bluuebunny/crossref_metadata_2025 huggingface dataset:
Macrophage migration inhibitory factor has a role in controlling colorectal cancer (TUM4P.918)
Authors: Rodriguez-Sosa Miriam , Pacheco-Fernández Thalia , Juárez-Avelar Imelda , Chirino Yolanda , León-Cabrera Sonia , Perez-Plasencia Carlos , Avila-Moreno Federico , Terrazas Luis I.
Published: January 2023

Chronic Inflammation has been related with cancer development by counteracting and unbalancing the immune response. The inflammatory cytokine macrophage migration inhibitory factor (MIF) is overexpressed in many carcinogenic tumors, stimulates angiogenesis and metastasis promoting proliferation and survival in malignant cells. In the inflammatory disease ulcerative colitis, MIF is early expressed and favors the inflammatory cytokine production like TNF-α and IL-1β. As intestinal chronic inflammation may proceed to colorectal cancer, we are interested in determining the role of MIF on development and pathology on colorectal cancer. To elucidate this point we used MIF-KO and WT mice, both with BALB/c genetic background, to develop a colorectal cancer model of Dextran Sodium Sulfate and Azoxymethan. As expected, MIF-KO mice showed low levels of serum inflammatory cytokines TNF-α, IL-1β and IFN-γ compare to WT mice. Strikingly, we found significantly more tumors and in a higher development stage on MIF-KO mice (24.4+/-2) compared to WT mice (12.6+/-2). Higer numbers of CD4+ cells were detected in intestinal epithelial cells on WT mice compared to MIF-KO mice. Therefore, although MIF has been recognized as a promoter of inflammation, which could favor the development of cancer, our results suggest that MIF is involved in controlling the development of colorectal cancer, possibly by its chemokine properties.
Tumor-derived alpha-fetoprotein impairs the differentiation and T cell stimulatory activity of human dendritic cells (TUM4P.929)
Authors: Pardee Angela , Shi Jian , Butterfield Lisa
Published: January 2023

Alpha-fetoprotein (AFP) is a well-established serum biomarker used in the diagnosis of several epithelial cancers and abnormalities of prenatal development. It has been proposed that AFP, an oncofetal antigen that is re-expressed particularly in hepatocellular carcinomas (HCC), plays an immunoregulatory role. In this study, human peripheral blood monocytes were cultured in the presence of cord blood-derived normal AFP (nAFP) or HCC tumor-derived AFP (tAFP). We unexpectedly observed that low doses of tAFP, but not nAFP, interferes with DC differentiation. These tAFP-conditioned DC expressed diminished levels of DC maturation markers, retained a monocyte-like morphology, and exhibited limited production of inflammatory mediators. Moreover, the ability of tAFP-conditioned DC to induce a robust T cell proliferative response to both alloantigen and cognate antigen was markedly compromised. Further analysis revealed that low molecular weight species that co-purify with AFP must be present in order for tAFP-mediated DC dysfunction to occur. Notably, in HCC patients exhibiting high levels of serum AFP, we have observed a lower ratio of myeloid-to-plasmacytoid circulating DC in comparison to AFPlow patients, suggesting that elevated levels of tAFP in cancer patients impair DC differentiation and function on multiple levels. Therefore, novel therapeutic approaches that antagonize tAFP regulatory circuits will be critical to improve clinical outcomes.
IL-17 regulates Toll-like receptor-mediated activation of dendritic cells (TUM4P.905)
Authors: Terada Satoshi , Wakita Daiko , Sumida Kentaro , Kaneumi Shun , Masuko Kazutaka , Ohtake Junya , Kishikawa Takuto , Ohno Yousuke , Kita Toshiyuki , Kitamura Hidemitsu
Published: January 2023

We have proposed that IL-17 produced by γδ T cells promoted angiogenesis and subsequent tumor progression in tumor microenvironment. However, the precise effect of IL-17 on immune system was remained unclear. Since dendritic cells (DCs) play a crucial role in immune regulation, we investigated the impact of IL-17 on DC function. Bone marrow-derived DCs were treated with IL-17 for 16 h and then stimulated with various toll-like receptor (TLR) ligands. IL-17 treatment significantly reduced IL-12, TNF-α, or IL-6 production by the DCs in a dose dependent manner. Interestingly, we found that IL-12 production was most clearly decreased compared to the other cytokines. In addition, IL-17 markedly suppressed the upregulation of MHC class II, CD40, and CD86 expressions on DCs after the stimulation. To evaluate effects of IL-17 on T cell-stimulating activity, IL-17-treated DCs were co-cultured with OVA antigen-specific T cells in the presence of OVA peptides or OVA proteins. We confirmed that IFN-γ production and T cell proliferation were suppressed by the IL-17-treatment. To determine effects of IL-17 on in vivo DC function, we established a novel tumor-bearing model using IL-17-producing cancer cells. As a result, expression levels of MHC class II and costimulatoly molecules on DCs were reduced in this model. These findings clearly indicated that IL-17 would inhibit activation of DCs including cytokine production and T cell activation in tumor microenvironment.
Attenuated listeria monocytogenes reprograms M2-polarized tumor-associated macrophages in ovarian cancer leading to iNOS-mediated tumor cell lysis (TUM4P.908)
Authors: Lizotte Patrick , Baird Jason , Stevens Cynthia , Lauer Peter , Green William , Brockstedt Dirk , Fiering Steven
Published: January 2023

