bluuebunny/crossref_metadata_2025 huggingface dataset:
CMV and inflammation contribute to decreased B cell vaccine response in the elderly (LYM6P.777)
Authors: Blomberg Bonnie , Diaz Alain , Romero Maria , Landin Ana Marie , Frasca Daniela
Published: January 2023

We previously showed intrinsic age-related defects in the in vivo and in vitro B cell responses to the seasonal influenza vaccine. Serum antibody responses were associated with the in vitro B cell response to the vaccine, measured by AID (activation-induced cytidine deaminase). Both responses decrease with age and are significantly correlated. In order to develop predictive markers for a beneficial humoral immune response, we measured markers of systemic and B cell-intrinsic inflammation. We hypothesized that the increased pro-inflammatory status of the elderly (including CMV) would directly impact B cell function, thus impairing the capacity of the individual to respond to vaccination. Our results show that aged B cells themselves make TNF-α before vaccine challenge, which positively correlates with serum markers of inflammation, but negatively correlates with B cell function, measured by AID expression after B cell stimulation. From the last 2 years (2011/2012 and 2012/2013) we show that CMV seropositivity is associated with lower in vivo and in vitro vaccine responses in both young and elderly. Recent studies will also be presented on the CD4 and CD8 TEMRA shown to be associated with lower antibody response. Our results reveal new molecular mechanisms which contribute to reduced antibody responses in aging and suggest that these will have an impact for crucial development of effective vaccines to protect the elderly from infectious and other debilitating diseases.
Distinct roles for TLR7 and TLR9 in regulation of B cell signal strength and differentiation (LYM6P.766)
Authors: Nundel Kerstin , Mande Purvi , Shaffer Art , Sindhava Vishal , Cancro Michael , Marshak-Rothstein Ann
Published: January 2023

BCR signal strength plays a key role in the fate decision of B cells. Strong BCR signals promote plasma cell differentiation and weaker BCR signals drive germinal center B cell fate. Signals through toll-like receptors (TLRs) also influence B cell differentiation, but it is unclear how the BCR and TLR signaling cascades interface to modulate B cell responses and differentiation. To address this question, we used a BCR sdTg mouse model, AM14, in which all mature B cells have a naïve follicular phenotype. The AM14 BCR recognizes IgG2a-bound immune complexes (ICs) and AM14 B cell activation depends on TLR7 and/or TLR9. ICs formed by the anti-nucleosome antibody PL2-3 can activate AM14 B cells through either TLR7 or TLR9, allowing us to dissect signaling pathways unique to each TLR. We find that activation of AM14 TLR7-deficient B cells leads to sustained activation of the NF-κB pathway and p38 phosphorylation, subsequently leading to cell death. In contrast, TLR9-deficient AM14 B cells activated with ICs are unable to sustain the initial NF-κB and p38 signaling response. Intriguingly, these TLR9-deficient AM14 B cells differentiate into short-lived plasmablasts as characterized by increased levels of plasma cell associated proteins such as IRF-4, Blimp-1 and CD138. Plasma cell development is not observed in IC activated TLR7-deficient AM14 B cells. Our results suggest divergent roles for TLR7 and TLR9 in B cell activation and plasma cell differentiation.
ENPP1-positive B cells and activation in human peripheral blood (LYM6P.772)
Authors: Yoon Jeong Heon , Wang Hongsheng , Morse Herbert
Published: January 2023

ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1), also known as PC1 (plasma cell alloantigen 1), is a homodimeric type II transmembrane glycoprotein that mediates nucleotide recycling by breaking down ATP to AMP. Previous studies of mouse B lineage cells showed that levels of PC1 expression could distinguish two distinct subsets of peritoneal B -1a cells. In addition, PC1 expression increases progressively on germinal center B cells and splenic plasma cells with highest levels on bone marrow plasma cells. To determine if PC1 might also help define human B1-like cells, we have used a mouse mAb to human PC1 to characterize its expression on human cells. The frequency of PC1-positive cells from cord blood (mean 6%, n=10) was higher than that from adult peripheral blood (1.34%, n=15). These PC1-positive cells are mostly derived from CD27- naïve B cells expressing CD38, IgM and IgD, and were detected in plasmablast/plasma cells in peripheral blood. In contrast to B-1a cells in mice, human PC1-positive cells did not secrete natural Abs or produce IL10. Expression of PC1 was greatly increased on purified peripheral blood B cells stimulated with anti-CD40, IL4 and IL21. These activated PC1-positive cells expressed low levels of CD5, HLA-DR, surface IgM, and high levels of B cell activation markers and CD138. Our results indicated that PC1-positive B cell populations in humans are different from either CD20+CD27+CD43+ B1 cells in human, or CD5+ B-1a cells in mice.
Recognition of the cyst life cycle stage induces a robust immune response to the opportunistic fungal pathogen Pneumocystis murina (MPF3P.814)
Authors: Evans Heather , Garvy Beth
Published: January 2023

