bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-15/IL-15Rα improves immune cell survival but does not reduce disease severity following pulmonary infection with the intracellular bacterium Francisella tularensis (56.27)
Authors: Schmitt Deanna , O'Dee Dawn , Brown Matthew , Horzempa Jospeh , Russo Brian , Nau Gerard
Published: January 2023

The potent anti-apoptotic cytokine IL-15 regulates the development and function of a wide range of innate and adaptive immune cells. Treatment with IL-15 in combination with IL-15Rα prevents apoptosis and immunosuppression in mouse models of sepsis. Inhalation of Francisella tularensis results in an acute pneumonia that demonstrates many of the hallmarks of sepsis including bacteremia and hypercytokinemia. We and others have observed widespread cell death in the lung following infection with virulent F. tularensis strain Schu S4. The predominant cell populations depleted are lymphocytes and neutrophils. Cell numbers declined in Schu S4-infected mice despite high levels of chemokines in the lung and correlated with an increase in TUNEL-positive cells. This supports the conclusion that immune cell depletion was due to cell death, and not a lack of recruitment. To determine whether this cell depletion was contributing to Schu S4 pathogenesis, mice were treated with IL-15/IL-15Rα prior to infection. IL-15/IL-15Rα treatment significantly increased the number of viable lung cells in Schu S4-infected mice compared to mock-treated controls, however, no difference in bacterial burden or disease severity was observed. These data suggest that depletion of immune cells by F. tularensis does not contribute to the morbidity and mortality associated with pneumonic tularemia. Moreover, IL-15/IL-15Rα may not be generally applicable to treat sepsis of different etiologies.
A JAK3 pathway may be involved in the production of IL-6 by human monocytes stimulated with BAFF. (57.16)
Authors: Yoshimoto Keiko , Tanaka Maiko , Ogata Hideko , Kojima Masako , Abe Tohru , Takeuchi Tsutomu
Published: January 2023

Background and Purpose: It is known that BAFF plays an important role in the pathogenesis of Sjögren’s syndrome (SS). In our previous study, we found that soluble BAFF (sBAFF) remarkably induced the production of IL-6 by peripheral SS monocytes. In the present study, we investigated a possible pathway which is involved in the transduction of signals triggered by BAFF for the production of IL-6 by monocytes. Materials and Methods: Peripheral monocytes were prepared from SS patients and normal individuals and stimulated in vitro with sBAFF in the presence of several kinase inhibitors. The production of IL-6 by the cells was measured by ELISA. Results and Discussion: The production of IL-6 by monocytes was strongly suppressed by a JAK3 inhibitor in a dose dependent manner. On the other hand, inhibitors for JAK2, JNK and p38MAPK had only marginal effects. In addition, real-time PCR analysis indicated that the expression level of JAK3 in SS monocytes was abnormally elevated upon stimulation with sBAFF as compared with normal monocytes. These data strongly suggest that JAK3 plays a pivotal role in the production of IL-6 by monocytes, and the expression of JAK3 is dysregulated in SS monocytes. Taken together, aberrant production of IL-6 by SS monocytes upon stimulation by sBAFF may be attributed to the abnormal expression of JAK3 in the cells.
Development of a novel, murine, peritoneal leukocyte recruitment model to overcome the lack of C5a receptor cross reactivity between human and mice (60.5)
Authors: MacLeod Heather , Serpa Megan , Zhang Qin , McAllister Kathryn
Published: January 2023

The ability to test target-specific small molecules in vivo can be hampered by a lack of species cross reactivity. In the absence of knock-in animals, novel methods to evaluate in vivo efficacy by a target-specific mechanism can be developed. In vivo models of peritoneal recruitment of leukocytes are often used to evaluate cell migration and inflammation by different pro-inflammatory stimuli. An example of a potent chemoattractant capable of inducing migration of inflammatory cells into the peritoneum is the anaphylatoxin C5a which binds to the C5a receptor (C5aR) on many cell types. The aim of this study was to develop a novel mouse peritoneal leukocyte recruitment model using C5a as the pro-inflammatory stimulus and a cell line expressing the human C5aR. Mouse B lymphocytes (L1.2 cells) were stably transfected to express human C5aR and were fluorescently labeled with Qdot® nanocrystals. Labeled cells were injected into mice i.v. followed by an i.p. injection of human C5a. At multiple time points following C5a administration, the peritoneum was lavaged and the recovered cells subjected to flow cytometry to quantify the number of the labeled cells that migrated to the peritoneum. Our data show that cells expressing the human C5aR are recruited to the peritoneum 24 hours following human C5a administration. This assay demonstrates a novel mouse model that can detect human receptor-driven chemotaxis in mice.
PD-1:PD-1 ligand interactions suppress B-1b cell responses to T cell-independent type 2 antigens (61.7)
Authors: Haas Karen
Published: January 2023

B-1b cells play a key role in producing antibodies (Ab) in response to T cell-independent type 2 (TI-2) antigens (Ags). However, the factors regulating Ab production by this unique B cell subset are not well understood. In this study, the role of the PD-1 immunoinhibitory receptor in regulating B-1b cell responses to TI-2 Ags was investigated in normal mice. Following TNP-Ficoll immunization, Ag-specific B-1b cells transiently expressed PD-1, which suppresses BCR-induced B-1b cell proliferation when ligated in vitro. In vivo administration of an Ab that blocks PD-1 from interacting with its ligands during the first 3 days following TNP-Ficoll immunization significantly enhanced Ag-specific B-1b cell expansion and differentiation relative to mice that had received control Ab. Serum levels of Ag-specific IgG3, the major IgG isotype produced in response to TI-2 Ags, remained significantly elevated in PD-1 mAb-treated mice for at least 6 weeks post-immunization. Remarkably, PD-1 mAb administration during the first few days following immunization resulted in significantly increased numbers of both splenic and bone marrow Ag-specific IgG3, but not IgM, secreting cells at both early (day 5) and late (6 week) timepoints. Thus, PD-1:PDL interactions occurring shortly after initial TI-2 Ag encounter play a critical role in suppressing Ag-specific B-1b cell expansion and the development of long-term IgG3-producing bone marrow and spleen cells.
Structural and functional studies on PD-1: from crystals to mice (63.17)
Authors: Lazar-Molnar Eszter , Ramagopal Udupi , Almo Steven , Nathenson Stanley
Published: January 2023

