bluuebunny/crossref_metadata_2025 huggingface dataset:
HCV impairs human T cell responses via MDSC induction. (154.2)
Authors: Tacke Robert , Hahn Young
Published: January 2023

Hepatitis C virus (HCV) is highly efficient in establishing persistent infection, which leads to the development of chronic liver disease and hepatocellular carcinoma. Impaired T cell responses with reduced IFN-γ production have been reported to be associated with HCV persistent infection. Extracellular HCV core is a viral factor known to cause HCV-induced T cell impairment via its suppressive effect on the activation and induction of pro-inflammatory responses by antigen presenting cells (APCs). Recently, myeloid derived suppressor cells (MDSC) have been shown to be crucial regulators of T cell responses. In this report we examine the accumulation of MDSCs in human PBMCs following exposure to extracellular core. We show that HCV core induces the activation of STAT3 in human APCs. Importantly, co-cuture of HCV core-treated CD33+ APCs with CD4 T cells suppresses autologous CD4 T cell proliferation and IFN-γ production in response to TCR stimulation. This suppression is partially rescued by STAT3 inhibitor WP1066, as well as by reactive oxygen species (ROS) inhibitor, catalase. In contrast, inhibitors to arginase-1 and iNOS do not affect T cell responsiveness. These results suggest that HCV core -mediated STAT3 activation promotes in the accumulation of CD33+ MDSC, resulting in ROS-mediated suppression of T cell responsiveness. The accumulation of these MDSC may facilitate the establishment of HCV chronic infection.
CIITA enhances HIV-1 attachment to CD4+ T cells leading to enhanced infection and cell depletion (154.27)
Authors: Porter Kristen , Kelley Lauren , Nekorchuk Michael , Jones James , Hahn Amy , de Noronha Carlos , Harton Jonathan , Duus Karen
Published: January 2023

Activated CD4+ T cells are more susceptible to HIV infection than resting T cells; the reason for this remains unresolved. Induction of CIITA and subsequent expression of the MHC class II isotype HLA-DR are hallmarks of CD4+ T cell activation; therefore, we investigated the role of CIITA expression in T cells during HIV infection. CIITA-expressing SupT1 cells display enhanced virion attachment in a gp160/CD4-dependent manner, resulting in increased HIV infection, virus release, and T cell depletion. Although increased attachment and infection of T cells correlated with HLA-DR surface expression, Ab blocking, transient expression of HLA-DR without CIITA, and short hairpin RNA knockdown demonstrate that HLA-DR does not directly enhance susceptibility of CIITA-expressing cells to HIV infection. Analysis of the remaining MHC class II isotypes, HLA-DP and HLA-DQ, MHC class I isotypes, HLA-A, HLA-B, and HLA-C, and the class II Ag presentation genes, invariant chain and HLA-DM, demonstrate that these proteins likely do not contribute to CIITA enhancement of HIV infection. Finally, we demonstrate that in activated primary CD4+ T cells as HLA-DR/CIITA expression increases there is a corresponding increase in virion attachment. Overall, this work suggests that induction of CIITA expression upon CD4+ T cell activation contributes to enhanced attachment, infection, virus release, and cell death through an undefined CIITA transcription product that may serve as a new antiviral target.
Granzyme B inhibits translation of viral proteins and viral replication through proteolytic cleavage of the eukaryotic initiation factor 4 gamma 3 (154.35)
Authors: Marcet-Palacios Marcelo , Duggan Brenda , Shostak Irene , Barry Michele , Wilkins John , Yanagiya Akiko , Sonenberg Nahum , Bleackley R.
Published: January 2023

Cytotoxic T lymphocytes (CTL) are the major killer of virus-infected cells. Granzyme B (GrB) from CTL induces apoptosis in target cells by cleavage and activation of substrates such as caspase-3. However, while undergoing apoptosis, cells can still produce infectious viruses unless a mechanism exists to specifically inhibit viral production. Using proteomic approaches, we found a novel GrB target that plays a major role in protein synthesis: eukaryotic initiation factor 4 Gamma 3 (eIF4G3). We hypothesized a novel role for GrB to arrest translation of viral proteins by targeting eIF4G3, which contains the GrB recognition sequence IESD1408S. We showed that GrB specifically cleaves eIF4G3 since the mutant eIF4G-Δ, with Ala substituted for D1408, was resistant to GrB degradation. Translational inhibition by GrB was reversed by the inclusion of eIF4G-Δ, showing that eIF4G3 is the critical GrB substrate involved in translation. In Jurkat cells, GrB or CTL treatment resulted in reduced rate of translation. We infected Jurkat cells with vaccinia virus (VV) and showed that viral protein synthesis and viral replication was inhibited in response to GrB or CTL. Transfection of eIF4G-Δ reversed the halt in protein synthesis induced by GrB. This demonstrated for the first time that GrB could prevent the production of VV by targeting the translational machinery of the host. These results represent a major new insight into what constitutes an effective CTL response to a virus-infected cell.
Two functionally-distinct subsets of CD4+CD39+ Treg in patients with HIV-1 infection (154.36)
Authors: Schuler Patrick , Macatangay Bernard , Schilling Bastian , Harasymczuk Malgorzata , Rinaldo Charles , Whiteside Theresa
Published: January 2023