Pneumocystis species are opportunistic fungal pathogens that cause severe pneumonia in immunocompromised hosts. The life cycle of Pneumocystis species consists of a vegetative trophic stage and an ascus-like cystic stage. We demonstrate that infection of wild-type adult or neonatal mice with a mixture of trophic and cystic P. murina life forms elicits a more robust CD4+ and CD8+ T cell response than infection with the trophic form alone. β-1,3-glucan is a predominant structural component in the cyst wall, but is not produced by the trophic stage. Depletion of cysts with the β-1,3-glucan synthase inhibitor anidulafungin prevents expansion of the remaining trophic organisms in immunocompromised adult mice. Depletion of cysts in immunocompetent adult mice results in fewer infiltrating CD11b+ cells in the bronchoalveolar lavage fluid. In addition, bone marrow-derived dendritic cells pulsed with mixed P. murina express more IL-1β and induce greater in vitro CD4+ T cell interferon-γ production than cells pulsed with the trophic form alone. Dendritic cells have been previously reported to activate CD4+ T cells in response to Dectin-1 stimulation by purified Pneumocystis β-1,3-glucan. We propose that dendritic cells stimulated by β-1,3-glucan on cystic Pneumocystis direct a robust pro-inflammatory response, while stimulation with the trophic form induces a less exuberant immune response that is sufficient for clearance in immunocompetent individuals.
Mycobacterium tuberculosis lipoprotein LprG binds lipoarabinomannan and determines its localization in the cell wall envelope and affects phagolysosomal fusion (MPF3P.818)
Authors: Shukla Supriya , Richardson Edward , Athman Jaffre , Shi Libin , Wearsch Pamela , McDonald David , Banaei Niaz , Boom W. , Jackson Mary , Harding Clifford
Published: January 2023

Genetic deletion of the lipoprotein LprG reduces the virulence of Mycobacterium tuberculosis (Mtb), but the function of LprG remains unclear. We report that LprG expressed in Mtb binds to lipoglycans, e.g. lipoarabinomannan (LAM). Lipoglycan binding to LprG was dependent on both insertion of lipoglycan acyl chains into a hydrophobic pocket on LprG and a novel contribution of lipoglycan polysaccharide components outside of this pocket. An lprG null mutant (MtbΔlprG) had lower levels of surface-exposed LAM, revealing a novel role for LprG in determining Mtb cell envelope structure. Furthermore, this mutant allowed accelerated Mtb phagosome-lysosome fusion, consistent with the role for LAM in blocking phagosome maturation. We propose that LprG binding to LAM facilitates its transfer from the plasma membrane into the cell envelope, increasing surface-exposed LAM, enhancing cell envelope integrity, allowing inhibition of phagosome-lysosome fusion and enhancing Mtb survival in macrophages
Bacterial membrane vesicles mediate the secretion of immunomodulatory factors during Mycobacterium tuberculosis infection (MPF3P.804)
Authors: Athman Jaffre , Wang Ying , McDonald David , Harding Clifford , Wearsch Pamela
Published: January 2023

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that infects lung macrophages and releases microbial factors to promote immune evasion and survival. These factors include the lipoglycan LAM which blocks phagosome maturation and Mtb lipoproteins which modulate MHC-II antigen presentation and cytokine production via TLR2 signaling. It is unknown how lipoproteins and lipoglycans are shed from the Mtb cell wall, but they are dispersed throughout Mtb-infected macrophages and released within extracellular vesicles. It was initially presumed that Mtb lipoproteins and LAM trafficked into exosomes, small vesicles (50-150 nm) of endocytic origin, but studies have shown that bacteria can secrete membrane vesicles of similar size (50-250 nm). Using imaging and biochemical approaches, we show that Mtb produces bacterial vesicles within infected macrophages that are subsequently released into the extracellular environment. These Mtb vesicles are enriched for cell wall components (LAM, TLR2 agonists) and are distinct from exosomes labeled with host cell markers (CD9, CD63). Our data suggest that bacterial vesicles are the mechanism employed by Mtb to transfer cell wall components and impair effector functions within the infected macrophage. Furthermore, we propose that released Mtb vesicles, not exosomes, circulate bacterial components beyond infected cells to further modulate immune responses. These mechanisms may promote the survival of Mtb and pathogenesis of infection.
Microbiota enhances intestinal immunity through JNK/ROS pathways (MUC4P.838)
Authors: Chen Lee-Wei , Wu Ying-Ying , Chung Pei-Hsuan , Hsu Ching-Mei
Published: January 2023

Microbiota maintains normal mucosal immunity through complex mechanisms. To examine the role of JNK signaling pathway in microbiota-related gut immunity, we assessed the microflora in ileal lumen and mucosa, the antibacterial protein expression, and the RORγt cells of lamina propria in WT and JNK-/- mice. The bacterial counts in ileal lumen and mucosa show a significant decrease of Lacobacilli in JNK-/- mice. Moreover, JNK-/- mice demonstrated a significant decrease of RORγt cells in lamina propria and an increased translocation of Klebsiella pneumoniae to the portal vein as well as blood. The generation of reactive oxygen species (ROS) in intestinal mucosa was also significantly decreased in JNK-/- mice as detected by using 2',7'-dichlorodihydrofluorescein diacetate. Oral supplementation of fructooligosaccharides (FOS) or L-NAME but not D-NAME increased the production of ROS, IL-1β, IL-23 as well as RORγt cells in intestine, and decreased the translocation of K. pneumoniae in JNK-/- mice compared to WT mice. N-Acetylcysteine, an antioxidant, treatment inhibited ROS production and reversed the effect of FOS on intestinal immunity in JNK-/- mice. Collectively, JNK pathway plays an important role in microbiota-induced intestinal immunity through the induction of ROS generation, and FOS or iNOS inhibitor increase the numbers of RORγt cells and the expression of IL-1β as well as IL-23 in JNK-/- mice through enhanced production of ROS.
Mucosal antimicrobial protection toward enteroaggregative Escherichia coli infection is modulated by dietary tryptophan through an IL17-dependent mechanism (MUC4P.846)
Authors: Philipson Casandra , Bassaganya-Riera Josep , Viladomiu Monica , Carbo Adria , Eden Kristin , Hontecillas Raquel
Published: January 2023

