bluuebunny/crossref_metadata_2025 huggingface dataset:
Distinct transcriptome of regulatory T cells in prostate cancer (162.18)
Authors: Arredouani M. Simo , Kissick Haydn , Dunn Laura , Sanda Martin
Published: January 2023

The inhibitory role of regulatory T-cells (Tregs) in cancer is now well established. Furthermore, inhibition of Treg function has been shown both experimentally and clinically to improve the immune response towards a variety of cancers. Developing new and more effective strategies interfering with the function of Tregs in cancer requires a deep understanding of Treg suppressor machinery, and a thorough dissection of the molecular elements that orchestrate their differentiation from T cells. With this in mind, we sorted Tregs and non-Treg CD4 cells from the prostate-draining LN or spleens of prostate tumor-bearing male mice and their control age-matched controls. Gene expression profiling was performed and data were analyzed to identify differential gene expression and major dysregulations in biological pathways, functions and networks. These changes encompassed key pathways such as T cell differentiation, TREM1, IL-17 and IL-10 signaling, as well as many important biologic functions that include inflammation, cellular movement, immune cell trafficking and cell-to-cell interaction. In addition, the most affected network is centered on TGF-β. Interestingly, prostate cancer Tregs exhibit many markers that are distinctive of macrophage and B cell lineages. This work paves the way for a better understanding of Treg biology in the context of prostate cancer and offers new avenues for Treg manipulation toward improving the outcome of immunotherapy.
Evidence for a BCR-ABL-induced anergic state of iNTK cells in chronic myeloid leukemia patients at diagnosis (162.32)
Authors: Levescot Anais , Rossignol Alexis , Jacomet Florence , Robin Aurélie , Basbous Sara , Giraud Christine , Roy Lydia , Guilhot François , Turhan Ali , Barra Anne , Herbelin André , Gombert Jean-Marc
Published: January 2023

Chronic myeloid leukemia (CML) is a myeloproliferative disorder, caused by the presence of the Philadelphia chromosome and the consecutive chimerical BCR-ABL oncoprotein, with deregulated tyrosine kinase (TK) activity. Whether invariant NKT (iNKT) cells, which are documented for their anti-tumoral activity, are affected during the disease remains unknown. Here, we addressed this issue by showing that proliferation rate of blood iNKT cells in response to their cognate ligand α-galactosylceramide is altered in CML patients at diagnosis, as compared to healthy subjects. This anergic state of iNKT cells was associated with both intrinsic and APC-dependent alterations. Furthermore, expression of perforin and CD95-L as well as that of PLZF, a transcription factor that is requisite for maintenance of functions of iNKT cells, were also decreased in patients at diagnosis. Importantly, all these functional impairments were corrected in patients who achieved a complete cytogenetic remission with the TK inhibitor imatinib mesylate (IM) or IFN-γ therapy. Together, these data demonstrate that chronic phase CML is associated with an acquired but potentially reversible defect in iNKT cells. Given the critical role of iNKT cells in tumor surveillance, it is tempting to propose that iNKT cell dysfunctions associated with CML thus could be one way used by tumors to escape from the anti-CML immune response while IM and IFN-γ exert their anti-leukemic effects in part in targeting iNKT cells.
Title: HMGB1 both enhances and blocks myeloid-derived suppressor cell potency Katherine H. Parker, Suzanne Ostrand-Rosenberg Department of Biological Sciences, University of Maryland Baltimore County, Baltimore MD 21250 (162.40)
Authors: Parker Katherine , Ostrand-Osenberg Suzanne
Published: January 2023

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells that accumulate in individuals with cancer and inflammation and play a pivotal role in tumor immunity by suppressing T-cell activation and secreting proinflammatory molecules. MDSC potency is mediated by immune suppressive factors such as inducible NO synthase (iNOs), arginase, and reactive oxygen species (ROS). MDSC also impair tumor immunity through crosstalk with macrophages in which IL-10 production increases while IL-12 production decreases, causing a shift in innate and adaptive immunity towards a type 2 tumor-promoting phenotype. Nuclear protein, High Mobility Group Box1 (HMGB1) is the second most abundant protein within a cell and is released from myeloid cells as a danger response to sepsis, infection, or arthritis. It’s release promotes inflammatory responses. HMGB1 signals through a multitude of receptors including TLR4 and RAGE, which are expressed by MDSC. In contrast to other inflammatory mediators which increase MDSC potency, HMGB1 reduced the suppressive mechanisms of TLR4-\- and wildtype MDSC, by reducing ROS in TLR4-\- MDSC and iNOs levels in wildtype MDSC. Glycyrrhizin, an HMGB1 inhibitor, decreased IL-10 in co-cultures of macrophages and MDSC. These findings suggest that HMGB1 plays both anti- and pro-inflammatory roles in regulating MDSC suppressive activity.
Total body irradiation enhances the anti-tumor efficacy of adoptively transferred CD8+ T cells stimulated ex vivo with IL-12 (162.41)
Authors: Johnson C. Bryce , Cloud Colleen , Rubinstein Mark , Cole David
Published: January 2023

