bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of PI3K-Akt in crosstalk between Notch and CD28-CD80/86 pathways in dendritic cell “backsignaling” (162.2)
Authors: Koorella Chandana , Nair Jayakumar , Carlson Louise , Lee Kelvin
Published: January 2023

Survival of myeloma cells is characterized by their interaction with bone marrow stromal cells, which produce critical soluble factors including IL-6 and IDO. Targeting pathways involved in production of factors which are important in myeloma cell biology, provides novel therapeutic targets for a disease which otherwise remains incurable. At the cellular level, dendritic cells (DC) in the bone marrow microenvironment and at the molecular level the CD28-CD80/86 and Notch-Jagged pathways were separately implicated by us in myeloma induced IL-6 production. While Notch signaling leading to IL-6 production in DC is well understood, the mechanism of “backsignaling” via CD80/86- ligands with short cytoplasmic tails, remains largely uncharacterized. We demonstrate that inhibiting Notch signaling leads to a significant decrease in CD28 mediated IL-6 and IDO production by DC, suggesting crosstalk between Notch and CD28-CD80/86 pathways. IL-6 production by dendritic cells, upon ligation of CD80/86 by CD28-Ig, involves the PI3K-Akt-FoxO3a axis, which has not been previously established. Furthermore, we demonstrate a novel crosstalk between Notch and CD80/86 signaling involving regulation of the PI3K-Akt pathway by casein kinase and PTEN, which in turn are regulated by Notch signaling. Targeting pathways involved in IL-6 and IDO prompts not only clinical evaluation to improve MM patient outcome but also extends to advancing knowledge in T-cells and normal plasma cell biology.
Synapse-directed delivery of immune-stimulants using T-cell-conjugated nanoparticles (162.30)
Authors: Stephan Matthias , Bak S. , Stephan Sirkka , Chen Jianzhu , Irvine Darrell
Published: January 2023

Regulating molecular interactions in the T cell synapse to prevent autoimmunity or, conversely, to boost anti-tumor immunity has long been a goal in immunotherapy. However, delivering therapeutically meaningful doses of immune-modulating compounds into the synapse represents a major challenge. Here, we report that covalent coupling of maleimide-functionlized nanoparticles (NPs) to free thiol groups on T cell membrane proteins enables efficient delivery of compounds into the T cell synapse. We demonstrate that surface-linked NPs are rapidly polarized toward the nascent immunological synapse (IS) at the T cell/APC contact zone during antigen recognition. To translate these findings into a novel therapeutic application we tested the NP delivery of NSC-87877, a dual inhibitor of Shp1 and Shp2, key phosphatases that downregulate T cell receptor activation in the synapse, in the context of adoptive T cell therapy of cancer. Conjugating NSC-97977-loaded NPs to the surface of tumor-specific T cells just prior to adoptive transfer into mice with advanced prostate cancer greatly promoted T cell expansion at the tumor site, relative to co-infusing the same drug dose systemically, leading to enhanced survival of treated animals. Altogether, our studies support the application of T-cell-linked synthetic NPs as efficient drug delivery vehicles into the IS, as well the broad applicability of this new paradigm for therapeutically modulating signaling events at the T-cell/APC interface.
The importance of type I interferons in radiation-mediated antitumor responses (162.33)
Authors: Lim Joanne , Gerber Scott , Lord Edith
Published: January 2023

Interferon alpha (IFNα) has been FDA-approved for treatment of high-risk melanoma since 1995. Besides having direct antitumor effects, IFNα and IFNβ, the other major type I IFN (IFN-I), have been found to act through enhancing multiple pathways within the host immune system. However, the mechanisms by which IFN-Is mediate these responses remain unclear. Our previous data demonstrated that treatment of in vivo B16 melanoma with a single high dose local radiation induced an increase in IFNγ levels within the tumor microenvironment. Interestingly, this increase is abolished in the absence of IFNα/β signaling within host cells, as observed using IFNα/β receptor knockout mice (KO). Importantly, the induction of downstream IFNγ-dependent genes such as the CXCR3 family of chemokines is also affected. Since CXCR3 chemokines have been shown to be crucial chemoattractants for T cells in tumors, it is not surprising that the lack of IFNα/β receptor also resulted in decreased recruitment of CD8 T cells to tumors in response to radiation treatment. Corresponding to these effects, radiation-treated KO mice exhibit poor tumor growth control compared to wild type mice. Taken together, our data suggest that IFN-Is play a critical role in radiation-mediated antitumor responses. An important mechanism through which this occurs depends on the ability of IFN-Is to enhance IFNγ responses, which in turn upregulates CXCR3 chemokines in the tumor, resulting in enhanced recruitment of CD8 T cells.
Immunoglobulin Fc fragment tagging allows strong activation of endogenous CD4 T cells to re-shape the tumor milieu and enhance the antitumor effect of lentivector immunization (162.4)
Authors: He Yukai , Hong Yuan , Peng Yibing , Xiao Haiyan , Mi Michael , Munn David
Published: January 2023

