bluuebunny/crossref_metadata_2025 huggingface dataset:
CD4 T cells license dendritic cells to enhance polyfunctional anti-viral T helper cell responses (P6094)
Authors: Olson Matthew , Turner Stephen
Published: January 2023

CD4 T cell help is critical for the generation of optimal B cell and CD8 T cell responses after immunization or infection. Robust and polyfunctional CD4 T cells are also generated after exposure to antigen, however, the factors that govern the development of potent CD4 T cell immunity are less clear. We demonstrate here that augmenting CD4 T cell help by adoptive transfer of increasing numbers of influenza A virus (IAV)-specific T cell receptor transgenic T cells resulted in enhanced polyclonal IAV-specific CD4 T cell responses after infection. Virus-specific CD4 T cells in mice that received augmented help also exhibited a greater capacity to produce multiple cytokines as compared to controls, indicating that CD4 T cell help is a major factor that dictates CD4 T cell polyfunctionality. Mechanistically, CD154 expression on CD4 T cells and CD40 on dendritic cells (DCs) were both necessary and sufficient to induce optimal CD4 T cell responses after infection, suggesting that helper T cells “license” DCs to enhance CD4 T cell responses. Interestingly, augmenting CD4 T cell help also resulted in enhanced IAV-specific CD4 and CD8 T cell memory responses after secondary infection. Taken together, these data define a CD4 T cell-DC circuit that promotes the priming of robust and polyfunctional CD4 T cell responses. These data also suggest that enhancing CD4 T cell help during vaccination could be used as a therapeutic approach to promote overall T cell immunity.
Th9 cells secrete BAFF (P1447)
Authors: Awe Olufolakemi , Jabeen Rukhsana , Kaplan Mark
Published: January 2023

The B-cell activating factor (BAFF) is essential for proper B-cell development. Cells of the innate immune system including macrophages and dendritic cells are the primary source of BAFF. The BAFF protein has two forms, soluble and membrane bound. The membrane bound form of BAFF can become soluble following furin convertase-mediated digestion.. Through microarray analysis comparing the gene profile of Th9, Th2 and T-regulatory cells we found that expression of the gene encoding BAFF (Tnfsf13b) was enriched within the Th9 subset. Using qPCR, intracellular staining, and ELISA, we have confirmed that Th9 cells express BAFF both at the mRNA and protein level. Although Tnfsf13b is expressed at the greatest level in Th9 cells, Th2 cells also express detectable mRNA. Yet, BAFF secretion is only detected in supernatants from Th9 cells. We hypothesized that the absence of soluble BAFF in Th2 supernatants could be due to a difference in the level of Furin expressed by Th9 and Th2 cells. Indeed Th9 cells express a higher level of Furin compared to Th2 cells. Moreover, blocking Furin activity diminishes BAFF secretion from Th9 cells. Together, these data suggest that BAFF production from Th9 cells results from the coordinated expression of both the gene encoding BAFF, and the enzyme required to generate soluble BAFF. Furthermore, Th9 cells may be an important source of BAFF during the development of humoral immunity.
Combined TLR/CD40 vaccination produces robust T lymphocyte memory by suppressing mTOR. (P4498)
Authors: Cross Eric , Pennock Nathan , Kedl Ross
Published: January 2023

A long-sought goal of vaccine development is to produce robust cytotoxic T cell responses to prevent future infection. This can be accomplished via live-attenuated vaccines, with the caveat of poor stability and pathogenic reversion concerns. However, a combined TLR agonist/CD40 agonist adjuvant mix (TLR/CD40 vaccination) pioneered by our lab produces potent and protective CD8+/CD4+ T cell memory. However, the mechanistic underpinnings of the protective memory are unknown. Recent work suggests that the metabolic state of a T cell plays a crucial role in the cell fate, effector or memory, realized. While glycolysis is primarily utilized in effector T cells, FAO and catabolic processes are extensively used in naïve and memory T cells. Rapamycin and metformin treatment skews T cell fate decision toward memory by inhibiting mTOR and the respiratory chain complex 1 respectively. Interestingly, CD8+ T cells induced in the presence of these agents have a phenotype identical to that generated by TLR/CD40 vaccination suggesting that our vaccination may influence the metabolic pathways utilized by the responding T cells. Consistent with this, phospho-mTOR levels are suppressed in T cells generated by TLR/CD40 vaccination as compared to those responding to Listeria monocytogenes infection. Ongoing work is establishing the means by which TLR/CD40 vaccination and related costimulatory molecules and cytokines (IL-27 and CD27) influence the metabolic processes in T cells post vaccination.
Vaccine-induced salivary IgA reduces SIV viremia in neonatal rhesus macaques. (P4501)
Authors: Jensen Kara , Wilson Robert , Ranganathan Uma , Van Rompay Koen , Jacobs, Jr. William , Fennelly Glenn , Larsen Michelle , Kozlowski Pamela , Abel Kristina
Published: January 2023

