Previous studies employing the generation of MHC-incompatible embryonic chicken chimaeras by injecting MHC-incompatible stem cells resulted in an unexpected finding. Chimaeras made late in gestation developed as adults a severe autoimmune syndrome resembling the human syndrome of Systemic Lupus Erythematosus. Work in our laboratory aims to understand the role of CD8 T cells in immunity and/or autoimmunity. We have tested a three-cell model of CD4 T cell activation and differentiation during the development of the immune response specific for MHC transplantation antigens in one way mixed lymphocyte reactions. Our model proposes that whether Th1 or Th2 immunity is generated depends on both the ratio of CD4:CD8 T cells specific for antigen at the initiation of the immune response and on the ability of antigens to coordinately induce both CD4 and CD8 T cells. Previous studies employing parent into F1 models of graft-versus-host disease in mice have shown that the injection of parental cells results in two distinct outcomes. Parental cells which do not have a sufficient number of CD8 T cells present produce an autoimmune syndrome characteristic of systemic lupus erythematosus and a chronic graft-versus-host disease mediated by a Th2 response. Conversely, the presence of an adequate number of CD8 T cells results in a Th1 immune response and acute graft-versus-host disease resulting in the death of the F1 host. Our findings indicate that the ratio of the number of CD4 T cells to the number of CD8 T cells present in the spleen is crucial in whether naive CD4 T cells differentiate into Th1 or Th2 cells. We refer to this ratio as the CD4:CD8 T cell ratio or CD4:CD8 ratio. Thus, the differentiation of naive CD4+ T cells towards a differentiated Th1 phenotype is critically dependent on the concomitant induction of CD8 T cells by the same antigen, driven by a low CD4:CD8 ratio. In contrast, inefficient induction of CD8 T cells during the initial priming of lymphocytes greatly facilitates the differentiation of CD4 T cells towards the Th2-type lineage, and occurs when the CD4:CD8 ratio is high. Given our findings on the significance of the ratio of CD4:CD8 T cells in the decision making process of CD4 differentiation stimulated by antigen, we hypothesized that different CD4: CD8 ratios at different stages of development might contribute to the immune response generated at these stages. We tested this hypothesis in mice by comparing the CD4:CD8 ratio in adults and neonates and the Th1/Th2 responses generated in vitro. This CD4:CD8 T cell ratio is significantly higher in neonates than adults resulting in predominant Th1 responses by adult spleen cells and Th1/Th2 responses by neonatal spleen cells as demonstrated by the ELISPOT assay. We have compared the CD4:CD8 T cell ratio of a large number of adult and neonatal spleens in several mouse strains and have studied it systematically in BALB/c and C57BL/6 mice by flow-cytometry. We have consistently found a 3-5 fold higher CD4:CD8 T cell ratio in neonates as compared to adults in the strains tested. Furthermore, we found that neonatal spleen cells generate a predominant Th2 response whereas adult spleen cells generate CD4 and CD8 Th1 immunity when activated under the same conditions. We have further studied the role of CD8 T cells in CD4 T cell differentiation by reconstructing the adult CD4:CD8 T cell ratio in neonatal spleen cells with age-matched, isolated CD8 T cells. We found that in these “CD4:CD8 ratio-reconstructed cultures”, the Th2/IL-4 immunity is suppressed with concomitant generation of Th1/IFN-γ immunity upon activation by allo-antigen. Additionally, we have characterized the phenotype of the T cell mediating Th1/IFNγ immunity in the “CD4:CD8 ratio reconstructed cultures” and we found that while CD8 T cells produce exclusively IFN-γ, CD4 T cells now produce IFN-γ rather than IL-4. We suggest that physiologically distinct CD4:CD8 ratios at different stages of life should be considered in designing protocols of neonatal vaccination against pathogens that are contained by Th1-type immunity upon infection as adults. Moreover, as elaborated in the discussion, our studies might be pertinent in understanding by which mechanism autoimmunity arises in some cases.

No description available.

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.

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.

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.

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.

