bluuebunny/crossref_metadata_2025 huggingface dataset:
CpG vaccination induces cytolytic T cells to the lung and heterosubtypic protection against lethal IAV infection (P4289)
Authors: Vogel Alexander , Condon Shirley , Brown Deborah
Published: January 2023

With the emergence of pandemic influenza A virus (IAV) strains such as H1N1 and H5N1, the development of a universal vaccine has become more necessary than ever. In contrast to the highly mutable outer coat proteins, internal proteins of IAV are more conserved among different strains and are therefore a target for developing vaccines that induce strong T cell responses. Using a Toll-like receptor 9 agonist with unmethylated CpG motifs, we have shown that intranasal immunization with CpG and inactivated x31 (H3N2) IAV protects mice following a heterosubtypic challenge with a lethal dose of PR8 virus (H1N1). Vaccination with CpG and inactivated IAV cause a stronger inflammatory response than IAV infection alone, with significant levels of antiviral cytokines such as MIP-1β, CXCL 10, and IL-6 produced in the lungs. Furthermore, CpG immunization induces a substantial influx of CD11b+ cells into the draining lymph nodes as well as a population of CD11c+ cells that express high levels of the costimulatory molecule CD86, providing favorable conditions for T cell activation. At 6 days post immunization, effector CD4 and CD8 T cells expressing Granzyme B and IFN-γ are observed in the lungs of vaccinated mice. These experiments suggest that TLR agonists can act as potent immune adjuvants to generate robust T cell immunity capable of combating IAV and other viral infections where successful vaccines have not yet been developed.
Network analysis of lung tissue repair processes in response to influenza-induced damage identifies tissue remodeling factor TIMP-1 in recovery (P3180)
Authors: Sanders Catherine , Sharma Shalini , Vogel Peter , Doherty Peter , Thomas Paul
Published: January 2023

Mild and severe influenza infections clearly differ in disease resolution, likely due to variations in the host’s repair response to lung tissue damage. Over 60 protein factors involved in lung tissue repair at multiple stages of a mild and a severe influenza infection (early, mid-stage, viral clearance, and after viral clearance) were quantified and organized into an interaction network. Factors characteristic of all phases of tissue repair—coagulation, inflammation, migration and proliferation, and remodeling—were up-regulated in mouse whole lung tissue after both types of infections. Differences in intensity and timing of these factors, particularly in the later three stages of wound repair, were observed. Network analysis allowed for identification of a key node in the repair pathway important in tissue remodeling, TIMP-1. TIMP-1-/- mice were more susceptible to PR/8 infection independently of viral titer or inflammatory immune responses. However, respiratory analyses showed exacerbated responses to both infections, suggestive of dysregulated lung function during attempted resolution of disease. We demonstrate the utility of systems biology for understanding complex, multi-layered, integrated biological processes such as wound healing, and in identification of previously overlooked factors involved in such processes. We also demonstrate the contribution of TIMP-1, a protein not involved in viral control, coagulation, or inflammation, in recovery from influenza infection.
Establishment of non-circulating, tissue-resident anti-viral memory T cells in specific niches of the lung (P3198)
Authors: Turner Damian , Bickham Kara , Farber Donna
Published: January 2023

Virus-specific memory T cells residing in mucosal sites play an important role in conferring immune protection against recurrent infections. Here we describe the specific retention of memory T cell populations within distinct lung niches following influenza virus (IAV) infection. We used intravenous infusion of fluorescent antibody to differentiate between T cells in the lung interstitium (protected) and those accessible to circulation (labeled). While naïve mice had a negligible frequency of CD4 T cells in the protected niche, after IAV infection 50-70% of total lung CD4 T cells segregated into the protected niche with 30-40% stably maintained within this niche as memory T cells after viral clearance. IAV-specific memory CD4 and CD8 T cells were highly enriched in the protected population compared to circulating T cells. Protected T cells did not recirculate, unlike labeled memory T cells which continuously circulate through lymphoid tissues and are significantly reduced in the lung when lymphoid egress is inhibited. Confocal microscopy showed that effector and memory CD4 T cells in the protected niche were clustered around airways and bronchovascular bundles, while the labeled CD4 T cells were dispersed randomly within the capillaries of the alveoli walls. Our results demonstrate that, following IAV infection, virus-specific memory T cells are retained in spatially distinct lung niches which may be important for protective immunity to respiratory pathogens.
The role of CXCR6 in lung airway memory CD8 T cell recruitment and maintenance (P3202)
Authors: McMaster Sean , Wilson Jarad , Wang Hong , Kohlmeier Jacob
Published: January 2023

Tissue-resident memory T (TRM) cells are uniquely positioned to provide an initial line of defense against invading microorganisms; airway-resident memory CD8 T (TARM) cells have been shown to confer protective immunity against a number of respiratory pathogens, including influenza virus and M. tuberculosis. Unlike TRM at other anatomical sites, TARM are not maintained via cytokine-driven homeostatic proliferation in the airways but must be continuously recruited from the circulation. We show that there exists a defect in TARM cell recruitment in mice infected with influenza virus via either intramuscular or intraperitoneal route as compared to the intranasal route. However, the mechanism(s) that enable intranasally-primed memory T cells to preferentially migrate to the airways have not been identified. Based on our preliminary data, we hypothesize that the candidate chemokine receptor, CXCR6, is responsible for steady-state trafficking of influenza-specific TARM cells to the airways from the general circulation. Our results show that CD8 T cell recruitment to the lung airways is significantly restricted in CXCR6 KO mice as compared to WT, while CD8 T cell numbers in the spleen remain constant. This study could help advance the field of cell-mediated vaccines to promote pathogen-specific T cell recruitment to the lung airways, enhancing protective immunity to respiratory pathogens.
Resistance to invasive pneumococcal infection in mice with acute allergic lung inflammation (P3228)
Authors: Sanfilippo Alan , Metzger Dennis
Published: January 2023