A major problem with current cancer vaccines is that the induction of CD8 immune responses is rarely associated with antitumor benefits mainly due to the multiple immune suppressions in the established tumor lesions. In this study, we investigated if and how activation of endogenous CD4 T cells could be achieved to relieve the suppressive tumor milieu. We engineered a lentivector to express a nominal fusion Ag composed of HBsAg and IgG2a Fc fragment (HBS-Fc-lv) to increase the magnitude of CD8 response, but more importantly, to induce effective CD4 co-activation. We found that, remarkably, HBS-Fc-lv immunization caused significant regression of established tumors. Immunological analysis revealed that HBS-Fc-lv immunization markedly increased the number of functional CD8 and CD4 T cells and the level of Th1/Tc1-like cytokines in the tumor, while substantially decreased Treg ratio. The favorable immunologic changes in tumor lesions and the improvement of antitumor effects from HBS-Fc-lv immunization were dependent on the Fc receptor mediated CD4 activation. Adoptive transfer of the CD4 T cells from the HBS-Fc-lv immunized mice could activate endogenous CD8 T cells via IFNgamma and CD40L dependent manner. We conclude that endogenous CD4 T cells can be activated by lentivector expressing Fc tagged Ag to provide another layer of help, i.e. creating a pro-inflammatory milieu within the tumor lesion to help the effector phase of immune responses to enhance the antitumor effect.
Aire regulates immune tolerance towards melanoma (162.43)
Authors: Zhu Menglei , Nagavall Anil , Su Maureen
Published: January 2023

Effective recognition of tumor antigens by circulating T cells constitutes an important part of successful immunosurveillance against cancer growth. Immune tolerance towards tumor antigens, therefore, is a barrier toward an effective anti-tumor immune response. Autoimmune regulator (Aire) is a transcriptional regulator that is critical for the development of central T cell tolerance by inducing self-antigen expression in the thymus. Aire has a well-known role in preventing autoimmunity, and Aire deficiency leads to multi-organ autoimmunity in both human patients and mice. Recently, Aire polymorphisms have been shown to be associated with melanoma development. Whether Aire may mediate immune responses against tumors and its mechanism, however, is not clear. We utilized a mouse harboring a dominant negative point mutation in Aire (Aire G228W) that results in hypomorphic Aire function to investigate the role of Aire deficiency in melanoma. Aire G228W mice develop more robust immune response against melanoma which leads to suppressed tumor growth. This enhanced immune response results from defective negative selection of T cells recognizing melanoma. Aire expression in the thymus is sufficient to mediate immune tolerance against melanoma. Our findings provide a biological mechanism for the epidemiological association between Aire polymorphisms and risk for melanoma and suggest that modulation of central tolerance mechanisms could be a potential strategy for cancer therapy.
Inhibition of prostate tumor growth by potentiating antitumor immune response using natural product (162.46)
Authors: Singh Shailesh , Singh Rajesh , Lillard, Jr. James , Grizzle William
Published: January 2023

It is now well accepted that tumors evade the patient's defense system during progression and growth at distant sites. Mounting evidences suggest immunological defects in PCa patients with higher numbers of functionally suppressive CD4+CD25+FOXP3+ T cells (Treg) and lower number of effectors T cells. Furthermore, tumor microenvironment exerts a profound immunosuppressive effect on effectors T cells by secreting TGF-β and IL-10. Therefore, modulating TGF-β and IL-10 using natural and dietary components constitutes an innovative approach for prostate cancer prevention and treatment. In this study, we have shown that Withaferin-A (WA), a bioactive compound isolated from Withania Somnifera also called “Ashwagandha” has the ability to modulate immunosuppressive cytokines (IL-10 and TGF-β) produced by the PCa cells. Additionally, we show selective inhibition of Treg proliferation and induction of apoptosis in Tregs cells in immune competent PCa mouse model treated with WA compared to controls. Our results suggest that WA have potential to fine-tune immunosuppressive phenotype of tumor-associated lymphocytes by modulating immuno-suppressive cytokines. Significant inhibition of tumor growth in immune competent PCa mouse model as compared to immune deficient xenograft suggest potential role of WA on reversal of anti-tumor immune response from immune suppression to immune competence, which is required to fight against prostate cancer.
Regulation of SHP2 by PTEN/AKT/GSK-3β signaling facilitates IFN-γ resistance in hyperproliferating gastric cancer (162.8)
Authors: Tseng Po-Chun , Huang Wei-Ching , Chen Chia-Ling , Sheu Bor-Shyang , Shan Yan-Shen , Tsai Cheng-Chieh , Wang Chi-Yun , Chen Su-O , Hsieh Chia-Yuan , Lin Chiou-Feng
Published: January 2023

In addition to oncogenic activation, and tumor suppressor gene inactivation escape from immune surveillance, such as IFN-γ resistance, is critical for cancer cell growth and survival. In this study, we investigated the crosstalk signaling between IFN-γ resistance and signaling of hyperproliferation in gastric cancer cells. IFN-γ inhibited the cell growth of MKN45 cells but not hyperproliferating AGS cells. AGS cells did not respond to IFN-γ because of a decrease in STAT1 but not due to dysfunctional IFN-γ receptors. Signaling of PI3K/AKT, as well as MEK/ERK, was required for the hyperproliferation; notably, PI3K/AKT alone mediated the IFN-γ resistance. Aberrant Src homology-2 domain-containing phosphatase (SHP) 2 determined IFN-γ resistance but unexpectedly had no effects on hyperproliferation or ERK activation. In the IFN-γ resistant cells, inactivation of glycogen synthase kinase (GSK)-3β by PI3K/AKT was important for SHP2 activation but not for hyperproliferation. An imbalance of AKT/GSK-3β/SHP2 caused by a reduction of PTEN was important for the crosstalk between IFN-γ resistance and hyperproliferation. PI3K is constitutively expressed in AGS cells and immunohistochemical staining showed a correlation between hyperproliferation and expression of SHP2 and STAT1 in gastric tumors. These results demonstrate the effects of PTEN/AKT/GSK-3β/SHP2 signaling on IFN-γ resistance in hyperproliferating gastric cancer cells.
The generation of TH17 cells in response to bacterial infection requires IL-6 and dendritic cell-produced TGF-β1, but not IL-1 (163.14)
Authors: Linehan Jonathan , Dileepan Thamotharampillai , Cleary P. , Jenkins Marc
Published: January 2023