A principal mechanism by which tumors evade immune-mediated elimination is through immunosuppression. Previous approaches to tumor immunotherapy have focused on modifying the immunosuppressive environment with immune checkpoint inhibitors, cytokine therapy, and other modalities with the intent to generate T-cell based anti-tumor immunity. We hypothesized that transformation of the suppressive ovarian cancer microenvironment could be achieved by introduction of the attenuated ΔactA/ΔinlB strain of Listeria monocytogenes. ΔactA/ΔinlB introduced into the microenvironment of the aggressive ID8-Defb29/Vegf-A murine ovarian carcinoma is preferentially phagocytosed by tumor-associated macrophages (TAMs) and reprograms that population from one of suppression to immunostimulation. TAMs in the peritoneum upregulated their co-stimulatory molecules CD80 and CD86, increased transcription of inflammatory cytokines, and downregulated transcription of suppressive effector molecules. Surprisingly, therapeutic benefit was not mediated by T- or NK-cell activity. ΔactA/ΔinlB-induced repolarization of TAMs activated direct tumor cell lysis via Nos2 production of nitric oxide. Modulation of the immunosuppressive nature of the ID8-Defb29/Vegf-A microenvironment, specifically by reprogramming of the TAM suppressive population from M2 to M1 polarization, is critical for our observed immune-mediated survival benefit.
Broadly neutralizing hemagglutinin stalk-specific antibodies require FcγR interactions for protection against influenza virus in vivo (VAC6P.951)
Authors: DiLillo David , Tan Gene , Palese Peter , Ravetch Jeffrey
Published: January 2023

Neutralizing antibodies (Abs) against influenza viruses have traditionally been thought to provide protection exclusively through their variable region; the contributions of mechanisms conferred by the Fc domain remain controversial. We investigated the in vivo contributions of Fc interactions with their cognate receptors for a collection of neutralizing anti-influenza Abs. Whereas five broadly neutralizing monoclonal Abs (bNAbs) targeting the conserved stalk region of hemagglutinin (HA) required interactions between the Ab Fc and Fcγ-receptors for IgG (FcγR) to confer protection from lethal H1N1 challenge, three strain-specific monoclonal Abs (mAbs) against the variable head domain of HA were equally protective in the presence or absence of FcγR interactions. Although all Abs blocked infection, only anti-stalk bNAbs were able to mediate cytotoxicity of infected cells, which accounts for their FcγR dependence. Immune complexes generated with anti-HA stalk mAb efficiently interacted with FcγRs, but anti-HA head immune complexes did not. These results suggest that FcγR binding capacity by anti-HA Ab was dependent on the interaction of the cognate Fab with antigen. We exploited these disparate mechanisms of mAb-mediated protection to reengineer an anti-stalk bNAb to selectively enhance FcγR engagement to augment its protective activity. These findings reveal a previously uncharacterized property of bNAbs and guide an approach toward enhancing mAb-mediated anti-viral therapies.
Vaccine-mediated B cell responses to Staphylococcus aureus alpha-Hemolysin (Hla) and effect of different boosting intervals on functional antibodies (VAC7P.979)
Authors: Lofano Giuseppe , Fiorillo Maria Teresa , Bagnoli Fabio , Bertholet Sylvie
Published: January 2023

Primary exposure to an antigen generates, along with antibody-secreting cells (ASCs), a heterogeneous B-cell memory compartment that changes over time and that may respond in different ways when re-encountering the cognate antigen. The goal of this study is to characterize B-cell responses after priming and the effect of different boosting intervals on the differentiation and function of ASCs and memory B cells. We used an immunization strategy based on the Hla protein from S. aureus, adjuvanted with Alum, as it was previously reported to induce protective antibodies in several animal infection models. In this study we performed a time course analysis following one or two immunizations, two weeks apart. Mice immunized twice had increased magnitude and quality of the Hla-specific humoral responses compared to mice immunized once. Increasing the boosting interval from two weeks to 1 month or 4 months, Hla-specific humoral responses increased in term of total IgG, affinity, and Hla-neutralizing titers. Hla-specific ASCs and memory B cells persisted for 4 months after the last immunization. We recently set up a method to identify Hla-specific B cells by flow cytometry (FACS).In the future we intend to further characterize their phenotype and to extend these analyses to Hla-specific memory B cells induced by different adjuvant formulations. This work will provide new insights in the memory B-cell compartment that might benefit the design of new vaccination strategies.
PLGA nanoparticle-entrapped inactivated porcine reproductive and respiratory syndrome virus vaccine coadministered intranasally with a potent adjuvant elicits cross-protective immunity in the pig respiratory system (VAC7P.981)
Authors: Binjawadagi Basavaraj , Dwivedi Varun , Manickam Cordelia , Ouyang Kang , Torrelles Jordi , Gourapura Renukaradhya
Published: January 2023

Porcine reproductive and respiratory syndrome (PRRS) is caused by a highly mutagenic RNA virus, PRRS virus (PRRSV). Currently used vaccines have failed to prevent PRRS outbreaks induced losses of estimated one billion dollar annually to the US pig farmers. The PRRSV infects primarily the lung macrophages. Because of safety advantage, we attempted to strengthen the immunogenicity of killed PRRSV vaccine antigens (KAg) in the pig respiratory system. We entrapped the KAg in biodegradable PLGA nanoparticles (NP-KAg) and coadministered the NP-KAg with a potent mucosal adjuvant, whole cell lysate of Mycobacterium tuberculosis (M. tb WCL), twice intranasally to growing pigs and challenged with a heterologous virus. Our results indicated that formulation of NP-KAg and unentrapped soluble M. tb WCL significantly cleared detectable replicating infective PRRSV with a 10-fold reduction in viral RNA load in the lungs, associated with substantially reduced microscopic lung pathology. Immunologically, enhanced virus neutralizing antibody titers by high avidity antibodies and augmented populations of IFN-γ secreting CD4+ and CD8+ lymphocytes, and reduced secretion of TGFbeta was detected in the lungs. Currently, we are in the process of identifying PRRSV epitope-specific T-cell response and frequency of apoptotic/necrotic immune cells in the lungs of vaccinated pigs. In conclusion, our vaccine formulation elicited broadly cross-protective anti-PRRSV immunity in the pig respiratory system.
HLA restricted CD8+ T cell responses following vaccination with monovalent and tetravalent dengue live attenuated vaccines (VAC7P.983)
Authors: Weiskopf Daniela , Angelo Michael , Bangs Derek , Sidney John , de Silva Aruna , Lindow Janet , Durbin Anna , Whitehead Stephen , Kirkpatrick Beth , Sette Alessandro
Published: January 2023

