bluuebunny/crossref_metadata_2025_split huggingface dataset:
Interleukin 27 (IL-27): an immunoregulatory and antiviral cytokine at the maternal-fetal interface during congenital infection
Authors: Merlino Madeline S , Clements Rebecca L , Lopez Alexandra H , Hunter Christopher A , Jurado Kellie Ann 
Published: August 2023

Abstract
Cytokines maintain homeostasis throughout pregnancy and have diverse responses to pathogens at the maternal-fetal interface. Interleukin 27 (IL-27) is a cytokine that is highly expressed in the placenta, however its functional role during pregnancy is unknown. In other contexts, IL-27 is a potent regulator of inflammation and has been shown to mediate antiviral immunity in the skin. Therefore, our objective was to determine how IL-27 contributes to immune responses at the maternal-fetal interface during congenital viral infection. To begin, we utilized an immunocompetent mouse model for Zika virus (ZIKV) infection to assess pregnancy and infection outcomes in the presence and absence of IL-27 signaling. At gestational day 13.5, we observed that the fetuses of IL-27-competent and -deficient dams had similar ZIKV burdens. However, we observed a significantly higher incidence of fetal pathology (resorption) in IL-27-deficient dams. To identify potential effectors of pathology, I evaluated IL-27 receptor (IL27RA) expression in placental tissues using flow cytometry and immunofluorescence and detected IL27RA+ immune cells throughout the maternal-fetal interface. Together these findings suggest that fetal pathology is independent of ZIKV viral burden in IL-27-deficient mice, and that IL-27 signaling may dampen pathologic immune cell responses during pregnancy. Interestingly, the placental tissues of infected, IL-27-deficient dams had significantly higher ZIKV burdens relative to IL-27-competent dams, indicating a potential antiviral role for IL-27 in the placenta. In conclusion, these data support novel antiviral and immunoregulatory roles for IL-27 at the maternal-fetal interface during congenital viral infection.
This work was supported by the University of Pennsylvania Cell and Molecular Biology Training Grant T32 GM-07229. Additional funding provided by the Linda Pechenik Montague Investigator Award and the Pew Charitable Trusts Biomedical Scholars Program.
Mechanism(s) of long-term humoral immunity and immune suppression induced by wildtype Measles virus in rhesus macaques
Authors: Liu Liting , Bartlett Maggie , Rubens Jessica , Ghimire Shristi , Brockhurst Jacqueline , Chan Kwan Pui , Larman H Benjamin , Griffin Diane E 
Published: August 2023

Abstract
Measles virus (MeV) causes a highly contagious systemic viral infection that remains an important cause of morbidity and mortality in children worldwide. While recovery from measles infection induces life-long protective immunity, it also causes immune amnesia which can lead to higher susceptibility to other infections. To better understand the long-term immune responses induced by the MeV vaccine versus wildtype (WT) MeV, first, we studied rhesus macaques for six months after WT MeV or Measles-Mumps-Rubella (MMR) inoculation. We quantified total antibody secreting cells (ASC) and MeV-specific ASCs in peripheral blood mononuclear cells (PBMCs) using well-characterized in house methods for ELISpot; and assessed MeV-specific IgG, avidity, and plaque reduction neutralization titers. Second, we assessed whether the loss in immune memory post WT MeV infection is linked to eviction of B cells from the bone marrow (BM) into circulation. BM and PBMCs from live attenuated MeV (LAMV) vaccinated monkeys were cultured and supernatant were assessed for isotype and antigen specificity by ELISA. PBMC cultures produced detectable amount of MeV-specific antibody and antibody recognition profiles will be compared via Virscan, a phage-display technology for epitope mapping. A novel panel of non-human primate (NHP) pathogen peptides representing Herpesviruses, Retroviruses, Adenoviruses, Pneumoviruses, and Enteroviruses were developed and expressed using the system with plans to use them to characterize changes in antibody production. We hypothesis that the robust immune response to WT MeV is accompanied by eviction of long-lived plasma cells from BM niches resulting in a loss of Ab diversity that does not occur after infection with LAMV.
Supported by grants from NIH (5R01AI153140-03)
STAT1 expression controls CD4 T cell Th1 and Th17 effector functionality during influenza virus infection
Authors: Finn Caroline M , Prokop Emily , Dhume Kunal , McKinstry Kai 
Published: August 2023

