bluuebunny/crossref_metadata_2025 huggingface dataset:
Circulating T helper cells with T follicular helper cell phenotype renders the autoantibody generation in patients with systemic lupus erythematosus (HUM7P.304)
Authors: Zhang Xin , Lindwall Elvira , Gauthier Carl , Chen Minzi , Khan Omar , Choi Yong Sung , Zakem Jerald , Quinet Robert
Published: January 2023

SLE is a chronic autoimmune disease characterized by autoreactive B cells and autoantibody production. Follicular helper T (Tfh) cells, a T cell subset located in the germinal center (GC) of lymphoid organs, is a key participant regulating B cell tolerance and antibody production. To identify the role of circulating Tfh (cTfh) cells in SLE, we examined their phenotype and function on supporting antibody production, and their correlation with autoantibody level and disease activity. Peripheral blood was collected from 29 SLE patients and age/gender-matched healthy donors. Tonsils from non-SLE controls were used to extract GC-Tfh cells. Tfh cells were defined by their signature surface markers (CXCR5, ICOS, CD57, and PD-1) via flow cytometry. IL-21 expression was measured by real-time PCR and intracellular staining. Tfh cells, non-Tfh cells were purified by MACS columns and co-cultured with B cells. IgG in the culture supernatant was detected by ELISA. Our results showed that cTfh cells in SLE patients expressed GC-Tfh signature molecules and IL-21 and were capable of driving B cells differentiate into plasma cells in vitro. The elevated frequency of cTfh cells correlated positively with levels of circulating plasmablasts, serum anti-dsDNA Ab and ANA. Our data suggests that the increased cTfh cells in SLE patients share phenotypic and functional properties with GC-Tfh cells. Tfh cells may serve as perpetrator to the pathogenesis in SLE patients and target for novel treatment.
Ubiquitination modulates expression of the serine arginine-rich splicing factor 1 (SRSF1) in activated T cells and in SLE patients (HUM7P.305)
Authors: Moulton Vaishali , Gillooly Andrew , Tsokos George
Published: January 2023

T cells from patients with systemic lupus erythematosus (SLE) are poor producers of the vital cytokine interleukin (IL)-2. We recently showed that SLE T cells express lower levels of the serine arginine- rich splicing factor 1 (SRSF1), more so in patients with active disease. Forced expression of SRSF1 restores IL-2 production, indicating that SRSF1 is important in T cell function, and its reduced expression is a potential molecular defect in SLE T cells. The goal of this study was to determine the mechanism/s regulating SRSF1 expression in human T cells and in SLE patients. T cell activation increased mRNA expression of SRSF1 by 3- to 4-fold within 24 hours, while protein expression did not alter much. This discrepancy between mRNA and protein expression was not due to reduced RNA stability. Activating T cells in presence of specific proteolysis inhibitors revealed that proteasome- but not lysosome-mediated mechanisms contribute to reduced SRSF1 expression. Accordingly, immunoprecipitation (IP) studies showed increased ubiquitination of SRSF1 in activated T cells compared with resting cells. More interestingly, SRSF1 showed increased ubiquitination in SLE patients compared to healthy controls and rheumatoid arthritis patients. Our results identify a novel mechanism of SRSF1 protein regulation in human T cells and suggest that increased ubiquitination and proteasome degradation represent a molecular mechanism that contributes to reduced SRSF1 expression in SLE T cells.
Acute lymphoblastic leukemia induces caspase-1 activation in human bone marrow monocyte/macrophages and is associated with impaired CD8 function. (HUM7P.315)
Authors: Shand Jessica , Mariano John , Toia Liana
Published: January 2023

B-acute lymphoblastic leukemia (ALL) is a malignancy of bone marrow-derived precursor-B cells and represents the leading cause of pediatric cancer death. Suppressive signals induced in the marrow of ALL patients may explain both the failure of host CD8 surveillance and inconsistent responses to adoptive CD8 therapy. We hypothesize that ALL induces a caspase-1 driven inflammasome in bone marrow monocyte/macrophages (BM-MM) that modulates CD8 function and contributes to relapse. To determine whether BM-MM could produce soluble factors that suppress CD8 function in the setting of ALL, we treated CFSE-labeled CD8 from healthy donor PBMC with supernatants from cocultures of primary human ALL and either CD14+ BM-MM or stromal fibroblast controls. CD8 exposed to supernatants from ALL cultured alone or with stromal fibroblasts maintained 100% proliferation, while those exposed to BM-MM cocultures failed to proliferate, regardless of whether ALL was cultured directly or in transwell with BM-MM. Cleaved caspase-1 was detected, both by qPCR (2.2-fold expression over endogenous control) and Western blot from BM-MM cultured with ALL, but not stromal fibroblasts Similar levels of soluble IL1B were detected from stromal fibroblast and BM-MM cocultures. Current studies are determining whether the reduced proliferative capacity observed in CD8 corresponds to impaired antileukemia killing and identifying specific signals downstream of caspase-1 that may be targeted to reverse this phenomenon.
Stable individual immune response gene expression signatures in peripheral human CD4 cells foreshadow response to stimulation (HUM7P.324)
Authors: Pease Larry , Zhang Yugi , Felts Sara , Scheid Adam , Van Keulen Virginia , Pankratz V. Shane
Published: January 2023

