bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular control of human regulatory T cell suppression for tumor immunotherapy (P2106)
Authors: Peng Guangyong , Ye Jian , Hsueh Eddy , Zhang Qunyuan , Ma Chunling , Zhang Yanping , Varvares Mark , Hoft Daniel
Published: January 2023

Immunotherapy is a promising approach for treating patients with malignant tumor, but immunosuppressive microenvironments induced by regulatory T cells (Tregs) present a major barrier to successful anti-tumor immunotherapy. A better understanding of the suppressive mechanisms utilized by Tregs is essential for the development of novel strategies to treat human cancer. Here we report that human Tregs can induce senescence in responder naïve and effector T cells in vitro and in vivo. Senescent responder T cells induced by human Tregs changed their phenotypes and cytokine profiles, and possessed potent suppressive function. Furthermore, Treg-mediated molecular control of senescence in responder T cells was associated with selective modulation of p38 and ERK1/2 signaling and cell cycle regulatory molecules p16, p21 and p53. We further revealed that human Treg-induced senescence and suppressor function could be blocked by TLR8 signaling and/or by specific ERK1/2 and p38 inhibition in vitro and in vivo in animal models. Our studies identify a novel mechanism of human Treg cell suppression that induces targeted responder T cell senescence, and provide new insights relevant for the development of strategies capable of preventing and/or reversing Treg-induced immune suppression for tumor immunotherapy.
Vγ9+ γδ T cells of glioblastoma multiforme (GBM) patients expanded ex vivo kill GBM cell lines and secrete IFNγ in vitro, suggesting their therapeutic potential. (P2108)
Authors: Bank Ilan , Peshes-Yaloz Naama , Garelick Daniella , Cohen Zvi , Malina -Marcu Victoria
Published: January 2023

GBM is the most common primary malignant brain tumor, carrying an ominous prognosis (< 2 years survival).Vγ9+ γδ (Vγ9+) T cells mediate major histocompatibility non restricted killing of tumor cells, suggesting Vγ9+ T cell- mediated adoptive immunotherapy may be of benefit in this disease. We established a protocol for ex-vivo expansion of Vγ9+ T cells from the peripheral blood (PB) of GBM patients, in whom Vγ9+ T cells consisted 0.55±0.58%(n=16) of the PB mononuclear cells (MC) just prior to surgical resection of the tumor (vs 0.91±0.42% in 9 healthy donors (HD), p<0.08). We found no correlation between % Vγ9+ T cells in patient PBMC and survival following surgical removal of the tumor. Nevertheless, short term culture of PBMC in the presence of a bisphosphonate, zoledronate (2μM) and 100 IU IL-2, expanded Vγ9+ T cells of all patients 522±466 fold, to consist 54±21% of the cultured MC (not significantly different from HD). The cultured cells were cytotoxic and expressed cytokines in response to GBM cell lines and autologous tumor derived cells. Furthermore, pre-treatment of the tumor with zoledronate, as well as with the clinical anti-GBM reagent temozolomide (TMZ), enhanced Vγ9+ T cell tumor recognition. These data suggest that adoptive immunotherapy employing autologously derived ex vivo activated and expanded Vγ9+T cells, in combination with zoledronate andTMZ, may lead to enhanced therapeutic responses in GBM patients.
An agonist of Toll-like receptor 5 sustains hepatic natural killer and dendritic cell recruitment to the liver while eliciting anti-cancer effects against liver metastases (P2110)
Authors: Brackett Craig , Kojouharov Bojidar , Gitlin Ilya , Johnson Christopher , Burdelya Lyudmila , Gudkov Andrei
Published: January 2023

Toll-like receptors (TLRs) are key components of innate immunity that act as sensors of conserved microbial patterns. To elucidate the mechanisms behind differences in physiological responses mediated by different TLRs, we compared tissue specificity of NF-κB activation in mice by agonists of three cell-surface TLRs: TLR5 agonist CBLB502, a pharmacologically optimized Salmonella flagellin derivative; TLR4 agonist, LPS; and TLR2/6 agonist CBLB612, a mycoplasma lipopeptide mimic. While the liver was defined as a major primary target organ of all three TLR agonists, the cellular response to each agonist was distinct. Thus, liver hepatocytes directly respond to TLR5 and TLR2/6 but not to TLR4, while Kupffer macrophages respond to TLR2/6 and TLR4 but not to TLR5 agonists. Mobilization of NK and DCs to the liver was transient (<24 h) in response to CBLB612 and LPS and sustained (>120 h) following CBLB502 administration. Differences in the liver innate immune response to these three TLR agonists are likely a reflection of cell type-specific expression and differences in molecular mechanisms triggered by different TLRs. Durable hepatic NK cell response by CBLB502 elicits strong TLR5 agonist-dependent effects against colon and breast cancer liver metastases. These long-lasting, TLR5-mediated changes within the liver provide important groundwork for further clinical development of CBLB502, which is currently in clinical trials as a potential radioprotector and immunotherapeutic agent.
Meningeal mast cells facilitate effector T cell function in a murine model of multiple sclerosis. (P4095)
Authors: Walker- Caulfield Margaret , Brown Melissa
Published: January 2023

