bluuebunny/crossref_metadata_2025 huggingface dataset:
The expression of PD-1/PDL-1 on CNS cells following Theiler’s virus infection is elevated by IL-6 and inhibited by type I IFN (P6365)
Authors: Jin Young-Hee , Hou Wanqiu , Kang Hyun Seok , Koh Chang-Sung , Kim Byung S.
Published: January 2023

Theiler’s murine encephalomyelitis virus (TMEV) infection in the central nervous system (CNS) of susceptible mice results in an immune-mediated demyelinating disease, which is considered as a relevant viral model for human multiple sclerosis. We previously showed that the expression of positive costimulatory molecules is higher on microglia of TMEV-resistant B6 mice compared to TMEV-susceptible SJL mice. In this study, we analyzed the expression levels of negative costimulatory molecules, PD-1 and PDL-1, in the CNS of TMEV infected SJL and B6 mice. Our results indicated that TMEV infection induces the expression of both PD-1 and PDL-1 on microglia and macrophages in the CNS but not in the periphery. The expression of only PD-1 on CNS-infiltrating macrophages, but not on resident microglia, was considerably higher in TMEV-infected SJL mice compared to TMEV-infected B6 mice. We further showed that the presence of IL-6 is necessary to induce the upregulated expression of PDL-1 after TMEV infection. TMEV infection appears to provide both IL-6 and other undefined signal(s) for the upregulation. The expression of PD-1 and PDL-1 in the CNS of TMEV-infected mice was increased in the absence of signaling by type I IFNs, TLR3 or MDA5-mediated pathway. Taken together, these results strongly suggest that the expression of PD-1 and PDL-1 in the CNS is upregulated by IL-6 plus an unknown signal(s) following TMEV infection, and downregulated type I IFNs induced via TLRs and MDA5 pathways.
Crosspresentation with epitope-tagged antibodies targeted to different molecules on the dendritic cell surface (P5032)
Authors: Stewart Danielle , Prechl Jozsef , Schreiber Heidi , Sandor Matyas
Published: January 2023

Tuberculosis (TB) is one of the most prevalent global health crises in the world today and is caused by the bacterium Mycobacterium Tuberculosis (Mtb). While CD4+ T cells are known to be critical in the protective response against Mtb, the importance of CD8+T cell responses are gaining attention. The current vaccine BCG has limited and variable efficacy. BCG induces a strong CD4, but weak CD8+ T cell response. Targeting epitopes to specific dendritic cell receptors is an efficient way to induce T cell immunity. We created recombinant antibodies that recognize the pro-phagocytic receptor DEC205 (CD205), the co-stimulatory molecule CD40, and the phagocytic/activating Fc receptors 2/3 (FcR II/III) (CD16/32). Fused to the Fc portion of the antibodies is an Mtb immunodominant peptide, as well as a CD8+ T cell epitope. Individually, or in combination, these antibodies induce robust CD4+, antigen specific immune response in vivo. Here, we report that these DC receptor-specific recombinant antibodies cross present and cross prime antigen-specific CD8+ T cells. These responses were measured by activation and proliferation both in vitro and in vivo studies. These recombinant antibodies give us a new tool to understand how DC surface receptors influence the processing and cross-presentation of antigens to T cells. In future studies, we will use these recombinant antibodies in combination with BCG in a murine mycobacterium infection model to induce protective immunity against Mtb.
HLA-F and MHC-I open conformers are ligands for natural killer cell receptor KIR3DL2. (P5004)
Authors: Geraghty Daniel , Goodridge Jodie , Burian Aura , Lee Ni
Published: January 2023

Killer Immunoglobulin-like receptors (KIR) are innate immune receptors expressed by NK and T cells classically associated with the detection of missing-self through loss of their respective MHC ligand. Some KIR specificities for allelic classical class I MHC (MHC-I) have been described, while other KIR receptor-ligand relationships, including those associated with non-classical MHC-I, have yet to be clearly defined. We report here that KIR3DL2 and the non-classical antigen HLA-F, expressed as a free form devoid of peptide, physically and functionally interact. We further extend those results to include classical MHC-I open conformers as ligands, defining new allelic relationships with KIR3DL2 and MHC-I. The data collectively suggest a broader, previously unrecognized interaction between MHC-I open conformers - with HLA-F as a prototypical example - and KIR receptors, acting in an immunoregulatory capacity centered on the inflammatory response.
How does HLA-DM catalyze peptide exchange? Insights into the molecular mechanism of HLA-DM action from structural and biochemical studies (P5012)
Authors: Stern Lawrence
Published: January 2023

Efficient antigen presentation in the class II MHC pathway requires the non-classical MHC-II protein HLA-DM to catalyze peptide exchange on classical MHC-II molecules. Recent crystal structures of HLA-DM bound to the peptide-loading inhibitor HLA-DO and to a tethered MHC-peptide complex have clarified the nature of the HLA-DM / MHC-II interaction and revealed substantial MHC-II conformational lability around the N-terminal side of the peptide binding site. However, the molecular mechanism by which HLA-DM binding to a MHC-peptide complex induces peptide release still is not clear, and several conflicting models have been proposed. We evaluated the roles of various MHC-peptide and MHC-DM interactions in equilibrium binding and peptide exchange kinetics studies. The results suggest a mechanism in which DM binding induces MHC conformational changes that result in loss of key peptide main-chain hydrogen bonding interactions in the vicinity of the P1 residue, and alteration of peptide side-chain interactions in the P1 pocket, leading to release of peptide and formation of a meta-stable DM-MHC complex. Peptide binding driven by interactions all along the binding site can reverse these conformational changes. Thus resistance to HLA-DM mediated peptide exchange and ultimately peptide immunogenicity is related to the strength of MHC-peptide interactions throughout binding site and not simply occupancy of the P1 pocket.
Monoclonal antibodies to transmembrane proteins (P4022)
Authors: Brown Michael , Chambers Ross , Onisk Dale , Joaquim Tony , Stafford Joe , Lindpaintner Klaus , Keter Daniel , Stave James
Published: January 2023