The complexity of molecular networks regulating the immune response presents challenges to measuring immune response potential in human populations. We measured gene expression in CD4+ peripheral blood cells from healthy subjects using RNAseq. RNA expression was assessed twice, one month apart to identify the subset of genes expressed stably within individuals over time yet discriminating individuals in a cohort. This immune response signature gene set then was used to identify and successfully order the immune response phenotypes of independent, unrelated subjects. Following a brief 4 hr stimulation of freshly isolated cells with anti-CD3/C28 antibody-coated beads, changes in gene expression were measured using a similar approach, defining individual response profiles and clustering response patterns. Examination of the RNA expression signatures of unstimulated CD4 cells revealed a gene subset which when analyzed for gene expression levels correctly aligned independent test subjects into the defined response-phenotype clusters following stimulation. Therefore, there exists a subset of genes stably expressed over time in circulating CD4+ cells which discriminate among individuals in a manner associated with immune response potential. Thus a systems approach can be used to describe the complex immune phenotypes of individuals in ways that might be useful in determining how individuals will respond to complex stimuli or in disease and treatment settings.
Evaluation of PBMC processing in the Precision Bioservices laboratory network (HUM7P.325)
Authors: Bahunde Faith , Neidinger Erik , Awoyode Rotimi , Johnson Netasha , Osunsan Tai , Tambwe Celestine , Waddington Michael , Shea Katheryn
Published: January 2023

Cryopreservation of peripheral blood mononuclear cells (PBMC) is a good alternative to testing fresh PBMC in clinical trials as functional immunologic assays may be batched to increase efficiency. Functional assays are essential for accurately evaluating the performance of vaccines, antiviral and anticancer agents. Optimal retention of PBMC functionality during cryopreservation depends on optimized and standardized cell isolation, freezing and storage conditions. Collection and analysis of PBMC is a significant technical challenge in multi-center clinical trials due to pre-analytical variability in processing, freezing and storage. It is vital that all centers participating in a multi-center collaboration operate with consistent protocols. To this end, Precision has developed a Laboratory Network (PLN) to ensure quality PBMC processing across multi-center trials. The current study was performed within the PLN to ensure samples processed at each laboratory achieved similar results. We report the recovery, viability and functionality of PBMC obtained from participating laboratories. Phenotypic characterization of the major cell subset was also analyzed and the percent obtained was comparable across laboratories. This data provides confidence that PBMC processing in a multi-center clinical trial operating with the same standard operating procedures (SOPs) produces comparable data, thus accelerating drug and vaccine development.
ASC inflammasome mediates host protection against rickettsiae via a NLRP3-independent signaling pathway (INC7P.403)
Authors: Fang Rong , Zhao Xuemei , Smalley Claire , Saito Tais , Brouwer Dedeke , Ismail Nahed , Walker David
Published: January 2023

Rickettsiae escape from the vacuole into the cytosol, where they replicate and inflammasomes recognize the cytosolic infectious pathogens via Nod-like receptors leading to secretion of IL-1β and IL-18. However, the interaction of rickettsiae with inflammasomes has never been investigated. In this study, we found that secretion of mature IL-1β and IL-18 from Rickettsia australis-infected macrophages is caspase-1 and ASC-dependent, suggesting that Rickettsia activates ASC inflammasome. All R. australis-infected ASC-/- mice succumbed to infection (100% mortality) while all infected wild type (WT) mice survived (0% mortality). Rickettsial loads in liver in infected ASC-/- mice were significantly increased with less cellular infiltration compared to infected WT mice. In vivo production of IL-18 was completely dependent on ASC in R. australis-infected mice as evidenced by abolished IL-18 production in infected ASC-/- mice compared to infected WT mice. R. australis-induced-IL-1β and IL-18 concentrations were only reduced, but not abolished in macrophages from NLRP3-/- mice compared to WT controls. Host survival and rickettsial loads in lung and liver of NLRP3-/- mice were comparable with those of WT mice, suggesting a negligible role of NLRP3 in host defense. Taken together, our results suggest that R. australis are recognized by ASC inflammasome in vitro and in vivo via a NLRP3-independent pathway. ASC inflammasome mediates host protection against this intracellular bacterium.
Lack of functional selectin ligand interactions enhances inherent neutrophil function leading to increased resistance to systemic Listeria monocytogenes infection (INC7P.408)
Authors: Agbayani Gerard , Sad Subash , Krishnan Lakshmi
Published: January 2023

Leukocyte recruitment towards sites of infection involves coordinated ligand-receptor interactions between leukocytes and the vascular wall. Selectins transiently bind to selectin ligands to enable leukocyte tethering and rolling on the vascular wall prior to leukocyte adhesion and transmigration into tissues. These interactions occur following post-translational modifications of selectin ligands, including terminal fucosylation by Fucosyltransferase-IV and -VII. In mice, deficiency in both enzymes (FtDKO) leads to loss of leukocyte tethering and rolling, increased circulating neutrophils and impaired T cell trafficking. However, the role of functional selectin ligand interactions in modulating immunity to bacterial infections remains unclear. We analyzed the kinetics of infection and modulation of immune responses in FtDKO mice infected with Listeria monocytogenes, an intracellular bacterium. FtDKO mice controlled systemic infection more rapidly relative to wild-type mice. This was correlated to increased number and functionality of neutrophils. Adoptive transfer of bone marrow cells from FtDKO mice into irradiated wild-type mice resulted in expansion of neutrophil populations and increased resistance to infection in a neutrophil-dependent manner. Functional selectin ligand deficiency on neutrophils was correlated to increased resistance to cell death. Thus, functional selectin ligand expression is a potential target for enhancing innate immunity against bacterial infection.
Intratracheal transfer of lung eosinophils reduces influenza virus burden in mice (INC8P.438)
Authors: Samarasinghe Amali , Woolard Stacie , LeMessurier Kim , Melo Rossana , McCullers Jonathan
Published: January 2023

