bluuebunny/crossref_metadata_2025_split huggingface dataset:
B1 B cell-derived bone marrow plasmacytes are the primary source of natural IgM (109.11)
Authors: Reynolds Alexander , Kuraoka Masayuki , Kelsoe Garnett 
Published: January 2023

Abstract
Natural IgM is a unique and important component of humoral immunity. It provides early protection against microbial pathogens and helps to maintain tissue homeostasis. Though B1 cells are known to be the primary source of natural IgM, the developmental stage and anatomical location of the cells that secrete natural IgM antibody remain controversial. In order to answer this question, we first confirmed earlier observations that the spleen and BM are the primary tissues in which IgM secreting cells reside. We next examined two lines of VDJ knockin mice and found that both exhibited 10-fold reductions in serum IgM levels despite the presence of normal IgG concentrations. Whereas knockin animals had normal numbers of IgM secreting cells in the spleen and numbers of splenic and BM IgG plasma cells that were indistinguishable from normal controls, the numbers of IgM plasmacytes in the BM of knockin mice was ≤20% of normal animals. In addition, the vast majority of peritoneal B1 cells were missing in both knockin strains whereas all B2 populations remained unaffected. By sorting BM cells, we determined that only those with the CD138high IgM+ phenotype secreted IgM in vitro; analyses of the two knockin mouse strains revealed this BM population to be significantly reduced in concordance with the peripheral B1 B-cell populations. We conclude that natural IgM antibody is produced by differentiated BM plasmacytes that are derived from peripheral, B1 B-cell populations.
Diversification of Toxoplasma gondii-specific cytotoxic T lymphocytes by IL-12 (110.15)
Authors: Shah Suhagi , Grotenbreg Gijsbert , Yap George 
Published: January 2023

Abstract
The obligate intracellular parasite Toxoplasma gondii elicits a robust and uniquely IL-12 dependent CD8+ T cell immune response. We have previously shown that IL-12 is required for the differentiation of KLRG1+ CD8+ T effector cells following T. gondii infection. We have also shown that in the absence of IL-12, the frequency of KLRG1- memory precursor effector cell (MPECs) differentiation is increased at the expense of KLRG1+ short-lived effector cells (SLECs). Therefore, IL-12 may be a critical signal for the early phase bifurcation of naïve CD8+ T cells into KLRG1+ SLEC and KLRG1- MPEC-fated lymphocytes. To determine if SLECs and MPECs are generated in the earliest phase of CD8+ T cell activation, we enumerated T. gondii reactive CD8+ T cells by magnetic bead enrichment of Kb-tgd057 tetramer binding cells. We have found that the initial proliferation of tgd057-specific CD8+ T cells in the spleen occurs between day 3 and day 5 following T. gondii infection. Concurrently, KLRG1+ and KLRG1- CD8+ T cells can be observed in both the spleen and the effector site of the peritoneum, suggesting early diversification of tgd057-specific CD8+ T cells. Current efforts are geared towards visualizing the events leading to the initial bifurcation of the CD8+ T cell response and determining how IL-12 regulates this decision-making process.
Genomic analysis of CD8 T cells during the early phases of the immune response (110.25)
Authors: Blair David , Mayya Viveka , Madar Aviv , Best J. Adam , Bonneau Richard , Dustin Michael 
Published: January 2023

Abstract
The generation of long-lived memory CD8 T cells is critical for the development of effective vaccines and adoptive immunotherapy strategies. However, the molecular mechanisms by which a T cell transitions from naïve to memory remains poorly understood. In an effort to better understand the molecular pathways involved in the differentiation of CD8 T cells, we have analyzed ovalbumin-specific OTI CD8 T cells during the very early phases of the immune response at the genomic level. Following infection with Listeria monocytogenes expressing ovalbumin, OTI CD8 T cells from infected mice were sorted and subjected to microarray analysis. We show that while cell surface phenotype of OTI T cells remained largely unchanged shortly after activation, large scale changes occurred at the level of transcription. Expression levels of many families of genes involved in signaling networks in T cells, in addition to multiple immune response factors, were differentially expressed shortly after T cells were activated, but prior to the first division. Notably, expression of IL-21, a cytokine implicated in T cell memory, was transiently expressed with peak expression occurring at 12 hours post-stimulation. This was confirmed by intracellular staining for the IL-21 protein. Similarly, the peak of STAT3 expression coincided with IL-21, a known activator of STAT3. We are currently investigating autocrine IL-21 signaling in CD8 T cells and its role in memory development.
B cell Transcription Factors Define a Novel Tumor Suppressor Gene Network In Acute Lymphoblastic Leukemia (111.11)
Authors: Heltemes Harris Lynn , Kornblau Steven , Farrar Micheal 
Published: January 2023