Streptococcus pneumoniae is the most common cause of bacterial pneumonia which results in 175,000 hospitalizations as well as 40,000 deaths in the United States annually. Recent work has identified a link between asthma and invasive pneumococcal disease (IPD) prompting the Advisory Committee on Immunization Practices to recommend that all adults with asthma receive vaccination. To study the underlying mechanism responsible for an increased risk of IPD for individuals with asthma, we used an acute model of ovalbumin (OVA)-induced allergic lung inflammation (ALI) and hypothesized that mice with acute ALI would be more susceptible than naive mice to a pulmonary S. pneumoniae infection. Surprisingly, mice with acute ALI were more resistant to infection than naïve controls and this increased resistance was found to be greatest 10 days following the final OVA challenge. While inflammatory cell infiltration and cytokine levels in the bronchoalveolar lavage (BAL) fluid had returned to levels similar to naïve controls by day 10, this time point also correlated with the peak of IgA expression in the BAL fluid, suggesting a critical role for non-specific IgA in protection against IPD. Indeed, protection against IPD was lost in IgA KO mice with ALI as well as naïve IgA KO mice.
Thymic stromal lymphopoietin directly influences the respiratory CD8 T cell response to influenza virus infection. (P3230)
Authors: Shane Hillary , Klonowski Kimberly
Published: January 2023

Mucosal surfaces are tightly regulated immune microenvironments with unique anatomical structures, profiles of cells and cytokines. These aspects, coupled with infection derived inflammatory signals, can differentially modulate downstream immunological responses. Thymic stromal lymphopoietin (TSLP) is a cytokine expressed in mucosal surfaces where its role in CD4 T cell immunity has been well studied. However, whether TSLP directly affects anti-viral mucosal CD8 T cell responses is less understood. Here, we show that influenza induces TSLP production in a lung epithelial cell line and infected mice. To test the direct role of TSLP on mucosal CD8 T cell responses we used a competitive adoptive transfer system where equal numbers of OVA-specific OT-I and TSLP receptor deficient OT-I cells are co-transferred into recipient mice infected with influenza expressing OVA. We found that TSLPR-/- OT-I CD8 T cells proliferate less early post infection (pi) and have decreased accumulation in the respiratory tract both at days 8 and 50pi. Interestingly, the lack of TSLP signaling imparted a higher level of CD62L expression on TSLPR-/- OT-I memory cells. Together these data suggest that TSLP signaling may directly drive the expansion and development of CD8 effector phenotypes while concomitantly limiting long term responses. Thus, TSLP participates in the regulation of immune responses in the respiratory tract and modulation of TSLP levels may promote long term immunity at mucosal sites.
The induction of humoral immune responses to the orally administered Dukoral vaccine occurs independently of TLR signaling and DC maturation (P3188)
Authors: Kumar Ashok , Majithia Jay , Azizi Ali , Sirskyj Danylo
Published: January 2023

We investigated the involvement of TLR signaling in the induction of humoral and cell-mediated immune responses (CMIR) following oral and intramuscular (IM) immunization with Dukoral (killed whole-cell V. cholerae and cholera toxin B subunit, CTB) or its components by using TLR-2-, TLR-4-, MyD88- and Trif-deficient mice. We show that wild type and all groups of TLR-deficient mice generated similar levels of V. cholerae- and CTB-specific IgG1and IgG2c serum antibodies, fecal IgA antibodies and agglutinating antibodies, following oral immunization with Dukoral or CTB alone. However, IM immunization with Dukoral or CTB alone required MyD88 signaling for the induction of CTB-specific IgG1and IgG2c serum antibodies. In addition, our results show that CD4+ T-cell and CD19+ B-cell proliferation, maturation of DCs in response to vaccine components, as measured by CD80, CD86, CD40, and MHCII expression, and cytokine secretion including IFN-g occurred in a MyD88-dependent manner in response to V. cholerae or CTB stimulation. In contrast, in response to CTB stimulation, Trif negatively regulated both CD4+ T-cell and CD19+ B-cell proliferation. Overall, our results suggest that despite the impaired ability of MyD88-/- DCs to mature, humoral responses, following oral immunization with Dukoral, occurred independently of TLR signaling, while CMIR were TLR-dependent. However, TLR-independence is lost in the generation of humoral responses when the vaccine is administered parenterally.
Differential requirement of CD8α+/CD103+ dendritic cells for bacterial dissemination versus T cell priming following mucosal infection with Listeria monocytogenes (P3060)
Authors: Princiotta Michael , Laird Renee , Wolf Benjamin , Hayes Sandra
Published: January 2023