Eosinophils, commonly known for anti-parasitic immunity and a controversial role in allergic asthma, may exhibit antiviral immunity against respiratory viruses. We have shown previously that mice subjected to a fungal asthma model were protected from morbidity associated with influenza A virus (IAV). Furthermore, we have also shown that mice studied in the model following resolution of allergic/eosinophilic inflammation and were more susceptible to influenza. We hypothesized that eosinophils promote viral clearance from the lungs. Lung eosinophils harvested from “allergic” mice were transferred directly into H1N1 influenza (A/CA/04/2009) infected mice. Recipients had improved lung compliance and reduced viral burden, although there was no change in weight compared to controls. The inflammatory response was equivalent between groups at day 3. There were fewer cells in airways of recipients by day 7, however, there were significantly more virus-specific CD8+ T cells present. The enhanced viral clearance was associated with increased IgA content in the airways of recipients. When mice which had achieved resolution of allergic inflammation prior to IAV infection were supplemented with eosinophils, viral burden also decreased, but the inflammatory profile was unaffected. Bone marrow-derived eosinophils killed IAV by a mechanism that appeared to be piecemeal degranulation. Taken together, these data suggest a novel function for eosinophils in altering the immune response to IAV.
Neonatal oxygen supplementation in mice leads to persistent modifications in NK cell functions during influenza A virus infection (INC8P.436)
Authors: Reilly Emma , Martin Kyle , Jin Guangbi , Yee Min , O'Reilly Michael , Lawrence B. Paige
Published: January 2023

Supplemental oxygen is used to treat preterm infants in respiratory distress. However, this treatment can disrupt normal lung development and alter responses to respiratory insults. Similarly, exposure of newborn mice to 100% oxygen leads to alveolar simplification and altered host resistance to respiratory infection. Upon challenge with influenza A virus, adult mice exposed to neonatal hyperoxia exhibit higher morbidity and mortality, and survivors display hallmarks of pulmonary fibrosis, indicative of atypical repair. Natural Killer (NK) cells are key players in antiviral immunity; identifying and killing infected cells. Emerging evidence suggest they also help maintain homeostasis in peripheral tissues, including the lung, by promoting epithelial cell regeneration via IL-22. We tested the hypothesis that adult mice exposed to hyperoxia as neonates have modified NK cell responses to infection. We found that mice exposed to neonatal hyperoxia had fewer IL-22 producing pulmonary NK cells during influenza virus challenge, but an increase in IFN-γ producing NK cells. In reciprocal bone marrow chimera mice, diminished IL-22+ NK cells aligned with hyperoxia-exposed donors, irrespective of the host’s exposure status. Overall, our findings suggest that neonatal hyperoxia leads to long-term changes in the reparative versus cytotoxic nature of NK cells, due in part to intrinsic changes in hematopoietic cells. These differences may contribute to altered host responses to infection.
Liver sulfatide-reactive type II NKT cells recognize endogenous phospholipids. (INC9P.445)
Authors: Kato Shingo , Terabe Masaki , Berzofsky Jay
Published: January 2023

NKT cells are T cells recognizing lipids presented by CD1d. Type I and II subsets recognize distinct antigens. Type I NKT cells recognize alpha-galactosylceramide (aGC). aGC-loaded CD1d tetramers are widely used to study them. Type II NKT cells and their antigens are much less well characterized than type I due to lack of widely available tools to study them. Sulfatide (3-o-sulfo-beta-D-galactosylceramide), an endogenous lipid, is the only lipid proven to be recognized by type II NKT cells in vivo. Recently, phosphatidylglycerol (PG) and phosphatidylinositol (PI) were reported to be recognized by type II NKT cell hybridomas. Here, PG-or PI-loaded CD1d-tetramer-reactive cells were detected in liver mononuclear cells as a distinct population from aGC-loaded CD1d-tetramer-reactive type I NKT cells, consistent with type II NKT cells. Simultaneous staining with PG-, PI-, and sulfatide-loaded CD1d-tetramers indicated, surprisingly, that these three populations were largely identical, even though the phospholipids have structures quite distinct from that of sulfatide. IFN-gamma and IL-4 production by spleen cells was detected after stimulation with sulfatide, but not with PG or PI. These findings suggest that although PG and PI, when loaded onto CD1d, bind the same cells as do sulfatide-CD1d tetramers, their ability to induce cytokine production was distinct. Whether these phospholipids are weak agonists or antagonists or induce other functional activities is under investigation.
Dual variable domain autoantibodies to analyze the distinct role of BCR-TLR7 and BCR-TLR9 co-engagement on B cells (INM9P.455)
Authors: Pawaria Sudesh , Moody Krishna , Marshak-Rothstein Ann
Published: January 2023

Self-constituents can aberrantly engage pattern recognition receptors and result in autoimmune diseases. The activation of autoreactive B cells by DNA- and RNA- immune complexes through TLR9 and TLR7 respectively, has been implicated in the development of systemic lupus erythematosus (SLE). Interestingly, TLR9-deficient autoimmune-prone mice develop more severe disease and TLR7-deficient autoimmune-prone mice develop less severe disease. Previous studies in the lab were limited to using primary B cells from an autoreactive IgG2a-specific BCR transgenic line (AM14) together with monoclonal IgG2a autoantibody-autoantigen complexes to dissect the TLR7/9 dichotomy. We have developed bifunctional dual variable domain autoantibodies (autoDVDs) to analyze the differences between BCR-TLR9 versus BCR-TLR7 signaling in polyclonal (non-transgenic) mouse and human B cell populations. The autoDVDs consist of conventional heavy and light chains fused in tandem by a short linker, and then connected to either murine or human C1 constant region domains. These autoDVDs provide a covalent bridge between an anti-BCR and either a DNA-reactive or RNA-reactive antibody. Initial results suggest that the DNA DVDs are functionally similar to natural DNA immune complexes on non-transgenic B cells from different murine strains. The autoDVDs provide us an important tool to target autoantigens to human BCR and also provide a high-throughput screen for identifying TLR-targeted therapies.
Role of NKG2D in the induction of mouse natural killer cell exhaustion (IRC8P.473)
Authors: Alvarez Maite , Negrin Robert
Published: January 2023