Transmembrane proteins, including multipass transmembrane proteins like GPCRs and ion channels, are important targets for therapeutic monoclonal antibody (mab) discovery. Therapeutic antibodies to this class of proteins are generally targeted to extracellular domains displayed on the surfaces of living cells. Challenges associated with developing antibodies to this class of targets are small numbers of extracellular amino acids, membrane-dependent protein conformation, difficulty in expression at high levels, high amino acid sequence homology of human and mouse proteins, and post-translational modifications. DNA immunization strategies with full-length constructs and high throughput flow cytometry screening of mab binding to transfected and control cells was used to generate and identify large numbers of mabs to CXCR4 and ADORA2A (GPCRs) and CD20. Panels of mabs were generated for all 3 targets with low numbers of hybridoma fusions. For each target the mab gene sequences were shown to be unique and contain levels of somatic hypermutation comparable to existing benchmark therapeutic antibodies. Functional assays including apoptosis and receptor modulation (calcium flux and cAMP modulation) further demonstrated that the technical approach generated diverse panels of antibodies that exhibit functional activity as good or better than existing benchmark therapeutic antibodies.
Functional impairment of regulatory T cell in multiple low dose streptozotocin induced murine diabetes (P3099)
Authors: Singh Kailash , Kadesjö Erik , Lindroos Julia , Sandler Stellan , Thorvaldson Lina
Published: January 2023

Despite intense research on the role of the regulatory T cells (Treg) , the kinetics of regulatory T cells in the early development of T1D is still not clear. We have examined the proportions of CD4+CD25+Foxp3+ regulatory T (Treg) cells and CD4+ T helper (Th) cells at close intervals in the multiple low dose streptozotocin (MLDSTZ) murine diabetes model using flow cytometry, PCR and immunostaining. The Foxp3 mRNA levels were elevated in the spleen, pancreas and PDLNs of MLDSTZ treated mice. Foxp3 immunostaining in pancreas and spleen suggested that the infiltration of Treg cells increased in pancreas and spleen of MLDSTZ treated mice. The proportions of CD4+CD25+FoxP3+ Treg cells increased in MLDSTZ treated mice from day 7 in pancreatic draining lymph nodes (PDLNs) and day 21 in spleen. The proportions of CD4+ Th cells in MLDSTZ treated mice decreased on day 7 but increase from day 10, at which time the numbers of Tregs increased suggesting that the increase in Tregs fail to control the ongoing immune insult. Impaired mRNA expression levels of IL-35 and IL-10 in PDLNs and spleen of MLDSTZ suggest a functional impairment of the Tregs. Elevated IL-17 mRNA level in spleen of MLDSTZ mice on day 21 further provide evidence of a functional defect in the Tregs. In conclusion Tregs are upregulated in the development of MLDSTZ. However, the upregulation does not protect from hyperglycemia possibly because of a functional deficiency in the Treg population in MLDSTZ treated mice.
Disease exacerbation in multiple sclerosis patients is characterized by loss of terminally differentiated neuroantigen-specific CD8+ Tregs (P3115)
Authors: Cunnusamy Khrishen , Baughman Ethan , Franco Jorge , Ortega Sterling , Greenberg Benjamin , Frohman Elliot , Karandikar Nitin
Published: January 2023

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system (CNS) that afflicts more than 400,000 people in the US. Although the etiology of the disease is unknown, pathogenic T cells are thought to underlie MS immune pathology. In contrast to the current paradigm, we recently showed that MS patients harbor CNS-specific CD8+ T regulatory cells (CD8 Tregs) that are deficient during disease relapse. In the current study, we demonstrate that the neuroantigen-specific CD8 Tregs were cytolytic and eliminated pathogenic CD4+ T cells. Sorting of CD8+ T cells using an array of surface cellular markers revealed that the CD8 Tregs were terminally differentiated (CD27-, CD45RO-). The CD8 Treg-mediated suppression was perforin, granzyme B, and interferon-γ-dependent. Interestingly, we found that MS patients with acute disease exacerbation displayed a significant loss (averaging 25%) in the terminally differentiated CD8+ T cells, with a concurrent loss in perforin and granzyme B expression. In order to restore the regulatory potential of impaired CD8 Tregs during exacerbation, we pre-treated exacerbation-derived bulk CD8+ T cells with the cytokine IL-12 and significantly increased the suppressive capability of the cells by ~48% through upregulation of granzyme B and perforin. Our studies uncover the immune suppressive mechanism of neuroantigen-specific CD8 Tregs, and may contribute to the design of clinically relevant immune therapies for MS patients.
Prognostic immunological markers in patients with relapsing-remitting multiple sclerosis: a prospective magnetization transfer brain imaging study (P3117)
Authors: Markovic-Plese Silva , Tao Yazhong , Zhang Xin , Weinstock-Guttman Bianca , Hayward Brooke , Dangond Fernando , Zivadinov Robert
Published: January 2023