The frequency of infection with any of the four dengue viruses (DENV 1-4) has increased dramatically in the last few decades, and the lack of a vaccine has led to significant morbidity and mortality worldwide. Correlates of protection from severe dengue disease and vaccine efficacy have not yet been established. A recent comprehensive analysis of T cell response against dengue virus suggested an HLA-linked protective role for CD8+ T cells, predominantly targeting the non-structural (NS) proteins. Here, we characterize for the first time CD8+ T cell responses after experimental vaccination and compare it to responses observed in natural infection with dengue virus (DENV). We have collected full blood donations from study participants receiving a dose of the monovalent or tetravalent live attenuated DENV vaccine (DLAV) developed by the NIH. CD8+ T cell responses in vaccinees were readily detectable with magnitude and breadth similar to natural dengue infection. Interestingly, while broad responses to all proteins have been observed after monovalent vaccination we have observed a dramatic focus of the T cell responses to the highly conserved proteins NS3 and NS5. Epitopes from these proteins are highly represented in a vast variety of field isolates and are able to elicit multifunctional T cell responses. Detailed knowledge of the T cell response may contribute to identify robust correlates of protection in natural immunity and vaccination against DENV.
Bivalent gp120/mAb complex vaccines to induce broadly reactive antibody response against HIV-1 (VAC7P.987)
Authors: Kumar Rajnish , Tuen Michael , Hioe Catarina
Published: January 2023

HIV-1 envelope gp120 is a key target for neutralizing antibodies (NAbs) against the virus. Our work focuses on improving immunogenicity of cross-reactive neutralizing epitopes on gp120 antigen. We demonstrated that immunogenicity of gp120 and especially its neutralizing V3 epitopes was enhanced when gp120 was administered with the anti-CD4 binding site monoclonal antibody (mAb) 654 as immune complex (IC) vaccine. The enhanced V3 immunogenicity correlated with greater V3 antigenicity and was mediated mainly by the Fab activity of mAb 654. Here, we further explored the use of gp120/654 to elicit V3 Ab responses with greater breadth. A bivalent IC vaccine made of two gp120 proteins with immunogenically distinct V3 loops, gp120JRFL and gp120LAI-N448Q (a LAI derivative), were compared with the individual ICs. Individually, these ICs elicited V3 Abs with complementary reactivity: gp120JRFL/654 induced V3 Abs reactive with many subtypes B and subtype C viruses but not LAI, whereas gp120LAI-N448/654 induced V3 Abs specific for LAI. Administration of the ICs as a mixture or sequentially did not expand the breadth of Ab response. However, injection of the ICs s.c. at different sites resulted in induction of Abs reactive to LAI and the other subtype B viruses. Neutralization was also detected against Tier I subtype B HIV-1 pseudoviruses. These results demonstrate the potential of multi-valent vaccines and the importance of the delivery methods to broaden Ab responses against HIV-1.
Immunogenicity of Clostridium difficult DNA-based vaccine in an aging mouse model (VAC7P.959)
Authors: Bernui Mariana , Baliban Scott , Jacobson Jeffrey , Sardesai Niranjan , Weiner David , Kutzler Michele
Published: January 2023

Clostridium difficile infection (CDI) is a major source of morbidity and mortality, costing our healthcare system ~ $1 billion/year. Over 90% of CDI-related deaths occur in individuals >85yrs. Protection against severe C. difficile-associated disease and recurrent CDI is mediated by host antibody responses against C. difficile toxins. Our goal is to determine the immunogenicity of a DNA based anti-toxin vaccine in an aging mouse model. Young (4 mo.) and old (>18mo.) mice were immunized with our optimized DNA vaccine encoding toxin A and toxin B receptor-binding domain of C. difficile (A-RBD; B-RBD). Day 12 after the first immunization the younger mice had significantly higher IgG titers against A-RBD than older mice (25 ug/ul vs. 5 ug/ul anti-toxin serum IgG). However 10 days post second immunization the both groups had comparable IgG titers (~500 ug/ul IgG) against A-RBD. Anti toxin B-RBD IgG responses in older mice remained lower when compared to young following the second immunization. This suggests our DNA vaccine platform can generate an antibody response against C. difficile toxin in older mice but defects in vaccine priming may be occurring leading to lower titers. Day 14 post second immunization, mice cellular responses were analyzed. Higher frequencies of CD4+/CD44+ (activated) T cells and toxin-specific CD4+ T cells secreting pro-inflammatory cytokines (IFN-gamma, IL2) and Th2 cytokines (IL4, IL10) were generated in older mice.
FcγRIIB limits protection against F. tularensis LVS challenge induced by inactivated F. tularensis (VAC7P.964)
Authors: Franz Brian , Li Ying , Bitsaktsis Constantine , Iglesias Bibiana , Pham Giang , Gosselin Edmund
Published: January 2023