Ectonucleotidases CD39 and CD73 are expressed on the surface of regulatory T cells (Treg) and cleave ATP to immunosuppressive adenosine. CD39 could potentially serve as a new functional marker for Treg isolation. The frequency and absolute numbers of CD4+CD39+ T cells were determined in 32 HIV+ patients and in 10 healthy donors (HD). CD4+ T cells were negatively selected by magnetic beads PBMC. CD39+ Treg cells were then isolated by biotin-conjugated anti-CD39 Abs and anti-biotin magnetic beads. T cells were phenotyped by flow cytometry for CD39+, CD73+, FOXP3+, CCR4+, CD127neg, CD49dneg, and CD121a+. CFSE-based proliferation assays and ATP hydrolysis were used to measure Treg functions. Cytokine expression in CD4+CD39+ T cells was determined by Luminex. HIV-patients on and off anti-retroviral regimen had a significantly lower absolute number of CD39+CD25+ Treg and CD39+CD25neg T cells (p<0.05) than HD. The CD4+CD39+ T cell populations isolated on immunobeads contained two cell subsets: FOXP3+CD25+ Treg hydrolyzed exogenous ATP and suppressed CD4+ T cell proliferation, which was reversed by blocking ectonucleotidase activity. In contrast, the FOXP3negCD25neg T cells were CD127+CD49d+ and expressed IFNg, TNFa, and IL-10. The presence of two distinct CD4+CD39+ cell subsets in PBMC is a general phenomenon seen in normal donors, as well as patients with HIV and cancer. This suggests that phenotypic and functional plasticity characterizes the human Treg subset.
Self MHC class I licensed NK cells enhance adaptive CD8 T cell viral immunity (154.5)
Authors: Stadnisky Michael , Xie Xuefang , Coats Ebony , Bullock Timothy , Brown Michael
Published: January 2023

MHC class I (MHC I) is essential to NK and T cell effector and surveillance functions. However, it is unknown if MHC I polymorphism influences adaptive immunity through NK cells. Previously, we found that MHC I Dk, a cognate ligand for the Ly49G2 inhibitory receptor, was essential to NK control of murine (M)CMV infection. Here we assessed the significance of NK inhibitory receptor recognition of MCMV on CD8 T cells in genetically defined MHC I Dk disparate mice. We observed that Dk-licensed Ly49G2+ NK cells stabilized and then enhanced conventional dendritic cells (cDC) recovery following infection. Further, licensed NK support of cDC recovery was essential to enhance the tempo, magnitude and effector activity of virus-specific CD8 T cells. Minimal cDC and CD8 T cell number differences after low dose MCMV in Dk disparate animals further implied that licensed NK recognition of MCMV imparted qualitative cDC changes to enhance CD8 T cell priming.
Soluble flagellin, FliC, induces an Ag-specific Th2 response, yet promotes T-bet-regulated Th1 clearance of Salmonella typhimurium infection (155.5)
Authors: Cunningham Adam , Bobat Saeeda , Flores-Langarica Adriana , Hitchcock Jessica , Marshall Jennifer , Kingsley Robert , Gil-Cruz Cristina , Letran Shirdi , López-Macías Constantino , Alexander James , MacLennan Ian
Published: January 2023

Clearance of disseminated Salmonella infection requires bacterial-specific Th1 cells and IFNg production and Th1-promoting vaccines are likely to help control these infections. Consequently, vaccine design has focused on developing Th1-polarizing adjuvants or Ag that naturally induce Th1 responses. In this study we show that, in mice, immunization with soluble flagellin (sFliC) induces Th2 responses as evidenced by Ag-specific GATA-3, IL-4 mRNA and protein induction in CD62Llo CD4+ T cells without associated IFNg production. Despite these Th2 features, sFliC immunization can enhance the development of protective Th1 immunity during subsequent Salmonella infection in an Ab-independent, T cell-dependent manner. Salmonella infection in sFliC-immunized mice resulted in augmented Th1 responses, with greater bacterial clearance and increased numbers of IFNg-producing CD4+ T cells, despite the early Th2 features induced to sFliC. The augmented Th1 immunity after sFliC immunization was regulated by T-bet although T-bet is dispensable for primary responses to sFliC. These findings show there can be flexibility in T cell responses to some subunit vaccines. These vaccines may induce Th2-type immunity during primary immunization yet promote Th1-dependent responses during later infection. This suggests that designing Th1-inducing subunit vaccines may not always be necessary since this can occur naturally during subsequent infection.
SA-4-1BBL and monophosphoryl lipid A as an adjuvant system for the development of effective therapeutic cancer vaccines (156.10)
Authors: Srivastava Abhishek , Sharma Rajesh , Zhao Hong , Yolcu Esma , Shirwan Haval
Published: January 2023

TAAs based subunit vaccines have compromised efficacy due to their weak immunogenic nature and various immune evasion mechanisms employed by the progressing tumors. To overcome these limitations, we herein used costimulatory molecule SA-4-1BBL and toll-like receptor 4 agonist MPL with distinct modes of action as a novel adjuvant system. A single immunization with both adjuvants and E7 TAA resulted in eradication of established E7 expressing TC-1 tumors in 100% of mice. Similarly, a single vaccination with survivin, a bona fide self-TAA, resulted in control/eradication of established 3LL pulmonary metastases. Combined adjuvants had better therapeutic efficacy over the individual adjuvants, while SA-4-1BBL as monotherapy significantly outperformed MPL. The therapeutic efficacy of combined adjuvants and SA-4-1BBL as monotherapy was correlated with potent Th1 responses and increased intratumoral CD8+ Teff/CD4+FoxP3+ Treg cell ratio. Surprisingly, MPL as monotherapy did not improve the intratumoral CD8+ Teff/Treg cell ratio as compared to controls, and depletion of Treg cells improved the efficacy of MPL from 50 to 100%. Taken together, these data demonstrate the utility of SA-4-1BBL/MPL as a promising adjuvant system for the development of therapeutic cancer vaccines with significant clinical potential.
Intranodal injection of tumor-derived autophagosomes (DRibble) in conjunction with anti-OX40 showed potent anti-tumor efficacy in murine 4T1 breast cancer (156.7)
Authors: Li Yuhuan , Cui Zhihua , Pang Puiyi , Shu Suyu , Weinberg Andrew , Fox Bernard , Urba Walter , Hu Hong-Ming
Published: January 2023

