bluuebunny/crossref_metadata_2025 huggingface dataset:
Loss of STING eliminates proinflammatory gene induction in TrexI-/- deficient mice. (P4091)
Authors: Konno Hiroyasu , Ahn Jeonghyun , Barber Glen
Published: January 2023

Patients defective in the 3’ repair exonuclease 1 (Trex1) suffer from Aicardi-Goutieres Syndrome (AGS) which instigates lethal encephalitis characterized by high levels of type I IFN production being present in the cerebrospinal fluid. Trex1 deficient mice exhibit a median life span of approximately 9 weeks since as yet uncharacterized self-DNA, presumably normally digested by Trex1, activates intracellular DNA sensors which triggers cytokine production and causes lethal inflammatory aggravated myocarditis. STING is essential for triggering cytoplasmic DNA-dependent innate immune signaling that comprises the production of type I IFN. Loss of TREX1 was observed to augment ssDNA and dsDNA-mediated induction of type I IFN in a STING-dependent manner. The replication of HSV1 was greatly reduced in hTERT-BJ1 or MEF lacking TREX1 due to elevated STING-dependent cytokine production. Loss of STING was observed to significantly extend the lifespan of Trex1-/- due to prevention of myocarditis. Microarray analysis showed dramatically reduced levels of proinflammatory genes in the hearts of Sting-/- Trex1-/-, Sting-/+ Trex1-/- compared to Trex1-/- mice. Anti-nuclear autoantibody (ANA) observed to be highly prevalent in the sera of Trex1-/- mice was almost completely absent in the sera of Sting-/- Trex1-/- mice. These data indicates that STING is responsible for pro-inflammatory gene induction in Trex1 deficient mice and plausibly may be involved in AGS.
Epstein-Barr virus, CD40 functional mimic, latent membrane protein-1 drives cellular molecular mimicry in the presence of Epstein-Barr virus nuclear antigen-1 in a novel murine model of lupus-like disease (P4063)
Authors: Munroe Melissa , Anderson Jourdan , Gross Timothy , Stunz Laura , Bishop Gail , James Judith
Published: January 2023

Immune dysregulation underlies SLE pathogenesis and EBV infection and abnormal latent protein expression are associated with SLE. EBNA-1 acts as a molecular mimic with SLE-autoantigens, while LMP1 acts a functional mimic of CD40 in an enhanced and dysregulated manner. Mice expressing a mCD40-LMP1 transgene (Tg) have autoantibodies by 2-3 months, but no overt autoimmune disease. This study evaluates the immune response to EBNA-1 in mCD40-LMP1 Tg mice and whether the addition of a molecular mimic accelerates lupus pathogenesis. Although anti-EBNA-1 levels are comparable in EBNA-1 immunized mCD40-LMP1Tg and mCD40 WT congenic mice (compared to adjuvant control and naïve mice), EBNA-1 specific proliferative and inflammatory cytokine responses, including IL-6, IL-17, IFN-γ, and TNF-α, are significantly enhanced (p < 0.0001) in mCD40-LMP1 Tg mice. mCD40-LMP1 Tg mice immunized with EBNA-1 exhibit enhanced cellular responses (p < 0.0001) to the EBNA-1 antigenic epitope PPPGRRP and the Sm B’ cross-reactive sequence PPPGMRPP. Enhanced cellular immune dysregulation with mCD40-LMP1 EBNA-1 immunization is accompanied by splenomegaly, increased serum BUN and creatinine levels, and elevated anti-dsDNA and ANA levels (p <0.0001 compared to mCD40 WT mice, p < 0.01 compared to adjuvant and naïve mCD40-LMP1 mice). These data suggest that expression of EBNA-1 and LMP1 may contribute to immune dysregulation that leads to pathogenic autoantigen-specific lupus inflammation.
Dendritic cells regulate the differentiation of inflammatory monocytes to affect Japanese encephalitis (P6118)
Authors: Eo Seong Kug , Choi Jin Young , Kim Seong Bum , Han Young Woo , Kim Bum Seok , Kim Jin Hyoung
Published: January 2023

Dendritic cells (DCs) as professional antigen-presenting cells have been known to play critical role in the initiation and modulation of the adaptive immune responses. Here, we will discuss the roles of CD11c+ DCs and functional modulation of inflammatory monocytes CD11b+Ly-6C+ in the progression of acute viral encephalitis caused by infection with Japanese encephalitis virus (JEV). The ablation of DCs exacerbated acute viral encephalitis and neuroinflammatory reactions following JEV infection. Also, the ablation of CD11c+ DCs induced early infiltration of inflammatory monocytes CD11b+Ly-6C+ in the central nervous system (CNS) and provided activated microglia and macrophages. This severed neuroinflammatory reaction was accompanied with early production of huge amount of pro-inflammatory cytokines, but type I IFN expression was not blunted in CD11c+ DC-depleted mice. In particular, the ablation of CD11c+ DCs induced immatured phenotype and morphology of CD11b+Ly-6C+ monocytes in lymphoid and inflammatory tissues. CD11b+Ly-6C+ monocyte developed in DC-depleted mice showed reduced antigen-presenting capacity and recovered phenotype when CD11c+ DCs were increased. These results suggest that CD11c+ DCs could regulate the differentiation of inflammatory monocytes CD11b+Ly-6C+ to affect clinical severity of JE.
Cytosolic interaction between MHC-I molecules and Derlin-1 is required for human cytomegalovirus US11-induced degradation of MHC-I molecules (P6122)
Authors: Jun Youngsoo , Cho Sunglim , Lee Miriam
Published: January 2023