We have previously demonstrated that intranasal (i.n.) immunization with inactivated Francisella tularensis (Ft) live vaccine strain (LVS) targeted to Fcγ receptors (FcγR) can induce partial protection against a lethal mucosal challenge with Ft SchuS4. This protection is antibody (Ab) and interferon gamma (IFNγ) dependent. FcγRIIB is the only inhibitory Fc receptor (FcR) in the FcγR family, and has been reported to regulate IgG production, which plays a significant role in protection against Ft LVS and Ft SchuS4 challenge. Thus, we sought to determine the impact of FcγRIIB on immune protection against Ft infection. We utilized inactivated Ft (iFt) as an immunogen. Naïve or iFt-immunized FcγRIIB knockout (KO) versus wildtype (WT) C57BL/6 mice were challenged with varying doses of Ft LVS. We observed no significant difference in survival between naïve KO versus WT mice or in basal levels of Ft-specific IgG or IgM. However, KO mice immunized with iFt showed significantly increased survival as compared to iFt-immunized WT mice. Furthermore, Ft-specific IgG and IgA production were elevated in the serum from iFt-immunized KO versus WT mice. IFNγ levels were also enhanced in iFt-immunized KO versus WT mice. In summary, our studies demonstrate that FcγRIIB influences the level of protection against Ft challenge generated by iFt immunization by limiting the production of Ft-specific Ab and IFNγ, which have been shown to play significant roles in protection against Ft infection.
DC-specific HMGB1 KO mice clear vaccinia viral infection more effectively (VIR5P.1018)
Authors: Wang Wenqian , Vernon Philip , LI Guanqiao , Billiar Timothy , Sampath Padmavathi , Stephen Thorne , Morelli Adrian , Liang Xiaoyan , Lotze Michael
Published: January 2023

High-mobility group box 1 (HMGB1), an evolutionarily ancient and abundant DNA-binding protein within the nucleus, acts as a Damage Associated Molecular Pattern (DAMP) molecule extracellularly to promote immunity. It also functions in the cytosol to promote autophagy, a critical mechanism in viral clearance. HMGB1 is required for dendritic cell (DC) maturation and chemotaxis but how HMGB1 regulates DC function particularly in viral clearance is unclear. We constructed mice with HMGB1-knockout (KO) DCs (DCH) by crossing HMGB1 floxed mice with CD11c Cre mice. Although KO DCs differentiate normally from the bone marrow, they have diminished lymphocyte stimulatory capacity and polarize naive T cells towards Th17 and Treg phenotypes while wide type (WT) DCs induce IFNγ and IL-4 secretion at a ratio of 1:1 in allogeneic assays (p<.05). We infected DCH and WT mice with luc+ vaccinia and assayed viral infection over 9 days. Surprisingly, the virus is cleared more rapidly in the DCH cohort (p<.05). This is associated with increased T cells (p<.01) and CD11c+ cells (p<.05) within the spleen. We hypothesize that the proliferation of vaccinia depends on intact autophagy which may be impaired in KO DCs. We also find DCs release HMGB1 through exosomes, which are membrane vesicles enriched in MHC molecules and responsible for virus transmission. These results suggest that HMGB1 is not only critical for function but also contributes to the persistance of vaccinia virus in this murine model.
Increased pathology in herpes stromal keratitis results from differences in local, and not systemic, immune responses (VIR5P.1028)
Authors: Sullivan Brooke , Rowe Alexander , Hendricks Robert
Published: January 2023

Harboring latent herpes simplex virus type 1 (HSV-1) in the trigeminal ganglion (TG) and periodic shedding of virus at the corneal surface can be asymptomatic or can trigger an immunopathological response called herpes stromal keratitis that leads to progressive scarring and visual impairment. Our goal is to understand the events that differentiate an asymptomatic from a pathological HSV-1 infection. To this end we infected mice with two different strains of HSV-1: RE that causes progressive corneal opacity, and KOS that induces no overt corneal pathology. The kinetics of viral clearance and the latent viral load in the TG at 30 days post-infection (dpi) were similar following corneal infection with the two HSV-1 strains. However, the pathogenic RE strain induced a stronger CD4 and CD8 T cell response in the draining lymph nodes and TG at 8 dpi that persisted in the TG through 30 dpi. Interestingly, bilateral infections with RE in one eye and KOS in the other resulted in pathology only in the RE infected eye, suggesting that the increased immunopathology caused by the RE strain reflects local differences in the cornea or ipsilateral TG, and not differences in the systemic immune response. We are currently testing the hypothesis that greater viral gene expression by the RE strain during latency results in a larger T cell infiltrate in both the cornea and the TG.
Regulation of lupus-antigen specific B cells by follicular dendritic cells (BA12P.113)
Authors: Das Abhishek , Dwivedi Nishant , Chatterjee Priyadarshini , Carroll Michael
Published: January 2023

Humoral responses are central to many autoimmune diseases such as systemic lupus erythematosus (SLE). Yet little is known about the cells and factors that are vital for maturation and sustenance of autoreactive B cells. In this study, we used a B cell receptor (specific for RNP complexes) knock-in strain 564Igi to demonstrate an important role of follicular dendritic cells (FDCs) in loss of tolerance of autoreactive B cells. Breeding the lupus-prone strain to a C4-deficient background resulted in escape of tolerance of auto-reactive idiotype+ (Id+) B cells. Total ablation or in vivo de-differentiation of FDCs in the C4-/- 564Igi mice resulted in a dramatic decrease in auto-reactive B cells and germinal centers suggesting their role in promoting loss of tolerance. Gene expression analysis of FDCs isolated from C4 -/- 564Igi mice show an increased expression of IFN-alpha (IFN-a) and downstream response genes. Treatment of the mice with anti-IFN-a receptor (IFNAR) resulted in restoration of tolerance. Blockade of myeloid cell IFNAR was identified as the critical pathway as mixed marrow chimeras of C4-/- 564Igi and IFNAR- deficient bone marrow into C4-/- recipients have a significantly lower frequency of auto-reactive B cells, which underlines the importance of IFN-a signaling in myeloid cells. Hence, we propose a model in which IFN-a secreted by activated FDCs act on dendritic cells, which in turn supports auto-reactive B cells by producing factors like IFN-a and BAFF.
Elucidating the role of TNF-like weak inducer of apoptosis (TWEAK) in the pathogenesis of cutaneous lupus (BA12P.114)
Authors: Doerner Jessica , Wen Jing , Xia Yumin , Friedman Adam , Michaelson Jennifer , Burkly Linda , Wu Lan , Putterman Chaim
Published: January 2023