Stimulation of T cells with IL-12 improves their cytolytic capabilities and ability to persist in vivo. Despite these enhanced functional characteristics, we have found that these cells can only eliminate established tumors in mice preconditioned with cyclophosphamide (CTX). While this result demonstrates the importance of lymphodepletion in adoptive cell transfer (ACT) regimens, it is unclear if other forms of lymphodepletion would also enhance the efficacy of IL-12 primed cells. Since the most successful lymphodepleting regimen in metastatic melanoma clinical trials uses total body irradiation (TBI), we sought to determine if TBI preconditioning combined with ACT of IL-12 stimulated pmel-1 CD8+ T cells might be similarly synergistic. When we adoptively transferred pmel-1 CD8+ T cells primed with antigen and IL-12 (pmelIL-12) into mice preconditioned with TBI, we saw significantly elevated numbers of donor T cells compared with mice adoptively transferred with pmel-1 CD8+ T cells primed with antigen alone (pmelsham). Importantly, the increase in donor T cell numbers seen with the combination of TBI + pmelIL-12 translated to significant delays in melanoma outgrowth. Strikingly, the addition of pmelsham to TBI preconditioning resulted in no improvement in anti-tumor immunity compared to TBI alone. Thus both CTX and TBI host preconditioning greatly enhance the anti-tumor efficacy of transferred T-cells stimulated with IL-12.
Gut commensal bacteria promote anti-tumor innate immune responses in distant tumors after immunotherapy and chemotherapy (162.5)
Authors: Iida Noriho , Stewart Charles , Goldszmid Romina , Dzutsev Amiran , Trinchieri Giorgio
Published: January 2023

Experimental evidence in models of autoimmunity and infection indicated that systemic innate and adaptive immune responses are influenced by gut commensal bacteria and impaired by treatment with antibiotics. This study aimed to determine the effect of commensal flora on tumor-associated inflammation and responsiveness to immunotherapy and immunogenic chemotherapy. To deplete the gut commensal flora, C57BL/6 mice were administered an antibiotic cocktail including neomycin, vancomycin and imipenem in the drinking water, resulting in a 3-log reduction in fecal bacteria. In mice bearing subcutaneous MCA38 colon carcinoma or B16 melanoma, this antibiotic treatment suppressed immunotherapy-induced tumor necrosis following intratumoral CpG treatment in combination or not with systemic anti-IL-10R antibody injection. TNF is a key effector molecule for tumor destruction in CpG-based therapy. Antibiotic-treated mice showed reduced frequency of tumor-infiltrating TNF-producing myeloid cells after therapy. Administration of single antibiotics revealed that tumor TNF production correlated with the remaining number of gut bacteria. Oral administration of LPS restored TNF production in antibiotics-treated mice. Finally, the chemotherapeutic effect of the immunogenic cytotoxic drug oxaliplatin was impaired in antibiotic-treated mice. Altogether, these results suggest that optimal immunotherapeutic and chemotherapeutic responses against cancer may require an intact commensal flora.
Antigen persistence regulates the functional characteristics and stability of secondary Th1 responders (163.10)
Authors: Jay David , Kim Chulwoo , Williams Matthew
Published: January 2023

CD4+ memory T cells exhibit a skewing towards high functional avidity during memory maintenance. We find that heterologous rechallenge of LCMV-induced CD4+ memory T cells from LCMV-immune mice with a recombinant Listeria monocytogenes that secretes the LCMV-derived glycoprotein peptide GP61-80 (Lm-gp61) preserves the high functional avidity of secondary Th1 effector cells and their subsequent stability within the memory pool. Conversely, adoptive transfer of CD4+ memory T cells into naïve mice prior to rechallenge results in a loss of functional avidity by secondary Th1 effector cells and poor maintenance of secondary memory. Loss of function is not due to the transfer itself, as transfer into LCMV-immune hosts allows secondary Th1 effector cells to maintain their enhanced function after Lm-gp61 challenge. LCMV-immune mice clear an Lm-gp61 infection more quickly than naïve mice, suggesting that the infectious environment of the secondary challenge is critical for the acquisition of secondary Th1 effector function. Artificially extending the inflammatory response alone following secondary challenge does not adversely affect the functional avidity of CD4+ memory T cells into LCMV-immune recipients. Instead, our findings indicate that upon rechallenge, secondary Th1 effector and memory cells are exquisitely susceptible to functional fine-tuning based on the persistence of infectious antigen.
T helper 17 cell lineage commitment is dictated by DC populations resident in priming tissues (163.23)
Authors: Hu Wei , Troutman Ty , Pasare Chandrashekhar
Published: January 2023

Activation of pattern recognition receptors on dendritic cells (DCs) and macrophages leads to secretion of cytokines that control activation and differentiation of CD4 T cells. While IL-12 is critical for Th1 lineage development, IL-6 and TGF-β play an important role in differentiation of Th17 cells in vitro. IL-1 has also been implicated in regulating Th17 differentiation. In the present study, we examined the relative contribution of IL-6 and IL-1 for in vivo Th17 differentiation and found that requirements for Th17 polarization depend entirely on the site of priming. We found that IL-6 plays a critical role in Th17 lineage priming in the skin and the mucosal tissue such as the gut and the lung, but is completely dispensable for Th17 priming in the spleen. IL-1 is however necessary for Th17 priming in all tissues. We discovered that differential cytokine requirements for Th17 lineage commitment are guided by differential population of DCs resident in different tissues. We found that CD103+ DCs that are present in the skin and intestinal lamina propria, but not in the spleen, favor the generation of iTreg cells while suppress Th17 priming, and IL-6 is required to overcome the effects of CD103+ DCs. Th17 lineage commitment is therefore differentially regulated in systemic and mucosal immune systems. Further work will focus on the dynamic regulation of CD103+ DC by the intestinal microbiota, and the role of CD103+ DCs in controlling intestinal immune homeostasis.
Detrimental effects of T regulatory cell-promoting IL-2-anti-IL-2 complexes in staphylococcal toxic shock syndrome. (164.13)
Authors: Rajagopalan Govindarajan , Tilahun Ashenafi
Published: January 2023