Tumor-derived autophagosomes, referred as DRibbles, are novel cancer vaccines that enable efficient cross-presentation of a variety of tumor-associated antigens including SLiPs and DRiPs. We have previously showed that DCs loaded with DRibbles is therapeutic in treating mice with B16F10 melamoma and 3LL Lewis lung cancer. Here we assessed the effect of route of DRibble administration and engagement of OX40 signaling on T-cell activation. We developed an effective DRibble and anti-OX40 combination immunotherapy regimen to treat 4T1 breast cancer. We report that intranodal(i.n.)administration of DRibbles was 10~100 folds more effective to induce T-cell expansion than the subcutaneous route(s.c.).Engagement of OX40 further enhanced antigen-specific CD8+ T-cell proliferation,survival,and acquisition of effector T-cell phenotype (KLRG1 expression). When DRibble i.n. was combined with anti-OX40 to treat BALB/c mice bearing 13-day established 4T1 tumor, all of the mice showed regression, and 60% of the mice were cleared of tumor. Robust TAA-specific immune response was detected in mice cured after vaccination with DRibble and anti-OX40. Anti-OX40 increased frequency of tumor antigen-specific T cells in the spleen, and CD8+/CD4+ T cells in the PBMC of peripheral blood. Thus DRibble vaccine in combination with anti-OX40 is an effective immunotherapy regimen to treat breast cancer. Clinical trials of the DRibble vaccine have begun in our cancer center.
IFI16 acts as a nuclear danger sensor and induces the inflammasome in response to Kaposi’s sarcoma associated herpesvirus (KSHV) infection (157.8)
Authors: Kerur Nagaraj , Veettil Mohanan , Sharma-Walia Neelam , Bottero Virginie , Sadagopan Sathish , Otageri Pushpalatha , Chandran Bala
Published: January 2023

Inflammasomes sense diverse classes of foreign molecules in the cytoplasm and induce caspase-1 activation and IL-1β maturation. Whether such a sensing mechanism exists in the nucleus is not known. Nuclear replicating herpesvirus KSHV is associated with Kaposi Sarcoma (KS) that is characterized by a microenvironment of inflammatory cytokines including IL-1β. How the caspase-1 inflammasome, which is required for IL-1β maturation is induced during KSHV infection is not known. Here we demonstrate that during de novo KSHV infection of endothelial cells, interferon gamma-inducible protein 16 (IFI16) interacts with ASC and procaspase-1 to form a functional inflammasome. This complex was initially detected in the nucleus and subsequently in the peri-nuclear area. Caspase-1 activation by KSHV was reduced by IFI16 and ASC silencing but not by AIM2 knockdown. Our studies reveal a new function of IFI16 as a nuclear danger sensor and demonstrate that the inflammasome’s danger sensing functions extend into the nucleus.
Targeting inhibitors of NOD2 signaling to allow productive inflammation in genetic inflammatory disease (157.9)
Authors: Marinis Jill , Homer Craig , McDonald Christine , Abbott Derek
Published: January 2023

The intracellular bacterial sensor, NOD2, coordinates innate immune signaling pathways. Maintaining appropriate activation of NOD2 is paramount in immunologic homeostasis as dysregulation of NOD2 signaling causes a variety of inflammatory diseases including Crohn’s disease (CD). We have identified the atypical TRAF family member, TRAF4, as a key inhibitor of NOD2 signaling. We show that this inhibition occurs through two mechanisms - through IKK phosphorylation and activation of TRAF4 and through direct binding of TRAF4 to NOD2. Further work shows that TRAF4 also binds CD-associated NOD2 variants and we hypothesize that competing TRAF4 from these NOD2 variants could allow a productive inflammatory response. To this end, we have generated cell permeable peptides that mimic the region of NOD2 that binds TRAF4 in hopes of competing TRAF4 from NOD2 in vitro and in vivo to prevent the inhibitory effect of TRAF4. This may partially revert loss-of-function CD-associated NOD2 variants and could serve as a platform for future therapeutics in Crohn’s disease patients harboring these mutations.
Production and characterization of polyclonal antibodies against CD38 (159.3)
Authors: Trujillo Ocampo Abel , Vences Catalan Felipe , De la Cruz Hernandez Hernandez Fidel , Santos Argumedo Leopoldo
Published: January 2023

CD38 is a 42 kDa transmembrane glycoprotein widely expressed in hematopoietic cells. Anti-CD38 agonistic antibodies induce proliferation, migration, and apoptosis of B lymphocytes. Some of the available monoclonal antibodies do not recognize CD38 by Western blot. Then, we decide to prepare antisera that can be equally useful in biochemical and functional assays. To reach this goal, we used the extracellular domain of murine CD38 fused with a V5 epitope and a Histidin tag or fused with GST as antigens. These proteins were characterized by western blot, enzymatic activity and by reactivity with the mAB NIM-R5, which recognizes a conformational epitope. We immunized New Zealand rabbits and a female goat with the CD38-V5-Histidin tag fusion protein. Antibodies from both species stained cells from C57BL/6 but not from CD38 deficient mice. In addition, the polyclonal antibodies recognized in extracts from splenic cells a band of the expected weight. Finally, these antibodies induced B lymphocyte proliferation of mouse splenic cells. These results demonstrated the production of functional reagents that can be used in the analysis of CD38 in B cells.
Multicolor flow cytometric analysis of mouse T cell subsets in aging (159.4)
Authors: Ernst David , Gao Guo-jian , O'Donnell Erika , Elia Jeanne , Wu Xiao-Wei , Chronopoulou Efthalia , Sepulveda Homero , Hingorani Ravi
Published: January 2023

