bluuebunny/crossref_metadata_2025 huggingface dataset:
Pulmonary Th17 immunity is regulated by regenerating islet-derived III-gamma and the gut microbiome (MUC4P.826)
Authors: McAleer Jeremy , Nguyen Nikki , Chen Kong , Armentrout Rachel , Pociask Derek , Ricks David , Binnie Matthew , Hein Aaron , Yu Amy , Vikram Amit , Bibby Kyle , Sheppard Dean , Ouyang Wenjun , Hooper Lora , Kolls Jay
Published: January 2023

Antimicrobial proteins provide an innate immune barrier against pathogen invasion. Here we identify a function for the bactericidal lectin regenerating islet-derived III-gamma (RegIIIγ) in shaping CD4 T cell polarization. Gram-positive bacteria in the intestine induce RegIIIγ expression, which modulates the immune tone of the gut, resulting in decreased Th17-type immunity during pulmonary fungal infection. This was associated with RegIIIγ inhibiting intestinal colonization with segmented filamentous bacteria (SFB), a pro-inflammatory commensal that augments Th17 differentiation. Vancomycin drinking water inhibited IL-17 production in lungs of RegIIIγ-/- and Il22-/- mice, demonstrating that intestinal Gram-positive commensals contribute to systemic inflammation. Reconstituting Il22-/- mice with IL-22 decreased the SFB/Clostridium ratio, while gastrointestinal delivery of recombinant RegIIIγ decreased inflammatory gene expression in lung tissue and protected Il22-/- mice from weight-loss during Aspergillus fumigatus infection. Therefore, intestinal antimicrobial proteins influence the development of adaptive immunity by altering the balance of pro- and anti-inflammatory intestinal commensal species.
Persistence of cognate antigen dictates effector T cell localization, phenotype, and function during pulmonary tuberculosis (MUC5P.860)
Authors: Moguche Albanus , Larson Ryan , Shafiani Shahin , Clemons Corey , Urdahl Kevin
Published: January 2023

How Mycobacterium tuberculosis (Mtb) evades eradication from the lung despite an apparently robust Th1 response remains poorly understood. We addressed this question using MHCII tetramers to monitor Mtb-specific CD4 T cells in the mouse model. We found that the location, phenotype, and functional capacity of T cells specific for distinct Mtb epitopes differ, and depend upon the expression profile of the specific Mtb antigen recognized. Terminally differentiated Th1 cells (defined by their high expression of the transcription factor T-bet, the inhibitory receptor KLRG1, and an enhanced ability to produce IFN-γ) specific for Mtb ESAT-6, an antigen expressed throughout infection, were surprisingly found to reside in the lung-associated vasculature rather than the lung parenchyma. Those ESAT-6-specific effector T cells within the parenchyma had a reduced capacity to proliferate and produce IFN-γ. In contrast, T cells specific for Mtb Ag85B, an antigen with limited expression during chronic infection, were found to reside primarily within the lung parenchyma and to proliferate and produce IFN-γ robustly. Our results refute the long-held belief that Mtb persists despite an abundance of functional Mtb-specific Th1 cells within the lung, and suggest that only T cells that recognize Mtb antigens with limited expression during chronic infection retain their functional capacity. These findings have obvious implications for vaccine and immunotherapeutic approaches for tuberculosis.
Irgm1 regulates the pulmonary innate immune response (MUC5P.864)
Authors: Azzam Kathleen , Madenspacher Jennifer , Lai Lihua , Gowdy Kymberly , Clayton Natasha , Cunningham Willie , Janardhan Kyathanahalli , Taylor Gregory , Fessler Michael
Published: January 2023

Irgm1 is an IFN-inducible GTPase that mediates host resistance to a variety of intracellular pathogens, and also modulates the LPS response in cultured macrophages. Whether Irgm1 regulates innate immune lung responses and controls host defense against extracellular pathogens is undetermined. We hypothesized that Irgm1 would regulate host defense against extracellular bacteria that cause pneumonia. Compared to wildtype, naïve Irgm1-/- mice had elevated expression of cytokines and chemokines in the lung parenchyma and bronchoalveolar lavage fluid, consistent with steady state inflammation. Moreover, naïve Irgm1-/- lungs displayed abnormal bronchiolocentric lymphocytic infiltration. Compared to wildtype, Irgm1-/- mice had increased influx of PMNs into the airspace after exposure to LPS and S. pneumoniae, but not K. pneumoniae. Bronchoalveolar lavage fluid chemokines, cytokines, and protein concentration (marker of microvascular injury) were all higher in Irgm1-/- mice after LPS and S. pneumonia. Irgm1-/- mice had lower lung CFUs after infection with S. pneumoniae, indicating enhanced pathogen clearance. Irgm1-/- mice also had increased survival after lung infection with S. pneumoniae but not K. pneumonaie. In conclusion, Irgm1-/- mice have an enhanced lung response to LPS and S. pneumoniae, but not K. pneumonaie, implicating a selective role for Irgm1 in host defense. Whether these responses derive from the Irgm1-/- basal lung phenotype is yet to be determined.
Therapeutic efficacy of baicalin on experimental autoimmune encephalomyelitis through down-regulation of JAK-STAT and NF-κB signaling pathways (THER3P.876)
Authors: Zhang Guang-Xian , Zhang Yuan , Li Xing
Published: January 2023