Menstrual or non-menstrual toxic shock syndrome (TSS), caused by the superantigen exotoxins of Staphylococcus aureus or Streptococcus pyogenes, is characterized by a robust activation of the immune system, systemic cytokine/chemokine storm, multiple organ failure and possibly death, if not treated promptly. We tested whether increasing the numbers of Treg in vivo by administration of IL-2-anti-IL-2 complexes (IL2C) could be beneficial in TSS. Administration of IL2C [prepared by incubating 1µg of IL-2 (Peprotech) and 5µg anti-IL-2 (eBioscience, clone-JES6-1A12) in a total 200 µl at 37oC for 30 min] on days 0, 1 and 2, caused a 3 to 4-fold increase in CD4+CD25+FoxP3+ Treg numbers in the spleens of HLA-DR3 transgenic mice by day 5. However, upon challenge with staphylococcal enterotoxin B (SEB, 50µg), compared to untreated HLA-DR3 transgenic mice, IL2C-treated mice produced significantly higher levels of IFN-γ (1714±1365 versus 6642±2253 pg/ml), MIP-1α (782±23 versus 2305±376 pg/ml,), MIP-1β, RANTES and TNF-α. SEB-induced mortality was also significantly increased in the IL2C treated group. While only 1/5 mice died in untreated group, 6/11 mice died in the IL2C treated group. In conclusion, even though administration of IL2C significantly increased the Treg numbers, such a treatment also increased their susceptibility to TSS. Hence, the benefits of T reg-promoting IL-2-anti-IL-2 complexes in TSS or related conditions need more stringent evaluation.
CD73-generated adenosine limits immunopathology during acute Toxoplasma gondii infection (164.17)
Authors: Mahamed Deeqa , Mills Jeffrey , Bynoe Margaret
Published: January 2023

As an obligate intracellular pathogen, the apicomplexan parasite Toxoplasma gondii elicits robust innate and adaptive immune responses that must be limited to prevent immunopathology. The breakdown of extracellular ATP to adenosine by the cell-surface enzyme CD73 plays an important role in the resolution of inflammation. The role of extracellular adenosine in the immune response to T. gondii infection has not been previously elucidated. In comparison to wildtype C57BL/6 mice, CD73-knockout mice infected with T. gondii by interperitoneal injection were highly susceptible to immune-mediated pathology, with significantly increased morbidity and mortality associated with marked infiltration of neutrophils and T cells into the peritoneal cavity. Peritoneal exudate cells from infected CD73KO mice generated higher levels of the inflammatory mediators nitric oxide, TNF-alpha, and IL1-beta, without enhanced parasite killing or clearance. The increased morbidity and mortality seen in CD73KO mice could be rescued by treating mice with the broad spectrum adenosine receptor agonist 5'-N-ethylcarboxamido adenosine. In addition, mice deficient in the adenosine receptor A2A were more susceptible to immunopathology during intraperitoneal infection with T. gondii compared to wildtype mice. Thus extracellular adenosine is a key molecule that regulates the immune response to an intracellular pathogen and promotes host survival.
The role of PKCθ in Francisella tularensis infection in mice (164.4)
Authors: Korzekwa Katy , Lovchik Julie , Wu Terry , Cannon Judy
Published: January 2023

Francisella tularensis is a facultative intracellular bacteria that causes tularemia in animals and humans. F. tularensis is highly virulent and can be lethal. T cells play a critical role in Francisella immunity. It has been shown that mice succumb to sublethal infections when all T cells are depleted; however individual subsets including CD4, CD8 and an unusual CD4-CD8-NK1.1- population can be protective. While T cells control infection by producing IFNγ and TNFα, other less well-characterized mechanisms also play a role. PKCθ is a protein kinase involved in T cell activation; PKCθ is important in IFNγ production and PKCθ-/- mice are protected against both TH1 and TH2 mediated diseases. To investigate the role of PKCθ in F. tularensis infection, we infected wild-type C57Bl/6 (WT) mice and B6.PKCθ-/- mice with a lethal dose of LVS (live vaccine strain) which is commonly used to study F. tularensis infection. While WT mice died within 2 weeks of infection, surprisingly, over 50% of the PKCθ-/- mice survived past 3 weeks. We found that infected PKCθ-/- mice had more CD4-CD8- T cells in liver and lung than WT mice. In contrast, PKCθ-/- mice had fewer CD4+ T cells in lymph nodes, spleen, liver, and lung. We found no difference in numbers of activated T cells in liver and lung of infected WT and PKCθ-/- mice. These results show that PKCθ may be important in the expansion of CD4+ and CD4-CD8- T cell subtypes and this shift in the absence of PKCθ may protect against LVS infection.
Combinational dasatinib and dendritic cell-based therapy improves therapeutic protection against melanoma growth (165.11)
Authors: Lowe Devin , Bose Anamika , Poudel Ravin , Taylor Jennifer , Storkus Walter
Published: January 2023

The solid tumor microenvironment (TME) is characterized by an abnormal vasculature system that promotes interstitial pressure, hypoxia, and acidity - diminishing the efficacy of standard single agent therapeutics. We hypothesize that combinational strategies that effectively target cancer cell growth and vasculature development will improve the clinical outcome of patients with malignant disease such as melanoma. To address this hypothesis, C57BL/6 mice were challenged subcutaneously with B16 melanoma cells and tumors allowed to progress 7 days. Animals were left untreated or received daily doses of dasatinib (an FDA-approved receptor tyrosine kinase inhibitor drug [TKI]) for one week with/without intratumoral injections of dendritic cells (DCs) transduced with IL-12 (DC.IL12) on days 7 and 14 post-tumor inoculation. Our pre-clinical results indicate synergistic benefits from co-treatment with dasatinib/DC.IL12 therapy based on the magnitude of anti-tumor immune responses promoted and the level of protective T cells recruited into treated tumor lesions. Dasatinib modified the aberrant TME in vivo by diminishing Treg (CD4+Foxp3+) and MDSC (CD11b+Gr1+) frequencies and reducing expression of markers associated with hypoxia, angiogenesis, and cancer stem cells. Overall, these data highlight the advantage of “sensitizing” the host using an anti-vasculature drug to instill an environment conducive to recruiting and sustaining vaccine-initiated anti-tumor immune effector cells.
Enhanced MHC Class I and II expression induced following staggered combination treatment of human tumor cells with Hsp90 inhibitors and Interferons. (165.16)
Authors: Kurnick James , Haggerty Timothy , Rose Lenora , Dunn Ian , Newton Estelle , Pandolfi Franco
Published: January 2023