Dendritic cells (DCs) are essential in priming CD8+ T cells to bacterial pathogens, such as Listeria monocytogenes (Lm). Interestingly, recent studies suggest that DCs also function in systemic dissemination of Lm, following an i.v. route of infection, as evidenced by the 2 log lower bacterial burden in BatF3-/- mice, which lack both CD8α+ and CD103+ DCs, than in wild-type (WT) mice. Lm infection, however, typically occurs via the mucosal route in humans through the binding of Lm internalin A (InlA) to E-cadherin on the surface of epithelial cells. Since murine E-cadherin does not bind InlA, we generated a “murinized” Lm strain that expresses a modified form of InlA that binds murine E-cadherin in order to test whether DCs are required for bacterial dissemination following a mucosal route of infection. In agreement with the previous study, we found that the bacterial burden was significantly reduced in BatF3-/- mice relative to WT mice, when infected i.v. with murinized Lm. By contrast, there were no significant differences in bacterial burden in BatF3-/- and WT mice following mucosal infection (intragastric and intranasal) with murinized Lm. Surprisingly, despite similar bacterial burden, BatF3-/- mice were unable to prime an Lm-specific CD8+ T cell response. Together, these data demonstrate that, following mucosal Lm infection, CD8α+ and CD103+ DCs are dispensable for systemic dissemination, but are indispensable for priming a CD8+ T cell response.
The interaction of classical complement component C1 with parasite and host calreticulin mediates Trypanosoma cruzi infection of human placenta (P3062)
Authors: Ferreira Arturo , Castillo Christian , Duaso Juan , Maldonado Ismael , Valck Carolina , Rosas Carlos , Aguilar Lorena , Ramirez Galia , Galanti Norbel , Kemmerling Ulrike
Published: January 2023

13 million people are infected with Trypanosoma cruzi in Latin America, plus more than 300,000 in the USA, Canada, Europe, Australia, and Japan. An important number develops circulatory or digestive syndromes. While in poor countries transmission is mainly through arthropod vectors, transplacental infection prevails in developed ones. T. cruzi calreticulin (TcCRT) translocates from the endoplasmic reticulum to the area of flagellum emergence, acting as a virulence factor because it binds maternal complement C1. As measured by qPCR in human placenta chorionic villi explants (HPCVE), C1q mediated up to a 4-fold increase in infectivity. Because anti-TcCRT and anti-HuCRT F(ab')2 antibody fragments are devoid of their Fc-dependent capacity to recruit C1q, they completely reverted the C1q-mediated infectivity by respectively preventing its interaction with cell-bound parasite and HPCVE CRTs. This was corroborated by using competing fluid-phase recombinant TcCRT or human CRT and by a high expression of fetal CRT on free villi. Increased C1q-mediated infection is paralleled by fetal syncytiotrophoblast damage, that is reverted by anti-TcCRT F(ab')2 fragments or fluid-phase HuCRT. Most likely, C1q bridges CRT on the parasite with its placenta orthologue, thus facilitating their first encounter. The placental explants assay used in these studies is the closest ex vivo available correlate now revealing a plausible new mechanism of human congenital T. cruzi infection.
Increased innate immune recognition and viral clearance modulates the immune potential of an engineered CD8 T-cell vaccine (P4463)
Authors: Pavelko Kevin , Bell Michael , Pease Larry
Published: January 2023

We have engineered an attenuated picornavirus vaccine using Theiler’s murine encephalomyelitis virus (TMEV) and are exploring its usefulness in immunotherapy. We find that two different insertion sites within the leader protein of the TMEV genome allow for effective production of virus vaccine strains. One site is at the amino terminal end of the protein after the third amino acid position (p3); the other is at the XhoI restriction site further downstream. We find that vaccine antigen insertion at p3 disrupts the ability of the virus to inhibit type I interferon production by infected cells, whereas XhoI insertion does not. Further, the p3 modified virus can be cleared from animals lacking functional T and B-cells but replicates to high titer in IFNα/β receptor knockout mice, suggesting that innate immune mechanisms are sufficient for viral clearance. In spite of rapid clearance, p3 vaccines readily stimulate proliferation of transgenic T-cells and induce de novo responses to T-cell antigens in wild-type mice, although to a lesser extent than the XhoI modified vaccines strains. The use of either vaccine however effectively delays tumor growth and enhances survival in mice bearing melanoma and breast tumors. This vaccine provides a safe and effective vaccine strategy for the induction of CD8+ T-cell immunity directed towards cancer and provides support for the development of picornaviruses as vaccine vectors for treating human disease.
MyD88, a central innate immune adaptor, is dispensable for spontaneous autoimmune demyelination in a mouse model of multiple sclerosis (P4077)
Authors: Waldner Hanspeter , Wexler Aaron , Frielle Christine , Berry Gregory , Budgeon Lynn , Baccon Jennifer , Christensen Neil
Published: January 2023

Multiple sclerosis (MS) is an autoimmune disease that is mediated by myelin-reactive T cells resulting in CNS demyelination. Mice that are transgenic for a myelin proteolipid protein (PLP)-specific T cell receptor (TCR) spontaneously develop experimental autoimmune encephalomyelitis (EAE), the animal model of MS. MyD88 is the key adaptor protein in Toll-like receptor (TLR), IL-1R and IL-18R signaling, resulting in the activation of innate immune cells, including DCs. Although, MyD88-signaling is critical for T cell responses against microbial agents, it is unknown whether it is required for priming of myelin-reactive T cells in spontaneous EAE or MS. Here, we investigated the requirement of MyD88-signaling for the pathogenesis of spontaneous EAE in PLP TCR transgenic mice that genetically lacked MyD88. We demonstrate that MyD88 is dispensable for the development of spontaneous EAE and autoimmune demyelination in these mice. MyD88-independent EAE was associated with activated DCs that showed superior ability to promote PLP-specific Th1 and Th17 responses in vitro compared to DCs from healthy counterparts. In addition, we provide evidence indicating that the MyD88-independent TRIF signaling pathway was induced in EAE-diseased mice. Taken together, our data suggest that MyD88-independent innate immune signaling critically contributes to priming of myelin-reactive T cells and initiation of spontaneous EAE in our MS model.
Self dsDNA induces IL-1β production from human monocytes by activating NLRP3 inflammasome in the presence of anti-dsDNA antibodies (P4083)
Authors: Shin Min Sun , Kang Youna , Lee Naeun , Kim Sanghyun , Kang Ki Soo , Lazova Rossitza , Kang Insoo
Published: January 2023