Baicalin (Ba) is a bioactive flavonoid compound derived from the root of Scutellaria baicalensis. It is widely used for the treatment of various inflammatory diseases in traditional Chinese medicine. In this study, we demonstrated that Ba was efficacious in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Treatment with Ba led to amelioration of disease severity in myelin oligodendrocyte glycoprotein-induced EAE, which correlated with reduced spinal cord inflammation and demyelination. The underlying mechanism of Ba-induced effects involved prevention entry of immune cells into the CNS, inhibition of Th1 and Th17 cell differentiation through JAK/STAT and NfκB signaling pathways, and reduction of proinflammatory molecular and chemokine expression. Ba treatment completely abolished the pathogenic Th17 cells responses to MOG35-55 in an adoptive transfer EAE model, and the same treatment significantly inhibited the ability of MOG 35-55-reactive Th1 cells to induce EAE. This study provides new evidence that natural compounds, such as Ba, are of great value in the search for novel anti-inflammatory agents and therapeutic targets for autoimmune diseases.
Ursolic acid ameliorates experimental autoimmune encephalomyelitis by regulating Th1 and Th17 cell differentiation (THER3P.877)
Authors: Zhang Guang-Xian , Li Xing , Zhang Yuan
Published: January 2023

Ursolic acid (UA) is a plant-derived triterpene with potent anti-inflammatory and immunomodulatory activities. In this study we investigated the potential therapeutic effect of UA on the experimental autoimmune encephalomyelitis (EAE), an accepted experimental model of multiple sclerosis. Treatment with UA at initiation or after the disease onset reduced clinical severity and incidence of EAE, which was associated with a decrease in inflammatory infiltrates in spinal cord and in the subsequent demyelination. UA decreased the presence/activation of encephalitogenic Th1 and Th17 cells in both periphery and nervous system, accompanied by down-regulated various inflammatory cytokines and chemokines. The effect of UA was attributable to its selective inhibition of IFN-γ and IL-17 production in CD4+ T cells, mediated via alteration of the STAT pathway and the transcription factors T-bet and RORγt/ROR α. More important, UA has been found novel properties of directly inhibiting Th1 and Th17 cell differentiation in vitro, and was also effective in reducing ongoing paralysis induced by adoptive transfer of either pathogenic Th1 or Th17 cells. These findings indicate that UA exerts potent anti-inflammatory effects through selective modulation of pathogenic Th1 and Th17 cells by targeting critical signaling pathways.
A mechanistic understanding of the selective clearance of encephalitogenic T cells (THER3P.882)
Authors: McNally Jonathan , Elfers Eileen , Millin Scott , Terrell Catherine , Jordan Michael , Hildeman David , Katz Jonathan
Published: January 2023

Most therapeutic approaches to control multiple sclerosis (MS) target autoimmune effector cells via broad spectrum immunosuppression. In contrast to this approach we have recently described a therapeutic intervention for the MS model experimental autoimmune encephalomyelitis (EAE) that selectively eliminates encephalitogenic T cells, while sparing protective naive and memory T cell responses. Here we report the mechanistic pathway by which our selective therapy acts. The specificity of action that targets encephalitogenic T cell is through the action of the drug, etoposide, which acts by targeting the double stranded break repair mechanism associated with highly activated T cells. In combination with differential expression of Bcl-2 family member leads to the preferential deletion of effector T cells over memory and naive T cells clearance. Based on our understanding of etoposide-mediated clearance of encephalitogenic T cells we expanded our findings to include other genotoxic and non-genotoxic agents that synergize to afford more specific and less toxic means of controlling autoimmunity. This study further defines the mechanism for the selective clearance of self-reactive T cells by establishing a mechanistic understanding and rational approach to the use of agents that target autoreactive T cells in vivo and result in novel and enhanced therapy for autoimmune diseases.
Myeloid-conditioning using Toll-like receptor agonists to restore immune potency against melanoma. (TUM4P.926)
Authors: Foureau David , Carpenter Kendall , Amin Asim , Guo Fei , White Richard , Salo Jonathan
Published: January 2023

Suppressor cells of myeloid origin contribute to the immune imbalance observed in advanced melanoma. Toll-Like Receptor (TLR) signaling regulates myeloid differentiation and maturation. This study sought to determine the distribution of myeloid suppressor cell subsets in B16F10 melanoma-bearing mice, and modulate their immunosuppressive phenotype by employing a regimen of intracellular TLR agonist(s). Tolerogenic dendritic cells (tDC) and granulocytic myeloid-derived suppressor cells (gMDSC) are primarily observed in the periphery (spleen, PBMC, draining lymph node) of B16F10-bearing mice whereas M2 macrophages (M2) are noted to be intratumoral. Toll-Like Receptor-expression analysis by quantitative PCR showed TLR9 was ubiquitously expressed on tDC, gMDSC and M2, while TLR3 was restricted to tDC and M2. Myeloid conditioning regimens employing TLR3 and 9 agonists were tested in vitro on bone marrow-derived tDC (CD11c+, MHCII+, IL-10+) and M2 (F4/80+, CD206+, Arg1+). Combinations of TLR3+9 agonists or Type A+B TLR9 agonists decreased IL-10/Arg1 mRNA expression while triggered IL-12 expression. In vivo, subcutaneous injection of type A+B TLR9 agonists reduced circulating tDC and gMDSC prevalence by 67% and 82% respectively, intratumoral M2 dropped by 35%. In conclusion, we demonstrate combining type A+B TLR9 agonists promoted immune potency by reducing prevalence of peripheral tDC and gMDSC, as well as tumoral M2 in vivo, and triggering IL-12 production by tDC and M2 in vitro.
Tumor-derived exosomes differentially modulate the adenosine pathway in human resting vs activated regulatory T cells (Treg) (TUM4P.927)
Authors: Muller Laurent , Mitsuhashi Masato , Jackson Edwin , Whiteside Theresa
Published: January 2023