Enteroaggregative E. coli (EAEC) is a predominant extracellular pathogen that causes inflammatory diarrhea in individuals worldwide, especially in persons suffering from malnutrition. We previously reported that protein-energy malnutrition abrogates protective Th17-dependent antimicrobial immunity in a murine model of EAEC infection. However, mechanisms whereby malnutrition enhances host-susceptibility to EAEC colonization remain unreported. This study presents evidence that tryptophan is crucial for antibacterial protection against EAEC infection. Mice fed tryptophan-free diet had reduced antimicrobial peptide production coinciding with significantly high pathogen levels. Likewise, bacterial burden occurred in a dose dependent manner when mice were supplemented with glycly-L-tryptophan dipeptide. Low serum tryptophan directly correlated with hampered gene expression levels of cytokines regulating effector IL-17A responses (e.g. IL-23 and IL-6). Mechanistically, orally administered tryptophan enhanced IL-17A and IL-17C production at the colonic and ileal mucosa facilitating antimicrobial defense and protection against EAEC colonization. IL17a-/- and IL17ra-/- mice receiving nourished diets or tryptophan supplements were unable to recover from disease due to impaired intestinal epithelial barrier function. Thus, our data suggests that tryptophan modulates IL17-dependent antimicrobial responses and is required for preventing EAEC colonization and pathogenic disease.
Microbial metabolites modulate GI mucosal damage from graft versus host disease (GHVD). (MUC4P.850)
Authors: Mathewson Nathan , Mathew Anna , Oravecz-Wilson Katherine , Wu Julia , Toubai Tomomi , Cummings Emily , Pennathur Subramaniam , Reddy Pavan
Published: January 2023

The microbiome of the GI tract is known to affect GI mucosal responses and regulate disease processes. GI tract damage is the major cause for graft versus host disease (GVHD) mortality. Studies have shown the microbiome affects the course of allogeneic bone marrow transplant and modulates GVHD. However, little is known about the microbial metabolites crucial for achieving the effects of the microbiome on immune responses, specifically GVHD. We examined the metabolome of naïve mice and murine recipients of syngeneic (B6→B6) and allogeneic (B6→BALB/c) BMT, in an unbiased way. We made a surprising observation that short chain fatty acids (SCFA), including butyrate, were altered in the intestinal epithelium but not in the luminal contents of allo-BMT recipients. Butyrate, a known metabolite of the microbiome was previously shown to have HDAC inhibitor (HDACi) and salutary effects on the intestinal epithelium. Additionally, recent data showed systemic chemical inhibition of HDAC protects GI epithelium from GVHD severity. The decrease of butyrate in the epithelial cells of the small intestine (P<0.03) suggested poor uptake of butyrate following allo-BMT. Consistent with this notion, we found that treatment of epithelial cells with butyrate confers protection from irradiation and decreases allo-induced cell death. Our results suggest that the microbiome regulates the damage from allogeneic GVHD due to the reduction in the tissue level of the SCFA-metabolite and HDACi, butyrate.
Understanding the effect of early-life pulsed antibiotic treatment on mucosal T-lymphocyte populations (MUC4P.827)
Authors: Ruiz Victoria , Gottwick Cornelia , Livanos Alexandra , Li Huilin , Blaser Martin
Published: January 2023

Broad-spectrum antibiotics are frequently prescribed for children. The gut microbiota have functional roles in the development and differentiation of the host immune system. Early-life antibiotic use may have dynamic effects on the host microbiota, promoting long-term immunologic dysregulation and subsequent allergic and autoimmune pathology. Using a model of early-life antibiotic use, we hypothesize that early-life pulsed antibiotic treatment (PAT) -induced perturbation of the intestinal microbiota leads to alterations in tissue-specific and systemic T-cell populations. To test this hypothesis, we characterized splenic and ileal T-cell populations in control and PAT-treated C57BL/6 mice. Flow cytometric analysis demonstrated that early-life PAT significantly (p<0.05) decreased the frequency of splenic CD4+ and CD8+ T-cells and ileal CD4+ IL-17A+ Th17 populations. In contrast, ileal CD4+ Tbet+ (Th1) T cells and CD4+ Foxp3+ regulatory T-cell populations were significantly increased in PAT-treated mice. Alterations in immune cell populations were associated with microbial compositional changes, including significantly higher relative abundance of Enterobacteriaceae and Akkermansia muciniphila in PAT-treated mice. These results provide evidence that early-life perturbation of the intestinal microbiota by PAT modulates systemic and mucosal T-cell populations, phenotypes that may persist after microbial recovery, promoting life-long consequences to host immunity.
Hyper Th17 responses in IL-17R knockout is regulated by segmented filamentous bacteria (SFB) (MUC4P.857)
Authors: Kumar Pawan , McAleer Jeremy , Elsegeiny Waleed , Armentrout Rachel , Pociask Derek , Kolls Jay
Published: January 2023

Blockade or genetic deletion of IL-17RA resulted in marked increases in IL-17 response by T-cells. We hypothesize that the hyper Th17 response in IL-17 deficient mice is due to alterations of the gut microbiome particularly overgrowth of segmented filamentous bacteria (SFB). Our data shows that Il17ra-/- and Il17rc-/- mice have overgrowth of SFB (10 fold higher than WT.Taconic), suggesting a critical role of IL-17 signaling in SFB colonization. Higher SFB colonization in Il17ra-/- and Il17rc-/- mice results in expansion of IL-17A and IL-22 producing Th17 cells. As further evidence that this expansion was not T-cell intrinsic, we observed similar frequencies of IL-17 producing cells in WT and Il17ra-/- when naïve T-cells were polarized in vitro to Th17 cells. Furthermore, vancomycin depletion of SFB in Il17ra-/- mice resulted into fewer Th17 cells in the lamina propria and spleen, suggesting gut microflora responsible for hyper Th17 response. SFB-colony free WT mice with intact IL-17 signaling can control SFB overgrowth but IL-17 deficient mice could not following SFB inoculation. To further define the role of IL-17 in SFB colonization, we have generated SFB-free Il17ra conditional knockout (Il17rafl/flxe2a cre) mice. SFB-free Il17rafl/flxe2a cre mice are devoid of hyper Th17 responses, and SFB inoculation resulted into acquisition of hyper Th17 phenotype as observed in Il17ra-/-. Our data suggest that IL-17 signaling regulates SFB colonization and associated Th17 responses.
Parenteral immunization with double-mutant heat labile toxin engages CD103+ dermal dendritic cells and redirects T cell immunity to the gut mucosa. (MUC4P.833)
Authors: Frederick Daniel , McLachlan James , Freytag Lucy
Published: January 2023