We have previously shown that Hsp90 inhibitors (iHsp90) can enhance both differentiation antigens and MHC Class I in melanomas and gliomas. To extend these findings, we treated a series of tumors including cancers of the breast, cervix, osteosarcoma and lymphomas to examine the effects of iHsp90 on both Class I and II MHC. Class I MHC was enhanced in most tumors, but induction of MHC Class II antigens was NOT induced in MHC Class II-negative tumors. Since both Types I and II interferon’s (IFN) are known to enhance MHC antigen expression, we wished to determine they would synergize with iHsp90s. However, since iHsp90s can inhibit IFN-signaling, we varied the time of exposure of the tumor cells to the IFNs and iHsp90s to determine if we could circumvent the counteracting effects. While IFN-gamma stimulated strong Class II MHC expression, the simultaneous addition of iHSP90 largely prevented this MHC Class II expression. However, pretreatment for 24 hrs with IFN-gamma allowed iHsp90 to further enhance MHC Class II expression. Similarly, synergistic enhancement of MHC Class I expression was noted if either IFN-beta or IFN-gamma were added prior to addition of iHsp90. Staggering treatment with interferon’s and iHsp90s yielded strong induction of both Class I and II MHC antigens. As both IFN and iHsp90 increased differentiation antigens and MHC, the further increase in MHC expression from the drug combination could be of particular utility in cancer immunotherapy.
Effects of age on the immune response in baboons to the Y. pestis F1 vaccine antigen. (166.24)
Authors: Bonnel Caroline , Arteaga-Cortes Lourdes , Ench Yasmin , Leland M. , Dube Peter , Kraig Ellen
Published: January 2023

The ability to mount effective immune responses declines with age, which correlates clinically with decreased vaccine efficacy and increased susceptibility to infection and cancer. As a result, new vaccines should be tested for efficacy in older individuals. Towards this end, young (5-6 years of age) and old (17-22 years) baboons were immunized with the LcrV candidate vaccine antigen from Yersinia pestis. Surprisingly, older baboons had a strong humoral response [JI 181:109] suggesting either that the baboon immune system is less affected by aging or that LcrV acts like a recall antigen. Thus, a second Y. pestis vaccine antigen, the F1 protein, was tested; F1 is unique to this bacterial species and would be seen as a “new” antigen. The antibody response to F1 did decline with age, but the T cell response did not. Thus, an analysis of the effects of aging on T cell fine specificity was undertaken. T cell proliferation and IFN-γ ELISpots were used to map which of 32 overlapping synthetic F1 peptides stimulated T cells from the immunized baboons. A Th1 response was observed for a few individual animals in response to whole both F1 antigen and specific peptides. ELISpots specific for IL-4 and IL-5 cytokines are currently being used to follow Th2 cells specific for F1. Future efforts will focus on generating F1-specific T cell clones using herpesvirus papio transformed B cell lines as antigen presenting cells.
A novel human Neonatal Tissue Construct (NTC) models age-specific immune responses to Bacille Calmette-Guérin (BCG) vaccine (166.27)
Authors: Sanchez-Schmitz Guzman , Stevens Chad , Baecher-Allan Clare , Levy Ofer
Published: January 2023

Newborns have a distinct immune system that impairs immune responses to many conventional pediatric vaccines rendering them susceptible to preventable infections. Since birth is a reliable point of healthcare contact worldwide, effective single-dose birth immunization could help reduce global morbidity and mortality. However, vaccine development has been largely ad hoc and empiric. Modeling age-specific human immune responses could help predict safety and efficacy of adjuvants and vaccines targeting the newborn. Using human primary endothelial cells, human extracellular matrix, newborn monocytes and 100% autologous intact plasma we assembled the Neonatal Tissue Construct (NTC). In this tri-dimensional micro-vascular interstitium extravasated CD33+ monocytes autonomously differentiated into HLA-DRhigh CD86high and CCR7+ mature Dendritic Cells (DCs) in response to the live mycobacterial BCG vaccine known to be safe and effective at birth, and to induce effector T cell responses. BCG-pulsed NTC-derived DCs induced autologous naïve CD4+ T cell proliferation and activation (CD45RO+, TNF-α), as efficiently as DCs from an Adult Tissue Construct. While newborn and adult DC:T cell co-cultures secreted IL-2 and TNF-α in response to BCG, only newborn cells demonstrated constitutive IL-4 production and a marked impairment in IFN-γ production. In recapitulating neonatal responses to BCG, the NTC demonstrates its potential as a novel platform to study age-specific vaccine responses.
Interferon-γ-activated human leukocyte antigen-G gene expression in placenta-derived multipotent cells involves the epigenetic control and transcriptional regulation (167.12)
Authors: Chen Pei-Min , Hsu Pei-Ju , Wei Chung-Fan , Liu Ko-Jiunn , Sytwu Huey-Kang , Hsu Ping-Ning , Yen B. Linju
Published: January 2023