The pathogenic hallmark of systemic lupus erythematosus (SLE or lupus) is the autoimmune response against self nuclear antigens, including dsDNA. The increased expression of the pro-inflammatory cytokine IL-1β has been found in the cutaneous lesion and peripheral blood mononuclear cells from lupus patients, suggesting a potential involvement of this cytokine in the pathogenesis of lupus. IL-1β is produced primarily by innate immune cells like monocytes and can promote Th17 cell response, which is increased in lupus. IL-1β production requires cleaving pro-IL-β into IL-1β by the caspase-1-associated multiprotein complex called inflammasomes. Here we show that self dsDNA induces IL-1β production from human monocytes dependently of serum or purified IgG containing anti-dsDNA antibodies by activating the NLRP3 inflammasome. Reactive oxygen species (ROS) and K+ efflux were involved in this activation. Knocking down the NLRP3 or inhibiting caspase-1, ROS and K+ efflux decreased IL-1β production. Supernatants from monocytes treated with a combination of self dsDNA and anti-dsDNA antibody-positive serum promoted IL-17 production from CD4+ T cells in an IL-1β dependent manner. These findings provide new insights in lupus pathogenesis by demonstrating that self dsDNA together with its autoantibodies induces IL-1β production from human monocytes by activating the NLRP3 inflammasome through inducing ROS synthesis and K+ efflux, leading to the increased Th17 cell response.
Influence of drinking water pH on gut microbiota and type 1 diabetes (P4069)
Authors: Sofi Mohammed , Gudi Radhika , Karumuthil-Melethil Subha , Perez Nicolas , Johnson Benjamin , Vasu Chenthamarakshan
Published: January 2023

Type 1 diabetes (T1D) is an autoimmune disease which occurs due to the interaction of genetically determined and environmental factors. T1D is characterized by progressive destruction of insulin secreting beta-cells by T cells leading to hyperglycemia. The non-obese diabetic (NOD) mouse spontaneously develops T1D similar to that in humans and thus widely used as a model for understanding the immunological basis and treatment of the disease. Incidence of TID in NOD mice is thought to be influenced by the degree of cleanliness of the mouse colony. Recent studies have shown a role for gut microbiota in promoting as well as preventing the disease. In this study, we show that pH of drinking water can have a profound influence not only on the type of commensal bacteria colonized in the gut, but also on the rate of disease progression. NOD mice that were on acidic pH water developed insulitis and hyperglycemia rapidly compared to those on neutral water. Interestingly, segmented filamentous bacteria (SFB) positive fecal transplant resulted in a profoundly suppressed insulitis and T1D incidence in NOD mice that are on acidic pH water. Microbial community analysis by pyrosequencing shows a significant difference in the type of gut microflora among the mice that were on acidic and neutral pH drinking water and this profile was altered by SFB+ fecal transplant. Our study shows that T1D incidence in NOD mice is influenced by the type of gut flora, but not the degree of cleanness alone.
Deficiency in SHP-1 triggers a cutaneous autoinflammatory disease that is provoked by IL-1α (P4071)
Authors: Lukens John , Kanneganti Thirumala-Devi
Published: January 2023

Mice homozygous for an Y208N amino acid substitution in the tyrosine phosphatase SHP-1 (referred to as Ptpn6spin mice) spontaneously develop a severe autoinflammatory syndrome that resembles neutrophilic dermatosis in humans and is characterized by persistent footpad swelling and suppurative inflammation. Deficiency in SHP-1 provokes dysregulated production of granulopoietic cytokines and neutrophilia. Genetic abrogation of IL-1R protected mice from cutaneous inflammation, suggesting that IL-1-mediated events potentiate disease. Surprisingly, inflammasome activation and IL-1β-mediated events were dispensable for SHP-1-mediated footpad disease. In contrast, we report a central role for IL-1α in chronic autoinflammation in Ptpn6spin mice. Microabrasion injury further exacerbated inflammatory responses and resulted in elevated cytokine production, neutrophilia, and unremitting tissue damage in Ptpn6spin mice. Bone marrow-derived cells were responsible for the autoinflammatory syndrome and the excessive inflammatory phenotype was rescued by genetic ablation of IL-1α. In contrast, genetic deletion of RIP3 failed to provide protection, suggesting that IL-1α drives spontaneous autoinflammation and aberrant wound-induced cytokine production in Ptpn6spin mice independently of RIP1/RIP3-mediated necrosis. This implicates that dysregulated SHP-1 activity triggers chronic autoinflammatory disease through IL-1α, and independently of inflammasomes or RIP1/RIP3-driven necrosis.
Vaccinia virus preferentially binds to protein enriched in lipid rafts on the surface of leukocytes (P6143)
Authors: Byrd Daniel , Amet Tohti , Hu Sishun , Lan Jie , Yu Qigui
Published: January 2023

