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    Roles of aging-associated Treg cell accumulation in experimental autoimmune encephalomyelitis (EAE) — a model of late-onset multiple sclerosis (MS)
    (2021) Wang, Weikan; Thomas, Rachel; Oh, Jiyoung; Su, Dongming
    Purpose: MS, a T cell-mediated autoimmune demyelinating disease of the central nervous system (CNS), has not been sufficiently studied in the elderly, despite instances of late-onset MS. Regulatory T (Treg) cells play an ameliorative role in MS. Given that the aged immune system exhibits accumulated peripheral Treg (pTreg) cells, the role of Treg cell in aged MS patients remains to be elucidated. Methods: We immunized young and aged mice to induce EAE and investigated the disease courses and Treg cell associated mechanisms. Results: We found that the aged mice have two types of late-onset EAE. In Type I, EAE onset was delayed in the age mice, but the disease became more severe once after onset; Alternatively, in type II, some aged mice never developed into as severe symptoms as the young mice until a second antigen challenge was given, which led to more progressive disease courses than their young counterparts. This phenotype was potentially associated with a lower proportion of CNS-specific Treg cells in the aged CNS, even though the aged mice had a high proportion of polyclonal pTreg cells. Transient inhibition of polyclonal pTreg cells partially ameliorated the disease severity in the aged mice, accompanied with increased CNS-specific Treg cells in the CNS. Conclusions: Therefore, we suggest that accumulated polyclonal pTreg cells in the aged periphery are detrimental for CNS repair processes during neuronal inflammation in aged MS mice, potentially due to hampering trafficking of CNS-specific Treg cells into the CNS.
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    Natural Killers: Targeting NK Cells in Cancer Immunotherapies
    (2021) Buller, Casey W.; Mathew, Stephen O.
    Natural Killer (NK) cells are innate lymphoid immune cells that have garnered attention for their pivotal role in tumor surveillance and their ability to recognize and clear cancerous cells. NK cells' delicate balance of activating and inhibitory receptors and those receptors' engagement with target cancer cells determines their effector function. NK cells show promise in the treatment of solid and hematologic tumors. Additionally, targeting NK cells to clear cancerous cells offers advantages over CD8+ T cells, such as not relying on major histocompatibility complex (MHC) I. Successful use of NK cell therapies does have challenges that must be overcome. Several strategies include the use of monoclonal antibodies, chimeric antigen receptor (CAR) NK cell therapy, bi- and tri-specific killer engagers (BiKES and TriKES), and immune checkpoint molecules.
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    Suppression of Adaptive Immunity by Borreliella burgdorferi: An Investigation of Bacterial Immune Avoidance Mechanisms
    (2021) Williams, Megan; Zhang, Yan; Allen, Michael
    Purpose: Infection with Borreliella burgdorferi, the causative agent of Lyme Disease, induces broad suppression of the host adaptive immune response. It has been shown that germinal centers form in the lymph nodes shortly after infection, but then collapse after one month. Additionally, when a group of Borreliella-infected mice were given an influenza vaccine, they mounted a significantly abrogated influenza-specific antibody response when compared to an uninfected group that received the same vaccine. A better understanding of how B. burgdorferi manipulates host immunity can help enhance serological testing for Lyme Disease. We aim to characterize how this suppression of host immunity changes over the course of infection with B. burgdorferi. Methods: We will randomly assign mice to 5 groups (n=4). One group will be inoculated with the Vanguard H3N2/H3N8 canine influenza virus (CIV) vaccine. The remaining 4 groups will be infected with B. burgdorferi and will receive the CIV vaccine at Day 0, 7, 28, and 45 post-infection. The CIV-specific antibody response will be measured using enzyme-linked immunosorbent assays (ELISA) at different stages of infection with B. burgdorferi. Results: This study has not yet been completed. The CIV bivalent vaccine was shown to be safe and with no adverse effects when tested in 15 BALB/cByJ mice. Conclusion: Although conclusions cannot yet be drawn, preliminary evidence demonstrates that the Vanguard CIV bivalent vaccine is safe for use in mice and may be used to address our question regarding the duration of Borreliella-induced immune suppression.
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    Thoracic Duct Lymph Reduces the Production of TNF-alpha IFN-gamma by Pulmonary Leukocytes in vitro
    (2021) Vo, Russell
    Purpose: Streptococcus pneumoniae, a cause of community acquired pneumonia, accounts for nearly one million hospitalizations in the U.S., annually. The lymphatic pump technique (LPT) is a manipulative medicine technique used by osteopathic physicians to mobilize lymph and treat pneumonia. Our objective was to identify the biological effect of thoracic duct lymph (TDL) mobilized with LPT on the immune response against S.pneumoniae. We hypothesized that lymph mobilized during LPT would suppress the in vitro activity of lung leukocytes in mice infected with S.pneumoniae. Methods: TDL was collected from dogs during 4min of baseline, 4min of LPT, and 10min post-LPT. Mice were intranasally infected with 5x10^5 CFU of S. pneumoniae. Lung leukocytes were isolated from healthy and infected mice (24hr post-infection.) Leukocytes were cultured with media plus 5% saline, 5% baseline TDL, 5% LPT TDL, or 5% post-LPT TDL, and co-cultured with/without LPS. The TNFa and TNFg were measured in supernatants after 6, 12, and 24hrs. Results: When cultured with LPS, the addition of baseline LPT, LPT, or post-LPT lymph decreased TNFa and TNFg production by leukocytes from healthy mice. Leukocytes from infected mice did not produce cytokines even when stimulated with LPS, suggesting expended biological activity in vivo. There were no differences in TNFa and TNFg production by leukocytes cultured with baseline LPT, LPT, or post-LPT lymph. Conclusion: TDL reduced inflammatory cytokine production by lung leukocytes. Mobilization of lymph during LPT may release protective factors that limit inflammation and protect the lungs from pulmonary disease.
