bluuebunny/crossref_metadata_2025 huggingface dataset:
Pseudomonas aeruginosa infection after burn injury polarizes innate immune cells towards an alternatively activated phenotype (55.19)
Authors: Neely Crystal , Kartchner Laurel , Mendoza April , Cairns Bruce , Maile Robert
Published: January 2023

Severe burn injury causes profound immunosuppression resulting in prevalent bacterial infections. We hypothesized that bacterial infection after burn injury polarizes macrophage (Mo) and neutrophil (No) responses into an alternatively activated (M2/N2) phenotype. To test this, we used a murine model of burn injury and bacterial challenge. Female C57BL/6 mice underwent either a 20% TBSA full thickness contact burn or control sham procedure. Twenty-four hours later mice were either given a subcutaneous injection of Pseudomonas aeruginosa or control saline and sacrificed two days later to determine bacterial burden in skin, liver and lung. Serum cytokine levels were measured by cytometric bead array. Splenocytes were cultured in the presence of Brefeldin-A for intracellular cytokine analysis, and Mo and No were identified by surface staining. Burn mice had bacterial dissemination indicated by positive cultures from all tissue samples. No bacteria were recovered from sham mice. Burn injury, but not infection, induced a significant increase in serum IFN-γ and TNF-α. IL-6 was also significantly increased after burn injury alone but was significantly higher in infected burn mice. IL-10 was elevated in infected burn mice only. Flow cytometry confirmed expression of IL-10 protein within Mo and No. These data suggest that after burn injury, bacterial infection polarizes serum cytokine and innate cell phenotype into an alternatively activated phenotype allowing systemic infection.
Fibrosis and expression of mast cells peptidase: chymase and tryptase in oral paracoccidioidomycosis lesions (55.25)
Authors: Rodrigues Denise , Araujo Marcelo , Alves Polyanna , Pereira Sanivia , Lima Lilian , Silva Marcelo , Rodrigues Virmondes
Published: January 2023

Mast cells belong to the innate immune system, which provides the first line of defense against tissue damage. Tryptases and chymases are mast cells peptidases that promote inflammation, matrix destruction, and tissue remodeling by several mechanisms. They also modulate immune responses by hydrolyzing chemokines and cytokines. Oral lesions in Paracoccidioidomycosis(PCM), microscopically shows granulomatous inflammatory reaction with giant cells, macrophages, lymphocytes, plasma cells and mast cells. Mast cells were seen in PCM, but their participation and interaction with other cells remains to be determined. This study evaluated in situ expression of mast cell peptidase and fibrosis on oral lesions of PCM patients. Fifteen oral biopsies from patients with clinical and histopathological diagnosis of oral PCM and thirteen biospies from normal oral mucous (control group) were analyzed. Fibrosis was significantly higher in disorganized granuloma when compared to controls. Indirect immunohistochemistry was used to evaluate the expression of peptidase chymase and tryptase. The number of immunostained cells for chymase and tryptase was significantly higher in control group, when compared to the Pmycosis patients (p<0.025). The down modulation of chymase and tryptase expression and the increase fibrosis in PCM oral lesion may be a consequence of inflammatory process induced by the presence of fungus and also are potentially involved in the regulation of immune response.
Ageing and expression of TLR pathway associated genes in macrophages to Porphyromonas gingivalis challenge. (55.26)
Authors: Shaik-Dasthagirisaheb Yazdani , Shen Steve , Genco Caroline , Gibson III Frank
Published: January 2023

Background: Periodontal disease is a chronic inflammatory disease that leads to progressive loss of soft and hard tissues supporting the teeth. Porphyromonas gingivalis (Pg) is a bacterium closely associated with periodontal disease. Toll-like receptors (TLR) and their intracellular signaling pathways play roles in host response to Pg. The focus of the current study was to define the expression profile of TLR pathway associated genes in macrophages (MØ) cultured with Pg, and to define changes in expression of these genes as a consequence of ageing. Methods: Bone marrow MØ from 2 months and1 year old wild type (WT), TLR2 knockout (KO), TLR4KO, MyD88KO and LPS2 mice were cultured with Pg. RNA was harvested from unchallenged and Pg challenged cells and expression of TLR pathway associated genes was measured by qPCR microarrays. Results: Pg challenge of WT MØ induced expression of a large set of TLR pathway associated genes. MØ gene expression data revealed 19, 48, 45 and 56 genes differentially regulated in TLR2KO, TLR4KO, MyD88KO and LPS2 mice respectively as compared with WT in response to Pg. We identified 19 genes that are commonly regulated in all four mutant genotypes. Age had little effect on the differential regulation of these genes by mutant MØ as compared with WT. Conclusions: Regulation of TLR pathway associated genes by MØ was highly responsive to Pg challenge. Pg challenge and mouse genotype had the strongest effects on gene expression in this model.
Selective Activation of Antigen-Specific T Cells by Anti-CD3 Constraining Nano-particles (58.15)
Authors: Lo Ying-Chun , Edidin Michael , Powell Jonathan
Published: January 2023