Programmed death-1 (PD-1) inhibits T and B cell responses upon binding to its ligands, PD-L1 and PD-L2, and has crucial role in maintaining peripheral tolerance. We have recently reported the crystal structure of the complex between PD-1 and PD-L2. Performing structure-based mutagenesis studies, high-affinity, and PD-L2-specific PD-1 mutants were identified. High-resolution crystal structures of the high-affinity PD-1 mutant with PD-L1 and PD-L2, respectively, provide the atomic details of the tighter receptor-ligand binding. The crystal structure of the complex between the PD-L2-specific PD-1 mutant and PD-L2 provides the atomic details of the selective interaction of this mutant with PD-L2. Soluble Ig fusion proteins of the two PD-1 mutants were designed to modulate the PD-1 pathway for immunotherapy. Finally, these mutations were introduced into mice by generating “knock-in” mouse strains, which provide novel animal models to study the role of PD-1 in immunity. Current work is focused on analyzing the functional effects of these mutations in PD-1 mediated immune responses. In summary, our data demonstrate the value of structural studies in developing novel immunotherapy and animal models for in vivo studies.
Stat3 activation plays a role in mTEC development (64.20)
Authors: Jain Manju , Lomada Dakshayani , Reddy Madhava , Kang Rhea , DiGiovanni John , Richie Ellen
Published: January 2023

The thymic medulla is central to the establishment of central tolerance. Development of medullary thymic epitthelial cells (mTECs) in the fetal thymus requires lymphoid tissue inducer cells, whereas in the adult, signals from positively selected thymocytes engage different components of NF-κB signaling pathway to regulate the formation of an optimal medullary stromal niche. Thus, mTEC development is impaired in RAG-1-/- and RAG-2-/- mice due to the early block in thymocyte differentiation. We find that introduction of a constitutively active Stat3 transgene regulated by a keratin 5 promoter (K5.Stat3C) in Rag 2-/- mice rescues mTEC development bypassing the requirement for positively selected thymocytes. Although K5.Stat3C mice exhibit thymus hypoplasia, the medullary compartment is expanded. Immunohistochemical analysis demonstrates K5+K14+ and UEA+ mTECs. However, FACS analysis reveals an increase in immature mTECs based on low expression of MHC class II and CD80 markers, as well as a higher mTEC to cTEC ratio. The results suggest a partial block in the terminal differentiation of the expanded immature mTEC population. Conditional deletion of Stat3 in mTECs, achieved by crossing Stat3fl/fl mice with K5.Cre mice, leads to a decrease in mTEC cellularity and a reduced mTEC to cTEC ratio. These findings implicate a novel role for Stat3 signaling in molecular regulation of the thymic mTEC compartment.
FlowCAP: critical assessment of flow cytometry population identification methods (65.2)
Authors: Scheuermann Richard , Aghaeepour Nima , Brinkman Ryan , Gottardo Raphael , Mosmann Tim , Qian Yu , Schoenfeld Jill
Published: January 2023

Traditional methods for flow cytometry (FCM) data processing have relied on manual gating of cell events to define cell populations for statistical analysis. However, this approach has become increasingly problematic with the advances in instrumentation and reagents that allow for evaluation of larger numbers of cell properties. Recently several groups have developed computational methods for automatically identifying cell populations in multidimensional FCM data obviating the need for manual gating. In order to compare the performance of these methods, the Flow Cytometry: Critical Assessment of Population Identification Methods (FlowCAP) competition was established to make available a common set of FCM data together with manual gating results for comparative analysis. The first FlowCAP competition included 5 different data sets with data from 12-30 samples containing 5000-100,000 cell events stained with 3-10 fluorochrome markers. We received 36 analysis result submissions from 14 research groups. Both model fitting and density-based clustering methods were found to perform well in comparison with manual gating by domain experts as the gold standard, using statistical tests to measure and rank algorithm performance. In addition, combining results using a computational “ensemble” method was found to outperform all individual methods. These results suggest that, in the near future, automated computational methods may become an integral part of routine FCM data analysis.
Optimiser™ microplate and OptiMax™ reagent systems enable femtogram/ml level sensitivity employing standard ELISA equipment and protocols (65.32)
Authors: Kai Junhai , Santiago Nelson , Puntambekar Aniruddha , Lee Se Hwan , Sehy David , Durnell Randy , Schultheis Ron , Han Jungyoup , Ahn Chong
Published: January 2023