Human cytomegalovirus (HCMV) US11 exploits Derlin-1-dependent endoplasmic reticulum-associated protein degradation (ERAD) to degrade major histocompatibility complex class I (MHC-I) molecules for immune evasion. US11 captures MHC-I molecules in the ER and convey them to Derlin-1, which then mediate their dislocation into the cytosol for degradation. The cytosolic tail of MHC-I molecules was shown to be required for dislocation to the cytosol, yet its precise role in the process remains unknown. Here, we found that the cytosolic tail of MHC-I molecules, although dispensable for interaction with US11, was required for their tight association with Derlin-1 in the presence of US11. Surprisingly, deletion of the C-terminal single amino acid of the cytosolic tail markedly disrupted the interaction between MHC-I molecules and Derlin-1, thereby rendering the mutant MHC-I molecules resistant to US11-induced degradation. Consistently, when MHC-I molecules were forced to interact with Derlin-1, through the transmembrane domain of US11 that had replaced that of the MHC-I molecules, the cytosolic tail or the C-terminal amino acid of the hybrid MHC-I became dispensable for dislocation. Thus, these results suggest that the cytosolic regions of Derlin-1 play a key role in interaction with the cytosolic tail of MHC-I molecules, and that this interaction is essential for Derlin-1 to dislocate them into the cytosol for degradation.
T cells utilize lamellipodia and filopodia for optimal intraluminal path finding (P5097)
Authors: Doh Junsang , Song Kwang Hoon , Kwon Keon Woo
Published: January 2023

Immune cells circulating in inflamed blood vessels undergo series of adhesion cascades to leave out of blood vessels to infiltrate into inflamed tissues. It has been shown that leukocytes crawl a considerable distance before they undergo transendothelial migration (TEM), but how they steer their crawling direction to find TEM sites is not well understood. To determine whether flow direction or EC orientation has predominant roles on the direction of T cell crawling, we fabricated well-aligned EC layers by culturing ECs on nanogrooved surfaces and applied shear flow either parallel or perpendicular to the direction of EC orientation. Regardless of flow direction, T cells crawled along the EC orientation. Then, we further identified guiding cues on ECs and found out that T cells tend to crawl along the valleys of topographical landscapes of EC layers while avoiding nuclei of ECs. By using pharmacological inhibitors for Arp 2/3, which nucleate actin filament branching to form lamellipodia, and cdc42, a small GTPase regulating filopodia formation, we found out that T cells utilize lamellipodia to sense topography of EC layers and filopodia to sense nuclei of ECs. Importantly, inhibitor-treated T cells crawled much longer distances than untreated T cells before they underwent TEM, suggesting that lamellipodia and filopodia of T cells are critical for optimal intraluminal path finding.
Consequences of dendritic cell necroptosis on the immune system (P5105)
Authors: Young Jennifer , He Tina , Reizis Boris , Winoto Astar
Published: January 2023

Dendritic cells (DCs) are essential in eliciting protective responses against pathogens, thus appropriate DC functions are essential in maintaining proper immune responses. The maintenance of DC homeostasis relies heavily on apoptosis to prevent disease development, and inhibition of DC apoptosis has been previously reported to induce systemic autoimmune disease. Fas-associated death domain-containing protein (FADD) is a critical adaptor protein that interacts with caspase-8 to transmit apoptotic signals downstream of the tumor necrosis factor family of death receptors. More recently, an alternative form of cell death, termed necroptosis, was reported to be downstream of the death receptors. Necroptosis is caspase-independent and requires the kinase activities of receptor interacting serine/threonine protein kinase 1 (RIP1) and RIP3. To investigate the role of FADD in DC death, DC-specific FADD conditional knockout mice were generated. Interestingly, these mice developed an inflammatory phenotype. We further elucidated the cause of inflammation by crossing the mice to RIP3-/-, MyD88-/- and MyD88flox/flox mutant mice. Our recent findings based on the analysis of these mice will be discussed.
“Bundling” of SIRPα into plasma membrane microdomains is essential for effective monocyte and macrophage binding interactions with CD47 and signaling (P5117)
Authors: Ha Binh , Liu Yuan
Published: January 2023

SIRPα is an ITIMs-containing, cell surface signaling receptor expressed on myeloid leukocytes. Through binding interactions with CD47, a universally expressed counter-receptor, SIRPα regulates important immunological processes including neutrophil and monocyte transmigration to inflammatory loci and macrophage recognition of phagocytic targets. While their crucial roles have been demonstrated, the mechanisms underlying the dynamic interactions between SIRPα and CD47 remain to be characterized. By cell adhesion assays, we found that freshly isolated peripheral monocytes and cultured monocytic cells (THP-1 and U937) express abundant SIRPα, but do not effectively bind to CD47. In contrast, monocytes that had transmigrated across endothelia, and macrophages differentiated from THP-1 or obtained from murine tissues, displayed a high avidity of SIRPα binding. Labeling of SIRPα on the cell surface observed a diffuse distribution pattern on naïve monocytes, but highly punctate patterns on transmigrated monocytes and macrophages. Protein crosslinking and sucrose density co-sedimentation confirmed that SIRPα in latter cells form oligomerized complexes through which SIRPα achieves high avidity binding to CD47. Furthermore, such SIRPα complexes contain SHP-1 and are localized in cholesterol-rich lipid domains in the plasma membrane, and their dynamic formation involves Src-family tyrosine kinase-mediated phosphorylation.
IL-5 produced by natural helper cells suppresses neutrophil function during influenza infection. (P4239)
Authors: Gorski Stacey , Braciale Thomas
Published: January 2023