Vertical transmission of HIV in breast milk remains a primary route of pediatric infections. While neutralizing antibodies are important for preventing viral entry, the role of mucosal antibodies for virus neutralization is still not well described. Neonates produce less mucosal IgA than adults and are thus more susceptible to oral pathogens. Here we tested whether an oral pediatric SIV vaccine could induce salivary IgA and protect against oral SIV acquisition. Rhesus macaques were immunized orally at birth with a live, attenuated Mycobacterium tuberculosis strain engineered to express SIVgag, pol and env proteins and boosted at 3 and 6 weeks with rMVAgag/pol/env. Starting at week 9, animals were orally challenged using a regimen of low-dose SIVmac251 (5000 TCID50) weekly. Although the vaccine did not prevent infection, vaccinated infants with higher SIV-specific salivary IgA antibodies at the time of challenge had lower peak viremia (p=0.034). A similar inverse correlation was observed between peak viremia and fecal IgA (p=0.0019). Salivary IgA concentrations correlated with fecal IgA (p=0.018) but not with plasma IgA (p=0.911). In fact, both plasma IgA and IgG showed trends towards a positive association with viremia. Thus, mucosal, but not systemic, antibodies efficiently controlled virus replication. Vaccine strategies that promote the development of mucosal IgA antibodies will likely improve the efficacy of pediatric vaccines to prevent oral HIV acquisition.
Hantaviruses employ multiple mechanisms to evade NK cell recognition (P6298)
Authors: Braun Monika , Klingström Jonas , Gupta Shawon , Björkström Niklas , Ljunggren Hans-Gustaf
Published: January 2023

Rodent-borne hantaviruses, such as Andes virus (ANDV) and Hantaan virus (HTNV) cause hantavirus pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS) respectively, two human diseases with high case-fatality rates and no specific treatment. Patients suffering from HPS or HFRS exhibit massive lymphocyte activation, including high numbers of responding CD8 T cells and NK cells, leading to hyperinflammation and vascular leakage. Interestingly, while endothelial cells are the main targets of hantaviruses, no direct cytopathic effects of the virus on the vascular endothelium have been found. Here, using an in vitro infection model with primary human endothelial cells, we show that hantavirus renders infected endothelial cells resistant to NK cell cytotoxicity by two mechanisms: first, by decreasing NK cell degranulation in an HLA class I-dependent manner and second, downstream of cytotoxic granule-release, by specifically inhibiting granzyme B and caspase 3 activity in infected endothelial cells. Although resistant to NK cell cytotoxicity, infected cells promote an highly activated NK cell phenotype and increased functional capacity. As clinical symptoms in HPS/HFRS most likely stem from the massive immune activation, a more complete understanding of how NK cell activation occurs and the role these activated cells may play in the pathogenesis of disease will be crucial to improve the clinical outcome of these, often fatal, infections.
Defining the immune correlates of pathogenicity of Simian Hemorrhagic Fever virus in Rhesus macaques (P6314)
Authors: Asefa Ben , Jett Catherine , Janosko Krisztina , Bernbaum John , Dodd Lori , Johnson Reed
Published: January 2023

The Clinical Laboratory at the Integrated Research Facility investigates correlates of immunity associated with lethality in nonhuman primates challenged with hemorrhagic fever viruses in high containment. In an intramuscular challenge study with simian hemorrhagic fever virus (SHFv) in rhesus macaques (Macaca mulatta) , we evaluated the factors associated with SHFv and opportunistic bacterial infections (e.g., abscesses) resulting from SHFv-induced immunosuppression. Elevated concentrations of the proinflammatory cytokines MCP-1 and IL-6 and severe septicemia were characteristic of most nonsurvivors of SHFv infection. Other factors associated with morbidity were elevated serum concentrations of AST, ALP, and ALT and decreased concentrations of albumin. Electron microscopy indicated that macrophages and endothelial cells were preferentially infected. Follow up in-vitro studies of SHFV-infected dendritic cells by immunophenotyping demonstrated that mature HLA-DR+ CD163+ cells were rapidly ablated within 48 h of infection with SHFV. Infection with SHFV, a biosafety level-2 (BSL-2) agent, is strikingly similar to infection with Ebola virus, a BSL-4 agent, in hemorrhagic presentation, clinical manifestations, and cellular targets. Further investigation is needed to elucidate the mechanisms of pathogenicity of SHFV infection, compare pathogenic pathways of these hemorrhagic fever virus infections, and identify therapeutic modalities for treatment of viral hemorrhagic fevers.
Age-associated B cells as a connection between anti-viral response and autoimmunity. (P4355)
Authors: Rubtsov Anatoly , Rubtsova Kira , Marrack Philippa
Published: January 2023

Recently our group has discovered a subset of B cells, called age-associated B cells (ABCs), which appear in elderly female mice and autoimmune prone mice of either gender. Our experiments suggest that these B cells can produce autoantibodies and contribute to autoimmune diseases. It has been shown that ABCs develop from follicular B cells and their appearance depends on TLR7 signaling. Here we demonstrate that ABCs also appear at the peak of the response to viral infection. These cells secrete antiviral class-switched (IgG) antibodies and are the major contributors in anti-viral humoral response. Appearance of ABCs during viral infection depends on TLR7 and IFN gamma signaling since mice deficient in these genes fail to accumulate ABCs during infection. Overall, our data suggest a new role of ABCs as a major contributor to anti-viral response. Moreover, ABCs can serve as a new connection between anti-viral response and autoimmunity as they are capable to secrete antibodies with broad reactivity against both self and viral antigens.
Type 2 innate lymphoid cells promote Th2 cytokine production by CD4+ T cells and antibody responses by B cells (P4335)
Authors: Drake Li , Kita Hirohito
Published: January 2023

Type 2 innate lymphoid cells (ILC2s) are part of a large family of ILCs that are important effector cells in innate immunity. Recent studies suggest that lung ILC2s play critical roles in host immunity against helminth infection and in maintaining tissue homeostasis after respiratory viral infection. However, little is known as to whether and how these cells influence adaptive immunity. Here we report that lung ILC2s enhanced proliferation of naïve CD4+ T cells and promoted production of T helper type 2 (Th2) cytokines including IL-4, IL-5 and IL-13. ILC2s regulated T cell function by direct contact through OX40/OX40L interaction. Furthermore, IL-4 produced by ILC2s when cultured with CD4+ T cells played an important role in Th2 cytokine upregulation. We further revealed that lung ILC2s promoted IgA, IgE, IgG1 and IgM antibody production by both B1 and B2 cells, and that this stimulatory effect of ILC2s was mediated at least in part by ILC2 production of cytokines IL-5 and IL-13. We conclude that ILC2s promote both T cell- and B cell-mediated immune responses and propose that ILC2s may play roles in regulating adaptive immunity in addition to their established roles in innate immunity.
Activation and proliferation of innate immune NK cells by adaptive immune B cells (P1082)
Authors: Kim Kwanghee , Lim Seon Ah , Kim Tae-Jin , Lee Kyung-Mi
Published: January 2023

