bluuebunny/crossref_metadata_2025_split huggingface dataset:
An unconventional T cell population is critical for protection from repeat pulmonary infection with Klebsiella pneumoniae
Authors: Smith Catherine Diane Morffy , Wasbotten Rachel , Twentyman Joy , Rosen David A. 
Published: January 2023

Abstract
Due to emerging antibiotic resistance, the nosocomial pathogen Klebsiella pneumoniae is an increasing public health threat. The extent to which survivors are protected against re-infection, or potential correlates of such protection, are poorly understood. We have explored the immunological mechanisms required for protection against K. pneumoniae re-infection using a nonlethal murine model of pulmonary K. pneumoniae challenge. Mice that survive K. pneumoniae TOP52 infection are protected from subsequent re-infection by an adaptive immune response. Genetically modified mice lacking mature lymphocytes or mature T cells were equally susceptible to primary and secondary infection, suggesting that T cells are required for protection. Intriguingly, neither CD4+ nor CD8+ T cells were absolutely required for either the establishment or execution of a protective memory response as depletion of CD4+ and/or CD8+ T cells during either primary or secondary infection did not impair protection against re-infection. However, we observe an expansion of γδ T cells following primary exposure to K. pneumoniae and a profound amplification of this population following secondary infection. Experiments using genetically modified mice deficient in γδ T cells or intact mice treated with γδ T cell depleting antibodies suggest that this T cell subpopulation is important for protection against re-infection. Thus, we hypothesize that K. pneumoniae exposure drives the development of a resident memory-like population of γδ T cells that are maintained in the lung and contribute to the efficient control of secondary K. pneumoniae infection.
Neonatal Deficiencies in NK cell-Activating Cytokines in the Context of Bordetella pertussis Infection
Authors: Jordan Cassandra Martinez , Mitchell Ashley E , Carbonetti Nicholas H 
Published: January 2023

Abstract
Whooping cough, caused by Bordetella pertussis, has seen a resurgence in recent years, in part due to the switch from the whole cell to acellular vaccine. Although both adults and infants can suffer from severe paroxysmal cough, infants can experience a fatal health decline. Disease progression in infant and adult mouse models show some parallels to human disease. Adult mice exhibit severe lung pathology, but overcome infection, while infant mice experience mild lung pathology, develop leukocytosis, bacterial dissemination, and succumb to infection. We hypothesize that this discrepancy in response to infection is due to a deficiency in neonatal Interferon gamma (IFN-y) production by Natural Killer (NK) cells, which can activate protective T-helper 1 (Th1) cells. We further hypothesize that this deficiency is in part due to the inability of neonatal antigen presenting cells to produce the NK cell-activating cytokines Interleukin (IL) 12 and IL18 when exposed to B. pertussis. To test this hypothesis, we isolated splenocytes from infected and uninfected infant and adult mice, incubated with heat killed B. pertussis, and analyzed for mRNA expression of cytokines via qRT-PCR. While adult splenocytes responded robustly with upregulated expression of IL12 and TNF-α, infant splenocytes failed to upregulate expression of these cytokines in response to bacterial stimulation. Moreover, analysis of lung tissue from infected mice revealed robust upregulation of IL12, IL18 and TNF-α in adult mice but not in infant mice. These results indicate that infant mouse cells fail to upregulate expression of NK cell-activating cytokines in response to B. pertussis infection, leading to a lack of protective IFN-y responses.
Staphylococcus epidermidis facilitates intracellular pathogen clearance through upregulation of antimicrobial protein perforin-2 (P-2) in the human skin gamma delta T cells
Authors: Strbo Natasa , O’Neill Katelyn E. , Head Cheyanne R. , Padula Laura , Stojadinovic Olivera , Pastar Irena , Tomic-Canic Marjana 
Published: January 2023