Interleukin (IL)-5 is a prototypical type II cytokine that is essential for eosinophil maturation and egress out of the bone marrow. We observed that eosinophil numbers progressively increase in the lungs during the course of experimental influenza A virus (IAV) infection in mice as a result of high level production of IL-5 by natural helper cells recruited to the infected lungs. Mice genetically deficient in IL-5 (IL-5 KO) demonstrate increased lung neutrophil numbers and decreased viral titers during the acute phase of IAV infection. However, this phenotype is not recapitulated in mice deficient in eosinophils (PHIL mice) suggesting an eosinophil independent function of IL-5 during infection. Unexpectedly, we found that like eosinophils, >98% of neutrophils express the IL-5 receptor alpha subunit (CD125) in the lung throughout infection. Unlike eosinophils, where ligation of the IL-5R by IL-5 induces phosphorylation of STAT1 and STAT5, engagement of the IL-5R on activated neutrophils results in suppressed pSTAT5 and pSTAT1 signalling as well as decreased expression of the activation marker CD11b. Furthermore, neutrophils treated with IL-5 undergo accelerated cell death in vitro suggesting a potential mechanism for the enhanced neutrophil numbers seen in the IAV infected IL-5 KO. Overall these data suggest that IL-5 may serve a regulatory role during the acute phase of IAV infection by controlling the number (viability) and activation state of recruited neutrophils.
CD14/TRIF pathway regulates macrophage polarization and Th2 immune responses (P4257)
Authors: Tundup Smanla , Srivastava Leena , Nagy Tamas , Harn Donald
Published: January 2023

Stimulation of macrophages with LPS and/or IFNγ promote an M1 phenotype, whereas the Th2 cytokines IL-4/IL-13 or helminth products drive an M2 phenotype. How macrophage polarization is regulated at the signaling level is not well understood. We tested the ability of macrophages to undergo polarization when exposed to M1 activating agents (LPS or poly I:C) and the M2 activating agent IL-4 at the same time. Co-stimulated macrophages failed to polarize towards M2 as determined by flowcytometric measurement of Fizz1 expression. Inhibition of M2 polarization was CD14 dependent as CD14-/- macrophages did polarize to M2 in response to IL-4-LPS co-stimulation. We found that the CD14/TRIF/STAT1 pathway, in addition to mediating M1, negatively regulates IL-4R-STAT6 mediated M2 polarization. Examination of schistosome infected CD14-/- mice showed increases in expression of CD4+ specific IL-4, IL-5 and IL-13 cytokines as well as significant increases in the recruitment of M2 macrophages in liver granulomas, associated with greater granuloma size and collagen deposition compared to infected WT mice. By adoptive transfer, we confirmed that macrophages are indeed responsible for the CD14 mediated negative regulation of alternative activation in-vivo in response to schistosome eggs. Our data demonstrated a unique role of the CD14-TRIF pathway in regulation of macrophage M1/M2 plasticity and in regulating CD4+ T cell biasing and alternative activation of APCs during helminth infection.
Bacillus anthracis cell wall peptidoglycan activates human dendritic cells to promote naïve TH17 and TFH cell differentiation (P4207)
Authors: Kovats Susan , Roach Kimberly , Raisley Brent , Bajana Sandra , Turner Sean , Roberts Virginia , James Judith , Coggeshall K.
Published: January 2023

In cutaneous and inhalational Bacillus anthracis infection, innate responses by tissue dendritic cells (DC) will direct adaptive immunity. To dissect these responses, human DCs were incubated with killed vegetative bacteria or highly purified cell wall peptidoglycan (PGN) of the B. anthracis ΔSterne strain. Both vegetative bacteria and PGN stimulated the activation of human DCs, which involved upregulation of surface HLA-DR, the T cell costimulatory molecules CD40 and CD86, the lymph node homing chemokine receptor CCR7, and CD83. B. anthracis PGN and vegetative bacteria elicited production of TNFα, IL-6, and IL-23, but not IL-12p70 or IL-10. To determine the effect of this cytokine profile on CD4+ T cell differentiation, we compared the ability of B. anthracis PGN- and Escherichia coli LPS-stimulated DCs to induce the differentiation of naïve CD4+ T cells into T helper (TH) subsets. While LPS-exposed DCs induced the polarization of CD4+ T cells into TH1 cells producing IFNγ, PGN-exposed DCs directed the differentiation of TH17 cells producing IL-17. Exposure of DCs to either PGN or LPS induced TFH cells producing IL-21. In sum, B. anthracis PGN is a highly stimulatory cell wall component that activates human DCs to display T cell costimulatory molecules and secrete IL-6 and IL-23. Thus, in vivo, PGN-exposed DCs are likely to promote the differentiation of proinflammatory TH17 cells important for extracellular bacteria infections and TFH cells important for humoral immunity.
A role of interleukin-17A in modulating intracellular survival of Mycobacterium bovis BCG in murine macrophages (P4217)
Authors: Ling Wai Lim , Wang LiangJie , Pong John C. H. , Lau Allan S. Y. , Li James C. B.
Published: January 2023

Interleukin-17A has been demonstrated to participate in the pathogenesis of mycobacterial infections. Whether interleukin-17A regulates innate defense mechanisms of macrophage against mycobacteria remain to be elucidated. In the present study, we investigated the effects of interleukin-17A on modulating the intracellular survival of Mycobacterium bovis BCG in RAW264.7 macrophages. Our data revealed that the clearance of intracellular BCG in macrophages was significantly enhanced by interleukin-17A pretreatment. Moreover, interleukin-17A-enhanced clearance of intracellular BCG could be abrogated by addition of inducible nitric oxide synthase inhibitor. We observed that interleukin-17A pretreatment was able to synergistically enhance both nitric oxide production and inducible nitric oxide synthase expression in BCG-infected macrophages in dose- and time-dependent manner. We also revealed that interleukin-17A was able to specifically enhance BCG-induced phosphorylation of JNK, but not ERK1/2 or p38 MAPK. By using specific JNK inhibitor SP600125, the production of nitric oxide in BCG-infected macrophages with interleukin-17A pretreatment was significantly suppressed. Taken together, we confirmed the involvement of JNK pathway in interleukin-17A-enhanced nitric oxide production in BCG-infected macrophages. In conclusion, our study revealed an anti-mycobacterial role of interleukin-17A through priming the macrophages to produce nitric oxide in response to mycobacteria infection.
Respiratory syncytial virus strains induce differential innate immune and CD4 T cell responses (P4039)
Authors: Weiss Kayla , Varga Steven
Published: January 2023