Abstract
Most CD4 T cells primed by influenza A virus (IAV) express the transcription factor T-bet, the Th1 ‘master regulator’, and are marked by phenotypic and functional Th1 hallmarks. The extent to which STAT1 and STAT4 activation, both needed for maximal T-bet induction in vitro, are required to direct IAV-primed CD4 T cell responses is not clear. Here, we transferred STAT-deficient (STAT−/−) or wildtype CD4 T cells recognizing IAV to congenic wildtype mice, challenged them with IAV, and analyzed the donor cells in the infected lungs 7 days later. STAT1−/− cells were barely detected in host mice but were rescued when NK cells were depleted in host mice prior to infection. Functional analysis revealed compromised Th1 identity in STAT1−/− effectors mirroring that of T-bet−/− cells. However, while T-bet−/− cells develop Th17 functions, STAT1−/− cells do not. We show that this is due to alternative receipt of type I IFN signals by the STAT1−/− cells, as blocking type I IFN receptor in IAV-primed mice promotes robust Th17 identity in STAT1−/− but not in WT cells. In contrast, STAT4−/− donor cells closely resemble WT cells. To ask if IAV-primed CD4 T cells can respond to STAT4-activating signals we treated mice receiving STAT4−/− donor cells with IL-12 during IAV infection. IL-12 dramatically increased T-bet and Th1 cytokine production of wildtype, but not of STAT4−/− or T-bet−/− cells. Our findings thus indicate that STAT1 activation is sufficient to direct prototypical antiviral effector identity, but that this response is not fully Th1-polarized without therapeutic STAT4 activation. This work impacts vaccine strategies that aim to promote protective T cell immunity against IAV by shaping the priming environment with STAT-activating cytokine signals.
Kidney disease impairs B cells response against infection via inhibiting germinal center formation and antibody titers
Authors: Peroumal Doureradjou , Jawale Chetan V , Antos Dani , Li De-dong , Wang Shuxia , Alcorn John F , Biswas Partha Sarathi 
Published: August 2023

Abstract
Immunity to infections is crucial to healthy life. However, recent Covid-19 pandemic has shown that comorbidity such as kidney disease and associated uremia impairs immunity against infections. Uremic patients show high susceptible to infections and also exhibit poor antibody responses to vaccine. The mechanisms by which uremia negatively impacts antibody response is unknown. Using multiple mouse models of uremia, we assessed B cells response to model antigen NP-KLH in alum. We show that uremia inhibits both canonical germinal center (GC) and non-canonical extra-follicular B cells response following NP-KLH immunization. Immunized uremic mice demonstrated compromised affinity maturation, isotype switching, antigen-specific antibody secreting cells, antibody titer and T follicular helper cells response. Consequently, uremic mice exhibited diminished GC and antibody response following prime-boost immunization. B cells showed increased cell cycle arrest, apoptosis and impaired migration between light and dark zone in the uremic GCs. We identified uremic toxin hippuric acid as a major driver of loss of mitochondrial membrane potential leading to increased apoptosis in mouse and human B cells. Finally, we show that GC B cells, T follicular helper cells and antibody response were similarly diminished in uremic mice during influenza virus infection, a major cause of mortality in patients with kidney disease. These results shed light on how uremic toxin(s) suppress antimicrobial immunity and vaccine response in individuals with kidney disease. Knowledge gained from this study may pave the path for developing effective therapeutic and preventive strategies in uremic patients against infections including SARS-CoV-2.
R01AI142354, R01AI162616 and R21AI159058
CRISPR/Cas9 screen of B cell differentiation pathways
Authors: Mulpur Roy Neal , Patterson Dillon , Scharer Christopher D , Boss Jeremy M 
Published: August 2023

Abstract
To provide immunity to pathogens, naïve B cells undergo differentiation to several states such as plasma cells and memory B cells. The transcription factors and epigenetic modifiers that program a naïve B cell to adopt a cell fate have not been fully characterized. During differentiation, B cells undergo extensive changes in transcription factor networks and gene expression that are underpinned by changes in the epigenome. One such example is a transition from expression of glycolysis related genes to increased expression of genes related to oxidative phosphorylation metabolism pathways. We previously demonstrated that in vivo, naïve B cells take 8 cell divisions to reach the plasma cell terminal differentiation and that at each division, the B cells also go through a hierarchy of reprogramming before becoming plasma cells after exposure to T independent antigens. Our data also showed that at division 3–4 a second branch emerged that expressed genes associated with the memory B cell fate. In order to find genes that play a key role in committing a naïve B cell to the plasma cell or memory B cell fate, cells committed to the two branches after T independent antigen exposure were sorted and analyzed with RNA sequencing. Analysis of this data revealed 67 genes involved in transcriptional and epigenetic regulation that were significantly differentially expressed between the two branches. In order to determine the role these genes play in B cell lineage path commitment, retrovirus libraries containing gRNAs for each of the 67 genes were constructed and tested in a targeted CRISPR/Cas9 knock out screen. This study identifies mechanisms of early memory B cell fate commitment.
This work was supported by NIH R01 AI123733, P01 AI125180 and T32 GM008490 to JMB
Thymus antibody-secreting cells possess an interferon-responsive gene signature and are comprised of a CD154(CD40L) dependent plasmablast population
Authors: Pioli Peter , Pioli KimAnh 
Published: August 2023