Whether TGF-β1, IL-1, and IL-6 are required for TH17 cell development and maintenance in vivo remains controversial. For example, a recent study suggested that TGF-β1 is not necessary for TH17 differentiation, whereas another proposed that a T cell-derived source is required. Other studies have implicated varying roles for IL-1 and IL-6 in TH17 differentiation as well. We investigated the requirement for TGF-β1, IL-1, and IL-6 in TH17 differentiation using tetramers of peptide and major histocompatibility complex II (pMHCII) to track an endogenous, pMHCII-specific TH17 response to intranasal infection with Streptococcus pyogenes. We show that TGF-β1 and IL-6 were absolutely necessary for TH17 differentiation in vivo, whereas IL-1 was dispensable. Additionally, deletion of the Tgfb1 gene from CD11c+ cells abrogated TH17 cell differentiation, while Tgfb1 deletion from T cells had no effect. These results identify an essential role for IL-6 and dendritic cell-produced TGF-β1 in promoting the differentiation of TH17 cells, during intranasal infection with a pathogen.
SLAM/ SAP signaling positively regulates the differentiation of TH17 cells and experimental autoimmune encephalitis (163.15)
Authors: Huang Yu-Hsuan , Chung Brian , Priatel John , Tan Rusung
Published: January 2023

Mutations in SAP (SLAM-associated protein) underlie the majority of cases of X-linked lymphoproliferative disease (XLP), a rare congenital disorder that often leads to fatal complications upon infection with Epstein-Barr virus. The small SH2-containing adaptor SAP transmits signaling via SLAM (signaling lymphocytic activation molecule) family receptors and has been shown to be critical for development and function of multiple immune cell types. Here, we have investigated the role of SLAM and SAP signaling in regulating the differentiation of naïve CD4 T cells into IL-17 secreting effectors (TH17 cells), a pro-inflammatory lineage implicated in host defense as well as autoimmune diseases. T cell receptor activation along with co-stimulating SLAM antibodies was found to augment TH17 cell differentiation in wild type but not SAP-deficient splenic T cells under IL-17 polarizing conditions. Furthermore, SAP-/- mice were protected from experimental autoimmune encephalomyelitis (EAE), exhibiting greatly decreased numbers of CNS-infiltrating TH17 cells and dramatically reduced disease severity. Collectively, these results suggest that SLAM-SAP interactions promote the differentiation of IL-17 secreting effector CD4 T cells both in vitro and in vivo.
Metabolic control of T cell fate decision: the HIF1α-glycolysis axis in the differentiation of TH17 and iTreg cells (163.17)
Authors: Shi Lewis Zhichang , Wang Ruoning , Green Douglas , Chi Hongbo
Published: January 2023

Activated T cells mainly use glycolysis to suffice their bioenergetic and biosynthetic demands. However, whether glycolysis actively controls the differentiation of activated T cell is not fully understood. As a master regulator of metabolism, HIF1α regulates multiple rate-limiting glycolytic enzymes. We previously reported that HIF1α deficiency inhibited TH17 differentiation and promoted induced Treg (iTreg) generation (Shi et al, J Exp Med 2011). To further explore the roles of the glycolytic pathway in T cell differentiation, we combined genetic and pharmacological approaches to examine T cell metabolism and signaling. Inhibition of glycolysis by multiple agents diminished TH17 differentiation and promoted iTreg induction, while supplementation of the products of the glycolytic flux partially rescued these defects. Importantly, blocking glycolysis ameliorated the pathogenesis of autoimmune neuroinflammation both prophylactically and therapeutically, suggesting that the glycolytic pathway is a potential therapeutic target for autoimmune pathogenesis elicited by TH17 cells. Mechanistic studies further revealed that downregulation of IL-23R expression contributed to the diminished TH17 differentiation in HIF1α-deficient T cells, as forced expression of IL-23R restored the TH17 defects. In conclusion, our results demonstrate that the HIF1α-mediated glycolytic pathway links T cell metabolism and signaling, thereby dictating the cell fate decision between TH17 and iTreg cells.
Complex role for IL-2 Inducible T cell kinase (Itk) in T helper 1 and T helper 2 differentiation and function (163.18)
Authors: Kannan Arun , Sahu Nisebeta , August Avery
Published: January 2023

T helper responses are critical for a productive immune response but an inappropriate response results in inflammatory and autoimmune disorders. There are still considerable gaps in our knowledge of pathways that regulate differentiation of T helper subsets. IL-2 Inducible T cell kinase (Itk) is primarily expressed in T cells and is critical for their development, activation and function. Here, we show that Itk regulates two distinct signals during T helper differentiation. The first signal represses IFNγ independent of Tbet and maintains naïve T cells in an unbiased state, until it comes in contact with cognate antigen. This signal controls epigenetic processes that maintains the IFNγ locus closed and is critical for induction of Th2 transcriptional program. The second signal occurs following T cell activation, and is critical for secretion of Th1, Th2 and Th17 cytokines. As a result, Itk-/- mice are not only defective in Th2 responses, but are also more susceptible to infection by Th1 inducing intracellular pathogen T. Gondii. More importantly, these signaling pathways are conserved in humans, as loss of Itk in human PBMC derived T cells and Jurkat cells results in a substantial reduction in the secretion of a number of effector cytokines, at least partly due to a failure to activate pERK. This work has implications for understanding T helper differentiation programs, and Itk as a therapeutic target for Th2 mediated inflammatory and Th1/ Th17 mediated autoimmune disorders
A proteomic study of early signaling events regulating the Fas-FasL Death Inducing Signaling Complex (163.20)
Authors: Ramaswamy Madhu , Do Thao , Barden Mary , Cruz Anthony , Siegel Richard
Published: January 2023