Natural Killer (NK) cells have been used as a cancer therapy against multiple cancers. Despite initial promises, NK-based immunotherapy has resulted in limited success and therefore, new approaches are needed. Our group described the induction of NK cell exhaustion (NCE) upon tumor exposure that resulted in reduced cytotoxic function associated with lower expression of several molecules such as NKG2D and the transcription factors Eomesodermin and T-bet. Here we show that NCE induced by tumor cells is MHC-I independent and is not mediated by the expression of NKG2D ligands (NKG2DL) on tumor cells. However, when NKG2D-deficient NK cells were exposed to tumor cells limited proliferation and higher IFNg production was observed suggesting a role for NKG2D in NCE. Moreover, NCE patters were observed at later stages of in vitro IL2-activation that were reduced in NKG2D-deficient NK cells. Interestingly, concomitant downregulation of NKG2D and upregulation of NKG2DL (MULT1, Rae-1) was also detected. Upregulation of NKGDL expression was particularly seen after the cells were acid-stripped which was shown to disrupt MHC-I-Ly49s cis-interaction complexes. NCE was also observed after prolonged in vivo IL2 stimulation that impaired NK cell maturation. In conclusion, we demonstrated a role for NKG2D in the induction of NCE that could represent a self-regulatory mechanism important in controlling NK cell activation utilized by tumor cells to prevent NK cell anti-tumor responses.
Down-regulation of CD73 expression favors host protection during Intracellular foodborne bacterial infections (IRC8P.496)
Authors: Alam Mohammad , Costales Matthew , Williams Kristina
Published: January 2023

Salmonella & Listeria infections cause foodborne illness. Adenosine is an immunomodulatory molecule produced by the enzymes CD39 & CD73. We examined the effect of bacterial infections on the host’s ecto-enzyme (CD73) expression & its role in regulating immune responses. After oral infection of B6 mice with Salmonella or Listeria, we tested the expression of CD39/CD73 in tissues, in T-cell & in RAW cells treated with antigen (Ag=bacterial-sonicate) & inflammatory cytokine expression. The function of CD73 was tested in Ag-activated RAW cells after addition of 5’-AMP with or without CD73 inhibition (APCP) & in splenocytes from CD73-KO mice after treatment of Ag in vitro. Inflammatory cytokine mRNA was tested by RT-PCR & cytokine proteins were tested from supernatant by ELISA. Infection, or Ag treatment, markedly downregulated CD73 mRNA in tissue with concomitant rise of the inflammatory cytokine TNF-α. In Th cells, Ag treatment or infection significantly reduced expression of CD73+ & CD39+CD73+ double positive cells compared to unstimulated/uninfected cells. Ag-treated & CD73-inhibited cells produced more anti-bacterial mediators (NO). Splenocyte derived Th cells from CD73-KO mice produced significantly more IL17A than wildtype mice when Ag treated. Also, Treg cells from Salmonella-infected wildtype mice expressed reduced CD73. CD73 downregulation during intracellular bacterial infection modulates host responses & may favor host protection.
Formation of mucosal resident memory CD8 T cells is controlled by mTOR. (IRC9P.705)
Authors: Marzo Amanda , Sowell Ryan , Rogozinska Magdalena , Nelson Christine , Vezys Vaiva
Published: January 2023

Mucosal tissues are sites of frequent pathogen exposure and major routes for the transmission of infectious diseases. Migration of virus-specific memory CD8 T cells into mucosal tissues such as the small intestine and vaginal mucosa is highly restricted to non-circulating tissue resident memory CD8 T cells. Resident mucosal memory CD8 T cells are sufficient to protect against the establishment of mucosa-acquired viral infections, and thus it is essential to develop vaccine strategies that elicit long-lived mucosal CD8 T cells. CD8 T cells residing in mucosal tissues are phenotypically distinct from those in secondary lymphoid tissues and factors critical for their formation are poorly defined. It is also unclear whether mucosal CD8 T cells possess a unique requirement for their generation and maintenance from that of circulating memory CD8 T cells. We report that unlike in the secondary lymphoid tissues, the formation of virus-specific memory CD8 T cells in the small intestine and vaginal mucosa is dependent on mTOR signaling. In addition, we demonstrate using a CD8 T cell mediated model of intestinal autoimmune disease that inhibiting mTOR results in a loss of CD8 T cells in the small intestine and protects mice from lethality. Furthermore, we will discuss data that targets mTOR signaling intrinsically within the CD8 T cells to provide mechanistic insight into how the mTOR pathway specifically controls the generation of mucosal CD8 T cells in response to viral infections.
Overexpression of suppressor of cytokine signaling-4 impairs T cell development and homeostasis (LYM7P.723)
Authors: Park Jung-Hyun , Feigenbaum Lionel , Luckey Megan
Published: January 2023

