bluuebunny/crossref_metadata_2025 huggingface dataset:
Lack of CD4 help induces tolerance of CD8 T cell response for H60, a dominant minor histocompatibility antigen. (169.46)
Authors: Ryu Su Jeong , Jung Kyung Min , Choi Eun Young
Published: January 2023

Mismatch on minor histocompatibility (H) antigens is a major obstacle for the success of transplantation between MHC-matched individuals, generating CD4 and CD8 T cell responses specific for the disparate allo-minor H antigens. H60 is a dominant minor H antigen, for which specific CD8 T cell response dominates over those for other minor H antigens in B6 anti-BALB.B response. In our previous study, the H60-specific CD8 T cell response was found to be Th dependent, requiring CD4 T cell activation for both the primary and memory expansion. Here, we show that CD4 help deficiency during the induction of the H60-specific CD8 T cell response not only forbids the primary blast expansion but also prevents the specific memory response development, implying the tolerance induction of the H60 specific CD8 T cells under helpless condition. H60-specific CD8 T cells activated under the helpless condition were more prone to cell death than those under helped condition. Tolerance induction on the H60-specific CD8 T cells rendered the total CD8 T cell immunity of B6 mice to BALB.B to be diminished, while it remained unaffected by matching on H60. The results demonstrate that CD4 help provision influences CD8 immunity against minor H antigens and suggest that tolerance of CD8 T cell response to dominant antigen would be applicable for controlling transplantation rejection or GVHD.
Characterization of adaptive immune receptors in hagfish (170.13)
Authors: Guo Peng , Gartland Lanier , Li Jianxu , Hirano Masayuki , Alder Matthew , Herrin Brantley , Sides Jeffrey , Kasahara Masanori , Cooper Max
Published: January 2023

Instead of the immunoglobulin based antigen receptors of jawed vertebrates, jawless fish use variable lymphocyte receptors (VLRs) comprised of leucine-rich-repeat (LRR) segments for antigen recognition. Two VLR genes, VLRA and VLRB, have been identified in both lampreys and hagfish. A third VLR, so called VLRC was recently discovered in lampreys. An extensive VLR repertoire is generated through the insertion of diverse LRR sequences into an incomplete germline VLR gene by a gene conversion mechanism. Earlier studies of sea lampreys have shown that VLRB lymphocytes resemble B cells of jawed vertebrates, whereas VLRA lymphocytes are similar to T cells. For the present studies, monoclonal and polyclonal antibodies against the invariant stalk regions of hagfish VLRA and VLRB were produced to identify the VLR-bearing lymphocytes and their products. Flow cytometric analysis indicated that VLRA and VLRB are expressed by separate lymphocyte populations, with VLRA lymphocytes being the dominant population. Western blot and gel-filtration analyses indicated that hagfish VLRB antibodies are released into the circulation as multimeric proteins that are formed by non-covalent linkage of four or five disulfide-bonded VLRB dimers. Conversely, soluble VLRA is not detected in hagfish plasma. These findings indicate that distinctive T-like and B-like lineages are a common feature of the adaptive immune system in both lampreys and hagfish, in keeping with their monophyletic relationship.
Response gene to complement 32 mediate TGF-beta induced production of extracellular matrix by astrocytes (44.16)
Authors: Tegla Cosmin , Cudrici Cornelia , Singh Anil , Vlaicu Sonia , Nguyen Vinh , Rus Violeta , Rus Horea
Published: January 2023

Later stages of multiple sclerosis (MS) are characterized by a reduced number of inflammatory cells and an excessive accumulation of extracellular matrix (ECM) components, which leads to tissue fibrosis. Profibrotic cytokines such as TGF-beta stimulate the synthesis of ECM by astrocytes. However, the intracellular mechanism by which TGF-beta stimulates the production of ECM is not completely understood. We have identified Response Gene to Complement (RGC)-32 as a factor induced by complement activation, growth factors, cytokines and TGF-beta. In view of the important role of TGF-beta in promotion of gliosis, we sought to determine whether RGC-32 plays a role in TGF-beta induced gliosis. RGC-32 was expressed at low levels in unstimulated astrocytes. After 3 h of TGF-beta stimulation, mRNA expression was significantly increased and after 18 h of treatment both the RGC-32 mRNA and protein expression levels were robustly increased when compared with controls. RGC-32 knockdown resulted in a significant reduction in procollagen I, procollagen II and fibronectin expression induced by TGF-beta. In addition, using chromatin immunoprecipitation assay we have shown the in vivo binding of RUNX1 to the RGC-32 promoter after TGF-beta stimulation. Taken together, our data demonstrate for the first time that RGC-32 plays a critical role in TGF-beta-induced ECM production in astrocytes. Our study suggests that RGC-32 can be a new target for therapeutic intervention to prevent gliosis in MS.
Deficiency of leukocyte NADPH oxidase increases joint inflammation in serum-induced arthritis model (44.27)
Authors: Huang Ya-Fang , Nigrovic Peter , Shieh Chi-Chang
Published: January 2023