Tumor-derived exosomes (TEX) suppress functions of human immune cells, while up-regulating functions of others. Here, we show that TEX isolated from supernatants of human cultured tumor cells or plasma of cancer patients differentially modify gene and protein expression of CD39, CD73 and adenosine receptors (ADORs) in Treg, influencing production of suppressive nucleosides. Resting or pre-activated (anti-CD3/CD28 Abs) CD4+CD39+ Treg were co-incubated overnight with TEX. Cellular mRNA was analyzed by qRT-PCR for expression of CD39, CD73, A1R, A2aR, A2bR and A3R genes. Protein expression was studied by flow cytometry and western blots. Nucleoside production by Treg ± TEX in the presence of 20µM ATP was measured by mass spectrometry. In resting Treg + TEX, mRNA expression of CD39/CD73 and ADORs was reduced relative to no TEX baseline (>1 log) except for A1R mRNA expression which was elevated (> 3 logs). CD39 protein expression was up-regulated, suggesting enhanced translation and signaling via A1R. Treg + TEX + ATP produced inosine only (7900ng/mL) and no 5’-AMP or ADO. In activated Treg +TEX, CD39/CD73 mRNA expression and protein levels were elevated (p< 0.05) as was mRNA expression for all ADORs, especially A2aR, and ADO was produced. Thus, TEX differentially regulated gene expression and protein levels of the ADO pathway components in resting vs activated Treg. The TEX-induced shift from inosine to ADO production implies changes in Treg-mediated immunosuppression.
Novel pneumocystis antigen discovery and validation using RNA sequencing and proteomics (VAC7P.977)
Authors: Eddens Taylor , Elsegeiny Waleed , Zheng Mingquan , Cai Yang , Kolls Jay
Published: January 2023

Pneumocystis (PC) is a fungus that causes pneumonia in immunocompromised individuals and remains the most common serious opportunistic infection in individuals diagnosed with HIV. Furthermore, PC has re-emerged in the HIV-negative population, as patients on immunosuppressive therapies for transplantation or malignancy are increasingly susceptible to PC pneumonia. As the at-risk population increases, the discovery of conserved PC antigens could lead to the development of vaccines, enhanced diagnostic tests and monoclonal antibody therapies. To that end, we have used RNA sequencing of separated PC life forms and surface biotinylation of whole PC to identify differentially expressed surface antigens. Histone 2B (H2B) appears to be an extracellular antigen with increased expression in the cyst life form, while a GPI-anchored protein (GPI) is more abundant on the troph life form. We used DNA vaccination to assess the immunogenicity of these antigens followed by CD4 depletion and challenge with live PC. H2B vaccination resulted in a significant increase in PC-specific IgG. After PC challenge, H2B vaccinated mice had a significant half-log reduction in PC lung burden when compared to unvaccinated controls. Similarly, GPI vaccinated mice had an increase in PC- and GPI-specific IgG, while also having a half-log reduction in PC lung burden. Taken together, these results suggest that H2B and GPI are novel PC antigens with vaccine potential in need of further exploration.
Ablation of reactive oxygen species intensifies diabetogenic CD4 T cell effector responses in type 1 diabetes. (BA14P.210)
Authors: Padgett Lindsey , Tse Hubert
Published: January 2023

In Type 1 diabetes (T1D), infiltrating leukocytes secrete reactive oxygen species (ROS) and pro-inflammatory cytokines that collectively participate in β-cell destruction and enhance autoreactive T cell responses. Our lab previously demonstrated that superoxide-deficient Non-Obese Diabetic (NOD.Ncf1m1J) mice were T1D-resistant due to skewed T cell responses. To dissect the role of ROS on autoreactive T cells, we generated the NOD.BDC-2.5.Ncf1m1J mouse, possessing diabetogenic, superoxide-deficient CD4 T cells and hypothesized that like NOD.Ncf1m1J, dampening ROS would diminish autoreactive T cell responses. Surprisingly, IFN-γ was upregulated 2-fold by NOD.BDC-2.5.Ncf1m1J T cells compared to NOD.BDC-2.5 with polyclonal stimulation (p=0.0045). Transfer of activated NOD.BDC-2.5.Ncf1m1J T cells was more diabetogenic, as 63% of NOD.scid recipients (n=11, p=0.0662) by 9 days post transfer were hyperglycemic, in contrast to 17% of wild-type recipients (n=7). To mechanistically explain the exacerbated Th1 cytokine response and diabetogenicity, phosphorylation of lymphocyte-specific protein tyrosine kinase (P-Lck (Y505)) and linker for activation of T cells (P-LAT (Y191)), redox-sensitive T cell receptor (TCR) signaling molecules, was enhanced 2-fold by NOD.BDC-2.5.Ncf1m1J CD4 T cells compared to NOD.BDC-2.5. Future experiments will dissect the redox-sensitive cues that control TCR adaptor molecule activation, as unraveling these signals may pinpoint novel targets for T1D prevention.
The role of CdK5 in the activation and effector function of T cells in experimental autoimmune uveitis (BA14P.216)
Authors: Liu Qi , Kim Byung-Gyu , Lam Eric , Choi Sung Hee , Doller Catherine , Zhang Zili , Letterio John
Published: January 2023

The activation and survival of T cells are both essential for the development and progression of uveitis. Cyclin-dependent kinas 5 (Cdk5) is a unique member of Cdk family that forms a complex with p35 (CDK5R1) and modulates the function of many proteins through proline-directed serine threonine phosphorylation. Our previous study showed that Cdk5 expression and activity is induced during T cell activation, but it is unclear whether CdK5 is implicated in experimental autoimmune uveitis (EAU). In this study, we found that CD4+CD44+ T cells have a higher level of Cdk5 expression. Inhibition of CdK5 by a small molecule inhibitor (roscovitine) led to the decrease in the number of activated CD4+CD44+ T lymphocytes, which coincided with increased T cell apoptosis. We observed increased expression and activity of CdK5, p35 (and its cleaved product p25) in the eyes and lymphocytes of C57BL/6 mice induced to develop EAU by immunization with interphotoreceptor retinoid-binding protein peptide 1-20 (IRBP1-20). In addition, roscovitine significantly suppressed proliferation of and cytokine production in T cells from mice with the disease in vitro. These results demonstrate that CdK5 plays a critical role in T cell activation during the pathogenesis of uveitis and suggests that targeting CdK5 may be a novel therapeutic strategy for treating T cell-mediated uveitis.
Delineating the role of cardiac myosin-specific CD8 T cells in autoimmune myocarditis (BA15P.220)
Authors: Massilamany Chandirasegaran , Steffen David , Reddy Jay
Published: January 2023