Objective: To identify predictive immunological biomarkers in patients with relapsing-remitting multiple sclerosis (RRMS) correlating with brain magnetization transfer imaging measures. Background: Baseline immunological biomarkers may help predict response to treatment. Methods: Intracellular cytokine staining and gene expression studies were performed on CD4+ and CD8+ T cells derived from 23 patients with RRMS and 15 healthy controls (HCs) at baseline (BL) and 6 months after subcutaneous interferon (IFN) β-1a treatment. The correlation between the changes in volume of normal-appearing brain tissue (NABT) and T2 lesion volume (LV) measured by voxel-wise magnetization transfer ratio (VW-MTR) with BL immunological parameters was investigated. Results: The percentage of BL IL-22-positive T cells was higher in patients than in HCs (CD8+, p=0.008; CD4+, p=0.039). A higher percentage of IL-22-positive CD8+ cells at BL correlated with a greater amount of decreasing VW-MTR T2 LV (p=0.031) in treated patients at 6 months, consistent with more prominent demyelination in T2 lesions, and a higher percentage of IFN-γ-positive CD8+ cells at BL correlated with a greater amount of decreasing VW-MTR NABT volume (p=0.007), consistent with more prominent demyelination in NABT. Conclusions: Production of IL-22 and IFN-γ by CD8+ cells is predictive of more prominent demyelinating changes.
Abnormalities in regulatory T cells and natural killer cells in major depressive disorder (P3127)
Authors: Savitz Jonathan , Tan Chibing , Taylor Ashlee , Drevets Wayne , Teague Kent
Published: January 2023

Major depressive disorder (MDD) is associated with increased levels of acute phase proteins, pro-inflammatory cytokines, and monocytosis, indicative of immune activation. However, in apparent contrast to evidence of depression-associated inflammation, a reduction in natural killer cell (NKC) activity is also a well-replicated finding, leading to the hypothesis that MDD is an “immunosuppressive” disorder. Here used FACS analysis to identify key subtypes of T cells and NKC in 36 healthy controls (HC) and 34 physically healthy, unmedicated patients with MDD matched for age and body mass index. After controlling for group differences in gender, we found a greater percentage of monocytes and Treg cells (relative to total live cells) in the MDD group. In addition, there was a smaller percentage of putative regulatory NKC (CD56+CD16-) but not cytotoxic NKC (CD56+CD16+) or NK T-cells (NKT) in the MDD patients. In wild-type mice, a microglia-driven, T cell-dependent protective response has been shown to be restricted by Treg cells, raising the possibility that the elevation of Treg cells in the MDD group may impair the resolution of acute pathophysiological changes leading to chronic inflammation. The depression-associated reduction in putative regulatory NKC but not effector NKC favors the hypothesis that MDD is associated with inflammation rather than immunosuppression.
The role of MMP-9 and serine proteases in the cleavage of surfactant protein D by stimulated PMNs and in sputum from CF patients (P3131)
Authors: Bratcher Preston , Gaggar Amit
Published: January 2023

Although the ability of serine proteases to cleave surfactant protein D (SP-D) has been well characterized, it has recently been reported by our group that matrix metalloprotease 9 (MMP-9) is able to cleave SP-D as well. However, our initial study examined MMP-9-mediated cleavage of SP-D in vitro using purified proteins. In order to expand upon this study in a more physiological context, we collected supernatants from primary polymorphonuclear leukocytes stimulated with LPS and fMLP and incubated them with purified SP-D in the presence of a general serine protease inhibitor (PMSF) and/or specific inhibitors against MMP-9 and neutrophil elastase (NE). All inhibitors were able to decrease the amount of detectable cleavage of SP-D, and none of them alone were able to completely inhibit cleavage. We were only unable to detect cleavage when a combination of PMSF and the MMP-9 inhibitor were used, suggesting that MMP-9 is the only non-serine protease released from PMNs with the ability to cleave SP-D. In addition, we examined the cleavage of endogenous SP-D in sputum samples from patients with cystic fibrosis (CF), and found that the MMP-9 inhibitor also significantly attenuated SP-D degradation seen when CF sputum was incubated at 37°C over time. These experiments suggest that MMP-9 is able to cleave SP-D in vivo, and that MMP-9 degrades SP-D in the airways of CF patients.
Increased circulating Th17 cells and elevated serum levels of TGF-beta and IL-21 involved in human non-segmental vitiligo development (P3141)
Authors: Zhou Li , Shi Yuling , Li Kai , Hamzav Iltefat , Lim Henry , Mi Qing-Sheng
Published: January 2023

Although it is widely believed that non-segmental vitiligo (NSV) results from the autoimmune destruction of melanocytes, the detailed immune mechanisms remain poorly understood. Th17 cells, a subset of CD4+ helper T cells, have been identified to be implicated in a variety of human autoimmune diseases. To comprehensively evaluate the role of Th17 cells and the pathways driving these T cell differentiations in NSV, we had compared the frequencies of peripheral blood Th17 cells and other IL-17 producing T cells by flow cytometry between 42 active NSV patients with 45 race-, gender- and age-matched healthy control subjects. Serum levels of IL-17A and Th17 cell differentiation related cytokines including TGF-beta1, IL-6, IL-23, IL-22 and IL-21 were detected by ELISA. Our results showed the increased circulating Th17 cell frequencies and elevated serum IL-17A, TGF-beta1 and IL-21 levels in NSV patients. Further more, the increased Th17 cell frequencies are positively correlated with serum TGF-beta1 levels and the body surface area (BSA) in NSV patients. These results indicate the involvement of Th17 cells in the development and progression of NSV, and more importantly, our data highly suggest that elevated serum TGF-beta and IL-21 could contribute to enhanced Th17 cell differentiation in NSV.
Calpastatin prevents NF-κB mediated hyperactivation of macrophages and attenuates colitis (P3151)
Authors: Rose Aaron , Huang Zhi , Hoffmann Fukun , Denk Tobias , Hashimoto Ann , Hoffmann Peter
Published: January 2023