Upon antigen (Ag) recognition, TCRs form nanoscale clusters on the T cell surface. We hypothesized that the spatial differences of surface TCR between naïve and previously activated cells might be exploited by using anti-CD3 coated quantum dots (Qdots) to selectively activate Ag-specific cells. Rag-/- Pigeon Cytochrome c (PCC) specific CD4+ T cells were activated with APC +/- PCC with or without anti-CD3 Qdots. Anti-CD3 Qdots markedly enhanced proliferation of T cells incubated with low dose PCC but did not drive proliferation of T cells incubated with APC alone, though such cells proliferated to soluble anti-CD3. Additionally, anti-CD3 Qdots increased cytokine production of such cells in TH1 and TH2 cultures. We also explored the effect of anti-CD3 Qdots on heterogeneous cultures containing Rag+/+ hemagglutinin peptide (HA) specific 6.5+ CD4+ T cells and bystander 6.5- CD4+ T cells. Soluble anti-CD3 resulted in proliferation of both 6.5+ and 6.5- T cells. Cultures including low dose HA induced only 6.5+ T cells to proliferate, and addition of anti-CD3 Qdots further selectively enhanced proliferation of 6.5+ T cells but not 6.5- T cells in the same culture. This effect was again seen using CD8+ TCR transgenic T cells specific for class I HA. In vivo, anti-CD3 Qdots also increased the recovery of 6.5+ cells in a 6.5 adoptive transfer and HA vaccination model. Thus, constraining anti-CD3 on a nanoparticle confers an Ag-specific “nano boost” to T cell proliferation and function.
Regulating immune responses to biologics: Epitope prediction and applications (58.23)
Authors: Cousens Leslie , Moise Lenny , Ardito Matthew , McClaine Elizabeth , Terry Frances , Martin William , De Groot Anne
Published: January 2023

Immune responses directed against protein therapeutics can directly impact drug safety and efficacy. The ability to predict and reduce immunogenicity of a potential protein therapeutic presents tremendous benefits at every stage of drug development. While many factors contribute to protein immunogenicity, T cell-dependent responses play a critical role. We report two approaches to mitigate therapeutic protein immunogenicity, one involving epitope modification and the other inducing antigen-specific tolerance induction. Rational epitope modification applied to either Factor VIII or botulinum toxin demonstrates reduced immunogenicity when following a systematic process of in silico epitope mapping, in vitro and in vivo validation of computational predictions, iterative in silico modification of immunogenic epitopes and experimental validation of carefully selected modified sequences. Tolerance induction involves co-delivery, chemical or recombinant linkage of regulatory T cell epitopes (Tregitopes) to therapeutic proteins. We have demonstrated that Tregitope incorporation leads to lower immune responses against target epitopes identified in insulin, Factor VIII and botulinum toxin. The ability to analyze, predict, and modify the immunogenicity of a protein expands the available strategies for mitigating therapeutic protein immunogenicity, enables quality by design for drugs in development and improvement of licensed biologics to create biobetters.
Antigen-induced release and retroviral subversion of TCR-enriched microvesicles at the CD4+ T cell immunological synapse (58.5)
Authors: Choudhuri Kaushik , Llodra Jaime , Kam Lance , Stokes David , Dustin Micheal
Published: January 2023

The immunological synapse (IS) is a specialized junction that forms between T cells and antigen presenting cells, which is critical for effective T cell antigen recognition and effector functions. Its spatiotemporal evolution results in laterally segregated supramolecular domains composed of a peripheral adhesive ring of the β2 integrin LFA-1 and its ligand ICAM-1, and centrally accumulated T cell receptors (TCR). The molecular and cell biological basis for supramolecular domain organization at the IS remains unclear. Here we show, using novel correlative optical-electron microscopy methods in conjunction with electron tomography, that centrally accumulated TCR is located on the surface of microvesicles that are shed at the IS center. The ESCRT I member TSG101 sorts TCR into microvesicles. TCR-enriched microvesicles (TEMs) are transferred from T cell to B cell during cognate antigen recognition, and B cells initiate intracellular signals in response to TEMs, suggesting their involvement in intercellular communication between immune cells. Expression of the HIV GAG structural polyprotein in human CD4+ T cells, displaces TCR from microvesicles at the IS, resulting in the release of GAG-containing virus-like particles. These findings provide an ultrastructural basis for supramolecular domain organization at the IS, and identify an ESCRT-dependent mechanism for production of stimulatory TEMs, which may be co-opted during infection for the release of enveloped viruses.
A novel monoclonal antibody against the human IL-17AF heterodimer for flow cytometry (59.11)
Authors: Schifano Matthew , Araji Soha , Li Shourong , Ruiz Natalie , Schrantz Nicolas , Huang Qiulong , Just Peggy
Published: January 2023

The IL-17 family members, IL-17A and IL-17F, are signature Th17 cytokines. Although they were both originally found to be produced as disulfide-linked homodimers, recent studies have confirmed the production of IL-17AF heterodimers in in vitro-differentiated and polarized Th17 cells. While ELISA-based assays have become available for the study of these heterodimers, flow antibodies have been generated only to the monomeric subunits, making discrimination between the homo- and heterodimer complexes difficult. Here, for the first time, we use a novel monoclonal antibody to identify producers of the IL-17AF heterodimer by flow cytometry. Under our polarization conditions, the majority of human lymphocytes that stain for both IL-17A and IL-17F also stain for the IL-17AF heterodimer. These results suggest that this population produces mostly IL-17AF heterodimers and not the IL-17A and IL-17F homodimers alone. This novel reagent will help expand our knowledge of the IL-17/Th17 axis and contribute to the elucidation of its role in the immune response.
Adaptive immune response in pulmonary hypertension (59.13)
Authors: Grunig Gabriele , Chen Wen-Chi , Hoffman Carol , Gordon Terry , Park Sung-Hyun
Published: January 2023