Enteroviruses like coxsackievirus B3 are common suspects in patients with myocarditis/dilated cardiomyopathy. Previously, it was shown that autoreactive cytotoxic T lymphocytes, also called CD8 T cells, have a role in the mediation of myocarditis induced by coxsackievirus B3. But identification of their target antigens in cardiac tissues continued to be a challenge. We have discovered that CD8 T cells have the ability to induce myocarditis in A/J mice by recognizing cardiac myosin heavy chain-alpha (Myhc) as the target antigen. We have localized two peptides designated, trunk 4 and trunk 5 within Myhc that can stimulate CD8 T cells and CD4 T cells. Furthermore, expectedly, CD8 T cells recognizing the Myhc peptides produced predominantly, interferon-gamma and tumor necrosis factor-alpha, but not interleukin-17, and A/J mice immunized with the CD8 T cell epitopes, develop myocarditis by active immunization. It has been widely believed that Myhc-induced autoimmune myocarditis is a CD4 T cell-mediated disease. Our discovery creates a paradigm shift in the understanding of how cardiac damage can occur by multiple immune cells that recognize the same target antigens but through different mechanisms.
Modulating the tumor necrosis factor-α induced inflammatory response in human colonic epithelial cells by inhibiting NF-κB signaling (CCR5P.258)
Authors: Rivard James , James Amy , Wegner Greta , Reagan Kevin
Published: January 2023

In colonic epithelia, tumor necrosis factor alpha (TNF-α) secreted by inflammatory cells is a key mediator of inflammatory bowel disease. TNF-α is a well-characterized activator of the NF-κB signaling pathway in intestinal epithelial cells and is known to up-regulate the expression of a subset of chemokines and cytokines. In this study, we used antibody arrays to profile the relative changes in chemokines secreted upon activation of the human colon carcinoma cell line HT-29. Elevated levels of CXCL1, CXCL7, CXCL8, CXCL10 and CCL20 were observed in conditioned media after TNF-α treatment of HT-29 cells. To better understand the contributions of the classical NF-κB signaling pathway to TNF-α activation of HT-29 cells, we used an inhibitor with high selectivity for IKK2 over IKK1, TPCA-1. In addition, both classical and the alternative NF-κB signaling pathways were inhibited at a level downstream of IKK1 and IKK2 by using the NEDD8 activating enzyme (NAE)-inhibitor MLN4924. TPCA-1 and MLN4924 both attenuated TNF-α activated secretion of these chemokines in a dose dependent manner in HT-29 cells. Confirmation of these array data by quantitative ELISAs supports the use of arrays for analysis of inhibitor biomarkers.
PLZF supports the acquisition and maintenance of the Th17 phenotype in human cells (CCR5P.250)
Authors: Singh Satya , Zhang Howard , Tsang Hsinyi , Gardina Paul , Myers Timothy , Farber Joshua
Published: January 2023

All human Th17 cells express the chemokine receptor CCR6, making CCR6 a potential therapeutic target. We hypothesized that the variable levels of CCR6 expression on blood CD4+ T cells reflect a continuum of Th17 differentiation, and that regulators of Th17 differentiation could be identified by evaluating global changes in gene expression in cells with increasing levels of CCR6. We found that ZBTB16, which encodes the BTB-ZF transcription factor PLZF, was expressed at increasing levels from the CCR6- to CCR6bright subsets and was poorly expressed in Th1, Th2, and TFH cells. Knockdown of ZBTB16 mRNA down-regulated CCR6 and other Th17-associated genes such as RORC, IL17A, IL22, and IL23R in CCR6+ memory Th cells from blood and in naïve cells activated under Th17 conditions ex vivo. In naive Th cells from cord blood, ZBTB16 expression was limited to the CD161+, Th17 cell precursors. ZBTB16 was not expressed in mouse Th17 cells and Th17 cells could be made from luxoid mice. Using ChIP for modified histones, p300, and Pol II to analyze CCR6, we identified regulatory regions at -10 and -14 kb from the TSS. We detected PLZF binding at these sites in the CCR6+ cells, which was diminished in cells transfected with ZBTB16 siRNA. These studies demonstrate a novel role for PLZF as a transcriptional activator important both for Th17 differentiation and for the maintenance of the Th17 phenotype in human cells, and an expanded role for the BTB-ZF family in the human immune system.
Differential requirements of IL-17 family cytokines in host defense against oral candidiasis (CCR6P.272)
Authors: Whibley Natasha , Mamo Anna , Conti Heather , Traggiai Elisabetta , Kolbinger Frank , Vogel Beate , Kammueller Michael , Siebenlist Ulrich , Iwakura Yoichiro , Gaffen Sarah
Published: January 2023

Antibodies against IL-17A and IL-17RA are in clinical trials for autoimmunity. Mutations in IL-17 signaling genes (IL17RA, IL17F, ACT1) cause susceptibility to mucocutaneous infection by the fungus Candida albicans. IL-17A, IL-17F and IL-17C all signal via IL-17RA, but their individual roles in controlling candidiasis are poorly defined. Accordingly, in vivo Ab neutralization of cytokines or knockout mice were evaluated in a model of oropharyngeal candidiasis (OPC). Following C. albicans exposure, tongue fungal burden after 4 d, weight change and target gene expression were assessed. Anti-IL-17A-treated mice showed modestly impaired clearance compared to WT at d4, whereas α-IL-17F-treated mice were not affected. However, IL-17RA-/- and Act1-/- mice were far more susceptible to OPC than α-IL-17A treated mice at 4 and 14 d, indicating contributions to immunity beyond IL-17A. Susceptibility was associated with reduced expression of IL-17-regulated genes encoding chemokines and defensins. In contrast to α-IL-17A-treated mice, IL-17A-/- mice showed no significant susceptibility to OPC. This discrepancy was not explained by contributions from IL-17F or IL-17C, because α-IL-17F-treated IL-17A-/- mice and IL-17C-/- mice cleared the infection as well as WT mice. The increased susceptibility of IL-17RA-/- and Act1-/- mice vs. those lacking specific cytokines implies compensation between IL-17 family cytokines and possibly additional antifungal effectors. Funded by Novartis and NIDCR.
Synergistic effect of HIV virion and bacterial LPS on memory B cell apoptosis (HUM8P.341)
Authors: Jiang Wei
Published: January 2023