To elucidate the role of reactive oxygen species (ROS) in modulating tissue inflammation, we established an arthritis model by intraperitoneally injecting the K/BxN serum into wild type and ncf1-/- CGD mice, which have deficiency in p47phox of NOX2. By comparing the joint inflammation index ankle thickening, we found that the serum-induced arthritis were more severe in CGD mice than in wild type mice (30.2 mm ± 4.6 vs. 19.6 mm ± 0.19 on the day 10). These data suggested that lack of leukocyte-produced ROS interferes with the control of the neutrophil-dominant joint inflammation. By depleting neutrophils with anti-Ly6G antibodies, we found that serum-induced arthritis was abrogated in both ncf1-/- and wild type mice, which confirms previous reports that neutrophils play an important role in the induction and progression of serum-induced arthritis. However, the joint reaction in serum-induced diseased ncf1-/- mice, different from wild type mice, could not be reversed by depleting neutrophils. We further analyzed the proinflammatory cytokines, chemokines, and neutrophil-related proteases in ncf1-/- mice and wild type. We also analyzed the redox modulation status of redox response proteins to elucidate the possible mechanisms involved in ROS-mediated innate immunity in regulating the joint inflammation. These data suggest that ROS play an important role in modulating immune system and controlling the tissue inflammation in joints.
Quantitative imbalance and functional defects of myeloid-derived suppressor cells in the NOD mouse model of autoimmune diabetes (44.39)
Authors: Adeegbe Dennis , Ricordi Camillo , Serafini Paolo , Inverardi Luca
Published: January 2023

Myeloid-derived suppressor cells (MDSCs) contribute to tumor escape by inhibiting tumor-specicif T cell response and accumulate at peripheral sites during inflammation or tissue damage. While accumulating evidence supports MDSC’s ability to suppress T cell responses against auto antigens, it is currently unknown whether they exhibit intrinsic defects in autoimmune disease conditions. Thus, the present study evaluated quantitative and qualitative properties of MDSCs in diabetes-prone NOD mice relative to healthy wild type (wt) control animals. FACS analysis revealed a higher fraction of Gr-1+CD11b+ MDSCs that further increased with diabetes incidence in the peripheral blood of NOD mice compared to controls. Although NOD MDSCs were similar phenotypically to their wt counterparts, an imbalanced ratio between the monocytic (LY6C+) and granulocytic (Ly6Ghi) subsets was prevalent in the NOD mice (1:15 vs 1:1.5 in controls). Importantly, MDSCs isolated from NOD mice demonstrated diminished suppressive capability in vitro compared to wt cells, consistent with their expression of lower levels of iNOS mRNA. In a diabetes transfer model, wild type but not NOD MDSCs significantly delayed disease occurrence in NOD.Scid recipients. Collectively, these data indicate a quantitative perturbation in MDSC subsets in NOD mice and suggest qualitative defects that may represent a previously un-identified mechanism in the etiology or progression of disease in this animal model of type 1 diabetes.
Anti-lipid response in Systemic Lupus Erythematosus (44.4)
Authors: Jovanovic Vojislav , Ng Ai Poh Amanda , Ho Xin Pei Eliza , Lim Yan Ting , Abdul Aziz Nurhuda , McKinney Eoin , Lyons Paul , Smith Kenneth , MacAry Paul , Kemeny Michael , Wenk Markus
Published: January 2023

Systemic Lupus Erythematosus (SLE) is a chronic, multisystem, autoimmune disorder with a broad range of clinical presentations. One of the main factors, proposed to contribute to the development of clinical manifestations of SLE is accumulation and impaired clearance of immune complexes. The aim of the present study was to investigate lipid and anti-lipid antibody profiles in SLE patients’ plasma using ELISA and gas chromatography-mass spectrometry (GC-MS). The SLE cohort employed in this study is composed of 24 subjects and disease monitoring was undertaken with serial BILAG disease scoring. The blood was collected at three time points: at the moment of flare; and 3-months/12-months after treatment had started. For oxysterols (7α-hydroxycholesterol, 7β-hydroxycholesterol, 7-ketocholesterol) we report a trend where lipid levels and corresponding anti-lipid IgG concentrations are significantly reduced during the course of treatment. For phospholipids (oxidized phosphatidylcholine, cardiolipin) we also show high levels of anti-lipid IgG during flare or active disease. Using GC-MS and ELISA we have confirmed an association of high lipid levels and high anti-lipid IgG level with disease flare in comparison to 12 months after the commencement of treatment. We propose that auto anti-lipid IgGs should now be considered a component of the accumulated immune complexes in SLE and thus may contribute to disease pathogenesis.
Non-invasive imaging of arthritic inflammation and therapeutic response with 19F MRI (44.42)
Authors: Wesa Amy , Balducci Anthony , Helfer Brooke , Ahrens Eric , O'Hanlon Charlie
Published: January 2023

Non-invasive imaging of inflammation to diagnose diseases such as rheumatoid arthritis (RA) and to monitor responses to therapy is critically needed. V-Sense, a perfluorocarbon tracer agent that preferentially labels inflammatory cells, which are then recruited out of systemic circulation to sites of inflammation, enables detection by 19F MRI. With no 19F background in the host, detection is highly-specific, yielding a quantitative marker of the degree of inflammation present. Collagen-induced arthritis in rats, a model with many similarities to human RA, was used to study the ability of V-Sense to measure the accumulation of inflammation over time. Disease progression in the rat hind limbs was monitored by caliper measurements and 19F MRI on days 14, 21 and 28, including the height of clinically symptomatic disease. Naïve rats served as controls. The capacity of V-Sense to assess the effectiveness of therapy was studied in a cohort of rats administered oral prednisolone on days 14 to 28. This study may support the use of V-Sense to clinically quantify and monitor the severity of inflammation, and to assess the effectiveness of treatments in RA and other diseases with an inflammatory component
The role of Reactive Oxygen Intermediates in B cell activation and differentiation (45.12)
Authors: Crump Katie , Liu Chun , Konopitski Andrew , Poole Leslie , Haas Karen , Grayson Jason
Published: January 2023

