Animal & Avian Sciences Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2741
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Item DIFFERENTIATION AND REGULATION OF BOVINE TH2 CELLS(2024) Kandel, Anmol; Xiao, Zhengguo Zhengguo; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Memory CD4+ T cells, specifically type-2 (Th2) cells, are pivotal in defending against infections caused by extracellular pathogens, including several economically important parasites. However, whether interleukin-4 (IL4) expression is a signature feature of bovine Th2 cells likewise in mice and humans is unclear. Pasture-raised cattle, routinely exposed to extracellular parasites such as Ostertagia ostertagi (OO), are likely to develop a typical Th2 memory response. Therefore, using cytokine induction assay, we evaluated the circulatory memory bovine T cell profile of these cattle and also analyzed if the expression of presumptuous memory marker, CD45RO, is reliable in identifying memory bovine T cells. Surprisingly, the majority of the memory CD4+ T cells dominantly produced interferon-gamma (IFNγ), with only a small fraction co-expressing IL4, and memory bovine T cell identification did not correlate with CD45RO expression. Results suggested that cattle naturally exposed to extracellular parasites do not develop typically IL4 dominant Th2 response. To further investigate these results, resting CD4+ T cells isolated from healthy cattle blood were cultured under simple in vitro Th2 culture. Analysis of differentiated cells through flow cytometry revealed limited IL4 protein detection, which was in line with the lack of upregulation of IL4 and its master regulator GATA3 transcripts shown by the quantitative polymerase chain reaction (qPCR) assay. To validate whether differentiated cells were actually Th2, unbiased proteomic analysis was conducted. Based on differentially expressed 397 proteins between differentiated cells and naïve phenotype, bovine Th2 differentiation was validated; nonetheless, the process was not found to be associated with IL4 induction. Moreover, despite using published strategies from mice and humans, such as reducing T cell receptor (TCR) stimulation strength and adding exogenous recombinant bovine IL4, the expression of IL4 could not be significantly enhanced. Interestingly, differentiated bovine Th2 cells proliferated in the presence of OO antigens, suggesting that extracellular parasites could influence bovine Th2 differentiation, at least in vitro. To validate the results from pathogen-infected tissues and in vitro culture, a panel of anti-parasitic CD4+ single T cell clones was established from five pasture-raised cattle that were infected with OO. Evaluation of memory responses exhibited by the anti-parasitic CD4+ single T cell clones strongly supported IFNγ dominant memory response, and only 20% of them co-expressed IL4 through a small subset of IFN γ + cells. All the data pointed out that bovine CD4+ T cell differentiation is partially distinct from those in mice and humans, and IL4 expression is not a hallmark feature of the bovine Th2 cells.Item BOVINE NEUTROPHILS RELEASE EXTRACELLULAR TRAPS UPON STIMULATION WITH OSTERTAGIA OSTERTAGI(2018) Mendez, Jonatan; Xiao, Zhengguo; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ostertagia ostertagi is a widespread parasite that causes significant production losses in the cattle industry. Recently discovered neutrophil extracellular traps (NETs) have been demonstrated as important effector mechanisms of neutrophils against various pathogens including parasitic worms. Exposure of neutrophils to O. ostertagi extract resulted in a significant release of extracellular DNA and co-localization of NET associated proteins histone and neutrophil elastase confirmed these structures of DNA as NETs. In response to both live and heat-killed O. ostertagi larvae, there was a similarly strong release of NETs. O. ostertagi induced NETs were significantly diminished by inhibition of enzymes NADPH oxidase, neutrophil elastase, and myeloperoxidase. Interestingly, NETs were also released in response to non-pathogenic nematode C. elegans indicating a potential conserved response to nematodes. Mouse neutrophils demonstrated a similar NET response to O. ostertagi however there was no response to C. elegans. Surprisingly, these NET responses did not appear to be dependent on production of reactive oxygen species (ROS) as has been previously reported. This is the first report indicating O. ostertagi-induced NET formation and indicates a potential role for NETs in the response against O. ostertagi infection.Item CTL-derived exosomes enhance the activation of CTLs stimulated by low affinity peptides(2018) Wu, Shu-Wei; Xiao, Zhengguo; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Cytotoxic T cell (CTL) is the key to induce an effective immune response against infections caused by intracellular pathogens, such as virus and cancer. CTLs with low affinity can induce and maintain a substantial population during an adaptive immune response, although CTLs with a highest-affinity receive competitive activation signals. Low-affinity CTLs are important to induce effector response and maintain a diverse memory repertoire. However, the mechanism of generating and maintaining the expansion of lower affinity CTLs is still unknown. Here, we showed that the presence of exosome (Exo) enhanced the CTL survival and increased the cell proliferation especially in CTLs treated with the low-affinity peptide. Exo together with peptides