Experimental autoimmune encephalitomyelitis (EAE) is a rodent model of the CNS demyelinating disease, multiple sclerosis. In EAE, autoreactive T cells are the major orchestrators of myelin damage. Recent data suggests that after priming in secondary lymphoid organs, these T cells are reactivated prior to entry into the CNS. This reactivation is thought to enhance effector function, however the details describing this mechanism are lacking. The meninges, tissues surrounding the CNS, have recently been appreciated as sites of immune responses in early EAE. Mast cells (MCs) residing in the meninges are activated within a day of disease induction and release neutrophil recruitment factors essential for the generation of severe disease. T cells also traffic to the meninges in early EAE suggesting the potential for T cell-MC interactions. Within 24 hours of adoptive transfer, MOG-specific effector CD4+ T cells accumulate in the meninges, an event dependent on the presence of MCs, and acquire the ability to produce the encephalitogenic cytokine, GM-CSF. T cell transfer also results in meningeal MC production of IL-1β. In vitro, co-culture of effector T cells with either wild type or IL-1β-/- MCs confirm that T cells can directly stimulate IL-1β production by MCs, which in turn elicits T cell expression of GM-CSF. These data support the concept that T cell-MC interactions in the meninges facilitate the acquisition of pathogenic effector functions necessary for severe disease.
Autoimmune linkage between the gut and the central nervous system (P4123)
Authors: Mindur John , Boppana Sridhar , Hong Derek , Ito Naoko , Ito Kouichi
Published: January 2023

Multiple sclerosis is an autoimmune disease that causes demeylination and axonal loss in the central nervous system. Although immune-linkage between the central nervous system and the gut is not well understood, recent studies indicated that activation of myelin-specific T cells by commensal bacteria initiated the development of autoimmune diseases in the central nervous system, suggesting that there is an autoimmune linkage between the gut and the central nervous system. We found that golli-myelin basic protein is expressed in the gut epithelial cells and myelin basic protein-specific T cells develop in the gut of transgenic mice expressing the HLA-DR2a and myelin basic protein-TCR genes. We also found that commensal bacteria-derived peptides can serve as an agonist/antagonist of HLA-DR2a-restricted myelin basic protein-specific T cells. This data suggests that commensal bacteria may play an important role in the development of myelin-specific pathogenic T cells.
Changes in CD4+ T cell affinity for MOG do not correlate with symptom relapse or remission in secondary progressive demyelinating disease (P4109)
Authors: Kersh Anna , Evavold Brian
Published: January 2023

We investigated the role of CD4 T cell frequency and affinity in the nonobese diabetic (NOD) mouse model of EAE. This model is clinically relevant in its MHC-class II-associated proclivity to autoimmune disease and its tendency to follow a secondary progressive disease course that resembles that seen in the majority of MS patients. Using the NOD model, we quantified the affinity of CD4 T cells for myelin oligodendrocyte glycoprotein (MOG) at initial peak of disease, symptom remission and at chronic disease time points. We used the micropipette adhesion frequency assay to obtain a sensitive and physiologically relevant two dimensional measurement of T-cell receptor affinity for peptide:MHC in a cell-anchored system. This assay allows us to quantify both frequency and affinity of antigen-specific CD4 T cells that are minimally detected by MHC-class II tetramer-based assays. Our data demonstrated that CD4 T cells isolated from the CNS at the initial peak of disease have similar affinities to those isolated at remission and chronic time points. However, CNS-infiltrating Tregs were roughly twice as frequent at disease remission as compared to initial peak of disease (p<0.0001). Additionally, changes in the distribution of cytokine-producing CD4 T cells seemed to contribute to the remission of symptomatic disease. Thus, Treg frequency and effector cytokine profile determined remission and relapse of symptoms and this progression was independent of changes in T cell affinity.
Role of superantigens in methicillin-resistant Staphylococcus aureus pneumonia (P3314)
Authors: Parker Dane , Prince Alice
Published: January 2023

Staphylococcus aureus is an important cause of respiratory infection in both hospital and community settings. T cells have been found to contribute significantly to pathology after USA300 MRSA pulmonary infection. We postulated this response was due to superantigens. USA300 expresses the superantigens SEK and SEQ. In response to S. aureus USA300 infection in C57Bl/6J mice we demonstrate significant activation of CD4+ T cells in the lung (CD25, CD69 and CD134 activation markers), mediastinal lymph nodes (CD69, 2.38 to 12.56%, P<0.01), and selective activation of CD4+ T cells expressing Vbeta 8.1/8.2 and 8.3, a pattern consistent with staphylococcal superantigens. Similar activation was also observed in human cells. Sterile bacterial supernatant significantly upregulated CD25 (1.7 vs 87%) and CD69 (1.1 vs 55%) (P<0.001) in the Jurkat human T cell line after 24 h of exposure and analysis of Vbeta chain expression in peripheral blood monocytic cells (PBMC) with supernatant showed selection of Vbeta 5, 6, 18 and 21 (4, 24, 5 and 8-fold increases respectively). This pattern of Vbeta expression is consistent with the known patterns for the superantigens SEK (5 and 6) and SEQ (5, 6 and 21). Our data from these models of acute infection indicate the interaction between S. aureus superantigens and T cells contributes significantly to pulmonary pathology. Modulation of the T-cell response to S. aureus may provide a therapeutic option to improve outcomes in a clinical setting.
Involvement of IL-17A-producing TCR γδ T cells in late protective immunity against pulmonary mycobacterial infection (P3324)
Authors: Umemura Masayuki , Touyama Seigo , Fukui Masayuki , Yahagi Ayano , Nakae Susumu , Iwakura Yoichiro , Matsuzaki Goro
Published: January 2023

Interleukin (IL)-17A is a cytokine originally reported to induce neutrophil-mediated inflammation and anti-microbial activity. The CD4+ TCR αβ+ T cells which produce IL-17A have been well characterized as Th17 cells. On the other hand, IL-17A-producing TCR γδ+ T cells have been reported to participate in the immune response at an early stage of infection with intracellular bacteria such as Listeria monocytogenes, Mycobacterium bovis bacilli Calmette-Guérin (BCG) and M. tuberculosis in mice. However, the involvement of IL-17A in protective immunity was not clearly demonstrated in the chronic stage of M. tuberculosis-infected mice. In this study, we found that TCR γδ+ T cells are a primary source of IL-17A, but that mycobacterial antigen-specific Th17 cells were hardly detected even at the chronic stage of M. tuberculosis infection. IL-17A-deficient mice showed a decreased survival rate, and increased bacterial burden in the lungs, livers and spleen on days 30, 60, 90 and 120 after the infection when compared to the wild-type C57BL/6 mice. Furthermore, a histological analysis showed an impaired granuloma formation in the infected lungs of IL-17A-deficient mice, which was considered to be due to a decrease of IFN-γ and TNF at the chronic stage. These data suggest that the IL-17A-producing TCR γδ+ T cells, rather than the Th17 cells, in the infected lungs are an indispensable source of protective immunity against M. tuberculosis infection.
CD36 regulates the malaria-induced pro-inflammatory cytokine responses and activation of NK and T cell by dendritic cells (P3298)
Authors: Gowda D. Channe , Gowda Nagaraj , Wu Xianzhu
Published: January 2023