Clearance of activated T cells by the Fas-FasL pathway is a critical mechanism of peripheral CD4 T cell tolerance. This process is preferentially higher in the effector subset of memory CD4+ T cells due to efficient recruitment and formation of the Fas death inducing signaling complex (DISC). In order to elucidate mechanisms of this heightened apoptosis sensitivity, we tested for differential DISC interacting proteins in Fas sensitive and Fas resistant CTCL cell lines using 2D-DIGE and Mass Spectrometry. We found several candidate proteins and invitro studies were done to confirm Fas receptor interactions through overexpression and Flourescence Energy Transfer (FRET) studies. One of these targets, Drebrin, is an actin binding protein previously found to play a role in T cell synapse formation by facilitating CXCR4 recruitment. Overexpression in 293 indicates that Drebrin interacts with Fas receptor. Further, endogenous drebrin preassociates with Fas receptor and remains associated with the Fas DISC even after Fas ligation only in Type I SKW cell line. Interestingly, Fas DISC associated Drebrin gets cleaved after Fas treatment and invitro caspase cleavage studies indicate that drebrin is a specific downstream target of caspase-8. Our studies propose Drebrin as a novel Fas interacting protein. Investigations are currently underway to study the mechanisms by which Drebrin modifies the Fas-DISC dynamics and the significance of the caspase-cleaved Drebrin in this process.
Effect of Selective Aryl Hydrocarbon Receptor Modulators on a murine model of Th17 driven Hypersensitivity Pneumonitis (HP) (163.21)
Authors: Mohinta Sonia , Perdew Gary , August Avery
Published: January 2023

The physiological role of Aryl Hydrocarbon Receptor (AhR) in immune response remains a key question after the discovery that AhR is an essential transcription factor for Th17 differentiation. To study the role of AhR in T cell differentiation we examined the effects of Selective AhR Modulators (SAhRMs) on antigen specific T cell response in vitro. We found that as expected, an AhR agonist enhances IL-17A production whereas a SAhRM suppresses it. However, we also found that AhR agonists suppressed production of IL-4, which was induced by the SAhRM. By contrast IFNγ was inhibited by both compounds. We also examined the effect of SAhRMs on a well established Th17 driven murine model of hypersensitivity pneumonitis mediated by a thermophile Staphylopolyspora rectivirgula (SR). Treatment of mice that have developed SR driven airway inflammation with an AhR partial antagonist or an antagonist, suppressed AhR activation in the lung. However, these AhR ligands also increased the production IL-4 by T cells in the lung, with an increase in the percentage of T cells that produce both IL-17A and IL-4. By contrast, these ligands did not suppress IFNγ or IL-17A production. Analysis of cytokine transcripts in lungs confirmed these data. This suggests that although AhR activation is required for Th17 differentiation, once cells commit to Th17 lineage, inhibiting AhR may not affect Th17 cytokine production, but may enhance the ability of these cells to produce IL-4.
High IL-4 expression and IL-4 gene polymorphisms are associated with susceptibility to human paracoccidioidomycosis (164.21)
Authors: Rodrigues Virmondes , Sawan-Mendonça Monica , Peraçoli Terezinha , Ribeiro Silvio , Silva-Vergara Mario
Published: January 2023

Paracoccidioidomycosis is caused by the dimorphic fungus Paracoccidioides brasiliensis. After invasion, the fungi are immediately destroyed or overcome the local defenses of the host and begin multiplication, causing initial injury. Previous studies have demonstrated that the severity of disease is associated with a Th2 immune response characterized by high IL-4 production. In the present study, we analyzed two polymorphisms in the IL-4 gene, -590 C/T and the intron-3 microsatellite, in 76 patients with paracoccidioidomycosis and in 73 control individuals from an endemic area. A significant correlation was observed between the RP2/RP2 intron-3 genotype and infection with P. brasiliensis (p=0.011), while the RP1/RP1 genotype was correlated with resistance. The IL-4 promoter polymorphism showed a significant correlation of the presence of T allele with the disease (p=0.030). Furthermore, high IL-4 expression observed on the patient group compared with control and was associated with TT genotype. This result suggests that IL-4 polymorphisms are associated with the ability of the host to control P. brasiliensis infection. The potential biological role of this polymorphism is supported by our observation that patients with disease produce high levels of IL-4 after mitogen or antigen stimulation and the association of high IL-4 levels with TT genotype. IL-4 gene is located in the cytokine cluster region of chromosome 5, where other polymorphisms have been described.
Type II interferon gamma is detrimental to the host in Streptococcus suis infections caused by an epidemic strain (164.6)
Authors: Lachance Claude , Segura Mariela , Pereyra Gerber Pehuen , Bergman Eugenia , Lacouture Sonia , Xu Jianguo , Gottschalk Marcelo
Published: January 2023