Calpain enzymes proteolytically modulate cellular function and have been implicated in inflammatory diseases. In this study, we found that calpain levels did not differ between intestinal tissues from inflammatory bowel disease (IBD) patients and healthy controls, but IBD tissues showed increased levels of the endogenous calpain inhibitor, calpastatin (CAST). To investigate the role of CAST in the immune system during IBD, mice were x-ray irradiated and reconstituted with either CAST knockout (KO) or wild-type (WT) bone marrow and subjected to dextran sulfate sodium (DSS)-induced colitis. CAST KO recipients with induced colitis exhibited more severe weight loss, bloody diarrhea, and anemia compared to WT controls. Histological evaluation of colons from KO recipients with colitis revealed increased inflammatory pathology. Macrophages purified from the colons of KO recipients had higher IL-6, TNFα, and IFNγ mRNA levels compared to WT controls. Mechanistic investigations using siRNA and KO bone marrow to generate CAST deficient macrophages showed that CAST deficiency during activation with bacterial PAMPs including heat-killed E. faecalis or CpG DNA led to increased IκB cleavage, NF-κB nuclear localization, and IL-6 and TNFα secretion. Thus, CAST plays a central role in regulating macrophage activation and limiting pathology during inflammatory disorders like IBD.
CXCL9 and γδ T cells are increased in patients with allergic conjunctivitis (P3159)
Authors: Jimenez-Martinez Maria C , Alonso Miguel , Estrada-Garcia I Iris , Estrada-Parra Sergio , Perez-Tapia Mayra , Garfias Yonathan
Published: January 2023

Allergic conjunctivitis (AC) is a frequent disease that affects the ocular surface, The study of immune physiopathology of allergic conjunctivitis is focused on T helper cells. Recently it has been suggested that γδ T lymphocytes are necessary for the maintenance of the ocular allergic response, however there are not studies in humans about the role of γδ T lymphocytes in human AC. The aim of this study was to evaluate the frequency of γδ T cells and chemokines in tears of AC patients. Patients: 31 patients were included (16 men and 15 women), AC diagnosis was based on clinical history, physical and eye examination. PBMC were obtained from patients and healthy controls and frequency of γδ T cells was evaluated by flow cytometry; tear samples were also collected to determine chemokine concentrations by CBA. Results: γδ T lymphocytes were 11.9 times increased in AC-patients (22.63 ± 2.9%) compared to healthy donors (1.9 ± 1.9%) (p=0.002), chemokine determination in tears showed increased MIG/CXCL9 in AC patients (1604 ± 1404 pg/ml) when compared with healthy subjects (568 ±841pg/ml) p< 0.02. Conclusion: γδ T cells could be also involved in human allergic conjunctivitis as is has been demonstrated in animal models, it is possible that γδ T cells could migrate to conjunctiva due to several chemokines such as CXCL9, significant increased in AC patients, sustaining the local inflammation in conjunctiva
Interferon and TNF family soluble inflammatory mediators are detected prior to and increase at Systemic Lupus Erythematosus classification in serum samples from military personnel acquired from the Department of Defense Serum Repository (P3087)
Authors: Munroe Melissa , Anderson Jourdan , Niewold Timothy , Tsokos George , Keith Michael , Harley John , James Judith
Published: January 2023

Processes that lead to clinical illness in SLE years before diagnosis are not well characterized. This study evaluates the temporal relationship between autoantibody production, cytokine levels and the onset of SLE. Serial sera from 60 SLE cases before diagnosis with no ACR criteria to SLE classification (average timespan=4.4 years) and matched healthy controls (HC) were obtained from the DODSR. Sera samples were tested for autoantibodies (BioPlex2200), C-reactive protein (hs-CRP), Von Willebrand’s Factor (VWF) and 32 soluble inflammatory mediators, including cytokines, chemokines, and soluble receptors. Patient samples before ACR classification had significant (p < 0.01) alterations in 16 soluble mediators of inflammation, as well as VWF and hs-CRP. TNF Receptor levels, TNFRI, TNFRII, BLyS, and APRIL, dramatically increased as lupus classification approached (p < 0.001). The increase in TNFR shedding parallels the accumulation of autoantibodies in SLE cases. Interferon (IFN)-associated mediators of inflammation, IFN-γ, IP-10, MIG, and MIP-1α (p < 0.01), were also increased leading up to classification. That these alterations are present prior to the transition to active SLE suggests that multiple perturbations in immune-mediated inflammatory processes occur long before clinical classification and suggest that high-risk, pre-clinical individuals, can be identified before their illness is clinically manifested and damage occurs.
Expansion of a marginal zone B cell-like population in lymph nodes of mice developing collagen-induced arthritis (P4042)
Authors: Palm Anna-Karin , Kleinau Sandra
Published: January 2023

The importance of B cells in collagen-induced arthritis led us to investigate whether particular B cell subsets participate in the autoimmune response to collagen type II. We show here that MZB cells in the spleen are naturally self-reactive to collagen type II and are the first cells to expand after collagen type II immunization. In contrast, the autoimmune collagen type II response in lymph nodes develops one week later. Due to this time lapse we hypothesized that the MZB cells may be involved in initiating the autoimmune response in the lymph nodes. We did indeed find a small MZB-like B cell population in the lymph nodes that expressed B220+CD23loCD1dhi, thereby being distinguishable from FOB cells. The MZB-like cells expanded almost two-fold after immunization with collagen type II but not the control antigen ovalbumin, making this phenomenon specific to the autoimmune response. The MZB-like cells displayed an antigen activated phenotype with increased cell surface expression of MHCII, FcγRIIb and CD70. Moreover, after immunization the MZB-like cells increased their expression of CXCR5, implying that they are migrating cells. Further investigation of the specific function of the MZB-like cells is warranted. In conclusion, MZB cells in the spleen trigger the autoimmune collagen type II response in collagen-induced arthritis, and a MZB-like population in the lymph nodes is specific for the autoimmune reaction and may be of importance for disease pathogenesis in this model.
Delivery of a genetic cancer vaccine containing a small molecule inducible immune adjuvant by in vivo electroporation (eVac) (P4332)
Authors: Collinson-Pautz Matthew , Seethammagari Mamatha , Decker William , Spencer David , Levitt Jonathan
Published: January 2023