Background: The effect of heightened microbial translocation on B cells in HIV infection is unknown. Methods: We examined the effects of HIV and LPS on CD27+IgD- memory B cell (mB) apoptosis in healthy controls. In vivo analysis was conducted on a cohort of 47 healthy and 49 HIV+ donors. Results: In vitro, exposure to LPS and HIV virus cooperated to induce mB death in PBMCs (P< 0.05, n=21). The median percentage of annexin V binding mB was 22 (17.1-30.8) in medium, 23.5 (19.3-29.8) in HIV, 22.5 (16.5-28.8) in LPS, and 35.5 (26.8-44) in HIV plus LPS. This synergistic effect was abolished in pDC-depleted PBMCs (P<0.05, n=5) or in the presence of neutralizing Ab against Fas or FasL (P<0.05, n=7). HIV induced FasL in pDCs (P>0.05, n=4). LPS and LPS plus HIV induced Fas on mB (P<0.05, n=15) through mDCs. The induction of Fas was related to the susceptibility of mB to apoptosis by HIV and LPS (r=0.76, P=0.01). In vivo, in 46 HIV- and 56 HIV+ donors, mB apoptosis, Fas expression and plasma LPS were increased in HIV+ ART- and ART+ patients compared to controls (P<0.05), but plasma FasL were similar in controls and patients (P=0.94). Correspondingly, a positive correlation was found between plasma FasL and HIV RNA (r=0.67, P<0.0001), and the Fas on mB and plasma LPS (r=0.55, P<0.0001) in HIV+ donors, but not in controls. Conclusion: This study suggests a novel mechanism of mB apoptosis mediated by LPS and HIV through the Fas/FasL pathway. DCs play an important role in this process.
Co-expression network analysis reveals immune response pathways important for pathogenesis of Chlamydial pelvic inflammatory disease (PID). (HUM8P.329)
Authors: Zheng Xiaojing , O'Connell Catherine , Nagarajan Uma , Wiesenfeld Harold , Hillier Sharon , Darville Toni
Published: January 2023

Processes driving development of immune pathology after Chlamydia trachomatis infection are poorly understood. To provide insight into the pathways that may be underappreciated by individual gene analysis, weighted gene co-expression network analysis was performed to identify network modules using blood transcriptional profiles of women with chlamydial PID (N=15) and asymptomatically infected women (N=84). Modules, defined as centrally connected genes, that were significantly associated with chlamydial PID were further accessed via Ingenuity Pathway analysis. Type I and type II interferon (IFN) signaling pathways were significantly up regulated in women with chlamydial PID. Up regulation of both of these pathways has been detected in the transcriptomes of patients with active tuberculosis, suggesting a parallel role in PID. Up regulation of aryl hydrocarbon receptor, agranulocyte/granulocyte adhesion and diapedesis pathways was also noted, while EIF2/EIF4, mTOR and ILK signaling, granzyme A/B signaling, cross talk between dendritic cells and natural killer cells, signaling in T helper cells and T cell receptor pathways were down-regulated. Pathways likely to contribute to immunopathogenesis, including migration of leukocytes and inflammation were activated in chlamydial PID, while those involved in cell growth/proliferation and clearance of infected cells were decreased. Bioinformatics provides a new avenue to improve understanding of chlamydial disease mechanisms.
Differential developmental requirement and peripheral regulation for dermal Vγ4 and Vγ6 IL-17-producing γδ T cells (IRC8P.479)
Authors: Cai Yihua , Fleming Christopher , Yan Jun
Published: January 2023

Dermal γδ T cells are professional IL-17-producing γδ T cells and play a critical role in skin inflammation. However, their development and peripheral regulation are still largely unknown. In this study, we demonstrated that dermal γδ T cells developed from fetal thymus and underwent homeostatic proliferation after birth with diversified TCR repertoire, mainly including Vγ4 and Vγ6. Thymus is absolutely required for dermal γδ T cell development. IL-17-producing Vγ6 T cells were bona fide resident in dermis and were reconstituted from fetal thymus while IL-17-producing Vγ4 dermal γδ T cells were mainly from bone marrow, suggesting precursors of dermal Vγ4 T cells may require extrathymic environment for maturation and gaining skin-homing property. In addition, we found that Vγ6 T cells were pathogenic to induce skin inflammation. Mice reconstituted with Vγ6 developed psoriasis-like skin inflammation upon IMQ application whereas Vγ4 T cells were preferentially expanded and the major IL-17 producer in IMQ-treated naïve mice. Although IL-23 and IL-1β were capable of driving dermal Vγ4 and Vγ6 T cell proliferation, IL-17 production was mainly from Vγ4 and IL-1 signaling was essential. These studies reveal a differential developmental requirement and peripheral regulation for IL-17-producing dermal Vγ6 and Vγ4 T cells. This work is supported by a grant from the National Psoriasis Foundation.
Repeated antigen exposure reverses endogenous CD8 T cell tolerance and is potentiated by IL-4 (MUC8P.804)
Authors: Thompson Emily , Nelson Christine , Vezys Vaiva
Published: January 2023