In falciparum malaria, the scavenger receptor CD36 plays multiple roles: (i) mediates the sequestration of parasites in the microvascular endothelia through the adherence of parasite-infected erythrocytes, (ii) supports phagocytic clearance of parasites by macrophages, and (iii) modulates immune responses to parasites. While the function of CD36 in the parasite sequestration and clearance has been extensively studied, its role in malaria immunity remains poorly understood. We studied the role of CD36 in cytokine responses to malaria parasite, Plasmodiun falciparum, by DCs and in DC function. Blocking of CD36 with anti-CD36 antibodies resulted in the substantial decrease in the levels of infected erythrocyte internalization and pro-inflammatory cytokine responses by DCs. Consistent with these results, DCs deficient in CD36 internalized lower levels of infected erythrocytes and produced decreased levels of pro-inflammatory cytokines. The observed CD36-depedent internalization and cytokine responses by DCs are evident at relatively low doses of infected erythrocytes; at higher doses, the contribution of CD36 become masked due to the increased rate of infected erythrocyte uptake by various other receptor-mediated phagocytosis. Furthermore, IFN-γ production by NK and T cells incubated with IRBC-internalized DCs was dependent on CD36. Overall, our data demonstrate that CD36 plays a critical role in the development of immunity to malaria.
Neonatal thymectomy prolongs the permeability of enteric antigens and promotes the strong activation of peripheral CD4 T cells (P3258)
Authors: Frimpong-Boateng Kwesi , Surh Charles
Published: January 2023

Thymectomy (Tx) during the neonatal period (days 2 to 4) leads to the development of a spectrum of organ-specific autoimmune diseases in several strains of adult mice. The etiology of this intriguing phenomenon is yet to be fully elucidated, although it is widely believed to be a consequence of two controversial mechanisms: (1) production of pathogenic CD4+ T cells from neonatal-specific defect in central tolerance, and (2) abolition in the production of regulatory T cells (Tregs), which are believed to develop later in ontogeny than typical T cells. We hereby suggest an additional mechanism that may contribute to the onset of autoimmunity, perhaps by instigating the chronic activation of autoreactive T cells. We found that neonatal C57BL/6 mice display a defect in their ability to induce efficient tolerance to enteric antigens during the first several days of their life, and neonatal thymectomy significantly prolongs the period of this defect. Hence, while enteric antigens induce strong activation of peripheral CD4+ T cells only during the first several days in euthymic neonatal mice, such responses continue for several weeks in d3 thymectomized mice. The unregulated responses to enteric antigens in return may lead to inadvertent activation of autoreactive T cells.
Intestinal Lin-c-Kit+NKp46-CD4- population highly produces IL-22 upon IL-1β stimulation (P3260)
Authors: Lee Youngae , Kumagai Yutaro , Jang Min Seong , Kim Jung-Hwan , Yang Bo-Gie , Akira Shizuo , Jang Myoung Ho
Published: January 2023

Small intestinal innate lymphoid cells (ILCs) regulate intestinal epithelial cell homeostasis and prevent pathogenic bacterial infections by producing IL-22. NKp46+ ILC (ILC22) and CD4+ ILC (CD4+ LTi) are known as main sources for IL-22 in the small intestine. We found that Lin-c-Kit+NKp46-CD4- (CD4- LTi), ILC22, and CD4+ LTi cells comprised 0.30 ± 0.03%, 1.10 ± 0.12%, and 0.07 ± 0.01%, respectively, of the total lymphocytes in the small intestinal lamina propria of adult mice (BALB/c). We next compared the induction of IL-17F and IL-22 among CD4- LTi, ILC22, and CD4+ LTi cells under stimulation with IL-1β or IL-23 or IL-1β and IL-23 stimulation, by real-time PCR, Bio-Plex analysis, and intracellular staining. We found here that CD4- LTi cells that expressed RORγt and IL-7Rα strongly expressed the transcripts for Il17f and Il22, and produced the proteins for IL-17F and IL-22 after stimulation with IL-1β and IL-23 stimulation compared with ILC22 and CD4+ LTi cells. However, none of CD4- LTi, ILC22, and CD4+ LTi cells produced IL-17F and IL-22 after stimulation with Pam3CSK4 (TLR2 ligand), Poly(I:C) (TLR3 ligand), or LPS (TLR4 ligand) for 24 hrs. Taken together, these findings indicate that the Lin-c-Kit+NKp46-CD4- population may control intestinal homeostasis and provide intestinal protection by producing high levels of IL-17F and IL-22 under the stimulation with IL-1β and IL-23.
Tlr4 regulates IFNγ and IL-17 production by both natural and induced Foxp3+ Tregs in the inflamed intestine during intestinal inflammation (P3276)
Authors: Cao Anthony , Yao Suxia , Stefka Andrew , Qin Hongwei , Elson Charles , Nagler Cathryn , Cong Yingzi
Published: January 2023