The use of dendritic cell vaccines to treat cancer has significant potential, however that potential has yet to be achieved in treating patients. Fortunately, pioneering biotechnologies are paving the way for more effective cancer vaccines. Our lab has developed a drug inducible MyD88/CD40 (iMC) composite adjuvant that promotes robust cytotoxic T cell priming in iMC activated DCs. Despite the iMC innovation, the use of patient-tailored ex vivo DC vaccines is impractical for widespread use, due to issues of scalability and cost. We have therefore begun to address the delivery of DC vaccines as “off-the-shelf” therapies using in vivo electroporation (EP) of plasmid DNA (pDNA) encoding both the iMC adjuvant and tumor antigen (eVac). Intradermal EP of pDNA encoding the model antigen LacZ in mice suggests that LacZ-specific T cell responses were induced, as mice receiving LacZ + EP prophylactically were either protected from establishment of B16/LacZ tumors or demonstrated significantly slower tumor growth compared to controls. In mice bearing pre-established B16/LacZ tumors, LacZ-eVac, but not EP with LacZ antigen alone significantly reduced tumor burden and increased survival. Taken together, these data indicate that eVac with iMC-antigen is an effective “off-the-shelf” cancer vaccine. These findings warrant elucidation of the underlying immunological mechanisms responsible for the anti-tumor responses observed with eVac treatment to guide further refinement of the vaccine.
Altered adaptive and TNF-superfamily soluble inflammatory mediators mark impending Systemic Lupus Erythematosus disease flare in European-American lupus patients after influenza vaccination (P6282)
Authors: Munroe Melissa , Anderson Jourdan , Merrill Joan , Guthridge Joel , Roberts Virginia , Air Gillian , Thompson Linda , James Judith
Published: January 2023

SLE is a waxing and waning disease characterized by major organ involvement, disease flares and immune dysregulation. Understanding mechanisms and identifying biomarkers of flare would help prevent damage and improve management. Plasma samples were obtained from SLE patients on the day of influenza vaccination (BL) and at 6 and 12 weeks post (FU) in 28 European American SLE patients who exhibited SELENA-SLEDAI defined disease flare at 6 (n=13) or 12 (n=15) weeks post-vaccination. Each SLE patient was matched to a unique SLE patient who did not flare (NF), and to a healthy control (HC). Samples from 13 SLE patients with flare were compared to samples from a non-flare year (self nonflare, SNF). Plasma samples were tested for 52 soluble inflammatory mediators using xMAP technology. SLE patients who exhibited disease flare had significantly (p < 0.01) higher levels of BL pro-inflammatory mediators, including Th1, Th2, and Th17-type cytokines compared to NF/SNF SLE patients and HC. Regulatory cytokines, including IL-10 and TGF-β were increased (p < 0.01) at BL in SLE patients not exhibiting disease flare (NF/SNF) compared to SLE flare and HC. TNFR family members, TNFRI, TNFRII, Fas, FasL, CD40L, were increased at BL in SLE flare patients. Select serum cytokines and chemokines are elevated in patients before SLE disease flare, providing important pathways to dissect for mechanism of flare, therapeutic selection or development.
The central role of dendritic cell-produced IL-6 signals in reversing age-related CD4 T cell defects (P4226)
Authors: Brahmakshatriya Vinayak , Rymeski Barbara , Kuang Yi , Swain Susan
Published: January 2023

The response of aged CD4 T cells to vaccines is less than that of young T cells resulting in ineffectiveness of many current vaccines and contributing to the greater susceptibility of the aged to new infections. We find that activation of DC by TLR ligands (*DC), used as APC for naive CD4 T cells, induces their production of high levels of IL-6 that improve aged CD4 T expansion, cytokine production and survival. Importantly, this enhances their ability to differentiate into T follicular helper cells that promote long-term B cell responses and sustained antibody production, a key to vaccine-induced immunity. The enhanced helper function of aged CD4 T cells is lost if the *DC cannot make IL-6, while effects of *DC on young CD4 T cells are not IL-6 dependent. This suggests that loss of responsiveness to IL-6 is central to the aged defects of CD4 T cells. We find that in vitro IL-6 signaling of the aged is blunted such that the dose of IL-6 required to achieve a particular level of IL-6R internalization and phosphorylation of STAT3 is higher in the aged vs. young naïve CD4 T cells. Clearly, the ability of aged CD4 T cells to respond to the strong *DC-mediated IL-6 signal during their interaction is still intact and can restore their function to that comparable to young cells. This *DC-based vaccine strategy to localize pro-inflammatory cytokines to the Ag-specific CD4 T cells, also avoids counterproductive and often-dangerous bystander effects of adjuvants given systemically.
Host DNA released in response to aluminum adjuvant enhances MHCII mediated antigen presentation and prolongs CD4 T cell interactions with dendritic cells. (P4244)
Authors: McKee Amy , Burchill Matthew , Michael Munks , Jin Lei , Kappler John , Friedman Rachel , Jacobelli Jordan , Marrack Philippa
Published: January 2023