Elucidating the induction, maintenance, and reversal of self-tolerant CD8 T cells is imperative for understanding the development and progression of autoimmunity, as well as cancer. Using a mouse model that permits examination of responses to small intestinal self-antigen, we can evaluate endogenous polyclonal self-specific T cells that have undergone tolerance induction. This established CD8 T cell tolerance can be reversed after repeated antigen delivery, generating large numbers of activated, functional endogenous self-specific T cells. Interestingly, this response is reduced in IL-4 limiting conditions suggesting a mechanistic role for IL-4 in self-reactive CD8 T cells responses. We hypothesize that IL-4 is required for self-specific CD8 T cell selection, survival and/or responses to cognate antigen. We show that while IL-4 does not act directly on CD8 T cells, it does impact their proliferation, especially during Ag recognition. Interestingly, we can detect self-reactive T cells that have escaped thymic selection in unmanipulated mice, but these remain naïve, despite the presence of large amounts of cognate MHC:Ag complexes. Future studies will focus on elucidating the function and origin of IL-4 in self-specific responses and understanding properties of endogenous, polyclonal self-specific CD8 T cells in negatively selecting environments.
Inhibition of TWEAK signaling preserves the glomerular filtration barrier and ameliorates spontaneous murine lupus nephritis (THER6P.845)
Authors: Xia Yumin , Herlitz Leal , Gindea Simona , Wen Jing , Pawar Rahul , Misharin Alexander , Wu Lan , Wu Ping , Michaelson Jennifer , Burkly Linda , Putterman Chaim
Published: January 2023

The TNF superfamily member TWEAK acts through its receptor, Fn14, to mediate several key pathological processes known to be involved in lupus nephritis. To explore the potential for renal protection by targeting this pathway, we introduced the Fn14 null allele into the MRL/lpr lupus mouse strain. At 26 to 38 weeks of age, female MRL/lpr Fn14 knockout (KO) mice (backcross #8) had significantly lower levels of proteinuria, BUN, and creatinine as compared to Fn14 wild type (WT) mice. Furthermore, Fn14KO mice had significantly improved renal pathology, accompanied by attenuated glomerular and tubulointerstitial inflammation and less glomerular immune deposits. There were no detectable differences between the strains in either serum autoantibody levels or in splenic immune cell subsets, although less lymphadenopathy was observed in Fn14KO mice. Importantly, Fn14KO mice displayed substantial preservation of podocytes in kidney glomeruli. Moreover, we demonstrated that TWEAK signaling via Fn14 directly damaged the integrity of the glomerular filtration barrier through effects on both podocytes and glomerular endothelial cells. Inhibition of TWEAK signaling due to deficiency of the Fn14 receptor significantly improves renal disease in a spontaneous lupus nephritis model, likely through prevention of the direct injurious effects of TWEAK on the filtration barrier. Thus, blocking the TWEAK/Fn14 axis is a novel therapeutic target in immune-mediated proliferative glomerulonephritis.
Pharmacologic inhibition of RORγt regulates Th17 signature gene expression and suppresses cutaneous inflammation in vivo (THER6P.855)
Authors: Yang Jianfei , Skepner Jill , Ramesh Radha , Trocha Mark , Schmidt Darby , Baloglu Erkan , Lobera Mercedes , Carlson Thaddeus , Hill Jonathan , Miller Lias , Barnes Ashley , Boudjelal Mohamed , Sundrud Mark , Ghosh Shomir
Published: January 2023

IL-17-producing Th17 cells, Tc17 cells and γδ T cells play critical roles in the pathogenesis of autoimmune psoriasis. RORγt is required for the differentiation of Th17 cells and expression of IL-17. Here we describe a novel, potent, and selective RORγt inverse agonist (TMP778), and its inactive diastereomer (TMP776). This chemistry, for the first time, provides a unique and powerful set of tools to probe RORγt-dependent functions. TMP778, but not TMP776, blocked human Th17 and Tc17 cell differentiation and also acutely modulated Th17-signature gene expression. In addition, TMP778 but not TMP776 inhibited IL-17 production in both human and mouse γδ T cells. IL-23-induced IL-17 production was also blocked by TMP778 treatment. In vivo targeting of RORγt in mice via TMP778 administration reduced imiquimod-induced psoriasis-like cutaneous inflammation. Further, TMP778 selectively regulated Th17-signature gene expression in mononuclear cells isolated from both the blood and affected skin of psoriasis patients. In summary, we are the first to demonstrate that RORγt inverse agonists (1) inhibit Tc17 cell differentiation, as well as IL-17 production by γδ T cells and CD8+ Tc17 cells; (2) block imiquimod-induced cutaneous inflammation; (3) inhibit Th17 signature gene expression by cells isolated from psoriatic patient samples; and (4) block IL-23-induced IL-17A expression. Thus, RORγt is a tractable drug target for the treatment of cutaneous inflammatory disorders.
IL-15 mediates alloreactive CD8 memory T cell resistance to CTLA4-Ig blockade. (TRAN3P.891)
Authors: Gorbachev Anton , Traitanon Opas , Baldwin William , Poggio Emilio , Fairchild Robert
Published: January 2023

Objective: The increased presence of alloreactive memory CD8 T cells in the peripheral blood of transplant patients is a risk factor for acute graft rejection and resistance to CTLA4-Ig immunosuppression. The mechanisms mediating this resistance are poorly understood. These studies tested the ability of IL-15, a cytokine produced in grafts during post-surgical inflammation, to confer memory CD8 T cell resistance to CTLA-4Ig blockade. Results: Addition of IL-15, but not IL-2 or IL-7, to co-cultures of purified CD28- or CD28+ memory CD8 T cells and allogeneic B cells induced proliferation of the CD28- memory cells. Proliferating CD28- memory CD8 T cells produced high amounts of IFN-g and TNF-a and up-regulated the cytolytic marker CD107a. IL-15 also induced rapid release of perforin and granzyme B by alloantigen-stimulated CD28- or CD28+ memory CD8 T cells. While CTLA4-Ig inhibited alloantigen-induced proliferation of CD28+ memory CD8 T cells, addition of IL-15 rescued this proliferation. CD28+CD8 memory T cells rendered resistant to CTLA4-Ig blockade by IL-15 down-regulated CD28, suggesting the loss of CD28 expression as one mechanism of resistance. Conclusions: IL-15 activates CD28- memory CD8 T cell to proliferate and to express effector functions in response to alloantigen and provokes down-regulation of CD28 on CD28+ memory CD8 T cells conferring resistance of these T cells to CTLA4-Ig mediated costimulation blockade.
Do farnesyltransferase inhibitors modulate immune responses by blocking cytokine secretion from alloreactive T cells? (TRAN3P.899)
Authors: Field Kenneth , Hayes Michael , James McMichael , Dieck Chelsea , Gaylo Alison , Crosby Erika
Published: January 2023