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by a low platelet count (less than 100 x 10^9 platelets/L) and an increased risk of bleeding. ITP is difficult to diagnose and manage due to the deficiencies in our understanding of the pathophysiological mechanisms leading to thrombocytopenia. Anti-platelet autoantibodies are believed to be the primary mechanism of thrombocytopenia in ITP. In this thesis, I demonstrate that autoantibodies can only be detected in half of all ITP patients; therefore, other mechanisms should be investigated. CD8+ T cells have been implicated as a mechanism of disease in ITP, but platelet-specific CD8+ T cells have yet to be identified. I have characterized CD8+ T cells in ITP patients and found that platelet-specific CD8+ T cells can be detected in ITP patients. These platelet-specific CD8+ T cells can also be detected in healthy individuals, so they are not specific to ITP. However, regulatory defects were observed in ITP patients and CD8+ T cell activity was elevated in ITP patients relative to healthy individuals and thrombocytopenic non-ITP patients. Investigating whether platelet-specific CD8+ T cells can actively participate in platelet destruction and underproduction will be an essential step towards better understanding the role of CD8+ T cells as a disease mechanism in ITP, which will lead to improvements in the management of ITP. / Thesis / Doctor of Philosophy (PhD) / Platelets are small blood cells that are involved in minimizing blood loss at the site of a wound by forming a plug. In a disease called immune thrombocytopenia (ITP), patients have a low platelet count, which can result in bleeding. The bleeding symptoms of ITP decrease the quality of life for ITP patients and can be life-threatening in rare cases. It is believed that ITP is caused by proteins produced by the immune system called antibodies. I found that the antibodies that cause ITP can only be detected in half of all ITP patients. Therefore, there are probably additional causes of ITP. It is suspected that CD8+ T cells might cause ITP in some patients. CD8+ T cells are part of the immune system and they typically destroy other cells that are cancerous or infected by viruses. CD8+ T cells might also destroy healthy cells, like platelets. My goal was to characterize CD8+ T cells in order to determine their role in ITP. I found that CD8+ T cells from ITP patients can target platelets, and that healthy people have these CD8+ T cells as well. In regard to CD8+ T cells that target platelets, the difference between ITP patients and healthy people appears to be related to immune system regulation and CD8+ T cell activity. In the future, we should focus on understanding how platelet-specific CD8+ T cells can cause a low platelet count in order to improve the clinical management of ITP.

The extent to which the progeny of one primary memory CD8 T cell differs from the progeny of one naïve CD8 T cell of the same specificity remains an important question. In order to explore cell autonomous functional differences between naïve and memory CD8 T cells that are not influenced by differences in the priming environment, an experimental model has been developed in which physiological numbers of both populations of cells were co-transferred into naïve host before antigen-stimulation. Interestingly, naïve CD8 T cells expand in numbers more than primary memory CD8 T cells after various infections or immunizations. The intrinsic ability of one naïve CD8 T cell to give rise to more effector CD8 T cells than one memory CD8 T cell is independent of the number of primary memory CD8 T cells present in vivo. The sustained proliferation of primary, but not the increased death of secondary effectors was shown to contribute to the differences in the observed magnitudes of expansion. In addition, longitudinal analysis of primary and secondary CD8 T cell responses revealed that the ability of naïve CD8 T cells to generate long-lived progeny (`memory generation potential') is better than for primary memory CD8 T cells despite the differences in overall kinetics of both responses after infection. Taken together, the data presented here revealed previously unappreciated differences between naïve and memory CD8 T cells and will help further define the functional potential for both cell types. The goal of immunization is to generate memory CD8 T cells of sufficient quality and quantity, and it has been shown that the naïve to primary memory CD8 T cell differentiation in vivo is controlled, at least in part, by the amount and duration of inflammation present early after the initiation of the response. In experiments where naïve CD8 T cells were co-transferred with increasing numbers of primary memory CD8 T cells, we observed a negative correlation between the number of primary memory present and the magnitude of primary CD8 T cell responses. Interestingly, the conversion of newly recruited (either TCR-Tg or endogenous) primary CD8 T cells into CD8 T cells with the phenotype (CD62Lhi, CD27hi) and function (tissue distribution, Ag-driven proliferation, cytokine production) of long-term memory was facilitated when they were primed in the presence of memory CD8 T cells of the same or unrelated specificity. Therefore, these data suggest that the presence of anti-vectorial immunity will not necessarily decrease the efficacy of CD8 T cell vaccination since newly recruited CD8 T cells, despite their decreased magnitude of expansion, might differentiate into functional memory cells faster.

Previous studies have shown that cytotoxic CD8+ T cells are important mediators of immunity against the bovine intracellular protozoan parasite T. parva. The present study set out to determine the role of granule enzymes in mediating killing of parasitized cells, first by characterising the granzymes expressed by bovine lymphocytes and, second, by investigating their involvement in killing of target cells. Experiments using the perforin inhibitor concanamycin A confirmed that CD8+ T cell killing of T. parva-infected cells is dependent on granule exocytosis, a process that involves release of granzymes into the target cell, resulting in activation of apoptotic pathways. Analysis of the bovine genome sequence identified orthologues of granzymes A, B, H, K and M, as well as another gene O, most closely related to granzyme A. The genes were found within 3 loci in the genome. Using specific PCR assays, all of these granzymes were shown to be expressed in Theileria-specific CD8+ T cells. Further studies were undertaken to study the role of granzyme B in killing. DNA constructs encoding functional and non-functional forms of bovine granzyme B were produced and the proteins expressed in COS cells were used to establish an enzymatic assay to detect and quantify expression of functional granzyme B protein. Using this assay, the levels of killing of different T. parvaspecific CD8+ T cell clones were found to be significantly correlating with levels of granzyme B protein expression. Moreover, the granzyme B inhibitor III, Z-IETDFMK was shown to inhibit killing by CD8+ T cell clones.