Cutaneous manifestations are very common in the systemic autoimmune disease systemic lupus erythematosus (SLE). TWEAK is a soluble cytokine member of the TNF superfamily that binds to a sole receptor, Fn14. TWEAK/Fn14 signaling is involved in cell survival, apoptosis, cytokine production, and angiogenesis, and as such has been found to be important in both tissue repair and inflammatory diseases. Whether TWEAK is involved in autoimmune skin disease is not known. We found that lupus-prone MRL/lpr mice deficient for Fn14 (Fn14KO) had attenuated cutaneous disease as compared to Fn14WT mice. MRL/lpr Fn14KO mice had significantly less epidermal acanthosis and follicular plugging, and there was decreased infiltration of T cells and macrophages as compared to Fn14WT mice. In vitro, TWEAK treatment of murine keratinocytes stimulated the secretion of RANTES (via Fn14), and promoted apoptosis. Parthenolide decreased production of RANTES, indicating that this effect of TWEAK is mediated via NF-KB. Ultraviolet light, specifically UVB, is recognized as a potent trigger of cutaneous lupus. We found that TWEAK co-treatment exacerbates UVB light- induced keratinocyte apoptosis and increases the amount of RANTES secreted, when compared to cells treated with UVB alone. These synergistic effects of TWEAK+UVB on keratinocytes were likely due to upregulation of Fn14 expression by UVB. Our data strongly implicate TWEAK/Fn14 signaling in the pathogenesis of the cutaneous manifestations of lupus.
Reciprocal interaction between NK cells and DCs regulates the Th17 response by controlling the innate IFN-γ/IL-27 axis (BA13P.124)
Authors: Chong Wai Po , Chen Jun , Silver Phyllis , Caspi Rachel
Published: January 2023

IFN-γ is a pathogenic cytokine involved in inflammation. Paradoxically, its deficiency exacerbates experimental autoimmune encephalomyelitis, uveitis and arthritis. We previously showed that it is innate IFN-γ that is protective, whereas adaptive IFN-γ is pathogenic. Here we demonstrate by immunological and imaging methods, using IFN-γ-deficient mice repleted with IFN-γ-sufficient NK cells, that innate production of IFN-γ from NK cells is necessary and sufficient to limit autoimmunity and to reduce the number of IL-17A and GM-CSF producing effector T cells in the eye. Mechanistic studies demonstrated that, following immunization for uveitis, DC recruited IFN-γ-producing NK cells to the draining lymph node (DLN) and interacted with them in a CXCR3-dependent fashion. During the interaction, IFN-γ from NK cells induced DCs to produce IL-27, which in turn enhanced IFN-γ production by NK cells, forming a positive feedback loop. The NK-DC-dependent IL-27 inhibited development of the adaptive pathogenic IL-17 response and induced Tr1-like cells, which upon adoptive transfer ameliorated disease in an IL-10-dependent manner. Our data reveal that an early NK-DC interaction controls the adaptive Th17 response and limits tissue-specific autoimmunity through an innate IFN-γ/IL-27 axis.
Autoimmunity in interferon-gamma AU-rich element deleted-mice is associated with loss of marginal zone B cells and macrophages. (BA15P.217)
Authors: Hodge Deborah , Berthet Cyril , Coppola Vincenzo , Kastenmüller Wolfgang , Buschman Matthew , Shirota Hidekazu , Schaughency Paul , Anver Miriam , Reynolds Della , Sanford Michael , Kaldis Philipp , Young Howard
Published: January 2023

We generated a mouse model with a 162 nt AU-rich element (ARE) region deletion in the 3’ untranslated region (3’UTR) of the interferon (IFN)-gamma gene that results in chronic circulating serum IFN-gamma levels. Mice homozygous for the ARE deletion present both serologic and cellular abnormalities typical of patients with systemic lupus erythematosus (SLE). Marginal zone B (MZB) cells and marginal zone macrophages (MZMs) are absent in ARE-deleted (ARE-Del)-/- mice. ARE-Del+/- mice retain both MZB cells and MZMs and develop no or mild autoimmunity. However, low dose clodronate treatment in ARE-Del+/- mice specifically eliminates MZMs and promotes anti-DNA antibody development and glomerulonephritis. Our findings demonstrate the consequences of a chronic IFN-gamma milieu on MZB cell loss and MZM function in autoimmunity. Furthermore, similarities between disease states in ARE-Del-/- mice and SLE patients suggest that IFN-gamma may not only be a product of SLE but may be critical for disease onset and progression.
Sustained PI3K activation destabilized tTreg (BA15P.227)
Authors: Zhou Xuyu
Published: January 2023