Natural killer (NK) cells belong to innate immune system and consist of circulating lymphocytes which provide innate inspection against infectious disease and cancer. NK cells co-exist with adaptive immune cells, such as T and B cells in secondary lymphoid organ, for example, the lymph nodes and spleen. Though NK cells are likely to interact with B cells, the functional consequence of this interaction remains uncertain. To address this question, , we co-cultured primary NK cells with freshly isolated B cells from the spleen and investigated the influence of B cells on NK cells. Here we show that co-presence of B cells in cultures facilitated NK cell expansion after suboptimal stimulation condition. NK cells cultured with B cells exhibited higher expression of CD69, B220, CD25 activation markers, suggesting that B cells drastically facilitated NK cell activation. In addition, In an in vivo sepsis model, a higher death rate was observed in nude and SCID mice compared to wild type mice after poly I:C treatment. Furthermore, expression of CD25 and CD69 activation markers on NK cells were elevated in nude mice compared to SCID and wild-type mice. Furthermore, this synergistic effect of B cells on NK cell functions could also be detected in humans. Given the fact that NK cells can exert a regulatory role in cancer or infection, co-presence of B cells can potentiate the role of NK cells by promoting their proliferation and cytotoxicity.
The 16 day administration of artemisinin for the long-term cure of malaria in Haiti (P3134)
Authors: Thornthwaite Jerry , Shah Hare , Thornthwaite Bonita , Respess Henry
Published: January 2023

Artemisinin is a natural compound that has been advocated by our Institute in cancer treatment and for its intended purpose in the treatment of malaria. Both malaria parasites and tumors accumulate iron, specifically in the mitochondria, which reacts with the Artemisinin to create a free radical complex that destroys the host malarial parasite or cancer cell. To date, over 50 patients in Haiti, have been treated with our protocol, which encompasses using 100mg orally per day for 16 days. Children under 12 years of age were given 50mg/day, while six babies were given 25 mg/day. Many of these malaria victims had previously experienced multiple recurrences with poor performance and side effects from the conventional malaria treatments. None of the patients treated ever had a recurrence with some over six years after the single treatment (n=17).These cures occurred in spite of repeated mosquito bites of 800-1,000 per year (African estimates). The interesting immunological question is why is a single treatment over 16 days so effective? There must be an in vivo vaccination occurring where the parasites are destroyed so that “super antigens”, regardless of the stage of the cell cycle, are produced. The 16 day treatment assures the transition between early IgM to IgG development. We are currently in the process to secure a collaboration to study antibody and even cytokine levels in these patients during and after treatment to attempt to explain these results.
The costimulatory ligand CD86 promotes resolution of disease after influenza virus infection (P6202)
Authors: Moser Emily , Hufford Matthew , Braciale Thomas
Published: January 2023

Influenza A virus (IAV) infection can produce severe pulmonary inflammation and injury. The host immune response to infection mediates virus clearance but also contributes to pulmonary inflammation and injury, marking the immune response as a potential therapeutic target after severe infections. Host factors mediating IAV clearance and lung inflammation are well-studied, however, factors controlling recovery from disease following IAV clearance are ill-defined. The costimulatory ligand CD86 mediates interactions between immune cells via its receptors CD28 and CTLA-4. We observed in infected mice that blockade of CD86 signaling in vivo after IAV clearance delays recovery and promotes accumulation of neutrophils in the respiratory tract. Furthermore, CD86 blockade reduces both IL-2 and T regulatory cells (Treg) in the lungs. Depletion of Tregs using DREG mice or Treg depleting antibody after IAV clearance also delays recovery and leads to pulmonary neutrophilia. These data suggest that Tregs may play a crucial role in regulating pulmonary inflammation after IAV infection and are in turn dependent on CD86 ligand engagement. We are continuing to explore the role of CD86 in recovery and resolution of inflammation after IAV infection with the long-term goal of exploring the therapeutic potential of optimizing CD86 signaling to promote Treg function in recovery from infection.
Inhibition of reactive oxygen species production attenuates highly pathogenic avian influenza H5N1-induced inflammation via up-regulation of suppressor of cytokine signalling 3 (P6188)
Authors: Ye Siying , Lowther Sue , Stambas John
Published: January 2023