The vaccinia virus (VV) has been found to rely on monocytic cell-mediated viremia to spread to tissues distant from initial infection sites. We have found that VV only infects monocytes to a significant degree among human leukocytes; however, VV binds to monocytes, B cells, activated T cells, and, to a lesser extent, neutrophils. Here we investigated the role of lipid raft-specific proteins in VV binding of leukocytes. When incubating fluorescent VV with leukocytes under binding conditions, we found that VV co-localizes with rafts on the surface of monocytes, B cells, activated T cells, and neutrophils. Also, VV strongly co-localizes with rafts when leukocytes are induced to polarize using various chemokines that induce rafts to move to cell uropods. This suggests that VV receptor proteins are enriched in rafts. It was previously found that mouse sera derived from human monocytes can block VV binding to human activated T cells. Using this principle, we created mouse sera from raft-enriched detergent-resistant membranes (DRM) or crude membrane (CM) extracts from monocytes, activated T cells, or resting T cells. Pretreating cells with sera derived from DRM blocked virus binding to the highest degree, but CM sera blocked to a lesser extent. Sera derived from resting T cells had no effect on virus binding. Our data indicates that leukocytes have protein virus receptors enriched in rafts on the cell surface, and that these cells share the expression of VV receptor proteins.
Neonates develop heterologous immunity but with altered crossreactivities (P6151)
Authors: Kenney Laurie , Carter Erik , Selin Liisa
Published: January 2023

The neonatal immune system is immature, lacks memory and is undergoing tolerization. This is also the age when children receive multiple vaccines. Previously, we have shown that sequential infection with unrelated pathogens can alter disease outcome. This is defined as heterologous immunity and is mediated by crossreactive T cells. In adult mice previous exposure to LCMV protects mice from lethal vaccinia virus (VV) infection. Crossreactive LCMV-specific memory cells recognize VV epitopes, are reactivated and expand during VV infection. This causes faster viral clearance and a skewing of the LCMV-specific immunodominance hierarchy. However, some LCMV-immune mice also develop acute fatty necrosis (AFN) of the abdominal fat pads during a VV infection. We questioned if mice immunized as neonates would have protective heterologous immunity during VV challenge. Mice immunized as neonates had comparable reduction in VV load and induction of AFN indicating that heterologous immunity is established during viral infections early in life. Interestingly, the LCMV-specific memory populations that expanded in mice immunized as neonates differed from that of mice immunized as adults. In adult mice 50% of the mice have an expansion of LCMV-NP205-specific CD8 T cells while the majority of neonates expanded the LCMV-GP34-specific CD8 T cell pool. This alteration in dominant crossreactivities may be due to the limited T cell receptor repertoire of neonatal mice.
Controlling viral immunoinflammatory lesions by using a mAb that specifically targets soluble and membrane bound lymphotoxin (P6105)
Authors: Veiga Parga Tamara , Gimenez Fernanda , Mulik Sachin , Chiang Eugene , Grogan Jane , Rouse Barry
Published: January 2023

Herpes simplex virus 1 (HSV-1) infection of the eye can result in stromal keratitis (SK), a chronic immunoinflammatory lesion and a significant cause of human blindness. A key to controlling the severity of SK lesions is to identify cell and molecular events responsible for tissue damage and to modulate their function. This report evaluates the role of LT-α, a proinflammatory cytokine that could be involved in mediating tissue damage during SK, and the use of a mAb that targets it. We show that in response to HSV infection, LT-α is upregulated and derived from proinflammatory cells such as neutrophils, γδ-T cells and T cells. We also show that LT-α, when expressed as LT-α3 (soluble) or LT-α1β2 (membrane bound), has a proinflammatory role in vitro in a corneal cell line. Moreover, mAb treatment during the clinical phase of the disease, which specifically targets cells expressing LT-α1β2 and its soluble version LT-α3, significantly attenuated SK lesions. In these mice, corneas expressed reduced levels of proinflammatory molecules, chemokines, and infiltration of proinflammatory cells when compared to control mice. However, treatment in vivo with a mutant version of the mAb, which only binds to LT-α3 did not decrease SK scores, indicating that the inhibitory activity may be dependent on targeting cells expressing LT-α1β2. We discuss our results in terms of the potential therapeutic value of managing SK in a clinical setting with a mAb targeting cells that express LT-α1β2.
Insoluble substances produced from human cytomegalovirus-infected cells induce deaths of nearby cells through non-classical death signal pathway (P6116)
Authors: Hwang Eung Soo , Kim Jung Heon
Published: January 2023

Human cytomegalovirus (HCMV) has many strategies to evade the host immune system, and eventually resides in some cells in latency. Although the involvement of Fas ligand in T cell death was reported in the pathogenesis of retinitis with HCMV infection to retinal pigment epithelial cells, the cell death near other lesions caused by HCMV infection was not studied in detail by now. The insoluble substances derived from HCMV-infected fibroblasts, induce the deaths of variety of cell lines including Jurkat, HL-60, K562, U937 and THP-1 cells, but not of fibroblasts, HEK293 and U373MG. They were resistant to treatment with 56°C for 30 min to lose death induceability to Jurkat cells. Cell death of Jurkat cells by these substances were not inhibited by the simultaneous treatment with neutralizing antibodies to Fas ligand or TRAIL, or caspase inhibitors. Insoluble substances induced cleavages of poly ADP-ribose polymerase-1 into 89 kDa fragment, but not through caspase 3, 7 or 9 pathway in Jurkat cells. AIF was detected in the cytoplasmic fraction, but not in the nuclear fraction of Jurkat cells from 6 hours following exposure to them. This result suggested AIF was not translocated from mitochondria to nucleus in Jurkat cells by the stimulation with insoluble substances. Collectively human cytomegalovirus infection would induce the death of myeloid and lymphoid cells near the infection site through non-classical death signal pathway.
LPA and autotaxin regulate T cell migration and homing (P5095)
Authors: Knowlden Sara , Chapman Timothy , Emo Jason , Rezaee Fariba , Capece Tara , Kim Minsoo , Georas Steve
Published: January 2023

