bluuebunny/crossref_metadata_2025 huggingface dataset:
IFN-γ suppresses antigen presentation by dendritic cells after infection (130.6)
Authors: Thacker Robert , Jordan Michael
Published: January 2023

IFN-γ has repeatedly been shown to increase antigen presentation in macrophages and dendritic cells (DCs). In the course of our studies investigating the influence of IFN-γ on antigen presentation during acute LCMV infection, we discovered novel suppressive effects of this cytokine on antigen presentation. We observed that after infection, the administration of an anti-IFN-γ ab on day 3 unexpectedly enhanced the ability of day 6 DCs to activate CD8+ and CD4+ T cells in vitro. In the presence of DCs isolated from these mice, T cells produce 5-10 times more IFN-γ, as compared to controls. Presentation of exogenously administered antigens is also heightened. Differences are not due to variability in viral load or antigen levels. Loading DCs with peptide reduced the differences in IFN-γ production down to 2 fold, suggesting variable costimulation played a minimal role in the observed phenomena. Inhibition and knockout studies have led us to conclude that neither IFN-γ induced production of 2,3 IDO nor NO production is responsible for the observed suppression. Evidence suggests the suppression may be due to an increase in SOCS-1 but further investigation is required. While these findings are at odds with the conventional understanding of the role of this cytokine as a promoter of antigen presentation (via upregulation of MHC molecules and immunoproteasome expression, etc.) they potentially explain much of its poorly understood disease and immune response-limiting effects.
Concurrent up-regulation of activation/maturation receptors and IFN-α by plasmacytoid dendritic cells in response to HSV and HIV (130.8)
Authors: Awoyomi Temitayo , Singh Sukhwinder , Fitzgerald-Bocarsly Patricia
Published: January 2023

Plasmacytoid dendritic cells (pDC) produce high levels of interferon-α (IFN-α) when stimulated with viruses; this viral stimulation also leads to modulation of functional surface receptors crucial for antigen presentation. These receptors, which include CD40, CD62L, CD80, CD83 and CD86, are important for pDC trafficking and T cell activation. In this study, we investigated whether the same pDC that produce IFN-α also modulate these surface receptors or whether they represent distinct subpopulations. pDC were stimulated with HSV-1 or HIV-1 and using flow cytometry, we observed a time-dependent increase in the co-expression of these activation/maturation markers and IFN-α expression. Up-regulation of CDs 40, 80, 83 and 86 were concurrent with IFN-α production in a subpopulation of pDC, while pDC shed CD62L and expressed IFN-α in response to virus. We also found that some pDC do not concurrently produce IFN-α and up-regulate surface receptors: they either produced IFN-α or expressed the activation markers. It is unclear whether these cells that carry out a single function represent distinct subpopulations of pDC or whether they were captured along a continuum: i.e., pDC that first produce IFN-α then mature into non-IFN-producing antigen presenting cells. Further studies will be done to tease apart the progression of expression of these markers, their dependence on IFN-α production and implications for diseases like HIV that suppress pDC function and alter pDC phenotype.
Contribution of autophagy to MHC-II mediated presentation of viral antigens (130.9)
Authors: Comber Joseph , Eisenlohr Laurence
Published: January 2023

Conventionally, major histocompatibility complex (MHC) class II molecules present peptides derived from exogenous sources of antigen. However, it is now accepted that endogenous antigen can also be processed for MHC class II peptide presentation. Several pathways of endogenous presentation have been reported, including macroautophagy. Here we used siRNA mediated knockdown to determine whether autophagy plays a role in the MHC class II-restricted immune responses to two different viral infections. Reduction of Atg7, a key ubiquitin- like enzyme catalyzing autophagosome expansion, did not significantly alter global MHC class II mediated presentation of epitopes derived from influenza or vaccinia viral infections. As a positive control, the autophagy protein LC3 was fused to the influenza specific MHC class II restricted epitope “Site-1”. In this context, presentation of the epitope was reduced by both chemical and siRNA mediated inhibition of autophagy. Taken together the results indicate that although individual epitopes can be presented on MHC class II via autophagy, the contribution of this pathway to the total MHC class II restricted response to viral infection may be limited.
Mam-A2.4 DNA vaccination leads to generation of effector phenotype CD8+ T cells and results in tumor regression. (131.1)
Authors: Lerret Nadine , Rogozinska Magdalina , Jaramillo Andres , Marzo Amanda
Published: January 2023

Mammaglobin-A (Mam-A) is a breast cancer-associated antigen expressed by approximately 80% of human breast tumors. Recently, seven epitopes derived from Mam-A were identified that bind HLA-A2 and are naturally presented by breast tumors, making these potential targets for immunotherapy. We show that epitope-specific, rather then full-length Mam-A DNA vaccination induces more effective tumor regression in vivo. Spleen cells from C57Bl/6 HLA-A2 mice, vaccinated with either full-length Mam-A DNA or epitope DNA (Mam-A2.1, Mam-A2.2, Mam-A2.4 and Mam-A2.6), were transferred into SCID-beige mice bearing tumors derived from the AU-565 (HLA-A2+/mammaglobin-A+) breast cancer cell line. Our results show that antigen-specific CD8+ T-cells derived from both full-length and epitope-specific DNA vaccination were initially capable of causing regression. However, only vaccination with Mam-A2.4 leads to long-term tumor regression, which can be further enhanced by low-dose total body irradiation of the tumor-bearing host. Furthermore, vaccination with Mam-A2.4 leads to the generation of CD8+ T-cells with an effector phenotype. These data demonstrate the potential of Mam-A single epitope DNA vaccination combined with low-dose total body irradiation as a therapy for the treatment and/or prevention of breast cancer.
Adoptive co-transfer of tumor specific CD4+ T cells awakens host protective immunity to MHC-II- ovarian cancer through CD40-mediated licensing of dendritic cells and CCL5 secretion. (131.14)
Authors: Nesbeth Yolanda , Martinez Diana , Toraya Seiko , Scarlett Uciane , Cubillos-Ruiz Juan , Rutkowski Melanie , Conejo-Garcia Jose
Published: January 2023