also improved the production of IFN-γ and GZB. The exosomal stimulation in CTLs was relative to up-regulation of CD25 expression, which enhanced the IL-2 survival signals. Moreover, Exo derived from an early stage of activation had a similar but weaker function comparing with Exo derived from a late stage of activation in activating CTLs. This study identified the important role of the exosome derived from CTLs stimulated by the highest-affinity peptide in activating the naïve T cells stimulated by the low-affinity peptide.Item Lineage reprogramming of tumor-infiltrating cytotoxic T lymphocytes using protein stem cell transcription factors(2015) BhaduriHauck, Anjuli Lucija; Xiao, Zhengguo; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Adoptive cell transfer therapy (ACT) is one of the most promising immunotherapies against cancer. However, this treatment regimen requires the expansion of a small population of effector cells, known as tumor infiltrating lymphocytes, into the billions in order to overcome the immunosuppressive tumor microenvironment. The cytotoxic T lymphocytes (CTLs) within this invading immune cell population are the most critical components to kill the growing cancer cells. Nonetheless, the rapid expansion of already exhausted tumor-infiltrating cytotoxic T lymphocytes (TICTLs) may further push them to a terminally differentiated state that reduces their proliferative response upon antigen stimulation. Recently, induced pluripotent stem cells (iPSCs) generated from TICTLs have been suggested as a way to create a renewable source of rejuvenated tumor-specific CTLs, but retroviral reprogramming is inefficient, and can lead to an increased chance of tumorigenesis. To improve the expansion of TICTLs, we used transient protein exposure to SOX2, OCT4, and NANOG (SON) in order to push these exhausted TICTLs to a less differentiated stage, preferably stem cell-like memory CTLs (Tscm). These three transcription factors were transiently delivered using a nuclear protein delivery system. We found only the TICTLs treated with SON (STICTLs) exhibited an increased proliferation rate and extended survivability, independent of additional cytokines and antigen stimulation both in vitro and in vivo; effector CTLs did not respond to the SON regimen. These highly proliferative STICTLs could be associated with up regulation of certain genes related in cell cycle control, such as cyclin D1. Though these STICTLs still express a T cell receptor (TCR), as well as many critical downstream components, they were unable to elicit a reaction against antigen exposure. Though clearly not iPSCs, it is possible that the SON treatment had pushed the TICTLs into a state similar to an early double negative thymocyte. Our findings indicate that TICTLs are uniquely responsive to protein SON compared to naïve and effector CTLs; suggesting TICTLs may also be sensitive to regulation by other more lineage specific transcription factors, thus present new avenue for cancer immune therapy.Item The role of interleukin-12 for mTOR regulation of memory CTLs(2014) Garcia, Karla; Xiao, Zhengguo; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)A major goal of vaccines is to induce functional immune memory, and efforts to improve the efficacy of vaccines targeting memory CTLs have revealed an important immunoregulatory role of rapamycin, a specific mTOR inhibitor. While inflammatory cytokines are critical for memory CTLs formation, it is unknown if cytokines such as IL-12 mediate rapamycin's regulation during infection. Inhibition of mTOR by rapamycin represses CTL expansion but enhances central memory during vaccinia virus infection in mice. Without IL-12, immunoregulatory effects of rapamycin on CTL expansion and subsequent memory formation are diminished, yet present compared to CTLs not treated with rapamycin. In infected mice, rapamycin directly enhances IL-12 signaling in WT CTLs by upregulating IL-12 receptor-B2 and STAT4 phosphorylation. Furthermore, secondary expansion of rapamycin-regulated memory CTLs in IL-12 receptor knockouts is impaired and resultant secondary memory CTLs are abolished. This indicates that interplay between cytokines and adjuvants should be considered during vaccine design.Item Generating memory cytotoxic T lymphocytes through repetitive peptide boosting(2012) Smyth, Kendra; Xiao, Zhengguo; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Cytotoxic T lymphocytes (CTLs) play a critical role in controlling intracellular pathogens and cancer cells, and induction of memory CTLs holds promise for developing effective vaccines against critical virus infections. However, generating memory CTLs remains a major challenge for conventional vector-based, prime-boost vaccinations. Thus, it is imperative that we explore nonconventional alternatives, such as boosting without vectors. We show here that repetitive intravenous boosting with peptide and adjuvant generates memory CD8 T cells of sufficient quality and quantity to protect against infection in mice. The resulting memory CTLs possess a unique and long-lasting effector memory phenotype, characterized by decreased interferon-gamma but increased granzyme B production. These results are independent of the specific adjuvant applied and are observed in both transgenic and endogenous models. Overall, our findings have important implications for future vaccine development, as they suggest that intravenous peptide boosting with adjuvant following priming can induce long-term functional memory CTLs.