Abstract
STAT5 is activated in a subset of patients with ALL and high levels of STAT5 activation prior to treatment predicted poor response to therapy. In mice, we identified synergy between constitutive activation of STAT5 (STAT5b-CA) and Ebf1 or Pax5 haploinsufficiency that rapidly induced progenitor B-ALL in 100% of the mice. The leukemic cells displayed 2-fold reduced expression of Ebf1 and Pax5 that affected a small subset of EBF1 or PAX5 target genes, including tumor suppressor genes and oncogenes. To test whether compound haploinsufficiency of Ebf1 and Pax5 is sufficient to cause transformation we generated Ebf1+/- x Pax5+/- mice. Over 80% of these mice developed progenitor B-ALL. Although these mice lack the Stat5b-CA transgene, the leukemic cells exhibit increased STAT5 activation. In preliminary studies we observed similar results in Ebf1+/- x Ikzf1+/- and Pax5+/- x Ikzf1+/- mice. Our findings suggest a model in which small perturbations in a self-reinforcing network of transcription factors critical for B cell development cooperate with STAT5 to initiate ALL and that EBF1, PAX5 and IKZF1 form an interrelated network of transcription such that loss of any two alleles in the network results in a loss of tumor suppressor function. This hypothesis represents a departure from the conventional idea that transformation is a result of deletion at both alleles of one tumor suppressor gene, and may explain why mutations in Pax5, Ebf1 and Ikzf1 are so common in human ALL.
Characterization of human thymocyte behavior in situ (111.46)
Authors: Melichar Heather , Halkias Joanna , Ross Jenny , Taylor Kayleigh , Yen Bonnie , Robey Ellen 
Published: January 2023

Abstract
Thymocyte development occurs through the coordinated movement of developmental intermediates to defined anatomical locations within the thymus during which thymocytes sample surrounding cells and the microenvironment for signals to promote differentiation. The development of an experimental model in which purified, labeled cell populations migrate into a 3-dimensional living thymic slice gives us the first opportunity to visualize and monitor the behavior of human thymic subsets in situ. Double negative (DN) progenitors and CD4+CD8+ double positive (DP) human thymic subsets primarily localize to the cortex, whereas CD4+ and CD8+ single positive (SP) subsets localize appropriately to the medulla. In many respects, the migration of human thymic subsets mirror that of their murine counterparts: immature DP and DN cells move randomly with slow motility rates, DP cells migrate slower than DN, and both SP populations are fast and exhibit some directional movement presumably toward the medulla. These similarities between human and mouse thymocyte motility occur despite significant differences in the development and microenvironment of the thymus between the species. We are also examining cross-talk between mouse and human factors as well as the sensitivity of human thymic subsets to chemokine signals. The suitability of the thymic slice model to study human thymocyte development is a powerful system to study and manipulate processes that are otherwise inaccessible to researchers.
Role of protein tyrosine kinase seven in human recent thymic emigrant cell survival (111.50)
Authors: Acharya Swati , Ramachandran Vasavi , Dionis Kira , Lewis David 
Published: January 2023

Abstract
Recent thymic emigrants (RTEs) are antigenically naïve T cells that have recently completed intrathymic maturation and emigrated from the thymus to the periphery. Our lab has shown that PTK7 is a novel marker for human RTEs of the MHC class II-restricted CD4+CD8- T-cell lineage and is highly expressed during human and murine intrathymic development. PTK7, a ‘pseudokinase’, is a member of the receptor tyrosine kinase family previously implicated in the planar cell polarity pathway of neuroepithelium. While viable CD4+ RTEs can be identified based on PTK7 surface staining to define the phenotype of RTEs in humans, the function of PTK7 in human T-cell development remains unclear. To decipher a functional role of PTK7 in RTEs, we performed lentiviral small hairpin sh RNA or small interfering si RNA-mediated gene silencing in T and non-T lineage cell lines, primary human adult and neonatal peripheral T cells and thymocytes. In all these cellular contexts RNAi-mediated knockdown of PTK7 consistently decreased cell survival alongside caspase 3 activation. Also, T cells with reduced PTK7 levels showed increased susceptibility to apoptosis inducing agents, such as camptothecin and staurosporin, with apoptosis dependent on caspase 3. Survival that was dependent on AKT phosphorylation downstream of PTK7 was also reduced by PTK7 RNAi knockdown. Together, these observations suggest an essential role for PTK7 in survival of RTEs, and most likely, in developing thymocytes.
TSLP expands the production of human B lineage cells in vitro and in a novel human TSLP expressing xenograft model (111.54)
Authors: Payne Kimberly , Su Rui-jun , Baez Ineavely , Francis Olivia , Weldon Abby , Dovat Sinisa , Milford Terry-Ann 
Published: January 2023