Abstract
Antibody-secreting cells (ASCs) consist of short-lived plasmablasts and post-mitotic mature plasma cells. While the major function of these cells is the production of antibodies, ASCs have been shown to regulate hematopoiesis and as well as the immune response in a context dependent manner. For years, it has been known that ASCs accumulate in the thymus of individuals with autoimmune disorders such as myasthenia gravis. In these instances, thymus ASCs persist long-term and generate pathogenic autoantibodies. However, it remains unclear how these cells are produced and regulated in the thymus under healthy conditions. Understanding these basic concepts may provide clues as to the emergence of select autoimmune diseases. As such, we have investigated the mechanisms of generation and the transcriptional landscape of thymus ASCs and compared these features to their counterparts in the bone marrow and spleen. Examination of ASC numbers demonstrated an organ-specific sex bias which manifested as increased numbers in the female thymus relative to males. Flow cytometric analysis for the proliferation marker Ki-67 demonstrated the presence of Ki-67+ plasmablasts. The administration of CD154(CD40L) blocking antibodies led to significant reduction in Ki-67+ ASCs as well as Ki-67+ B cells in the thymus. As CD40 expression was nearly exclusive to thymus B cells, the effect of CD154(CD40L) blockade was most likely mediated on upstream B cells. Finally, single cell RNA-sequencing revealed that thymus ASCs possessed an interferon-responsive gene signature which was validated at the protein level. Overall, these data provide insights as to the generation and regulation of thymus ASCs which can be leveraged for future studies.
This work was supported by the National Institute on Aging of the National Institutes of Health under Award Number R03AG071955.
Quantifying how TCR sequence variation affects T cell fate at single-cell resolution
Authors: Lagattuta Kaitlyn , Kwun Mujin , Raychaudhuri Soumya 
Published: August 2023

Abstract
Mucosal-Associated Invariant T (MAIT) and Natural Killer T (NKT) cells demonstrate that some T cell transcriptional fates are driven by the T cell antigen receptor (TCR). Though activation through the TCR is integral to T cell differentiation, the contribution of TCR sequence features to other T cell transcriptional fates remains yet to be defined. In this study, we identify how αβ V(D)J recombination affects T cell differentiation at single-cell resolution. By applying Canonical Correlation Analysis (CCA) to 340,557 TCR clones collected from 256 individuals, we define a set of TCR scoring functions that quantify transcriptional fate predispositions conferred by the TCR. Unsurprisingly, the strongest fate predispositions correspond to cognate peptide presentation molecules: MR1- or CD1d-restricted PLZFhigh innate-like (MAIT/NKT) transcriptional fate (72.6-fold increase in fate likelihood for top percentile compared to bottom percentile TCRs, AUC = 0.85) and MHC class I-restricted CD8 fate versus MHC class II-restricted CD4 fate (18.7-fold increase, AUC = 0.75). However, our results also uncover that hydrophobic CDR3 residues promote regulatory T cell transcriptional states in both the CD8 and CD4 lineages, and that TCR sequence features preferred by thymic positive selection continue to promote memory formation in the periphery. Applying our TCR scoring functions to 10× dextramer-labeled T cells reveals that not all antigen-specific TCRs are equally capable of mounting an effector response. Even among T cells that recognize the same antigen, biophysical variation in the TCR sequence directs which T cells undergo transcriptional reprogramming to form immunological memory.
Supported by grant from NIH (T32GM007753)
Characterization, functional validation, and analysis of the cross-reactive T cell repertoire in the context of heterosubtypic influenza A virus infection
Authors: Gaevert Jessica A , Duan Susu , Minervina Anastasia , Pogorelyy Mikhail , Mayer-Blackwell Koshlan , Kirk Allison , Crawford Jeremy , Thomas Paul 
Published: August 2023