Suppressor Of Cytokine Signaling molecules (SOCS) are key players in regulating cytokine signaling. SOCS inhibits cytokine-induced JAK/STAT signaling by directly binding to JAK or by competing with cytokine receptor downstream signaling molecules. So far, eight SOCS family members have been identified, but only a few are known to play roles in the immune system. Here we identified SOCS4 as a SOCS family member whose expression is regulated during thymocyte development, with high mRNA and protein expression at the CD4, CD8 double positive stage and downregulation upon positive selection. To assess the role of SOCS4 during thymocyte development, we generated transgenic mice overexpressing a murine SOCS4 cDNA under the control of a human CD2 mini-cassette. Interestingly, SOCS4 overexpression resulted in reduced thymocyte numbers but did not affect CD4/CD8 lineage choice. Surprisingly, SOCS4 overexpression did not inhibit cytokine signaling. Instead, we found that SOCS4 transgenic T cells showed decreased ability to upregulate CD69 and CD5 following TCR stimulation. Moreover, we found that SOCS4 overexpression impaired positive selection in the thymus and interfered with T cell homeostasis in HY TCR transgenic mice. These results suggest a new role for SOCS4 in T cell development and homeostasis, independent of cytokine signaling.
Essential role of Cavβ2 in T Cell development and homeostasis. (LYM7P.715)
Authors: Jha Mithilesh , Jha Archana , Singh Ashish , Weissgerber Petra , Freichel Marc , Flockerzi Veit , Flavell Richard
Published: January 2023

Although many of the molecular components that regulate TCR signaling and thymocyte development are well characterized, the interactions between these molecules and their assembly in signaling complexes have remained elusive. Here, we report a novel and intriguing role of β2 regulatory subunit of voltage gated calcium channels (Cavβ2) during T cells development in thymus. We found that the T cell specific ablation of Cavβ2 resulted in severe reduction in thymocytes. Cavβ2-deficient DN and DP thymocytes were prone to die spontaneously. Consequently, the lack of Cavβ2 led to substantially lower peripheral T cell numbers, which became even more prominent in a competitive environment, thus emphasizing the crucial function of Cavβ2 in maintaining the peripheral T cell pool. Nicardipine, a selective Cav1 channel blocker, abolished the constitutive Lck phosphorylation in thymocytes and impacted the sustained calcium influx upon TCR-stimulation. The in vivo treatment of nicardipine resulted in a dramatic reduction in number of thymocytes and T cells but not bone marrow cells. Our findings demonstrate that Cavβ2 and Cav1 channels are critical components required during development and homeostasis of T cells.
Understanding Plasmodium-specific B cell dynamics during blood stage malaria infection (MPF6P.742)
Authors: Krishnamurty Akshay , Perez-Mazliah Damian , Pepper Marion
Published: January 2023

Immunity to malaria is acquired gradually and comes at the expense of 1 million deaths per year. Antibodies help confer clinical immunity and longitudinal studies of those in endemic areas show that B cell mediated protection develops slowly and is maintained only with constant re-exposure. These studies, however, do not give insight to the types of B cells responding and which are most protective. Using an antigen based enrichment method we observed the dynamics of Plasmodium-specific B cells in response to blood stage Plasmodium infection against Merozoite Surface Protein 1 (MSP1), a blood stage specific protein. We find at the peak of infection the emergence of short-lived plasmablasts and as parasitemia begins to decline later arising germinal center (GC) B cells form that seed a GC-derived memory B cell population. Surprisingly, there is an emergence of GC-independent B cells, which also form a stable memory population to levels similar to that of GC-derived cells. Together these data show that in response to infection, three heterogeneous populations of Plasmodium-specific B cells emerge with two stable memory populations maintained over time. We are currently assessing the functional contributions of each of these memory populations to protective immunity. These studies represent the first glimpse of the endogenous B cell response to Plasmodium with the hopes of defining optimally protective B cell responses.
IL-10R blockade enhances effector and memory CD4+ T cell responses during persistent bacterial infection (MPF6P.733)
Authors: Singh Abinav , Thirumalapura Nagaraja
Published: January 2023

Several bacterial pathogens have evolved to establish persistent infection in their hosts through suppression of host immune responses. IL-10 has emerged as a key immune-regulator during infection with various pathogens. Ehrlichia are tick transmitted bacteria that establish persistent infection in their natural reservoir hosts. In the present study, we analyzed the role of IL-10 in the development of immune responses and subsequent long-term protection against persistent ehrlichial infection in a murine model. We demonstrate that IL-10 secretion leads to reduced inflammatory responses and bacterial clearance and it also inhibits antigen-specific effector and memory CD4+ T cell responses during persistent bacterial infection. We also found that IL-10 secretion had no significant effect on the antigen-specific CD8+ T cell population. We observed that therapeutic blockade of IL-10R enhanced the magnitude and functional capacity of antigen-specific effector CD4+ T cells that translated into increased and more effective memory responses and secondary recall response after bacterial re-challenge. Our results provide new insights into the mechanisms of bacterial persistence and support the idea that alternative immune-based strategies may have applications in persistent bacterial infections.
TNF receptor 2 on CD8 T cells and TNF receptor 1 on non-CD8 T cells mediate upper genital tract pathology following primary genital Chlamydia muridarum infection (MUC7P.764)
Authors: Li Weidang , Manam Srikanth , Thomas Joshua , Schripsema Justin , Ramsey Kyle , Murthy Ashlesh
Published: January 2023