The vast majority of infectious agents enter hosts through mucosal compartments. Despite this, most current vaccines are administered via parenteral injection, which induce vigorous systemic but only limited mucosal immune responses. Studies have suggested that certain adjuvants can induce mucosal antibody responses regardless of route; however, there is minimal evidence that these injections direct antigen-specific effector T cells to the mucosa, where they are likely needed for protection against mucosal pathogens. We sought to explore the potential of the mucosal adjuvant dmLT (double-mutant heat-labile toxin) to drive antigen-specific T cells to the mucosa after parenteral immunization. By using a combination of peptide:MHCII-specific antibodies and tetramers, we can directly observe the impact of intradermal delivery of dmLT plus antigen, on both antigen-presenting cells (APCs) and antigen-specific T cells. Here we show that this immunization results in T cell upregulation of the gut mucosal homing marker, α4β7, in local draining lymph nodes and mucosal sites when compared to immunization with CpG. Additionally, we observed dmLT preferentially recruits skin CD103+ dendritic cells to the draining lymph nodes after parenteral immunization. These results show that administration of a mucosal adjuvant in the skin results in recruitment of mucosal-like APCs and induce T cell responses in remote, mucosal compartments.
The role of type I interferon signaling in the maintenance of resident memory T cell populations in the lung (MUC5P.867)
Authors: Turner Damian , Farber Donna
Published: January 2023

We have recently described populations of memory T cells that establish residence within specific niches of the lung following respiratory influenza virus (IAV) infection. Lung resident CD4 and CD8 memory T cells (TRM) are highly enriched with IAV-specific cells and exhibited increased CD11a and CD69 expression, which is characteristic of tissue resident populations. Confocal microscopy further revealed that lung TRM are clustered around airways and blood vessels within bronchovascular bundles. We sought to investigate the environmental factors that are involved in the retention of lung TRM. To investigate the role of tonic type I interferon (IFN-α/β) signaling in the maintenance of lung TRM we treated mice (previously infected with IAV) with interferon receptor (IFNAR1) blocking antibody and found that short-term blockade led to the down regulation of CD69 without a reduction in the number of lung TRM. Blocking IFNAR1 for 8 days resulted in a dramatic reduction in the numbers of CD4 and CD8 TRM in the lung as well as lung γδ T cells. We also found that, over time, fewer IFNAR1 deficient T cells were retained within the lung resident niche than wildtype T cells following IAV infection. These results suggest that constitutive IFN-α/β signaling may play a role in the maintenance of lung TRM populations, and may have therapeutic implications for respiratory diseases.
A method for detecting protein expression in single cells using the C1™ Single-Cell Auto Prep System (TECH2P.874)
Authors: Egidio Camila , Ooi Aik , Holcomb Ilona , Ruff Dave , Boutet Stephane , Wang Jing , Ramakrishnan Ramesh
Published: January 2023

Recent improvements in microfluidics and biochemistry have enabled single-cell molecular analysis, providing new insight into the heterogeneity of cell populations. The C1 Single-Cell Auto Prep System is an automated platform that streamlines the isolation and processing of 96 individual, live cells for RNA and DNA analysis. Single-cell protein profiling is a direct complement to genomic analysis as it provides additional insights into key molecular mechanisms and system biology. To enable this, we adapted a highly multiplexed protein detection system (Proseek Multiplex Oncology, Olink Bioscience) for use on the C1 System. We analyzed 192 individual cells from two human promyelocytic leukemia cell lines (HL60 and K562) and compared these result against 16 FACS-sorted cells in tube format. These results indicated that four key proteins (i.e. CASP3, CSTB, CD69 and MPO) are differentially expressed between the two cell lines and vary from cell-to-cell. For example, the human protein MPO, produced by neutrophils for their microbicidal activity, was differentially expression between individual HL60 and K562 cells, suggesting varying levels of neutrophil activation within the cell populations. This work demonstrates highly-multiplexed protein detection at the single-cell level and shows promise to have sufficient sensitivity, reproducibility and scale to effectively interrogate a statistically significant numbers of individual cells.
MicroRNA-dependent modulation of histone acetylation in multiple sclerosis (THER3P.875)
Authors: Guan Hongbing , Singh Narendra , Singh Udai , Rao Roshni , Mrelashvili Davit , Nagarkatti Prakash , Nagarkatti Mitzi
Published: January 2023

Inhibitors of histone deacetylases (HDACs) are of interest for treatment of MS. However, global modifications of DNA and chromatin structure are found to be associated with toxicities and adverse effects. For example, HDAC inhibitor, Trichostatin A, was capable of ameliorating the development of a mouse model of MS (EAE). However, the clinical trials in MS patients showed exacerbation of symptoms. This discrepancy may have been due to the non-specific nature produced by the drug or additional genetic changes. Thus, novel epigenetic approaches capable of targeting a specific set of genes in encephalitogenic T cells are desirable for controlling MS while minimizing adverse effects. We recently found that encephalitogenic CD4+ T cells present with a specific miRNA and HDAC signature characterized by increased expression of let-7e, HDAC-3, -5, -9 and reduced miR-140-5p and the rest of HDAC. Predicted let-7e targeted genes include HDAC-2, -10, and -11. We confirmed that let-7e regulated expression of HDAC-10. Using lentivirus or synthetic oligonucleotides to modulate the expression level of let-7e, we found that down-regulated HDAC-10 accompanied with up-regulation of acetyl-histone-3 and -4, which led to increased encephalitogenic Th1- and Th17-cell development and EAE. Our results indicated that HDAC-10 contributed to the pathogenesis of MS. Thus, use of let-7e oligonucleotides may constitute an alternative to chemical HDAC inhibitors in the treatment of MS.
T cell repertoire renewal following autologous stem cell transplantation for multiple sclerosis (THER3P.879)
Authors: Robins Harlan , Muraro Paolo , Malhotra Sachin , Howell Michael , Phippard Deborah , Desmarais Cindy , de Paula Alves Sousa Alessandra , Griffith Linda , Lim Noha , Nash Richard , Turka Laurence
Published: January 2023