Abstract
The T cell receptor (TCR) repertoire is extremely diverse and plays an instrumental role in fighting off pathogens individuals encounter in a lifetime. A key aspect of the immune system combatting the diverse array of pathogens an individual sees may be TCR cross-reactivity, where a TCR can recognize more than one peptide displayed on an MHC-I molecule. Specifically, cross-reactive TCRs may play a key role in allowing the immune system to adapt to rapidly evolving pathogens, such as influenza A virus (IAV). Utilizing 6 variant influenza viruses for priming and secondary challenge, our preliminary data has shown that cross-reactive TCRs seen in primary infection with one IAV can mount a robust and effective memory response to a different IAV, across a range of mutations. However, selection for cross-reactive receptors over multiple infections may create a narrowed repertoire consisting of a few clonotypes, covering a narrow range of antigenic space representing the mutant epitopes encountered. Our data shows that cross-reactive TCRs are more similar to each other than single epitope specific cells and that cross-reactivity can be replicated during peptide stimulation when cross-reactive TCRs are transduced into TCR null cell lines invitro. Given the narrowing nature of cross-reactive responses elicited over different infections, selection for cross-reactivity may provide a substantial escape opportunity with future antigenic variations. Ongoing work focuses on how repeated IAV challenges shape the repertoire, and how previous infections inform future responses by selected cross-reactive T cell pools.
Supported by NIH Grant R01112404040 and the St. Jude Graduate School of Biomedical Sciences
Venous plexus-associated lymphoid hubs support meningeal humoral immunity
Authors: Zanluqui Nagela Ghabdan , Fitzpatrick Zachary , Rosenblum Jared S , Tuong Zewen Kelvin , Chandrashekhar Vikram , Negro-Demontel Maria Luciana , Allinson Kieren S J , Mastorakos Panagiotis , Chittiboina Prashant , Maric Dragan , Donahue Danielle , Ferdinand John R , Portet Anais , Penalver Ana , Gillman Eleanor , Zhuang Zhengping , Clatworthy Menna R , McGavern Dorian B 
Published: August 2023

Abstract
There is increasing interest in how immune cells in the meninges, the membranes that surround the brain and spinal cord, contribute to homeostasis and disease in the central nervous system. The outer layer of meninges, the dura mater, contains both innate and adaptive immune cells and functions as a site for B cell development. In this study, we identified previously undescribed lymphoid structures surrounding fenestrated venous plexi in the dura mater. We found the most elaborate immune organization, including lymphatic vessels, surrounding the rostral-rhinal confluence of sinuses. We termed this structure that interfaced with the skull bone marrow and a comparable venous plexus at the skull base, the rostral-rhinal venolymphatic hub. This hub emerged during development in mice at P8/9 before formation of the superior sagittal sinus. Single cell RNA sequencing demonstrated that rostral rhinal hub hosts a diverse array of resident innate and adaptive immune cells during steady state. Immune aggregates were also present in this structure during homeostasis and expanded with age or following challenge with systemic or nasal antigens. Following intranasal VSV infection, the rostral-rhinal hub supported local germinal center (GC) reactions consisting of T follicular helper cells as well as GC B cells that underwent proliferation, somatic hypermutation, class switch, and conversion into plasma cells locally. These data demonstrate lymphoid architecture around vascular plexi in the dura mater that can sample antigens and rapidly elaborate local humoral immune responses to protect the meninges and underlying brain parenchyma from pathogens.
This research was supported by the intramural program of the National Institute of Neurological Disorders and Stroke, NIH
Differential T Cell Responsiveness to Proliferative Stimuli Assessed with a Novel, Homogeneous Ki-67 Immunoassay
Authors: Lazar Dan F , Kupcho Kevin R , Niles Andrew L , Cali James J 
Published: August 2023