Generating a protective humoral immune response requires proper B cell activation. Ligation of the BCR increases the level of reactive oxygen intermediates (ROI). While they have been implicated in oxidative stress, recent studies suggest ROI act as secondary messengers facilitating normal cellular function. Our focus is on the mechanisms by which ROI control B cell function. Following BCR ligation in vitro, the levels of the first oxidation product of cysteine, sulfenic acid, increased in the proteome. Using 5,5-dimethyl-1,3-cyclohexanedione (dimedone), a compound that covalently reacts with sulfenic acid to prevent further oxidation or reduction, we determined reversible cysteine sulfenic acid formation is critical for sustained tyrosine phosphorylation, calcium flux, and proliferation of B cells. To address the role of ROI in vivo, lymphocytic choriomeningitis virus (LCMV) infected mice were treated with Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), a superoxide dismutase mimetic, from days 0-8 postinfection. On day 38, LCMV specific IgG antibody secreting cells (ASC) were 5-fold lower in the bone marrow. In contrast, the number of LCMV specific memory B cells was unaffected. The ASC decrease was not detected when mice were only treated from days 0-4 or 4-8 postinfection or during secondary viral infection. These data demonstrate ROI and reversible cysteine sulfenic acid formation are critical to B cell function and differentiation.
CD28: a novel negative regulator of plasma cell function and survival. (45.16)
Authors: Njau Modesta , Kim Jin , Chappell Craig , Jacob Joshy
Published: January 2023

CD28 is a cell surface protein that is critical for the activation of naive T cells. Interestingly, CD28 is also expressed on plasma cells. Plasma cells occur in two versions - short lived ones that appear in the secondary lymphoid organs shortly following activation and the long-lived ones that reside in the bone marrow where they produce antibodies for a life time. Here we examined the role of CD28 expression on long-lived plasma cell function and longevity. To generate a host that lacks CD28 expression only on plasma cells, we adoptively transferred CD28-/- or syngeneic wildtype B cells into B cell deficient, μMT mice. We subsequently immunized the recipient hosts and monitored their serum antibody levels, plasma cell frequencies and phenotype for six months. We observed significantly higher antigen-specific IgM and IgG levels in the recipients of CD28-/- B cells in comparison to wildtype counterparts. Consistent with the higher antibody titers, CD28-/- plasma cells produced more immunoglobulin per cell and were present in greater numbers than wildtype plasma cells. Interestingly, CD28-/- plasma cells expressed higher levels of plasma cell survival factors as compared to wildtype plasma cells. Collectively these results suggest that CD28 is a negative regulator of plasma cell function and survival. Insight on the factors that modulate plasma cells can be applied to improve vaccine-induced antibody responses or regulate self-reactive plasma cells in autoimmunity.
CTLA-4 activates the hippo pathway to regulate terminal differentiation of the CD8+ T cell. (46.17)
Authors: Thaventhiran James , Hoffmann Anja , Fearon Douglas
Published: January 2023

Replicating, antigen-specific CD8+ T cells must not commit to terminal differentiation until there has been sufficient clonal expansion. The Hippo pathway of organ size control mediates this requirement by linking expression of the differentiation-inducing transcription factor, Blimp-1, to contact between replicating cells, which would be dependent on their frequency. TCR and IL-2R stimulation assemble the Hippo pathway in the CD8+ T cell by inducing expression of WW45, Mob1, Lats1, and YAP, the transcriptional co-activator that mediates organ growth. Contact between activated CD8+ T cells triggers the Hippo pathway, causing serine phosphorylation and degradation of YAP. This is suppressed by addition of naïve CD8+ T cells, indicating that the ligand-receptor pair triggering the Hippo pathway is expressed only by activated cells. The ligand was identified by suppressing YAP degradation with blocking CD80/86 antibody, and the receptor was defined by inducing YAP degradation by crosslinking CTLA-4. That YAP regulates differentiation was shown by ectopically expressing a non-phosphorylatable, stable form of YAP in activated CD8+ T cells, which suppressed Blimp-1 expression in vitro, and the differentiation/senescence marker, KLRG1, in vivo. This role for CTLA-4 was confirmed by the presence of YAP in T cells from CTLA-4-/-, but not CTLA-4+/-, mice. Therefore, in a process that resembles quorum sensing, the Hippo pathway regulates terminal differentiation of the CD8+ T cell.
Defining the role of mTOR in effector and memory CD8+ T cell differentiation (46.20)
Authors: Pollizzi Kristen , Delgoffe Greg , Waickman Adam , Powell Jonathan
Published: January 2023

Recent studies implicate mTOR in the generation of CD8+ T cell memory. Here, we demonstrate that mTORC1 is required for CD8+ T cell effector function during the activation of naïve CD8+ T cells. We generated mouse T cell-specific knockouts of Rheb, a TORC1 component; TSC2, a TORC1 negative regulator; and Rictor, a TORC2 component. We show both in vivo and in vitro that Rheb-/- CD8+ T cells have diminished effector capabilities, specifically reduced cytokine secretion and proliferation after stimulation or infection. However, these cells persist for over 30 days and retain memory cell characteristics. Conversely, TSC2-/- antigen-specific CD8+ T cells display robust effector function with increased proliferation and cytokine secretion relative to wildtype (WT). The majority of these TSC2-\- cells are unable to transition to a memory phenotype and perish during the contraction phase. Interestingly, TORC2 is not required for effector function as Rictor-/- CD8+T cells are capable of cytokine secretion and proliferation during a primary immune response. Loss of TORC2 may in fact promote memory responses, as we found Rictor-/- CD8+ T cells to have more potent antigen recall in adoptive transfer compared to WT cells. We use this novel approach to selectively probe specific mTOR functions in T cells. Ultimately, our findings highlight the divergent roles of TORC1 and TORC2 in directing CD8+ T cell effector and memory function.
Rapamycin inhibits generation of memory CD8 T cells that localize to mucosal tissues (46.23)
Authors: Sowell Ryan , Rogozinska Magdalena , Marzo Amanda
Published: January 2023