Abstract
Staphylococcus epidermidis (SE), nonvirulent Gram-positive (G+) bacterium, is a member of the normal human skin microbiota with beneficial relationship with the host. Gamma delta (GD) T cells as the major T cell population in epithelial tissues have been implicated in maintaining tissue integrity, regulating inflammation and defending against pathogens. Perforin-2 (P-2) is a recently described antimicrobial protein responsible for clearance of intracellular G+ and G− bacteria. We examine the relationship between SE and P-2 expression in the human skin.
Healthy human skin tissue was used for flow cytometric and bacterial infection analyses. We analyzed P-2 expression at a single cell resolution using an amplified fluorescence in situ hybridization (FISH) technique for detection of P-2 mRNA in combination with immune-phenotyping. Methicillin resistant Staphylococcus aureus (MRSA) intracellular killing assay was performed on the skin cells that were pretreated with S. epidermidis for 24 h.
We found increase in the frequency of skin GDT cells and induction of P-2 transcripts in GDT cells after S. epidermidis infection. Incubating skin cells with S. epidermidis for 24h prior infection with MRSA resulted in rapid intracellular clearance of MRSA. Our findings reveal a novel P-2 mediated mechanism by which skin commensal bacteria may exert their beneficial role in modulating host innate immune response. In contrast, establishment of S. aureus biofilm resulted in suppression of P-2 expression, revealing a mechanism by which S. aureus escapes cutaneous immunity to cause persistent biofilm wound infections.
IgM+IgD− B cells in Human GALT Harbor Characteristics of B1 B cells
Authors: Bukhari Ameera M 
Published: January 2023

Abstract
During embryogenesis, B cell lymphopoiesis occurs in fetal liver (FL) and bone marrow (BM). B cells derived from these organs differ in their Ig VH gene usage, their morphological and functional characteristics, and they home to different organs in the mature fetus. Although in mice, FL-B cells are known to home to the peritoneum and GALT, little is known of these cells in humans. We identified a FL-like B cell population representing 10–20% of total B cells in human GALT (appendix and tonsil), but only 1–2% in peripheral blood. These cells express IgM, but not IgD on their surface, spontaneously secrete antibodies in culture, and preferentially class-switch to IgA upon LPS stimulation, three characteristics of FL-B cells. The GALT IgM+IgD− B cells are negative for immature B cell markers CD10 and CD5, plasma cell markers CD38hi and CD138, the activation marker CD43 and the transitional B cell marker CD24hi as expected for FL B cells. In infants and children less than 10 years of age, most (70%) of these IgM+IgD− cells are CD27- non-memory B cells. These data support the hypothesis that the GALT IgM+IgD− B cells are of FL origin. Consistent with this idea, approximately 50% of the B cells in an appendix from a 1-day-old neonate were IgM+IgD−. Deep BCR sequence analysis of these cells will establish their developmental origin.
Role of Granzyme B in mucosal immune responses
Authors: Hoek Kristen L , Nazmi Ali , Singh Kshipra , Piazuelo M. Blanca , Wilson Keith , Olivares-Villagómez Danyvid 
Published: January 2023

Abstract
Granzyme B is a serine protease initially described in cell-mediated cytotoxicity. Recently, granzyme B has been found to be produced by non-cytotoxic cells, and elevated levels of granzyme B or cells expressing it have been implicated in many disease states. To investigate the role of granzyme B in mucosal immune responses, we utilized two models of intestinal inflammation: infection with Citrobacter rodentium, a colon-specific bacterium that induces intestinal inflammation in mice; and a T cell transfer model of colitis. Following infection, C. rodentium colonization was observed in the colon of both wt and grzB−/− animals at 14d post-infection. While wt mice displayed no overt signs of infection, severe weight loss as well as disease-associated changes in appearance and colon pathology were observed in grzB−/− mice at 10–14d post-infection. Similarly, adoptive transfer of grzB−/− CD4+CD45RBhi naïve effector T cells into rag2−/− recipients led to severe weight loss and disease-associated changes in appearance by 21d post-transfer, while rag2−/− recipients of wt CD4+CD45RBhi T cells were relatively unaffected at this early time point. Interestingly, disease in recipients of grzB−/− T cells corresponded with increased IL-17+ CD4+ T cells in the mesenteric lymph nodes and colon lamina propria relative to recipients that received wt T cells. These data suggest that granzyme B functions in a protective manner during intestinal inflammation, possibly playing a role in T cell differentiation by holding generation of IL-17-producing T cells in check.
Th17 immunity in the colon is controlled by two novel subsets of colon-specific mononuclear phagocytes
Authors: Huang Hsin-I , Youssef Nourhan , Jewell Mark , Huang Min-Nung , Hammer Gianna 
Published: January 2023