Abstract
Proliferative capacity is an important component of T cell fitness and a relevant parameter for characterization of CAR T cells and T cell activating agents. Ki-67 protein is a well-validated marker of cell proliferation. We used Lumit™ technology to develop a homogeneous, luminescent immunoassay for determination of intracellular Ki-67 levels. Purified CD8+ T cells from different human donors were plated in 96-well format, then treated with a panel of T cell activators +/− IL-2, including αCD3/αCD28, αCD3/αCD28/αCD2, phytohemagglutinin, concanavalin A, and a cocktail of PMA and ionomycin. An optimized lytic reagent was added to control and treated cells, followed by two anti-Ki-67 monoclonal antibodies labeled with SmBiT and LgBiT subunits, respectively. Upon coincident binding of these labeled antibodies to Ki-67 protein molecules, the resultant proximity of SmBiT and LgBiT reconstitutes NanoBiT luciferase. Addition of Lumit Detection Reagent produces light proportional to the level of Ki-67 expression, with a total assay time of 2 h. It was determined that stimulation of Ki-67 levels varied markedly between T cell activators tested, with the maximum responses observed exceeding 13-fold. Notably, T cell responses varied significantly between donors, including with respect to the degree of IL-2 dependence. This assay system was compatible with same-well fluorescent determination of cell death. In parallel treated wells, Lumit cytokine immunoassays were used to assess concomitant increases in cytokine levels. This simple, no-transfer, no-wash immunoassay for quantitative determination of Ki-67 levels provides an improved workflow for rapid and efficient comparative assessments of T cell proliferative responses.
Salivary gland response to type I interferons is independent of circulating cytokine levels: Implications for understanding Sjögren’s syndrome pathogenesis
Authors: Bagavant Harini , Pyclik Marcelina , Papinska Joanna , Durslewicz Justyna , Deshmukh Umesh S 
Published: August 2023

Abstract
Sjögren’s syndrome (SS) is a chronic autoimmune disorder affecting the salivary glands. Viral infection-mediated activation of innate immunity and elevated type I IFNs are considered risk factors for SS. However, the frequency of individuals developing SS is far lower than those infected with viruses. To address the role of genetic susceptibility for SS development, we treated 9 inbred strains of mice covering a diverse genetic repertoire with the TLR3 agonist poly (I:C). Serum cytokines were measured by bead-based assays, and salivary gland immune cell populations were studied by spectral flow cytometry. Primary salivary gland cell cultures were stimulated with IFNα, and the expression of IFN-responsive genes was analyzed. Poly (I:C) induced circulating cytokine levels were widely different between the mouse strains, and they correlated with the presence of a CD11b+ macrophage population in the peritoneum. All poly (I:C) treated mice showed significantly increased frequencies of salivary gland NKp46+ (NK and ILC1) innate immune cells compared to saline-treated controls. IFNα-treated primary salivary gland cultures showed strain-specific differences in IFN-responsive gene expression. Surprisingly, the IFN-responsive gene signature score and elevated frequency of NKp46+ cells in salivary glands did not correlate with the levels of circulating cytokines. These data show that the magnitude of salivary gland responses to type I IFNs are not concordant with the systemic innate immune response. Our study suggests that following a viral infection, genes dictating salivary gland response to type I IFNs might play a critical role in SS development. Identifying such genes will lead to a better understanding of SS etiopathogenesis.
Supported by grants from NIH (NIDCR DE031166)
Acquired Lipodystrophy is Mediated by a Treg Specific Deletion of Helios
Authors: Thornton Angela M , Penna Vinay , Gavrilova Oksana , Shevach Ethan M 
Published: August 2023

Abstract
The selective deletion of the transcription factor, Helios, in Regulatory T Cells (Treg) leads to systemic immune activation characterized by a Th1 phenotype, hypergammaglobulinemia, and enhanced germinal center formation. Strikingly, we also observe acquired lipodystrophy, hepatic steatosis, and insulin resistance. Further analysis of the lipodystrophy had revealed a significant lymphocytic infiltrate in both the inguinal and perigonadal adipose tissue, indicating autoimmune mediated destruction of the white adipose tissue (WAT). We show here that the destruction of adipocytes is dependent on CD8+ T cells, as Heliosfl/fl × Foxp3Cre mice crossed to B2m deficient mice (lacking Class I MHC) maintain normal adipose tissue volume. Flow cytometry analysis of the infiltrate demonstrates that the destruction of the adipose tissue is mediated by CD8+ T cells through both cytotoxic and Fas-L dependent mechanisms.
Preliminary analysis of the adipose tissue Tregs suggests that the Treg fail to acquire or maintain an effector state in the absence of Helios. Thus, Helios deficiency in Treg disrupts immune homeostasis in the adipose tissue, leading to the expansion and activation of CD8+ T cells, the destruction of the tissue and metabolic disease. This work was supported by the Intramural Research Program of NIAID, NIH.
This work was supported by the Intramural Research Program of NIAID, NIH.