Respiratory syncytial virus (RSV) infects the respiratory tract causing acute pulmonary disease in both children and the elderly. The host immune response contributes to disease severity. However, it is not currently clear to what extent RSV strain differences influence the host immune response and disease severity. To determine the impact of viral strains on the severity of RSV-induced disease, we compared several different RSV A isolates. Following infection with the line 19 strain of RSV, we observed increased weight loss and decreased airway function between days 1-4 p.i. as compared to RSV strains A2 or 2-20. On day 2 p.i., the total number of macrophages and neutrophils was increased following RSV line 19 infection. In addition, IL-1β and IL-6 protein levels were increased at day 2 p.i.. Inflammasome activation leads to secretion of mature IL-1β following RSV infection. Our results suggest that there is increased priming and activation of the inflammasome following RSV line 19 infection. Coinciding with the increased priming and activation of the inflammasome, we observe an increased Th17 response following RSV line 19 infection as compared to RSV A2 and 2-20. Neutralization of IL-1β and IL-6 results in a decreased Th17 response following RSV line 19 infection. Overall, our results demonstrate that RSV strains differ in their capacity to elicit a robust innate immune response, and subsequent CD4 T cell response, contributing to differences in disease severity.
Ptpn22 promotes immunization responses to Influenza A (P4043)
Authors: Crabtree Juliet , Verma Vikram , Wang Yaya , Liang Yuying , Peterson Erik
Published: January 2023

A variant allele of PTPN22 is a potent susceptibility factor for autoimmune diseases, including Rheumatoid Arthritis and Type I Diabetes. Ptpn22 is required in myeloid cells for selective promotion of Toll-Like Receptor signaling leading to Type I IFN production. Since Type I IFN are essential for anti-viral immunity and for efficient responses to viral immunization, we investigated the role of PTPN22 in response to vaccination. We tested the ability of Ptpn22 knockout mice to mount a protective immune response to vaccination against Influenza A virus. We found that Ptpn22 is required for efficient generation of both influenza-specific cytotoxic T lymphocyte and neutralizing humoral responses after vaccination with inactivated influenza. Further, upon challenge with live influenza, vaccinated Ptpn22 KO animals exhibit increased mortality, suggesting that defects in antibody and CTL responses associated with Ptpn22 deficiency result in decreased protection engendered by inactivated influenza vaccination. These findings indicate a requirement for Ptpn22 in protective immune responses to influenza vaccine. Moreover, they suggest that further studies of vaccine responsiveness should be mounted in human carriers of the disease-associated PTPN22 variant, since such carriers exhibit diminished Toll-Like Receptor-induced Type I IFN production by myeloid cells.
Effect of self antigen specific CD4+ T cell precursor frequency on the anti-tumor immune response: Implications for adoptive T cell therapy (P2040)
Authors: Malandro Nicole , Budhu Sadna , Murphy Judith , Rizzuto Gabrielle , Houghton Alan , Merghoub Taha , Wolchok Jedd
Published: January 2023

Current approaches to cancer immunotherapy aim to enhance the natural T cell response against tumors; however, these treatments may fall short due to the negative selection of tumor antigen specific T cells. To determine how frequency of CD4+ T cells may shape an anti-tumor response we have developed a model in which precursor number can be manipulated in mice bearing implantable melanoma through the adoptive transfer of tumor specific T cells. We have previously shown that lower frequencies of CD8+ T cells specific for melanoma antigen gp100 generate the most productive response due to intraclonal competition at higher concentrations. Unlike CD8+ T cells, the adoptive transfer of greater quantities of CD4+ T cells specific for melanoma antigen TRP-1 results in better tumor regression. The activation and expansion of CD4+ T cells at high precursor frequencies is diminished compared to lower frequencies, which indicates the occurrence of intraclonal competition. We believe the observed differences in tumor eradication are due to increased infiltration of TRP-1 CD4+ T cells into the tumor when present at high precursor frequencies with lower precursor frequencies favoring the preferential accumulation of TRP-1 specific Foxp3+ regulatory T cells both in the tumor and periphery. Further understanding of how antigen specific regulatory T cells develop and shape the effector response may have implications for the use and improvement of CD4+ T cells in adoptive cell therapy.
Characterizing the effects of the BrafV600E inhibitor vemurafenib on CD8 T cell responses to Braf-mutated murine melanoma (P2046)
Authors: Steinberg Shannon , Malik Brian , Zhang Peisheng , Bosenberg Marcus , Turk Mary Jo
Published: January 2023

Vemurafenib, a small molecule inhibitor of the BrafV600E mutation present in 60% of patient melanomas, has lead to promising results in the clinic. Unlike broad spectrum MEK pathway inhibitors, which have significant off-target effects, specific inhibition of BrafV600E does not impair host immunity, raising the interesting idea that vemurafenib may in fact enhance the host immune response to Braf-mutation bearing melanomas. To test this hypothesis, we have utilized the Tyr-CreERT BrafCA Ptenlox/lox (Braf/Pten) mice, an inducible model of BrafV600E-mutated melanoma. These mice provide an immune competent system in which to characterize the effects of BrafV600E-inhibition on the host immune response to melanoma. Our work demonstrates that treating Braf/Pten tumor-bearing mice with vemurafenib completely arrests the growth of established tumors, and significantly enhances the proportion of CD8 T cells infiltrating dermal melanomas. BrafV600E-inhibition also significantly decreases the proportion of immunosuppressive Treg cells infiltrating the tumor, resulting in a more favorable ratio of CD8 effectors to Tregs than in untreated tumors. Furthermore, CD8 T cell depletion studies have confirmed that these CD8 T cells provide potent long-term tumor control even after BrafV600E-inhibition is discontinued. This work serves as a proof-of-principle that vemurafenib, and possibly other targeted molecular therapies, can enhance endogenous anti-tumor immunity.
Panobinostat in combination with azacitidine reduces the frequency of TNFR2+ regulatory T cells in acute myeloid leukemia patients. (P2048)
Authors: Govindaraj Chindu , Tan Peter , Wei Andrew , Spencer Andrew , Plebanski Magdalena
Published: January 2023