Many vaccines include aluminum salts (alum) as adjuvants in spite of little knowledge of alum’s functions. Host DNA rapidly coats injected alum. Here we further investigated the mechanism of alum and DNA’s adjuvant function. Our data show that DNAse co-injection reduces CD4 T cell priming by intramuscularly injected antigen plus alum. This effect is partially replicated in mice lacking STING, a mediator of cellular responses to cytoplasmic DNA. Others have shown that DNAse treatment impairs alum induced DC migration from the peritoneal cavity to the draining LN. However our data shows that DNAse does not affect accumulation of, nor expression of costimulatory proteins on, antigen loaded dendritic cells in lymph nodes draining injected muscles, the site by which most human vaccines are administered. DNAse does inhibit prolonged T cell-DC conjugate formation and antigen presentation between antigen+ DCs and antigen specific CD4 T cells following intramuscular injection. Thus, from the muscle, an immunization site that does not require host DNA to promote migration of inflammatory DC, alum acts as an adjuvant by introducing host DNA into the cytoplasm of antigen-bearing DCs, where it engages receptors that promote MHC II presentation and better DC-T cell interactions.
Dexamethasone inhibits tumor cell lysate induction of a pro-inflammatory dendritic cell phenotype and function modulating T cell cytokine profiles (P4246)
Authors: Falcon Cristian , Tempio Fabian , Perez Claudio , Behrens Carolina , Riberos Ana-Adelia , Falcon Felipe , Salazar-Onfray Flavio , Lopez Mercedes
Published: January 2023

Recently, we showed the effectiveness of a DCs-based immunotherapy for improving long-term survival in patients with melanoma using an allogeneic melanoma cell lysate. Glucocorticoids are used in cancer patients being implicated in immune suppression. Here, we aim to study the effect of dexamethasone on the inhibition of the pro-inflammatory phenotype and immunogenicity capacity of melanoma cell lysate-loaded DCs. Monocytes were differentiated to DCs as previously described. Dexamethasone was given at day 1 and 2 to obtain tolerogenic DCs. DCs maturation markers and TH1, TH17 phenotype were determined by flow cytometry. T cell cytokines were measured by ELISA and proliferation evaluated by CFSE dilution assay. We show that dexametasone stimulus elicits a semi-mature DC phenotype (low MHC II, CD83, CD80 and CD86) sharing mature and immature DCs characteristics, secreting low levels of IL1β, IL-6 and IL-12 and high levels of IL-10, but not affecting DC phagocytosis capacity. TolDCs inhibited T cell proliferation and IFNγ, IL-17 and TNF-α release by T helper cells. At contrary, an augment of Treg cells could be observed. The used glucocorticoids affects the maturation of newly differentiated monocytes-derived DCs in presence of immunogenic tumor cells, affecting the activation of a correct adaptive anti tumor immunity.
Scavenger receptor-mediated dendritic cell internalization and activation by Polyguanine-conjugated antigen (P4234)
Authors: Narciso Jo Erika , Toussaint Helene , Erdos Geza , Carey Cara , Watkins Simon , Falo, Jr. Louis
Published: January 2023

Targeting specific antigens to antigen presenting cells for efficient antigen processing and immune activation is an essential design consideration for vaccine development. Our group has developed polyguanine (Poly(dG))-conjugated antigens with intrinsic antigen targeting and adjuvant properties. These antigen conjugates can potentially increase vaccine efficiency by transforming soluble antigens into aggregated particulates that are targeted to the scavenger receptor in dendritic cells (DCs), resulting in efficient internalization, activation, and cross-presentation. We have successfully conjugated Poly(dG) residues to the model Ag ovalbumin (OVA) and found that Poly(dG)-OVA can induce robust antigen-specific helper, cytotoxic, memory T cell, and antibody responses. In this study, we measure DC internalization and demonstrate that Poly(dG)-OVA is internalized several times more efficiently than OVA alone. This internalization is specifically inhibited by scavenger receptor ligands. Treatment with Poly(dG)-OVA also resulted in upregulation of CCR7 expression and secretion of MCP-1, TNF-α, and IL-6 by DCs in vitro. These results suggest that coupling Poly(dG) to antigens enables efficient DC targeting and activation through the scavenger receptor. This vaccine design can be applied to viral or tumor protein or peptide antigens for the production of more efficient antiviral and antitumor vaccines without the need for additional adjuvants.
Enhanced cancer immunotherapy using nanoparticle-based modulation of SOCS1 genes in dendritic cells (P4242)
Authors: Lim Yong Taik , Heo Min Beom , Noh Young-Woock
Published: January 2023

Although dendritic cells have important roles in cancer immunotherapy, their therapeutic efficiencies were limited due to the presence of immunosuppressive factors. Here, we report the fabrication and use of polymer nanoparticles for the simultaneous delivery of antigens and small interference RNA (siRNA) of immune suppressor genes into dendritic cells. The polymer nanoparticles containing ovalbumin (OVA, as a model antigen) and siRNA of suppressor of cytokine signaling 1 (SOCS1) were fabricated by double emulsion solvent evaporation method. The encapsulation of siRNA was about 57.6 % when OVA was present during the preparation, while that was only 2 % without the OVA. The polymer nanoparticles containing OVA and siRNA of SOCS1 were efficiently taken up by the dendritic cells and showed no detectable toxicity. The knockdown of SOCS1 genes in dendritic cells induced pro-inflammatory and anti-inflammatory cytokine expression. The polymer nanoparticles are expected to be potent nanotechnology platform for targeted delivery of siRNA and antigens into dendritic cells, and use as CD8+ T cells-based cancer immunotherapy.
A novel ligand-independent peptide inhibitor of TREM-1 modulates the inflammatory response and improves survival in septic mice (P4210)
Authors: Sigalov Alexander
Published: January 2023