Autologous hematopoietic stem cell transplantation (HSCT) is commonly employed for hematologic and non-hematologic malignancies and also has been used in clinical trials for severe autoimmune diseases to “reset” the immune system and produce a new, hopefully non-autoimmune repertoire. While it has long been feasible to eliminate the vast majority of mature T cells, it has been very difficult to accurately and quantitatively determine the relationship of the regenerated repertoire to the baseline repertoire. Here, in a phase II study of HSCT for poor prognosis multiple sclerosis (MS) patients, we have used high throughput deep TCRβ chain sequencing to directly assess millions of individual TCRs per patient sample. We find that HSCT has distinctive, and separate, effects on the CD4 and CD8 repertoires. In the case of CD4 T cells, the dominant TCR clones present pre-treatment were not detectable following reconstitution, and patients largely developed a new repertoire. In contrast, dominant CD8 clones were not effectively removed, and the reconstituted CD8 repertoire was created by clonal expansion of cells which were present pre-treatment. Importantly, patients who failed to respond to treatment had less diversity in their T cell repertoire early during the reconstitution process. This approach may enable monitoring of pathogenic or protective T cell clones following HSCT and or cellular therapies.
CD11b regulates immunity to tumor and mediates tumor promotion of macrophages recruited by mesenchymal stem cells (TUM4P.922)
Authors: Zhang Jimin , Yuan Zeng-rong , Ren Guangwen , Zhang Liying , L'Huillier Andrew , Shi Yufang
Published: January 2023

Here we report that systemic gadolinium chloride, or GdCl3, treatment, a potent macrophage inhibitor, significantly suppressed melanoma development in mice, which was related to the reduction of CD11b expression. We then directly examined the role of CD11b in tumorigenesis in mice by using CD11b-/- mice, or by blocking CD11b function with anti-CD11b antibody. We found that knockout or blockage of CD11b significantly suppressed the development of melanoma. Histological analysis of melanoma tissue demonstrated that abundant inflammatory cells infiltrated the surrounding region of tumor in CD11b-/- mice but not in wild type mice. This result is consistent with the increased expression of inflammatory cytokines, including TNFα, IL-6, IL-12 and IL-23, in GdCl3-treated macrophages in vitro. More IL-17 was produced in CD4+ T cells co-cultured with GdCl3-treated macrophages compared with no GdCl3 treatment, and administration of a low amount of IL-17 suppressed melanoma growth in mice in vivo, showing that CD11b-mediated immune tolerance in tumor may be through inhibition of IL-17 production in T cells. Furthermore, we reported that tumor-educated or TNFα-pretreated mesenchymal stem cells promoted tumor development by recruiting macrophages, thus we examined the effect of GdCl3 treatment or CD11b knockout and found that the tumor promoting effect was abolished. Therefore, our findings demonstrate that CD11b provides a tolerogenic signal during the immune response to tumor.
The central role of tumor-evoked Bregs in breast cancer metastasis as inducers of metastasis-promoting Tregs and myeloid-derived suppressive cells. (TUM4P.903)
Authors: Biragyn Arya , Bodogai Monica , Lee-Chang Catalina
Published: January 2023

We recently found that, by utilizing metabolites of 5-lipooxygenase, highly metastatic 4T1 breast cancer induces the generation of unique subset of regulatory B cells, tBregs1,2. The role of tBregs in metastasis is that they induce conversion of FoxP3+ regulatory T cells (Tregs) that protect metastasizing cells by inactivating antitumor effector NK cells and CD8+ T cells 1,3. The absence of tBregs or a B-cell deficiency in μMT mice is sufficient to disable the ability of 4T1 cancer cells to metastasize into the lungs. Although in this model the myeloid-derived suppressive cells (MDSC) have been shown to also promote metastasis, surprisingly we failed to associate their importance in metastasis. MDSC are normally expanded (an assumed pro-metastatic requirement) without apparent phenotypic changes in B-cell deficient mice or if tBregs are inactivated. However, adoptive transfer of MDSC from tumor-bearing WT mice, but not μMT mice, is able to restore lung metastasis of 4T1 cancer in μMT mice, suggesting that tBregs may be needed for the education of MDSC. Here we provide the mechanistic proof that tBregs indeed render MDSC pro-metastatic. tBregs target and use relatively small subset of MDSC. Thus, our data not only uncover previously unknown regulatory pathway, but also suggest a cautious new interpretation on the importance of MDSC in metastasis. 1 Olkhanud, P. B. et al. Cancer Res. 2011. 2 Wejksza, K. et al. J.Immunol. 2013. 3 Olkhanud, P. B. et al. Cancer Res. 2009.
Novel small molecule nucleotide homologs (SMNH) activate human macrophages to kill intracellular Mycobacterium tuberculosis. (VAC6P.949)
Authors: Khan Arshad , Singh Christopher , Pandey Rajendra , Sheri Anjaneyulu , Padmanabhan Seetharamaiyer , Iyer Radhakrishan , Jagannath Chinnaswamy
Published: January 2023