Influenza A virus infection causes a rapid influx of inflammatory cells, resulting in increased ROS production and acute lung injury. Pro-inflammatory stimuli induce intracellular ROS by activating NADPH oxidase (NOX). Nox2 is the catalytic subunit of the phagocyte NOX. It has been shown that Nox2-containing NOX is the major source of ROS induced by influenza A virus infection in mice. Apocynin, a NOX inhibitor, reduced virus replication and accumulation of inflammatory cells in the lungs of X31- or PR8- infected mice. As hypercytokinemia is thought to contribute to severe disease pathogenesis of high pathogenicity avian influenza (HPAI) H5N1 infection, it is hypothesized that apocynin could reduce HPAI H5N1-induced cytokine production. A panel of airway epithelial and immune cells from mammals and chickens were infected with low pathogenicity avian influenza H5N3 (A/duck/Victoria/0305-2/2012), HPAI H5N1 (A/chicken/Vietnam/0008/2004), or PR8 H1N1 virus in the absence or presence of apocynin. Quantitative real-time PCR showed that apocynin inhibited influenza-induced cytokines (IL-6, IFN-β, CXCL10 and CCL5) and ROS production, although viral replication was not significantly altered. Interestingly, apocynin treatment significantly increased SOCS3 expression, enhancing negative regulation of cytokine signaling. Therefore, apocynin or its derivatives could be used as a therapeutic agent for reducing hypercytokinemia, resulting in amelioration of disease.
High and low molecular weight hyaluronan use TSG-6 or CD44 signaling pathways respectively in activation of different subsets of dendritic cells thereby affecting the development of encephalitogenic T effector cells and autoimmune encephalomyelitis (P5172)
Authors: Guan Hongbing , Nagarkatti Prakash , Nagarkatti Mitzi
Published: January 2023

Microglia and astrocytes can overexpress hyaluronan in demyelinated lesion of brain in multiple sclerosis (MS), which affects the remyelination of axons. However, the exact role of hyaluronan in the development and treatment of MS is not clear. This becomes more complicated because the prototype hyaluronan is degraded into fragments of various sizes in MS. Previous studies using high molecule weight hyaluronan (HMW-HA) or low molecule weight hyaluronan (LMW-HA) have so far resulted in confusing or contradictory results. In this report, we found that HMW-HA and LMW-HA used different receptors depending on the subsets of dendritic cells (DC), which affect the activation of DC and subsequent development of effector T cells. HMW-HA (950KDa) bound to tumor necrosis factor-6 (TSG-6) receptor on myeloid DC (mDC) and induced a Th2 biased response. LMW-HA (12Kda) bound to CD44 receptors on plasmacytoid DC (pDC) and lymphoid DC (lDC) and induced a Th17 biased response. Accordingly, systemic administration of HMW-HA inhibited the development of experimental autoimmune encephalomyelitis (EAE), whereas systemic administration of LMW-HA exacerbated the development of EAE. We also found that gamma-interferon-inducible lysosomal thiol-reductase (GILT) was required for the therapeutic activity while HMW-HA and LMW-HA regulated GILT activity differentially. Our study, for the first time, elucidated the distinct role of hyaluronan in the development and treatment of MS.
Targeting CD44 ameliorates autoimmune diabetes by reducing pancreatic infiltrates and improving Treg efficacy (P5180)
Authors: Li Cheng-Rui , Mueller Erin , Bradley Linda
Published: January 2023

In type 1 diabetes (T1D), Th1 cells are a major population orchestrating the disease pathology. We showed previously that signals through CD44 are needed for Th1 cell survival and memory development. Therefore, we hypothesized that blocking CD44 could impact progression to overt diabetes. To test this, we treated recently hyperglycemic NOD mice with a short course of anti-CD44 and found a significant delay in the spontaneous development of TID that was associated with a reduction in islet infiltrates. However, previous studies suggested that targeting CD44 might enhance Treg function. To test whether this could contribute to the anti-CD44-mediated effect, we transferred polyclonal in vitro differentiated adaptive Treg cells (aTregs), which we showed can protect ~50% of newly hyperglycemic mice from overt diabetes, together with anti-CD44 treatment. We found a significantly increased the success rate of diabetes prevention. The islet infiltrates in these mice had a higher frequency of CD4+ cells producing IL-10 and IL-2 as well as of endogenous Tregs that displayed enhanced FoxP3 expression. However, anti-CD44 treatment did not alter cytokine profiles or Foxp3 expression of the transferred aTregs. The data therefore suggest that anti-CD44 treatment can alleviate inflammation in the target organ, and can functionally enhance the endogenous Treg population, thereby improving the establishment of long-term control by aTregs, which form a protective memory population.
In situ detection of autoreactive CD4 T cells in the mouse models of autoimmune diseases (P4550)
Authors: Massilamany Chandirasegaran , Jia Ting , Kang Guobin , Zhou You , Li Qingsheng , Reddy Jay
Published: January 2023

We report here the utility of major histocompatibility complex (MHC) class II dextramers for detecting self-reactive CD4 T cells in the target tissues using two mouse models of autoimmunity namely, experimental autoimmune encephalomyelitis and experimental autoimmune myocarditis. We determined the conditions for dextramer staining using brain sections obtained from SJL mice immunized with proteolipid protein (PLP) 139-151 by staining the sections with a cocktail of IAs/PLP 139-151 (specific) or Theiler’s murine encephalomyelitis virus (TMEV) 70-86 (control) dextramers conjugated with the fluorescent dyes, and CD4 antibody. The sections were examined by Olympus Fluoview laser scanning microscope, and the analysis revealed PLP 139-151 dextramer-positive cells to be colocalized to CD4-expressing cells, but such reactivity was lacking with TMEV dextramers, suggesting that the staining obtained with PLP dextramers was specific. We next extended these observations to detect cardiac myosin-specific T cells by deriving IAk dextramers for cardiac myosin heavy chain-α (Myhc) 334-352 and ribonuclease 43-56 (control). Expectedly, heart sections prepared from A/J mice immunized with Myhc 334-352 on day 21 postimmunization showed infiltrations of Myhc-specific CD4 T cells. The data suggest that MHC class II dextramers can be potentially used to enumerate the precursor frequencies of antigen-specific CD4 T cells in situ directly, without having to amplify the fluorescent signals.
Growth hormone effect on type 1 diabetes development (P4546)
Authors: Kakabadse Schmidt Dimitri , Villares Ricardo , Gomez Lucio , Martinez Carlos , Mellado Mario
Published: January 2023