A limitation of current vaccine strategies is the inability to generate effective memory CD8 T cell responses in locations relevant to disease transmission. There is a gap in our knowledge of how memory CD8 T cells are generated and maintained in vivo in different tissues. In response to vaccination, CD8 T cells expand, differentiate into effectors and then undergo contraction. The end result is a population of memory CD8 T cells that survive this process. The mammalian target of rapamycin (mTOR) kinase is a critical regulator of many cell processes including proliferation, differentiation, and survival. The drug rapamycin is a selective inhibitor of mTOR and while it is widely used as an immunosuppressant to inhibit allograft rejection, it has also been shown to increase the number of viral memory CD8 T cells found within secondary lymphoid tissues. The factors that account for the discrepancy observed with rapamycin treatment on pathogen-specific versus graft-specific T cells are unclear. Here we demonstrate that despite enhancing generation of memory CD8 T cells in secondary lymphoid tissues, rapamycin inhibits generation of virus-specific memory CD8 T cells found within non-lymphoid tissues such as the lamina propria of the small intestine and vaginal mucosa. Taken together these data suggest that rapamycin treatment results in an increase in functional memory CD8 T cells residing in secondary lymphoid tissues at the expense of memory CD8 T cells in non-lymphoid sites.
The C3a receptor defines a CD8+ memory T cell subset that produces IFN-γ and TNF-α (46.3)
Authors: Moyron-Quiroz Juan , Ly Minh , Yang Xifeng
Published: January 2023

Complement is an important component of the innate immune system, but is also required for the generation of an optimal adaptive immune response. C3a is a product of the complement activation and is a potent mediator of inflammation. The C3a receptor (C3aR) is involved in chemotaxis, cell activation and granule enzyme release. Previous reports described that C3aR is not expressed on resting T cells, but is inducible on activated T cells. In this study, we describe a subset of C3aR+ T cells present in peripheral blood from normal healthy donors. We found that over 80% of the cells in this population are CD8+ and with an effector memory phenotype (CD45RO+, CD45RA-, CD62L-). Further analysis revealed that the CD8+C3aR+ cells express high levels of CCR4, CCR5, CCR6, CXCR3, and CXCR6, all of which are the receptors of pro-inflammatory chemokines. In addition, this cell population also expresses the adhesion molecules CD11a (LFA-1α), CD18 (β2), CD29 (β1), CD50 (ICAM-3) and CD51/61 (αV/β3), which are involved in costimulation and homing to inflamed sites. Comparing with CD8+C3aR- cells, the CD8+C3aR+ cells produce more IFN-γ and TNF-α. We hypothesize that this memory CD8+ T subset is recruited to inflamed sites, either in response to C3a or chemokines. These cells likely play a pivotal role in protective immune responses.
Genome-wide association study of inbred murine strains discovered nuclear factor of activated T-cells Nfatc4 as a major gene controlling severity of inflammatory arthritis. (47.14)
Authors: Ramez Mohamed , Forde Toni , Chow Chi-Wing , Adarichev Vyacheslav
Published: January 2023

Nuclear factor of activated T cells (NFAT) is a family of transcription factors critical in regulating gene expression in development and disease. NFAT is an important target for cyclosporine immunosuppression in transplant rejection. The NFAT pathway is also important in inflammatory and autoimmune diseases such as rheumatoid arthritis. Using genome-wide association study (GWAS) of antibody-mediated arthritis in inbred murine strains, we found NFATc4 within the major arthritis severity peak at 56.35-56.46 Mb of chromosome 14 with genetic association of log(1/p) > 5.5. To confirm the genetic linkage and functional significance of the NFATc4 gene, we performed collagen antibody-induced arthritis (CAIA) and found that NFATc4-deficient mice exhibited significantly reduced inflammation as compared to wild-type counterparts (55% downregulation, p < 0.01). Immunoblotting analysis demonstrated the presence of NFATc4 in articular cartilage and chondrocytes derived from femoral heads of arthritic and naïve mice. Notably, NFATc4 in chondrocytes isolated from arthritic mice was hypo-phosphorylated as compared to naïve cells, indicating activation of NFATc4. NFATc4, as expected, was not found in splenocytes. We also performed genome-wide Affymetrix oligonucleotide array studies in CAIA experimental arthritis model using wild type and NFATc4-deficient mice. Taking together, we uncovered a novel role of NFATc4 in inflammatory arthritis in chondrocytes.
The role of CTSH in narcolepsy susceptibility and autoimmunity (47.16)
Authors: Weiner-Lachmi Karin , Faraco Juliette , R Kornum Birgitte , Lin Ling , Steinman Lawrence , Mignot Emmanuel
Published: January 2023

Narcolepsy, a disease associated with HLA-DQB1*0602, is caused by the selective autoimmune destruction of 70,000 hypocretin neurons in the brain. Recently we identified Cathepsin-H as a novel Narcolepsy susceptibility locus. SNP rs3825932 was associated with Narcolepsy in our set of 807 Caucasian cases vs. 1074 controls (p= 0.002 OR 1.24). The same SNP marker, was previously reported to be associated Type 1 Diabetes in two studies. Cathepsin H is a member of the papain-like family, and participates in intracellular protein degradation. CTSH have also been suggested to be involved in apoptosis and neurodegeneration. CTSH expression levels in peripheral white blood cells are strongly correlated with CTSH genotype at rs1036938 (p<0.0001). Presence of the disease associated C allele is correlated with a lower expression of the CTSH, in a dose dependent manner, in CD8 (T lymphocytes, NK) and CD4 (TH, regulatory, monocytes, macrophages, dendritic) cells, but not in other peripheral blood mononuclear cell types. The low expression variant is also associated with lower CTSH specific activity levels (p=0.029). The genetic association, and results from expression and activity studies strongly suggest a role for CTSH in the development of Narcolepsy. CTSH is implicated in at least two highly selective autoimmune diseases, therefore understanding the role of CTSH in Narcolepsy susceptibility may yield important insights into the development of that and other autoimmune diseases.
Altered IRF5 exon usage and increased expression of IRF7 targets in cells with an allele associated with risk for lupus (47.27)
Authors: Poole Brian , Clark Daniel , Henrie Brandon , Sloan Jason , Baumann Doug , Heap Casey , Bills Trieste , Greenhalgh Kathryn , Read Daniel , Bergsten Sean , Roznik Marinya
Published: January 2023