We recently demonstrated that pathogenesis in the upper genital tract (UGT) following primary vaginal Chlamydia muridarum infection in mice is dependent upon TNF-alpha production and antigen-specific CD8 T cells. In this study, we evaluated the contribution of TNF receptor 1 (TNFR1) and TNFR2 to UGT pathogenesis following primary intravaginal C. muridarum infection (5X104 IFU/mouse), using depo-provera ® treated mice deficient in TNFR1 (TNFR1 KO), TNFR2 (TNFR2 KO), or TNFR1 and 2 (TNFR1/2 DKO), and the corresponding C57BL/6J wild type (WT) mice. All groups (n=8) of mice displayed comparable chlamydial clearance, antigen-specific cytokine response, and serum anti-chlamydial antibody following primary bacterial inoculation. On day 80 after primary inoculation, TNFR1 KO, TNFR2 KO, and TNFR1/2 DKO mice displayed significantly reduced oviduct pathology (hydrosalpinx), when compared to WT mice, suggesting that both TNFR1 and TNFR2 contribute significantly to oviduct pathology. Repletion of wild type or TNFR1 KO CD8 T cells did not enhance pathology significantly in TNFR1 KO mice. On the other hand, repletion of WT, but not TNFR2 KO, CD8 T cells enhanced oviduct pathology to WT levels in TNFR2 KO mice. Collectively, these results suggest that TNFR2 on CD8 T cells and TNFR1 on non-CD8 T cells mediate UGT pathology following primary genital chlamydial infection.
Programmed death-ligand 1 expressed on corneal epithelium inhibits the local innate and adaptive immune response in the herpes simplex virus-1-infected cornea (MUC7P.751)
Authors: Jeon Sohyun , Harvey Stephen , Hendricks Robert
Published: January 2023

Constitutive expression of the PD-L1/B7-H1 ligand for the inhibitory receptor Programmed Death-1 (PD-1) appears to help protect the cornea from damaging inflammation, but might also limit clearance of pathogens from this mucosal surface. Indeed, mice lacking B7-H1 expression or those treated locally in the cornea with anti-PD-L1 mAb exhibited enhanced clearance of herpes simplex virus type 1 (HSV-1) from the cornea from 1 - 4 days post infection (dpi). PD-L1 expression peaked in the cornea on both CD45+ and CD45- cells at 4 dpi. Increased early viral clearance (1 dpi) was associated with significantly increased infiltration of CD45+ Gr-1+(presumed neutrophils) and increased production of the neutrophil chemokines CCL3 and CXCL1. Bone marrow chimeras that expressed PD-L1/B7-H1 only on bone marrow derived cells exhibited enhanced late viral clearance at 4 dpi in association with increased numbers of CD69+ CD3+ cells, but did not show enhanced early viral clearance at 1 dpi. Our findings suggest that PD-1 interacting with PD-L1/B7-H1 on CD45+ cells impairs early viral clearance from the cornea by inhibiting neutrophilic infiltration at 1 dpi, whereas PD-1 interacting with parenchymal cells (presumably corneal epithelial cells) inhibits late viral clearance by inhibiting T cell infiltration and/or activation at 3 dpi. Importantly, increasing viral clearance by inhibiting PD-1/PD-L1 interaction did not result in observable inflammatory damage to the cornea.
Renal monocyte-derived dendritic cells extend processes into the intravascular lumen (MUC7P.754)
Authors: Yatim Karim
Published: January 2023

Dendritic cells (DCs) have been shown to extend cellular processes that sample antigen (Ag) at barrier surfaces such as the epidermis and gut mucosa. Here, we tested the hypothesis that DCs in the kidney, a non-barrier organ, extend cellular processes into the blood vessel lumen, potentially allowing them to sample intravascular Ag. Flow cytometric analysis of the mouse kidney revealed multiple DC subsets (Lineage-(CD19,CD90,NK1.1,Ly-6G)CD45+CD11c+MHCII+cells). Among these, monocyte-derived DCs (mono-DC: CD11b+F4/80- DC) were the majority at ~68%. Two-photon intravital microscopy (2-PIM) of CD11c-eYFP (DCs express YFP) and CX3CR1-GFP (monocytic-lineage cells express GFP) mouse kidneys showed identical cellular networks located between the endothelial and epithelial cell layers. 2-PIM of CD11c-eYFP x CX3CR1-GFP mouse kidneys, where mono-DCs express GFP and YFP, revealed that this network is mainly composed of mono-DCs. The mono-DC network encased renal post-capillary venules. High-resolution image stacks of mouse kidneys showed DCs extending processes into the lumina of post-capillary venules. Intravascular filling defects, as well as capture of intravascularly retained, anti-CD11c-coated beads by mono-DCs, confirmed the above finding. Our data indicate that in the kidney, mono-DCs extend processes into the lumina of postcapillary venules. This suggests a role for the kidney in immune surveillance of the blood.
Regulation of plasma cell IgA production through the B cell maturation antigen receptor (MUC8P.802)
Authors: Wade Nekeithia , Loo William , Erickson Loren
Published: January 2023

B Cell Maturation Antigen (BCMA) is a receptor expressed on plasma cells (PCs) and is important for the survival of long-lived PCs in the bone marrow. Our laboratory has shown that autoimmune-prone mice deficient in BCMA (lpr.BCMA-/-) develop an exacerbated autoimmune phenotype, including increased numbers of PCs, T cells, and serum autoantibody IgG titers. We recently determined that these mice also have increased total and auto-antigen specific serum IgA titers. Because IgA is normally produced in the mucosa-associated lymphoid tissue, this observation suggests a loss of tolerance in the intestinal mucosa. Based on these data, we hypothesized that BCMA signaling regulates homeostasis between commensal bacteria and the protective intestinal IgA response. We found that lpr.BCMA-/- mice have increased serum secretory IgA and commensal-specific IgA levels compared to controls. However, this was not due to increased intestinal permeability as assessed by a serum FITC dextran assay. To determine if commensal bacteria regulate peripheral IgA responses, lpr.BCMA-/- mice were placed on a one month antibiotic regimen. This regimen ameliorated autoimmune disease and reduced IgA-producing PCs, including both autoantibody and commensal-specific IgA. These results suggest that BCMA is important for the regulation of intestinal mucosal tolerance in our models, elucidating a previously unknown role for BCMA in maintaining the balance between commensals and the adaptive immune response.
T-bet controls intestinal chronic inflammation via regulation of IL-10 production by CD4 T cells (MUC8P.806)
Authors: Shin Boyoung , Poholek Catherine , Yeh Wen-I , Harrington Laurie
Published: January 2023