The effect of adoptive cell transfer (ACT) on endogenous anti-tumor immunity remains largely unknown. We previously showed that transferring tumor-primed T cells into tumor-bearing mice awakened endogenous antitumor mechanisms and induced the rejection of otherwise lethal ovarian cancers when coupled with depleting tumor-associated immunosuppressive dendritic cells (DCs). Here we elucidate this arousal of host responses to be due to the licensing of host DCs by co-transferred CCL5-secreting CD4+ T cells that recruit host antitumor immune cells. Tumor-primed CD4+ T cells significantly delayed tumor progression and directly activated host tumor-resident DCs which subsequently activated tumor-exposed C8+ T cells. These effects were long-lived, as host T cells obtained from mice treated with ACT were protective against secondary challenge with the same tumor. Disrupting CD40-CD40L interactions during ACT impaired DC maturation and ability to stimulate tumor-infiltrating CD8+ T cells. Similarly, abrogating CCL5 secretion reduced DC maturation and the survival increases obtained from ACT. The absence of CCL5 and CD40 further reduced the protective phenotype of the host cells against secondary challenge with the same tumor. Our data unveil a CD40-dependent mechanism of transferring immunity from adoptively transferred CCL5-secreting CD4+ T cells to host immune cells, causing sustained antitumor effects, and advance the field towards developing more potent anti-tumor therapies.
Nanoparticle [Gd@C82(OH)22]n induces dendritic cell maturation and promotes Th1 immune responses (131.2)
Authors: Yang De , Zhao Yuliang , Guo Hua , Li Yana , Tewary Poonam , Zhang Ning , Oppenheim Joost
Published: January 2023

[Gd@C82(OH)22]n, a fullerene-based nanoparticle of approximately 22 nm in saline solution, has been previously shown to exhibit anti-tumor effect in mouse tumor-bearing models with little toxicity, however, the underlying anti-tumor mechanism(s) remains elusive. Here we report that [Gd@C82(OH)22]n could induce phenotypic maturation of dendritic cells (DC) by stimulating DC production of cytokines including IL-12p70, upregulating DC costimulatory (CD80, CD83, and CD86) and MHC (HLA-A,B,C and HLA-DR) molecules, and switching DCs from CCL5-responsive to CCL19-responsive phenotype. In addition, [Gd@C82(OH)22]n-treated dendritic cells became functionally mature as illustrated by their capacity to activate allogeneic T cells. When mice were immunized with ovalbumin in the presence [Gd@C82(OH)22]n, [Gd@C82(OH)22]n enhanced ovalbumin-specific Th1-polarized immune responses as evidenced by predominantly increased production of IL-1β, IL-2, and IFNγ. Thus, [Gd@C82(OH)22]n nanoparticle is a potent activator of dendritic cells and Th1 immune response, which contributes to its potent anti-tumor effect.
Vaccination with survivin as a self TAA and SA-4-1BBL is effective in eradicating established lung carcinomas in CD8+ T cell and NK cell dependent manner (131.29)
Authors: Sharma Rajesh , Srivastava Abhishek , Yolcu Esma , Shirwan Haval
Published: January 2023

Costimulatory ligands of TNF family have great potential as adjuvant for therapeutic vaccines due to their critical role in the expansion of T cells, survival, and long-term memory. Particularly, 4-1BBL has the most potent T cell activity among the TNF costimulatory members. However, the use of 4-1BBL as the immunomodulatory component of conventional therapeutic vaccine has been limited due to its lack of activity in soluble form. We have recently generated a chimeric 4-1BBL with streptavidin (SA-4-1BBL) that exists as tetramers/oligomers with potent activity in soluble form. We herein formulated a subunit protein based vaccine containing SA-4-1BBL and survivin as an autologous tumor associated antigen. The therapeutic efficacy of this vaccine was tested in the transplantable survivin expressing 3LL metastatic lung tumor model. Vaccination with the survivin and SA-4-1BBL resulted in the eradication of established 3LL tumors in 75% of the animals. Importantly, animals with successful immunotherapy developed a robust NK cell cytotoxicity, antigen specific T cell proliferation, and IFN-γ and IL-2 responses. Depletion of various immune cell types demonstrated the indispensable role of CD8+ T cells and important role of NK cells for vaccine efficacy. Chimeric SA-4-1BBL hence prime CD8/NK cell axis for tumor eradication and has potential to be used as a specific immunological adjuvant to develop therapeutic vaccines against tumors with well characterized tumor associated antigens.
RNA nanoparticle vaccines: combining cytokine activation with antigen and CTL peptide delivery to improve vaccine efficacy (131.4)
Authors: McCormick Alison , Wykoff-Clary Sherri , Kaur Hardeep
Published: January 2023

We have used an RNA nanoparticle system to test the hypothesis that co-expression of a tumor antigen-cytokine fusion, with or without a co-delivered tumor CTL epitope, can improve anti-tumor immunity to a self antigen. Our initial studies demonstrated that a Semliki Forest Virus (SFV) bGal RNA expression vector could be modified to allow for in vitro coat protein RNA encapsidation by a distant alphavirus family member, Tobacco Mosaic Virus (TMV). Encapsidated RNA stimulated robust targeted immune cell uptake and activation, including dendritic cells. In response to vaccination, encapsidation significantly improved humoral and cellular immune responses compared to naked RNA, including IFNg activation , and protection from challenge with a lethal dose of bGal expressing tumor. More recently, our goal is to test efficacy of encapsidated RNA in a Non-hodgkin’s murine lymphoma tumor self-antigen system using the A20 expressing tumor. We have constructed a variety of SFV A20 scFv antigen-cytokine fusions, and are co-developing an A20 CTL surface antigen fusion. In vitro expression will be confirmed, and mice will be immunized and tested by ELISA and ELISpot for functional B and T cell immune activation. This nanoparticle RNA system generates vaccine compositions that are easy to customize, easy to produce, and facilitates intracellular delivery of unique nucleic acid/protein antigen combinations that should improve vaccine efficacy against a weakly immunogenic tumor.
Pre-transplant treatment with a GM-CSF-secreting myeloid leukemia cell vaccine combined with autologous BMT significantly improves the graft-versus-leukemia response (131.43)
Authors: Zilberberg Jenny , Friedman Thea , Korngold Robert
Published: January 2023