Aging is accompanied by profound changes in the function and subset makeup of peripheral CD4 and CD8 T cells. Using the Violet Proliferation Dye 450, we found significantly fewer splenic CD4 and CD8 T cells from aged mice progressed through cell cycle in response to CD3/CD28 cross linking. However, intracellular cytokine staining showed higher percentages of total T cells in older mice that produced IFN-γ and IL-17 on stimulation with little difference in their signaling pattern. Additionally, the age-associated responses were accompanied by dramatically increased proportions of memory (CD44hiCD62Llo/hi) and decreased fractions of naïve (CD44loCD62Lhi) T cell subsets. To determine if the altered T cell subset proportions could be responsible for functional differences, phospho-specific flow cytometry was used to evaluate alterations in naïve and memory T cells from young vs. old mice. Interestingly, many signaling responses were reduced in memory T cells relative to naïve T cells, including the phosphorylation of STAT1 (pY701) and STAT3 (pY705) in response to stimulation with exogenous IL-6 or IFN-γ and the phosphorylation of SLP-76 (pY128) and AKT1 (pS473) in response to CD3/CD28. However, signaling responses were largely similar within equivalent T cell subsets from old and young mice. Our results suggest that the altered T-cell functions associated with advanced age may primarily result from the dramatic shifts in naïve and memory CD4 and CD8 T cell subset representations.
Nasopharyngeal colonization by Streptococcus pneumoniae elicits long-lasting immune responses that confer protection from invasive lung infections (161.19)
Authors: Maung Nang , Goodwin Aaron , Muñoz-Elías Ernesto , Camilli Andrew , Gerstein Rachel , Leong John
Published: January 2023

Streptococcus pneumoniae can cause life-threatening pulmonary and systemic infection, but resides usually harmlessly in the nasopharynx of many people. Humoral immunity is thought to be the primary defense against serious pneumococcal infection, and we hypothesized that nasopharyngeal colonization of mice may result in the generation of an antibody response that provides long-term protection against lung infection. In fact, we found that intranasal colonization elicited pneumococcal-specific antibody responses and protected from a S. pneumoniae lung challenge that is lethal in naive mice. B cells were required for protection, and transfer of immune sera from colonized mice protected naive mice. CD93-/- mice, which are deficient in long-term antibody secreting cells (ASC), had fewer pneumococcal-specific bone marrow ASC after intranasal colonization and did not survive lung challenge. T cells were required to generate a fully protective response, but elimination of CD4+ T cells just prior to lung challenge did not compromise. These data are consistent with the hypothesis that CD4+ T cells promote the development of post-colonization immunity but are not needed to sustain the response that provides protection from lung challenge. These results stand in contrast to the T-cell independent antibody responses that are sufficient to protect mice from systemic infection, and have implications for development of effective vaccines.
Intestinal epithelial cells modulate CD4 T cell responses via the thymus leukemia antigen (161.8)
Authors: Olivares-Villagómez Danyvid , Scott Algood Holly , Singh Kshipra , Parekh Vrajesh , Ryan Kaitlyn , Piazuelo Maria , Wilson Keith , Van Kaer Luc
Published: January 2023

The thymus leukemia (TL) antigen is a non-classical MHC class I molecule primarily expressed on intestinal epithelial cells (IEC) that interacts with CD8αα, which is primarily expressed by intestinal intraepithelial lymphocytes. The close proximity of cells expressing these two molecules suggests that TL-expressing IEC can regulate CD8αα-expressing lymphocytes. Here, we report that TL influences the cytokine profile and pathogenic potential of CD4+ T cells expressing CD8αα. We show that TL+ IEC regulates IL-17 production by CD4+ T cells. Consequently, TL expression enhances development of colitis in an adoptive transfer model and confers resistance to Citrobacter rodentium infection by allowing the generation of an adequate Th17 response. Thus, TL expressed on IEC modulates CD4+ T cell responses in the intestinal mucosa.
Adenosine synthesized by CD73 and CD39 regulates inflammation during murine Salmonella infection (162.10)
Authors: Alam Mohammad , Gaines Dennis , Kuo Jennifer , Pereira Marion , Bigley Elmer , Ernst Peter , Williams Kristina
Published: January 2023

Salmonella spp. is a leading cause of hospitalization & death in the US associated with foodborne illness. Salmonella causes acute gut inflammation. Adenosine is an antiinflammatory mediator that limits tissue damage during inflammation. Surface enzymes CD39 & CD73 mediate the synthesis of adenosine. We studied the expression of CD39 & CD73 in liver, spleen, & CD4+ Th cells after infection of C57BL/6 mice with Salmonella enterica serotype Typhimurium (ST) & evaluated the role of CD73 in regulating ST-induced inflammation. Proinflammatory cytokine mRNA expression was assayed by RT-PCR arrays & cytokine proteins were tested by FACS & ELISA after ST infection or ST antigen (Ag) treatment. Function of CD73 was tested by examining proinflammatory cytokine production with or without CD73-inhibitor treatment of activated splenocytes exposed to ST Ag. Inflammatory responses to ST infection in CD73-/- mice were evaluated in liver & spleen tissue in H&E sections & in activated CD4+ Th cells stained for intracellular IL17a & IFN-γ. ST Ag-treated & CD73-inhibited splenocytes produced more proinflammatory cytokines (IL17A). Splenocytes from ST-infected CD73-/- mice produced more IL17a cytokine & Th17 cells & infected CD73-/- mice also had greater infiltration of inflammatory cells in the liver. Extracellular adenosine synthesized by CD73 may regulate host inflammation during murine Salmonellosis.
Toll-like Receptor 2 signaling in human endothelial cells (162.5)
Authors: Wilhelmsen Kevin , Mesa Kailin , Hellman Judith
Published: January 2023