Lysophosphatidic acid (LPA) has been suggested to regulate lymphocyte entry into lymph nodes because autotaxin (ATX), the enzyme that generates LPA, is constitutively expressed by lymph node high endothelial venules. However very little is known about the effects of LPA on T cell migration and homing. We studied the effects of LPA (16:0 and 18:1, 1-10 µM) on naïve mouse CD4+ T cells. Using chemotaxis assays, we found that LPA induces CD4+ T cell chemorepulsion (1.5±0.5 fold migration away from 1 µM LPA, n=26, p <0.0001) but not chemotaxis. In addition, we found that LPA increases the quality of naïve CD4+ T cell migration on ICAM-1/CCL21 coated plates as shown by increased track length, displacement, and velocity of cells in the presence of LPA (p<0.001). We next investigated the expression of LPA receptor mRNA on resting and anti-CD3/CD28 activated CD4+ T cells using qRT-PCR. We found that LPA2, LPA5, and LPA6 are highly expressed on naïve CD4+ T cells, and the expression of these decreases over the course of cell activation. In preliminary adoptive transfer experiments using gene-targeted mouse models, we found that LPA2 deficiency does not inhibit the recovery of transferred CD4+ cells from recipient lymph nodes after 42 hours, whereas fewer wild-type CD4+ cells were recovered from autotaxin-deficient mice (ATX+/-) compared to wild-type controls. Our results have uncovered previously unsuspected roles for LPA and autotaxin in regulating T cell migration and homing.
Targeting junctional adhesion molecule C improves the protective Th1 immunity through enhanced dendritic cell recruitment in response to Leishmania major infection (P5101)
Authors: Ballet Romain , Emre Yalin , Tacchini-Cottier Fabienne , Imhof Beat
Published: January 2023

Dendritic cells (DCs) are recruited to sites of infection where they process antigens and migrate to the draining lymph node in order to trigger specific T cell responses. In the C57BL/6J mouse model of cutaneous Leishmaniasis, monocyte-derived dendritic cells were shown to initiate the protective Th1 response against the parasite. Junctional adhesion molecule C (JAM-C), a tight junction protein expressed on endothelial cells and fibroblasts, was described in the past few years to control leukocyte migration during inflammation. Here we showed that targeting JAM-C with a monoclonal antibody improves the development of cutaneous lesions after Leishmania major infection. Indeed, we observed a two-fold increase in the number of neutrophils and monocyte-derived dendritic cells recruited one-day post infection without affecting the number and function of resident macrophages. In addition, analysis of the draining lymph node populations at several time points after infection showed a higher number of migratory dendritic cells. This enhanced antigen presenting cells recruitment boosted the activation of the Th1 response as shown by the increased number of IFN-γ-producing CD4 T cells. Overall, targeting JAM-C could represent a new way to enhance T cell response in Leishmaniasis as well as other infection diseases.
Drastic increase of DC movement from immunized site and rapid replacement of antigen-carrying DC was induced in the draining LN during initiation of T cell proliferation (P5103)
Authors: Tomura Michio , Sawano Asako , Miyawaki Atushi , Kanagawa Osami
Published: January 2023

Detailes of spatiotemporal regulation of antigen-carring DCs migrated from immunized site and antigen-specific T cell prolifeaion in the dLN is unknown. Kaede-Tg mice can monitor cellular movement by labeling cells by changing color from green to red by violet light and Fucci-Tg mice can visualize cell-cycle progression. Footpads of Kaede-Tg mice were immunized with CFA/OVA and labeled cells in the footpad and followed their migration to the dLN. After immunization, 100 times of MHC class IIHighDC migrated from immunized footpad compared with that in the steady state and lasted more than 14d. Whereas, MHC class IIInt DCs and pDCs increasead in dLN, which was sustained by increase of precursor immigration from blood circulation, migration from immunized site and proliferation. However, almost all migrated DCs from immunized site in the dLN replaced within less than 24h. Thus rapid replacement of DCs enable to control fine-tune of immune responses and spatiotemporal regulation of DCs under immune response is like “Large supply and mass consumption”. Although antigen-specific T cell proliferation already terminated 7d after immunization, skin-derived DCs continued to carry antigen from immunized site and dLN had capability to induce proliferation of re-transferred naive T cells. Thus termination of antigen-specific T cell proliferation is controlled by T cell intrinsic manner, not for the result of the absence of antigen-presenting DCs in dLN.
Tuning tumor immunity through integrin transregulation (P5077)
Authors: Cantor Joseph , Slepak Marina , Ginsberg Mark
Published: January 2023