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    An updated model for maternal separation with early weaning to study early life stress on immune competency
    (2021) Choe, Jamie Y.; Jones, Harlan
    Epidemiological data suggests early life stress (ELS) has negative effects on long-term health and a role in immune-mediated diseases. A significant challenge of studying ELS in the basic sciences has been difficulty developing a reliable mouse model with robust, reproducible stress effects. Although variations of maternal separation in rodents have been published, to date no mouse variant has demonstrated predictable effects on peripheral corticosterone. We describe an updated version of the maternal separation with early weaning (MSEW) paradigm and investigate how psychosocial stress during a defined perinatal window impacts the cytokine profile of select primary and secondary lymphoid tissues. Pups were produced through in-house breeding procedures and subjected to our modified MSEW procedure. Euthanasia occurred at postnatal day (PD) 14 or 21 for blood collection via cardiac puncture. Serum corticosterone and catecholamine levels were detected using commercially available ELISA. Corticosterone was significantly increased in stressed males at PD21 (P< 0.001). Thymocytes of stressed females showed increased secretion of IL-10 (P< 0.001), while stress males exhibited elevated IL-17A (P=0.003) and IL-10 (P< 0.001). Splenocytes of stressed males produced decreased levels of IFN-gamma (P< 0.001) relative to the control. This pilot study demonstrates our updated MSEW model is capable of inducing increased peripheral corticosterone in C57BL/6 pups at PD21 and shows ELS skews cytokine production within select lymphoid tissues at PD21. Preliminary data also suggests sex-dependent effects of ELS on secretion of cytokines critical for T helper 17 and regulatory T cell phenotypes.
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    HIV-1 impairment via UBE3A and HIV-1 Nef interactions utilizing the ubiquitin proteasome system
    (2021) Park, Inwoo
    HIV-1 life cycle regulation has thus far focused on viral gene stage-specificity, despite the quintessence of post-function protein elimination processes in the virus life cycle and consequent pathogenesis. Our studies demonstrated that a key pathogenic HIV-1 Nef interacted with ubiquitin-protein ligase E3A (UBE3A/E6AP), suggesting that interaction is integral to regulation of viral and cellular protein decay and thereby the competing HIV-1 and host cell survivals. In fact, Nef and UBE3A degraded reciprocally, and UBE3A-mediated degradation of Nef was significantly more potent than Nef-triggered degradation of UBE3A. Further, UBE3A degraded not only Nef but also HIV-1 structural proteins, Gag, thus significantly inhibiting HIV-1 replication in Jurkat T cells only in the presence of Nef, indicating that interaction between Nef and UBE3Awas pivotal for UBE3A-mediated degradation of the viral proteins. Nef and UBE3A were specific and antagonistic to each other in regulating proteasome activity and ubiquitination of cellular proteins in general, wherein specific domains of Nef overlapping with the long terminal repeat were essential for the observed actions. Further, Nef itself reduced the level of intracellular Gag by degrading a transcription regulator, Tat, demonstrating a broad role for Nef in the regulation of the HIV-1 life cycle. These data demonstrated that the Nef and UBE3A complex plays a crucial role in coordinating viral protein degradation and hence HIV-1 replication, providing insights into the nature of pathobiologic and defense strategies of HIV-1 and HIV-infected hosts.
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    Age-related thymic atrophy impairs development and function of an antigen-specific tTreg clone
    (2021) Thomas, Rachel; Oh, Jiyoung; Wang, Weikan; Su, Dongming
    Purpose: The atrophied thymus generates an increased ratio of polyclonal thymic Regulatory T (tTreg) cells to thymic conventional T (tTcon) cells, and peripheral Treg (pTreg) cells accumulate during aging. So, why are pTregs in the elderly unable to effectively suppress age-related inflammation ("inflammaging")? Methods: We utilized a mock self-antigen (autoimmune pancreatitis) chimeric mouse model, in which irradiated rat insulin promotor (RIP)-driven mOVA mice received mixed OT-II TCR transgenic and wild-type bone marrow. Thus, we can easily visualize the generation and activation of an antigen-specific Treg cell clone. Additionally, our mOVA mice carry a FoxN1-floxed gene for induction of conditional thymic atrophy, analogous to the aged thymus. Results: The chimeric mice with thymic atrophy exhibited significant decline in central and peripheral OVA-specific (OT-II) Tregs, but not total (pan) Tregs. Further, intrinsic Treg changes in FoxP3 expression was observed, suggestive of reduced suppressive capacity. This was confirmed via functional assays showing that OVA-specific Tregs were significantly less able to suppress antigen-specific stimulation of Teffs in vitro. Additionally, we performed TCR sequencing and observed a trend for decreased TCR diversity in tTregs in mice with thymic atrophy. Conclusion: These data indicate that although the effects of age-related thymic atrophy do not affect pan-Treg generation, certain tissue-specific Treg clones may experience abnormal thymic selection, creating holes in Treg-mediated immunoregulation. This, combined with enhanced pan-pTreg cells, may help explain inflammaging.