Abstract
Immune responses in the intestine are coordinated by specialized populations of mononuclear phagocytes. Although the functions of intestine-resident mononuclear phagocytes and the different cell subsets that constitute these populations are thought uniform across the entire intestine, it is clear that the anatomy, microbes, and immunological demands are distinct for small and large intestine. Whether these distinctions also include organ-specific mononuclear phagocyte populations, or that these have organ-specific roles in immunity are unknown. Here we identify two novel subsets of colon-specific mononuclear phagocytes, a dendritic cell (DC) subset and a macrophage subset, both of which are exclusive to the colon and are not found in small intestine. These colon-specific DCs and macrophages co-expressed CD14 and CD24, markers which are generally singularly expressed by intestinal DCs and macrophages and commonly used to distinguish between these two cell types. Colon-specific CD14+CD24+ DCs and macrophages failed to express macrophage markers CX3CR1, F4/80, CD64 and CD88, and surprisingly, both the DC and macrophage subset were dependent on the transcription factor IRF4. We further find that colon and small intestine have distinct antigen presenting cell (APC) requirements for Th17 immunity and that while colon-specific CD14+CD24+ APCs were essential for Th17 immunity in the colon, this role in small intestine was mediated by different APC subsets. Our findings reveal unappreciated organ-specific diversity of intestine-resident mononuclear phagocytes and organ-specific requirements for Th17 immunity.
The role of JAML-CAR costimulation in gamma delta T cell-mediated repair of the intestinal epithelium
Authors: Johnson Margarete Daisy , Witherden Deborah , Havran Wendy L 
Published: January 2023

Abstract
Tissue-resident γδ T cells are key players in the maintenance of the body’s epithelial barrier surfaces and perform an essential role in promoting tissue repair. Although γδ IELs are important coordinators of repair in the intestinal epithelium, relatively little is known about how these cells recognize and respond to damage. Recognition of skin wounds by γδ T cells requires costimulation through the junctional adhesion molecule-like protein (JAML) by ligation of coxsackie and adenovirus receptor (CAR), raising the possibility that these molecules may have a similar function in the gut. Here we evaluated the role of JAML-CAR costimulation in the activation and function of γδ IELs using the dextran sulfate sodium (DSS) model of intestinal injury and repair. Following DSS treatment we found that γδ IELs upregulated their surface expression of JAML and observed a shift in the isoforms of CAR expressed by intestinal epithelial cells. In addition, JAML−/− mice exhibited more severe weight loss and epithelial damage than wildtype animals in response to DSS treatment. While we saw that JAML−/− mice had lower numbers of IELs in the colon, this difference did not account for the more severe response to DSS-induced injury in these animals. Importantly, the activation of γδ IELs following DSS treatment was reduced in the absence of JAML-CAR costimulation. In addition, we found evidence of impaired γδ IEL production of IL-17, a cytokine known to protect against DSS-induced epithelial injury. Taken together our results suggest that JAML-CAR costimulation may be needed for the activation of tissue repair functions in γδ T cells across barrier tissues and supports targeting this pathway for therapeutic approaches for the acceleration of healing.
Analysis of tissue specific mechanisms that control the maintenance of Foxp3+ regulatory T cells in the intestine
Authors: Morales Elisa Cruz , Hart Andrew , Laufer Terri M 
Published: January 2023

Abstract
Our lab studies the tissue specific pathways implicated in the maintenance of intestinal Foxp3+ regulatory T cells (Tregs). Previously we used K14 transgenic mice lacking peripheral TCR-MHCII to show that the small intestine lamina propria (siLP) is a niche for natural Tregs that can be filled and maintained independently of Ag-specific signals. K14 siLP Tregs display an effector phenotype (CD62loCD44hiCD103+ICOS+) similar to WT. Effector Tregs are known to be largely peripheral tissue-resident cells that require continued TCR and costimulatory signals for their maintenance. In contrast, we find that in K14 mice the blockade of the CTLA4/CD28—but not ICOS—pathway reduces the proliferation of siLP Tregs while preserving their effector phenotype (CD62loCD44hi). Thus, our results suggest that a population of thymically derived effector Tregs that depend on costimulatory signals can be maintained in the siLP independently of Ag-specific signals. B7-1/B7-2 and CD28/CTLA4 are cell surface molecules that interact through cell-cell contact. By microscopic analyses we show that a population of siLP Tregs localize inside of organized lymphoid structures known as isolated lymphoid follicles (ILF). B cells are the largest lymphocyte population within ILFs. Interestingly, our preliminary data suggest that B cells play a role in maintaining siLP Tregs in steady state, as the depletion of B cells with Rituximab reduces Treg numbers in the intestine of huCD20 mice. Additional imaging studies show ILF Tregs in intimate contact with B cells, suggesting that B cells might influence Tregs numbers by a cell-cell contact dependent mechanism. Thus, our data highlight the intricate signaling network that regulates intestinal Treg homeostasis.
Biological Evaluation of the MILLIPLEX® Human Cytokine/Chemokine/Growth Factor Panel A for Disease Profiling of 48 Immune Factors
Authors: Keith Robert H , Lokshin Anna , Prosser Denise , Pepin Danielle , Xiao Qiang 
Published: January 2023