S. suis is a major swine pathogen that can be transmitted to humans causing severe symptoms. A large human outbreak was described in China, where approximately 25% out of 215 infected humans developed an unusual streptococcal toxic shock-like syndrome (STSLS). Albeit increased expression of inflammatory mediators following infection by the Chinese S. suis strain was suggested as responsible for STSLS case severity, the mechanisms involved are still poorly understood. Therefore, we investigated the host innate immune response to infection by either one of 3 strains of S. suis: 89-1591 (Canadian, intermediate virulence), P1/7 (European, high virulence), and SC84 (Chinese, epidemic strain). Using Illumina microarray, qPCR, and Luminex assay, infected mice showed elevated expression of mainly pro-inflammatory chemokine and cytokine genes. Generally, pro-inflammatory genes were expressed at a higher level in mice infected with strain SC84 > P1/7 > 89-1591. Interestingly, IFNγ was expressed at much higher levels only in mice infected with strain SC84, which could potentially explain some of the STSLS symptoms. IFNγ-KO mice infected with epidemic SC84 strain showed significantly better survival than WT mice while no difference was observed in mice infected with highly virulent P1/7 strain. Overall, our results show an important role of IFNγ in S. suis infections and might explain in part the increased virulence of the epidemic SC84 strain responsible for the outbreak in China.
The negative feedback mechanism between SLPI and IFNγ is impaired in tuberculosis patients (164.7)
Authors: Tateosian Nancy , Pasquinelli Virginia , Hernandez del Pino Rodrigo , Garcia Veronica , Chuluyan Eduardo
Published: January 2023

Mycobacterium tuberculosis increases IFNγ secretion which plays an important role in granuloma formation and clearance of it. Also, the exposure of macrophages to M. tuberculosis increases secretory leukocyte protease inhibitor (SLPI), a serine protease inhibitor with antimycobacterial activity. Therefore, the aim of the present study was to determine the role of a putative interaction between IFNγ and SLPI in PBMC derived from healthy donors (HD) and tuberculosis patients (TB). Peripheral blood mononuclear cells (PBMC) from TB and HD were treated with Mtb antigen and IFNγ and SLPI was determined by ELISA. Our results showed that the stimulation with Mtb of PBMC from HD, it decreased SLPI (p<0.01) while increased IFNγ secretion. Furthermore, the blockage of IFNγ reversed the effect of the Mtb antigen on SLPI secretion in PBMC-derived HD. Surprisingly Mtb antigen did not modify the SLPI secretion in PBMC-derived from TB, although it increased IFNγ. Furthermore, the mAb against IFNγ did not affect the SLPI secretion. When plasma levels of SLPI and IFNγ were measured, we found a positive correlation between them in TB but not in HD. Moreover, tuberculosis patients showed more SLPI in plasma, being the levels higher with the severity of the disease. Overall, these results suggest that there is a negative cross talk between IFNγ and SLPI in HD that it is not present in TB patients. Therefore, the higher concentration of IFNγ and SLPI might contribute to control the infection.
A novel iNKT-mediated vaccine maneuver overcomes PD-1-mediated T cell exhaustion in cancer (165.13)
Authors: Varghese Bindu , Lynch Lydia , Vriend Lianne , Kissick Haydn , Dunn Laura , Varghese Sharlin , Arredouani M. Simo , Balk Steven , Exley Mark
Published: January 2023

Our immune system plays an important role in preventing cancer. However, tumor-derived immuno-suppression hinders effective anti-tumor immunity. T cell dysfunction or exhaustion in tumor-bearing hosts is one such mechanism. Numerous studies have identified PD-1 and Tim-3 as markers of exhausted T cells, and antibodies blocking PD-1 and Tim-3 have been shown to partially restore T cell function. We have found that both Tim-3 and PD-1 are expressed on CD8+ tumor-infiltrating lymphocytes (TILs) in an animal model of B cell lymphoma and that these cells represent the predominant fraction of T cells infiltrating tumors. We find that iNKT stimulation using the ligand, alpha-galactosylceramide, combined with TLR9 stimulation using the ligand, CpG, can down-regulate Tim-3 and PD-1 expression in TILs, restore TIL function as measured by proliferation and production of TNF and IFNg in response to tumor, and induce long-lasting therapeutic immune responses in mice with large advanced (>1cm2) tumors.
Markers of effective anti-tumor CTL populations for adoptive cell therapy (165.5)
Authors: Yang Jianping , He Shujie , Peng Lifeng , Rawson Pisana , Perret Rachel , Jordan Bill , Ronchese Franca
Published: January 2023

Adoptive transfer of in vitro activated, tumor-specific CTL is a useful strategy for cancer immunotherapy. We compared in vitro culture conditions for ability to generate tumor-specific CTL that reject tumours in vivo, and for expression of molecular markers associated with effective anti-tumor activity. Specific CTL were obtained in short-term in vitro cultures of naïve CD8+ T cells and bone marrow DC loaded with peptide antigen, and were expanded in IL-2 for 2 days before transfer in vivo. The conditions of DC activation did not appear to affect the recovery, phenotype or effector function of cultured CTL, but had a substantial effect on the CTL’s ability to prevent tumour growth in vivo. Proteomic analysis of the total and surface proteome of different CTL populations was carried out using liquid chromatography and tandem mass spectrometry. Among more than 1,000 identified proteins, several were expressed with different abundance in CTL populations with varying anti-tumour activity. The functional significance of proteins showing the greatest differential expression is currently being examined using a combination of gene silencing and in vivo transfer studies. The identification of markers associated with effective anti-tumor function will provide useful means to understand and predict the functional activity of CTL populations for in vivo transfer.
Intratumoral delivery of liposome-anchored anti-CD137 and IL-2 induces highly potent local and systemic anti-tumor immunity while minimizing toxic inflammatory side effects (165.6)
Authors: Kwong Brandon , Liu Haipeng , Irvine Darrell
Published: January 2023