Tuberculosis is a major cause of death in mankind and drug resistant tuberculosis has posed a problem for therapy. Immunotherapy is an option for better control of tuberculosis. We discovered two types of novel SMNH compounds which were active as adjuvants since they activated respectively NOD2 and TLR-7 receptors in macrophages. Since these cytosolic pattern recognition pathways lead to bactericidal mechanisms, we tested the efficacy of the SMNHs to activate macrophages to kill mycobacteria. Methods: Human THP1 macrophages were treated with either NOD-2 (SB44, SB44-1) or TLR-7 (SB9922) SMNH followed by infection with M. tuberculosis. Three days later macrophages were lysed and plated for bacterial counts. Cytokines from BMs were estimated using sandwich ELISA and autophagic markers were evaluated using monodansyl cadaverine and fluorescence microscopy. Results: Both NOD2 and TLR-7 activating SMNH compounds reduced the numbers of M. tuberculosis in human macrophages by 1-log10 over 3 days of culture. These SMNHs also reduced the numbers of M. tuberculosis within mouse BMs between 1-2log10 over 3 days. SMNHs activated autophagic processes in macrophages underscoring a new mechanism to kill intracellular M. tuberculosis. Since the main scaffold of SB44 has been used as an adjuvant in human trials, we anticipate that SB44 and SB9922 like compounds can be used as adjuncts to drugs for treating ongoing infections of M. tuberculosis in humans.
Heterologous prime-boost with Ad35/AERAS-402 and MVA85A elicits potent CD8+ T cell immune responses in a phase I clinical trial (VAC7P.969)
Authors: Hokey David , O'Dee Dawn , Graves Andrew , Douoguih Macaya , Pau Maria , Versteege Isabella , Sheehan Sharon , Harris Stephanie , Landry Bernard , Sadoff Jerald , McShane Helen
Published: January 2023

Developing an effective vaccine to prevent pulmonary tuberculosis represents a public health priority. AERAS-402 (adenovirus expressing a fusion of M.tuberculosis antigens 85A, 85B, and TB10.4) and MVA85A (Modified Vaccinia Anakara expressing Ag85A) are TB vaccine candidates that have been independently evaluated in adult and infant studies and shown to be safe and immunogenic. Despite robust immune responses in adults, infant trials have revealed lower than expected immunogenicity for these vaccine platforms, possibly contributing to the failure of the MVA85A vaccine in a recent phase II efficacy trial. Complimentary studies of malaria and HIV vaccines report enhanced immune responses using heterologous vaccine approaches with combinations of adenoviral and pox virus vectors. Here we present the preliminary immunogenicity data from a phase I clinical trial conducted in healthy BCG-vaccinated adults that were vaccinated with one or two doses of AERAS-402 and subsequently boosted with MVA85A. Preliminary data indicate that MVA85A boosts both CD4+ and CD8+ T cell responses. Interestingly, MVA85A, which primarily stimulates CD4+ T cell responses when given alone,robustly boosted AERAS-402 CD8+ T cell responses to Ag85A, with average responses reaching approximately 0.5% to 1% of CD8+ T cells. These data suggest that this vaccine combination is highly immunogenic in adults. Further studies are needed to assess the clinical efficacy of this vaccine combination.
Development of a recombinant BCG based vaccine that prevents the infection with the human metapneumovirus stimulating Th1 immunity (VAC7P.975)
Authors: Palavecino Christian , Céspedes Pablo , Gomez Roberto , Kalergis Alexis , Bueno Susan
Published: January 2023

The human metapneumovirus (hMPV) is one of the leading causes of childhood hospitalization and a major health burden worldwide. Unfortunately, due to an inefficient immunological memory, hMPV infection provides limited immune protection. The inadequate immune response causes severe lung inflammation leading to airway obstruction. It is likely that an effective clearance of hMPV require a balanced Th1 type immune response, involving the participation of activated, IFN-γ secreting, T cells. A recognized inducer of Th1 immunity is Mycobacterium bovis bacillus Calmette-Guerin (BCG), which has been used for decades to protect newborns from tuberculosis. Here, we show that a re-infection worsens the viral illness in Balb/c mice, questioning the safety of vaccines based on attenuated virus. We also observed a Th1 pattern of cytokines animals infected as normal infection curse. The immunization with our recombinant BCG strains expressing the Phosphoprotein (P) from hMPV induces a protective Th1 immunity. The mice were protected from clinical disease such us weight loss, inflammatory cells recruitment in airway and viral replication in the lungs. Our recombinant vaccine also induced the activation of hMPV-specific T cells producing IFN- γ and IL-2, which protect from hMPV infection when transferred to naïve mice. These data strongly support the notion that recombinant BCG induces protective Th1 immunity and could be considered as an efficient vaccine against hMPV.
Optimizing oncolytic vaccinia virus and anti-CTLA4 combination therapy to treat cancer (VAC8P.997)
Authors: Rojas Juan , Sampath Padmavathi , Hou Weizhou , Stephen Thorne
Published: January 2023

Monoclonal antibody therapies, such as anti-CD25 or anti-CTLA4, have proven effective in reducing immunosuppressive cells and allowing T-cell responses in tumors. In addition, clinical trials with Oncolytic Vaccinia vectors are showing promising results thanks to the their ability to directly destroy tumors and elicit a potent immune response against cancer cells. Here we examined the possibility for combining both therapies in the treatment of tumor models. Firstly, we compared anti-CD25 and anti-CTLA4 therapies for synergistic effects in combination with Oncolytic Vaccinia. While both combinations were able to reduce tumor growth, anti-CTLA4 combination therapy produced the greatest antitumor response. Interestingly, when anti-CTLA4 antibody and Oncolytic Vaccinia where administered at the same time, immune stimulation driven by the monoclonal antibody facilitated the recognition of the virus, depleting viral replication and reducing antitumor activity. Instead, a two step approach resulted in enhanced anti-tumor effects: firstly, Oncolytic Vaccinia virus is injected and selective-replication occurs in the tumor site; once the virus reached maximum distribution throughout the tumor, anti-CTLA4 monoclonal antibody was added, resulting in enhanced recognition of tumor cells by the immune system. This combination resulted in potent antitumor activity in several orthotopic mouse models, demonstrating the requirement for both immune stimulation and viral replication.
Glycerol channel aquaporin-9 plays a critical role in the formation of memory CD8 T cells after viral infection (VIR4P.1006)
Authors: Cui Guoliang , Kaech Susan
Published: January 2023