Abstract
IL7 and TSLP are important in murine B cell development, however their role in human B lymphopoiesis is unclear. In contrast to other cytokines that act on B cell progenitors, TSLP lacks species cross reactivity and activates different intracellular signaling pathways in mice and humans. Thus, human models are essential for understanding the role of TSLP in human B lymphopoiesis. We developed a novel human-only model of in vitro B cell development that allows selective cytokine stimulation and used this model to show that IL-7 is essential for human B cell production where it increases B cell progenitors by over 60 fold (JI 2009 82:4255). Here we use this model and a novel xenograft model system to study human B lymphopoiesis from CD34+ HSCs in umbilical cord blood (CB). BrdU incorporation shows that TSLP induces proliferation of human B lineage cells to produce in vitro expansion of B cell progenitors similar to that observed for IL7. TSLP acts directly on human B cell precursors to induce STAT5 phosphorylation and upregulate PAX5. To evaluate the role of TSLP in vivo we developed a novel xenograft model system consisting of human TSLP+ (hTSLP+) and hTSLP- immune deficient NSG mice. Using the hTSLP+/- model system we show that hTSLP augments existing IL7 effects to expand human B lineage cells (from 37% to 68% total B lineage chimerism) in NSG mice, a strain that facilitates human B cell production. These data suggest that TSLP is important in human B lymphopoiesis.
Differential thymocyte motility and microenvironmental localization during normal and neoplastic T cell development (111.55)
Authors: Ehrlich Lauren , Cardenas Kim , Jones Jessica , Selden Hilary 
Published: January 2023

Abstract
As thymocytes develop, they modulate expression of receptors, such as chemokine receptors, that promote migration into distinct thymic microenvironments. Within these thymic regions, developing thymocytes have the opportunity to interact with unique stromal cells that provide survival, selection, and/or differentiation signals. Thymocyte: stromal interactions are essential for proper thymocyte development, implicating the molecules involved in regulating intrathymic migration as key regulators of T cell development. Previously, we have used 2-photon microscopy to study real-time migration of thymocytes within live thymic slices. These studies revealed that immature thymocyte progenitors were restricted to the cortex while only post-positive selection thymocytes were able to enter the medulla. The ability to migrate within cortex versus medulla was regulated by multiple mechanisms, including G-protein coupled receptor mediated chemotaxis and substrate accessibility. We hypothesize that the molecular cues that mediate normal intrathymic migration may be disrupted during thymic lymphomagenesis, contributing to the initiation and/or progression of disease. Consistent with this hypothesis, we find that the thymic microenvironment is disorganized in a murine model of thymic lymphoma. Furthermore, using 2-photon microscopy, we find that migration of lymphoma cells within thymic tissue is abnormal, suggesting that aberrant thymocyte: stromal interactions may contribute to disease.
Enhancing host defense against bacterial pore-forming toxin by suppressing HMG-CoA reductase pathway in airway epithelial cells (112.13)
Authors: Kao Cheng-Yuan , Hung Li-Yin , Chang Ching-Yun , Lim Jae-Hyang , Li Jian-Dong , Wu Reen 
Published: January 2023