Immunostimulatory cytokines and antibodies induce potent anti-tumor immune responses in animal models, but also elicit dose-limiting systemic inflammatory toxicity, limiting their effectiveness in the clinic. To address this limitation, we developed a strategy to anchor immunotherapeutics to the surface of lipid nanoparticles (NPs), thus restricting their biodistribution following local injection. We previously demonstrated that NP-coupled anti-CD40 and CpG oligos significantly inhibited growth of s.c. B16F10 tumors, while minimizing systemic exposure to these agents and eliminating the symptoms of inflammatory toxicity associated with soluble agent delivery. Here we have modified this strategy to deliver NP-conjugated anti-CD137 and IL-2. Following local injection into established B16F10 tumors, NP-coupled anti-CD137 + IL-2 therapy enabled complete cure of the primary tumor in nearly 50% of mice and significantly delayed tumor progression in all mice, without systemic inflammatory effects. Importantly, anti-CD137/IL-2-NP therapy also delayed the growth of simultaneous distal untreated tumors, and induced the complete rejection of secondary tumor challenges in 100% of mice. Ongoing experiments are aimed at determining the cellular mechanisms of the combined local and systemic anti-tumor immune response. These results suggest that locally potent immunostimulation can drive systemic anti-tumor immunity without the severe toxicity typically associated with systemic therapy.
Immunomodulatory actions of lanthionine synthetase C-like protein 2-based drugs (165.9)
Authors: Lu Pinyi , Hontecillas Raquel , Viladomiu Monica , Pedragosa Mireia , Carbo Adri , Bevan David , Lewis Stephanie , Bassaganya-Riera Josep
Published: January 2023

Current inflammatory bowel disease (IBD) treatments are associated with significant side-effects. We identified lanthionine synthetase component C-like protein 2 (LANCL2) as a target for abscisic acid, a naturally occurring compound with potent anti-inflammatory effects. The goal of this study was to determine the role of LANCL2 as a therapeutic target for developing novel anti-inflammatory drugs. Structure-based virtual screening was performed using a compound library from National Cancer Institute Diversity Set II. To validate the anti-inflammatory efficacy of the top-ranking compound 61610, a series of in vitro and pre-clinical efficacy studies were performed using a mouse model of dextran sodium sulfate-induced colitis. Our findings showed that oral administration of 61610 (20 mg/kg/day) ameliorated experimental colitis by down-modulating colonic inflammatory gene expression and favoring regulatory T cell responses. We also investigated the cell specificity and molecular targets underlying the anti-inflammatory mechanism of 61610. Our in vivo findings indicate that anti-inflammatory efficacy of 61610 depends on macrophage expression of peroxisome proliferator-activated receptor γ, a receptor downstream of LANCL2. In summary, we used an integrated drug discovery platform consisting of molecular modeling approaches followed by experimental validation to confirm LANCL2 as a novel therapeutic target against IBD and demonstrated that 61610 is a novel anti-inflammatory drug.
Synthetic LbL microparticle vaccines containing the conserved T1BT* epitope of Plasmodium falciparum elicit protective antibody and cellular immune responses with minimal inflammation (166.11)
Authors: Powell Thomas , Tang Jie , DeRome Mary , Jacobs Andrea , Palath Naveen , Cardenas Edwin , Boyd James , Mitchell Robert , Nardin Elizabeth
Published: January 2023

Microparticle vaccines containing the conserved T1B repeat and T* epitopes from the P. falciparum CS protein were synthesized via layer-by-layer (LbL) fabrication on a solid core; microcapsules were prepared by dissolution of the particle core. Mice immunized with microparticles or microcapsules yielded T1B-specific antibody responses that neutralized Plasmodium parasite in vitro. The mice also developed T1BT*-specific cellular responses, including Th1, Th2, and CD4+ cytotoxic effector cells. When challenged with PfPb, a recombinant P. bergheii expressing the T1B repeats from P. falciparum, 70-80% of the mice immunized with microparticle, and 40-50% immunized with microcapsule, showed >90% reduction in liver parasite burden compared to naïve challenged mice. A minority of mice with reduced parasite burden did not have potent PfPb-neutralizing antibody activity. Since depletion of either CD4+ or CD8+ cells prior to challenge did not ablate protection, the vaccine-induced protection appears to be primarily associated with antibody responses but may also involve other mechanisms not yet defined. An examination of cytokine and chemokine gene expression in the liver post-challenge did not reveal any evidence of inflammatory responses following LbL vaccination and PfPb challenge. These results demonstrate that synthetic LbL vaccines bearing the conserved T1BT* epitopes can elicit protective immunity against Plasmodium without triggering unwanted inflammatory responses.
Modulation of protection against Mycobacterium tuberculosis by adjuvants that elicit different T cell responses. (166.18)
Authors: Baldwin Susan , Bertholet Sylvie , Reese Valerie , Ching Lance , Reed Steven , Coler Rhea
Published: January 2023

The use of an adjuvant within a vaccine can influence and direct the immune response to enable a desired outcome. A T helper 1 (Th1) response, including antigen-specific production of interferon-gamma (IFN-γ), is needed to protect against Mycobacterium tuberculosis. A successful subunit vaccine should include not only an appropriate antigen but also a proper adjuvant to ensure that a Th1 mediated cellular response is induced. Only a few adjuvants have been approved for use in human vaccines such as Alum and oil-in-water (o/w) based emulsions, including MF59 (Novartis), AS03 (GSK Biologics), AF03 (Sanofi) and liposomes (Crucell). These adjuvants primarily induce humoral responses. A new adjuvant in approved products is AS04, which combines the TLR-4 agonist monophosphoryl lipid A (MPL) with Alum. In this study we combine our candidate TB vaccine, ID93, with a synthetic TLR-4 agonist, glucopyranosyl lipid adjuvant (GLA) mixed with a stable o/w emulsion (SE). Both SE and GLA-SE induce potent cellular responses when combined with ID93 in mice. ID93/GLA-SE induced multifunctional CD4+ Th1 cell responses (IFN-γ, TNF-α, IL-2) in mice and protected both mice and guinea pigs against M. tuberculosis. In contrast, ID93/SE in the absence of GLA induced IL-5 and provided no protection, as assessed by bacterial burden, survival, and pathology. These results demonstrate the importance of properly formulating subunit vaccines with effective adjuvants for use against TB.
A zebrafish model to develop DNA-based vaccines - identification of cdh as a protective antigen against mycobacterial disease (166.19)
Authors: Rämet Mika , Oksanen Kaisa , Harjula Sanna-Kaisa , Halfpenny Nicholas , Milka Vuoksio , Pajula Elina , Parikka Mataleena
Published: January 2023