Panobinostat (LBH) and azacitidine (Aza) are currently being evaluated in the therapy of AML. The effect of this drug combination on T cells, particularly on regulatory T cells (Tregs) has not been previously investigated. The presence of Tregs in AML is detrimental, as these cells suppress anti-tumor immunity. Tregs expressing Tumor Necrosis Factor Receptor 2 (TNFR2) are associated with maximal immune suppressor activity. Here, we investigated the combined effect of LBH and Aza on effector T cells (Teff), and TNFR2+ and TNFR2- Tregs in newly diagnosed AML patients. Patients were treated with Aza on days 1-5 for each 28-day cycle combined with 7 doses of LBH on days 5-19. T cells within peripheral blood (PB) and bone marrow (BM) were analyzed via flow cytometry, at baseline and at the end of cycle 1 (EOC1). Specifically, TNFR2+ Tregs were present at high levels within patient PB when compared to controls and were significantly reduced by EOC1 of treatment. Interestingly, only patients who responded to treatment had this reduction in their BM. Mechanistic studies adding LBH alone or with Aza to PB cells during T cell activation in vitro indicated LBH might primarily drive the reduction in Tregs. Concomitant with Treg reduction, IFNγ and IL2 production by CD4 Teff and the ratio of CD4 Teff to TNFR2+ Tregs were enhanced by EOC1 in the BM of responder, but not non-responder patients. AML patients who responded to Aza and LBH treatment have a beneficial immunological outcome.
Host conditioning approach and choice of target epitope determine the success of adoptive T cell immunotherapy against oncogene-induced established tumors (P2068)
Authors: Ward-Kavanagh Lindsay , Cooper Timothy , Schell Todd
Published: January 2023

Host conditioning regimens can dramatically enhance adoptive T cell-based therapies for some cancers. We investigated the unique and combined contributions of whole body irradiation (WBI) and agonist anti-CD40 conditioning to successful adoptive immunotherapy in a mouse model of autochthonous cancer. Line Rip1-Tag4 mice develop pancreatic islet cell tumors due to expression of the SV40 large T antigen oncogene from the rat insulin promoter. Using transfer of T cell receptor transgenic CD8+ T cells specific for two unique determinants in T antigen (designated TCR-I or TCR-IV), we demonstrate that successful control of established tumors in these mice is both regimen- and epitope-dependent. WBI conditioning alone minimally enhanced T cell accumulation and persistence, while anti-CD40 alone transiently enhanced the subdominant TCR-I but not the immunodominant TCR-IV response. Combining anti-CD40 with WBI conditioning enhanced the magnitude and duration of both T cell responses, but only TCR-I cells accumulated and persisted at high levels in the pancreas. Mice that received combined conditioning plus TCR-I transfer had an increased life span, and showed histological signs of anti-tumor immunity. Lack of therapeutic impact by TCR-IV T cells was associated with development of an unusual differentiation phenotype following antigen recognition in vivo. The results indicate that target selection and conditioning regimen dramatically influence the efficacy of adoptive T cell therapy.
Chemoimmunotherapy provides protective immunity of murine pancreatic cancer model (P2070)
Authors: Ghansah Tomar , Vohra Nasreen , Kinney Kathleen , Sarnaik Amod , Pilon-Thomas Shari
Published: January 2023

Pancreatic cancer is an aggressive disease with grim prognosis. Immunotherapy is less effective in treating tumors partly due to Myeloid Derived Suppressor Cells (MDSC). In this study we evaluated tumor regression due to MDSC depletion with dendritic cell (DC) vaccination of pancreatic tumor bearing (TB) mice. Flow cytometry results showed a significant increase in the percentage of MDSC in tumors and spleens of TB mice compared to naïve mice. MDSC isolated from TB mice significantly suppressed antigen-specific T cell responses compared to naive mice. Gemcitabine (GM) is a chemotherapeutic drug for cancer therapy. Flow cytometry results showed a significant decrease in the percentage of MDSC in splenocytes from GM-treated TB and naïve mice. To study effects of DC vaccination combined with MDSC depletion on tumor rejection, mice were vaccinated with PANC02 lysate-pulsed DC or PBS, three times at seven-day intervals. One week after final vaccination, mice were challenged with PANC02 tumor cells. Mice in the vaccinated and control groups either received PBS or GM therapy. In vivo depletion of MDSC was performed three days prior to tumor challenge, continued until the end of the experiment and was confirmed using flow cytometry. A significant survival benefit was seen in mice receiving both DC vaccination and MDSC depletion. Results from this study strongly suggest that the depletion of MDSC significantly enhances protective immunity in a murine pancreatic cancer model.
Modulating CXCR3 chemokine expression in the tumor microenvironment as a therapeutic approach for skin cancers. (P2074)
Authors: Gorbachev Anton , Kish Danielle , Fairchild Robert
Published: January 2023