Immune responses have long been associated with pulmonary arterial hypertension. Previous studies in our lab have shown that a prolonged T helper 2 (Th2) response to inhaled antigen induces severe pulmonary arterial remodeling and that this is exacerbated by urban particulate matter (PM) from air pollution. The present study was designed to identify the role of the adaptive immune response (B cells, IL-13, IL-17) in pulmonary hypertension induced by antigen and urban PM. The respirable fraction of airborne PM (PM2.5) was collected in New York City. Th2 primed mice were challenged with soluble antigen (Ovalbumin) combined with urban PM2.5 intranasally. We determined pulmonary arterial remodeling by histology, right heart hypertrophy by ventricular weight measurements and right ventricular systolic pressures recorded in anaesthetized, spontaneously breathing mice. In contrast to wild type, B cell KO mice had no significantly increased right heart weights, or right heart systolic pressures in response to intranasal antigen and urban PM2.5. Reconstitution with anti-antigen antibody restored the development of pulmonary hypertension in these mice. Combined blockade of IL-13 and IL-17 significantly ameliorated pulmonary hypertension induced in wild type mice exposed to antigen and urban PM2.5. Our studies indicate that antigen-specific antibody, IL-13 and IL-17 are necessary for the development of pulmonary hypertension induced by the exposure to antigen and urban PM2.5.
Dysregulated production of IL-17 by γδ T cells in the absence of interleukin-15 receptor α (59.2)
Authors: Colpitts Sara , Pham Quynh-Mai , Lefrançois Leo
Published: January 2023

Interleukin (IL)-15 and the IL-15 receptor α-chain (IL-15Rα) are required for the homeostasis of certain subsets of γδ T cells. We sought to determine if IL-15 signaling was also required for the generation and maintenance of a unique subset of IL-17-producing (IL-17+) γδ T cells. Surprisingly, we found a significant increase in the percentage and total number of IL-17+ γδ T cells in IL-15Rα KO (RKO) mice, most notably in the spleen, mesenteric lymph node, liver, and thymus. By generating mixed chimeras from WT and RKO neonatal thymocytes, we determined that the effect was not cell-intrinsic since WT- and RKO-derived γδ T cells exhibited equivalent IL-17 production. Considering the dissemination of IL-17+ γδ T cells to peripheral tissues, we hypothesized that their migration could be altered in RKO mice. Using parabiosis, we found that IL-17+ γδ T cells did not equally distribute between joined WT mice, suggesting limited recirculation, but also observed unexpected recirculation of IL-17+ γδ T cells into the thymus. When comparing WT:WT versus RKO:RKO parabionts, there was a significant increase in the percentage of IL-17+ cells in the donor γδ T cell population of RKO pairs, suggesting increased recirculation in the IL-15Rα-deficient environment. These findings support the presence of a cell-extrinsic effect of IL-15Rα signaling in regulating the IL-17+ γδ T cell subset. Further studies will continue to dissect the mechanism(s) by which this regulation occurs.
IL-23 promotes autoimmune Th17 memory formation and function (59.6)
Authors: McGeachy Mandy , Haines Christopher , Chen Yi , Blumenschein Wendy , McClanahan Terri , Cua Daniel
Published: January 2023

IL-23 is essential for the differentiation of pathogenic effector Th17 cells, but little is known about its requirement in memory Th17 responses. To investigate autoimmune Th17 memory, we developed a variation of the experimental autoimmune encephalomyelitis model, in which CD4+ T cells from MOG(35-55)-immunized donors were transferred into naïve recipients and rested before re-challenge. Following secondary recall, memory Th17 cells rapidly expanded in lymph nodes before trafficking to the central nervous system in high numbers, resulting in earlier onset and increased severity of clinical signs. Th17 memory cells were generated from IL-17+ and RORγt+ precursors, and stability of the Th17 phenotype was dependent on time. IL-23 was required during the primary response for generation of Th17 memory cells During the recall response, IL-17 production was not directly impacted by neutralizing IL-23R at secondary challenge, but the generation of effectors that co- expressed T-bet was strongly impaired. In addition, IL-23R blockade resulted in decreased proliferation and reduced total numbers of Th17 memory cells in the secondary response.
B7x deficiency exacerbates renal injury in antibody mediated nephritis (60.7)
Authors: Pawar Rahul , Goilav Beatrice , Xia Yumin , Ghosh Kaya , Herlitz Leal , Zang Xingxing , Putterman Chaim
Published: January 2023

B7x (B7-H4, B7S1) is an important member of the B7/CD28 superfamily of T cell costimulatory proteins. B7x is a negative regulator of CD4+ and CD8+ T cell activation and proliferation, and is expressed in both hematopoietic and non-hematopoietic tissues. We hypothesized that B7x is instrumental in the pathogenesis of antibody mediated nephritis. To examine this question we induced nephritis in B7x-deficient (B7x-/-) (n=10) and WT B6 mice (n=10) by passive transfer (i.v.) of nephritogenic antibodies. Following induction of nephritis, we found a marked increase in the levels of serum IgG (p=0.023), IgG2b (p=0.055), and IgG1 (p=0.064) in B7x-/- mice compared to WT mice. Moreover, renal damage was more severe in B7x deficient mice. Serum creatinine and BUN were elevated in the B7x-/- group compared to the WT mice. Furthermore, B7x-/- mice demonstrated worse kidney histopathology, with an increase in infiltrating PMN cells in glomeruli (p<0.0001) and interstitium (p=0.004), crescent formation, and tubular atrophy/dilatation. Immunohistochemical studies revealed increased glomerular infiltration of CD3+ T cells (p=0.034) and CD68+ macrophages (p=0.003) in B7x-/- mice kidneys. At the mRNA level, IL-23 was significantly upregulated in kidneys of B7x-/- mice. Thus, B7x plays a protective role in the pathogenesis of antibody mediated nephritis. The relative contributions of systemic versus local immune effects of B7x in nephrotoxic nephritis are currently being investigated.
Low frequency of iNKT cells in Type 1 Diabetes Patients and their first degree relatives: A Mexican Cohort (60.9)
Authors: Beristain-Covarrubias Nonantzin , Canché-Pool Elsy , Vaal-Aguilar Mayra , Gómez-Díaz Rita , Ortiz-Navarrete Vianney
Published: January 2023