Background. The single nucleotide polymorphism rs2004640 within interferon regulatory factor 5 (IRF5) is a well established genetic risk locus for systemic lupus erythematosus (SLE). The overall effects on the cell, as well as the mechanisms through which this polymorphism enhances the risk for lupus remain elusive. Since an alternate first exon is created by this polymorphism, we hypothesized that the risk haplotype causes changes in splicing patterns. We further hypothesized that the risk allele may alter the expression levels of the transcriptional targets of either IRF5 or IRF7, which is modulated by IRF5. Methods. Peripheral blood mononuclear cells were isolated from lupus unaffected individuals after informed consent. DNA was extracted and genotyped for the allele at rs2004640. IRF 5 splice variant usage in EBV-transformed cells from nine race, sex, and age-matched pairs differing in rw2004640 genotype were compared using quantitative RT-PCR. Expression of genes regulated by IRF5 and IRF7 was also compared using quantitative RT-PCR. Statistical differences were evaluated using the Wilcoxon signed rank sum test. Results. Cells with the risk haplotype had more IRF5 mRNA (1.5-fold, p=0.05), but less exon 1C (1.9-fold, p=0.017) and less exon 1D (3.5-fold, p=0.012) than those with the protective type. Additionally, transcription of three targets of IRF7 was increased in risk cells, (average 1.5-fold increase, p<0.05).
Children with autism spectrum disorders experiencing neuropsychiatric symptoms, cognitive skills, and gastrointestinal symptoms exhibit distinct innate immune abnormalities and transcriptional profiles in peripheral blood monocytes (47.7)
Authors: Jyonouchi Harumi , Geng Lee , Streck Deanna , Toruner Gokce
Published: January 2023

ASD is considered a behavioral syndrome associated with multiple genetic and environmental factors. This study focuses on a subset of ASD children characterized by fluctuating behavioral symptoms and cognitive skills, triggered by immune insults, in addition to chronic GI symptoms (ASD-immune subtype; ASD-IS). The study included ASD-IS (N=19), control ASD (N=29), and normal control (N=26) children. We evaluated production of pro-inflammatory and counter-regulatory cytokines by peripheral blood mononuclear cells (PBMCs) in response to agonists of toll like receptors (TLRs), polyclonal T cell stimulants, and recall antigens (Ags), and transcriptional profiles of PB monocytes. ASD-IS PBMCs produced less pro-inflammatory (IL-1β, IL-6, IL-12, and IL-23) and counter-regulatory (IL-10 and sTNFRII) cytokines with TLR agonists and less Th1 cytokines (IFN-γ and TNF-α) with recall Ags than control cells. ASD-IS PBMC also produced higher amounts of CCL2 than controls. Transcript profiles of ASD-IS monocytes were distinguished by altered mRNA expression (>2x) in >900 genes compared with either ASD or normal controls. Among these, 392 genes overlapped in the two control groups, including genes regulating TLR signaling, inflammatory responses, cell migration/adhesion, chemokines (CCL2 and CCL7), and genes associated with risks for autism, sychizophrenia, and depression. Our findings indicate a role of innate immunity in the puzzling clinical features observed in ASD-IS children.
Tumor-specific CD4+ T cells enhance the therapeutic efficacy of adoptive immunotherapy with CD8+ T cells in the lymphopenic setting and establish a long-term memory response (48.10)
Authors: Church Sarah , Jensen Shawn , Fox Bernard
Published: January 2023

TCR-gene-modified CD8 T cells are used for cancer treatment. We examined if tumor-specific CD4 T cells would enhance persistence and function of adoptively transferred CD8 T cells. RAG1-/- mice bearing 3-day B16-D5 melanoma pulmonary metastases received both naïve tumor-specific Trp-1 TCR CD4 T cells and effector gp100-specific TCR CD8 transgenic T cells or CD8 T cells alone. Significantly fewer mice had pulmonary metastases when treated with both CD4 and CD8 T cells compared to CD8 T cells alone at day 10 (3/20 vs. 14/14 p<0.001) and 20 (7/20 vs. 13/14 p=0.001). CD4 T cells alone were ineffective. More effector CD8 T cells were observed in the blood of mice treated with both CD4 and CD8 T cells at day 20 (1.7-fold p<0.001). These cells had a tumor-specific type 1 cytokine profile (IFNγ, TNFα, IL-12 and GM-CSF). CD4 and CD8 T cell treated short-term (50 days) and long-term (120 days) survivors rejected subsequent tumor challenge (0/5 and 0/15 respectively, p<0.001). T cells in these long-term survivors maintained tumor-specific cytokine expression (IFNγ, Grz B and IL-2). Contrary to non-transgenic models, depletion of either CD4 or CD8 cells prior to challenge at day 120 did not reduce ability to reject tumor. While no mice grew tumor at the challenge site, 30% of mice developed amelanotic metastases. This tumor escape was associated with loss of T cell function. These data provide a strategy for augmenting the therapeutic efficacy of TCR gene therapy of patients with cancer.
Forced NF-κB in T cells leads to tumor rejection (48.24)
Authors: Evaristo Cesar , Gajewski Thomas , Alegre Maria-Luisa
Published: January 2023