Both natural Treg and induced Treg play crucial roles in maintaining intestinal homeostasis. Paradoxically, significant numbers of Foxp3+Treg accumulate in the inflamed lesions in experimental colitis and IBD patients. Treg production of the effector cytokines IFNγ and/or IL-17 may explain their ineffectiveness in suppressing intestinal inflammation. However, it is unknown whether both iTreg and nTreg produce effector cytokines, and how TLR signaling regulates this process. We report here that Foxp3+Treg cells were increased in the intestines of TLR4-/- mice and colitic IL-10-/- mice. Deficiency in both TLR4 and IL-10 resulted in more severe colitis and further increased Treg cells in the inflamed intestines. The majority of Tregs in spleen are Fox3+ Helios+ Npn1+ nTreg whereas most Tregs in intestinal LP are Fox3+ Helios- Npn1- iTreg. Unexpectedly, more nTreg expressed IFNγ and/or IL-17 than iTreg in both spleen and intestine, which was further increased with TLR4 deficiency and intestinal inflammation. LPS-TLR4 signaling in both T cells and APC inhibited Foxp3+Treg via MyD88-dependent, TRIF-independent pathway, which was negatively regulated by SOCS3. These data demonstrate that intestinal inflammation drives iTreg expansion and induces effector cytokines by both nTreg and iTreg. TLR4 not only regulates intestinal inflammation, but also Treg conversion into Th1 and/or Th17. Further, both nTreg and iTreg phenotypes exhibit plasticity in the inflamed intestinal environment.
Dimethyl fumarate stimulates cyclic AMP production and suppresses production of pro-inflammatory mediators in immune cells (P5143)
Authors: Salinthone Sonemany , Wilkinson Amelia , Carr Daniel
Published: January 2023

Multiple Sclerosis (MS) is a disease of the central nervous system that is characterized by chronic inflammation, demyelination, axonal damage and loss of oligodendrocytes. Current FDA approved therapies are only partially effective, have side-effects and are costly. Thus, there is a need for development of novel treatment alternatives for MS. Of interest is the investigational drug BG-12, also known as dimethyl fumarate (DMF). A phase III clinical trial showed that treatment with BG-12 reduced the annual relapse rate by 53% and decreased disability by 38%. Additionally, BG-12 reduced the number of gadolinium-enhancing lesions by 90%. However, the mechanisms of action of BG-12 are not fully understood. BG-12 is an activator of the nuclear factor (erythroid-derived 2)-like 2 (NRF2) transcriptional pathway. To determine if BG-12 is able to activate other signaling cascades, we treated human peripheral blood mononuclear cells with DMF. We discovered that DMF stimulates cAMP production by approximately 3.5 after 1 minute treatment in vitro. DMF also suppresses the production of interleukin (IL)-2, IL-17, regulated and normal T cell expressed and secreted (RANTES) and tumor necrosis factor (TNF)-α in these cells. Furthermore, inhibitor studies indicate that adenylyl cyclases mediate DMF induced cAMP production. These novel findings suggest that DMF may inhibit immune cell function via the cAMP signaling pathway and has implications for MS therapy.
Regulation of 4-1BBL-mediated inflammation at the late phase of TLR response (P1228)
Authors: Kang Young , Ma Jianhui , Shim Eun-Jin , Lu Jiawei , Eun So-Young , Croft Mick , Tobias Peter , Takeuchi Osamu , Akira Shizuo , Han Jiahuai , Karin Michael
Published: January 2023

Toll-like receptor (TLR)-mediated signaling leads to the expression of inflammatory genes, and this process is sustained for the duration of inflammatory response. The TNF superfamily member 4-1BB ligand (4-1BBL) plays an essential role in sustained TNF production in macrophages. 4-1BBL expression was induced by the initial TLR signaling, and 4-1BBL formed a complex with TLRs at a late phase to trigger the secondary signaling. TLR/4-1BBL-mediated late phase signaling was dependent on TIRAP and IRAK2, and the downstream TRAF6/TAK1/TAB1 complex formed and induced MAPK activation. During the early phase of TLR4 response, the TLR4, MyD88, TIRAP, and IRAK2 formed an initial complex, and then the secondary TLR4/4-1BBL/TIRAP/IRAK2 complex formation was followed to initiate the late phase signaling that sustains TNF production in macrophages. Inhibition of the late phase TIRAP activity downregulated sustained TNF production in macrophages and further ameliorated LPS-induced septic shock. Thus, the late phase TLR/4-1BBL/TIRAP/IRAK2 signaling is critical for sustained inflammation in innate immune cells.
Crucial role for TACI in TLR activation and macrophage phenotype determination (P1206)
Authors: Akkoyunlu Mustafa , Katsenelson Nora , Uslu Kadriye , Allman Windy
Published: January 2023

Transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI) regulates antibody responses to T cell independent type 2 (TI-2) antigens such as polysaccharides. However, it is not known whether TACI is important in generation of Ab response against T cell dependent (TD) antigens. In this study, we investigated the role of TACI in Ab responses against Neisseria meningitidis type C polysaccharide-tetanus toxoid conjugate (MCPS-TT) vaccine. We found that TACI-/- mice had lower amounts of Abs against MCPS and TT compared to WT mice. More importantly, even the potent adjuvant CpG did not augment Ab responses to MCPS-TT vaccine in TACI-/- mice. Stimulation of splenic cells or peritoneal macrophages of TACI-/- mice with CpG, LPS, or PolyI:C led to a significantly impaired production of pro-inflammatory cytokines and lower levels of co-stimulatory molecule upregulation, suggesting that TACI plays a key role in TLR signaling. Significantly lower expression of TLR9, TLR4, TLR3 and CD14 as well as impaired co-localization of the adaptor molecules, MyD88 and TRIF with the TLR receptors is likely to play a role in the impaired TLR response in TACI-/- mice. Further characterization of TACI-/- macrophages revealed that they manifest the characteristics of alternatively activated (M2) macrophages. Thus, M2 macrophage phenotype appears to be responsible for the impaired TLR activation and poor Ab responses to TD antigens in TACI-/- mice.
TLR4 as a TRIF-biased receptor (P1212)
Authors: Kolb Joseph , Casella Carolyn , SenGupta Shuvasree , Mitchell Thomas
Published: January 2023