Regulatory T cells (Treg) play a central role in immune homeostasis, whose dysfunction has been implicated in autoimmune disease pathogenesis. Two subsets Tregs have been defined in vivo: thymus derived tTregs and peripherally derived pTreg. The plasticity of Treg, especially the thymically derived tTreg remains considerable controversial. Since the conserved non-coding sequence 1(CNS1) of Foxp3 gene is only critical for the generation of induced pTreg cells, we generated a new BAC-transgenic mouse expressing a thy1.1 2A Cre fusion protein driven from the CNS1 deleted Foxp3 promoter (del CNS1 Foxp3-thy1.1-Cre). Characterization of these mice demonstrated the thy1.1 2A Cre fusion protein expressed exclusively in the Nrp1+Foxp3+ peripheral tTreg subset. Moreover, thy1.1 expression in the thymus was delayed compare with endogenous Foxp3. Stability of thy1.1+ Treg was tested by breeding the thy1.1 2A Cre Tg mouse line to a conditional YFP knock-in reporter mouse, termed ROSA26-stop-YFP. A subset range from 2% to 8% Cre mapped cells spontaneously lost Foxp3 expression in healthy animals, thus even the late developed tTreg cells have some extent of plasticity. Final, we found sustained PI3K activation by breeding with a conditional active PI3K Rosa26 knockin mouse, can significant destabilized late developed tTreg and generated pathogenic exFoxp3 cells in the homeostatic condition.
Development of alpha4beta1+MBP-speific T cells in the GALT (BA15P.225)
Authors: Ito Naoko , Yadav Sudhir , Mindur John , Ito Kouichi
Published: January 2023

Multiple Sclerosis (MS) is a CNS autoimmune disease caused by activated myelin-specific T cells. To examine how Myelin Basic Protein (MBP)-specific T cells spontaneously differentiate into encephalitogenic T cells, we created transgenic mice that express MS-associated HLA-DR, DRB5*0101, and MBP-specific TCR isolated from an MS patient. We found that the MBP-specific T cells differentiated into GM-CSF+ Th1 and Th17 cells in the thymus spontaneously and migrate into the periphery including mesenteric lymph nodes and the gut-associated lymphoid tissue (GALT). Interestingly, we found that alpha4beta1 integrin was up-regulated in the GALT of spontaneous EAE mice although up-regulation of alpha4beta1 was not observed in mice that did not develop spontaneous EAE. Furthermore, reduction of gut commensal bacteria with antibiotics reduced the development of spontaneous EAE. This data suggest that commensal bacteria-mediated up-regulation of alpha4/beta1 integrin on the MBP-specific T cells in the GALT may be associated with the development of spontaneous EAE.
Exacerbated tissue homing of neutrophils during sepsis and TLR2-induced cytokine production are regulated by integrin α3β1. (CAM5P.230)
Authors: Lerman Yelena , Sarangi Pranita , Hyun Young-Min , Lim Kihong , Falkner Kathleen , Pietropaoli Anthony , Kim Minsoo
Published: January 2023

Neutrophil migration to sites of infection is vital for pathogen clearance, but massive neutrophil infiltration during systemic inflammation leads to tissue injury and host mortality. While β2 integrins are well known to mediate neutrophil firm adhesion and crawling, the role of β1 integrins in vascular extravasation has been less explored. In this study, we evaluated the surface expression kinetics of β1 and β3 integrin heterodimers on neutrophils during sepsis using mouse models (CLP and endotoxemia) and human samples. We show that only integrin α3β1 becomes significantly upregulated. Moreover, human subjects with sepsis, but not SIRS (systemic inflammation w/o infection) upregulated α3β1. The increased expression of α3β1 on neutrophils correlated with their activation state. Administration of synthetic peptide (LXY2), which binds at the ligand binding site on α3β1, and conditional gene deletion of the integrin in granulocytes significantly reduced the number of extravasated neutrophils and improved mouse survival. Host bacterial clearance remained unaffected. Interestingly, LXY2 binding to α3β1 on neutrophils was associated with a reduction in TLR2-, but not TLR4-induced IL-10 secretion in vitro. Previous studies suggest a role for IL-10 as a regulator of the transition from mild sepsis to irreversible septic shock. Thus, integrin α3β1 upregulation promotes enhanced neutrophil migration and, in turn, could regulate sepsis progression by modulating neutrophil IL-10 release.
CD13 is a novel regulator of TLR4 endocytosis in dendritic cells (CAM5P.241)
Authors: Ghosh Mallika , Subramani Jaganathan , Rahman Mamunur , Shapiro Linda
Published: January 2023

Toll like receptors (TLRs) recognize antigens produced by infection or injury to induce innate immunity via cell surface or endocytic pathways. TLR4 sequentially triggers both pathways, leading to TLR4 activation of the pro-inflammatory NF-kB pathway from the cell surface followed by dynamin-mediated TLR4 endocytosis, activation of TRIF and production of anti-inflammatory IRF (Interferon Regulatory Factor)3-regulated genes. We found that myeloid cell surface peptidase CD13 regulates TLR4 signal transduction by controlling dynamin-dependent endocytosis. In response to LPS, CD13KO DCs showed enhanced activation of IRF3, increase in its target gene IFN-β and inducible nitric oxide activity in a dynamin dependent manner. Endogenous TLR4 ligands produced in response to ischemic injury provoked hyperactivation of pIRF3 and its target cytokines in CD13KO animals. CD13KO DCs internalize higher levels of cell-associated TLR4 ligands released by dying cells or the TLR4 ligand LPS, indicating that CD13 regulates inflammatory TLR4 responses in vivo by modulating DC antigen uptake. Our results further suggest that CD13 and the LPS binding protein, CD14, may potentially act as opposing regulators of TLR4 endocytosis in inflammation. We propose that in response to injury or infection, CD13 serves to balance the immune response by modulating TLR4 induced pro- and anti-inflammatory cytokine profiles and maintain inflammatory equilibrium that is critical to proper wound healing.
Lymphocytes rapidly infiltrate into the brain following cardiac arrest and cardiopulmonary resuscitation (CA/CPR) (CAM5P.246)
Authors: Carter Jessica , Deng Guiying , Wagner David , Herson Paco
Published: January 2023