Optimiser™ Microfluidic ELISA Plates dramatically enhance ELISA sensitivity through the incorporation of microfluidic channels into a standard ELISA plate configuration. Validation studies with Optimiser™ microfluidic ELISA plates demonstrate the Optimiser™’s ability to utilize available sandwich ELISA reagents, equipment, and protocols to attain unparalleled levels of sensitivity. Limits of Detection and Limits of Quantification are demonstrated for a variety of assays in the range of 30-100 fg/ml, representing a 20-1000 fold improvement over the same assays run in conventional ELISA plates. OptiMax™ ELISA Reagent protocols on Optimiser™ Microfluidic ELISA Plates employ the similar reagents, but abbreviated assay steps because, unlike conventional sandwich ELISAs, no traditional plate washing is required. Optimiser™ Microfluidic ELISA Plates are compatible with existing pipetting systems (manual or automated) and ELISA plate readers. Furthermore, the surface area to volume ratio of each microfluidic reaction chamber represents a 50-fold increase when compared to the well of a conventional 96-well immunoassay plate. The improvement in surface to volume ratio increases binding kinetics dramatically, enabling assays to be run in less than half the time of traditional ELISA assays and providing researchers with the flexibility to use dramatically smaller sample and reagent volumes (e.g., 5 ul).
The application and demonstration of a user-friendly, antibody-based biosensor for environmental monitoring (65.33)
Authors: Spier Candace , Vadas George , Unger Michael , Kaattari Stephen
Published: January 2023

Monoclonal antibodies (mAbs) are a valuable resource in both clinical and environmental health. In clinical applications, mAbs have been extensively used as a diagnostic tool for the rapid evaluation of specific diseases and in targeted anti-cancer therapies, among others. Recently, mAbs have been employed in environmental monitoring applications for the detection of oil, pesticides, and many other pollutants. MAb-based biosensors have become an excellent means to achieve rapid, sensitive monitoring of a specified target. Here I describe the application of a mAb employed in a portable, user-friendly, sensor format for the detection and quantification of polycyclic aromatic hydrocarbons (PAHs) in environmental samples. PAHs are carcinogenic and found widespread in the environment. One application of this biosensor included the monitoring of estuarine water during the dredging of PAH-contaminated sediments, revealing a plume of PAHs emanating from the dredge site. The second application was the near real-time monitoring of PAHs in stormwater run-off. The ease of technology transfer was demonstrated by the operation of the biosensor by high school students in two science programs: VIMS/NASA Governor’s School Summer Internship and NSF’s GK-12 Program. Overall, these opportunities demonstrated the utility of this biosensor to rapidly and precisely quantify PAHs in environmental samples. Moreover, this biosensor technology was successfully operated by non-professionals.
Novel methods for simultaneous assessment of CD4 and CD8 T cell mitochondrial potential changes (65.39)
Authors: Clor Julie , Tyagarajan Kamala
Published: January 2023

The study of mitochondrial membrane potential (ΔΨm) changes in immune cells is important in understanding immune cell apoptosis, mechanism of diseases, and evaluating the action of compounds and drugs that cause mitochondrial stress. Simultaneous identification of T cells along with ΔΨm changes is challenging due to the broad fluorescence of traditional mitochondrial dyes, such as JC-1, in multiple channels. Here we demonstrate results from a novel approach for the simultaneous evaluation of ΔΨm in CD4 & CD8 T cells using simplified cocktails and a mitochondrial responsive dye followed by microcapillary flow cytometry. PBMC’s were treated with a range of compounds including staurosporine and gambogic acid and evaluated for mitochondrial membrane potential changes and annexin V binding on T cells. Our results demonstrate that with most treatments, a greater % of cells exhibit earlier and more sensitive changes in ΔΨm, than Annexin V binding. Further, some donors treated with staurosporine, a significantly greater % of CD8 T cells demonstrated changes in ΔΨm vs. CD4 T cells. Gambogic acid demonstrated a dramatic reduction of ΔΨm and showed increased apoptosis on both CD4 and CD8 T cells simultaneously. The ability to measure mitochondrial membrane potential changes in CD4 and CD8 T cell simultaneously can allow more sensitive identification of immune cell perturbation and provide deeper insights into the mechanism of immune cell apoptosis in disease and drug discovery.
SMAD3 deficiency causes immune dysregulation, colitis and colon tumorigenesis (66.13)
Authors: Bommireddy Ramireddy , Ball Corbie , Cherukuri Durga , Nasr Michael , Doetschman Tom
Published: January 2023

Introduction: TGFβ plays important role in T cell development, differentiation, tolerance and autoimmune diseases. However, recent studies have shown an important role for TGF-β1 and β3 in pathogenic helper T-cell (Th17) differentiation in the presence of IL-1β, IL-6 and IL-23. SMAD3 is mediates TGFβ signaling in many cell types including lymphocytes. Smad3 /- mice develop inflammation-induced colon cancer upon Helicobacter hepaticus infection suggesting that TGFβ1 signaling through SMAD3 is essential to prevent inflammation and progression to colon cancer. In this study we investigated the role of SMAD3 in inflammation using mice deficient in Smad3. Methods: Splenocytes are cultured in the presence of anti-CD3/CD28 beads, TGFβ1 and IL-2 for 3 days. Cytokines are determined by ELISA, CBA and MSD methods. Cells are stained with antibodies for CD4, CD25, CD62L and intracellular FOXP3 and Helios. Results: Smad3-/- mice exhibit inflammatory response upon infection with commensal bacterium H. hepaticus. We find that while SMAD3 signaling is important for inducible Treg cell generation and for their maintenance, deficiency of SMAD3 increases IL-6 and IL-17 levels suggesting a regulatory role for SMAD3 in Th17 cell differentiation. Conclusions: While SMAD3 signaling is important for iTreg cell generation and maintenance, it is not required for Th17 cell differentiation. In addition, we find that SMAD3 signaling negatively regulates Th17 differentiation through induction of FOXP3.
Tumor-associated macrophages recruit CCR6+ regulatory T cells and promote the development of colorectal cancer via enhancing CCL20 production in mice (66.33)
Authors: Liu Jinlin , Wang Honglin
Published: January 2023