Type 1 diabetes (T1D) is an autoimmune disease that affects approximately 34 million people around the world. T1D is caused by the destruction of insulin-producing pancreatic β-cells by autoreactive T cells that lack effective immune regulation. One of the most common animal models for diabetes study is the NOD (non-obese diabetic) mouse strain. Using female NOD mice, which develop spontaneous T1D, we observed that growth hormone (GH) induces a protective effect on insulin-producing cells and as a result, inhibits T1D development. We generated a transgenic NOD mouse that expresses bovine GH and showed almost complete protection against diabetes development. Histochemical analysis demonstrated an increase in the number of pancreatic endocrine progenitor cells, overgrowth of islets of Langerhans, and although these mice showed peri-insulitis, infiltration was much reduced. The mechanism involved an increase in regulatory T cell number and activity, a polarization phenotype toward anti-inflammatory macrophages, and altered B cell responses with reduced antibody production. Our results identify a regulatory role for GH in immune cells and pinpoint a new target for therapeutic intervention on type 1 diabetes.
CD40L at the interface of the innate and adaptive immune responses. (P5206)
Authors: Ragheb Jack , Bushar Nicholas
Published: January 2023

Induction of CD40L expression on CD4+ T cells is a critical event in the generation of cellular and humoral immune responses. CD40L interacts with B cells via its cognate receptor to trigger immunoglobulin isotype class switching and promote Ig secretion and the differentiation of memory B cells. Through its induction of IL-12 and upregulation of costimulatory molecules on antigen presenting cells (APC), CD40L is also vital to the generation of Th1 cellular responses. We have previously demonstrated that a combination of cell-contact dependent and soluble signals act in concert to orchestrate the biphasic pattern of CD40L expression that underlie its complex role in promoting the adaptive immune response. We now report that CD40L likely plays an equally important role in the innate response. We show for the first time that TCR-dependent expression of CD40L on primary human T cells can be induced by interaction of β1 integrins on CD4+ T cells with extracellular matrix (ECM) in a monocyte and IL-2 independent fashion. We provide evidence in a model of vascular wall injury that ECM mediated induction of CD40L on CD4+ T cells plays a role in wound healing. These findings establish a pivotal role for CD40L at the interface of the innate and adaptive immune responses and suggest that therapeutic modalities directed towards CD40L may have benefit in modulating vascular pathophysiology.
B6.g7 mice reconstituted with BDC2.5NOD stem cells are protected against autoimmune diabetes. (P1392)
Authors: Rajasekaran Narendiran , Wang Nan , Hang Yan , Macaubas Claudia , Rinderknecht Cornelia , Beilhack Georg , Shizuru Judith , Mellins Elizabeth
Published: January 2023

In BDC2.5NOD mice, a spontaneous model of Type 1 diabetes, CD4+T cells express a transgene-encoded T-cell receptor with reactivity against a pancreatic antigen. This leads to a massive infiltration and destruction of the pancreatic islets and subsequent diabetes. When we reconstituted lethally irradiated lymphocyte-deficient B6.g7 (I-Ag7+) Rag-/- mice with BDC2.5NOD (ckit+Lin-Sca-1hi) hematopoietic stem and progenitor cells (HSPC), the recipients exhibited hyperglycemia and succumbed to diabetes. Surprisingly, lymphocyte-sufficient B6.g7 mice reconstituted with BDC2.5NOD HSPCs were protected from diabetes. In this study we investigated the factors responsible for attenuation of diabetes in the B6.g7 recipients. Analysis of chimerism in the B6.g7 recipients showed that although B cells and myeloid cells were 98% donor-derived, the CD4+ T cell compartment contained ~50% host-derived cells. These host-derived CD4+ T cells were enriched for conventional Tregs (CD25+Foxp3+) and also for host-derived CD25-Foxp3- CD4+ T cells that express markers of suppressive function, CD73, FR4 and CD39. Though negative selection did not eliminate donor-derived CD4+ T cells in the B6.g7 recipients, these cells were functionally suppressed. Thus host-derived CD4+ T cells that emerge in mice following myeloablation exhibit a regulatory phenoytpe and likely attenuate autoimmune diabetes. These cells may provide new therapeutic strategies to suppress autoimmunity.
Differential role of Bcl-2 in CD8+ and CD4+ T cell homeostasis (P1336)
Authors: Hildeman David , Raynor Jana , Tripathi Pulak , Sholl Allyson , Chougnet Claire
Published: January 2023

Regulated induction of apoptosis is critical for normal functioning of the adaptive immune system. Both TCR/MHC interactions and by soluble cytokines maintain T cells via their ability to modulate Bcl-2 family members, although the dependence of CD4+ and CD8+ T cells upon individual anti-apoptotic molecules remains unclear. Here, we used mice with a conditional allele of Bcl-2 crossed with dLck-Cre mice to assess the role of Bcl-2 in CD4+ and CD8+ T cell survival. We found that dlck-Cre-driven deletion of Bcl-2 resulted in a massive loss of naïve CD8+ T cells, but only a modest loss of naïve CD4+ T cells. We next tested if Bcl-2 was important for effector CD4+ T cell survival after infection with lymphocytic choriomeningitis virus (LCMV). Interestingly, the majority of LCMV-specific CD4+ T cells did not require Bcl-2 to survive contraction of the response. Further, treatment of mice with ABT-737, a Bcl-2, Bcl-xL, and Bcl-w inhibitor, only modestly increased the loss of effector CD4+ T cells, suggesting that neither Bcl-2, Bcl-xL, nor Bcl-w are required for survival of effector CD4+ T cells. Instead, we find that the Bcl-2 family member, A1 is increased in effector CD4+ T cells and we are currently investigating the role of A1 in the survival of naïve and activated CD4+ T cells. As the pro-apoptotic Bcl-2 family member Bim is critical to control naïve and effector CD4+ T cells, A1 may be an important Bim antagonist that promotes effector CD4+ T cell responses.
Host response in a vector transmission model of monocytotropic ehrlichiosis (P3353)
Authors: Saito Tais , Walker David
Published: January 2023