NF-κB activity has been reported to be reduced in T cells from tumor-bearing hosts. Our previous results indicate that reduced NF-κB activation results in impaired T cell survival, decreased Th1 and Th17 differentiation and increased iTreg differentiation. Mice with reduced T cell-NF-κB activity fail to reject cardiac and pancreatic islet allografts in absence of any pharmacological treatment. We hypothesize that forced NF-κB activation in T cells may have the opposite effect and promote T cell survival, facilitate Th1/Th17 differentiation and prevent iTreg differentiation, which would be beneficial to reject tumors. We generated mice with constitutively active (CA) IKKβ in T cells (henceforth CA-IKKβ mice). Ectopic CA-IKKβ expression resulted in NF-κB phosphorylation. Transgene expression was limited to CD4, CD8 and NKT cells and T cells showed increased NF-κB activation and nuclear translocation. T cell numbers were comparable to littermate controls, but CA-IKKβ mice had fewer Tregs and increased frequency of activated T cells that produced IFNγ upon re-stimulation. When B16-SIY melanoma cells were injected subcutaneously, tumors grew progressively in control littermates, but were rejected by mice expressing CA-IKKβ in T cells. Furthermore, CA-IKKβ expressing CD4+ and CD8+ T cells were necessary and sufficient for tumor control. Our results demonstrate NF-κB to be at the cross-roads of major T cell fate decisions that should uniquely synergize for control of tumor growth.
Survivin TCR transgenic mice develop T cell lymphoblastic lymphoma (48.25)
Authors: Cui Yongzhi , Garber Haven , Mackall Crystal
Published: January 2023

Survivin has been considered a potential tumor antigen due to high expression in most cancers and limited expression in normal tissues. To explore the potential for survivin reactive TCRs to mediate antitumor effects in mice, we generated several founders of TCR transgenic mice with specificity for the H-2b restricted immunodominant epitope of survivin. In transgenic mice, survivin reactive T cells were predominantly CD8+ and mediated specific immune reactivity toward survivin peptide pulsed targets. Some antitumor reactivity was observed, but it was not potent, and the survivin reactive transgenic T cells were unable to mediate objective tumor regression of survivin bearing tumors in vivo. Spontaneous T cell acute lymphoblastic leukemia (T-ALL) was observed at 4-6 months of age in offspring of all 3 founders and was found in both survivin TCR+Rag+/- and survivin TCR+Rag-/- mice. Flow cytometry demonstrated that these lymphoblastic cells were CD3+ and survivin TCR+, and were variably CD8+ or CD4-/CD8-. Analysis of alpha gene rearrangements revealed oligoclonality but the cell were malignant, grew continuously in vitro and induced tumors in C57BL/6 recipients. The occurrence of T-ALL in 3 founders suggests that the transgene itself, rather than insertional mutagenesis, is causative. We postulate that self-reactive TCR serves as an oncogene if expressed early during thymopoiesis. TCR signaling may cooperate with NOTCH activation and/or other events to generate the malignancy.
Over-expression of uridine cytidine kinase like-1 protects tumor cells from NK-mediated lysis in vitro and in vivo (48.26)
Authors: Ambrose Elise , Gullickson Gail , Kornbluth Jacki
Published: January 2023

NK lytic-associated molecule (NKLAM) plays a critical role in NK-mediated tumor cytotoxicity. Our hypothesis is that NKLAM and other granule proteins are released from NK cells upon interaction with tumor targets. Acting as a ubiquitin ligase, NKLAM ubiquitinates uridine cytidine kinase like-1 protein (UCKL-1) in tumor cells, leading to its degradation. Downregulation of UCKL-1 by RNA interference was previously found to increase tumor susceptibility to NK killing. Here we show that UCKL-1 over-expression protects tumor cells from NK killing and from apoptosis mediated by chemotherapeutic agents. Gene array analyses and protein expression studies indicate an involvement of UCKL-1 with the NF-κB and death receptor (DR) 3 pathways. Studies were performed to evaluate the role of UCKL-1 in tumor survival in vivo. Wild type and NKLAM-deficient mice were injected intravenously with RMA-S tumor cells expressing various levels of UCKL-1. Results to date indicate that RMA-S cells expressing the highest levels of UCKL-1 have the greatest survival in wild type mice. Tumor survival is potentiated in NKLAM-deficient mice, suggesting that the NKLAM/UCKL-1 interaction contributes to eradication of tumor cells in vivo. These findings indicate an involvement of UCKL-1 in tumor cell survival. Findings suggest an important role for UCKL-1 and its interaction with NKLAM in cancer control mediated by NK cells.
The role of amyloid precursor-like protein 2 in Ewing’s sarcoma resistance to immunity (48.3)
Authors: Peters Haley , Wang Xiaojian , Nordgren Tara , Gilling Christine , Joshi Shantaram , Yan Ying , Solheim Joyce
Published: January 2023

Metastasized Ewing’s sarcoma, a childhood cancer of the bone, has a dismal 20-30% 5-year survival rate, despite multi-modal therapy. Ewing’s sarcoma cells have been shown to escape elimination by the immune system. We have found that several Ewing’s sarcoma cell lines express substantial levels of amyloid precursor-like protein 2 (APLP2). We sought to characterize cellular changes associated with Ewing’s sarcoma cells that avoid immune recognition, with emphasis on the function of APLP2 in this process. Ewing’s sarcoma cells that survived exposure to lymphokine-activated killer (LAK) cells exhibited higher levels of APLP2 expression, suggesting that elevated APLP2 in these cells may contribute to resistance to apoptotic cell death mediated by immune effectors. Consistent with this theory, we demonstrated that LAK-challenged cells have a reduced tendency to arrest in G2/M and to undergo apoptosis following exposure to gamma-irradiation. Transient transfection of a Ewing’s sarcoma cell line with APLP2 reduced irradiation-mediated G2/M arrest and apoptosis. IFN-gamma (a cytokine produced by LAK, NK and T cells) was found to up-regulate APLP2 production in a Ewing’s sarcoma cell line in vitro. This effect of IFN-gamma may contribute to the known ability of IFN-gamma to protect tumor cells from LAK-mediated lysis. Overall, our findings from these studies increase our understanding of mechanisms regulating immune recognition of Ewing’s sarcoma cells.
T cells expanded in presence of IL-15 exhibit elevated cell surface thiols, intracellular glutathione and increased long term survival (48.34)
Authors: Kaur Navtej , Naga Osama , Norell Hakan , Al-khami Amir , Chakraborty Nitya , Johnson Christina Voelkel , Mukherji Bijay , Mehrotra Shikhar
Published: January 2023