Farnesyltransferase inhibitors (FTIs) have been shown to modulate alloreactive immune responses in mouse models of transplantation (A.E. Gaylo, et al., Transplant Immunology 20 (2009) 163-170) and graft versus host disease (A.-K. Hechinger, et al. Haematoligica 98 (2013) 31-40). We are testing the hypothesis that FTIs modulate immune responses by blocking cytokine secretion from alloreactive T cells. By blocking T cell cytokine secretion FTIs could affect the differentiation of activated CD4+ T cells into Th1, Th2, or other effector subtypes. We have examined the ability of FTIs to affect cytokine secretion from various T cell subsets and T cell polarization in mice during an allorejection response. Using qPCR we quantified cytokine gene expression in graft-draining lymph nodes (GDLNs) isolated from mice during the rejection of an MHC class II-mismatched skin allograft (bm12 donors to B6 recipients). Using ELISpot assays, we also measured the number of alloreactive Th1 (IFN-γ producing) and Th2 (IL-4-producing) cells in GDLNs. In vitro T cell polarization of polyclonally-stimulated CD4+ T cells isolated from spleen was measured using IFN-γ, IL-4, IL-17, and FoxP3 intracellular staining. Using these various approaches, we found that FTI treatment differentially affects cytokine secretion from the CD4+ T cell subsets studied. These effects lead to differences in Th1/Th2 cell polarization that could explain the immunomodulatory activity of FTIs.
Small molecule agonists of CD11b/CD18 reduce leukocyte activation and recruitment to promote kidney allograft survival (TRAN3P.909)
Authors: Khan Samia , Faridi Mohd , Guo Lingling , George James , Agarwal Anupam , Gupta Vineet
Published: January 2023

Allograft rejection in kidney transplantation is associated with leukocyte infiltration, including myeloid cells expressing β2 integrin CD11b/CD18. We reported that activation of CD11b/CD18 by a small molecule compound, leukadherin-1 (LA1), increases leukocyte cell adhesion to the inflamed endothelium, prevents transmigration and inhibits leukocyte tissue recruitment resulting in improved kidney function in a murine nephritis model. Here we test whether LA1 mediated activation of CD11b/CD18 enhances kidney allograft survival in a mouse model of fully MHC-mismatched orthotopic kidney transplant, where C57BL/6J (H-2b) recipients received a kidney allograft from Balb/c mice (H-2d). Control recipients were treated daily with cyclosporine (CsA) for 2 weeks, while the test group received standard CsA therapy and daily LA1 injections during week 1 and alternate days during weeks 2-8. Renal allograft rejection was considered when mice displayed signs of ill health and high serum creatinine levels. Combination therapy reduced leukocyte infiltration and neointimal hyperplasia and increased graft survival from 48.5% (CsA only) to 100% (CsA and LA1) on day 60. At week 5, CsA treated controls had serum creatinine values of 0.6 mg/dl while LA1 and CsA treated mice had baseline values of 0.2 mg/dl. These findings indicate a crucial role for CD11b/CD18 in the control of leukocyte migration to the transplanted kidney and identify leukadherins as therapeutic agents for renal transplantation.
TIGIT inhibits CD8+ T cell effector function during chronic viral infection and cancer (TUM7P.933)
Authors: Grogan Jane , Johnston Robert , Comps-Agrar Laetitia , Hackney Jason , Yu Xin , Huseni Mahrukh , Yang Yagai , Eaton Dan , Park Summer
Published: January 2023

Tumors constitute highly suppressive microenvironments in which infiltrating T cells are functionally suppressed by co-inhibitory receptor ligands. We identify T cell Immunoglobulin and ITIM domain (TIGIT) as a co-inhibitory receptor that critically limits CD8+ T cell responses to cancer and chronic infection. Expression of TIGIT was highly correlated with T cell infiltration in a broad range of solid tumors, and both human and murine tumor-infiltrating lymphocytes coordinately expressed TIGIT and PD-1, a key marker of T cell exhaustion. In mouse models of chronic viral infection and cancer, antibody blockades of TIGIT and PD-L1 synergistically and selectively enhanced CD8+ T cell effector function, resulting in significant viral clearance and tumor rejection respectively. Anti-TIGIT mediated anti-tumor immune responses were durable, as treated mice possessed antigen-specific and CD8+ T cell-dependent protection against tumor re-challenge. Strikingly, these effects were dependent on the activity of TIGIT’s complementary co-stimulatory receptor, CD226, whose dimerization and function was impaired by direct interaction with TIGIT in cis; and ablation of TIGIT was sufficient to enhance CD226 co-stimulation in CD8+ T cells. These results reveal a novel mechanism of action for co-inhibitory receptors and define a critical role for TIGIT as a regulator of anti-tumor and other chronic CD8+ T cell responses in vivo.
Role of signal transducer and activator of transcription 6 in the development of colitis associated colorectal cancer (TUM7P.959)
Authors: Leon-Cabrera Sonia , Molina Emmanuel , Delgado Yael , Becerra Mireya , Ledesma-Soto Yadira , Gitierrez-Cirlos Emma , Rodriguez-Sosa Miriam , Avila-Moreno Federico , Chirino Yolanda , Perez-Plasencia Carlos , Terrazas Luis I.
Published: January 2023