The dynamics of pro- and anti-inflammatory cytokine secretion by CD4 T cells is critical for the maintenance of intestinal homeostasis. Disruption of this equilibrium can result in chronic inflammation in the intestine, causing inflammatory bowel disease (IBD). Among the various subsets of CD4 T cells, the Th1 and Th17 subsets are increased in IBD patients and are proposed to be pathogenic. Therefore, understanding the mechanisms by which these cells are regulated is important to expand current treatments of IBD. Using the CD4 T cell dependent adoptive transfer model of colitis, we have investigated the Th1-associated transcription factor, T-bet, in the context of intestinal inflammation. Loss of T-bet results in Th17 skewed immune responses; however, despite wild-type Th17 CD4 T cells being colitogenic, transfer of T-bet deficient naïve CD4 T cells fails to induce colitis in a lymphopenic recipient. Surprisingly, we show that IL-10 production is significantly increased in CD4 T cells in the absence of T-bet. Furthermore, deficiency of both T-bet and IL-10 negates the protective effects of T-bet deficiency alone, as recipients of T-bet-/-IL-10-/- CD4 T cells develop colitis with similar weight loss and histological damage to recipients of wild-type CD4 T cells. Therefore, our data suggests that T-bet plays crucial role in the maintenance of intestinal homeostasis by regulating the intricate balance of effector and regulatory cytokines, in particular IL-10 levels.
Homeostatic IL-23R signaling limits TH17 response through IL-22-mediated containment of commensal microbiota (MUC9P.818)
Authors: Ghilardi Nico , Cox Jennifer , Shih Vincent
Published: January 2023

Mammalian hosts are colonized with commensal microbes in various mucosal and epithelial tissues, including the intestinal tract. In mice, the presence of segmented filamentous bacteria (SFB) is required for TH17 differentiation and the development of autoimmune disease. Here we demonstrate that the IL-23 pathway dynamically regulates the abundance of SFB as well as mucosal barrier function in the adult animal. Genetic or pharmacological inactivation of the pathway selectively perturbs abundance of a small group of commensals, including SFB, and results in an impaired mucosal barrier, leading to systemic microbial dissemination. Conversely, microbial dissemination leads to induction of the IL-23 pathway with dual consequences: IL-23 drives IL-22 production to reinforce mucosal barrier function and elicit antimicrobial activities. It also drives the differentiation of TH17 cells in an attempt to combat escaped microbes in the lamina propria and in distal tissues. Thus barrier defects generate a systemic environment that facilitates TH17 development.
In vivo miR-146a administration reverses Pristane-induced pulmonary hemorrhage in C57BL/6 mice (THER5P.836)
Authors: Liang Dong , Zhou Shiyu , Liu Zheng , Shan Zhengyuan , Brohawn Philip , Yao Yihong , Harley John , Shen Nan
Published: January 2023

Purpose: MiR-146a as a lupus susceptibility gene plays a critical role in resolving acute inflammation. This study explored a therapeutic potential of miR-146a to treat Pristane-induced pulmonary hemorrhage in a murine model of lupus. Methods: The C57BL/6 (B6) mice were intravenously injected with PBS or miR-146a via lateral tail veins. The peripheral bloods from 3, 7 and 14 days post Pristane injection were evaluated by real time PCR or gene expression profile analysis. For comparison, the B6 mice were categorized into 3 groups, receiving respectively 3 consecutive injections of PBS (control group) or miR-146a either 3 days before (prevention group) or 7 days after (treatment group) a single intraperitoneal injection of Pristane. The pulmonary hemorrhage was histologically investigated by HE staining. Results: The injections of miR-146a mimic (agomir) increased its peripheral blood expression in both the prevention and treatment groups. HE staining showed that miR-146a administration reduced the prevalence of complete pulmonary hemorrhage from 56% in control group to 25% in treatment group and further to 0% in prevention group. Gene expression profile analysis showed that miR-146a treatment substantially suppressed multiple inflammatory signaling pathways. Conclusion: MiR-146a plays a suppressive role in the Pristane-induced pulmonary hemorrhage in B6 mice. Interference with miR-146a may constitute a useful therapeutic intervention for SLE patients with pulmonary hemorrhage.
Metabolic inhibitors normalize CD4 T cell metabolism and functions, and reverse disease in a murine model of lupus (THER5P.839)
Authors: Yin Yiming , Brusko Todd , Perry Daniel , Seay Howard , Mathews Clayton , Sobel Eric , Morel Laurence
Published: January 2023