Ehrlichioses are emerging infectious diseases affecting veterinary and human hosts. Studies reveal potential pathogenic mechanisms of infection; however, not much is known about the role of the vector on disease establishment, as demonstrated by a needle-challenged vaccine but failed in field challenge by infected ticks. We have developed a tick transmission model for ehrlichial infection to study the vector-pathogen-host interaction. We infected mice by intravenous (IV) and intradermal (ID) routes and compared the subsequent infection with tick transmission of the newly described E. muris-like agent. We collected samples during the course of infection to evaluate bacterial levels in tissues and antibody production to characterize the differences induced by the route of infection. Our results showed bacterial levels and distribution patterns comparable between mice experimentally infected by the ID or IV route. Higher IgM antibody levels were observed in the early stage of infection in mice infected by tick transmission compared to ID and IV routes. Moreover, IgG antibody levels in tick-transmitted ehrlichiosis were higher than in ID inoculated mice and much higher than in IV infection, which showed only low levels during both early and later phases of infection. These initial results suggest important differences in host processing and presentation of ehrlichial antigen due to the route of infection, which will be further explored in our vaccine development research.
Identification of novel autoantibodies in systemic autoimmunity using 10,000-antigen proteome arrays (P4006)
Authors: Li Yajuan , Wang Ling , Zhou Jinchun , Mayeux Jessica , Lian Yun , Mohan Chandra , Li Quanzhen
Published: January 2023

Systemic erythematosus lupus (SLE) is an autoimmune disease characterized by presence of autoantibodies against a broad spectrum of self-antigens. In order to identify novel serum autoantibodies associated with SLE, we utilized a protoarray bearing 10,000 antigens to screen IgG and IgM autoAbs in sera of SLE patients. 446 IgG and 1218 IgM autoAbs were significantly increased in SLE compared with health controls (p<0.05), in which 367 autAbs overlapped in IgG and IgM. Except the autoAbs previously identified in SLE patients, such as anti-DNA, Ro, La, etc., the protoarray also uncovered a group of novel autoAbs against a broad range of antigens. The increased expression of 16 novel autoantibodies in SLE, including IgG autoantibodies against APEX1, AURKA, BAG3, CSNK1G1, EIF2C1, HMGB1, IFIT5, MAPKAPK3, PADI4, PKRKA, RALGPS1, RGS3, SRP19, STIP1, UBE2S and VRK1 were further confirmed by ELISA. Gene expression analysis revealed that some of the self-antigens also significantly upregulated on transcription level. In addition to uncovering ~500 novel autoantibody specificities, the novel arrays also shed light on some of the pathogenic pathways leading to disease in systemic lupus. In conclusion, protoarray provide a powerful tool for identification of novel autoantibodies in system autoimmune diseases.
Antigen cross-presentation via Sec61 is indispensable for the development of lupus nephritis: a novel ‘self-organized criticality theory’ explaining the cause of SLE (P4014)
Authors: Tsumiyama Ken , Shiozawa Shunichi
Published: January 2023

Objective: In our ‘self-organized criticality theory’ of autoimmunity, CTL develops via antigen cross-presentation and causes lupus tissue injuries. Here we show an essential role of a translocon Sec61 for antigen cross-presentation and lupus nephritis. Methods: Bone marrow-derived dendritic cell (BMDC) from BALB/c mice was cultured with fluorescent-labeled OVA. Early endosome antigen 1 (EEA1), calnexin and Sec61 were detected by immunofluorescent staining. For in vivo study, BALB/c mice were repeatedly immunized with OVA to induce SLE. The splenic DC (spDC) was also cultured with fluorescent-labeled OVA to examine localization of engulfed antigen. Further, exotoxin A was co-immunized with OVA to inhibit Sec61 in vivo, and IFNγ-producing CD8+ T cell and proteinuria were examined. Results: In BMDC, OVA was co-localized with an endosomal marker EEA1, and subsequently separated from EEA1. OVA was not co-localized with an ER marker calnexin. Instead, OVA was co-localized with Sec61. The same result was obtained using spDC, suggesting that OVA could be exported from endosome to cytoplasm via Sec61. After repeated immunization with OVA, the amount of Sec61 localized in endosome was increased compared with control. Further, treatment with a Sec61 inhibitor exotoxin A inhibited the generation of CTL and lupus nephritis. Conclusion: Direct export of antigen from endosome to cytoplasm via Sec61 is essential for antigen cross-presentation and the development of lupus nephritis.
Plasmacytoid dendritic cells correlate with fibrosis in patients with systemic sclerosis and contribute to fibrosis in a murine model (P3133)
Authors: Kafaja Suzanne , Divekar Anagha , Valera Isela , Khanna Dinesh , Saggar Rajan , Furst Daniel , Singh Ram
Published: January 2023

Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by fibrosis in multiple organs including skin and lung. Mechanisms that drive fibrosis in SSc remain unclear. Here, we report the role of dendritic cells (DC) in SSc, with a focus on plasmacytoid DC (pDC) subset. We found that whereas pDC were reduced in the blood of patients with SSc as compared to healthy controls, these cells accumulated in target organs such as skin and lungs. In the lung lavage fluid (BAL), pDC frequency correlated with CT scores of SSc-interstitial lung disease (ILD) and with a set of proteins involved in inflammation, leukocyte migration, and wound repair. Importantly, pDC were reduced in the BAL of SSc patients after treatment with imatinib mesylate that has been previously reported to improve, albeit modestly, skin score and lung function. To directly investigate the role of pDC in vivo, we depleted pDC in mice injected with bleomycin that induces SSc-like disease. pDC-depleted mice had reduced skin and lung disease compared to controls. Thus, we provide several lines of evidence in humans and mice supporting a role of pDC in SSc pathogenesis. Ongoing studies will investigate mechanisms that lead to increased migration of pDC from blood to target organs and/or their local expansion in humans and mice with SSc.
Neurokinin-1 receptor stimulated dendritic cell vaccines downregulate IL-10 and license host inflammatory dendritic cells to bias Th1 immunity (P4236)
Authors: Sumpter Tina , Janelsins Brian , Rojas-Canales Darling , Tkacheva Olga , Shufesky William , Morelli Adrian , Larregina Adriana
Published: January 2023

Substance-P (SP) is a neuropeptide that stimulates immunity by binding to the neurokinin-1 receptor (NK1R). As professional Ag presenting cells, dendritic cells (DC) are unique activators of naïve T cells. DC express functional NK1R, indicating that NK1R agonists may influence their T cell-stimulatory function. Relevant studies analyzing the relationship between neuropeptides and DC function are lacking. Here, we demonstrate that conditioning bone marrow derived DC (BMDC) with NK1R agonists leads to maturation and abrogation of IL-10 without inducing secretion of IL-12p70. Mechanistic analyses in vitro demonstrated that NK1R signaling blocks the CREB1/TORC2 pathway to inhibit the Il-10 promoter. Following subcutaneous administration as a cellular vaccine, Ag-loaded and NK1R-signaled BMDC homed to skin draining lymph nodes (sDLN), initiating inflammation and Th1 and Tc1 immunity in vivo. This type-1 immunity required IL-12p70 secreted in sDLN by host DC, including LN-resident, and recruited inflammatory DC. Utilizing CCR7KO BMDC and IL-12p35KO mice, we elucidated that endogenous DC subsets were activated in situ by NK1R-signaled-BMDC homing to sDLN. Moreover, reduced levels of IL-10 by NK1R signaled BMDC licensed IL-12 secretion by host DC and mediated type-1 immunity. Our data demonstrate that NK1R-signaling promotes immunostimulatory DC and provide a relevant insight into the mechanisms employed by neuromediators to regulate the outcome of cellular immune responses.
Development of genetically engineered TCR-transduced T cells for immunotherapy of chronic hepatitis B and C virus infections (P4222)
Authors: Park Su-Hyung , Park Heiyoung , Pan Warren , Rehermann Barbara
Published: January 2023

More than 350 million people worldwide are chronically infected with hepatitis B (HBV) or hepatitis C virus (HCV). While vigorous CD8 T cell responses are required to spontaneously clear acute HBV and HCV infections, these virus-specific T cells are typically diminished in number and functionally impaired in chronic HBV and HCV infections. Here, we set out to use adoptive therapy with TCR-engineered T cells to restore HBV and HCV-specific immune responses. Three HBV-specific and four HCV-specific TCRs with high avidity were cloned from patients and chimpanzees that cleared the respective infection, expressed in retroviral vectors and used to transduce lymphocytes of chimpanzees with chronic HBV and HCV infection. The transduced T cells gained a broad spectrum of antigen-specific effector functions (IFN-γ secretion, cytotoxicity and proliferation). They were expanded to high numbers via short-term anti-CD3 and IL-15/IL-21 stimulation, which resulted in a preservation of their differentiation status and a lower frequency of regulatory T cells than the conventional expansion method with anti-CD3 and IL-2. Collectively, we show that retroviral TCR gene transfer followed by short-term expansion in the presence of IL-15/IL-21 efficiently redirects the antigen specificity of resting primary T cells and generates a large number of functional effector T cells. These will be used in adoptive T cell therapy protocols to restore T cell function in chronic viral hepatitis.
Antigen-specific natural killer cell cytokine production in chronic hepatitis C infection (P6062)
Authors: Bowman Kathryn , Holz Lauren , Rehermann Barbara
Published: January 2023

Chronic hepatitis C virus (HCV) infection results in an inflammatory liver disease leading to fibrosis and cirrhosis. The progression of liver disease is thought to be immune-mediated because HCV itself is non-cytopathic. Given that HCV-specific T cells are diminished in number and functionally exhausted in chronic HCV infection, it remains unclear which cell population drives disease pathogenesis. Here, we investigated the function of natural killer (NK) cells, the major innate immune cell population in the liver. PBMCs from 15 patients with chronic HCV infection were stimulated for 8 hours in a whole blood activation assay with pools of overlapping 18-mer peptides comprising HCV structural (E1, E2) and nonstructural (NS3) proteins. Cytokine production by NK cells and T cells was assessed by multicolor flow cytometry. The frequency of IFN-γ+ NK cells was 5-10 fold greater than the frequency of IFN-γ+ T cells. A minority of IFN-γ+ NK cells co-produced TNF-α. NK cell responses to HCV peptides varied between subjects, but did not correlate with T cell responses or viremia. This study demonstrates that NK cells are activated in an antigen-specific manner in chronic HCV infection and respond to both structural and nonstructural HCV proteins. The mechanism of antigen-specific NK cell activation is currently under investigation.
An analysis of expression of lupus-associated miRNAs in splenic lymphocytes from male and female NZB/WF1 mice (P4074)
Authors: Dai Rujuan , McReynolds Savannah , Heid Bettina , Ahmed S.Ansar
Published: January 2023