Background: Tumor-associated macrophages (TAMs) remodel the colorectal cancer (CRC) microenvironment. Yet, findings on the role of TAMs in CRC seem to be contradictory compared with other cancers. Regulatory T (Treg)-cells dominantly infiltrate CRC. However, the underlying molecular mechanism in which TAMs may contribute to the trafficking of Treg-cells to the tumor mass remains unknown. Methodology/Principal Findings: CRC was either induced by MNU and H. pylori or established by subcutaneous injection of mouse colorectal tumor cell line (CMT93) in mice. Recruitment of FoxP3GFP+ Treg-cells was assessed using the IVIS Imaging System. A role for macrophages in trafficking of Treg-cells and in the development of CRC was investigated in CD11b diphtheria toxin receptor (CD11b-DTR) transgenic mice in which macrophages can be selectively depleted. Treg-cells remarkably infiltrated solid tumor, and predominantly expressed the homing chemokine receptor (CCR) 6 in the induced CRC model. Macrophages produced a large amount of CCL20, the sole ligand of CCR6 when co-cultured with CRC cells. Injection of recombinant CCL20 into tumor sites promoted its development with a marked recruitment of Treg-cells. Conditional macrophage ablation decreased CCL20 levels, blocked Treg-cell recruitment and inhibited tumor growth in CD11b-DTR mice. Conclusions/Significance: TAMs recruit CCR6+ Treg-cells to tumor mass and promote its development via enhancing the production of CCL20 in a CRC mouse model.
CD73: an emerging target for cancer immunotherapy (66.7)
Authors: Wang Long , Fan Jie , Thompson Linda , Shin Tahiro , Curiel Tyler , Zhang Bin
Published: January 2023

CD73 is over-expressed in many types of human and mouse cancers, and is implicated in the control of tumor progression. However, the exact contribution of tumor and host CD73 expression to tumor growth remains unknown to date. We here show that host CD73 promotes tumor growth in a T cell-dependent manner, and that the optimal anti-tumor effect of CD73 blockade required inhibiting both tumor and host CD73. Notably, CD73 on bone marrow (BM)-derived cells (CD4+CD25+ T regulatory cells), but not on nonhematopoietic cells, inhibited systemic anti-tumor T cell expansion and effector function. On the other hand, CD73 on non BM-derived cells (presumably endothelial cells) limited anti-tumor T cell homing to local tumors. Thus, CD73 has distinct roles in hematopoietic and nonhematopoietic cells in regulating anti-tumor T cell responses. In addition, pharmacological blockade of CD73 using its selective inhibitor α,β-methylene adenosine 5′-diphosphate or an anti-CD73 monoclonal antibody, inhibited tumor growth and completely restored efficacy of adoptive T cell therapy. These findings suggest that both tumor and host CD73 cooperatively protects tumors from incoming anti-tumor T cells, and point to a novel cancer immunotherapy strategy by targeting CD73.
Characterization of the mechanism by which L-asparaginase II of Salmonella enterica serovar Typhimurium induces T cell inhibition (99.12)
Authors: Torres AnnMarie , Kullas Amy , van der Velden Adrianus
Published: January 2023

T cells play a key role in controlling and clearing infection with the bacterial pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium). However, T cell-mediated immunity against S. Typhimurium takes months to develop and has been described as slow and inefficient. Previously, we have shown that S. Typhimurium have a direct inhibitory effect on T cells, down-modulating expression of the beta chain of the T cell receptor (TCR-beta) and inhibiting T cell proliferation. Furthermore, we have shown that a soluble, proteinaceous factor produced or induced by S. Typhimurium is responsible for this inhibition. Most recently, we have found that STM3106 is required for S. Typhimurium to inhibit T cells and that L-asparaginase II, which is encoded by STM3106, is present in the supernatants of T cells cultured in the presence of S. Typhimurium. In addition, we have found that L-asparaginase II produced by S. Typhimurium is both necessary and sufficient for the inhibition of T cells. With further characterization, this research should provide new insight into the mechanism by which S. Typhimurium inhibit T cells, avoiding immune clearance.
TRAIL-dependent suppression of naïve and memory CD8 T cell responses to secondary bacterial infection after sepsis (99.14)
Authors: Griffith Thomas , Gurung Prajwal , Rai Deepa , Badovinac Vladimir
Published: January 2023

Sepsis is the leading cause of death in most ICUs, and the death of septic patients usually does not result from the initial septic event but rather from subsequent nosocomial infections. Patients who survive severe sepsis often display severely compromised immune function. Using a cecal-ligation and puncture (CLP) model to induce intra-abdominal polymicrobial peritonitis, we recently established a link between the apoptotic cells generated during sepsis and the induction of sepsis-induced immune suppression. Using a clinically-relevant “two-hit” sepsis model that better reflects the delayed mortality seen during sepsis due to secondary infection, we have extended this work to investigate sepsis-induced immune suppression of naïve and memory Ag-specific CD8 T cell responses to an experimental heterologous bacterial (LM-OVA) infection. CLP-treated WT mice had a reduced ability to clear LM-OVA vs. sham-treated WT mice, which was paralleled by suppressed T cell responses in the CLP-treated mice. In contrast, bacterial clearance and T cell responses were similar in CLP- and sham-treated TRAIL-/- and DR5-/- mice. Administration of a blocking anti-TRAIL mAb to CLP-treated WT mice led to bacterial clearance and T cell responses like those seen in sham-treated mice. These data further suggest the importance of TRAIL in the induction of sepsis-induced immune suppression and that TRAIL neutralization may be a potential therapeutic target to restore cellular immunity in septic patients.
HIV triggers interleukin 21-mediated induction of granzyme B-secreting B cells with antiviral properties (105.18)
Authors: Kaltenmeier Christof , Dahlke Karen , Gawanbacht Ali , Hofmann Stefanie , Beyer Thamara , Härter Georg , Grüner Beate , Kern Peter , Kirchhoff Frank , Schrezenmeier Hubert , Jahrsdörfer Bernd
Published: January 2023