Abstract
Cytokine, chemokine and growth factor research plays a significant role in achieving a deeper understanding of the immune system and disease. We recently developed a MILLIPLEX® Human Cytokine/Chemokine/Growth Factor Panel A multiplex assay for simultaneous measurement of picogram levels of 48 human immune factors using Luminex® xMAP® technology in small volumes of serum, plasma or cell culture samples. Distinctly different stimulant-dependent cytokine responses were observed for challenged human PBMCs. Both LPS and Con-A induced similarly the secretion of 13 analytes, whereas LPS preferentially induced 10 analytes, including TNFα, IL-1α and IL-1β. Con-A preferentially induced 16 analytes including Th17 cell cytokines IL-17A and IL-17F. Plasma samples, from active (n=20) and benign (n=18) ovarian cancer patients (Dr. Richard Moore, U of Rochester Medical Center) were tested at UPMC. Fourteen analytes were significantly elevated in active patients vs. benign patients including: cytokines TNFα, IL-5, M-CSF; chemokines MCP-3, Fractalkine; Growth Factors FLT-3L, PDGF-BB (p&lt;0.05); cytokines IL-6, IL-18 (p&lt;0.02); cytokines IL-7, IL-27, IL-10, IL-13 (key role in cancer pathogenesis); chemokine MIP-1β (p&lt;0.01). Residual serum and plasma sepsis patient samples (n=16) were compared to those from healthy controls (n=20) and 7 analytes were significantly elevated in sepsis patients: cytokines IL-1RA, IL-6 (p&lt;0.05), IL-7, IL-27, MIP-1β (p&lt;0.02), M-CSF; chemokine MIG (p&lt;0.01). Twelve other analytes trended higher in sepsis samples. Thus, the Human Cytokine/Chemokine/Growth Factor Panel A is a useful tool for profiling 48 immune factors both in cell culture or blood samples for the study of human disease and inflammation.
Using super-resolution microscopy to detect and resolve the distribution of biomarkers that are relevant in a range of immuno-oncology studies
Authors: Burns Sheila , Senior Michael 
Published: January 2023

Abstract
Super-resolution imaging is a novel, Nobel prize-winning technology that can count individual receptors on the membranes of tumour cells and immune cells. It is ultra-sensitive, able to detect as few as 10 molecules per cell. Moreover, super-resolution provides the molecular coordinates of these receptors with nanometre precision, allowing unprecedented accuracy to investigate the interaction between multiple protein species.
We will highlight the ability of super-resolution to detect and resolve the distribution of biomarkers that are relevant in a range of immuno-oncology studies. Recently developed tools allowed us to parameterize the molecular distribution of a target such as checkpoint inhibitors. We further revealed the intermolecular distances between two targets of a novel bispecific proteins. These methods are more relevant than ever before, as ONI’s Nanoimager has packaged the necessary tools into a plug-and-play, desktop-compatible solution. This system shows dynamic imaging of live cell-cell interactions to immune synapse profiling at single molecule detection with nanometre resolution imaging that applies to fixed cells and tissue sections.
EVALUATION OF A MULTIPLEX ARBOVIRUS SEROLOGY ASSAY AS A TOOL FOR SEROSURVEILLANCE
Authors: Venkateswaran Neeraja , O’Brien Thomas W , Nelson Wiilam Max , Venkateswaran Kodumudi S 
Published: January 2023