Mycobacterium tuberculosis, the causative agent of human tuberculosis, is an intracellular pathogen killing nearly two million people annually. The current live vaccine is controversial and thus novel preventive approaches are needed. DNA vaccines represent an alternative for immunizing against tuberculosis. As a vertebrate model organism with a fully developed immune system, zebrafish (Danio rerio) presents itself as an attractive and ethical option for screening novel vaccine antigens. Here we show that antigens can be expressed in the adult zebrafish as DNA-based vaccines. Furthermore, introduction of DNA plasmids expressing a combination of three previously characterized mycobacterial antigens (Ag85B, ESAT-6, CFP-10) was protective against M. marinum challenge in adult zebrafish. Using a low-dose intra peritoneal infection with 19-28 bacteria, both the number of granulomas and the amount of affected organs were reduced. Likewise, using a lethal infection dose of 20,000 cfu, vaccination significantly reduced both mortality and bacterial counts. In addition, a putative diacylglycerol pyrophosphatase cdh was protective against lethal M. marinum infection with 25, 000 cfu decreasing mortality by more than 50% compared to controls. In summary, DNA-based vaccination protects adult zebrafish against experimental Mycobacterium marinum infection and cdh is a promising novel protective antigen in this model.
Oral polio vaccination failure in urban slum children is associated with malnutrition and evidence of chronic inflammation (166.2)
Authors: Newell Evan , Sigal Natalia , Haque Rashidul , Kidd Brian , Tse Tiffany , Petri William , Davis Mark
Published: January 2023

Oral vaccines for polio (OPV) and rotavirus have been shown to be much less effective in poor children in the developing world. The reason(s) for oral vaccine failure in this setting has been unknown. We hypothesized that failure of orally administered vaccines such as OPV was due to tropical enteropathy and resultant inflammation from endotoxin exposure. We tested this hypothesis in a cohort of children followed from birth in an urban slum of Dhaka Bangladesh. Response to oral poliovirus vaccine was measured in the children who received the recommended minimum of three doses of OPV by age 6 months. Diminished antibody responses to OPV were associated with malnutrition, serum endotoxin-specific antibodies, and shorter breastfeeding duration. In a subset of these children, we investigated the mechanism for vaccine failure using a systems-immunology approach, performing comprehensive cellular assays on peripheral blood mononuclear cells, including a single-cell mass-spectrometry based assessment of cellular phenotypes and functional capacities. From this analysis, children with vaccine failure exhibited globally reduced cellular responsiveness to a range of cytokine stimulations, as well as elevated pro-inflammatory cytokine expression. These data indicate that oral vaccine failure in this at-risk group is related to malnutrition, gut barrier dysfunction and shorter duration of breast-feeding in early childhood, and is associated with a chronic inflammatory immune phenotype.
Adaptive immunity against Francisella tularensis elicited by an adenovirus-based vaccine (166.21)
Authors: Zeng Mingtao , Kaur Ravinder , Chen Shan , Arévalo Maria , Xu Qingfu , Chen Yanping
Published: January 2023

Francisella tularensis is a highly infectious organism that causes tularemia. A live attenuated vaccine strain (LVS) has been developed, but it has not been licensed for public use by the FDA due to safety concerns. Thus, there exists a need for a safer and improved vaccine. In this study, we have constructed a replication incompetent adenovirus, Ad/opt-Tul4, encoding a codon-optimized gene for expression of the membrane protein, Tul4, of LVS. Its ability to protect against lethal challenge and its immunogenicity were evaluated in a murine model. An intramuscular injection of a single dose (1×107 PFU) of Ad/opt-Tul4 elicited a robust Tul4-specific antibody response. Assays suggest a Th1-driven response. A single dose elicited 20% protection against challenge with 100×LD50 LVS; two additional boosters resulted in 60% protection. In comparison, three doses of 5µg recombinant Tul4 protein did not elicit significant protection against challenge. Therefore, the Ad/opt-Tul4 vaccine was more effective than the protein vaccine, and protection was dose-dependent. Compared to LVS, the protection rate is lower, but an adenovirus vectored vaccine may be more attractive due to its enhanced safety profile and potential mucosal delivery. Furthermore, simple genetic modification of the vaccine may potentially produce antibodies protective against a fully virulent strain of F. tularensis. Our data support the development and further research of an adenovirus-based vaccine against tularemia.
Vaccination with a novel Yersinia pestis single component, two-valent LcrV-F1 vaccine protects against lethal intranasal challenge. (166.23)
Authors: Parent Michelle , Guth Kerri , Waters Stephanie , Chichester Jessica
Published: January 2023