We reported previously that the vaccine adjuvant monophosphoryl lipid A (MPLA) could behave as a TRIF-biased agonist of TLR4, with mostly intact TRIF-dependent outcomes but less MyD88/TRIF co-dependent activation of proinflammatory cytokines, and proposed that TRIF-bias was an important component of MPL’s success as a clinically safe immunostimulant. Taking a pharmacological approach, we have now found that synthetic MPLA (sMLA) has approximately 10% the potency of synthetic Lipid A (sLipid A) for all TRIF and MyD88/TRIF co-dependent signaling outcomes tested, revealing that sMLA is not a TRIF-biased agonist of TLR4. We observed instead that both agonists’ potencies for TRIF-dependent outcomes were markedly higher than MyD88/TRIF co-dependent outcomes. This suggests that the receptor, not the agonist, is TRIF-biased. TLR4’s TRIF-bias was not restricted to “highly-inducible” chemokines because potency measurements using TRIF-dependent CD86 showed the same result. Tests of tetra-acylated lipid IVa also showed TRIF-biased signaling by TLR4, indicating that the phenomenon was not unique to hexa-acylated structures such as sMLA and sLipid A. Lower potency TLR4 agonists like MPLA may be useful as vaccine adjuvants because they take advantage of a TRIF-biased receptor system, providing enough signal strength to activate TRIF-dependent adaptive priming with little MyD88/TRIF co-dependent inflammation.
Regulation of the expression of MKP-1 and MKP-2 by ERK MAP kinases in macrophages following LPS stimulation (P1214)
Authors: Liu Yusen , Xue Jianjing , Wancket Lyn , Shakibi Yasaman , Nelin Leif , Frazier W.
Published: January 2023

Although the MKP family plays an important role during host defense, the regulation of MKPs is poorly understood. We studied the expression of MKP-1 and MKP-2 in RAW264.7 cells during LPS stimulation. We found that pretreatment of cells with U0126 (a MEK1/2 inhibitor), substantially decreased MKP-1 and MKP-2 mRNA levels in LPS-stimulated macrophages. In contrast, SB203580 (a p38 inhibitor), had little effect on the levels of MKP-1 and MKP-2 mRNA. The combination of U0126 and SB203580 resulted in greater decreases in MKP-1 and MKP-2 mRNA levels, suggesting that p38 contributes to MKP-1 and MKP-2 induction in response to LPS. Interestingly, in the absence of LPS, SB203580 enhanced ERK and MEK1/2 activities, suggesting that p38 negatively regulates the ERK pathway by inhibiting a signaling event upstream of MEK1/2. LPS potently stimulated CREB phosphorylation mediated by p38, but not ERK, and enhanced phospho-CREB binding to MKP-1 and MKP-2 chromatins. Interestingly, LPS also enhanced the phosphorylation of RNA polymerase II at serine-2 at the MKP-1 chromatin in a manner dependent on CDK9 and ERK. At the protein level, both MKP-1 and MKP-2 underwent robust phosphorylation mediated by ERKs. Phosphorylation of both MKP-1 and MKP-2 substantially increased the stability of these proteins. Taken together, our studies suggest that the ERK pathway plays a predominant role in the regulation of MKP-1 and MKP-2 through both transcriptional and post-translational mechanisms.
Phenotypic and functional characteristic of newly identified CD8+Foxp3-CD103+ regulatory T cells (P1047)
Authors: Zheng Song Guo , Liu Ya , Wang Julie , Horwitz David , Quismorio Francisco , Xu Anping
Published: January 2023

TGF-β is crucial for induction of CD4+Foxp3+ Tregs and maintenance of immunologic tolerance. It is, however, unclear if TGF-β also induces the similar CD8+ Tregs. While CD8+ cells primed with TGF-β (CD8+ iTregs) displayed much lower Foxp3 expression compared with compartment CD4+ cells, their suppression activity in vitro and in vivo was equivalent or even better than CD4+ Tregs. These cells did not express Granzyme A, Granzyme B or Perforin and lacked cytotoxic activity on T response cells. Additionally, CD8+ iTregs generated from Granzyme B and Perforin KO mice still suppressed autoimmunity. Transwell experiments revealed that cell-contact is required for their suppressive activity. Adoptive transfer of the CD8+ iTregs markedly suppressed experimental colitis, EAE and cGVHD. We further found both Foxp3- and Foxp3+ subsets from TGF-primed CD8+ cells had suppressive activities. Among CD8+Foxp3- cells, we identified CD103 expression is crucial for their generation and function since CD8+ but not CD4+ iTreg production decreased on CD103 KO mice. Adoptive transfer of CD8+CD103+Foxp3- subset suppressed colitis, and IL-10 and TGF-β signal seem to be crucial for this therapeutic effect. TGF-β can induce CD8+Foxp3- and CD8+Foxp3+ iTreg subsets that displayed suppressive activity in cell contact-dependent, non-cytotoxic manner and have protective effects on autoimmune diseases. Generation of CD8+ iTregs may have considerable therapeutic potential on patients with autoimmune diseases.
Regulation of the expression of GARP/latent-TGF-β1 complexes on mouse regulatory T cells and their role in Th17 differentiation. (P1033)
Authors: Edwards Justin , Unutmaz Derya , Shevach Ethan
Published: January 2023