Chemokines can inhibit or promote tumor growth through multiple mechanisms such as inhibition or promotion of angiogenesis or recruiting effector or regulatory cells into tumor. Modulation of intra-tumor chemokine expression is considered to be a promising approach of cancer immunotherapy. CXCR3 chemokines (CXCL9/Mig and CXCL10/IP-10) are known as potent tumor suppressors that inhibit tumor angiogenesis and recruit Th1 and NK cells into the tumor. We used murine model of aggressive skin cancer (cutaneous sarcoma or melanoma) to test the induction of Mig expression within tumors as a therapeutic approach. Intra-tumor delivery of Mig DNA by retroviral vector or by plasmid suppressed growth of MCA205 sarcoma and B16 melanoma. This therapeutic effect was T cell-dependent and was mediated through enhancing the activation of tumor-specific T cells by tumor-infiltrating dendritic cells rather than through angiostatic functions of Mig. Surprisingly, the deficiency of tumor hosts in IP-10 resulted in the suppression of tumor growth and increased activation of tumor-specific T cells similar to the therapeutic effects mediated by overexpression of Mig. These results suggest that besides similar angiostatic and chemoattracting functions, Mig and IP-10 impose non-redundant and even antagonistic effects on anti-tumor immunity and that the induction of Mig expression and simultaneous inhibition of IP-10 in tumor hosts might be a potent therapeutic approach to treat aggressive skin cancers.
Combining anti-OX40 with an inhibitor of TGF-β signaling overcomes the immunosuppressive environment in established tumors (P2026)
Authors: Tucker Christopher , Triplett Todd , Garrison Kendra , Sun Lihong , Ling Leona , Akporiaye Emmanuel , Weinberg Andrew
Published: January 2023

OX40 (CD134, TNFRSF4), a member of the tumor necrosis factor receptor (TNFR) superfamily is expressed on activated CD4+ and CD8+ T cells. Engagement of OX40 with cognate ligand or soluble agonist enhances T cell proliferation, survival and cytokine production by memory T cells. Although αOX40 therapy has been effective at controlling incipient tumors in a variety of mouse tumor models, it has been less effective at eradicating large established tumors. Pervasive immunosuppression in the tumor microenvironment has been implicated as a potential mechanism of immune evasion by tumor cells. Here we show that combining agonist antibody (αOX40) with an orally bioavailable small molecule inhibitor of TGF-β signaling, results in complete regression of well-established tumors in ~85% of treated mice. Tumor infiltrating lymphocytes (TIL) from these dual treated mice produce increased levels of Granzyme-B and IFNγ and higher levels of the proliferation marker Ki67. Differences seen in STAT3 expression by the TIL led us to investigate the role of STAT3 in this anti-tumor effect. Using an OX40 promoter-driven CRE model to knock out the STAT3 gene in activated T-lymphocytes, we demonstrated that STAT3 is essential for the therapeutic efficacy of the combination treatment as a 50% drop in tumor cure rate was observed in STAT-3 deficient mice compared to STAT3 sufficient mice. Together these results demonstrate the anti-tumor efficacy of combining OX40 therapy with TGF-β inhibition.
Intra-tumoral administration of tumor membrane vesicles expressing GPI-anchored IL-2 inhibit tumor growth in a murine breast cancer model (P2080)
Authors: Bozeman Erica , Shashidharamurthy Rangaigh , Srivatson Sanjay , Selvaraj Periasamy
Published: January 2023

Cancer is the second leading cause of death in the US with breast cancer being among the most common cancers. Tumor membrane vesicles (TMVs) used a vaccine will present a variety of tumor antigens to the immune system. TMVs due to their size and particulate nature are likely to be processed and presented by dendritic cells. To enhance their immunogenicity, TMVs were prepared from homogenized 4T07 mouse breast cancer cell transfectants expressing GPI-IL-2 Expression of GPI-IL-2 was verified by flow cytometry analysis. To ensure functionality, IL-2 expressing TMVs were co-cultured with CTLL-2 cells, an IL-2 dependent cytotoxic T cell line, for 48hrs and proliferation of the CTLL-2 cells was assessed by thymidine incorporation. Mice were then challenged with live wild-type 4T07 cells and administered 50ug of TMVs intra-tumorally on days 10 and 17 post tumor challenge. We observed that GPI-IL-2 expressing TMVs are functionally active and capable of inducing proliferation of CTLL-2 cells. More importantly, intra-tumoral injection of GPI-IL-2 expressing TMVs is capable of significantly reducing overall tumor burden in a therapeutic setting. These studies provide evidence that intra-tumoral vaccination with TMVs expressing GPI-IL-2 could potentially be a viable immunotherapeutic strategy for breast cancers that cannot be surgically removed.
Sorafenib induces apoptosis through reactive oxygen species production, JNK/p38 MAPK activation, and suppression of Akt/NF-kB signaling in EBV-transformed B cells (P2088)
Authors: Choi Yunock , Park Ga Bin , Kim Ja Young , Kim Hae Jin , Kim Seonghan , Kim Yeong-Seok , Lee Hyun-Kyung , Cho Dae-Ho , Lee Wang Jae , Bae Ki Won , Hur Dae Young
Published: January 2023

Sorafenib (SRF), the multiple tyrosine kinase inhibitor, is known as a potential targeted drug that may have antitumor activity against various cancers, but the roles of SRF in EBV-transformed B cells remain unclear. Here, we investigated the in vitro cytotoxic effects of SRF on EBV-transformed B cells and the underlying mechanism such as the changes in oxidative stress and MAPKs signaling, which are closely associated with cell death-signaling transduction pathways, in EBV-transformed B cells treated with SRF. We first observed that SRF significantly decreased the cell viability of EBV-transformed B cells in a dose-dependent manner. SRF induced the generation of reactive oxygen species (ROS), translocation of Bax into mitochondria, disruption of mitochondrial membrane potential, activation of caspase-9, -3, and PARP, and subsequent apoptosis. Moreover, we found that SRF exposure activated the phosphorylation of JNK and p38 MAPK and suppressed the phosphorylation of PI3K p85 and Akt. In particular, SRF diminished nuclear localization of NF-κB in EBV-transformed B cells. These results demonstrate that SRF induces apoptosis through ROS production, ER stress, and mitochondrial-dependent pathway in EBV-transformed B cells. Taken together, these findings provide an overview of the molecular signaling pathway driving SRF-mediated cell death in EBV-transformed B cells and suggest SRF could be a potential therapeutic drug for the treatment of EBV-transformed B cells.
Human antigen-specific invariant NKT cells generated by the TIL 1383I T cell receptor gene transfer (P2098)
Authors: Nagato Kaoru , Scurti Gina , Moxley Kelly , Wang Xinxin , Seetharaman Aravind , Simms Patricia , Motohashi Shinichiro , Yoshino Ichiro , Nakayama Toshinori , Nishimura Michael
Published: January 2023