Type 1 Diabetes (T1D) is a chronic, autoimmune disease, characterized by specific destruction of the insulin producing pancreatic β cells. This destruction is mediated by auto-reactive T cells. Lymphocytes NKT play an important role in type 1 diabetes (T1D) pathogenesis. Data from the NOD mouse shows a numerically and functionally deficient NKT cell, and those produce subnormal levels of IL-4 and IFNγ when stimulated. Similar defects have been suggested in humans, although they are not conclusive, some studies predict a role for NKT cells in T1D development. Class I Restricted T Cell Associated Molecule (CRTAM) is expressed at the cell surface of activated human invariant NKT cells (iNKT), CD8 and CD4 T cells. Recently we analyzed a cohort of 42 T1D patients, 103 relatives (mother, father and sibling), and 40 healthy controls. We found a significant low percentage and absolute number for iNKT in T1D patients, and identified two cell populations among the iNKT cells (iNKThi and iNKTlo). High levels of Va24/Vb11 (TCR) were observed for the iNKThi whereas the iNKTlo expressed low levels of TCR and it was CRTAM positive.
Expression pattern of agonist antigen influences timing of thymic selection (63.5)
Authors: Ju Ji Min , Jeon Ji Yeong
Published: January 2023

MHC complexes with peptides from minor histocompatibility (H) antigens which are naturally occurring polymorphic proteins are recognized by T cells. The mismatches at the Minor H loci induce allo-reactive T cell activation, leading to graft-rejection, graft-versus-host disease, or graft-versus-leukemia under MHC-matched transplantation circumstances. T cell response specific for H60 has been characterized as a dominant. In order to investigate the effect of antigenic expression pattern on the outcome of bone marrow transplantation, we generated a TCR transgenic mouse (named J15), of which specificity is H60 peptide in complexes with H2-Kb, with TCR α and β genes were originated a H60-specific CTL clone. The thymocytes in the J15 mice mostly developed to CD8 T lineage and showed proliferation in response to H60-stimulation. The thymocytes bearing J15 TCR were deleted in the mice where H60 are expressed in hematopoietic cell-restricted pattern and ubiquitously, that is in the F1 mice after crossing of J15 to H60 congenic (J15TgH60C/-) or to H60 transgenic mouse (J15TgAct-H60Tg), respectively. But the deletion timing was different during the double negative stages between the two F1 mice. The different deletion timing was observed when the bone marrow chimeras were generated with H60 congenic mice and with Act-H60Tg mice as hosts into which bone marrow from the J15 were transferred. These results demonstrate that antigen expression pattern influences the thymic selection timing.
ShcA and c-Abl provide a new signaling axis regulating beta-selection (63.8)
Authors: Trampont Paul , Zhang Li , Giles Amber , Walk Scott , Pendergast Ann Marie , Ravichandran Kodi
Published: January 2023

Progression of immature CD4-CD8- thymocytes through β selection checkpoint is determined by signals from the pre-T cell receptor (pre-TCR) along with signals from Notch. Knockout of the adapter protein ShcA, or transgenic expression of a phosphorylation-defective mutant of ShcA (ShcFFF) leads to a severe block at β selection. Our studies to mechanistically understand the ShcA function showed that ShcA acts upstream of ERK, and the transcription factors Egr-1 and Egr-3 during β selection; however, phospho-ShcA also appeared to be linked to other signaling pathways. We performed a yeast three-hybrid screen, using as bait ShcA that was phosphorylated by Lck. This screen identified binding of c-Abl (via its SH2 domain) to phosphorylated ShcA. During pre TCR dependent signaling, phosphorylation of ShcA was required for optimal c-Abl phosphorylation. Using mice deficient either in c-Abl and/or its homologue Arg, we show that ShcA and c-Abl regulate DN thymocyte proliferation, and the proper localization of DN thymocytes near the thymic cortex. Ectopic expression of a constitutive active c-Abl allowed thymocytes to resume proliferation, differentiation, and CXCR4 dependent migration to SDF-1α. Collectively, these data reveal a previously unappreciated ShcA::c-Abl signaling axis that appears to critically influence thymic β selection.
The role of CD4+CD40+ cells in EAE mice. (65.1)
Authors: Carter Jessica , Wagner David
Published: January 2023

Auto-aggressive T cells are responsible for the onset and perpetuation of autoimmune diseases, including Type 1 Diabetes (T1D) and Multiple Sclerosis (MS). A subset of CD4 T cells, CD4+CD40+ cells, have an expansive role in the Non-Obese Diabetic (NOD) mouse model of T1D. Not only do CD4+CD40+ cells increase in number with the onset of disease, but they are necessary and sufficient in transferring T1D from NOD to NOD.scid mice. Because CD4+CD40+ cells have an imperative role in the development of disease in T1D, we considered that these cells might also be involved in Experimental Autoimmune Encephalomyelitis (EAE), a neurological disease which mimics human MS. When given myelin oligodendrocyte glycoprotein (MOG) and pertussis toxin (PT), C57Bl/6 mice develop neurological symptoms and impaired motor functions similar to those in human MS patients. Data shows an increase, not only in CD4 T cells, but also in CD4+CD40+ cells in mice receiving induction of EAE. This does not occur in their control counterparts. Further, purified splenic CD4+CD40+ cells from asymptomatic disease induced C57Bl/6 mice rapidly transfer EAE to C57Bl/6.scid recipients with only 1.2x106 cells, demonstrating that CD4+CD40+ cells alone can induce EAE. Also, we show cerebral infiltrates of immune cells in localized regions of the brain of the CD4+CD40+ recipient mice, suggesting that these lesions are similar to the lesions found in human MS patients.
Induced-tolerogenic Dendritic Cells promote tolerance and de novo differentiation of regulatory T cells (65.6)
Authors: Maldonado Roberto , Perdomo L. Carolina , Vascotto Fulvia , Bauquet Aurelie , Sharpe Arlene , Kuchroo Vijay , von Andrian Ulrich
Published: January 2023