Placenta-derived multipotent cells (PDMCs) are progenitor cells isolated from the human term placenta with differentiation capacity for all three germ layers as well as strong immunomodulatory properties. The placenta is central to maintaining fetomaternal tolerance, and one of the most important immunomodulatory molecules involved is the human leukocyte antigen-G (HLA-G). HLA-G is a non-classical major hisotocompatibility complex (MHC) class I molecule which exhibits a restricted tissue-specific expression and exerts multiple immuneregulatory functions for maintaining the tolerance of placenta. We have shown that PDMCs can resist natural killer (NK) cell-mediated cytotoxicity by interferon-γ (IFN-γ)-induced surface HLA-G expression, suggesting that the expression of HLA-G may be involved the cis-acting epigenetic control and transcriptional regulation. Recently, we found that IFN-γ induces the expression of IFN regulatory factor 1 (IRF-1) and phosphorylation of STAT-1 in PDMCs. We speculate that the control of HLA-G in PDMCs involved not only the IFN-γ cis-acting regulatory elements including the IFN-stimulated response element (ISRE) and IFN-γ activation site (GAS), but also DNA-methylation which synergistically render the HLA-G gene promoter highly responsive to IFN-γ. Taken together, our results indicate that IFN-γ-activated HLA-G gene expression in PDMCs is involved the cis-acting epigenetic control and transcriptional regulation through JAK/STAT-1 signaling pathway.
Engagement of Glucocorticoid-induced leucine zipper (GILZ) with Infectious Bursal Disease virus (IBDV) VP4 suppresses type I interferon expression and enhances IBDV growth in host cells (168.13)
Authors: Zheng Shijun , Wang Yongqiang , Li Zhonghua , Li Xiaoqi , Cao Hong
Published: January 2023

Infectious Bursal Disease (IBD) is an acute, highly contagious and immunosuppressive avian disease caused by IBD virus (IBDV). Although IBDV-induced immuno-suppression has been well-established, the exact molecular mechanism for such induction is unclear. We report here that IBDV VP4 is an interferon-suppressor by interacting with the Glucocorticoid-induced leucine zipper (GILZ) in host cells. We found that VP4 suppressed the expression of type I interferon in HEK293T cells with Tumor Necrosis Factor(TNF)-α treatment or Sendai virus (SeV) infection, and this suppression could be completely abolished by knockdown of GILZ by siRNA. Furthermore, knockdown of GILZ significantly inhibited IBDV growth in host cells. Thus, VP4-induced suppression of type I interferon is mediated by interacting with GILZ, a protein that appears to inhibit cell responses to IBDV infection.
Structural basis of MHCI antigen presentation sabotage by cowpox encoded protein CPXV203 (168.18)
Authors: McCoy William , Wang Xiaoli , Yokoyama Wayne , Hansen Ted , Fremont Daved
Published: January 2023

Viral dissemination can be greatly facilitated by immune evasion proteins that subvert MHCI antigen presentation, thereby conferring the ability to evade cytotoxic T-lymphocyte (CTL) detection and clearance. Surprisingly, ablation of MHCI-specific immune evasion proteins has been reported to be dispensable for both immediate and latent MCMV and RhCMV infections, while murine infection by the equivalent mutant cowpox virus (ΔCPXV012, ΔCPXV203) showed attenuated virulence in a CTL-dependent manner. Here we show that the cowpox virus encoded CPXV203 protein inhibits MHCI surface expression by directly binding mature MHCI heterodimers, thus bridging MHCI to the KDEL receptor (KDELR) rescue pathway via the viral protein’s C-terminal ER-retention sequence (KTEL). Similar to KDELR/KDEL binding, CPXV203/MHCI binding was found to be pH-dependent, insuring high-affinity association of KDELR/CPXV203/MHCI in the Golgi for retrieval to the ER. Ensuing structural studies of CPXV203/MHCI revealed that CPXV203 binds MHCI through conserved tapasin, CD8, & NKR interaction sites. Mutagenesis of the CPXV203/MHCI interface followed by cellular & biophysical studies identified the critical importance of CPXV203 recognition of the MHCI α3 domain and CPXV203 histidines that confer pH-dependent binding. These studies clarify mechanistically how CPXV203 coordinates with CPXV012 to effectively block antigen presentation through the targeting of distinct MHCI assembly states.
Ecto-5'-nucleotidase (CD73) regulates host inflammatory responses and bacterial persistence during murine Salmonellosis (169.10)
Authors: Alam Mohammad , Kuo Jennifer , Pereira Marion , Gaines Dennis , Bigley Elmer , Ernst Peter , Williams Kristina
Published: January 2023

Salmonella spp. is responsible for the vast majority of foodborne illnesses in the US. Adenosine is an antiinflammatory mediator that limits tissue damage during inflammation. Surface enzymes CD39 & CD73 mediate the synthesis of extracellular adenosine & can regulate immune responses. We studied the expression of CD39 & CD73 in liver & spleen after infection of C57BL/6 mice with Salmonella enterica serotype Typhimurium (ST) & evaluated the role of CD73 after oral infection of wildtype & CD73-KO mice, in regulating immune responses & bacterial persistence. Liver & spleen cytokine mRNA expression was tested by RT-PCR. Cytokines were assayed by ELISA from cultured supernatant of activated splenocytes. Splenocyte-derived Th cells were tested for intracellular cytokines by FACS. Inflammatory responses in infected CD73-/- mouse livers were tested in H&E sections. Bacterial count was done from liver tissues. After ST infection of wildtype mice, both CD39 & CD73 transcript levels declined in spleen & liver. Splenocytes from ST-infected CD73-/- mice produced significantly more IFN-γ & IL17a. Infected CD73-/- mice had higher expressions of IFN-γ & TNF-α mRNA in liver. Histological examinations of liver sections showed significantly greater number of “inflammatory-foci”. Liver bacterial load was significantly lower in CD73-/-mice. CD73 expression contributes to adenosine accumulation that attenuates inflammation during Salmonellosis & may impair immunity to favor bacterial persistence.
Linking IL-17 signaling to TRAF6 transcriptome activities during OC-or-/DDOC-mediated osteoclastogenesis (169.15)
Authors: Liu Yenchun , Chiang Min Shyuan , Wang Yan Hsiung , Teng Yen Tung
Published: January 2023