Certain lymphocyte subsets including plasmacytoid dendritic cells and regulatory T cells can secrete granzyme B (GrB), thereby suppressing T cell expansion. Recently, we found that B cells can also produce GrB in response to interleukin (IL) 21. Since HIV has been shown to be associated with elevated serum IL-21 levels, we hypothesized that GrB-expressing B cells may be induced during HIV infection. Here, we demonstrate for the first time, that infection of CD4+ T cells with HIV 1 (NL4-3), but not mock infection, induces strong expression of IL-21. We further demonstrate that such T cells induce GrB in co-cultured B cells in an IL-21-dependent fashion. In support of these data, serum levels of both IL-21 and GrB are significantly higher in HIV-infected patients before HAART as compared to healthy controls. Up to 60% of B cells (36.2 ± 12.9%) from patients infected with HIV, but not normal B cells, express GrB. Importantly, co-culture of HIV-infected CD4+ T cells with GrB+ B cells resulted in GrB transfer, and strongly suppressed both, proliferation of T cells and virus replication as indicated by significantly reduced p24 levels. The observed effects were enhanced by IL-21, and reduced by GrB inhibition. In summary, we demonstrate that HIV induces IL-21 in CD4+ T cells, thereby indirectly triggering the development of GrB-secreting B cells with antiretroviral properties. GrB-secreting B cells may play a so far unappreciated role in decelerating the expansion of HIV.
IL-15-activated bystander CD8+ T cells contribute to host injury by NK-like cytolytic activity in acute viral hepatitis. (105.21)
Authors: Chang Dong-Yeop , Lee Hyun Woong , Kang Wonseok , Cheon Doo-Sung , Kim Hyung Joon , Shin Eui-Cheol
Published: January 2023

In viral infection, activation of bystander CD8+ T cells and their role have been controversial. We investigated bystander activation of CD8+ T cells and their contribution to tissue damage in acute hepatitis A (AHA), which is caused by hepatitis A virus (HAV) and manifested by severe liver injury in adults. We show bystander activation of CD8+ T cells specific to irrelevant viruses such as CMV, EBV and influenza A virus during acute necroinflammatory phase. We also demonstrate an unexpected correlation of the bystander activation to the liver injury, indicating a role of activated bystander CD8+ T cells in the liver injury. In contrast, HAV-specific T cell response was associated with viral clearance rather than the liver injury. Intriguingly, IL-15 is produced by HAV infection, and expression of IL-15 and IL-15Rα is increased in AHA. Furthermore, in vitro IL-15 treatment of healthy donors’ lymphocytes recapitulated the activated phenotype of the bystander CD8+ T cells observed in AHA. CD8+ T cells activated in vitro by IL-15 or obtained from the AHA liver exerted NK-like cytolytic activity triggered by NKG2D and/or NKp30 without TCR engagement. In addition, hepatocytes of the AHA liver over-expressed NKG2D ligands, and therefore could be cytolytic targets. Taken together, we report here a human viral disease in which bystander CD8+ T cells are activated by IL-15 and contribute to tissue damage by NK-like cytolytic activity without TCR engagement.
Co-infection alters CD8 T cell immunity and immunoprotection (105.27)
Authors: Kenney Laurie , Cornberg Markus , Chen Alex , Trees Andrew , Emonet Sebastien , de la Torre Juan Carlos , Selin Liisa
Published: January 2023

Co-infection with multiple pathogens can result in increased pathology and difficulties with diagnosis and treatment. Moreover, interference between co-administered vaccines has resulted in lower antibody production. However, the effect of co-infections on T cell responses remains poorly understood. We have shown that sequential infection with unrelated viruses can alter T cell responses and disease outcome. This is defined as heterologous immunity, which is mediated by crossreactive T cells. We questioned if co-infection with two viruses containing a crossreactive epitope would alter the immune response and disease outcome upon rechallenge. Compared to mice infected with a single virus, rechallenged of mice co-infected with lymphocytic choriomenigitis (LCMV) and Pichinde viruses (PV) had a loss of immunoprotection and enhanced immunopathology. Each viral rechallenge identified a different potential mechanism to explain these changes. First, co-infection resulted in fewer LCMV-specific effector-like memory CD8 T cells leading to decreased protection and enhanced liver pathology. Secondly, co-infection caused some mice to have inappropriately enhanced crossreactive responses, which contributed to increased immunopathology. As combined vaccines or simultaneous immunizations are also co-infections, our data would suggest that T cell responses should be examined in the context of vaccine development.
Do fortuitous cross-reactive CD8 memory T cells dominate the response of aged mice to primary influenza virus infection? (105.37)
Authors: Lanzer Kathleen , Yager Eric , Blackman Marcia
Published: January 2023