Human invariant NKT cells (iNKT cells) are a novel lymphocyte population characterized by an invariant T-cell receptor (Vα24/Vβ11). iNKT cells activated with αGalCer and IL-2 have been shown to produce large amounts of cytokines such as IFN-γ and also have potent killing activity against various tumor cell lines. One major hurdle to studying the anti-tumor activity of iNKT cells is they don’t recognize antigens presented by classical MHC molecules and their tumor antigen specificity is unknown. Therefore, we expressed the MHC class I restricted TIL 1383I TCR into iNKT cells to better study their biology and anti-tumor activity. The 1383I TCR is a high affinity TCR that recognizes the melanoma antigen tyrosinase presented by HLA-A2. In this study, we report the production and function of the TIL 1383I TCR transduced human iNKT cells. iNKT cells were activated and expanded from normal PBMCs by stimulating them with αGalCer in the presence of IL-2 and IL-15. The iNKT cells were isolated using iNKT magnetic beads and transduced using retroviral vectors encoding the TIL 1383I TCR. The TIL 1383I TCR transduced iNKT expression was measured by flow cytometry (FACS) and their anti-tumor activity by intracellular cytokine staining. The TIL 1383I TCR transduced iNKT cells specifically produced IFN-γ in response to tyrosinase peptide loaded T2 cells and HLA-A2+/tyrosinase+ human melanomas. These results indicate that iNKT cells can be engineered to recognize normal tumor antigens.
Characterization of polyclonal type II NKT cells and their role in CpG oligodeoxynucleotides-mediated anti-tumor response (P2112)
Authors: Zhao Jie , Weng Xiufang , Bagchi Sreya , Wang Chyung-Ru
Published: January 2023

CD1d-restricted NKT cells are innate-like T cells with potent immunomodulatory function via rapid production of both Th1 and Th2 cytokines. NKT cells comprise well-characterized type I NKT cells which can be detected by α-GalCer (α-galactosylceramide) loaded CD1d tetramer and less-studied type II NKT cells which do not recognize α-GalCer. Here we characterized type II NKT cells on a polyclonal level by using an IL-4 reporter mouse model. We found type II NKT cells, like type I NKT cells, exhibit an activated phenotype and are dependent on the transcriptional regulator PLZF (promyelocytic leukemia zinc finger) and the adaptor molecule SAP (signaling lymphocyte activation molecule-associated protein) for their development. Compared to type I NKT cells, type II NKT cells produce lower levels of IFN-γ but comparable amounts of IL-13 in response to polyclonal TCR stimulation, suggesting they may play different roles in regulating immune responses. However, type II NKT cells can be activated by CpG oligodeoxynucletides to produce IFN-γ, but not IL-4 or IL-13. Importantly, CpG-activated type II NKT cells contribute to the anti-tumor effect of CpG in the B16 melanoma model. Taken together, our data reveal the characteristics of polyclonal type II NKT cells and their potential role in anti-tumor immunotherapy.
Potential roles of human chorionic gonadotropin in tumorigenesis and the development of novel vaccination strategies (P2116)
Authors: Khare Priyanka , Javed Saleem , Jain Swatantra , Singh Om , Pal Rahul
Published: January 2023

Human chorionic gonadotropin (hCG) is associated with poor prognosis in several cancers, but causative molecular events remain inadequately described. In this study, tumor cells were found to express message for both hormonal subunits. Along with increasing cell viability, exogenous hCG enhanced several key tumor-promoting mechanisms. It induced the transcriptional activation and secretion of the angiogenic factors VEGF and IL8. Matrix-degrading enzymes associated with invasion (MMP2 and MMP9) were also enhanced, as was invasiveness. hCG up-modulated secretion of the proteoglycan versican and the consequent secretion of the inflammatory cytokines IL6 and TNFα from macrophages. hCG up-regulated FOXP3 in tumor cells, leading to the increased secretion of the immunosuppressive cytokines IL10 and TGFβ and up-modulation of CTLA-4; co-culture hCG-stimulated tumor cells with mature BMDCs heightened the secretion of the tryptophan catabolizing enzyme indoleamine 2,3-dioxygenase. Anti-hCG antibodies restricted the growth of human tumor xenografts, and immunization with a novel anti-hCG vaccine formulation (βhCG-TT + Mycobacterium indicus pranii) synergistically attenuated tumor development and prolonged survival in syngeneic mice. By preventing autocrine and subsidiary paracrine hCG-induced effects on multiple pathways, new generation anti-hCG vaccines may therefore hold considerable promise as adjunct therapy in patients of gonadotropin-sensitive tumors.
Adoptively transferred TCR-engineered cytotoxic human T lymphocytes use TRAIL apoptotic pathway in killing melanomas: Pivotal role of DR5 and regulation of resistance by SAHA (P2124)
Authors: Jazirehi Ali , Economou James
Published: January 2023