Abstract
Arthropod-borne viruses (Arboviruses) constitute a serious threat to global health by causing significant mortality and morbidity. Recent years have seen the emergence of arboviruses due to climatic changes, the adaptation of mosquitoes, and other arthropods to a newer geographic area, urbanization, and increased global travel and trade. Currently, most serosurveillance programs focus on single pathogen testing that may miss the prevalent pathogen or misdiagnose due to the presence of cross-reacting antibodies from closely related arboviruses. In the wake of the 2015–2016 Zika virus outbreaks, we developed a multiplex suspension array for the detection of IgG antibodies and avidity to multiple analytes of arboviruses on the Luminex platform to overcome these limitations. We used optically coded magnetic microspheres to immobilize antigens from 10 different arboviruses, namely, Zika (ZIKV), Dengue all four serotypes (DENV 1–4), West Nile (WNV), Japanese Encephalitis (JEV), Yellow Fever (YFV), Tick-Borne Encephalitis (TBEV), and Chikungunya (CHIKV. This 18-plex assay also includes five different internal controls that assure the assay and reagent performance. Initial characterization of the platform was performed using experimentally infected primate samples collected longitudinally before and after infection with ZIKV. In this paper, we describe the results of the evaluation of this multiplex platform using over 400 well-characterized healthy and diseased state human serum and plasma samples from various commercial and collaborating resources. We conclude that this assay provides a relevant tool for efficient monitoring of population based arboviral antibodies in endemic areas and also for detection of outbreaks.
Evaluation of intrahepatic macrophages in patients with non-alcoholic steatohepatitis using multispectral imaging
Authors: Saldarriaga Omar A , Jiao Jingjing , Beretta Laura , Freiberg Benjamin , Krishnan Santhoshi , Rao Arvind , Booth Adam L. , Stevenson Heather L. 
Published: January 2023

Abstract
Patients with non-alcoholic steatohepatitis (NASH) have different rates of fibrosis progression. We hypothesized that this is due to intrahepatic macrophages. Kupffer cells (KCs) can affect the development of fibrosis by recruiting systemic macrophages that activate stellate cells. Liver biopsies were collected from patients with NASH and either minimal (n=6) or advanced (n=5) fibrosis. We used spectral imaging to evaluate KCs (CD68+), monocyte-derived (Mac387+), pro-fibrogenic (CD163+), pro- (CD14+), and anti- inflammatory (CD16+) macrophages. NanoString evaluated gene expression profiles in the same biopsy. t-distributed stochastic neighbor embedding (t-SNE) plots and phenotype matrices were used to visualize differences between groups. Mean normalized counts from ‘cell seg’ data were used to determine the percentage of phenotypes present. Results showed that patients have variable macrophage phenotypes in the liver depending on their propensity to develop hepatic fibrosis. Patients with minimal fibrosis showed increased CD14+ macrophages and more protective gene expression profiles. Patients with advanced fibrosis had significantly increased numbers of pathogenic pro-inflammatory/pro-fibrotic macrophages (MAC387+, CD163+) and increased expression of genes associated with mixed macrophage activation (IDO1, CD86, HLADR, IL1β, TNFα, COX-2, Dectin-1, CD209, PI3K/AKT3, IL10R, TGFB1, IRF4, CLEC7A/DECTIN-1). However, when we analyzed each patient’s liver biopsy on an individual basis, subtle variations were observed between the patients in each group. Thus, the various phenotypes of macrophages present in patients with NASH is complex and is only partially associated with fibrosis stage.
Gain a Better Understanding of Immunology using Single-Molecule Localization Microscopy
Authors: Burns Sheila 
Published: January 2023

Abstract
Studying protein organization and function at molecular level is a major technical challenge that limits our understanding from both a diagnostic and treatment perspective. Single-molecule localization microscopy (SMLM) is a Nobel Prize winning technique that allows quantitative imaging below the diffraction limit and can be applied to significantly improve our understanding of many biological phenomena. We provide a versatile imaging platform with unrivalled stability and flexibility to work on a lab bench. The data presented was acquired using two and three-color, super-resolution imaging of EVs and TCR alpha and beta receptors and reveal their spatial organization at 20 nm resolution. Additional experiments to show levels of expression on the cell surface, yielding the characterization of receptor distribution and ultimately activity and signaling. Experiments will show the wide-ranging applicability of our instrument as a tool for multiple biological analyses encompassing many fields within immunology.