Recent trials using adoptive cellular immunotherapy for cancer have shown the exciting potential of this approach. However, ex vivo-modified tumor-specific T cells are limited by poor homing to solid tumors. Surprisingly this problem has received relatively little attention. α4 integrins are crucial in governing the migration of T cells to sites of infection and inflammation. Blocking phosphorylation of α4 integrin by mutating Ser988 to Ala prevents release of the adaptor protein paxillin, and boosts LFA-1-dependent migration in trans. We thus generated a mouse with this mutation to test effects on leukocyte migration in vivo. Here we report that the α4(S988A) mutation, which boosts VLA-4/LFA-1 integrin transregulation, selectively increases migration of lymphocytes to both inflammatory and tumor sites; myeloid cell homing was unaffected. Melanoma tumor growth was greatly reduced, associated with increased migration of α4(S988A) T cells to tumors, suggesting that boosting integrin trans-regulation is a promising approach to improve adoptive cellular immunotherapy for cancer.
T cells autonomously disengage from Functionalantigen presenting cells after entering the G1 phase of cell cycle (P5089)
Authors: Ma Zhengyu , Han Huihui , Finkel Terri
Published: January 2023

Activation of naïve T cells requires prolonged engagement with antigen presenting cells (APCs). Once activated, however, T cells need to disengage from the APC, exit lymphoid organs, and migrate to the site of infection to fulfill their functions. Although much has been learned about how T cells stop and engage antigen-bearing APCs, it remains unclear how the interaction between T cell and APC ends. It has been generally assumed that T cell disengagement is a result of diminished antigen presentation by APCs or upregulation of certain chemokine receptors by T cells. Here, using Chinese Hamster Ovary (CHO) cells presenting antigens at a constant level, we demonstrate that T cell disengagement from APC is autonomous and independent of antigen presentation by APCs or exogenous chemokines. The motility of T cells gradually increases over time and T cells start to disengage from APCs before the T cell enters the S phase of the cell cycle. Using phase-specific cell cycle blockers, we confirmed that entering the G1 phase is required for T cell disengagement of APC. Our study suggests that T cells actively end interaction with APCs as soon as they are committed to cell division. This active disengagement may contribute to the efficiency of immune responses by making the APC available to other T cells and by initiating early migration to sites of infection. Our results also suggest a mechanism that links cell cycle control with the actin network driving T cell motility and migration.
Alveolar macrophages regulate the susceptibility of alveolar epithelial cells to influenza A virus infection and the outcome of infection. (P4241)
Authors: Cardani Amber , Kim Taeg , Braciale Thomas
Published: January 2023

Influenza A virus (IAV) is a major human pathogen. Patients with lethal primary influenza pneumonia exhibit severe diffuse lung inflammation, respiratory tract injury, and diffuse alveolar damage. While IAV is cytolytic, the host immune response has recently been appreciated to also be a significant cause of lung injury during IAV infections. To explore the role of alveolar macrophages (AMs) in virus clearance and lung injury/repair during IAV infection, we developed a novel mutant mouse strain with a selective deficiency in the AM compartment. We found that AM deficient mice die following a sub-lethal (in control mice) IAV infection in spite of a normal immune response and virus clearance. However, these mice undergo a rapid decline in O2 saturation at the onset of the adaptive immune response (5-6 dpi) and also demonstrate an increased susceptibility of alveolar type I epithelial cells to IAV infection. Destruction of this critical cell type necessary for oxygen exchange by virus or the adaptive response likely accounts for their loss of lung function and death. Thus AMs may have a critical function in controlling the susceptibility of alveolar epithelial cells to IAV infection and therefore the severity of infection.
Primed effector macrophages mediate rapid worm expulsion after N. brasiliensis infection (P4247)
Authors: Chen Fei , Wu Wenhui , Millman Ariel , Patel Nirav , Gause William
Published: January 2023

The innate immune cell populations that mediate macroparasite expulsion remain largely undefined. We examined the role of innate cells in the immune response to the nematode parasite, Nippostrongylus brasiliensis, hypothesizing that they may mediate the markedly accelerated CD4-independent worm expulsion occurring after secondary inoculation. By three days after secondary inoculation, immunofluorescence microscopy demonstrated a localized infiltrate of macrophages and eosinophils surrounding the L3 in the lung, and lung L3 showed reduced ATP, indicating impaired metabolism. Transfer of macrophages from N. brasiliensis primed WT mice to naïve recipients resulted in accelerated expulsion after primary inoculation, similar to WT mice given a secondary inoculation; this protective effect was blocked when primed macrophages were transferred from IL-4Rα-/- donor mice. Culture of L3 with sorted macrophages from primed WT, but not IL-4Rα-/-, mice resulted in macrophage adhesion to L3, decreased L3 metabolism and increased L3 mortality. Neutrophil (Ly6G+) depletion during priming blocked protective immunity after secondary inoculation and depletion of neutrophils in primed mice blocked protective effects of transferred macrophages in recipient mice and inhibited their in vitro binding to L3. These data indicate that neutrophils in the context of a type 2 cytokine environment prime effector macrophages that can directly bind parasites and mediate worm damage and expulsion.
Radiation-induced depletion of cutaneous dendritic cells: Functional consequences of a breakdown in the cutaneous immunological barrier (P4259)
Authors: Gerber Scott , Cummings Ryan , Ryan Julie , Haidaris Constantine , Lord Edith
Published: January 2023