Adoptive transfer of ex vivo engineered autologous lymphocytes encoding high-affinity MART-1/ A*0201-specific TCRα/β chains (F5 CTL), densely infiltrate into metastatic sites, mediating dramatic but partial responses in metastatic melanoma patients. We hypothesized that down-modulation of death receptors and aberrant apoptotic signaling confer resistance to F5 CTLs. To test this, we established a preclinical model of resistance from sensitive parental lines under serial F5 CTL-selective pressure. While surface peptide/MHC complex remains intact (F5 CTLs efficiently recognize and engage melanomas), R lines downregulate surface DR5 expression, exhibit constitutive NF-κB activation and overexpress resistant factors. The HDACi SAHA blocked antiapoptotic pathway, reduced expression of resistance factors, restored DR5 expression, and sensitized R lines to F5 CTL. At concentrations used, SAHA didn’t affect functionality and viability of F5 CTLs. Using blocking Abs, rhTRAIL and murine CTL hybridoma line, we found that TCR-engineered F5 CTLs mainly utilize TRAIL apoptosis pathway. Further, using Drozitumab (αDR5 mAb), pivotal role of DR5 and its regulation by SAHA was confirmed. These studies identify molecular determinants of resistance to TCR engineered CTL and suggest that adjuvant therapy with SAHA in immune-resistant tumors imposes a proapoptotic milieu by regulating apoptotic machinery and inducing death-receptor expression, thus, overcoming melanoma immunotherapy resistance.
The autoantibody-inducing CD4 T cell causing systemic lupus erythematosus belongs to CCR4+CD45RBlo122lo CD4 subpopulation (P4125)
Authors: Miyazaki Yumi , Tsumiyama Ken , Shiozawa Shunichi
Published: January 2023

Objective: We have shown that repeated immunization of mice with any antigen reproducibly led to development of SLE, in which a novel T cell type which we term an autoantibody-inducing CD4 T (aiCD4 T) cell was generated via TCR revision at periphery. The aiCD4 T cell induces varieties of autoantibodies and also help full maturation of CTL to induce lupus tissue injuries. We are trying tried to assign CD number on this aiCD4 T cell. Methods: BALB/c mice were repeatedly immunized with 12x OVA, KLH or SEB, and the expression of effector / memory markers on CD4 T cell was studied. CD4 T cells were isolated referring to CD45RB, CD27 and CD122 markers, and these cells were adoptively transferred into naïve mice. Autoantibodies in sera of recipient mice were measured 2 weeks after transfer. Microarray analyses were also performed using CD45RBlo122lo CD4 T cell immunized 12x with OVA. Results: The CD45RBlo, CD27lo and CD122hi CD4 T cells were significantly expanded in mice immunized 12x with either OVA, KLH or SEB. Adoptive transfer of CD45RBlo122lo CD4 T cells of mice immunized 12x with OVA showed that autoantibodies were significantly increased in naïve recipients. In microarray analysis, chemokine (C-C motif) receptor 4 (Ccr4) was increased >x4 in CD45RBlo122lo CD4 subset after 12x immunization with OVA. Conclusion: The aiCD4 T cell that induces SLE belongs to CCR4+CD45RBlo122lo CD4 subpopulation.
Islet-derived CD105+ stromal cells release autoantigens via exosomes secretion: activation of autoreactive T and B cells in NOD mice (P4131)
Authors: Rahman Muhammad , Regn Danielle , Bashratyan Roman , Dai Yang
Published: January 2023

Exosomes (EXO) are secreted, nano-size membrane vesicles that contain potent immunostimulatory materials. We have recently demonstrated that insulinoma-released EXO carry islet antigens and activate autoreactive T- cells in non-obese diabetic (NOD) mice, a spontaneous disease model for type 1 diabetes (T1D). To investigate whether primary islet cells in NOD can produce EXO, we isolated cells from the islet of Langerhans and cultured them in vitro. Interestingly, cultured islets release fibroblast-like, self-replicating cells that express mesenchymal stem cell makers such as CD105, Sca-1 and CD90 molecules. These islet stromal cells (iSC) release EXO that are highly immunostimulatory to activate lymphocytes and induce inflammatory cytokines and chemokines. The EXO-induced IFN-gamma production is clearly associated with the autoreactivity of the lymphocytes and the progression of T1D in the mice. These results support that iSC may release unique autoantigens via EXO secretion that may trigger islet-specific autoimmunity. Consistent with these observations, immune histological analysis of pancreata showed that CD105+ stromal cells restrict to the peri-islet area in normal islets but penetrate into beta cell area as lymphocyte infiltration occurs. In addition, serum EXO level in NOD mice is dramatically increased at prediabetic stage, possibly following the islet inflammation. Thus, EXO are autoantigen carriers with unique adjuvant activities and may activate autoreactive cells.
Dysregulated CD8+ T cells promote antibody-mediated autoimmune disease (P4105)
Authors: Hoyer Katrina , Gravano David , Brunello Jacob , Jiang Shuwei
Published: January 2023

IFNγ-producing CD4+ and CD8+ T cells accumulate during systemic autoimmune disease in IL-2-deficient mice. CD4+ T cells are necessary for disease progression, and we have previously shown that IL-2-KO CD4+ T cells can transfer autoimmunity to recipient mice. However, the importance of CD8+ T cells to initiation and propagation of disease is unclear. Several groups have demonstrated a protective role for CD8+ T cells in inflammation and autoimmunity, while others have suggested a pathogenic role. To define the function of CD8+ T cells during spontaneous autoimmune disease, we depleted CD8+ T cells in IL-2-KO mice and evaluated the pathogenesis of autoimmunity. Elimination of CD8+ T cells for only two weeks resulted in a significant augmentation of survival, reduction in the number and activation state of the CD4+ T cells and B cells, and a delay in the development of erythrocyte (RBC)-specific antibodies. In untreated IL-2-KO mice, both CD4+ and CD8+ T cells produced IFNγ in response to RBC antigens. This upregulation indicates that autoreactive CD8+ T cells specific for RBC-antigens are present during disease. Our results suggest that autoreactive CD8+ T cells contribute to the initiation or propagation of lymphoproliferation and pathogenesis of autoimmunity. We are currently addressing the importance of these autoreactive CD8+ T cells relative to self-reactive CD4+ T cells. We are also evaluating the mechanisms by which CD8+ T cells promote the autoimmune disease process.