Tolerance towards specific antigens is achieved by elimination of reactive T cells and/or their differentiation into regulatory T cells (Tregs). We screened several cell culture conditions known to confer potent tolerogenic capacity on Dendritic Cells (DC) in an attempt to obtain antigen-presenting cells that eliminate naïve CD4+ T cells (Tn) or induce Foxp3 expression, the master regulator of Treg differentiation and function. We identified Rapamycin and TGFβ as key factors that imprint DCs with strong tolerogenic function. TCR stimulation of Tn by these induced-tolerogenic DC (itDC) results in the selective reduction of antigen specific cells and the upregulation of Foxp3 expression in the remaining cells. The resulting adaptive regulatory T cells (aTregs) express high levels of CD25, GITR, CD152, CD62L and suppress T cell responses in vitro. Interestingly, aTreg differentiation induced by itDC was cell contact-dependent and occurred in absence of IL-10, TGFβ, IDO1 and FasL. In vivo injection of itDCs at the first clinical signs of MOG-induced Experimental Autoimmune Encephalomyelitis (EAE, Day 12) completely blocks the progress of the disease. Finally, monocyte-derived human itDC also induced sustained Foxp3 expression in human T cells that display suppressive function. These results suggest that itDCs may be a valuable tool to induce antigen-specific tolerance in vivo and represent a potential novel therapy for autoimmune disorders.
Neutrophils Suppresses Protective Immunity and Mediates Immunopathology in Mice during Fatal Bacterial Infection-Induced Toxic Shock (67.1)
Authors: Ismail Nahed , Ismaeel Nourhan , Yang Qin , Al-Hendy Omar , Ghose Purnima
Published: January 2023

Ehrlichia chaffeensis is an obligate intracellular bacteria that causes systemic infection and fatal toxic shock-like syndrome in immunocompetent patients. The balance between protective immunity mediated by CD4+Th1 cells, and immunopathology, which is mediated by NK and CD8+T cells, determines the outcome of Ehrlichia infection in mice. Early recruitment of neutrophils to the sites of infection is critical for the control of bacterial infection. However, the contribution of neutrophils to protective immunity or immunopathology during Ehrlichia infection remains undefined. Compared to innocuous ehrlichial infection, lethal high-dose Ehrlichia infection of wild type mice is associated with a substantial expansion of activated neutrophils at the main sites of infection and a substantial TLR2 signaling. Surprisingly, depletion of neutrophils from lethally-infected mice enhanced bacterial elimination, decreased immune -mediated pathology, and prolonged survival. Furthermore, neutrophil depletion in infected mice resulted in amelioration of pathogenic responses as evidenced by abrogation of cytokine and chemokine storm and decreased frequency of pathogenic TNF-alpha- producing CD8+T cells compared to lethally infected isotype controls. These results revealed an unexpected role of neutrophils in suppression of protective immunity and mediating immunopathology during infection with intracellular bacteria.
Serum amyloid A and apolipoprotein E alter the host innate immune response elicited by malarial hemozoin (68.14)
Authors: Kassa Fikregabrail , Shio Marina , Olivier Martin
Published: January 2023

Malaria is one of the most important infectious diseases worldwide and still remains to be the major cause of death in tropical countries. The intraerythrocytic stage of the malaria parasite results in the production and release of hemozoin (HZ). Recently we have identified that HZ specifically interacts with serum proteins such as serum amyloid A (SAA) and apolipoprotein E (ApoE). In our current work, we were interested to determine the impact of these proteins on the macrophage’s innate immune response triggered by HZ. Since HZ induces IL-1β production via the NLRP3 inflammasome, we looked at the production of IL-1β using PMA-differentiated THP-1 cells. IL-1β, along with TNF-α, is considered to be the major contributor for malaria pathology. Our results show that SAA bound to HZ induces the production of IL-1β in a dose dependent manner. Furthermore, HZ-SAA and hemozoin-ApoE complexes phosphorylate the MAPKs specifically ERK1/2 and JNK. This is in agreement with the observation that SAA activates downstream signaling pathways via LYN resulting in the activation of ERK1/2 and JNK. Apart from IL-1β production, HZ bound serum proteins also modulate ROS production and phagocytosis in THP-1 cells. Overall, our results show that serum inflammatory proteins adhering on HZ modify its capacity to stimulate phagocytosis, IL-1β and ROS productions by THP-1 cells upon stimulation, suggesting that such cooperation could greatly influence the development of malaria-related pathologies.
The oligoadenylate synthetase-like protein enhances cellular antiviral activity through RIG-I pathway (68.3)
Authors: Sarkar Saumendra , Zhang Yugen , Zhu Jianzhong , Forero Adriana , Ganapathiraju Madhavi , Hartmann Rune , Coyne Carolyn
Published: January 2023

Oligoadenylate Synthetases (OAS) are interferon (IFN) inducible enzymes, which contribute to the host anti-viral activity by inducing RNA degradation via classical OAS-RNaseL pathway. However, the OAS-like protein (OASL), which is strongly induced following virus infection, does not have enzymatic activity to synthesize 2’-5’ oligoadenylates. Therefore, the mechanism of the anti-viral activity of OASL has remained elusive. Here we describe the basis of anti-viral activity of OASL via its ability to modulate type I IFN induction through RIG-I pathway. OASL expressing cells show a strong anti-viral activity against a number of RNA and DNA virus infections in a RIG-I dependent manner. Expression of OASL enhances RIG-I mediated IFN induction. We show that OASL interacts with cytoplasmic viral RNA sensor RIG-I and enhance its activity through its C-terminal ubiquitin like domain. Poly ubiquitin binding to RIG-I is necessary and essential for RIG-I mediated induction of IFN. We demonstrate that following induction of OASL protein it provides the necessary poly Ub binding function of RIG-I. Furthermore, inactive RIG-I mutants, which are defective in ubiquitination and poly Ub binding can be rescued in presence of OASL. We show that OASL specifically enhances RIG-I mediated IFN induction without affecting MDA5, STING activation. Our findings suggest a novel mechanism by which OASL contributes to host anti-viral response.
Characterization of Effector Memory CD8+ T cells in the Synovial fluid of Rheumatoid Arthritis (69.1)
Authors: Kim Hang-Rae , Cho Bon-A , Sim Ji Hyun , Kim Hye Won , Yoo Wan-Hee , Kang Insoo , Lee Eung Bong
Published: January 2023

