease. We found that CD8 T cells may restrict ZIKV persistence in tissues but ultimately have a minimal role in protection to either primary or secondary challenge. However, we showed that immune manipulation, either naturally through pregnancy or artificially through depletion experiments, can skew metabolic and innate immune pathways in unexpected ways. While cellular immunity appeared to minimally impact ZIKV infection, such responses in SIV are important in controlling viral replication, which we inversely showed by tracking patterns of viral mutation to evade CD8 responses. We also identified transcriptional signatures in ZIKV infection that may underlie the development of neurologic diseases and found that different virus lineages have unique impacts on gene expression. Together, these experiments showcase the utility of profiling approaches in understanding the immune complexity that accompanies viral infection. / 1 / Blake Schouest

A memory response is established following primary antigen exposure that stays more or less constant. It appears to adopt a set-point in magnitude but upon re-exposure the response is quicker and better and there is an upward shift in memory frequency that varies with individuals based on the exposure pattern to other microbes or its components. Our investigations were designed to test such differences of non-specific stimulation by PAMPs in lowering the threshold of activation. Neonatal mice were pre-exposed to TLR-ligands intermittently and later analyzed for its resilience to challenge with virus during adult-life. Secondly, adult mice with pre-existing memory to virus were exposed to various TLR-ligands and analyzed for their quality of memory response. The TLR-ligands exposed animals were better responders to a new agent exposure compared to the animals kept in sterile surroundings. Moreover, immune memory recall and the viral specific CD8+ T cells response with TLR-ligands were comparable to the recall response with the cognate antigen. The results provide insights into the role of hyper-sanitized environment versus PAMPs mediated signaling in adaptive immunity and long-term immune memory.