We investigated the effect of vaccination with a GM-CSF-secreting myeloid leukemia cell line MMB3.19 (GM-MMB3.19) on graft-versus-leukemia (GVL) responses in the syngeneic C57BL6 (B6) → B6 bone marrow transplantation (BMT) model against MMB3.19 (B6 origin) challenge. Our approach of recipient vaccination prior to BMT differed from current treatment designs in that no post-BMT de novo donor immune reconstitution was required. The contribution of host vs. donor T cells in the development of GVL responses were dissected by treatment of some groups with anti-CD4 and CD8 mAb to deplete host T cells (HTD group) prior to injection s.c with irradiated GM-MMB3.19 cells. Mice were exposed to 850 cGy 7 days later and underwent BMT, with or without additional donor lymphocytes (DL). On day 1 post-BMT, mice were challenged with MMB3.19 cells (i.p). A single pre-BMT treatment with irradiated GM-MMB3.19 significantly improved the survival rate of tumor-challenged DL mice (50%), compared to DL alone (0-12%; p<0.01). Vaccinated mice receiving no DL also had increased survival vs. DL alone (33%, p<0.05), an advantage which was eliminated in the HTD group. Hence, a single pre-transplant dose of GM-MMB3.19 cells can significantly improve survival of MMB3.19 challenged mice undergoing syngeneic BMT, suggesting that both host immune cells surviving recipient lethal irradiation and mature donor T cells given at time of BMT constitute significant components of the GVL effect.
Integration of HSP90 inhibitors in combinational immunotherapy targeting receptor tyrosine kinases (RTKs) in cancer (131.45)
Authors: Rao Aparna , Kawabe Mayumi , Storkus Walter
Published: January 2023

Background: Receptor tyrosine kinases (RTKs) and HSP90 play important roles in tumor growth, survival and metastasis. An effective therapeutic approach would promote decreased expression of RTKs on tumor cells, while increasing presentation of RTK-derived peptides in tumor cell MHC class I complexes, thereby enhancing the efficacy of anti-RTK CD8+ T effector cells. Aim: We are assessing the safety and effectiveness of the HSP90 inhibitor 17-DMAG in enhancing anti-tumor immunotherapy with RTK-specific T cells in murine tumor models. Methods: We tested the effect of 17-DMAG on tumor cell viability, proliferation and expression of RTK and the MHC I antigen-processing machinery in vitro. We also assessed the effect of 17-DMAG on DC phenotype and function. Results: 17-DMAG facilitated a dose-dependent reduction in RTK expression, but had no adverse effects on the antigen-processing machinery of the tumors. Anti-RTK CD8+ T cells exhibited improved recognition of DMAG-treated tumor cells in vitro. However, 17-DMAG treatment of DC resulted in decreased co-stimulatory molecule expression, IL-12p70 production and allo-stimulatory capacity by these APC in vitro. Conclusion: 17-DMAG treatment promotes immunologically-preferred alterations in tumor cells, but potentially adverse effects on DC functions in vitro. In vivo studies are warranted to assess the net anti-tumor impact of combinational immunotherapies integrating 17-DMAG.
Expanding Dendritic Cell Life Span and Enhancing VLP Vaccine Efficiency by Ginkgolides via Neutralization of ROS and Inhibition of p-JNK (131.5)
Authors: Zhang Sheng , Cubas Rafael , Chen Changyi , Yao Qizhi
Published: January 2023

Dendritic cells (DCs) play a critical role in initiating immune activation and/or tolerance in the host. This is dependent on the generation of reactive oxygen species (ROS). However high levels of ROS trigger activation-induced apoptosis (AIA) of DCs. Ginkgolides are known, strong antioxidants and we wanted to determine whether, with the help of Ginkgolides, virus-like particle (VLP)-matured DCs could increase vaccine efficiency. The generation of ROS and the apoptosis of DC was determined. Flow cytometry analysis was used to assess the activation and maturation of DCs. Specific antigen presentation by DCs was measured by ELISA and CTL assays. We found that VLPs stimulate the activation and maturation of DCs as demonstrated by the increased expression of CD40, CD54, CD86, and I-A. Staining of DHE showed that VLPs trigger ROS production and JC-1 staining demonstrated that this activation induced the apoptosis of DCs. Ginkgolides were shown to decrease the levels of ROS, inhibit p-JNK, inhibit apoptosis, and promote the longevity of DCs. Ginkgolide treatment also demonstrated an enhancement of antigen presentation to CD4+ T cells and cross-priming to CD8+ T cells by DCs. Further evidence also showed that administration of Ginkgolides increased the efficiency of VLP vaccination in vivo. Our results suggest that Ginkgolides have a notable potential to protect DCs from AIA while expanding their life span to prolong antigen presentation and enhance VLP vaccine efficiency.
Development of tumor-associated antigen (TAA)-specific T cell responses in heavily-tolerized mice requires vaccination with peptide variants that target T cells with decreased responsiveness to TAA (131.7)
Authors: Kemmler Charles , Slansky Jill
Published: January 2023