A major contributing factor to the decline in immunity with age is reduced repertoire diversity, a consequence of thymic involution. The resulting decrease in naïve T cells and accumulating antigen experience leads to a dramatic increase in the ratio of memory to naïve peripheral CD8 T cells. We have described a major impact of age-associated reduced repertoire diversity on the response of aged mice to de novo influenza virus infection, in some cases resulting in a “hole in the repertoire.” We hypothesize that because of the enhanced ratio of memory to naïve cells in aged mice, fortuitously cross-reactive memory cells dominate the response to new infections. The implications are that responses in individual aged mice may be highly stochastic, of low avidity and with a restricted repertoire. We have confirmed the ability of fortuitously cross-reactive memory cells to respond to influenza virus by adoptive transfer experiments. CD8 memory (CD44high) T cells from influenza virus naïve mice were transferred into T cell-deficient recipients, which were then infected with influenza virus and the epitope-specific response measured 12 days later. Cumulative data show that transferred memory cells from 9 out of 15 mice were able to respond to influenza virus-specific epitopes. Experiments in progress will have profound implications for understanding the cellular immune response to new infections in aged mice, and for the development of vaccine strategies targeted to the elderly.
The role of IL-21 in rescuing CD8 T Cell responses in chronically infected HIV patients (105.38)
Authors: Yi John , Weinhold Kent
Published: January 2023

CD4 T cells are vital in regulating CD8 T cell responses during chronic viral infections. Notably, the hallmark of HIV infection is the severe loss of CD4 T cells resulting in defective CD8 T cell responses. One consequence of CD4 depletion following HIV infection is the loss of interleukin-21 (IL-21) production by the CD4 T cells, which has been demonstrated to be a critical factor in promoting viral control in murine studies. However, it is unknown whether IL-21 can rescue the exhausted CD8 T cells in chronically infected HIV+ patients. In this study we build upon novel murine studies to demonstrate a key role for IL-21 in improving CD8 T cell responses in PBMC from chronically infected HIV patients. To determine whether IL-21 can rescue exhausted CD8 T cells, PBMCs from HIV patients with viral loads > 10,000 RNA copies/mL were incubated with IL-21. Following re-stimulation with overlapping Gag-peptide, IL-21 enhanced the production of IFN-γ by CD8 T cells although no effect was observed on IL-2 or TNF-α production. In addition, an increase in the central memory (CD45RO+CD27+) population was observed with IL-21 culture along with higher MFI of Bcl-6 levels. Interestingly, IL-21 cultured CD8 T cells expressed higher MFI of PD-1 even though CD8 T cell function was enhanced. Significantly, these results provide an approach in which IL-21 can be potentially used to enhance CD8 T cell responses in chronically infected HIV patients, whose CD4 counts are low or defective.
Transmembrane domain sequences regulate the association of MHC- class II with lipid raft microdomains (106.19)
Authors: Khandelwal Sanjay , Roche Paul
Published: January 2023

MHC II is known to be present in cholesterol-dependent lipid raft plasma membrane microdomains, and this association is important for MHC II- dependent activation of CD4 T cells by Dendritic cells (DCs). Until now it is not clear what regulates MHC II lipid raft association. In the present study we attempt to define the signals present in the MHC II transmembrane (TM) domain that regulate MHC II association with lipid rafts. Palmitoylation of cysteine residues is a well-known modification that often results in lipid raft association of TM protein and each MHC II I-Ab subunit contains a TM cysteine. We therefore expressed MHC II-α and β-chains containing TM domain cysteine → serine mutants in DCs obtained from MHC II null mice and analyzed MHC II lipid raft association. These experiments showed that the mutation of the TM cysteine residues actually enhanced MHC II raft localization about 3 fold. We also examined the role of the entire MHC II TM domain in lipid raft association. Replacement of the MHC II-α and β-chain TM domains with that of the non-raft protein ICAM 1 reduces MHC II lipid raft association 5 fold. We are currently examining the role of TM domain sequences present in each MHC II subunit in an attempt to identify dominant raft localization sequences in MHC II.
Plasma Membrane Interactions that determine Antigen Presentation by Antigen-Presenting Cells (106.20)
Authors: Hari Aswin , Shi Yan
Published: January 2023

Antigen presentation is a critical process in host defense for initiating adaptive immune responses. The APC loads peptides derived from antigens onto major histocompatibility complex (MHC) molecules and exports them to the surface for T cells sampling. If the primary interaction between an APC and T cell is successful, i.e. the TCR binds an appropriate MHC-peptide complex, this leads to an adaptive immune response. Peptide antigens are presented on two types of MHC molecules, MHC I and MHC II via several distinct processing pathways. In turn, the MHC molecule dictates which type of T cell can be activated and thereby the nature of the adaptive immune response. Therefore, antigen loading onto MHC molecules is a critical determinant of the adaptive immune responses. Exogenous antigens can be presented on MHC I and II molecules. Antigen processing mechanisms have been elucidated in great detail, but the fundamental reason behind why an antigen is directed into a specific pathway is not known. One of the foremost aims for this project is to determine why particulate antigens are presented more often on MHC I and why soluble antigens are more efficiently presented on MHC II, by deciphering the interactions of plasma membrane with particulate versus soluble antigen. This fundamental sensing mechanism of the human body when elucidated can help in generating an antiviral response in diseases like HIV and in field of preventative medicine.
The transcription factor AhR modulates the balance of functionally distinct DC subsets during influenza virus infection (106.5)
Authors: Jin Guang-Bi , Lawrence B. Paige
Published: January 2023