Persistence of effector cytotoxic T lymphocytes (CTLs) during an immunological response is critical for successfully controlling a viral infection or tumor growth. Various cytokines are known to play an important part in regulating the immune response. IL-2 and IL-15 possess similar properties, including the ability to induce T cell proliferation. The IL-2 family of cytokines that includes IL-2 and IL-15 are known to function as growth and survival factors for antigen-experienced T cells. Whereas long term IL-2 exposure has been shown to promote apoptosis and limit CD8+ memory T cell survival and proliferation, it is widely believed that IL-15 can inhibit apoptosis and maintains a memory CD8+ T-cell population. Our data show that human T cells and CTLs specific for melanoma reactive antigen Mart-127-35 and influenza matrix protein MP58-66 expanded in vitro in the presence of IL-15 had higher expression of CD56 and increased survival due to higher levels of anti-apoptotic proteins bcl-2, bcl-xl, cell surface thiols, and intracellular glutathione. Our study suggests that increased longevity of CTL cultured in the presence of IL-15 might depend on increased expression of antioxidant proteins and a subset of a highly expressed CD56hi population. Efforts to modulate the redox pathway may increase the T-cell survival without compromising cell functionality and confer therapeutic benefit to T-cell-based immunotherapy protocols.
IFNγ-inducible chemokines involved in lymphocyte trafficking and tumor regression are induced by immunotherapeutic Listeria monocytogenes (48.39)
Authors: Guirnalda Patrick , Wood Laurence , Pan Zhen-Kun , Crespo Joel , Paterson Yvonne
Published: January 2023

Using a transplantable HPV immortalized tumor model, TC.1, and a Listeria monocytogenes based immunotherapeutic that expresses HPV-E7, we have examined the requirements for T cell homing and infiltration following immunotherapy. Pro-inflammatory cytokines are produced in response to vaccination with Listeria based vaccines and underlie mechanisms of vaccine efficacy. Lymphocyte migration and infiltration into tumors and vaccine induced tumor regression are dependent upon a functional IFNγ receptor on the tumor cells. We hypothesized that vaccine induced and IFNγ dependent tumor regression relies on the presence of tumor derived, and IFNγ regulated chemokines. We noted the upregulation of CXCR3, CCR4 and CCR5 ligands at the mRNA and protein level in IFNγ and TNFα stimulated TC.1 tumor cells compared to unstimulated TC.1 cells. Vaccination with Listeria vaccines induced (i) the upregulation of tumor CCL4, CCL5 and CXCL9 and (ii) tumor infiltration of T cells bearing their cognate receptors. Tumor infiltrating lymphocytes consisted of both tumor antigen specific and Listeria antigen specific T cells. Chemokine receptor expression patterns varied among tumor infiltrating T cell subsets. IFNγ and TNFα stimulated CXCR3 ligand upregulation is a common feature among a variety of tumor cell lines and across a variety of tissues. Tumor cells are a source of effector T cell chemoattractants under vaccine induced pro-inflammatory conditions.
Expression of CXCL9/Mig in the tumor microenvironment is critical for recruitment of CD8 T cells and suppression of cutaneous tumor growth (48.4)
Authors: Gorbachev Anton , Petro Marianne
Published: January 2023

The role of T cell chemoattractants CXCL9/Mig and CXCL10/IP-10 in the suppression of tumor growth is not entirely clear. Our recent studies demonstrated the presence of tumor cells deficient in the expression of CXCL9/Mig in cutaneous murine fibrosarcomas. These Mig-deficient tumor variants were more tumorigenic than parental Mig-expressing tumor cells as indicated by accelerated growth kinetics of tumors induced by these tumor cells. Mig-deficient and Mig-expressing tumor variants were used as a tool to study the contribution of tumor-derived Mig to the intra-tumor recruitment of T cells. Using adoptive transfer of primed T cells into mice bearing either Mig-expressing or Mig-deficient tumors we demonstrated that CXCR3-expressing CD8 T cells were recruited into tumors producing Mig, but were marginal to undetectable in Mig-deficient tumors. The ability of Mig-deficient tumors to express IP-10 did not substitute for the expression of Mig to recruit CD8 T cells. Furthermore, knocking down expression of Mig (but not IP-10) by specific shRNAs resulted in increased growth of Mig-producing tumor variants in immunocompetent mice. Overall, the results suggested that Mig expression in the tumor microenvironment is non-redundant for the recruitment of CD8 T cells and for the suppression of tumor growth in the skin. The appearance of Mig-deficient tumor variants in growing tumors may indicate loss of Mig expression as a mechanism of tumor escape from host’s T cell-mediated immunity.
Increased Myeloid Derived Suppressor Cell recruitment and skewed TH2 response during influenza A virus infection in the absence of TLR7. (49.21)
Authors: Jeisy Scott Victoria , Bowzard J. , Davis William , Gangappa Shivaprakash , Katz Jacqueline , Sambhara Suryaprakash
Published: January 2023