Sepsis is a serious medical condition characterized by systemic inflammation and is associated with high mortality and morbidity rates. Currently, no FDA-approved sepsis drugs are available and over 30 drug candidates have failed late-stage clinical trials, which highlights the urgent need for novel therapeutic approaches. Triggering receptor expressed on myeloid cells-1 (TREM-1) amplifies the sepsis-associated systemic inflammatory response. TREM-1 upregulation on peritoneal neutrophils has been found in human patients with sepsis and in mice with experimental lipopolysaccharide (LPS)-induced septic shock. However, the precise function of TREM-1 and the nature of its ligand are not yet known. Here we show that TREM-1 can be specifically silenced in vitro and in vivo using a novel ligand-independent peptide inhibitor, which was designed using a new model of immune signaling, the signaling chain homooligomerization (SCHOOL) model. Blockade of TREM-1 function using non-toxic and non-immunogenic SCHOOL peptide inhibitors: 1) protects mice against LPS-induced shock and 2) substantially decreases pro-inflammatory cytokine production in vitro and in vivo. In addition, macrophage-targeted delivery of SCHOOL peptides utilizing lipoprotein-mimicking nanoparticles significantly increased peptide half-life and dosage efficacy. Together, the results suggest that ligand-independent modulation of TREM-1 function using small synthetic peptides might be a suitable treatment for sepsis.
Identification of a new cell therapy candidate reveals immunological specialization and robust therapeutic efficacy against high-mortality sepsis (P4212)
Authors: Fletcher Anne , Elman Jessica , Knoblich Konstantin , Armant Myriam , Parekkadan Biju , Boyd Richard , Turley Shannon
Published: January 2023

Sepsis is an aggressive inflammatory syndrome, killing 7.3 million people per year. In mouse and human systems, we have identified highly differentiated, immune-specialized myofibroblasts called Fibroblastic Reticular Cells (FRCs) as a new cell therapy candidate for sepsis. One injection of ex-vivo expanded allogeneic FRCs significantly reduced mortality when administered at therapeutic timepoints. When administered 4 hrs after cecal ligation and puncture (CLP) in mice, FRCs prevented peritonitis progressing to blood sepsis, reducing deaths by 50%, while mesenchymal stromal cells (MSCs) were ineffective. FRCs also reduced lung pathology and lowered systemic concentrations of pathogenic cytokines. Rarely for putative sepsis therapies, we found that when FRCs were administered very late, as mice approached euthanasia endpoints (16 hrs), survival was still extremely robust: 44% of FRC-treated mice survived compared to 9% of controls. Production of the prominent vasodilator and immunosuppressant NOS2 was a primary molecular mechanism. Human FRCs showed strong similarities to mouse FRCs and exhibited desirable traits for a cell therapy candidate, including low variance between unrelated donors. Importantly, human FRCs constitutively secrete high levels of soluble cytokine decoy receptors against major mediators of sepsis. Together these data describe a novel anti-inflammatory cell type and provide robust pre-clinical evidence for therapeutic efficacy in multiple models of sepsis.
Metilprednisolone and dialyzable leukocytes extracts induce changes in frequency of CLA+ T cells in patients with atopic dermatitis (P4196)
Authors: Estrada Alejandro , Ramirez Erika , Toledo Mirna , Galicia Jorge , Estrada-Garcia I Iris , Jimenez-Martinez Maria , Perez-Tapia Mayra
Published: January 2023

Atopic dermatitis(AD) is a T cell-mediated skin disease associated with a Th2 local response and skin inflammation is associated with CLA expression. In this work we evaluated expression of CLA on CD4 and CD8 T cells in 2 groups of patients one of them received metilprednisolone(mP-Advantan-) as treatment(Group 1) and second group received mP and dialyzable leukocyte extracts(DLE-Transferon-) as immune modulator(Group 2). Methods: Double blind randomized placebo controlled study, approved by hospital ethical committee. PBMC were obtained before, during (14 day) and at the end of treatment (28 day) and CLA expression on CD4 and CD8 was evaluated by flow cytometry. Results: Group 1:Frequency of CD4+CLA+T cells before treatment 1%±0.6 vs 1.9%±0.9 p=0.02 after treatment. CD8+CLA+T cells before treatment 4.5%±0.9 vs 8.1%±1.9 p=0.0002 after treatment. Group 2: CD8+CLA+T cells before treatment 5.3%±2.1 vs 8.8%±1.8 p=0.005 after treatment. Conclusion: We treated 58 AD-pediatric patients with mP or with mP+DLE, in both groups clinical outcome was improved after therapy; mP-treated patients induced CLA on CD4+ and CD8+ Tcells, while DLE-treated patients induced only CD8+CLA+T cells. Despite CLA expression is associated with local skin inflammation, some authors have suggested that CLA expression is also induced in Tregs; it is possible that DLE- induced CD8+CLA+ T could be enriched Tregs since it has been reported a diminished frequency of AD activation after DLE immune modulation.
Antigen-nonspecific memory CD8+ T cells display phenotypic and functional parallels during cancer immunotherapy and influenza infection (P6064)
Authors: Zamora Anthony , Sckisel Gail , Tietze Julia , Baumgarth Nicole , Murphy William
Published: January 2023