The United States continues to be a prime target for attack by terrorist organizations in which nuclear detonation or dispersal of radiological material are legitimate threats. These types of attacks could result in devastating consequences to a multitude of individuals in the form of radiation injury to particular organ systems. One of these organs, the cutaneous system, which provides the first line of defense against invading pathogens, is particularly sensitive to ionizing radiation (IR) leaving the host vulnerable to cutaneous pathogen invasion. Our laboratory demonstrated that exposure to IR depleted murine cutaneous dendritic cells (cDCs); a network of cells responsible for forming a cutaneous immunological barrier that is critical in protecting the host against microorganism infection. This IR-induced depletion was caused by a migration of cDCs out of the skin responding to abnormal upregulation of cDC migratory factors, CCR7 and CCL21. As a result, this breakdown in the immunological barrier left the host susceptible to a cutaneous Candida albicans challenge where the pathogen was not contained locally but rather disseminated systemically to the kidneys. Interestingly, a subcutaneous treatment with IL-12 prevented the IR-induced depletion of cDCs offering a potential radiation countermeasure to preserve the cutaneous immunological barrier and perhaps prevent the lethal dissemination of opportunistic pathogens following radiation exposure.
Pregnane X receptor regulates the innate inflammatory response against intracellular bacterial infection (P4209)
Authors: Qiu Zhijuan , Mukherjee Subhajit , Schouten Jonathan , Acharya Nandini , Maher Leigh , Salazar Juan , Cervantes Jorge , Mani Sridhar , Khanna Kamal
Published: January 2023

Pregnane X receptor (PXR) is a member of the nuclear receptor family and is primarily expressed in stromal cells of liver and intestine. It’s well established that PXR regulates the metabolism of xenobiotics. Here we demonstrate that PXR signaling specifically in hematopoietic cells is also essential for regulating innate antimicrobial immune responses. Compared to wild type mice, Pxr-/- animals exhibited significantly higher inflammatory response to intracellular bacteria Listeria Monocytogenes (LM) infection. However, strikingly, despite the more robust innate immune response, Pxr-/- mice were unable to control LM infection. Our data demonstrated that the inflammatory monocyte response was significantly reduced in Pxr-/- mice, and these monocytes produced elevated amounts of TNF-α and reactive oxygen species that are known to induce cell death. We propose that the dysregulated inflammatory response in Pxr-/- monocytes results in their increased cell death and thus elevated bacterial burden. Surprisingly, increased activation of the TLR pathway in Pxr-/- mice was the singular determinant of the susceptibility since Pxr-/-Tlr4-/- mice were efficient in clearing LM. In conclusion, our study for the first time defines a novel role of PXR as a critical negative regulator of the innate immune response. Our results also illustrate the need for exquisite fine tuning and balance of the innate inflammatory pathways to ensure the clearance of an intracellular pathogen.
C-reactive protein exacerbates renal ischemia reperfusion injury (P4021)
Authors: Pegues Melissa , McCrory Mark , Zarjou Abolfazl , Szalai Alexander
Published: January 2023

Renal ischemia-reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) after kidney transplantation, hypotension, and cardiovascular surgery. Mortality from AKI is high due to incomplete knowledge of the pathogenesis of IRI and the lack of an effective therapy. Inflammation accompanies IRI and increases the blood level of C-reactive protein (CRP), a biomarker of worsened outcomes in AKI. To test if CRP is causal in AKI we subjected wild type mice (WT), human CRP transgenic mice (CRPtg) and CRP deficient mice (CRP-/-) to bilateral renal IRI (both pedicles clamped for 30 min at 37°C then reperfused for 24 h). Serum human CRP level was increased ~six-fold after IRI in CRPtg (11.58 ± 1.40 µg/ml at baseline versus 65.75 ± 7.90 µg/ml at 24 h) but was not elevated by sham surgery wherein kidneys were manipulated but not clamped. Compared to WT, serum creatinine and urine albumin was increased after IRI in CRPtg mice. Histological evidence of kidney damage was greater in CRPtg than in WT, whereas CRP-/- kidneys were protected. RT-PCR analysis of mRNA isolated from whole kidneys subjected to IRI revealed that expression of FcγRs increased in CRP-/- kidneys and markers of macrophage activation indicated a stronger classically activated (M1) macrophage response in CRPtg kidneys. Our finding that CRP exacerbates IRI induced AKI by shifting the balance of beneficial and detrimental FcγRs and macrophages, makes CRP a promising therapeutic target for the treatment of AKI.
The p110δ isoform of PI3K modulates TLR4 signalling and is crucial for survival from endotoxin and Escherichia coli infection (P4023)
Authors: Okeke Emeka , Okwor Ifeoma , Jia Ping , Uzonna Jude
Published: January 2023

Sepsis, a systemic immune response to severe bacterial infection, is a leading cause of death in critical care medicine caused by dysregulated immune response to bacteria endotoxin LPS which is recognised by TLR4. The p110δ isoform of PI3K is expressed mostly on leukocytes and has been shown to be important in leukocyte recruitment to sites of inflammation and infection. We report that pharmacological or genetic inhibition of p110δ signalling led to increased expression of TLR4 upon LPS stimulation an effect which was not observed for p110α and p110β. Differential cell counting showed higher numbers of inflammatory cells in the peritoneum of p110δ knock-in [p110δ(D910A)] mice compared to wild type mice. Macrophages from p110δ(D910A) mice induced higher proinflammatory cytokines compared to WT mice in response to LPS. In addition, absence of p110δ activity led to mortality in an otherwise non lethal dose of LPS accompanied by exaggerated production of proinflammatory cytokines. We authenticate our findings by showing that p110δ(D910A) mice are unable to recover from a non lethal dose of Escherichia coli infection. Thus modulation of p110δ signalling is a potential therapeutic target in acute inflammation and sepsis