Vaccination with peptide variants of a tumor-associated antigen (TAA) can induce T cell responses that cross-react with the TAA. Using the gp70(423-431, AH1) epitope of endogenous MuLV as our model TAA, we have developed peptide variants that, unlike AH1 itself, induce AH1-reactive T cell responses upon vaccination of BALB/c mice. Two of these variants, A5 and 39, induce robust AH1-reactive responses in young mice. These variants were used to vaccinate aging mice, which have increased expression of and tolerance to gp70 protein. Variant-39 consistently induced AH1-specific responses, regardless of levels of gp70 expression in individual mice. Conversely, variant-A5 did not induce AH1-specific responses in most aging mice tested. The inability of variant-A5 to induce AH1-specific responses in individual mice correlated with increased gp70 expression. We hypothesized that variant-39 expands AH1-specific cells that are less reactive to AH1 than those expanded by variant-A5, thus impeding tolerance by peripheral AH1 presentation. Analyzing variant-induced responses in young mice, we determined that T cells expanded by variant-39 indeed have diminished function upon stimulation with AH1 relative to those elicited by variant-A5. These findings suggest that tumor vaccines require TAA variants that target cross-reactive T cell repertoires with low enough reactivity to TAA to avoid tolerance in the periphery, yet sufficient reactivity to provide effective antitumor responses.
Homeostatic Proliferation (HP) induced CD8+ T cell memory is regulated by the energy sensitive kinase; mTOR (132.17)
Authors: Li Qingsheng , Rao Rajash , Shrikant Protul
Published: January 2023

The cell intrinsic mechanisms guiding Homeostatic Proliferation (HP) leading to CD8+ T cell expansion and functional maturation for memory remain unclear. We demonstrate that lymphopenia induced naïve CD8+ (OT-I) T cell proliferation (HP) and clonal expansion requires IL-7 induced mTOR activation. Moreover, HP-induced mTOR enhances T-bet expression for functional maturation; IFNg and CTL activity, as well as expression of CD122 to facilitate IL-15 dependent memory generation. Surprisingly, rapamycin mediated mTOR blockade inhibits HP-induced clonal expansion and functional maturation, but promotes memory generation; in an IL-15 independent manner. However, the generation of CD8+ memory by either pathway produces tumor immunity. These results demonstrate that mTOR is a central regulator of HP-induced CD8+ T cell memory generation and indicates that HP-induced memory but not clonal expansion and functional maturation are essential for tumor immunity. Supported by: NIH RO1 CA104645, Alliance Foundation of Roswell Park and OCRF (to PAS)
T-independent antigens induce the formation of long-lived plasma cells (132.18)
Authors: Bortnick Alexandra , Quinn III William , Cancro Michael , Allman David
Published: January 2023

Long-lived plasma cells (PCs) are terminally differentiated B cells that reside in the spleen and bone marrow where they secrete antibody for a life-time. Although PCs are the cellular arbiters of humoral immunity, the signals required for formation of long-lived antibody secreting PCs remain poorly defined. In addition, how long-lived PCs resist apoptosis in response to ionizing radiation (IR) is also not understood. We find that resistance to IR-induced apoptosis occurs exceptionally early, within three days, during PC differentiation. IR-resistance is even established in PCs following immunization with NP-LPS, a type 1 thymus-independent (TI-1) antigen thought to generate only short-lived PCs. We have exploited this finding by administering IR in vivo to investigate the lifespan of antigen-specific NP-LPS induced PCs months after immunization. Contrary to current models, we find immunization with NP-LPS, a response which neither requires T-cell help nor elicits germinal center formation, is sufficient to drive the formation of long-lived IR-resistant PCs. Further, we find that resistance to IR-induced apoptosis is a cell intrinsic property unique to PCs and is not shared by other activated B cells. These findings establish radioresistance as a novel property of very early PCs and challenge the popular view that TI-1 antigens are unable to elicit the formation of long-lived PCs.
The hypoxic microenvironment modulates monocytic and dendritic cell inflammatory gene expression (133.10)
Authors: Varesio Luigi , Bosco Maria Carla , Blengio Fabiola , Pierobon Daniele , Raggi Federica , Fardin Paolo , Giovarelli Mirella
Published: January 2023

Hypoxia is a condition of low oxygen tension occurring in inflammatory lesions which creates a special microenvironment conditioning cell physiology. We investigated the molecular bases underlying the immunoregulatory functions of monocytes (Mn) and of terminally differentiated dendritic cells (mDCs) within the hypoxic microenvironment both in vitro and in vivo. Mn and mDCs hypoxic transcriptome was assessed using high-density oligonucleotide microarrays. The molecular pathways underlying gene transcription regulation by hypoxia were evaluated by promoter-driven reporter studies and EMSA. Profound modulation of the gene expression pattern was detected following Mn and DC exposure to 1% O2. We identified a significant cluster of hypoxia-responsive genes with immunological relevance, among which the macrophage inflammatory protein-3α (MIP-3α/CCL20) and the cytokine/extracellular matrix protein, osteopontin (OPN). Hypoxic upregulation of both genes was confirmed in vitro in primary Mn, Mn-derived macrophages, and DCs and in vivo in Mn cell recruited to the synovial joints of patients affected by Juvenile Idiopatic Arthritis (JIA), and associated with hypoxia-inducible factor-1α (HIF-1α) expression and NF-kB transcriptional activation. These studies lead to new perspectives on the impact of hypoxia on mononuclear phagocyte functions and to the definition of the mechanisms linking low pO2 to the pathogenesis of chronic inflammation.
AV411 (Ibudilast) and AV1013 are non-competitive inhibitors of macrophage migration inhibitory factor: a novel induced-fit allosteric inhibition mechanism (133.11)
Authors: Cho Yoonsang , Crichlow Gregg , Vermeire Jon , Leng Lin , Du Xin , Hodsdon Michael , Bucala Richard , Cappello Michael , Gross Matt , Gaeta Federico , Johnson Kirk , Lolis Elias
Published: January 2023