One of the hallmarks of the adaptive immune system is the development of specificity towards antigens by immune cells. Memory T cells are an important component of acquired immunity, providing the host a great deal of protection due to their rapid acquisition of effector function and low requirement for co-stimulation. In addition to antigen-specific T cells, the presence of bystander activated T cells has been well documented in various pathogenic infections and autoimmunity. We have recently characterized a population of memory CD8+ T cell following systemic immunotherapy (IT) with agonistic anti-CD40/IL-2 that display increased expression of NKG2D but not CD25, indicating a lack of TCR engagement. Likewise, primary infection with different strains of influenza resulted in rapid expansion of memory CD8+ T cells displaying this same bystander phenotype. IT-based stimulation of memory CD8+ T cells resulted in antigen-nonspecific activation, with NKG2D ligation inducing MHC-unrestricted cytotoxicity and leading to anti-tumor effects. Further characterization of bystander activated versus antigen-specific CD8+ T cells during IT and infection has revealed differential expression of activation and co-inhibitory molecules depending on location in lymphoid or non-lymphoid tissues. These results suggest different functional as well as regulatory mechanisms may occur between these cells depending on their location.
A TH1 immune response efficiently clears rabies virus from the CNS in the absence of inflammation (P6073)
Authors: Lebrun Aurore , Kean Rhonda , Li Jianwei , Hooper Douglas
Published: January 2023

Little is known about CD4+ T cell entry into the central nervous system (CNS) in the absence of inflammation. Attenuated rabies viruses (RABV) are unique tools to study this process, as they spread from the site of inoculation to the CNS trans-axonally, without compromising the blood-brain barrier. Previous studies with live-attenuated RABV showed that CD4+ T cell entry into the CNS is associated with an increase in IFN-g mRNA in the brain and the production of IgG2a antibodies in the periphery, indicating a bias towards TH1 immunity. To further investigate the nature of the CD4+ T cells involved in the CNS response to RABV, we performed a temporal comparison of development of antiviral immunity in C57Bl6 and T-Bet knock-out (T-Bet-/-) mice. In both mouse strains, we observed that virus spread through the CNS is associated with elevated IFN-g mRNA, but that immune cell infiltration and antibody production in CNS tissues differs significantly. Immune cell infiltration into the CNS is significantly lower in T-Bet-/- mice at day 8 post-infection. Moreover, despite the fact that immunized T-Bet-/- mice produce a substantial amount of neutralizing antibody in the periphery and are fully protected against i.m. challenge with pathogenic RABV, they are unable to survive an i.c. challenge with the same virus. Together, these findings suggest that TH1 cells play a critical role in cell infiltration into the CNS and the clearance of RABV despite the absence of inflammatory pathology.
Virus-specific CD4 and CD8 T cell responses in the absence of Th1-associated transcription factors (P6052)
Authors: Harrington Laurie , Mollo Sarah , Ingram Jennifer , Kress Robert , Zajac Allan
Published: January 2023

The eradication of various types of pathogens is mediated by the coordinated efforts of both effector CD4 and CD8 T cells. Following pathogen control, the majority of CD4 and CD8 T cell effector populations undergo apoptosis, but a subset of these cells persists, giving rise to the memory T cell pool. The requirements for the generation of T cell memory have not been completely elucidated, hence we were interested in understanding importance of the Th1 transcription factors Tbet and STAT4 in this process. To interrogate this, we utilized lymphocytic choriomeningitis virus infection of BALB/c mice. Not surprisingly, we observed that in the absence of Tbet, the virus-specific CD4 T cell IFNγ response was ablated but the IL-2 response remained unperturbed. In contrast, we found that STAT4 was not required for IFNγ production by virus-specific CD4 T cells. Conversely, the functional virus-specific CD8 T cell response was intact in the absence of Tbet, STAT4, or both, although increases in the proportion of effector cells competent for IL-2 production were noted in mice that lacked Tbet expression. We did detect marked differences in the expression of CD127 and KLRG1 on virus-specific effector CD8 T cells, however this did not translate into alterations in the numbers of memory CD8 T cells after the resolution of the infection. Collectively, we discovered distinct roles for Tbet and STAT4 in shaping the phenotype and function of virus-specific T cell responses.
Calmodulin boosts Itk activity to induce sustained calcium responses in T cells (P1117)
Authors: Huang Yina , Wang Xinxin , Boyken Scott , Hu Jiancheng , Xu Xiaolu , Suleiman Hani , Shea Madeline , Shaw Andrey , Andreotti Amy
Published: January 2023

The Tec family tyrosine kinase Itk is an important transducer of T cell activating signals. Itk regulates the levels of two second messengers Ca2+ and diacylglycerol, which are required for initiating transcriptional programs important for T cell priming and differentiation. How T cells translate intracellular signaling cascades into distinct functional outcomes remains a fundamentally important question. We hypothesize that distinct mechanisms of Itk activation lead to Ca2+ patterns that result in divergent T cell responses. Itk activation requires recruitment via its Pleckstrin homology (PH) domain to its membrane ligand PIP3, which is generated by PI-3 kinase. Here, we report a role for Calmodulin (CaM) in sustaining Itk activity and Ca2+ responses. CaM is a downstream Ca2+ sensor that controls numerous cellular enzymes, including Calcineurin. We have identified a novel interaction between the Itk PH domain and CaM. NMR analyses revealed that CaM binds adjacent to the PIP3 binding pocket, leading us to hypothesize that CaM regulates PIP3 binding. CaM inhibition or genetic mutation of the CaM binding site resulted in decreased PIP3 association and Itk activation, resulting in less sustained Ca2+ responses. We propose a model in which initial Itk activation results in low Ca2+ increases that feedback to boost membrane recruitment of Itk via CaM. We speculate that T cell responses requiring sustained Ca2+ signals, such as T cell differentiation require this feedback loop.