Yersinia pestis, a category A select agent is the causative agent of pneumonic plague. Currently, an effective vaccine has yet to be developed to protect against pneumonic infection therefore, Y. pestis weaponization and aerosolization is considered a bioterrorism threat. Here, we investigated the efficacy of a novel plague vaccine containing the low-calcium-response V ( LcrV) and the Fraction 1 capsular (F1) proteins, expressed as fusions to the thermostable lechinase enzyme (LicKM) of Clostridium thermocellum and purified from Nicotiana benthamiana plants. BALB/c mice primed and boosted with 1μg of this single component two-valent LcrV-F1 vaccine in alhydrogel provided protection against a lethal intranasal challenge with Y. pestis KIM D27. We determined this protection was antibody mediated as vaccination generated high IgG antibody titers. Additionally, adoptive transfer of serum from vaccinated mice to naïve mice protected against a lethal challenge while treatment of this serum using a Protein G column negated this serum-mediated protection. In order to determine if this vaccination regimen also resulted in memory T cell production, CD4 T cells isolated from vaccinated mice and subjected to in vitro restimulation with either LcrV or F1-LicKM fusion resulted in IL-2 production. Here, we have determined that vaccination using this novel LcrV-F1 LicKM fusion results in humoral immunity and protection against lethal Y. pestis KIM D27 infection.
Adjuvanted influenza vaccines in young and aged BALB/c mice (166.25)
Authors: Yam Karen , Gupta Jyotsana , Allen Elizabeth , Ward Brian
Published: January 2023

Although the available influenza vaccines are generally safe, they are far from ideal. They have reduced efficacy in the most at risk groups: the very young and the elderly. To address such concerns, the vaccine industry is increasingly turning its attention to the use of adjuvants to enhance the immune responses generated by these vaccines. During the 2009 pandemic H1N1 influenza outbreak, an adjuvanted influenza vaccine was selected for administration to Canadians; it was formulated with the oil-in-water adjuvant AS03 and consisted of 1/4 the usual antigen dose of vaccine. This study investigated the immune responses generated following immunization with adjuvanted influenza vaccines in aged and young mice. We tested two ages of BALB/c mice (2 months, 1.4 years), two doses of Influenza A/Uruguay H3N2 split vaccine (0.75ug, 3ug), and two adjuvants (alhydrogel, AS03). The use of an adjuvant increased serum HAI titers compared to vaccination with unadjuvanted vaccine. We found no significant difference in using the high dose of vaccine antigen with both adjuvants. At the low dose of vaccine, the AS03 adjuvant gave significantly higher HAI titers in both age groups. Finally, aged mice given a low dose of the AS03-adjuvanted split vaccine showed significantly lower HAI titers than young mice. We are identifying additional differences between the immune responses of young and aged mice with an overall goal to be able to design more effective vaccines, especially for the elderly.
Identification of antigen-specific antibody responses associated with upper genital tract pathology in mice infected with Chlamydia muridarum (166.3)
Authors: Zeng Hao , Zou Quanming , Zhong Guangming
Published: January 2023

Urogenital infection with C. trachomatis in some women can lead to upper genital tract pathologies such as hydrosalpinx, potentially affecting fertility. In the current study, 27 of 40 mice intravaginally infected with C. muridarum developed visible hydrosalpinges in the oviduct while the remaining 13 did not although all infected mice displayed a similar infection time course. Antisera from the 40 mice recognized 130 out of 257 C. muridarum proteins as antigens and 17 as immunodominant antigens. Importantly, the 27 mice with hydrosalpinx preferentially recognized two C. muridarum proteins (TC0582 & TC0912; thus, designated as pathology-associated antigens) while the 13 mice with no hydrosalpinx preferentially recognized 10 proteins designated as non-pathology antigens. The preferential recognition was validated by absorption and independently confirmed in Western blot. A fragment of TC0912 was found to improve the differentiation of hydrosalpinx from non-hydrosalpinx mice. TC0582 is a highly conserved ATP synthase and it may contribute to chlamydial pathogenesis via similar mechanisms hypothesized for the highly conserved HSP60. Thus, we have identified chlamydial antigens and epitopes that are associated with either susceptibility or resistance to upper genital tract pathology, which will help us to further understand chlamydial pathogenesis and develop anti-Chlamydia subunit vaccines.
C1q engages leukocyte-associated inhibitory receptor (LAIR)-1 and inhibits monocyte-derived dendritic cell differentiation (167.1)
Authors: Son Myoungsun , Santiago-Schwarz Frances , Diamond Betty
Published: January 2023

Leukocyte-associated inhibitory receptor (LAIR)-1 is a collagen receptor. Cross-linking of LAIR-1 suppresses the differentiation of dendritic cells (DCs). We found that C1q, which is known to have an inhibitory role in the differentiation of DCs, maintained the expression of LAIR-1 in cells transitioning between monocytes and DCs. Since C1q contains a collagen-like tail, we hypothesized that a C1q/LAIR-1 interaction leads to DC lineage arrest. In order to assess interactions between C1q and LAIR-1, we explored three models. First, HEK293T cells were transfected with a plasmid encoding LAIR-1. Labeled C1q associated with LAIR-1 expressing cells in a dose specific manner; this binding was inhibited by soluble LAIR-2, which exhibits the same collagen binding capacity as LAIR-1. Second, THP-1 cells, a LAIR-1hi human monocytic leukemia cell line, exhibited a high degree of binding to C1q which was also blocked by sLAIR-2. Third, in primary human monocytes, sLAIR-2 blocked the effects of C1q on monocyte-derived DC differentiation. We confirmed a direct interaction between C1q and LAIR-1 by dot blot analysis. Moreover, LAIR-1 was phosphorylated in a C1q-dependent manner in primary monocytes, demonstrating that C1q is an inducer of LAIR-1/ITIM-specific phosphorylation. Collectively, these data indicate that C1q functions as a natural ligand for LAIR-1. These findings provide novel insights into how C1q may prevent unwarranted immune responses.