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor expressed in cells of the immune system. AhR activation by high-affinity ligands such as the pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin) has been shown to suppress immune responses and impair host resistance to infection. However, the mechanisms by which AhR activation modulates immune function remain poorly understood. Dendritic cells (DCs) are antigen-presenting cells (APCs) that play a key role in priming naïve T cells. We previously reported that AhR activation reduces the ability of DCs to prime influenza virus-specific naïve CD8+ T cells ex vivo. In this study, we show that AhR activation changes the number of functionally distinct DC subsets in the lung and mediastinal lymph nodes in influenza virus-infected mice. By adoptively transferring virus-specific naïve CD8+ T cells into recipient mice, we demonstrate that AhR activation in the recipients dramatically reduces the clonal expansion and differentiation of transferred naïve virus-specific CD8+ T cells. Moreover, using AhR mutant mice, which express an AhR protein that binds ligand but cannot interact directly with DNA, we found that alterations in DC phenotype require the ligand-activated AhR complex to bind DNA via AhR’s DNA binding domain. Our findings suggest that AhR activation modulates phenotypically-distinct DC subsets, which may contribute to altered susceptibility and response to respiratory viral infection.
MDA5 Inhibits the Replication of Hepatitis B Virus (108.13)
Authors: Lu Hsin-Lin , Liao Fang
Published: January 2023

Hepatitis B virus (HBV) infection causes a wide spectrum of liver diseases; however, the innate immunity against HBV infection has been rarely studied and remains elusive. Here, we investigated the involvement of pattern recognition receptors, RIG-I like receptors (RLRs), in HBV replication. We demonstrated that the expression of MDA5, but not RIG-I, was increased in Huh-7 cells transfected with HBV replicative plasmids. Consistent with the in vitro finding, the expression of MDA5, but not RIG-I, was also increased in mice receiving HBV replicative plasmids through hydrodynamic injection. To further determine the effect of RLRs on HBV replication, we cotransfected MDA5 or RIG-I with HBV replicative plasmids into Huh-7 cells and measured HBV replication. The results showed that with similar protein levels of MDA5 and RIG-I, only MDA5, but not RIG-I, significantly inhibited HBV replication. In line with these results, knockdown of MDA5 by siRNA in cells transfected with HBV replicative plasmids increased HBV replication while knockdown of RIG-I did not have any effect on HBV replication. Interestingly, activation of MDA5 by HBV significantly induces IRF3 phosphorylation and NF-κB translocation. This result suggests that HBV-mediated MDA5 activation may be responsible for the IRF3 and NF-κB activation. In summary, our results demonstrate that MDA5, a known cytosolic sensor for RNA virus, contributes to the innate immune response against HBV infection.
Inherent baseline levels of phosphorylated IRF7 impact pDC IFNα response to TLR7 stimulation (108.8)
Authors: Griesbeck Morgane , Doyle Erin , Lindsay Robert , Boucau Julie , LeGall Sylvie , Altfeld Marcus , Chang J. Judy
Published: January 2023

Background: We previously showed that plasmacytoid dendritic cells (pDCs) derived from females can produce significantly more IFNα in response to HIV-1 and HIV-1-encoded TLR7/8 ligands than pDCs derived from males, resulting in stronger secondary activation of CD8+ T cells. Given the crucial role of interferon regulatory factor 7 (IRF7) in the regulation of type I IFN production by pDCs, we hypothesized that IRF7is involved in the differences observed in IFNα production. Methods: Fresh PBMC were isolated from HIV-1-negative subjects enrolled at Massachusetts General Hospital. Levels of IRF7 were measured in pDCs by phospho-flow cytometry, In-Cell Western blotting and qRT-PCR at baseline and at different time-points after TLR7 stimulation. IFNα levels were quantified by flow cytometry, ELISA and qRT-PCR Results: We observed a higher percentage of pDCs expressing phosphorylated IRF7 at baseline in females than in males using flow cytometry (p<0.05). This difference persisted after TLR7 stimulation and was associated with higher IFNα production by females. Conclusion: These data show that sex differences in the baseline level of phosphorylated IRF7 in pDCs might account for described higher IFNα production upon TLR7 stimulation, and might provide new insights into the mechanisms underlying faster HIV-1 disease progression in females compared to males after controlling for viral load.
A Bruton’s Tyrosine Kinase inhibitor prevents antigen-driven B cell activation, germinal center persistence and memory B cell re-activation in vivo (109.10)
Authors: Rodriguez Varenka , Springer John , Schnute Mark , Dunussi-Joannopoulos Kyri , Douhan John , Ruzek Melanie , Benson Micah
Published: January 2023

The Tec family kinase, Bruton’s Tyrosine Kinase (BTK), is one of the key intracellular signaling components proximal to the B cell receptor (BCR). In this study, we utilized an orally-accessible BTK inhibitor (BTKi) to study the functional effects blocking this signaling pathway has on antigen-driven B cell activation in vivo. We report that BTK is required for anti-IgD driven B cell activation in vivo as evidenced by suppression of the surface activation markers CD86 and mRNA transcripts (e.g. c-Myc, Bcl-xL, CCL3, CD98, EBI2, EGR1, EGR2 and IRF4) otherwise rapidly induced upon engagement of the BCR with antigen. Using the T-dependent protein PE and SRBCs as model antigens, we tested for a requirement of BTK in generating antigen-specific IgM and IgG antibody titers and germinal centers, and find that BTK is required for germinal center maintenance. Lastly, we tested for a role of BTK in long-lived PE-specific memory B cell re-activation and report that BTKi significantly suppressed the induction of PE-specific IgM and IgG titers upon antigen re-challenge. The use of BTKi allowed us to bypass the impact a genetically inactivated BTK gene has on B cell development and to assess BTK inactivation in both a wild-type setting and in a temporal manner. These results provide insight into the role of BTK in BCR-driven B cell activation in various antigen naïve and experienced B cell populations.
Functional overlap between the mTOR and the unfolded protein response in the generation of mucosal plasma cells (109.2)
Authors: Tirosh Boaz , Benhamron Sandrine
Published: January 2023