AV411 (Ibudilast) is an anti-inflammatory drug that was initially developed for the treatment of bronchial asthma but which also has been used for cerebrovascular and ocular indications. It is a non-selective inhibitor of various phosphodiesterases (PDEs) and has varied anti-inflammatory activity. More recently, AV411 has been studied as a possible therapeutic for the treatment of neuropathic pain and opioid withdrawal through its actions on glial cells. As described herein, the PDE inhibitors rolipram, theophylline, isobutylmethylxanthine (IBMX), cilostazol, arofylline, AV411 and its PDE-inhibition compromised analog AV1013 inhibit the catalytic and chemotactic functions of the pro-inflammatory protein, macrophage migration inhibitory factor (MIF). Enzymatic analysis indicates that these compounds are non-competitive inhibitors of the p-hydroxyphenylpyruvate (HPP) tautomerase activity of MIF. The allosteric binding site of AV411 and AV1013 is identified by NMR and X-ray crystallography, and the structural basis of inhibition is reported using the crystal structure of the MIF/AV1013/HPP ternary complex.
IFN-γ inhibits and TGF-β promotes thyrocyte hyperplasia and proliferation (TEC H/P) by modulating pro- and anti-proliferative molecules (134.11)
Authors: Fang Yujiang , Braley-Mullen Helen
Published: January 2023

Our previous in vivo studies showed that IFN-γ-/-NOD.H-2h4 mice develop severe TEC H/P. Proliferating TEC produce TGF-β and IFN-γ inhibits TEC H/P. This study was undertaken to directly test the hypothesis that IFN-γ inhibits and TGF-β promotes TEC H/P in vitro and explore the underlying mechanisms. Varying concentrations of IFN-γ and TGF-β were incubated with cultured TEC for 3 days. Cell proliferation was evaluated by IHC staining for PCNA, while apoptosis was determined by TUNEL staining. RT-PCR was used to evaluate the effect of cytokines on pro- and anti-proliferative molecules. The results indicate that IFN-γ inhibits and TGF-β promotes TEC proliferation in a dose dependent manner, and both cytokines had little effect on apoptosis. The anti-proliferative effect of IFN-γ correlates with upregulation of the anti-proliferative molecule P21 and downregulation of pro-proliferative molecules cyclin D and PCNA. Studies to determine the mechanisms by which TGF-β promotes TEC H/P are in progress. These in vitro results are consistent with our previous in vivo studies and provide direct support for the hypothesis that IFN-γ inhibits, while TGF-β promotes, TEC H/P. This extends our previous study by providing evidence that cytokines regulate TEC H/P by modulating pro- and anti-proliferative molecules. Such studies may increase our understanding of mechanisms underlying development of epithelial cell hyperplasia in the thyroid as well as other tissues and organs (NIH RO1 AI074857).
Systemic IL-12 administration modulates dendritic cells to overcome the immunosuppressive microenvironment of the liver (134.14)
Authors: Chan Tim , Back Timothy , Subleski Jeffrey , Weiss Jonathan , Ortaldo John , Wiltrout Robert
Published: January 2023

The liver is an immunologically unique organ containing tolerogenic dendritic cells (DCs) maintaining an immunosuppressive microenvironment. Immunotherapeutic strategies, such as systemic IL-12 therapy activate NK and T cells improving host outcome to tumor. However, the feedback effect of IL-12-based treatments on splenic and hepatic DCs in their microenvironments and their contributing roles to an anti-tumor response has not been examined. In this study, we demonstrate systemic IL-12 therapy results in a marked 2-3 fold increase in total splenic and hepatic DC populations in naïve and RENCA-bearing mice with all DC subsets increasing except for hepatic plasmacytoid DCs (pDCs). Increased costimulatory molecule expression in splenic and hepatic DCs was observed following IL-12 treatment but absent in common gamma chain knockout mice deficient in NK cells. Furthermore, hepatic DCs from IL-12 treated mice produced higher levels of IL-12 and TNFα when combined with TLR agonists and on a per cell basis, demonstrated significantly greater T cell proliferative capacity to levels similar to splenic DCs. Furthermore, IL-12 administration preferentially increased hepatic T cell activation and IFNγ expression compared to splenic T cells. Collectively, the data shows IL-12 administration enables DCs to overcome inherent suppressive components of the hepatic environment and potentially impact the design of IL-12-based immunotherapeutic strategies targeting hepatic malignancies.
Supraphysiological levels of IL-15 accelerate immune reconstitution in lymphopenic mice (134.16)
Authors: Bergamaschi Cristina , Ganneru Brunda , Rosati Margherita , Usami Osamu , Valentin Antonio , Beach Rachel , Alicea Candido , Felber Barbara , Pavlakis George
Published: January 2023

Loss of lymphocytes and T cells are a significant medical problem in AIDS and other immunodeficiencies, as well as after various treatments and cytoreductive therapies. T cell recovery after lymphopenia is delayed and incomplete and this can lead to prolonged immune dysfunctions. Lymphoablated patients have rapid and transient increases of circulating homeostatic cytokines IL-7 and IL-15, suggesting a role for these factors in immune reconstitution. Analysis of sera from lymphoablated patients revealed that the main form of circulating IL-15 is in a complex with soluble IL-15Rα (IL-15sRα). We have shown that injection of DNA expressing supraphysiological levels of IL-15/IL-15sRα in normal mice led to dose-dependent increases in the number and frequency of NK and T cells. The effect of IL-15/IL-15sRα on immune reconstitution was tested in mice lymphodepleted by cyclophosphamide (CTX) treatment. A single administration of IL-15/IL-15sRα DNA accelerates the full recovery of NK cells in spleen and lung. Two administrations of IL-15/IL-15sRα DNA promote the recovery of T cells in spleen within 14 days, faster than CTX-treated mice (p=0.017 Wilcoxon). T and NK cells from mice treated with supraphysiological levels of IL-15/IL-15sRα show greater degranulation and IFN-γ production after in vitro stimulation. These results suggest that IL-15 can contribute to rapid immune reconstitution, and suggest the use of IL-15/IL-15sRα to accelerate recovery from lymphopenia.
Regulation of TLR4 signaling in macrophages by Macrophage Stimulating Protein, the ligand for the Ron receptor tyrosine kinase (134.20)
Authors: Yu Shan , Ray Manujendra , Sharda Daniel , Wilson Caleph , Liu Qingpin , Kaushal Naveen , Prabhu K. Sandeep , Hankey Pamela
Published: January 2023