Toll-like receptors (TLRs) recognize microbial components and initiate inflammation during infection. TLR2 is the receptor for microbial lipoproteins and other parts of Gram-positive and Gram-negative bacteria, and fungi. The endothelium expresses TLR2 and is important in sepsis-induced organ failure. We found that activation of TLR2 broadly affects endothelial cell (EC) functions, leading to upregulation of cytokine and chemokine production, increased neutrophil trafficking, altered expression of coagulation pathway intermediaries, and increased permeability. We probed the signaling pathways involved in TLR2-dependent activation of the EC by treating human umbilical vein EC (HUVECs) with lipopeptide TLR2 agonists and performed immunoblots, NF-κB EMSA’s, transcription factor arrays, and qRT PCR to assess responses. We found that TLR2 agonists transiently activate p38-MAPK and JNK at early time points and induce a sustained, albeit weak, activation of the transcription factor NF-κB. Through the use of siRNA knockdown and specific inhibitors, we determined that p38-MAPK, JNK, and NF-κB are necessary for TLR2-dependent upregulation of IL-6, IL-8 and TLR2 itself. Additionally, using mRNA human immune arrays, we found that markers of neutrophil trafficking and activation are most robustly upregulated by TLR2-dependent activation of the endothelium. We are currently investigating the mechanisms responsible for neutrophil trafficking and activation responses in more detail.
Ethanol consumption inhibits Th1 but not Th2 skewed contact hypersensitivity responses (163.11)
Authors: Brand Rhonda , Stottlemyer J. Mark , Cline Rachel , Falo, Jr Louis
Published: January 2023

Alcohol abuse is associated with suppression of both of innate and adaptive immune systems. Functionally, several studies suggest that ETOH consumption polarizes immune responses away from Th1 responses toward Th2 skewed immunity. Understanding the mechanisms by which alcohol alters immune function is critical to the development of strategies to reverse these changes. Contact Hypersensitivity reactions are dendritic cell dependent, antigen specific, T cell-mediated immune responses triggered by skin exposure to haptens that become immunogenic after binding to peptides or proteins. We examined the impact of alcohol consumption on the response to Th1 skewing (DNCB) and Th2 skewing (FITC) contact sensitizers. Our studies suggest that ETOH consumption significantly inhibits CHS responses to the Th1 skewing sensitizer DNCB. In contrast, alcohol consumption did not diminish the CHS response triggered by the Th2 skewing sensitizer FITC. Importantly, the differential immune effects of ETOH consumption occurred independently of ETOH associated liver damage, as the results were similar in mice fed alternative alcohol diets that that did or did not induce liver damage. These results demonstrate specificity in ETOH-induced immunomodulation, and suggest strategies to prevent and treat ETOH associated immune dysfunction relevant to vaccination and immune mediated ETOH induced pathologies.
Induction and suppression of allergic diarrhea and systemic anaphylaxis in a mouse model of food allergy. (163.21)
Authors: Kucuk Zeynep , Mahler Ashley , Strait Richard , Hogan Simon , Finkelman Fred
Published: January 2023

We evaluated the requirements for development of allergic diarrhea (AD) and anaphylactic shock (AS) in response to Ag ingestion and whether ingested Ag must be absorbed systemically to trigger these responses. TNP-BSA ingestion induced AS without AD in IgE anti-TNP mAb-primed, immunologically naïve wild-type mice and intestinal IL-9 transgenic mice, even though the latter mice have intestinal mastocytosis, but both AS and AD in IgE anti-TNP mAb-primed mice that had recently been immunized i.p., then p.o. with ovalbumin (Ova). Mice inoculated once i.p. with Ova/alum, then multiple times p.o. with Ova, develop both AD and AS in response to oral Ova challenge; a second i.p. Ova/alum inoculation prevents AS, but not AD. J chain- and pIgR-deficient mice, which have increased serum IgA and decreased gut IgA, are relatively resistant to development of AD. Treatment of IgE anti-TNP mAb-primed, Ova-immunized mice with i.v. IgG anti-TNP mAb inhibits both AD and AS responses to oral TNP-BSA challenge, with AS more sensitive to inhibition. We conclude that: 1) induction of AD, but not AS, by ingested Ag requires local Ag-induced changes to the intestinal immune system beyond mast cell and IgE induction; 2) ingested Ag must be systemically absorbed to induce both AD and AS; and 3) more ingested Ag must be absorbed systemically to induce AS than to induce AD. These observations suggest that induction of a systemic IgG Ab response can suppress established food allergy.
Plasmin plays an essential role in amplification of psoriasiform skin inflammation in mice (164.10)
Authors: Ke Fang , Wang Honglin
Published: January 2023

Background Although increased levels of plasminogen activators have been found in psoriatic lesions, the role of plasmin converted from plasminogen by plasminogen activators in pathogenesis of psoriasis has not been investigated. Methodology/Principal Findings Here we examined the contribution of plasmin to amplification of inflammation in psoriasis. We found that plasminogen was diminished, but that the amount and activity of its converted product plasmin were markedly increased in psoriasis. Moreover, annexin II, a receptor for plasmin was dramatically increased in both dermis and epidermis in psoriasis. Plasmin at sites of inflammation was pro-inflammatory, eliciting production of inflammatory factors, including CC chemokine ligand 20 (CCL20) and interleukin-23 (IL-23), that was mediated by the nuclear factor-κB (NF-κB) signaling pathway and that had an essential role in the recruitment and activation of pathogenic C-C chemokine receptor type 6 (CCR6)+ T cells. Moreover, intradermal injection of plasmin or plasmin together with recombinant MCP-1 resulted in induction of psoriasiform skin inflammation around the injection sites with several aspectsκof human psoriasis in mice. Conclusions/Significance Plasmin converted from plasminogen by plasminogen activators plays an essential role in amplification of psoriasiform skin inflammation in mice, and targeting plasmin receptor - annexin II - may harbor therapeutic potential for the treatment of human psoriasis.
Mammaglobin-A DNA vaccine increases the IFN-γ CD4+ICOS+ T- cells frequency in human breast cancer patients (165.26)
Authors: Tiriveedhi Venkataswarup , Fleming Timothy , Goedegebuure Peter , Naughton Michael , Ma Cynthia , Lockhart Craig , Gao Feng , Gillanders William , Mohanakumar T.
Published: January 2023