Little is known about the cellular characteristics of CD8+ T cells in rheumatoid arthritis (RA). We addressed this by investigating whether the frequency of the CD8+ T cell subsets and their phenotypic characteristics are altered in the peripheral blood and synovial fluid (SF) from patients with RA. In this study, CD45RA- effector memory (EM) CD8+ T cells were increased significantly in the SF, but not in the peripheral blood from RA patients, compared with healthy controls. This was accompanied by increased levels of IL-15. The synovial EM CD8+ T cells were activated phenotypes with high levels of CD80, CD86, and PD-1, and had a proliferating signature in vivo upon Ki-67 staining, whereas the Fas+ cells were prone to apoptosis. In addition, EM CD8+ T cells in the SF were less cytotoxic, as they expressed less perforin and granzyme B. In particular, the proportions of synovial fluid mononuclear cells that were CCR4+CD8+ T cells and IL-4-producing CD8+ T cells were significantly higher than those in peripheral blood mononuclear cells of patients with RA and healthy controls. In addition, the number of IL-10-producing CD8+ suppressor cells increased significantly in the SF of RA patients. Especially, CD8+ T cells were inversely correlated with disease activity. These findings strongly suggest that EM CD8+ T cells in the SF are increased, likely because of inflammation, and they may be involved in modulating inflammation, thereby affecting the development and progression of RA.
Proliferative dysfunction in chronically activated T lymphocytes from chagasic patients (70.1)
Authors: Giraldo Nicolas , Bolaños Natalia , Cuellar Adriana , Roa Nubia , Cucunubá Zulma , Velasco Victor , Rosas Fernando , Puerta Concepción , González John
Published: January 2023

Background: Trypanosoma cruzi persistence has been associated with cardiac and gastrointestinal tissue damage in nearly 30% of the infected individuals; however, the pathogenic mechanisms are yet unknown. This study’s goal was to compare the activation status and proliferative capacity of T lymphocytes among chronic chagasic patients and uninfected controls. Methodology: Twenty-seven chronic chagasic patients, 20 healthy individuals and 28 non-chagasic cardiomyopathy donors were analyzed. Peripheral blood cells were stained with CD3, CD4, CD8, CD28, HLA-DR and CD38. PBMCs were labeled with CFSE and co-cultivated with PHA or T. cruzi lysate; at the fifth day post-stimulation cells were stained for CD3, CD4 and CD8. Results: Chagasic patients displayed higher frequencies of CD4+ (P=0.0001) and CD8+ (P=0.0002) T cells co-expressing HLA-DR and CD38 in comparison with healthy and non-chagasic cardiomyopathy donors. Also, the former group exhibited lower percentages of CD8+/CD28+ T lymphocytes (P=0.0005). After 5 days of stimulation, the proliferation index was lower in both CD4+ (P=0.01) and CD8+ (P=0.04) PHA-stimulated T cells from chagasic donors when compared with both control groups. Conclusions: Despite their increased activation status, the T lymphocytes from chagasic donors displayed reduced proliferative capacity after mitogenic stimulation, corroborating a dysfunctional cellular immune response in the chronic stages of the disease.
The impact of Staphylococcus aureus infection on human B cell responses (70.5)
Authors: Pauli Noel , DeDent Andrea , Huang Min , Henry Dunand Carole , Kim Hwan , Zheng Naiying , Andrews Sarah , Frank Karen , Schneewind Olaf , Wilson Patrick
Published: January 2023

Staphylococcus aureus, S. aureus, is a growing concern in medicine due to the rise of antibiotic resistant strains, such as Methicillin Resistant Staphylococcus aureus (MRSA). Infection and clearance of this bacterium does not lead to immunity to future infections in humans. S. aureus utilizes a variety of virulence factors in order to evade and actively suppress an effective immune response; one of particular importance to humoral immunity is Staphylococcus Protein A (SpA). SpA has the ability to act as a B cell superantigen and anti-opsonin. We address the effects of S. aureus infection on human B cell populations and how the bacterium is able to prevent long lasting, protective immunity; analyzing the effect of infection in humans at the resolution of individual B cells. Focusing on human subjects that have experienced infections, we report on our initial efforts to characterize the kinetics and magnitude of B cell activation by flow cytometry and ELISPOT analysis. Further, we show the ability to derive S. aureus specific monoclonal antibodies (mAbs) from individually sorted B cells in the context of human infection; analyzing the responding B cell BCR repertoire to better understand the human response to this pathogen and how it prevents the generation of useful immunity. It is hoped that specific human monoclonal antibodies to S. aureus virulence factors could be used for therapeutic and prophylactic purposes, or identify epitopes to direct vaccine design.
Deficiency of Id3 (inhibitor of DNA binding 3) results in Inflammation in salivary glands and gamma-delta (gd) T cell lymphoma (71.11)
Authors: Kasagi Shimpei , Maruyama Takashi , Li Jun , Chen Wanjun
Published: January 2023