RANKL/RANK-OPG regulates osteoclastogenesis; whereas TRAF6 is an adaptor-protein associated -with bone-loss. Here, we investigate how these signal-interactions work between RANKL/RANK and cytokines’ to activate TRAF6-transcriptome and osteoclastogenesis in-vitro/in-vivo. Methods: A 1.1-kb mTRAF6-promoter-region was cloned into pCR-BluntII-TOPO-vector. Later, purified CD11c+dendritic-cell-derived osteoclasts(DDOC) & RAW264.7 stably transfected with pGL4-mTRAF6-Luc are stimulated in the presence/absence of RANKL/cytokines (i.e., IL-17, etc.) in-vitro, then measured the transcription via luciferase-activity, and correlated with mRNA/protein expressions by RT-PCR & western-blots, respectively, and TRAP-mediated resorptive pits (p<0.05). Further, EMSA-vs.-ChIP assays are employed to reveal transcriptional binding-motifs on mTRAF6-promoter. The results showed that: i) 1.1-kb of mTRAF6-promoter-upstream contain critical binding-motifs stimulated by RANKL and osteotropic cytokines; ii) while RANKL/IL-17 both mediate positive signaling on mTRAF6-transcriptome, IL-17 of different-dose also inhibited its responses; iii) there are 3-or-4 transcription binding-motifs critical for osteoclatsogenesis; iv) IL-17-depletion leads to inhibition of alveolar-bone-loss induced by anaerobes in diabetic-NOD -mice in-vivo. Conclusion: These findings suggest that RANKL can work alone or interactively with IL-17 (or TNF-α) via mTRAF6-transcriptome-machinery on OCp/DDOC-mediated bone -loss.
Effect of Statins in influenza (169.22)
Authors: Ghosh Sanjukta , Ezquerro Fernando , Kobzik Lester
Published: January 2023

Excessive inflammatory immune responses are a major cause of influenza morbidity and mortality. Some epidemiological studies find reduced mortality from influenza in patients on statins. This suggests that the anti-inflammatory properties of these drugs could be useful. We first tested the effect of acute administration of 100 mg/kg pravastatin i.p. per day for 3 days, starting either at 1 day prior to, 2 days after, or 6 days post infection. For all three regimens, we observed increased, not decreased, mortality compared to control groups (infection alone). We then sought to better model the epidemiologic data in which a majority of patients are taking statins before hospitalization for influenza. We used longer-term administration (10 days) prior to influenza of pravastatin or simvastatin, followed by continued therapy for 7 days after infection. Chronic statin treatment of mice suppressed early inflammatory responses measured in lung lavage at 36 hours (decreased TNF release by ~50%, and reduced protein leakage), but did not change these parameters later in the course (days 3 and 7). Long-term pretreatment with statin, continued through first week of influenza, did not result in the enhanced mortality observed with the acute treatments. The potential benefit, if any, of the early anti-inflammatory effect of statins in long-term users remains to be determined.
The innate immune response to cytomegalovirus: the kick-start (170.1)
Authors: Verma Shilpi , Fukuyama Satoshi , Ware Carl , Benedict Chris
Published: January 2023

Like all herpesviruses, cytomegalovirus (CMV, a β-herpesvirus) persists for the lifetime of its host. Primary infection is sub-clinical in healthy individuals, but can cause serious disease when immunity is compromised or naïve. We have previously shown the initial type I Interferon (IFNαβ) produced by the splenic stroma within hours after mouse CMV (MCMV) infection is dependent on lymphotoxin (LT)-LTβR signaling. We now show it is splenic marginal zone (MZ) stromal cells that are initially infected by MCMV and produce this initial IFNαβ. This “first wave” of IFNαβ is critical to restrict MCMV production from the MZ stroma, inhibiting the earliest measurable viral burst by 100-1000 fold. NK cells are key controllers of MCMV in the first few days of infection, but their role in restricting this first burst of MCMV is unclear. Depleting NK cells revealed them to contribute to initial control of MCMV production from the MZ stroma. Additional studies revealed NK cells can mediate their earliest effector function(s) independently of both IFNαβ and Ly49H, a key NK activating receptor that binds the MCMV m157 protein. Strikingly, the levels of MCMV produced from MZ stroma at the earliest time measurable are the highest observed at any subsequent time point prior to immune control of MCMV. These results highlight the importance of both IFNαβ and NK cells in controlling initial MCMV replication and spread, establishing a baseline for MCMV production over the course of acute infection.
FIV infection activates Regulatory T cells expressing GARP-bound TGFb complexes which suppress T helper cells and may contribute to immune dysfunction (170.12)
Authors: Miller Michelle , Tompkins Mary
Published: January 2023