Mammaglobin-A (Mam-A), a 10kD secretory protein, is over expressed in 80% of primary and metastatic human breast cancers. Previous studies in our laboratory using a HLA-A2/Human CD8 transgenic murine model have demonstrated that Mam-A DNA Vaccine elicit specific CD8+ cytotoxic T-cell (CTL) responses to Mam-A and induces tumour regression in SCID mice. In our current study we report the activation of T-helper cells with specific expression of ICOS (inducible costimulator), a T-cell specific activation marker, following Mam-A vaccination. Stage IV breast cancer patients were chosen for the study and vaccinated by Mam-A DNA vaccine on 0, 28 and 56 days. Six months following Mam-A vaccination (n=5), flow cytometry analysis demonstrated a increase in CD4+ICOS+ T-cell frequency from 5±2% to 23± 4% with a concomitant drop in the CD4+Foxp3+T-cell (Treg) frequency from 19±6% to 10±5%. However, the frequency of CD4+CD25+ICOS+ did not change following vaccination (23±5%). ELISpot analysis demonstrated that following vaccination, CD4+ICOS+ sorted T-cells demonstrated a cytokine phenotype switch from IL-10 to IFN-γ. We conclude that following Mam-A DNA vaccination, there is a specific activation T-cells with increase in the pro-inflammatory IFN-γ CD4+ICOS+T-cells along with decrease in Treg frequency. We propose that this switch in T-cell frequency following vaccination may be important in tumor regression in breast cancer patients.
A novel NOD.SCID mouse model suggests CD200-mediated regulation may be important for both growth and therapy of CLL (165.36)
Authors: Wong Karrie , Spaner David , Gorczynski Reg
Published: January 2023

Chronic lymphocytic leukemia is characterized by the accumulation of malignant CD5+CD19+ B cells that, like anergic B cells, typically fail to engraft in immunocompromised murine hosts. CD200, a type I transmembrane molecule with potent immunoregulatory functions, is known to be overexpressed on CLL cells. Using a CD200 sandwich-ELISA, we have identified a soluble form of CD200 (sCD200), whose expression is increased by >5-fold in the serum of CLL patients compared to healthy controls. Further analysis of sCD200 levels in CLL patient serum showed a correlation between sCD200 levels and Rai disease stage. Infusion of sCD200hi CLL serum into NOD.SCIDIL-2γ-/- mice receiving 1x108 CLL splenocytes enhanced engraftment of CLL cells, which were detectable in both the spleen and peritoneum of animals, in comparison to mice receiving sCD200lo normal serum. Enhanced engraftment using CLL serum was abrogated by depletion of sCD200 from the serum. When given at 21 days post-CLL splenocyte infusion, anti-CD200mAb was as effective as Rituximab in eliminating engrafted CLL. In vivo T cell depletion with anti-CD3mAb also suppressed CLL engraftment, despite ongoing CLL serum infusion, suggesting that T cells are important to maintain CLL survival and growth. Our studies show that infusion of sCD200hi CLL serum into NOD.SCID mice allows development of a novel xenograft model for CLL, and further suggest that anti-CD200 mAb may have value in treatment of disease.
Low dose γ-radiation can mitigate some deleterious effects of carcinogen injection in A/J mice. (165.42)
Authors: Gonzales Veronica , Gott Katherine , Makvandi Mackey , Scott Bobby , Wilder Julie
Published: January 2023

Low dose radiation (LDR) activates the immune response and may lead to cancer suppression. We investigated the acute effect of LDR and the carcinogen benzo(a)pyrene (BaP) on lung and spleen cell number and phenotype as well as spleen cell cytokine secretion in an A/J mouse model. Mice were given a single gamma radiation dose (10, 100, 1000 mGy) one day prior to intraperitoneal injection with BaP or Tricaprylin vehicle. Lung and spleen tissues were harvested at days (d) 2, 7, and 9 post-irradiation to determine viable cell counts and phenotype. Splenocytes were cultured for 48 hours +/- Concanavalin A (5 μg/ml) and cytokine secretion was measured by Luminex. BaP treatment decreased lung cell (d2) and splenocyte (d7) number while lung neutrophila was increased on d7 and 9. BaP also decreased activated (CD69+) CD4+ and CD8+ cells in both organs by d7. The reduction in activated CD8+ was attenuated in mice treated with 10 mGy radiation. BaP also decreased lung B cell and dendritic cell number. Treatment with 1000 mGy decreased B cells in both the lung and spleen. BaP treatment decreased Th2 cytokine production while increasing IL-1β and IL-17. 10 mGy treatment increased IL-2, IL-4, IL-12p70, VEGF, and IL-17 while IL-6 and IL-10 production was decreased by higher radiation doses on d2 post-radiation. These data suggest that treatment with BaP and LDR significantly affect the immune system with 10 mGy, but not 1000 mGy, treatment often mitigating the deleterious effect of BaP.
Peptide vaccines prevent tumor growth by stimulating a similar T cell repertoire as the native tumor antigen (165.46)
Authors: Slansky Jill , Jordan Kimberly , Buhrman Jonathan , Kappler John , Sprague Jonathan , Moore Brandon , Gao Dexiang
Published: January 2023