GARP/LRRC32 has previously been defined as a marker of activated human regulatory T cells (Tregs) that is responsible for surface localization of latent TGF-β1. We find that GARP and latent TGF-β1 are also found on mouse Tregs activated via TCR stimulation, but in contrast to human Tregs, GARP is also expressed at a low level on resting Tregs. The expression of GARP can be upregulated on mouse Tregs by IL-2 or IL-4 exposure in the absence of TCR signaling. GARP is expressed at a low level on Tregs within the thymus and Treg precursors from the thymus concomitantly express GARP and Foxp3 upon exposure to IL-2. The expression of GARP is independent of TGF-β1 and TGF-β1 loading into GARP and is independent of furin-mediated processing of pro-TGF-β1 to latent TGF-β1. Specific deletion of GARP in CD4+ T cells results in lack of expression of latent-TGF-β1 on activated Tregs. GARP-deficient Tregs develop normally, are present in normal numbers in peripheral tissues, and are fully competent suppressors of the activation of T conventional cells in vitro. Activated Tregs expressing GARP/latent-TGF-β1 complexes are potent inducers of Th17 differentiation in the presence of exogenous IL-6. Induction of Th17 producing cells is preferentially induced by Tregs expressing the latent-TGF-β1/GARP complex on their cell surface rather than by secreted latent-TGF-β1.
Characterization of a naturally occurring severe combined immunodeficiency in pigs (P6087)
Authors: Wyatt Carol , Cino Ozuna A. , Rowland Raymond , Dekkers Jack , Waide Emily , Tuggle Christopher
Published: January 2023

Severe combined immunodeficiency (SCID) refers to a group of inherited disorders in which two components of the immune system are defective. We have identified a naturally occurring, SCID in a line of pigs that were bred for increased feed efficiency. Affected piglets remain healthy while on the sow, but succumb to infections soon after weaning. SCID piglets have underdeveloped lymph nodes and spleen with a paucity of lymphocytes, they do not make antibodies to Porcine Reproductive and Respiratory Syndrome (PRRS) virus, and they accept human tumors. Further analyses showed that the defect has an autosomal recessive inheritance pattern. Blood was collected within 2 days after birth to titer serum immunoglobulins, perform lymphocyte counts and identify lymphocyte populations by flow cytometry. Affected piglets had no detectable serum immunoglobulins, and very low numbers of circulating lymphocytes. By flow cytometry, there were Natural Killer cells, but few to no T or B lymphocytes. At necropsy, thymus and small intestine were collected from each affected piglet and from control littermates. Histologic sections, stained with Hematoxylin and Eosin, were evaluated for cellularity. Affected piglet thymic remnants were small, containing Hassall’s corpuscles, but relatively few thymocyte-like cells. There were no detectable Peyer’s Patches in small intestine. These observations are consistent with T-/B- NK+ SCID.
Effect of antibiotic treatments on the expression levels of metabolizing and drug resistance proteins in immunological cells from salmon head kidney (P6071)
Authors: Cárcamo Juan , Arias Luis , Mancilla Edith , Yañez Alejandro
Published: January 2023

The massive use of drugs on fish farming could have an important effect on immunological status of these organisms. Here we showed the effect of antibiotics used in salmon farming on the protein expression levels involved in metabolism and drug resistance in immunological cells from salmon head kidney (SHK-1 cells). The protein expression levels of CYP1A, CYP3A, FMO, GST, Pgp and MRP1 were measured by Western blot after antibiotic treatments. Cell viability was measured by MTT assays. Significant fluctuations of expression in the most proteins analyzed as response to antibiotic treatments were evidenced, while no effect on cell viability were observed. Therefore, the antibiotic treatment changes the expression levels of metabolizing and drug resistance proteins in these salmonid cells, which may be associated with the induction of drug resistance phenomena and a immunological response diminished. This information allows a deeper understanding of the mechanisms involved in the immunological status from a fish specie belonging to one of the most important economic sectors in Chile.
Endogenous tumor-reactive CD8+ T cells are differentiated effector cells with high expression of CD11a and PD-1 but are unable to control tumor growth (P2089)
Authors: Gibbons Rachel , Liu Xin , Harrington Susan , Krco Christopher , Markovic Svetomir , Kwon Eugene , Dong Haidong
Published: January 2023

We utilized CD11a, an integrin up-regulated on effector and memory CD8+ T cells, as a biomarker to identify, measure, and monitor endogenous tumor-reactive cytotoxic lymphocytes (CTLs) in two mouse tumor models and in melanoma patients. CD11ahigh CD8+ T cells were detected in primary and metastatic tumor sites after tumor cell injection, as well as in spontaneous murine breast cancer tissues. In the peripheral blood of melanoma patients, tumor antigen-specific T cells were detected within the population of CD11ahigh CD8+ T cells that express high levels of PD-1. Infusion of lethally irradiated tumor cells failed to stimulate the expansion of CD11ahigh CD8+ T cells, suggesting that the access of tumor cells to target tissues is required to induce CD11ahigh CD8+ T cell responses. Phenotypic analysis revealed that the CD11ahigh CD8+ T cells detected in our studies were proliferating (Ki67+) activated T cells (CD62L-, CD69+). Although CD11ahigh CD8+ T cells were differentiated effector cells with CTL effector functions, they were typically unable to control tumor growth in our mouse models, possibly due to high expression of PD-1. Our results demonstrate that CD11a can be used as a biomarker to identify and track endogenous tumor-reactive CTLs.
The E3 ligase Itch and deubiquitinase Cyld act together to regulate Tak1 and inflammation (P2107)
Authors: Poojary Venuprasad , Ahmed Neesar , Zeng Minghui , Polin Lisa , Sinha Indrajit , Rathinam Chozhavendan , Flavell Richard , Massoumi Ramin
Published: January 2023