Here we identify on feline Treg cells Glycoprotein A Repititions Predominant (GARP), a cell surface anchor for TGFb, as a functional protein in Treg-mediated suppression. We demonstrate co-precipitation of mature TGFb with GARP from feline CD4+CD25+ cells. The GARP:TGFb complex is expressed on the surface of highly effective suppressors, as depletion of these cells from the Treg pool reduces suppression of CD4+CD25- targets as measured by IL2 inhibition. In vitro activation of CD4+CD25+ cells upregulates surface GARP:TGFb as measured by flow cytometry. Surface phenotyping of PBMCs from noninfected or chronically FIV-infected cats reveals a higher percent of GARP+TGFb+ Tregs and of TGFbRII+ T helper targets in FIV+ cats. At days 3 or 6 post in vitro FIV infection CD4+CD25+ cells upregulate GARP:TGFb and CD4+CD25- upregulate TGFbRII. In vivo infection studies were performed and expression of GARP, TGFb or TGFbRII was evaluated on PBMC for 13 weeks post infection. Acute FIV infection generated increased GARP:TGFb expression on CD4+CD25+ cells one week post infection and a corresponding increase in TGFbRII on CD4+CD25- consistent with in vitro study results. We conclude that GARP: TGFb is increased on the surface of Treg cells during acute FIV and contributes to the CD4+ T cell anergy via ligation of TGFbRII on the surface of CD4+CD25- T helpers. We hypothesize that FIV infects Treg cells in vivo, resulting in activation of these suppressor cells and chronic T helper dysfunction.
Immunomodulatory role of acute cigarette smoke exposure in pdmH1N1 and H9N2 infection (170.14)
Authors: Han Yan , Ling Manto , Mao Huawei , Tu Wenwei , Lau Yulung
Published: January 2023

Cigarette smoke has both immune-activating and suppressing activities. Mouse studies have demonstrated that smoke exposure increases the expressions of some factors likely to be important in the antiviral defense. However, there is no report about the role of cigarette smoke in 2009 pandemic H1N1 (pdmH1N1) and avian influenza A H9N2 (H9N2/G1) infection. In this study, C57/B6N mice were whole body exposed to 4% cigarette smoke 4 hours per day for 21 days and then infected with pdmH1N1 or H9N2 virus. Some mice were exposed to room air in parallel as the control group. Mortality, weight changes, lung virus loads and inflammatory cytokines and chemokines were analyzed. For pdmH1N1 infection, although no significant difference in the lung virus loads was shown between the two groups, smoke exposure group had a significantly lower mortality than the air exposure group, which may be caused by the lower inflammatory response in the smoke exposure group than in the air exposure group at day 5 post virus infection. Similarly, after H9N2 virus infection, the mice in smoke exposure group displayed a significantly milder disease, as evidenced by a less weight loss, as well as lower inflammatory response compared to the air exposure group. Our study provided the first in vivo evidence that acute cigarette smoke exposure may modulate the effect of influenza A virus infection. The underlying mechanism is under investigation.
Pathogenesis of Newly Discovered Severe Fever with Thrombocytopenia Syndrome Bunyavirus in Mice (170.19)
Authors: Jin Cong , Liang Mifang , Ning Junyu , Gu Wen , Jiang Hong , Zhu Hua , Qin Chuan , Li Dexin
Published: January 2023

Severe fever with thrombocytopenia syndrome (SFTS) was an emerging zoonotic disease identified since 2009 in multiple provinces in China. A novel phlebovirus in the family Bunyaviridae was demonstrated as the etiological agent of SFTS and named as severe fever with thrombocytopenia syndrome bunyavirus (SFTSV). However, lacking of knowledge on pathology and pathogenic mechanisms of SFTSV in human underscores the need to study pathogenesis of SFTSV using an animal model. The infectious C57/BL6 mouse model with hallmark symptoms of thrombocytopenia and leukocytopenia was established. Viral RNA and histopathological changes were identified in the spleen, liver, and kidney. Intriguingly, viral replication was only found in the spleen, which suggested spleen as the principle target organ of SFTSV. Focusing on spleen, we found that the number of macrophages and platelets dramatically increased in the infected spleen, and SFTSV co-localized with platelets in cytoplasm of macrophages in the red pulp of the spleen. In vitro cellular assays further revealed that SFTSV adhered to human platelets and facilitated the phagocytosis of platelets by human macrophages, which in combination with in vivo findings suggests that the SFTSV-induced thrombocytopenia was caused by clearance of circulating virus-bound platelets promoted by splenic macrophages. Thus, the study elucidated pathology and pathogenic mechanisms of thrombocytopenia in a murine model which resembles human SFTS disease.
Human High Mobility Group Box 1 Induced Human Lung Fibroblasts Migration Through Activation PI3 kinase, MAPK, and NF-κB Signaling Pathway (173.31)
Authors: Lee Chen-Chen , Tsai Yi-Ru
Published: January 2023

High mobility group box 1(HMGB1) is a nuclear protein that involves the interactions with DNA and chromatin regulation and transcription. HMGB1 is also a cytokine that can activate monocytes neutrophils involving in inflammation. In this study, we want to investigate the role of HMGB1 on cell migration on human fibroblast cell line -WI-38. After treated with HMGB1 1, 10, 100 ng/ml for 24h, we did not found obviously cytoxicity and proliferation effect on WI-38 cells by MTT and BrdU incorporation assay, respectively. We found that HMGB1 induced cell migration on WI38 by dose-dependent manner and caused MMP9 activation but not MMP2. Using specific inhibitor of protein kinases, we observed that extracellular signal related kinase (ERK) inhibitor- PD98059(2μM), c-Jun N-terminal kinase (JNK) inhibitor - SB203580(2μM), p38 mitogen activated protein kinase (p38) inhibitor-SP600125(2μM), phosphoinositide 3-kinase (PI3 kinase) inhibitor- LY294002(2μM), and NF-κB inhibitor-Bay117082(1μM) all showed the inhibition effect on HMGB1 induced WI-38 cells migration. We also found that HMGB1 induced ERK, JNK and p38 activated and increased the nucleus β-catenin expression by time-dependently. In the future, we will further investigate the mechanisms of HMGB1-induced cell migration on WI-38 including HMGB1 membrance receptors activation and related signaling pathway.
Putative roles of specific T cells in progression of obesity-associated inflammation in adipose tissue. (173.36)
Authors: Taniguchi Kaori , Miyazaki Toru
Published: January 2023