A major goal of immunotherapy for cancer is to focus and activate the T cell response to tumor-associated antigens. One strategy to stimulate these T cells is to immunize with specific peptides in combination immunostimulatory modalities. We are using an immunogenic mouse colon cancer model to characterize both the activating peptides and the responding T cells. We have identified peptides that interact with the restricting MHC molecule and a tumor-specific T cell clone that are either non-protective like the natural antigen, or protective against tumor challenge. The T cell repertoires responding to these peptides and the tumor are distinct, although they overlap with the natural antigen. Examination of the Vb regions expressed by the tumor-specific T cells, showed that the responses to the peptide variants were more focused than those to the natural antigen. Further ex vivo analysis showed that the T cells responding to the protective peptides and in the TIL (tumor infiltrating lymphocytes) were selected to express a CDR3b motif, which was also shared by the T cells responding to the natural antigen. We suggest that the increased signal delivered by the peptide variants that target the T cells naturally responding to the tumor such as those found in TIL result in more effective antitumor responses.
The effect of receptor-associated protein level on the immunogenicity of HSP-peptide complexes (165.7)
Authors: Zhou Yu , Binder Robert
Published: January 2023

Heat shock proteins (HSPs) serve as molecular chaperones in every type of cell. As HSPs are released from cells as a result of necrotic death, they are sampled by antigen presenting cells (APCs), which then prime T cell mediated immune responses. CD91, an endocytic receptor on APCs, has been shown to recognize HSPs in the extracellular milieu allowing for cross-presentation of HSP-chaperoned peptides. Various CD91 ligands, such as the ER-resident receptor-associated protein (RAP), compete with HSPs for receptor binding in vitro. Since the HSP-CD91 pathway appears critical for initiation of immune responses, we hypothesize that expression of elevated levels of RAP will negatively affect the immunogenicity of HSP-peptide complex, when RAP is released from necrotic tumor cells. To address this hypothesis, RAP was over-expressed in several tumor cell lines, and those cell lines were then tested for their immunogenicity by tumor challenge experiments. Tumor cells expressing RAP grew with faster kinetics than the non-RAP expressing counterparts in wild type mice despite maintaining identical proliferation rates in vitro. Additionally, the reduced immunogenicity of RAP-expressing cells is tested in prophylaxis of cancer in murine models and receptor competition is examined in vivo. The data suggest a potential novel mechanism of immune evasion for tumors expressing competing ligands for immune receptors.
CD103 blockade reduces CD8 T cell accumulation during GVHD, but does not attenuate GVL effects. (169.35)
Authors: Anthony Bryan , Hamadani Mehdi , Wang Jiao-Jing , Gaughan Alice , Hadley Gregg
Published: January 2023

Graft versus host disease (GVHD) remains the salient barrier to broader use of hematopoietic stem cell transplantation (SCT) as a curative therapy for hematological malignancy. Although multiple means exist to neutralize donor-reactive T cells, such strategies have the potential to inhibit graft-vs-leukemia (GVL) effects, leaving the host vulnerable to relapse. The goals of the present study were: 1) to define the role of CD103 in promoting accumulation of donor CD8 T cells in the host gut following SCT, 2) assess the role of CD103 in promoting GVL effects. Following CD4 depletion, donor specific CD8 T cells selectively accumulated in the host gut epithelium in either MHC-matched or MHC-mismatched SCT models. Moreover, CD103 expression by CD8 T cells increased dramatically over time in both models. BLI studies revealed that CD103 expression is required for efficient accumulation of donor CD8 T cells in the host gut. BLI studies using TCR transgenic CD8 T cells co-expressing luciferase revealed that host-specific CD8 T cells preferentially accumulate in the host gut (as compared to the host spleen) during GVHD. Using a model of human chronic lymphocytic leukemia (CLL), we found that CD103 was not required for effective clearance of malignant B cells. These data indicate CD103 deficiency selectively attenuates GVHD pathology without compromising GVT effects, identifying CD103 as a potential target for GVHD prophylaxis.
Latent membrane protein 1 of Epstein Barr Virus alters cellular microRNAs in B cell lymphomas (169.37)
Authors: Harris Aleishia , Lambert Stacie , Krams Sheri , Martinez Olivia
Published: January 2023

Epstein-Barr Virus (EBV) infection is usually benign but immunodeficiency can lead to B cell lymphomas such as Post-Transplant Lymphoproliferative Disease (PTLD). PTLD is associated the expression of the EBV oncogene latent membrane protein 1 (LMP1). MicroRNAs (miRs) are small, non-coding RNAs that regulate gene expression. We hypothesized that LMP1 signaling in EBV+ B cell lymphomas can modulate cellular miRNA expression as a mechanism to promote viral persistence and/or host cell survival. Using an inducible, chimeric LMP1 signaling system we analyzed miR expression in a B cell lymphoma cell line after activation of LMP1 variants isolated from PTLD patients and from the B95.8 form obtained from a mononucleosis patient. The expression of 747 miRs was assayed by qPCR array and 323 miRs were detected. B95.8 LMP1 activation modulated 50 total miRs including miR503, linked to inhibition of proliferation in epithelial cells. The PTLD-derived LMP1 variants modulated 59 distinct miRs. These included pro-apoptotic miR29b which was down-regulated by PTLD-derived LMP1 variants but not by B95.8 LMP1. These data indicate that PTLD-derived forms of LMP1 uniquely alter cellular miR as compared to B95.8 LMP1. Thus, study of the functional properties of LMP1 variants may provide insight into mechanisms of lymphomagenesis.
Human and mouse natural killer cell cytotoxicity towards embryonic stem cells (169.41)
Authors: Peter Anna , Ames Erik , Zhou Ping , Nolta Jan , Murphy William
Published: January 2023