Abstract
Pneumolysin and α-hemolysin are the main pore-forming toxins known to play critical roles in bacterial pneumonia caused by the infections of Streptococcus pneumoniae and Staphylococcus aureus, respectively. Airway epithelia are the initial and the primary targets of these air-borne bacterial infections. Several epidemiological studies have linked the use of statin to the improvement in pneumonia outcomes, but the nature of the mechanism is still unknown. Using primary normal human bronchial epithelial (NHBE) cells and an immortalized normal bronchial epithelial cell line, HBE1, we confirmed the effects of simvastatin pre-treatment in enhancing host defense against these bacterial toxins. The protective effects could be further demonstrated in vivo with simvastatin administration prior to intra-tracheal instillation of these toxins. We further found that the protection requires protein synthesis and is calcium dependent. Using siRNA gene silencing, pharmacological treatment with inhibitors, immuno fluorescence microscopy, Western blot and qRT-PCR approaches, we have delineated the protection mechanisms in airway epithelial cells through HMG-CoA-Reductase pathways. Our results also suggest that the inhibition of HMG-CoA reductase pathway may enhance host defense molecule synthesis in airway epithelium. It is plausible that this study may lead to the development of an adjuvant therapy against pore-forming toxin-related bacterial infections in lung.
IgG keeps virulent Salmonella from evading dendritic cell uptake (112.15)
Authors: Riquelme Sebastián , Bueno Susan , Kalergis Alexis 
Published: January 2023

Abstract
Salmonella enterica serovar Typhimurium (ST) can prevents Dendritic Cells (DCs) from activating antigen-specific T cells. In addition, it has been described that the Salmonella Pathogenicity Island 1 (SPI-1), which promotes phagocytosis of bacteria in non-phagocytic cells, can suppresses this process in DCs in a PI3K-dependent manner. Recent studies have shown that IgG-opsonization of Salmonella can restore the capacity of DCs to present antigenic pMHC complexes and prime T cells in a FcγRIII-dependent manner. This suggests that this receptor could counteract both antigen presentation and phagocytosis evasion by bacteria. Here we show that, despite IgG-coated Salmonella retains its capacity to secrete anti-capture proteins, DCs are efficiently capable to engulf high loads of IgG-coated bacteria. These results suggest that DCs employ another mechanism to engulf IgG-coated ST different from that used for free bacteria. Thus, we noted that DCs do not employ PI3K, actin cytoskeleton or dynamin to capture IgG-coated bacteria. Likewise, we observed that the capture is a FcγR-independent mechanism. Interestingly, these internalized bacteria were rapidly targeted for degradation within lysosomal compartments. Thus, our results suggest a novel mechanism in DCs that does not employ PI3K/actin cytoskeleton/dynamin/FcγRs to engulf IgG-coated Salmonella, is not affected by anti-capture SPI-1-derived effectors and enhance DC immunogenicity, bacterial degradation and antigen presentation.
The Membrane Attack Complex of complement is required for the development of murine Experimental Cerebral Malaria (112.2)
Authors: Ramos Theresa , Darley Meghan , Barnum Scott 
Published: January 2023

Abstract
Cerebral malaria (CM) is a severe complication of P. falciparum malaria with a 15-30% fatality and 10% of CM survivors have permanent neurological sequelae. Since inflammation is key to CM development, we examined the role complement in experimental cerebral malaria (ECM) development using the P. berghei model. Previous studies indicated that C5-/- mice were protected in ECM. To determine the role of complement activation, we examined mice deficient in pathway specific components, C4 and Factor B, as well as C3, which forms the C5 convertase. We found no differences in survival between wild type (WT) and C4 or Factor B deficient mice. We observed moderate protection in C3-/- mice, suggesting that blocking C5 cleavage is important in ECM development. We found that complement anaphylatoxin receptor deficient mice were not protected, suggesting that the mechanism of protection in C5-/- mice is not mediated through C5a-induced inflammation. We next examined C5b indirectly by treating WT mice with anti-C9 antibody and found significant increase in survival. We also treated C3-/- mice with anti-C9 antibody to determine if blocking MAC in these mice could abrogate disease, and found significant protection in treated mice compared to controls. Our results suggest that pathological effects of complement in ECM are primarily mediated at C5 through the terminal pathway and that this is occurring even in the absence of C3 suggesting an alternative mechanism for C5 cleavage in C3-/- mice.
Molecular mechanism for type I interferon induction by atypical strains of Toxoplasma gondii (112.6)
Authors: Melo Mariane , Nguyen Quynh , Saeij Jeroen 
Published: January 2023