Glycerol channel aquaporin 9 (AQP9) has been reported to be highly expressed in memory CD8 T cells post infection, but its function in memory CD8 T cells formation remains unknown. In this study, we report that AQP9 is vitally required for survival and differentiation of memory T cells after viral infection. AQP9 imports glycerol into cells for fatty acid esterification and triglyceride storage. Loss of AQP9 substantially impaired lipid storage and caused antiviral CD8 T cells sensitive to apoptosis. Interestingly, this defect could be rescued by ectopic expression of glycerol channels or triglyceride synthases. Furthermore, IL-7 induces AQP9 expression dependent on Foxo1 pathway. This study revealed an unexpected role of glycerol in CD8 T cell fate decision by regulating energy storage.
Broad alphaherpesvirus cross-reactivity of human CD4 and CD8 T-cells (VIR4P.1014)
Authors: Koelle David , Jing Lichen , Laing Kerry , Russell Ronnie , Barlow Russell , Haas Juergen , Johnston Christine , Wald Anna
Published: January 2023

Peptide-HLA diversity exceeds the TCR repertoire. The resultant T-cell cross-reactivity implies that prior antigen exposure could modulate the outcome of heterologous infections. The alphaherpesviruses HSV-1, HSV-2, and VZV have unexplained ranges of infection severity, and the reasons for partial efficacy of therapeutic live VZV vaccination are unknown. The viruses share 60 homologous ORFs. Using whole viruses, protein sets covering the viral ORFeomes, and peptides, we studied the cross-reactivity of polyclonal and mono-specific CD4 and CD8 T-cells. Effectors were enriched from PBMC using HSV or VZV by direct or cross-presentation (J. Clin. Invest. 122:654) or with tetramers. Cross-reactivity between HSV and VZV was readily detectable. For example, VZV-driven polyclonal CD4 T-cells lines showed a mean of 31% IFN-gamma/IL-2 reactivity to whole VZV and a mean of 11% reactivity to HSV-1, implying that 35% of T-cells were cross-reactive. Cross-reactivity was confirmed at the protein and peptide levels for both CD4 and CD8 T-cells. Cross-reactive CD8 T-cells directly recognized infected target cells, including non-IFN-treated VZV-infected dermal fibroblasts, despite VZV encoding a TAP-blocking protein. We hypothesize that cross-reactive alphaherpesvirus ORFs are rational subunit vaccine candidates to cover HSV and VZV, and that heterologous infections and resulting T-cell cross-reactivity may contribute to variability in infection severity and vaccine responses.
Inhibition of T cell response by Japanese encephalitis virus: a mechanism of immune evasion (VIR5P.1019)
Authors: Cui Min , Yuan Jiaolong , Wang Chong , Zhang Nan , Wang Ke , Wang Kunlun , Hu Pingdong , Lin Xianwu , Yang Hong , Fu Zhenfang
Published: January 2023

Japanese encephalitis virus (JEV) is a mosquito-borne neurotropic virus, which can escape from the peripheral immune response resulting in serious neuroinflammation. However, the mechanism of JEV immune evasion still remains unclear. In our study, it was found that co-inhibitory molecules CD279 (PD-1) and CD272 (BTLA) were upregulated on T cells in JEV infected mouse. The increase of CD279 on CD8+ T cells was more obvious than on CD4+ T cells in the early stage of infection. The expression of CD274 (PD-1 ligand 1) was also upregulated on dendritic cells and macrophages accordingly. As a consequence, both CD4+ and CD8+ T cell population dramatically decreased in the early stage of infection. Moreover, the CD4+CD25+FoxP3+ regulatory T cells (Tregs) increased in the peripheral immune organs after JEV infection. Splenic Foxp3+ Treg cells kept expanding during the disease pregression; instead, in the blood, Tregs only increased in the early stage. IL-10, which indicating the immunosuppression, also dramatically increased in the same location. Collectively, our data suggest that there are four strategies involved in the JEV mediated immune evasion: upregulating the co-inhibitory molecules on T cells and APCs, inducing T cell apoptosis, expanding the Treg cells, and producing inhibitory cytokines. As a consequence, JEV escapes the immune surveillance and causes fatal CNS infection.
Distinct roles of IFNβ and IFNγ in the production of proinflammatory and antiinflammatory cytokines in bone marrow-derived dendritic cells (BA12P.109)
Authors: Yen Jui-Hung , Kong Weimin , Hooper Kirsten , Kuo Ping-Chang , Ganea Doina
Published: January 2023