Influenza A virus (IAV) is recognized by the innate immune system through RNA sensing pattern recognition receptors. One such receptor, Toll like receptor 7 (TLR7), and its downstream adaptor MyD88, have been shown to play an important role in the adaptive immune response to IAV infection. We investigated the specific contribution of TLR7 on the innate immune response to IAV. C57/B6 (WT) and TLR7-gene knock-out (TLR7-/-) mice were inoculated intranasally with a sub-lethal dose of Influenza A/PR/8/34 virus (PR8). Lungs were collected on days 3, 5, and 7post-infection to characterize cellular infiltrates and determine viral titers. TLR7-/- mice displayed increased morbidity following infection, despite having similar lung viral titers to WT mice. By day 7 post-infection, TLR7-/- mice had increased numbers of MDSC, neutrophil, and dendritic cell recruited to the lungs. Furthermore, on day 10 post-infection, the CD4+ T-cells of TLR7-/- mice expressed significantly higher levels of IL-4 compared with those of WT mice. To investigate functional differences between WT and TLR7-/- MDSCs, we purified MDSCs from day 7 infected mice and co-cultured them with OT-2 T-cells in the presence of ovalbumin peptide. On day 3, we observed increased numbers of IL-10 producing OT-2 T-cells when cultured with TLR7-/- MDSCs. Taken together, we conclude that TLR7 plays an important role in modulating the magnitude and quality of MDSCs and TH1/TH2 polarization during IAV infection.
Interferon gamma limits the magnitude of CD8 T cell responses in the lung during influenza infection (49.24)
Authors: Prabhu P Nayana , Ho Adrian , Kemeny David
Published: January 2023

In response to a viral challenge, antigen specific CD8 T cells proliferate and gain effector functions while simultaneously acquiring the capacity to secrete cytokines such as interferon gamma (IFN-γ). IFN-γ is thought to play a role in the homeostatic control of antigen specific CD8 T cell numbers although the mechanism by which this occurs is still unclear. Infection of mice deficient for IFN-γ (IFN-γ-/-) with PR/8 influenza was associated with increased antigen-specific CD8 T cell counts in the lung and the BAL fluid when compared to wildtype (WT) C57BL/6 mice. Using in vitro experiments, we show that there is no difference in the rates of proliferation of the WT and IFN-γ-/- CD8 T cells. However upon stimulation, we observed increased cell death in WT CD8 T cells compared to their IFN-γ-/- counterparts. This effect could be reversed upon addition of IFN-γ to the IFN-γ-/- CD8 T cells in a dose-dependent manner. We also observed decreased cell death in the WT CD8 T cells after blocking IFN-γ signaling. These experiments suggest that increased antigen specific CD8 T cell counts in IFN-γ-/- mice is not the result of increased proliferative capacity but is instead due to the absence of IFN-γ-induced CD8 T cell death. Our results suggest that IFN-γ plays a role in promoting cell death that limits the CD8 T cell response during an influenza viral infection.
Increased IL-17 production in post-septic mice exacerbates respiratory syncytial virus induced immunopathology of the lung (49.25)
Authors: Mukherjee Sumanta , Lukacs Nicholas , Kunkel Steven , Carson IV William
Published: January 2023

Survivors of severe sepsis exhibit increased morbidity and mortality in response to secondary infections. Although bacterial secondary infections have been widely studied, there remains a paucity of data with viral infections post sepsis. Here, we show in an experimental mouse model of severe sepsis (cecal ligation and puncture, CLP) followed by respiratory syncytial virus (RSV) infection, exacerbated immunopathology in the lungs of CLP mice compared to RSV infected sham surgery mice. The immunopathology associated with the viral infection in CLP mice was in part due to increased mucus production in the lungs and impaired ability of septic mice to clear the virus. These findings correlated with increased IL-17 production in the lungs of RSV-infected CLP mice. RSV-infected CLP mice had increased levels of Th2 cytokines and reduced IFNγ in the lungs and lymph nodes compared to RSV infected sham mice. In addition, CD4 T cells from CLP mice ex vivo increased IL-17 production irrespective of the skewing conditions. Further, in vivo neutralization of IL-17 prior to RSV infection led to a significant reduction in virus induced mucus production and Th2 cytokines. Taken together, these data provide evidence that post septic CD4+T cells are primed toward IL-17 production, which may contribute to the immunopathology of a secondary viral infection.
Local inflammatory chemokines support the chronic activation of naïve influenza-specific CD8 T cells during the recovery from influenza infection (49.4)
Authors: Wu Tao , Suarez-Ramirez Jenny , Lee Young-Tae , Bouchard Keith , Cauley Linda
Published: January 2023

We previously found processed peptide antigens in the draining lymph nodes of the respiratory tract two months after primary influenza infection. These antigens were presented to naïve (but not central memory) CD8 T cells by CD11b+ DC. Many partially-activated F5 cells remained clustered in the paracortex of the MLN during late antigen presentation. Further studies show that the MLN remains enlarged >30dpi and contains larger numbers of CD11b+ DCs than resting LNs but the DC are not phenotypically mature. Consistently, RT-qPCR analysis shows very small quantities of cytokines in the lungs and DLNs during antigen persistence, which may contribute to the immature phenotype of the DCs and incomplete activation of naïve F5 cells. During this time mRNA levels for several inflammatory chemokines remain elevated in the lungs and DLNs including CCL8 and CXCL9. Consistent with this, naïve F5 cells lacking CXCR3 and CCR2 chemokine receptors show impaired proliferative responses when transferred to influenza infected mice 30dpi, as compared to wild type cells. These data indicate that a mild inflammatory response is induced during antigen persistence and supports chronic activation of naïve CD8 T cells through local chemokine expression.