STAT6 signaling pathway has important pathophysiologic implications in a variety of cell types and diseases. On cancer studies, mice lacking STAT6 display enhanced tumor immunity to both primary and metastatic mammary carcinomas. In humans, STAT6 protein has been found to be constitutively activated in several cancer types. However, the role of STAT6 in influencing the resistance or susceptibility of colon cancer cells to apoptosis and invasiveness/metastasis has not yet been validated during in vivo colitis-associated colon cancer (CAC).This study aims to identify the effects of STAT6 in tumorigenesis utilizing STAT6-KO mice in an experimental CAC model and evaluating changes in immune cell populations in the absence of this gene. CAC was induced in both STAT6-KO and WT mice by injection of 12.5 mg/kg AOM followed by three rounds of 2% DSS exposure to elicit colitis. On day 63 after CAC induction, mice were sacrificed. Colonic inflammation, proliferation and tumorigenesis were evaluated. Tumor numbers per mouse (0.8 versus 19.6; P=0.0001), and bowel weight (0.3 versus 0.6 g; P=0.05) were significantly decreased in STAT6-KO mice compared to WT mice. Furthermore, both inflammation and proliferation scores in colon from STAT6-KO mice were significantly lower than WT mice. Although additional studies are needed to clarify the mechanism(s) underlying improved anti-tumor immunity in STAT6-KO mice, these results may be a basis for an immunotherapy strategy in CAC development.
Cellular and molecular characterization of hagfish VLR-based adaptive immune system (VET2P.1043)
Authors: Li Jianxu , Guo Peng , Hirano Masayuki , Herrin Brantley , Cooper Max
Published: January 2023

Jawless vertebrates have an alternative adaptive immune system in which variable lymphocyte receptors (VLR) are somatically diversified through recombinatorial assembly of leucine-rich repeat cassettes during lymphocyte development. Three VLR loci (VLRA, VLRB, and VLRC) have been defined in lampreys and each is expressed by a distinct lymphocyte population. Lamprey VLRA and VLRC are expressed by T-like lineages of lymphocytes, whereas VLRB is expressed by a B-like lineage of lymphocytes, much like αβ T, γδ T and B cells in jawed vertebrates. Recently we have revised the nomenclature for the hagfish VLRs by defining a third VLR gene. Here, monoclonal and polyclonal antibodies were generated against invariant residues of hagfish VLRA, VLRB and VLRC and these were used to identify VLR-bearing lymphocytes and their products. We found that hagfish VLRA, VLRB and VLRC are expressed by three separate lymphocyte populations. Size-exclusion chromatography and western blot analysis indicated that multivalent VLRB proteins are released into the circulation, which suggests that hagfish VLRB+ cells are B-cell like. In contrast, neither VLRA nor VLRC was detectable in the plasma, supporting the hypothesis that these two lymphocyte populations are T-cell like. The demonstration of three lymphocyte populations in both lampreys and hagfish suggests that this tripartite lymphocytic differentiation program already existed in a common ancestor of the two cyclostome lineages around 480 Mya.
Leishmaniasis triggers changes in monocyte subsets in dogs. (VET2P.1048)
Authors: Goncalves Ricardo , Barbosa Vitor , Antonelli Lis , Mosser David , Tafuri Wagner
Published: January 2023

Peripheral blood monocytes are the largest group of circulating progenitor cells. Its functions have been revealed through the study of their subpopulations, especially in humans and mice in pathological processes. In Brazil, the parasite L. infantum is the cause of the canine visceral leishmaniasis. This study aimed to characterize subpopulations of peripheral blood monocytes from uninfected or L. infantum naturally infected dogs and compare their phenotypical and functional aspects. Blood samples were collected from uninfected and infected dogs, labeled by antibodies against different surface markers and analyzed by flow cytometry. It was observed two distinct populations according to CD14 expression: a population CD14High, constituting about 90% of the monocytes showing basophilic and horseshoe-shaped nucleus with greater phagocytic capacity, and another CD14Low, constituting about 10% of monocytes, showing less basophilic and kidney-shaped nucleus. The analysis of the other molecules showed different levels of expression between the two populations. In dogs with visceral leishmaniasis no difference in frequency was observed between populations, however there is an increase of CD14+ cells in infected dogs. We also noticed marked reduction of the expression of molecules related to the activation of monocytes, especially in the group of symptomatic dogs, indicating a possible inhibition on the capacity of these cells to control the disease.
Endogenously-processed influenza epitopes intersect with the classical MHCII presentation pathway via the GILT endosomal reductase (APP2P.104)
Authors: Veerappan Ganesan Asha Purnima , Miller Michael A , Eisenlohr Laurence C
Published: January 2023

CD4+ T cell activation is driven by recognition of pathogen-derived peptides (epitopes) displayed by MHC-II on the surface of antigen presenting cells. By convention, peptides derived from extracellular antigen are internalized and loaded onto MHC-II in the late endosome. However, our recent findings suggest that this is a minor contributor to influenza-derived peptide display. In order to elucidate alternative pathways, T cell hybridomas were developed to six different specificities derived from A/PR/8/34 influenza virus proteins. Fully half of the epitopes were found to be proteasome dependent, reflecting a major role for endogenous, cytosolic antigen processing. Intriguingly, endogenous processing of two epitopes, which are constrained within the neuraminidase protein (NA) by disulphide bonds, was strongly dependent on γ-interferon-inducible lysosomal thiol reductase (GILT), an enzyme that can be critical for exogenous processing. Presentation of these epitopes was also inhibited by primaquine (a compound that prevents trafficking from endosome to the plasma membrane) suggesting that the relevant GILT activity resides in the endosomal network. This interpretation was further supported by the observations that presentation of disulphide constrained synthetic peptides were also GILT dependent but responses to MHC-I restricted epitopes within NA are not. These results point to a novel alternative MHC-II pathway that includes both cytosolic and endosomal processing steps.