Chronic inflammation has been strongly associated with tumor progression, but the underlying mechanisms remain elusive. We demonstrated that ubiquitin ligase Itch and deubiquitinase Cyld formed a complex via interaction through the ‘WW-PPXY’ motifs. The Itch-Cyld complex sequentially cleaved Lys63-linked ubiquitin chains and catalyzed Lys48-linked ubiquitination on TGF-beta activated kinase 1(Tak1) to terminate inflammatory signaling. Itch or Cyld deficiency resulted in sustained Tak1 activation and elevated production of proinflammatory cytokines by bone marrow derived macrophages (BMDMs). Tak1 selective inhibitor, (5Z)-7-Oxozeaenol abrogated Tak1 activation and production of proinflammatory cytokines by Itch-/- and Cyld-/- BMDMs. Reconstitution of wild-type Cyld but not the mutant Cyld (Y485A), which cannot associate with Itch, blocked sustained Tak1 activation and proinflammatory cytokine production by Cyld-/- BMDMs. Deficiency in Itch or Cyld led to chronic production of tumor-promoting cytokines by tumor-associated macrophages and aggressive growth of lung carcinoma. Thus, we have identified an Itch-Cyld-mediated regulatory mechanism in innate inflammatory cells.
Identification and expansion of an endogenous polyclonal population of tumor antigen specific T cells with high affinity TCRs (P2109)
Authors: Moran Amy , Weinberg Andrew
Published: January 2023

Previously, the field of tumor immunology has relied on T cell receptor (TCR) transgenic mice and model tumor antigens to study the T cell and tumor cell interaction. However, these models have limitations. Namely, they do not reflect the natural affinity or precursor frequency of tumor antigen specific T cells. Recently, a novel Nur77GFP mouse was generated that allows us to study the antigen receptor signal strength and study the tumor immune response to endogenous tumor antigens. This mouse has a green fluorescent protein regulated by antigen receptor signaling. The stronger the antigen:antigen receptor interaction, the higher the Nur77GFP. Importantly, the reporter is sensitive to even very weak antigen receptor engagement as is speculated to be the nature of endogenous tumor antigens. Our preliminary data suggests that the co-expression of Nur77GFP and PD-1 identifies a subpopulation of polyclonal endogenous tumor infiltrating lymphocytes that are exclusively tumor antigen specific; produce more IFNγ upon re-stimulation with syngeneic tumors, and increases with anti-OX40 therapy. We have previously shown that treatment of tumor bearing hosts with anti-0X40 can promote tumor regression, therefore we propose that one mechanism of OX40 mediated immunotherapy is via the expansion of high affinity tumor antigen specific T cells within the tumor microenvironment.
Tumor-derived exosome alters the proliferation and function of T lymphocytes in nasopharygneal carcinoma (P2111)
Authors: Li Jiang , Ye Shu-Biao , Tang Xiao-Feng , Mo Hao-Yuan , Zeng Yi-Xin
Published: January 2023

Background: Tumor-derived exosomes take an important role in the inflammatory responses and anti-tumor immune responses in tumor microenvironments. Here, we used exosomes from NPC tumor cell line TW03-EBV+ (exosome1) and TW03-EBV- (exosome2) to evaluate the impact of exosome1 and exosome2 on the proliferation of CD4+ and CD8+ T lymphocytes, the differentiation of CD4+ naïve T cells mediated by NPC tumor cells, and the lysis function of tumor-infiltrating lymphocytes (TILs) to the tumor cells from NPC. Results: Exosome1 and exosome2 all could promote the proliferation of CD4+ and CD8+ lymphocytes stimulated by OKT3 or PHA through up-regulated the phosphorylation of Erk-1,2; however exosome1 and exosome2 inhibited the proliferation of CD4+ and CD8+ lymphocytes in IL-2 medium; Exosome1 and exosome2 inhibited the Th1 and Th17 cell differentiation mediated by NPC tumor cells through changing the phosphorylation of STAT1 and STAT3; Exosome1 and exosome2 inhibited the lysis function of TIL to EBV-positive NPC tumor cell line TW03. Conclusions: The tumor-derived exosomes can alter the cross talk between lymphocytes and tumor cells in NPC, and maybe be a potential tool for NPC immunotherapy in the near future.
p53-dependent chemokine expression by tumor cells supports NKG2D-dependent elimination of senescent tumors by natural killer cells (P2115)
Authors: Iannello Alexandre , Thompson Thornton , Ardolino Michele , Raulet David
Published: January 2023

The cellular senescence is a key mechanism by which p53 suppresses tumorigenesis. Using a mouse model in which p53 expression can be regulated in tumor cells, we confirmed that NK cells participate in the elimination of senescent tumors. We further demonstrate that tumor elimination is dependent on the function of NKG2D. Surprisingly, however, p53 induction did not increase the expression of any of the NKG2D ligands. Furthermore, tumor cells grown in vitro were sensitive to NK lysis, in an NKG2D-dependent fashion, but p53 induction did not increase the sensitivity of the cells to NK lysis. Instead, our data demonstrate that p53 restoration enhanced the recruitment of NK cells into the tumor. We found that p53 restoration in vitro and in vivo induced the secretion of various chemokines and cytokines. Among the chemokines induced in senescent tumor cells was CCL2. Blockade of CCL2 in vivo with antibodies inhibited NK cell recruitment to the tumor and reduced the rate of elimination of the senescent tumors. Our findings suggest that expression of NKG2D ligands occurs largely independently of p53 or senescence, but that recruitment of NK cells to the tumor, and consequently the efficient elimination of the tumor by NK cells, is dependent on chemokine expression associated with the induction of senescence. Thus, p53-induced immune cell recruitment acts cooperatively with other signals in tumor cells that induce NKG2D ligands to promote the elimination of tumors.
Tumor infiltrating lymphocytes are enriched for highly suppressive CD4+ Foxp3+ regulatory T cells compared to peripheral blood: a flow cytometric analysis of two distinct cancer types (P2123)
Authors: Kovacsovics-Bankowski Magdalena , Chisholm Lana , Vercellini Jonna , Newell Philippa , Ma Jun , Tseng Paul , Vetto John , Wolf Ronald , Hansen Paul , Weinberg Andrew
Published: January 2023