Id3-/- mice have been shown to develop a T cell-dependent autoimmune-like sjogren’s syndrome(SS). The dysregulated differentiation between Foxp3+ Tregs and Th17+ T cells in Id3-/- CD4+ T cells promoted us to study whether the autoimmune-like SS in Id3-/- mice was associated with the abnormal expression of Th17 cells in the target organs. We focused on the salivary glands and lachrymal glands, as they are main target tissues involving in SS. 50% of Id3-/- started to show proinflammatory CD4+ infiltrating cells in the salivary glands. Significantly higher frequency of Th1 and Th17 cells were observed in Id3-/- salivary glands, and higher frequency of Th17 and lower frequency of Tregs were observed in Id3-/- spleen. Notably, 26% mice Id3-/- mice developed gd T cell lymphoma. The gd T lymphoma cells were characterized by dysregulated IL-17 production and high expression of an oncogene, c-myc. Adoptive transfer of Id3-/- gd T lymphoma cells to Rag1-/- mice resulted in gd T lymphoma, suggesting that Id3 may regulates gd T cell tumor development.
Crosstalk between colonic epithelial cells and macrophages (71.7)
Authors: Kristek Maja , Rahman Arman , Loscher Christine
Published: January 2023

Macrophages are the most abundant mononuclear phagocytes in the intestine. Unlike other macrophage populations, intestinal macrophages seem to be in a state of partial activation showing no response to inflammatory stimuli, no production of pro-inflammatory cytokines and low expression levels of innate response receptors. Intestinal macrophages are thought to derive from circulating blood monocytes, which are fully responsive to inflammatory stimuli. Therefore it is probable that the intestinal environment itself is responsible for altering the phenotype of these macrophages upon arrival in the gut. In order to explore whether the intestinal environment can influence macrophage phenotype and function J774A.1 macrophages were conditioned with supernatants from the murine colonic epithelial cell line, CMT-93. Conditioning of J774 cells with CMT-93 media altered the expression levels of innate response receptors and cytokine production. Furthermore, conditioning with CMT-93 media modulated the macrophage response to Toll-like receptor stimuli. They acquired more of a regulatory phenotype with the down-regulation of CD80, CD40 and MHC class II expression and IL-12p40 production, along with increased production of TNF-α. This data implicates intestinal epithelial cells as one of the factors that can change and shape macrophage response, therefore it is important to investigate this crosstalk further in order to understand its role in the maintenance of intestinal homeostasis.
The Microbiota Limit CX3CR1+ Cell-Dependent Trafficking of a Pathogenic Bacterium to the Mesenteric Lymph Nodes. (71.9)
Authors: Diehl Gretchen , Longman Randy , Zhang Jing-Xin , Breart Beatrice , Schwab Susan , Littman Dan
Published: January 2023

The organization of the intestine makes regulating inflammation a unique challenge. The lumen of the intestine contains not only partially digested food, but also an enormous load of beneficial bacteria (up to 10^12 organisms/gram of contents). As commensal and pathogenic bacteria contain the same immunostimulatory molecules, it is unclear how the intestinal immune system can distinguish between the two. We hypothesized that recognition of commensals modulates intestinal immune responses to limit inflammation. To this end, we examined immune responses against non-invasive Salmonella Typhimurium in MyD88-deficient mice or wildtype mice treated with antibiotics to reduce their microbiota. We found that, in the presence of commensals, non-invasive Salmonella, a pathogen whose entry is limited, is unable to reach the mesenteric lymph nodes (MLN). However, in animals where the load of commensals has been reduced with antibiotics or in animals deficient for MyD88, non-invasive Salmonella trafficks to the MLN in a CCR7-dependent manner. This trafficking to the MLN results in the induction of IgA. Additionally, we found that CX3CR1+ mononuclear phagocytes, an intestinal cell population previously thought to be non-migratory, are responsible for this trafficking of non-invasive Salmonella. These findings indicate that recognition of commensals limits the amount of antigen reaching the MLN. This in turn limits induction of immune responses and serves to reduce intestinal inflammation.
Catenarin, an anthraquinone compound, inhibits CXCR4 and CCR5 pathways to protect against type 1 diabetes in NOD mice (72.1)
Authors: Yang Wen-Chin , Shen Ming-Yi , Jang Yu-Song , Chen Zeng-Weng , Chang Cicero
Published: January 2023

Inflammation contributes to leukocyte migration, termed insulitis, and β cell loss in type 1 diabetes (T1D). Naturally occurring anthraquinones are claimed as anti-inflammatory compounds; however, their actions are not clear. This study aimed to investigate the effect and mechanism of catenarin on the inflammatory disease, T1D. Catenarin and/or its anthraquinone analogs dose-dependently suppressed C-X-C chemokine receptor type 4 (CXCR4)- and C-C chemokine receptor type 5 (CCR5)-implicated chemotaxis in leukocytes. Catenarin, the most potent anthraquinone tested in the study, prevented T1D in non-obese diabetic mice. Mechanistic study showed that catenarin did not act on the expression of CCR5 and CXCR4. On the contrary, catenarin inhibited CCR5- and CXCR4-mediated chemotaxis via the reduction of the phosphorylation of mitogen-activated protein kinases (p38 and JNK) and their upstream kinases (MKK6 and MKK7) and calcium mobilization. Overall, this study demonstrates the preventive effect and molecular mechanism of action of catenarin on T1D, suggesting its novel use as a prophylactic agent in T1D.
CM3457, a potent and selective oral CRAC channel inhibitor, suppresses T and mast cell function and is efficacious in rat models of arthritis and asthma (72.3)
Authors: Ramos Stephanie , Grigoryev Sergey , Rogers Evan , Roos Jack , Whitten Jeffrey , Stauderman Kenneth , Velicelebi Gonul
Published: January 2023