Isolated ischemic episodes, such as occurs with stroke, result in long-term neural damage. It is known that lymphocytes along with other immune cells infiltrate brain tissue promoting that process. During cardiac arrest, global ischemia then reperfusion occurs. Although inflammatory mechanisms have been linked to neuronal injury following global cerebral ischemia, the presence of infiltrating immune cells remains understudied. We examined the brains of mice at different time points after global cerebral ischemia induced by cardiac arrest and cardiopulmonary resuscitation (CA/CPR) and characterized the influx of lymphocytes into the injured brain. We observed that CA/CPR caused a rapid influx of lymphocytes within 3 hours of resuscitation that was maintained for the duration of our experiments. The large majority of infiltrating lymphocytes were CD4+ T cells that expressed the Th1 characteristic cytokines TNFα or INFγ. Of the CD4+ infiltrating cells, a large portion of them were also CD40+ (Th40). Brain injury models attribute much of the neural damage to microglia and subsequent oxidative stress. We found that the CA/CPR injury model was largely dependent upon T cells, and the lack of functional T cells in TCRα knockout mice significantly reduced neuronal injury. This data indicate that studies investigating the neuro-immune response after global cerebral ischemia should consider the role of infiltrating T cells in orchestrating the acute and sustained immune response.
Changes in plasma angiopoietin-like protein 2 levels and macrophage migration associated with hibernation in thirteen-lined ground squirrels (CAM5P.232)
Authors: Taylor Bernadette , Hanson Erika , Shaheen Bayan , Cooper Scott
Published: January 2023

Thirteen-lined ground squirrels in the northern United States hibernate from around late September until March. In late summer they gain a substantial amount of fat that is gradually expended as fuel over the course of hibernation. Immune system changes during hibernation include a 90% drop in circulating leukocytes and a reduction in immune responsiveness. Adipose tissue in obese humans is associated with changes in leukocyte migration including macrophage infiltration. This study explored an adipose-associated inflammatory protein, angiopoeitin-like protein 2 (ANGPTL2) as a potential mediator of leukocyte migration in hibernating ground squirrels. Plasma ANGPTL2 in eight ground squirrels was measured by quantitative Western blot at non-hibernating, entering hibernation, hibernating, interbout arousal and post arousal time points. ANGPTL2 levels were higher in hibernation than all other time points and were significantly higher in hibernation compared to interbout arousal or post arousal time points. Macrophages were detected in adipose tissue of hibernating ground squirrels using a ground squirrel macrophage specific monoclonal antibody in immunoperoxidase stained frozen sections. Macrophage-staining in the spleen appeared to be unusually dense in venous sinuses of hibernating compared to non-hibernating squirrels.
Super-hydrophilic nanostructured titanium surface improves implant osseointegration by mediating macrophage polarization (CAM5P.233)
Authors: Chen Lihua , Ma Qianli , Fang Liang , Wang Ying , Liu Rongrong , Zhang Yusi , Jin Boquan , Zhang Yumei
Published: January 2023

Aims: The purpose of this study was to fabricate a nanostructured surface on a titanium (Ti) implant that can regulate the macrophage inflammatory response and thereby improve the performance of the implant. Materials and Methods: Pure Ti samples were anodized and then subjected to UV irradiation to generate super-hydrophilic, nanostructured TiO2 surfaces. Their surface characteristics were analyzed, and osteointegration as well as the inflammatory response of modified implants were evaluated in vivo and in vitro. Results: Both nanostructured TiO2 surfaces showed super-hydrophilic and multi-tubular surface topography with diameters of 30 nm (NT5) and 80 nm (NT20). Although the in vitro osteogenic activity of bMSCs was similar on the two surfaces, the NT5 surface showed more bone formation, less inflammation, and a reduced CD68+ macrophage distribution in vivo compared to the NT20 and polished Ti surfaces. Further experiments revealed that the NT5 surfaces suppressed macrophage secretion of inflammatory cytokines and induced M2 polarization, which is associated with improved healing. By contrast, promoted M1 polarization could further impair bone regeneration. Conclusion: The results demonstrate the dominant role of macrophage-related inflammation in bone healing around implants and that nano-scale surface topography may have an immune-regulating effect that can support the success of implants.
IL-13 heteroreceptor and Th1 cells: the race for control of neonatal immunity (CCR5P.256)
Authors: Miller Mindy , Dhakal Mermagya , Chen Weirong , Ellis Jason , Khairallah Marie-Therese , Ukah Tobechukwu , Zaghouani Habib
Published: January 2023

Neonates are susceptible to microbial infections because they lack Th1 cells, the major effectors against microbes. Also, newborns are prone to allergic reactions perhaps due to their tendency to develop Th2 biased immunity. Fine analysis of primary neonatal immunity indicated a balance among Th1 and Th2 cells but the former display an unusual expression of IL-13 receptor alpha 1 (IL-13Rα1). Furthermore, this chain heterodimerizes with IL-4Rα and the resulting IL-4α/IL-13Rα1 provides a death receptor through which IL-4 of Th2 cells drives apoptosis of Th1 counterparts during secondary immunity. Herein, we observed that IL-4 and IL-13 signaling through the heteroreceptor shifts antigen-induced activation towards the mitochondrial apoptotic pathway by recruiting interferon-regulatory factor 1 (IRF-1). Further investigation of heteroreceptor signaling of neonatal Th1 cells should yield information useful for the development of pediatric vaccines and drugs against allergies.
IL-23 Maintains Tc17 CD8 T Cell Phenotype but not Commitment to IL-17 production (CCR6P.282)
Authors: Yen Hung-Rong , Lin Bo-Shiou , Huang Wan-Chun , Liao Cha-Hui , Yeh Yueh-Chiao , Liang Kai-Li
Published: January 2023