The Ron receptor tyrosine kinase regulates the balance between classical (M1) and alternative (M2) macrophage activation. The ligand for Ron, macrophage stimulating protein (MSP), inhibits the expression of iNOS, while promoting the expression of arginase I in primary murine macrophages. Ron-/- mice express elevated levels of IL-12 following endotoxin administration, resulting in increased serum IFNγ levels and enhanced susceptibility to septic shock. Here, we demonstrate that MSP inhibits LPS-induced IL-12p40 expression in primary macrophages. MSP does not inhibit the MyD88-independent activation of IRF3 and production of IFNβ in response to LPS, but limits the IFNβ-dependent phosphorylation of Stat1 following LPS stimulation. While MSP does not inhibit LPS-induced Tak1 phosphorylation or Map kinase activation in primary macrophages, the induction of IκB kinase (IKK) activity, IκB degradation and DNA binding of NFκB following LPS stimulation are delayed in the presence of MSP. In addition, Ron inhibits serine phosphorylation of p65 and NFκB transcriptional activity induced by LPS stimulation of TLR4. Finally, MSP inhibits the NFκB-dependent upregulation of the nuclear IκB family member, IκBζ, while inducing expression of the Stat3-regulated nuclear IκB family member, Bcl-3. LPS also induces expression of Ron and Sf-Ron in primary macrophages, suggesting that the upregulation of Ron by LPS could play a role in limiting subsequent LPS responses in macrophages.
The region in the common gamma chain between amino acids (aa) 314-323 dictates efficient activation of the insulin receptor substrate (IRS)-2 pathway by interleukin (IL)-4. (134.4)
Authors: Heller Nicola , Qi Xiulan , Gesbert Franck , Keegan Achsah
Published: January 2023

Previously, we demonstrated that the γC subunit of type I IL-4 receptor was required for robust tyrosine phosphorylation of the downstream adapter protein, IRS-2, and the robust expression of mRNA and protein of a subset of genes characteristic of alternatively-activated macrophages. The type II IL-4 receptor did not have this robust activity. In addition to the unique ability to bind Jak3, the cytoplasmic domain of human γC contains a putative IRS-2 docking motif and other γC-utilizing cytokines also activate IRS-2. Thus, we hypothesized that the γC cytoplasmic domain provides a docking site for IRS-2 cooperating with the site in IL-4Rα. To test this, full-length (FL) and γC truncation mutants were stably co-expressed with wildtype (WT) IL-4Rα in CHO cells; signaling responses to IL-4 were examined. IL-4-induced IRS-2 and STAT6 phosphorylation was observed in CHO expressing FL γC and γC truncated at aa323 but not in cells expressing γC truncated at aa308 or 285. These data suggest robust activation of IRS-2 and STAT6 by IL-4 requires a 15 aa portion of γC between aa314-323. This interval did not contain Tyr325, part of the putative IRS-2 docking motif. Previous studies in other receptor systems suggested that aa323-335 was required for Jak3 engagement. The requirement for IL-4-induced JAK3 recruitment/activation will be examined in these CHO transfectants. Supported by HL096897 (NMH) and AI038985 (ADK).
Mechanistic study of leptin-enhanced B cell survival (134.7)
Authors: Lam Queenie , Lu Liwei
Published: January 2023

Regulation of apoptosis and cell cycle progression plays an essential role in the maintenance of B cell homeostasis because the fine balance of survival and death of B cells is critical to prevent occurrence of lymphocytic disorders. During the past decade, remarkable progress in understanding the process of B development has been achieved but much less is known on the survival niches that are critical to the maintenance of B cell homeostasis. Leptin has recently been recognized to play a crucial role in modulating the immune response but it has remained unclear whether and how leptin signaling is involved in modulating B cell physiology. In our studies we demonstrate that leptin promotes B cell survival by inhibiting apoptosis and enhancing cell cycle progression, which are associated with the induction of expression of Bcl-2 and Cyclin D1, respectively. We further show that leptin can induce the expressions of Bcl-2 and Cyclin D1 by two pathways; by activating their promoters directly and by suppressing miRNAs that target their putative 3’untranslated region to enhance their expressions indirectly. These findings provide insights into the important role of leptin in maintaining B cell homeostasis, which will lead to a fuller understanding of the regulation of adaptive immunity and be beneficial to the future design of alternative therapeutic strategies in B cell-derived disorders.
Vitamin D receptor deficiency alters the ability of CD8 T cells to induce inflammatory bowel disease. (135.14)
Authors: Bruce Danny , Cantorna Margherita
Published: January 2023