Autoreactive CD4 T cells are key effectors in Systemic Lupus Erythematosus (SLE). Cell metabolism is an important checkpoint for T cell activation, proliferation, and differentiation. We hypothesized that 1) SLE T cells have metabolic defects that cause abnormal T cell functions; 2) Targeting T cell metabolism may abrogate T cell inflammatory functions and reduce disease symptoms in SLE mice. Metabolism of CD4 T cells from lupus-resistant B6 mice and the congenic lupus model B6.Sle1.Sle2.Sle3 (B6.TC) were studied. Here it was observed that compared to B6, CD4 T cells from B6.TC mice have a stronger glycolysis and a higher mitochondrial oxygen consumption, as well as an enhanced mTOR activity. To normalize T cell metabolism in B6.TC mice, we used metformin, which activates the AMPK pathway and inhibits mitochondrial oxygen consumption, and 2-DG, an inhibitor of glycolysis. In vitro, metformin blocks IFNγ production by CD4 T cells and facilitates Treg development. 2-DG also blocks IFNγ production, but only after T cell activation. In vivo, a combined treatment with metformin and 2-DG of B6.TC mice normalized T cell metabolism and reversed disease phenotypes, including T cell activation and autoantibody production. Further, CD4 T cells from SLE patients have an enhanced metabolism as compared to healthy controls, and excessive IFNγ production was significantly reduced by metformin. Our research suggests that T cell metabolism is a novel target for SLE treatment.
Induction of immune tolerance by CGEN-15001, a novel immunomodulatory Fc fusion protein of the B7 family (THER5P.823)
Authors: Hecht Iris , Vaknin Ilan , Oren Anat , Toporik Amir , Tarcic Nora , Rotman Galit , Neria Eyal , Keren Aviad , Gilhar Amos , McNamee Kay , Williams Richard , Miller Stephen , Podojil Joseph
Published: January 2023

We discovered a novel immune checkpoint protein based on characteristics of the B7/CD28 family. CGEN-15001 is a fusion protein composed of the extracellular domain of this protein fused to IgG Fc. CGEN-15001 inhibits T cell activation, demonstrates immunomodulation by inhibiting pro-inflammatory Th1 and Th17 responses while promoting anti-inflammatory IL-10 and Th2 responses, and enhances iTreg differentiation. CGEN-15001 is efficacious in animal models of autoimmunity such as multiple sclerosis (MS), rheumatoid arthritis, type1 diabetes (T1D) and psoriasis. In the EAE model of MS and in the NOD model of T1D, short 2-week treatment with CGEN-15001 resulted in long term inhibition of disease. In the T1D model, mice treated with CGEN-15001 retained normal blood glucose levels18 weeks after cessation of treatment. To further study tolerance induction, the H-Y minor Ag mismatch bone marrow transplantation model was used. CGEN-15001 treatment resulted in graft survival and increase in Tregs, indicating donor specific tolerance induction. The prolonged therapeutic responses in models of autoimmunity and the prevention of graft rejection, imply establishment of immune tolerance by CGEN-15001. Taken together, the mode of action of CGEN-15001 that combines inhibition of autoreactive cells, immunomodulation and restoration of immune tolerance, suggests it should provide long standing and safe therapy for autoimmune diseases.
Deletional therapy for harmful immune responses: beyond chemotherapy (THER5P.828)
Authors: Jordan Michael , Hildeman David , Katz Jonathan , Millen Scott
Published: January 2023

Immune suppressive therapies may be categorized as either inhibitory (e.g. inhibitors of co-stimulatory pathways, intracellular signaling pathways, various cytokines, etc.) or deletional (e.g. anti-T or B cell antibodies, chemotherapy to kill activated lymphocytes, etc.). While inhibitory therapies may be tailored to a specific application, they are never truly immune selective because any immune pathway may be involved in both pathologic and protective immune responses. Similarly, depletional strategies may have non-specific effects on the immune system and significant off target toxicities (e.g. genotoxicity). We have recently reported the exquisite immune selectivity of etoposide, though this agent still has non-immune toxicities related to its non-specific DNA damaging mechanism. We have now found that the replicative stress experienced by recently activated T cells leads to a strong spontaneous DNA damage response (DDR), sensitizing them to DNA-damaging agents. Thus, we hypothesized that non-DNA damaging agents designed to therapeutically exploit the DDR would impair the ability of T cells to cope with intrinsic genotoxic stresses and would have therapeutic potential. We have now found a series of novel agents which impair cell cycle checkpoints and/or potentiate pro-apoptotic effects of the DDR which potently delete activated T cells in vivo while sparing resting naïve and memory T cells, thus implicating novel, antigen-specific forms of immune suppressive therapy.
High efficacy of Toll-like receptor 4 targeting in murine and humanized models of rheumatoid arthritis in comparison with IL-1 and TNF inhibitors (THER6P.860)
Authors: Abdollahi Shahla , Koenders Marije , Joosten Leo , van de Loo Fons , van den Berg Wim
Published: January 2023

Enhanced expression of Toll-like Receptor (TLR) 4 and its endogenous agonists in rheumatoid joints suggests involvement in rheumatoid arthritis (RA). We assessed therapeutic efficacy and downstream effects of TLR4 blockade in murine and humanized models of RA compared with interleukin-1 (IL-1) and tumor necrosis factor (TNF) inhibitors. TLR4 blockade using purified B. quintana LPS suppressed established collagen-induced arthritis, and reduced serum IL-1β and IL-6 concentrations to the same extent as IL-1 and TNF inhibitors. TLR4 targeting exceeded beneficial effects of TNF blocker by reducing serum TNFα and IL-17 along with synovial expression of IL-23p19, IL-17 and the Th17-related transcription factor RORγt, while Th1 markers and type II collagen-directed T cell proliferation and antibody responses remained unaffected. To translate the findings into human disease, RA synovial biopsies were engrafted into SCID mice. TLR4 inhibition in this model resulted in reduction of spontaneous IL-6 and IL-8 release by RA synovium and reduced synovial inflammation, thereby equaling anti-TNF therapy. In ex vivo cultures of RA synovial explants, high-density protein microarray revealed suppressed activation of multiple key signaling molecules including NFκBp65 phospho-Ser536 (27%), TAK1 phospho-Thr187 and -Ser412 (26 and 23%, resp.), JAK1 phospho-Tyr1022 (22%), IRF3 (51%), Foxo3a (39%) and Btk (24%). The data collectively suggest TLR4 as a potential therapeutic target in RA.