CD8 T cells that produce IL-17 is an unknown field with lots to explore. In our previous studies, we found a novel subset of IL-17 producing CD8 T cells persisted longer in a self-antigen autoimmune model versus conventional IFN-g producing Tc1 cells; furthermore, Tc17 can convert to an IFN-g producing phenotype in vivo. In the present study, IL-17 producing Tc17 cells were purified by the cytokine-secretion assay and re-stimulated with anti-CD3/CD28 monoclonal antibodies with exogenous IL-12 or IL-23 cytokine. We found that IL-23 maintains IL-17-producing Tc17 CD8 T cell phenotype. However, administration of IL-23 is not enough to maintain the Tc17 phenotype. Addition of other cytokines such as IL-12 repressed IL-17 production and gene expression. In summary, IL-23 can maintain the Tc17 phenotype, but cannot promote the commitment of Tc17 CD8 T cells to an IL-17-secreting lineage.
Analysis of the misregulation of RhoA and RhoH in autoimmune mice. (HUM7P.302)
Authors: Antoni Angelika , Ray Chelsea , Kohn Ryan , Andreyko Diana , Levine Jerrold
Published: January 2023

When cultured in the presence of apoptotic cells, cell bodies or lipids, RhoA is misregulated in macrophages from all 6 autoimmune mouse strains but none of the 7 normal strains tested. Misregulation of RhoA results in defective morphology, altered cytokine production, and increased adhesion. The same macrophages cultured without apoptotic cell lipids had properly regulated RhoA and behaved normally. Normal mouse macrophages cultured with the Rho inhibitor C3 toxin, had defective macrophage morphology and behavior. Therefore, we proposed that autoimmunity is due to an apoptotic cell recognition signaling defect and that normal macrophage function may be restored by a chemical reversal of the defect. Our new data demonstrate that lupus strain, MRL, macrophages cultured in the presence of C3 no longer had defects in morphology and function. The fact that a RhoA inhibitor restored RhoA function indicates that the misregulation in autoimmune macrophages involves a RhoA regulator also affected by C3 and not RhoA itself. RhoH is a known inhibitor of RhoA and it is only expressed in hematopoietic cells. Experiments comparing RhoH expression in BALB and MRL macrophages suggest at least four different sizes of RhoH protein are expressed in normal macrophages in response to lipid exposure and that lupus macrophages do not express the multiple forms of RhoH even in the presence of lipids. A lack of proper RhoH expression could be behind the autoimmune macrophage defect.
TCR-independent activation of T-cells through CD31 triggering as an etiology of cytokine production in juvenile idiopathic arthritis (JIA). (HUM7P.311)
Authors: Ferguson Ian , Griffin Patricia , Michel Joshua , Kietz Daniel , Rosenkranz Margalit , Kietz Daniel , de Vallejo Abbe
Published: January 2023

Pro-inflammatory cytokines in the blood and synovium are key actors in JIA pathology. We recently reported that JIA patients have an over abundance of prematurely senescent CD8+ T cells expressing CD31, an adhesion molecule expressed by granulocytes and endothelial cells. CD31 triggering alone is sufficient to activate such senescent T cells. This study examined whether cytokine profile in JIA may be derived from T cell activation through CD31 ligation independent of the TCR. Results showed a cytokine signature of JIA characterized by dominance of IL-6, IL-10 and TNFα. Notably, patients with oligoarticular JIA showed higher levels of IFNγ and IL-17 family cytokines than those with polyarticular JIA. All patients also had a novel subset of T cells deficient in CD4, CD8, and CD28, but expressing CD31. Cross-linking bioassays, using anti-CD31 or its ligand CD38, show high levels of induction of IL-2, IL-17, and IFNγ. This CD31-driven, TCR-independent cytokine production was also verified by using somatic T cell line mutants that lack TCR and TCR/CD3, respectively, but both are CD31+. The observed cytokine production was associated with the phosphorylation of the signaling substrates ZAP70, cAbl, and p65-NFΚB. TCR independent, CD31-driven induction of these cytokines suggests maladaptive T cell function in JIA. Further investigation on the relevance of IFNγ and IL-17 to disease activity, and the CD31 signaling pathway may allow for innovations in immunotherapy.
BTLA expression on CD4+ T cells negatively regulates the development of pulmonary fibrosis (HUM7P.319)
Authors: Casaos Joshua , Takahashi Stephenie , Blaine Kelly , Hrusch Cara , Noth Imre , Sperling Anne
Published: January 2023

The role of the adaptive immune response in idiopathic pulmonary fibrosis (IPF) has been an area of debate. Several studies have shown that patients with IPF had increased numbers of T cells in their lungs, suggesting that these cells may play a role in disease pathogenesis. We have found that decreased expression of an important co-inhibitory molecule, B- and T- lymphocyte attenuator (BTLA) on peripheral blood CD4+ T cells is correlated with IPF patients most likely to rapidly progress in their disease course. Based on these findings, the objective of our study was to determine the role of BTLA on CD4+ T cells in the development of pulmonary fibrosis by utilizing a mouse model of bleomycin-induced pulmonary fibrosis. We found that, similar to our patient studies, bleomycin treated WT mice express decreased levels of BTLA on CD4+ T cells compared to untreated WT mice. BTLA-/- mice exhibited significantly increased weight loss and pulmonary fibrosis compared to WT mice. However, CD4+ depletion in the BTLA-/- mice prior to bleomycin challenge protected these mice, as they lost less weight had decreased lung collagen deposition. These findings support a significant role for the loss of BTLA on CD4+ T cells in the pathogenesis of fibrotic disease progression.