Vitamin D receptor (VDR) KO mice are more susceptible to several different experimental models of inflammatory bowel disease (IBD) including the Rag KO transfer model when naïve VDR KO CD4 T cells are used to induce disease. CD8 T cells from WT mice do not induce IBD when transferred to Rag KO mice. Conversely, VDR KO CD8 T cells induce IBD in Rag KO recipients. There is no difference in the expression of CD62L, CD44, CCR9 and α4β7 or secretion of IFN-γ and TNF-α between WT and VDR KO CD8 T cells. Disease induced by VDR KO CD8 T cells is mild and the recipients do not lose weight or show outward signs of disease. Instead histological evidence of inflammation and increased numbers of CD8 T cells in the IEL characterize the disease. While WT CD8 T cells fail to induce IBD when transferred to Rag KO mice, they do induce a fulminating form of IBD when transferred to Rag/VDR double KO (DKO) mice. Therefore, the host expression of the VDR alters CD8 T cell development and or function. Experiments to probe the cause of CD8 T cell induced pathology in DKO mice are underway.
GM-CSF control of macrophage differentiation is a critical effector of Th17 responses in vivo (135.27)
Authors: Barin Jobert , Baldeviano G. , Talor Monica , Zheng Dongfeng , Čiháková Daniela , Rose Noel
Published: January 2023

Like many other tissue-directed models of autoimmune disease, experimental autoimmune myocarditis (EAM) is mediated by Th17-directed pathology. The composition of cardiac infiltrates in EAM is predominantly monocytic, indicating that these cells are most likely to be inflammatory effectors in this disease process. Like other Th17 autoimmune diseases, IFNγ-deficient mice develop very severe disease. We observed enhanced GM-CSF production from autoantigen-specific IFNγ-/- CD4+ cells, from the heart and spleen, suggesting that the induction of GM-CSF by Th17 cells may participate in disease pathophysiology. After generating IFNγ-/-IL17A-/- double-knockout mice, we observed that the production of GM-CSF in CD4+ is driven by IL17 signaling. Towards this end, we generated bone marrow-derived macrophages (BMMϕ) in vitro, in the presence of GM-CSF, rather than M-CSF. Following transfer, recipients of GM-CSF BMMϕ developed more severe disease than controls. This severe disease was accompanied by enhanced fibrosis and progression to dilated cardiomyopathy. This report links GM-CSF control of macrophage differentiation to the functional consequences of Th17-mediated autoimmunity
Tissue tropism of autoreactive memory CD4 T-cells (135.30)
Authors: Lees Jason , Farber Donna
Published: January 2023

The continual presence of antigen in site-specific autoimmune diseases, such as type 1 diabetes, makes it difficult to determine the relative roles of effector and memory CD4 T-cells in perpetuation of established autoimmunity. It is not known to what extent memory cells retain effector and tissue trafficking capacities of the primary autoimmune response that established them and the timing and mechanisms involved in imprinting of these attributes is unclear. In this study, we hypothesized that pancreas homing diabetogenic effector cells may develop into memory T-cells with distinct trafficking capacities. We used a mouse model of type 1 diabetes in which transferred influenza hemagglutinin (HA)-specific CD4 T-cells induce pancreatic islet destruction in mice that express HA via an insulin promoter. We examined the functional and trafficking capacities of memory T-cells derived in the absence of further antigen recognition by adoptive transfer of tissue resident effector T-cell populations recovered from the spleen or pancreas throughout diabetes development into wild type mice. Effector cells recovered from both spleen and pancreas during diabetes onset generated memory T-cells, but only memory T-cells derived from the pancreas were capable of retrafficking to the pancreas in the absence of antigen. These data demonstrate that tissue effector CD4 T-cells contain a memory precursor imprinted with tissue trafficking specificity prior to development of memory T-cells.
Kinetics of Th17 development in type 1 diabetes (135.35)
Authors: Li Cheng-Rui (Michael) , Bradley Linda
Published: January 2023

Type 1 diabetes (T1D) is an autoimmune disease. It has been established that Th1 cells play a central role in the autoimmune attack against islet β cells. The Th17 subset has recently been implicated in many autoimmune disease models. However, its role in T1D is still unclear. In diabetic NOD mice, we found elevated serum IL-17 levels and IL-17 producing infiltrating CD4+ T cells from islets. Adoptive transfer of in vitro differentiated islet-antigen-specific Th17 cells induced diabetes in recipient NOD.Scid mice with similar kinetics of that induced by Th1 cells. These data suggest a possible causal role of Th17 cell in T1D. To study the development of Th17 cells in vivo, we transferred naïve CD4+ T cells into neonatal NOD mice, and monitored phenotype and function of both transferred and endogenous T cells for up to 35 weeks. Surprisingly, we were able to identify islet infiltrating endogenous CD4+ T cells that expressed either IFNγ or IL-17 in 2-week-old NOD mice. Transferred naïve cells proliferated in recipients; they rapidly acquired effector memory phenotype and effector cytokine production. As expected, the frequency of IFNγ-producing CD4+ T cells increased over time in both endogenous and transferred populations. In contrast, the frequency of IL17-producing CD4+ T cells did not significantly change over time. These data suggest that, in T1D model, the Th17 cells may be a dynamic population with constant turnover rate, while contributing to diabetes pathogenesis.
Gamma-delta TCR+ T-cells in systemic sclerosis (135.37)
Authors: Nwaneshiudu Adaobi , Jung Weon-ju , Tsygankov Alexander , Rayevskaya Marina , Oleszak Emilia , Myers Allen , Platsoucas Chris
Published: January 2023