Embryonic stem cells (ESC) have tremendous potential for the treatment of numerous diseases but little is known regarding the immune response towards them. Human ESC (hESC) express very low levels of MHC class I, rendering them resistant to T cell-mediated rejection but making them likely targets of natural killer (NK) cells. As clinical use of MHC-matched ESC is unlikely, we examined the susceptibility of ESC to NK cell mediated killing. We tested the ability of activated human NK cells from several different donors to lyse the H9 hES cell line in vitro. Activated peripheral blood NK cells achieved significant H9 killing in a 4 hour chromium release assay. Similar experiments using mouse ES cell lines (JM8A3.N1, B6 background) with syngeneic (B6) or allogeneic (BALB/c) activated NK effector cells showed significant mouse ESC killing, regardless of NK cell source. This could be explained by very low MHC class I expression on the ESC targets leading to NK cell-mediated lysis due to lack of MHC class I, rather than allogeneic recognition. Experiments determining mouse ESC in vivo clearance by NK cells are under way. Together these data underscore the susceptibility of human and mouse ESC to NK cell-mediated lysis. This suggests that therapeutic applications of ESC are susceptible to host NK-cell mediated resistance.
Dendritic cells facilitate thymic recovery and hasten immune reconstitution after hematopoietic stem cell transplantation (169.44)
Authors: Langfitt Deanna , Busby Ken , Parish Colleen , Dallas Mari
Published: January 2023

The well-established role of dendritic cells in antigen presentation demonstrates their important role in development of T cells. In hematopoietic stem cell transplantation (HSCT) donor T cells are essential for engraftment, immune reconstitution, and graft vs. leukemia effect. Surprisingly, the role of Dendritic cells (DCs) in facilitating donor engraftment of hematopoietic stem cells has not been studied. Previously, we have demonstrated the importance of ex vivo expanded regulatory T cells (Tregs) in facilitating thymic engraftment after HSCT. Here we show the addition of dendritic cells given simultaneously with murine stem cells significantly improves engraftment after HSCT. Histological analysis demonstrated that thymic recovery was faster and more robust when dendritic cells were administered along with hematopoietic stem cells (HSC). Cell recovery and immune reconstitution was observed to occur more quickly in the thymus, spleen, bone marrow, and peripheral blood in experimental samples given additional DCs in HSCT. FACS analysis also confirmed immune reconstitution and donor cell engraftment was faster and increased survival was observed in experimental animals given DCs as well as stem cells. We hypothesize that the additional DCs establish themselves in the recipient thymus and assist in educating donor T cells, hastening immune reconstitution and improving cell engraftment.
Applying systems biology to develop T cell tolerance: Jurkat cell responses to knockdown hub genes using siRNA in-vitro transfection (169.47)
Authors: Lwin Wint , Park Ken , Wauson Matt , Gao Qin , Perkins David , Khanna Ajai
Published: January 2023

Introduction: Systems biology is increasingly gaining importance in studying complex genome wise associations and will, in future, integrate into personalized medicine. Aim: To study the effect of T cell lipotransfection by certain hub genes and evaluate their role in inducing T cell anergy. Methods: Using publically available gene-gene interactions, we constructed a network using an open bioinformatics software platform for visualizing molecular interaction networks to identify the following upregulated hub genes: ATP5C1, IL6ST, PRKCZ, MYC, FOS, JUN and MAPK1. We hypothesized that suppression of hub genes in the gene network would result in significant phenotypic effects on T cells resulting in T cell hyporesponsiveness and examined this in-vitro. Jurkat and HeLa cells were transfected with siRNA for the selected hub genes. Cell proliferation was measured using ATP luminescence and BRDU labeling. Results: We found a significant decrease in ATP production by Jurkat cells (P<0.05) when the hub genes ATP5C1 and PRKCZ were knocked down using siRNA transfection. No difference in ATP production was observed in siRNA transfected HELA cells. However, these cells showed a significant (P<0.05) decrease in cell proliferation when the genes MAPK1, IL6ST, ATP5C1, JUN and FOS were knocked down using siRNA. This experiment provides proof of principle of applying a systems biology approach in identifying hub genes for targeted therapeutics in transplant immunology.
CD200+ graft-infiltrating APCs are sufficient for induction of tolerance to skin allografts (169.8)
Authors: Gorczynski Reginald , Yu Kai , Chen Zhiqi , Khatri Ismat
Published: January 2023

CD200, a molecule of the immunoglobulin supergene family, increases graft survival and suppresses inflammation/acquired immunity after binding to its receptors (CD200Rs). Skin and cardiac allograft survival is increased in transgenic mice over-expressing CD200 under control of a doxycycline-inducible promoter, (CD200tg), along with increased intra-graft expression of mRNAs for genes which alter T cell subset differentiation. We found a decreased ability of CD11c+ or CD11b+ APCs from CD200tg recipients at days 7/14 to induce CTL from control lymph node cells in vitro, and increased induction of Foxp3+Treg by the same APCs. Focused gene array and real-time PCR measurements confirmed that with increased graft survival in CD200tg mice altered chemokines/cytokines expression occurred, in particular of IL-9, a mast cell growth factor. Mast cells infiltrated grafts of control animals also, but in these mice evidence of degranulation was apparent, and fewer Foxp3+Treg were present than in the case of CD200tg mice, where no degranulation was observed. Infusion of a competitive inhibitor of CD200:CD200R interactions, CD200tr, at high concentrations (50μg/mouse iv) caused rapid rejection of grafts in CD200tg mice, with evidence of mast cell degranulation within graft tissue. We conclude that allergic-type responses can break peripheral tolerance with induction of episodes of acute T-cell-mediated inflammation.