Plasma cells (PCs) are terminally differentiated B lymphocyte responsible for antibody secretion. Differentiation into PCs involves a remarkable expansion of the endoplasmic reticulum (ER) in a mechanism that is governed by the unfolded protein response (UPR). The transcription factor XBP-1, which is activated in the course of UPR, is necessary for PC differentiation. In the absence of XBP-1, ER expansion is impaired and little amounts of antibody molecules are secreted. mTOR is an evolutionary conserved serine/threonine kinase. When activated, mTOR promotes protein translation and lipid biosynthesis. Deletion of TSC1 or TSC2 renders mTOR constitutively active. We investigated whether activation of the mTOR can override the requirement for XBP-1 in PC differentiation. To this end we generated mice single and double knocked out for XBP-1 and TSC1 in their B cell lineage. A normal serum IgA levels was observed in double knockout (DKO) animals, while IgM and IgG were reduced similarly to what measured in XBP-1 deficient mice. Because IgA is primarily secreted by mucosal PCs we investigated the IgA secretion in gut B cells. IgA was expressed and secreted to a higher level in the DKO than in XBP-1 KO. Our data demonstrate that the program for mucosal PC development can utilize the mTOR pathway in lieu of the UPR. This is the first indication that the mTOR under certain conditions can compensate for lack of the UPR in promoting the differentiation of professional secretory cells.
Immunoglobulin heavy chain enhancer's (Eμ) role in cell selection at the pre-B to immature B cell transition (109.7)
Authors: Peng Cheng , Eckhardt Laurel
Published: January 2023

We have recently described a second check-point for allelic exclusion that operates at the pre-B to immature B cell transition and is dependent upon the IgH intronic enhancer Eμ. We suggest that Eμ’s function at this transition is to ensure that IgH chains are made at levels sufficient to signal positive selection, regardless of light chain sequences and despite mono-allelic expression of the heavy chain. Evidence leading to this hypothesis includes our findings that B cells expressing an Eμ-deficient allele undergo heightened light-chain editing (suggesting that only some light-chain partners can circumvent the effects of lower BCR levels), or express a second IgH allele (“double-producing” cells to achieve higher BCR levels). We now show that B cells expressing an Eμ-deficient IgH allele, consistent with inefficient positive selection, remain longer at the pre-B cell stage, resulting in a significantly diminished immature B cell compartment. To determine whether escape from this developmental stall is dependent upon light chain, we limited the available light chain repertoire in these animals by pre-assembled VL insertion. The result was an effect on both B cell development and the emergence of double-producing cells. Together with our earlier findings, these new results suggest that IgH levels at the pre-B to immature B transition have an important impact on the breadth of the BCR repertoire as well as on the maintenance of allelic exclusion.
Intrinsic role of FoxO3a in the contraction of CD8+ T cell response (110.14)
Authors: Sad Subash , Haddad Elias , Peng Stanford , Sekaly Rafick , Tzelepis Fanny
Published: January 2023

CD8+ T cells undergo rapid expansion during infection with intracellular pathogens, which is followed by swift and massive culling of primed CD8+ T cells. The mechanisms that govern the massive contraction of primed CD8+ T cells are not clear. We show that the transcription factor Foxo3a plays a key role in the contraction, not expansion, of CD8+ T cell response against Listeria monocytogenes (LM). FoxO3a-mutant OVA-specific CD8+ T cells isolated during peak of response displayed reduced expression of pro-apoptotic molecules BIM and PUMA, and underwent reduced apoptosis during contraction phase in comparison to WT cells. A higher number of memory precursor effector cells (MPECs) were detectable in FoxO3a-mutant mice compared to WT mice during contraction phase. Furthermore, FoxO3a-mutant OVA-specific CD8+ T cells upon transfer into normal mice underwent highly reduced contraction. These results show for the first time that FoxO3a acts in a cell intrinsic manner to promote the culling of primed CD8+T cells during the contraction phase.
IL-10-IL-21-STAT3 axis regulates memory CD8 T cell development (110.9)
Authors: Cui Weiguo , Liu Ying , Weinstein Jason , Craft Joseph , Kaech Susan
Published: January 2023

Developing memory CD8 T cells is a paramount goal of many vaccines that will fight infections and cancer. Many types of acute infections can generate robust memory T cells, and therefore there must be a mechanism for instilling memory cell properties into a small portion of the memory precursor effector CD8 T cells and “preserve” their potential to develop into memory cells. However, the signals and genetic pathways that govern this process remain poorly defined. Here we show that the IL-10-IL-21-STAT3 pathway was critical for memory CD8 T cell development after acute LCMV infection. Lack of either IL-10 and IL-21 or STAT3, virus-specific CD8 T cells retained terminal effector (TE) differentiation states and failed to mature into protective memory T cells that contained self-renewing central memory T cells. Expression of BCL-6 and SOCS3 was considerably reduced in STAT3-deficient memory CD8 T cells, and BCL-6- or SOCS3-deficient CD8 T cells also had perturbed memory cell development. Reduced SOCS3 expression rendered STAT3-deficient CD8 T cells hyperresponsive to IL-12, suggesting that the STAT3-SOCS3 pathway helps to insulate memory precursor cells from inflammatory cytokines that drive TE differentiation. Next, we began to dissect the cellular sources of IL-10 and IL-21 and the temporal role of these cytokines in effector and memory CD8 T cell differentiation. This will advance our understanding of cellular mechanisms of memory CD8 T cell fate commitment and maintenance.