Abstract
Host cells infected with Toxoplasma gondii undergo profound physiological and morphological changes as a result of the host cell’s response to the parasite and the parasite’s attempt to convert the host cell into a suitable growth environment and evade the immune system. T. gondii infection is known to affect cytokine induction by host cells through modulation of intracellular signaling pathways, including those controlled by NFκB and STAT3/6. To identify other relevant host cell signaling pathways altered by parasite infection, bone marrow-derived macrophages were infected with 26 different strains of Toxoplasma. Expression profiles of both parasite and host cells were then determined by highthroughput RNA sequencing. Initial data analysis revealed that host cell response varies considerably depending on the strain used for infection. We were able to identify a group of strains that can induce IFNβ production and the subsequent upregulation of interferon regulated genes, including viperin and IRF7. Type I interferons are usually produced upon recognition of nucleic acids by either surface or cytoplasmic pathogen-associated molecular pattern receptors, including Toll-like receptors, and the RNA helicases RIG-I and Mda5, triggering a signaling cascade that activates the transcription factors IRF3, IRF5 and/or IRF7, which induce transcription of the IFNβ gene. We are currently investigating the molecular mechanisms controlling IFNβ production in Toxoplasma-infected macrophages.
Large Scale T Cell Epitope Discovery in Yellow Fever Virus (113.12)
Authors: Kongsgaard Michael , Stryhn Anette , Buus Søren 
Published: January 2023

Abstract
This project involves a comprehensive, large-scale discovery effort directed at identifying T cell epitopes targeted in the acute immune response following vaccination with attenuated yellow fever (YF) virus in an out bred population of vaccinees. WHO recommends revaccination once every 10 years. Volunteers have donated blood before and about 2 weeks after vaccination and divided into groups of naïve and boosted donors. Initially, pre-and post PBMCs from the vaccinees were analyzed using multicolor flowcytometry to determine the frequency of activated T cells using intracellular and surface phenotypic markers. Clearly, boosted donors generated low frequencies of KI67+, Bcl-2-, CD8+ T cells (1.4%) and CD38+, HLA-DR+, CD8+ T cells (2.1%), resembling background levels (1.2% and 1.2%), while naïve donors generated higher frequencies of activated cells (5.1% and 5.1%). YF peptide-specific, HLA-restricted T cell responses were identified by ELISpot with a 30x30 peptide matrix and validated using multicolor flowcytometry to determine the nature of the YF specific T cell response (CD4, CD8 and CD38, functional markers such as IFNγ and TNFα and specificity markers (HLA tetramers)). The number of responses and the frequency of peptide-specific cells are clearly lower in boosted donors, than in naïve donors. This coincides with YF-specific antibody titers in boosted donors.
Modification of immune activation in HIV-1-infected humanized mouse model using TLR7/9 antagonists (113.19)
Authors: Chang J. Judy , Lindsay Robert , Doyle Erin , Vrbanac Vladimir , Seung Edward , Dudek Timothy , Precopio Melissa , Kandimalla Ekambar , Tager Andrew , Altfeld Marcus 
Published: January 2023

Abstract
Background: T cell immune activation is a strong predictor of HIV-1 disease progression, and IFNα production following TLR7 stimulation has been associated with elevated CD8+ T cell activation. We hypothesize that modulation of TLR7 stimulation could be used to manipulate IFNα production and subsequently reduce HIV-1-associated immune activation. Methods: Humanized BLT mice were generated by transplanting irradiated NOD/SCID/IL2Rγc-/- mice with human fetal thymus, liver tissue and human hematopoietic stem cells. After immune reconstitution, human DC, monocyte and T cell populations were detectable in the mice, and in vitro TLR responsiveness of DCs and monocytes reflected those observed in human PBMCs. Humanized mice were infected with HIV-1 and then treated with a TLR7/9 antagonist. The T cell activation markers were examined at each stage by flow cytometry. Results: BLT mice-derived human pDC responses to TLR7 stimulation were significantly blocked by in vitro treatment with the TLR7/9 antagonist (P&lt;0.05). HIV-1 infection of BLT mice led to increased expression of immune activation marker CD38 on human T cells in vivo, and treatment of infected mice with the TLR7/9 antagonist led to a significant reduction in CD38 expression. Conclusion: Treatment of HIV-1-infected humanized BLT mice with TLR7/9 antagonist resulted in significant reduction of HIV-1-associated immune activation, indicating an important role of the TLR7/9 pathway in mediating immune activation in HIV-1.