Calcium-release activated calcium (CRAC) channel inhibitors represent a new class of oral immunomodulatory agents with a potential safety profile suitable for chronic dosing to treat autoimmune disorders. Calcium entry through CRAC channels is a critical step in the functional responses of T cells and mast cells. Patients with mutations in CRAC channels are known to have a form of severe combined immunodeficiency, wherein the adaptive immune response is suppressed without major impairment of other organ systems. CalciMedica has discovered several novel small molecules that selectively inhibit CRAC channels, and is currently developing the first of these inhibitors, CM2489, for the treatment of psoriasis. We describe here the properties of CM3457, another selective CRAC channel inhibitor that is structurally distinct from CM2489. CM3457 potently inhibits CRAC channels, Th1, Th2 and Th17-derived cytokine production, T cell proliferation, and mast cell degranulation. Once-daily oral dosing of CM3457 significantly inhibits inflammation and joint histopathology in a rat collagen-induced arthritis model. CM3457 also inhibits lung inflammation and eosinophilia, as well as improves lung function, in a rat OVA-induced asthma model. These data provide evidence that CM3457 may be an effective therapeutic in the treatment of autoimmune disorders and asthma.
Novel insights into the effects of spingosine-1-phosphate receptor modulators on monocytes (72.6)
Authors: Lewis Nuruddeen , Haxhinasto Sokol , Anderson Shawn , Fogal Steve , Stefanopoulos Dimitria , Brown Maryanne , Modis Louise
Published: January 2023

Sphingosine-1-phosphate (S1P) receptors are critical for lymphocyte egress from secondary lymphoid organs and S1P receptor modulators effectively suppress lymphocyte circulation. FTY720, a novel S1P1,3,4,5 modulator, is currently approved for the treatment of multiple sclerosis. Despite the current progress in elucidating the function of S1P receptors on lymphocytes, the role of S1P receptors on innate immune cells are incompletely understood. S1P receptors are widely expressed on cells of the innate immune system and have been demonstrated to modulate cell trafficking, activation, cytokine production, organization in secondary lymphoid tissues, maturation, and antigen presentation. We determined the effects of FTY720 and an S1P1,4,5 modulator on innate immune cells under normal and inflammatory conditions using experimental autoimmune encephalitis in mouse and rat as our model systems. We will present our findings on the impact of these two S1P receptor modulators on monocytes. This data contributes to the current understanding of S1P receptors in rodents and provides novel insight into additional functions of S1P receptor modulators.
Decreasing immunogenicity of ESC derived cardiomyocytes (73.1)
Authors: Karabekian Zara , Idrees Sana , Jamshidi Aria , Sarvazyan Narine
Published: January 2023

One of the major obstacles to repair damaged myocardium with embryonic stem cell derived cardiomyocytes (ESC-CM) is their rejection by non-syngeneic hosts. We aimed to reduce the immunogenicity of these cells by downregulation of MHC class I molecules (MHC-I) on their surface. Cardiomyocytes were derived from mouse ESC in which puromycin resistance gene is driven by a cardiac-specific promoter. They formed beating cardiomyocyte layers and expressed cardiac specific proteins. Interferon gamma treatment was used to mimic the effects of inflammatory stimuli on the expression of MHC-I. Transduction of ESC-CM with beta-2-microglobulin shRNA decreased expression of MHC-I in both interferon gamma-treated and untreated samples. When MHC-I downregulated ESC-CM were used as in vitro targets, activation of cognate T-lymphocytes was significantly reduced. Moreover, in vivo, MHC-I downregulated cells exhibited improved survival when injected i.p. into allogeneic, but not to syngeneic mice. To confirm that this strategy can be used for different cell types, the results were verified using W19 fibroblasts. In the past, ESC-CM have been shown to improve ischemic myocardium in syngeneic or immunocompromised animals. Inhibition of MHC-I via shRNA offers a simple approach to minimize the immunogenicity of these cells for use in allo- or xenogeneic recipients. Extension of this methodology to ESC of larger mammals can provide a source of less immunogenic cardiomyocytes for human heart repair.
Increased anti-melanoma efficacy of hu14.18K322A by combining with αCD40 +CpG (74.1)
Authors: Alderson Kory , Luangrath Mitchell , Elsenheimer Megan , Gillies Stephen , Navid Fariba , Rakhmilevich Alexander , Sondel Paul
Published: January 2023

Targeted monoclonal antibodies (mAb) can be used therapeutically for tumors with identifiable antigens such as disialoganglioside GD2, expressed on neuroblastoma and melanoma. αGD2 mAbs have been clinically successful in patients with neuroblastoma. An important mechanism of mAb therapy is Antibody Dependent Cellular Cytotoxicity (ADCC). Combinatorial therapeutic strategies can dramatically increase the anti-tumor response elicited by mAbs. We combined a novel αGD2 mAb, hu14.18K322A, with an immunostimulatory regimen of agonist CD40 mAb and class B CpG-ODN 1826. This combination immunotherapy produced a synergistic anti-tumor response in a syngeneic model of melanoma with minimal tumor burden, resulting in long-term cure of mice. NK depletion in B6 mice showed NK cells were required for the anti-tumor effect. Anti-tumor responses were also observed in tumor-bearing SCID/beige mice; thus NK cell cytotoxicity was not essential. Interrogation of the myeloid population in the peritoneal cavity showed an increase in neutrophils and monocytes with high Fc receptor expression after treatment with αCD40 +CpG. Furthermore, plastic adherent peritoneal cells inhibited tumor cells in vitro in an antibody-dependent manner. These data highlight the importance of myeloid cells as potential effectors in immunotherapy regimens utilizing tumor-specific mAb and suggest further studies are needed to investigate the therapeutic potential of activated myeloid cells.