T-cell involvement may be central to the pathogenesis of systemic sclerosis (SSc). Like αβ TCR+ T-cells, γδ TCR+ T-cell diversity during T-cell development results in a repertoire of different T-cells each with a unique TCR. Therefore the probability of finding identical TCR on defined T-cell populations is negligible, except in the context of an antigen-driven T-cell response. To determine whether γδ TCR+ T-cells are clonally expanded in skin biopsies and/or peripheral blood of patients with SSc (n=7), γ-chain (VγI & Vγ9) and δ-chain (Vδ1 & Vδ2) TCR transcripts were amplified by gene-specific PCR, followed by cloning and sequencing. We report the presence of substantial proportions of identical VγI-chain transcripts (14.3%-37.2%; p<0.05), and Vγ9 transcripts (24%-83.3%; p<0.05) in skin biopsies and/or PBMC of patients with SSc. Likewise, there were multiple identical Vδ1-chain transcripts (25%-50%; p<0.05), and Vδ2-chain transcripts (18.1%-80%; p<0.05) in the samples analyzed. Extensive clonal expansions of γ- and δ-chain TCR transcripts were identified in skin biopsies and peripheral blood of patients with SSc, demonstrating the presence of oligoclonal populations of γδ TCR+ T-cells in these patients. These γδ TCR+ T-cells may have undergone activation and clonal expansion in vivo in response to as yet unidentified antigens. Future studies to identify the antigens recognized by these clonally expanded γδ TCRs will facilitate better understanding of SSc pathogenesis.
When Toll-like receptor and T-cell receptor signals collide: A mechanism for enhanced CD8 T-cell effector function (136.11)
Authors: Geng Degui , Zheng Liqin , Asprodites Nicole , Velasco-Gonzalez Cruz , Davila Eduardo
Published: January 2023

Emerging reports reveal that activating toll-like receptor-2 (TLR)-MyD88 signals in CD8 T-lymphocytes enhances cytokine production and cytotoxicity. In the present study, we examined the physiological-significance and molecular mechanisms involved in this process. We found that TLR2 engagement on T-cell receptor transgenic CD8 OT-1 T-cells increased T-bet transcription factor levels and consequently augmented effector gene transcript and protein levels both in vivo and in vitro whereas, TLR2 agonist did not costimulate TLR2-/-OT-1 or MyD88-/-OT-1 T-cells. Elevated T-bet level in TLR2-MyD88-stimulated T-cells was a consequence of increased biosynthesis resulting from the enhanced activation of the mammalian target of rapamycin (mTOR) pathway. Inhibition of mTOR or blockade of phosphatidylinositol-3-kinase (PI3K)-Akt or PKC in T-cells abolished the costimulatory effects of the TLR2 agonist. In vivo, activating TLR2-MyD88 signals within OT-1 T-cells increased the levels of effector molecules and enhanced the clearance of Listeria monocytogenes-Ova. These results help define a signaling pathway linking the TLR-MyD88 and mTOR pathway in an Akt and PKC signaling-dependent manner, highlighting a critical role for TLR-MyD88 signaling in T-cell activation and cytotoxicity. These findings offer the opportunity for improving the efficacy of vaccines and T-cell-based tumor-immunotherapies by manipulating TLR-MyD88 signaling within CD8 T-cells.
NLRP3 inflammasome-dependent HMGB1 release induced by a bacterial pore-forming toxin (136.15)
Authors: Chu Jessica , Alber Sean , Watkins Simon , Núñez Gabriel , Salter Russell
Published: January 2023

The NLRP3 inflammasome is a caspase-1-containing complex involved in the maturation of pro-inflammatory cytokines IL-1β and IL-18. NLRP3, independent of its inflammasome functions, also plays a role in a cathepsin B-dependent, caspase-1-independent necrotic cell death process known as pyronecrosis. We have previously shown that the cholesterol-dependent, pore-forming toxin tetanolysin O (TLO) triggers IL-1β release from LPS-primed murine bone marrow-derived macrophages (BMDM) in a NLRP3 inflammasome- and cathepsin B-dependent manner. The work contained herein supports an additional role for TLO in the necrosis of LPS-primed BMDM. Both a low and a high TLO dose induce release of lactate dehydrogenase (LDH) and high mobility group box 1 (HMGB1) and this release is NLRP3-dependent for the low toxin dose. Low dose TLO-induced HMGB1 release is also dependent on the activities of caspase-1 and cathepsin B, suggesting a novel form of cell death that is distinct from caspase-1-independent pyronecrosis. We also observe via fixed cell microscopy that TLO causes HMGB1 to move from the nucleus to the cytoplasm in LPS-primed BMDM and that HMGB1 release is dependent on NLRP3 activity. To our knowledge, this is the first study to visualize HMGB1 localization in cells lacking NLRP3. These results provide information on the regulation of HMGB1 release and could eventually lead to new therapeutic approaches to target HMGB1, which contributes to many inflammatory diseases.
Progesterone minimizes immunoprotective effect of CpG ODN against infection by skewing immune responses toward Th17 (136.2)
Authors: Butts Cherie , Wang Vivian , Puig Montserrat , Verthelyi Daniela
Published: January 2023

Oligodeoxynucleotides encoding CpG motifs (CpG ODN), which activate the innate immune system and promote a strong Th1 response, are currently being tested in therapies for allergies and infectious diseases, including cutaneous leishmaniasis. Steroid hormones (such as progesterone) are associated with suppression of pro-inflammatory immune responses and a shift away from Th1 responses - critical for clearing intracellular parasites. Our assessment of progesterone effects on immune responses by mouse splenocytes in vitro and in vivo demonstrated a shift in the responses to CpG ODN characterized by limited upregulation of MHC Class II molecules by antigen presenting cells, lower IL-12 and IL-6, and increased IL-23 production. This was associated with increased IL-17 and lower IFN-γ secretion by CD4+ helper T cells. Similarly, progesterone treatment of human PBMCs stimulated with CpG ODN limited upregulation of CD83 and CD86 and decreased IFN-γ and IP-10 production. Assessment of whether the shift from Th1 responses by progesterone reduces the immunoprotective effects of CpG ODN in mice challenged with Leishmania major showed that orchiectomized mice receiving progesterone developed larger lesions than those receiving placebo and were not protected from disease when treated with CpG ODN. These data indicate that progesterone is able to skew immune responses by CpG ODN towards Th17 and contribute to disease susceptibility in women.