The metabolic syndrome is known as a chain of illness, triggered by obesity followed by diabetes, hepatic steatosis and atherosclerosis. It is well known that adipose tissues in obesity are in a state of chronic inflammation, and that such inflammatory state is tightly associated with multiple metabolic disorders. Accumulating evidence has shown that the inflammation is mainly lead by the recruitment of macrophages into adipose tissues. Although it has recently been suggested that T cells may also play a role in establishment of inflammation in adipose tissue during the early phase of obesity, specificity and roles of them are poorly understood. In order to solve this issue, we first kinetically analyzed the repertoire of T cells recruited into adipose tissues along with the progression of obesity in mice. Interestingly, the adipose tissue T cells in mice fed with high fat diet for 9 wks harbored restricted repertoires of T cell receptor (TCR), indicating the presence of specific antigen(s) derived from fat tissues in obese individuals. Until now, we have created TCR transgenic mice in which majority of T cells may respond to a particular antigen exposed in obese adipose tissue. Our study will clarify the precise role of T cells in metabolic syndrome, and will contribute to development of a new therapeutic strategy.
Effects of bee venom-PLA2 on Regulatory T cells in asthma mice model (173.37)
Authors: Park Soojin , Choi Taewon , Lee Gihyun , Shin Minkyu Shin , Hong Moochang , Bae Hyunsu
Published: January 2023

CD4+CD25+Foxp3+ regulatory T cell (Treg) plays an important role in immune tolerance which can reduce inflammation and allergic reactions through its inhibitory functions. Bee venom (BV) has been widely used in the treatment of chronic inflammatory diseases such as Rheumatoid Arthritis. In this study, we hypothesized that phospholipase A2 (PLA2), major constituent of BV, could increase Treg expression and treat OVA-induced allergic asthma in mice. In vitro study, the percentage of Treg increased significantly by PLA2 treatment in CD4+T cell compared to saline treatment. In OVA induced allergic asthma model, PLA2-treatment significantly suppressed eosinophil and lymphocyte infiltration in BALF. In addition, PLA2 inhibited the production of IL-4, IL-13, INF-γ, IgE and OVA-specific IgE, along with suppressing airway hyper-responsiveness. Furthermore, CD4+CD25+Foxp3+ population was significantly increased by PLA2 treatment. Interestingly, these anti-asthmatic effects by PLA2 were totally abolished by Treg depletion via anti-CD25 antibody injection, suggesting pharmaceutical action of PLA2 was medicated by Treg. Our data suggest that PLA2 inhibits OVA-induced allergic airway inflammation by modulating Treg function.
Cleavage of the CD11b extracellular domain by the leukocyte serprocidins is critical for neutrophil detachment during chemotaxis (173.7)
Authors: Zen Ke , Guo Yalan , Bian Zhen , Liu Yuan
Published: January 2023

The β(2)-integrin CD11b/CD18 mediates the firm adhesion of neutrophils (PMNs) to epithelial monolayers, a key step in PMN transepithelial migration. To complete the transmigration process, adherent PMNs must detach from epithelial surfaces to move forward. The mechanism that governs the detachment of adherent PMNs, however, is not clear. Here, we present evidence that cleavage of the CD11b extracellular domain containing the ligand-binding I-domain by 3 structural and functional related serine proteases (elastase, proteinase-3 and cathepsin G) serves as a novel mechanism for PMN detachment after the initial cell adhesion. Kinetic studies showed that the cleavage of CD11b is positively correlated with PMN detachment and subsequent transmigration. Moreover, the results demonstrated that elastase, proteinase-3 and cathepsin G all cleaved the purified, functionally active form of CD11b in a pattern similar to the CD11b shedding that occurs during PMN transmigration. Their cleavage sites on purified CD11b were located at (761)Thr-Ala(762) (elastase/proteinase-3) and (760)Phe-Thr(761) (cathepsin G), respectively. CD11b cleavage and PMN detachment and chemotaxis, were impaired in elastase/cathepsin G-deficient Beige mice; this defect could be restored by the addition of extracellular elastase. By illustrating CD11b shedding by elastase, proteinase-3 and cathepsin G as a novel mechanism for PMN detachment, our study provides novel therapeutic targets for controlling inflammation.
Enhanced Claudin-9 expression is associated with lung cancer metastasis (173.8)
Authors: Sharma Rajesh , Jala Venkatakrishna , Satpathy Shuchismita , Haribabu Bodduluri
Published: January 2023

Typical metastasis models utilize intravenous injection of tumor cells, thus bypass the egression of malignant cells an obligatory step in metastasis of spontaneous cancers. We herein utilized a unique metastasis model by in vivo passaging the Lewis lung carcinoma (3LL) cells and maintaining them in vivo with 7-11 days of intermittent culture in between passages. When injected subcutaneously parental 3LL cells have minimal metastasis, while in vivo passaged cells show robust metastasis. Most interestingly, parental and passed cell lines if injected i.v., yielded similar metastatic burden suggesting that egression from the primary tumor is facilitated by in vivo passaging. Microarray analysis of RNA from these cells as well as the primary tumors derived from each revealed several novel markers associated with increased metastasis. Expression of a tight junction protein “claudin 9” is highly up regulated in passaged 3LL lines. Enhanced expression of claudin-9 in passaged 3LL lines and tumors derived from these cells was confirmed by real time PCR and Western blot as well as immuno-fluorescence microscopy. Passaged 3LL cells exhibited greater motility and invasiveness relative to parental 3LL cells. Taken together, these data indicate distinct association of claudin 9 expression with metastatic phenotype and suggest that claudin 9 may serve as a biomarker and a potential therapeutic target for preventing lung cancer metastasis.