Characterizing TIL composition allows monitoring of immunotherapies and can be a prognostic factor for clinical outcome. Here we analyze the phenotype and function of lymphocytes collected from peripheral blood and tumor infiltrating lymphocytes, immediately following resection, from 15 patients with colorectal cancer liver metastases (CRLM) and 21 ovarian cancer primary tumors (OVC). Samples were analyzed using a multi-color flow panel and by qPCR for cytokine production. The percentage of different lymphocyte subpopulations was similar in both patients. Highly suppressive Tregs expressing HLA-DR were significantly increased in TIL compared to PBL in CRLM: 69.4% vs 31.7% (p=0.0002) and in OVC: 74.8% versus 38.6% (p<0.0001). Enhanced expression of CD39, CTLA-4 and Helios on the surface of the Treg, confirmed that TIL are enriched for a highly suppressive subpopulation. The cytokine profile showed that IL-6, is uniquely detected in CD4+ T cells in TIL samples. Both TIL populations also contained a significantly higher percentage of activated CD8+ T cells (HLA-DR+/CD38+) compared to PBL (CRLM: 30.2% vs 7.7%, (p=0.0012), OVC 57.3% vs 11.9%, (p<0.0001)). This study demonstrates that multi-color flow of fresh tumor samples is an effective method to understand the activation state of TIL and may reveal subpopulations not observed in fixed tissue samples. TIL composition were remarkably similar and enriched for highly suppressive Tregs and activated CD8+ T cells.
Extracellular Proliferating Cell Nuclear Antigen is a novel marker for cancer stem cells and facilitates evasion of Natural Killer cell effector function (P2133)
Authors: Horton Nathan , Mathew Porunelloor
Published: January 2023

Natural Killer (NK) cell function is strictly regulated by inhibitory and activating receptors binding corresponding ligands on the surface of target cells, providing vital first line defenses against infections and cancer. NKp44, originally discovered as an activating NK cell receptor, was recently found to elicit inhibitory effects on NK cell effector function through recognition of cell surface Proliferation Cell Nuclear Antigen (PCNA). Other reports have pointed to potential associations between NKp44 and Human Leukocyte Antigen (HLA) I molecules. In this report, we have identified novel interactions between HLA I and PCNA on the surface of human tumor cells by confocal microscopy and immunoprecipitation. In addition to previous reports, we show PCNA on the cell surface where novel association with HLA I does not require the presence of NKp44 expressing NK cells and occurs with endogenous PCNA. The association of HLA I and PCNA forms the inhibitory ligand for NKp44, resulting in inhibition of NK effector function. Furthermore, extracellular PCNA was only found on tumor cells exhibiting a cancer stem cell phenotype. Thus extracellular PCNA is a novel marker for cancer stem cells, derived from conventional growth of tumor epithelial cell lines in tissue culture, and a mechanism by which cancer stem cells evade NK cell effector functions.
The adaptor molecule SAP plays essential roles during invariant NKT cell cytotoxicity and lytic synapse formation. (P2135)
Authors: Das Rupali , Bassiri Hamid , Peng Guan , Wiener Susan , Nichols Kim
Published: January 2023

The adaptor molecule SAP plays a critical role during invariant natural killer T (iNKT) cell ontogeny. As a result, SAP-deficient humans and mice lack iNKT cells. The strict developmental requirement for SAP has made it difficult to discern its possible involvement in mature iNKT cell functions. Using temporal Cre recombinase-mediated gene deletion to ablate SAP expression after completion of iNKT cell development, we demonstrate that SAP is dispensable for T cell receptor (TCR)-induced iNKT cell cytokine production and iNKT cell-dependent activation of bystander cells. In contrast, SAP is essential for TCR-induced iNKT cell cytotoxicity against T- and B-cell targets in vitro and for iNKT cell-mediated control of T-leukemia growth in vivo. These findings are not restricted to the murine system, as siRNA-mediated silencing of SAP in human iNKTs also significantly impairs TCR-induced cytolysis. Mechanistic studies reveal that iNKT cell killing requires the tyrosine kinase Fyn, a known SAP-binding protein. Furthermore, SAP is required within iNKTs to enable formation of conjugates with targets and support polarization of the microtubule-organizing center (MTOC) to the immunological synapse (IS). Collectively, these studies highlight a novel and essential role for SAP-associated signals during iNKT cell cytotoxicity and formation of a functional iNKT cell IS.
Human CD26hi Th17 cells eradicate large established mesothelioma (P2139)
Authors: Paulos Chrystal , Nelson Michelle , Huff Logan , Kundimi Sreenath , Goodyear Morgan
Published: January 2023

Human CD4+ T cells can differentiate into multiple effector subsets, but their role in anti-tumor immunity remains incompletely elucidated. Further, the costimulatory molecules that effectively bolster their antitumor activity are unknown. To uncover the role of CD28 versus ICOS costimulation, we sorted Th1 (CXCR3+), Th2 (CCR4+), or Th17 (CCR6+ or CD26hi) subsets from bulk human CD4+ T cells and stimulated them with CD3/CD28 or CD3/ICOS beads. We found that ICOS costimulation not only augmented human Th17 cells’ function, but also enhanced the function of human Th1 and Th2 cells superior to CD28 costimulation. Moreover, CD26hi Th17 cells were more polyfunctional than the other T cells subsets, as indicated by increased secretion of IL-17, IFN-γ, IL-22 and TNF-α. To determine the therapeutic potential of these subsets, we sorted Th1, Th2 and Th17 cells from human peripheral blood, expanded them with CD3/ICOS beads, and engineered them to recognize mesothelioma. We found that CD26hi Th17 cells mediated superior regression of large human mesothelioma in mice compared to CXCR3+Th1, CCR4+Th2 or CCR6+Th17 cells. The therapeutic effectiveness of CD26hi Th17 cells was associated with enhanced functionality and persistence in vivo. Taken together, these data indicate that the appropriate selection of effector CD4+ T cells is critical for successful tumor eradication. These findings will help guide next generation clinical trials for patients with advanced malignancies.