IFNβ and IFNγ play distinct roles in MS/EAE. IFNγ exacerbates whereas IFNβ ameliorates the disease of EAE. Dendritic cells (DC) paly a sentinel role in the immune system, and are recruited to the CNS to restimulate encephalitogenic T cells during the course of EAE. We investigate how DC are regulated differently by IFNβ and IFNγ on the production of proinflammatory cytokines IL-12 and IL-23 and of antiinflammatory cytokine IL-10. Moreover, the molecular mechanisms of IFNβ and IFNγ-mediated regulatory effects are also investigated. Our results show IFNβ inhibited the production of IL-12 and IL-23, whereas enhanced the production of IL-10 in activated DC. Although both IFNβ and IFNγ inhibited IL-23 production, IFNγ enhanced IL-12 but suppressed IL-10 production. IFNγ-induced STAT-1/IRF-1 activation is required for IL-12 upregulation and IL-23 downregulation. Although STAT-1 activation is responsible for IFNγ-mediated IL-10 downregulation, IRF-1 activation is not involved in STAT-1-mediated IL-10 downregulation. Similar to IFNγ, IFNβ induced STAT-1 activation to suppress IL-23 production, however, the effect is independent of IRF-1 activation. IFNβ-induced Akt activation and GSK3β inactivation might be responsible for IL-12 downregulation and IL-10 upregulation as GSK3β activation is linked to inflammatory responses. In conclusion, our results provide the molecular mechanisms by which IFNβ and IFNγ utilize distinct signaling pathways to regulate the cytokine productions in DCs.
Differential expression of the transcription factor ARID3a in lupus patient hematopoietic progenitors may affect function (BA12P.101)
Authors: Ratliff Michelle , Ward Julie , James Judith , Webb Carol
Published: January 2023

Autologous hematopoietic stem cell (HSC) transplants have been used for severe cases of autoimmune disease, but success rates for systemic lupus erythrematosus (SLE) patients are less than for other autoimmune diseases. We recently found >40% of SLE patients had increased numbers of peripheral B lymphocytes that express the DNA-binding protein ARID3a, and that numbers of ARID3a+ B cells were associated with increased disease activity. Because ARID3a is also expressed in HSCs, we queried if SLE CD34+ cells abnormally express ARID3a. Indeed, nearly 40% of SLE patients had increased numbers of CD34+ cells with higher expression of ARID3a compared to healthy controls. Progenitors with increased ARID3a expression showed enhanced proliferation in vitro relative to other SLE cultures, and those progenitors generated increased numbers of cells that retained CD34 expression. We hypothesize that variation in ARID3a expression in hematopoietic progenitors contributes to variability in engraftment and are testing that hypothesis using humanized mice. Together, these data suggest new functions for ARID3a in HSCs and autoimmunity.
The autoantibody-inducing CD4 T cell (aiCD4 T cell) causing to systemic lupus erythematosus (SLE) belongs to PD-1+CD45RBlo122lo CD4 subpopulation (BA13P.121)
Authors: Miyazaki Yumi , Tsumiyama Ken , Shiozawa Shunichi
Published: January 2023

Objective: We have shown that repeated immunization of mice with antigen reproducibly led to development of SLE, in which a novel T cell type which we term an autoantibody-inducing CD4 T (aiCD4 T) cell was generated via TCR revision at periphery. The aiCD4 T cell induces varieties of autoantibodies and also helps full maturation of CTL to induce lupus tissue injuries. We further showed that aiCD4 T cell seemed to belong to CD45RBlo122lo CD4 T subpopulation. We are trying to assign CD number on this aiCD4 T cell in more detail. Methods: BALB/c mice were repeatedly immunized with OVA. To investigate gene expression profiles of aiCD4 T cell, we performed microarray analysis of CD45RBlo122lo CD4 T cell after immunization 12x with OVA. CD45RBlo122lo CD4 T cells were isolated referring to programmed cell death 1 (PD-1) expression, and these fractionated cells were adoptively transferred into naïve recipients. Autoantibodies in sera of recipient mice were measured 2 weeks after cell transfer. Results: Under microarray analysis, we found that gene expression of PD-1 was increased x2 in the CD45RBlo122lo CD4 subset. Simultaneously, surface expression of PD-1 protein was also significantly increased in this subset. Adoptive cell transfer of PD-1+CD45RBlo122lo CD4 T cells from 12x immunized mice significantly increased both RF and anti-dsDNA antibody in the naïve recipients. Conclusion: The aiCD4 T cell that induces SLE belongs to PD-1+CD45RBlo122lo CD4 subpopulation.
Murine autoimmune cholangitis requires two hits: cytotoxic KLRG1+ CD8 effector cells and defective T regulatory cells (BA13P.122)
Authors: Huang Wenting , Kachapati Kritika , Adams David , Wu Yuehong , Leung Patrick , Yang Guo-Xiang , Zhang Weici , Ansari Aftab , Flavell Richard , Gershwin Merrill , Ridgway William
Published: January 2023

Primary biliary cirrhosis (PBC) is an enigmatic disease mediated by autoimmune destruction of cholangiocytes in hepatic bile ducts. Early immunological events in PBC are poorly understood; clinical signs of disease occur very late in the pathological process. We have used our unique murine model of PBC in dominant-negative TGF-β receptor type II transgenic mice to delineate critical early immunopathological pathways, and previously showed that dnTGFβRII CD8 T cells alone transfer biliary disease. Herein we report significantly increased numbers of hepatic terminally differentiated (KLRG1+) CD8 T cells in dnTGFβRII compared to B6; these cells are the major cytotoxic CD8 subset. Unexpectedly, the disease is not simply CD8 T cell intrinsic since mixed bone marrow chimeric mice were protected from biliary disease. Adoptive transfer studies showed that co-transfer of B6 but not dnTGFβRII Tregs prevented dnTGFβRII CD8 T cell mediated cholangitis; the protection was associated with decreased numbers of hepatic KLRG1+ CD8 T cells and with elimination of hepatic dnTGFβRII CD8 mediated cholangiocyte cytotoxicity. DnTGFβRII Treg cells were inferior in suppressing effector CD8 T cells in vitro compared to wild type B6 Tregs. In conclusion, these results emphasize that autoimmune cholangitis requires defects in both the T effector and regulatory compartments, which has important implications for understanding the early pathogenesis of human PBC.