Systemic Lupus Erythematosus (SLE) is a prototype, female predominant autoimmune disease. The functional importance and pathogenic contribution of microRNAs (miRNAs) to lupus are now highly recognized. We reported recently a common set of lupus-associated miRNAs including miR-182-96-183 cluster, miR-155, miR-31, and miR-127 in three spontaneous murine lupus models including NZB/WF1. To understand whether miRNAs are differentially expressed in males versus females, and thereby contribute to increased susceptibility to lupus in female NZB/WF1, we analyzed the expression of above miRNAs in splenic lymphocytes from male and female NZB/WF1 mice at different disease stages. Our data indicated that there is no significant difference in the expression of above lupus-associated miRNAs between female and male NZB/WF1 at 17-18wk old, an age before the onset of disease. However, by 23 weeks of age (when NZB/WF1 begin to develop mild lupus disease) the expression levels of miR-182, miR-183, miR-127, and miR-31 were significantly up-regulated in female NZB/WF1 mice compared to age-matched male controls. The expression levels of these miRNAs were further increased in 30 wk old females, following the progression of lupus disease. There was also a slight increasing trend of the above miRNAs in 30 wk old male NZB/WF1 mice when compared to 23 wk old male mice, however it was not significant. Taken together, our study further suggested the association of these miRNAs with lupus development.
Contribution of novel microRNAs in the pathogenesis of rheumatoid arthritis (P4078)
Authors: Singh Ram , Hahn Bevra , Hossain Awlad
Published: January 2023

Rheumatoid arthritis (RA) is a systemic inflammatory autoimmune disease with progressive joint destruction, lifelong disability, functional deterioration, and high mortality. The major effector cells in RA are T cells and synovial fibroblasts. Defects in T cells and synovial fibroblasts lead to activation of proinflammatory cytokines, which induce Th1 and Th17 cells and chronic inflammation. Recent studies provide further evidence of dysregulation of candidate microRNAs in inflammation and autoimmunity. In our preliminary studies we have found dysregulated candidate microRNA (miRNA21, miR25 miR95, miR124a, miR146a and miR186) expression in RA patients compared to healthy controls. Furthermore proinflammatory cytokines such as IL-6, IL-12, IL-17 and IL-21 are significantly increased in RA patients compared to healthy controls, and expression of these cytokines positively correlates with the expression of candidate microRNAs such as miR21 and negatively correlates with the expression of miR146a. Furthermore we found that miR21 and miR124a positively correlate, and miR146a negatively correlates, with DAS28 score in RA patients. Based on these preliminary data, we hypothesize that dysregulated microRNAs play an important role in RA pathogenesis. This study has translational potential and may identify novel therapeutic targets in patients with RA.
TNF receptor superfamily members drive thymic Treg development (P4430)
Authors: Mahmud Shawn , Manlove Luke , Schmitz Heather , Hogquist Kristin , Farrar Michael
Published: January 2023

CD4+FOXP3+ regulatory T cells (Tregs) express several TNF receptor superfamily (TNFRSF) members, but what role these molecules play in thymic Treg development is unclear. We found that CD25+FOXP3- Treg progenitors and CD25+FOXP3+ thymic Tregs highly overexpress OX40, GITR, and TNFR2 relative to conventional CD4SP thymocytes. Utilizing a novel Nur77GFPBac reporter mouse, which reports TCR signal strength via GFP, we found that OX40, GITR, TNFR2 and CD27 expression by Treg progenitors positively correlates with TCR signal strength. Importantly, the ligands for these receptors are expressed by APCs in the thymic medulla. Antibody-blockade of TNFRSF members in thymic organ cultures reduced expression of CD25 and FOXP3 by Tregs. Conversely, stimulation of sorted Treg progenitors with TNFSF agonists enhanced Treg differentiation ~3-4 fold and elevated CD25 and Bcl-2 expression. In vivo, GITR- or OX40-deficiency reduced Treg development by ~50%. Finally, dominant negative retroviral bone marrow chimeras designed to knock out up to three TNFRSF members simultaneously showed a dose dependent (up to ~6 fold) reduction in Treg development indicating that these receptors function redundantly. Our data support a model in which TNFRSF expression driven by TCR signaling enhances IL2 sensitivity and promotes survival in Treg progenitors to enhance thymic Treg differentiation.
A clinical, epidemiological and immunological characterization of a dengue cohort in a metropolitan city in Malaysia (P3027)
Authors: Sekaran Shamala Devi , Rathakrishnan Anusyah , Azliyati Azizan , Sathar Jameela , Sanchez-Anguiano Aurora , Kumari Santha
Published: January 2023

Dengue has neither licensed drugs nor vaccines and Its immunopathogenesis is elusive. The disease is widespread and imposes healthcare, economic and social burden on endemic countries like Malaysia. This study aims to understand and identify clinical and immunological markers of disease. Dengue suspected patients were recruited in 2 hospitals where 77% of the study population was categorized as dengue with warning signs. Diagnosis based solely on clinical symptoms is insufficient. Clinically, platelet counts of dengue patients were low and together with increased vWF and prolonged clotting time, retain thrombocytopenia in dengue as an excellent marker for disease progression but not as a differentiator for patients with and without warning signs. Liver enzymes, ALT and AST, were good indicators for dengue disease progression. Immunogenetically, HLA alleles A*24 and B*57 and A*03 were associated with Chinese, Indians and Malay dengue patients, respectively. Three non-structural proteins were found to be immunodominant via the IFN-γ T cell ELISpot assay while cytokines, TNF-β, MIF, SDF-1α and HGF had differential levels in patients with and without warning signs. Although we did observe any discriminative clinical markers, we detected differential levels of 4 cytokines between the groups of patients, which may, with further studies, serve as predictive markers for dengue patients developing warning signs.