bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential expression of two pathways of chemokine induction protect CD14- and TLR4-deficient mice from localized E. coli infections (94.16)
Authors: Goyert Sanna , Metkar Shalaka , Kim Kwang , Silver Jack
Published: January 2023

Sepsis, characterized by a systemic bacterial infection, is a life-threatening condition that can lead to multi-organ failure and death from septic shock. We have observed that CD14- and TLR4-deficient mice are resistant to death by sepsis from several different clinical isolates of E. coli injected intra-peritoneally. This resistance has been attributed to the 10-fold lower levels of the proinflammatory cytokines TNF and IL-6 induced in these mice. Surprisingly, however, neither TLR4- nor CD14-deficient mice were resistant to two closely related organisms that differed only in that they expressed a K1 polysaccharide capsule, even though these mice continued to express low levels of the proinflammatory cytokines. These and other observations suggest that the ability to limit bacterial dissemination may be equally or even more important than reducing the levels of these proinflammatory cytokines in preventing death from sepsis. We present evidence here, that the pathogenic role for the CD14/TLR4 pathway in severe infection, whereby it mediates the induction of proinflammatory cytokines, may be secondary to a more important insidious role whereby differential activation of local and systemic pathways of chemokine induction via CD14/TLR4-dependent and independent mechanisms generate chemokine gradients that prevent PMN transmigration from the blood to infected tissue and facilitates the dissemination of bacteria from a localized site of infection.
Costimulatory signals regulate the proliferative potential and effector function of NKT cells (95.14)
Authors: Sun Wenji , Oelke Mathias , Schneck Jonathan , Webb Tonya
Published: January 2023

Natural killer T (NKT) cells play a pivotal role in maintaining immune homeostasis. NKT cells rapidly secrete both proinflammatory and inflammatory cytokines following activation. However, the role costimulatory molecules during activation and their impact on effector functions have yet to be clearly defined. Our innovative system, which utilizes bead-based artificial Antigen Presenting Cells (aAPC), allows us to systematically evaluate the role of different costimulatory molecules on NKT cell activation and expansion. In this study, we have examined the effects of CD28, CD44, CD161, and OX-40 on NKT cell proliferation, phenotype and function. We found that the costimulatory molecules which increased NKT cell proliferation also enhanced cytokine production and cytotoxic function. These studies will enhance our knowledge of NKT cell biology and may aid in the development of novel NKT cell-based immunotherapeutic approaches for the treatment of infectious and autoimmune diseases, as well as cancer.
Regulation of anti-tumor immunity by p53 (95.2)
Authors: Singh Nagendra
Published: January 2023

Several pro-apoptotic molecules play an essential role in regulating oncogenesis as well as T-cell expansion during immune responses. Tumor suppressor molecule p53 regulates DNA repair, genomic integrity, cell proliferation and apoptosis. Mutations of P53 pathway is the most common genetic aberration in human tumors and as many as 50% of all the human tumors contain mutation in p53 pathway. Mice lacking p53 preferentially develops T-cell lymphomas and is susceptible to induced autoimmune dieases, suggestting a role for p53 in regulation of T cell responses. We found that p53 acts as a negative regulator of immune responses by inducing apoptosis and inhbiting T-cells proloiferation during immune responses. Furthermore, p53-deficient mice generates a robust anti-tumor cytotoxic T-cell responses and reject an implanted immunogenic tumor. These findings demonstrate an essential role of p53 in generating anti-tumor T-cell responses and suggest that a careful immunomodulation should be considered to achieve optimum results if p53 pathway is potential target of cancer chemotherapy.
Deletion of IL-6 in dnTGFβRII Mice Improves Colitis but Exacerbates Autoimmune Cholangitis (96.10)
Authors: Zhang Weici , Tsuda Masanobu , Yang Guo-Xiang , Tsuneyama Koichi , Rong Guanghua , Ridgway William , Ansari Aftab , Lian Zhe-Xiong , Flavell Richard , Gershwin M. Eric
Published: January 2023

The role of IL-6 in autoimmunity continues to attract attention, particularly because of the impending clinical usage of monoclonal antibodies designed to block IL-6 pathways, using antibodies to IL-6R. We have previously reported that dnTGFbRII mice develop both colitis and autoimmune cholangitis with elevated serum levels of IL-6. Based on this observation, we have generated IL-6 deficient mice on a dnTGFbRII background (dnTGFbRII IL-6-/-) and examined sera for the presence of anti-mitochondrial antibodies, levels of cytokines, as well as histopathology and immunohistochemistry of liver and colon tissues. As expected, based on reports of the use of anti-IL6R in inflammatory bowel disease, dnTGFbRII IL-6-/- mice manifest a dramatic improvement in their own inflammatory bowel disease. Importantly, however, autoimmune cholangitis in dnTGFbRII IL-6-/- mice was significantly exacerbated, including elevated inflammatory cytokines, increased numbers of activated T cells and worsening hepatic pathology. The data from these observations emphasize that there are distinct mechanisms involved in inducing pathology in inflammatory bowel disease compared to autoimmune cholangitis. These data also suggest that patients with inflammatory bowel disease may not be the best candidates for treatment with anti-IL-6R if they have accompanying autoimmune liver disease and emphasize the need for further studies.
A novel Highly Specific Small Molecule Inhibitor for BTK Suppresses Fc Receptor Function in Macrophages and Prevents Inflammatory Arthritis (96.18)
Authors: Reif Karin , Huang Tao , Barbosa Jim , Hymowitz Sarah , Gallion Steven , Hurez Vincent , Maciejewski Patricia , Dennis Jr. Glynn , Rong Hong , Shen Fang , Diehl Lauri , DeFroge Laura , Lee Wyne , Balazs Mercedesz , Currie Kevin , DiPaolo Julie
Published: January 2023

Activating Fc receptors and inflammatory cytokine production by macrophages have been implicated in the development and pathology of arthritis. However, the molecular components that mediate these events are not well characterized. Here we identify Bruton’s tyrosine kinase (BTK) as an essential player not only in B-cell dependent but also in myeloid-cell dependent inflammatory arthritis using a novel highly specific small molecule inhibitor of BTK (BTK SMI). In rodent models of collagen-induced arthritis, treatment with BTK SMI inhibits disease by at least two mechanisms: preventing autoantibody production and suppressing inflammatory cytokine production. Specifically, BTK SMI treatment prevents signaling and inflammatory cytokine production in response to immune complex activation in macrophages. Pathways found in rodents translate to human as BTK SMI prevents Fc receptor induced cytokine production in human monocytes. These data indicate that BTK SMI may be beneficial for the treatment of arthritis and that BTK contributes substantially to myeloid cell-dependent disease pathogenesis.
Novel peptides ameliorate symptoms of systemic lupus erythematosus in mice (96.2)
Authors: Wormser Uri , Proscura Elena , Brodsky Berta , Shapira Elena
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a loss of immunologic tolerance, production of auto-antibodies and inflammatory damage in multiple organs. We have tested the effect of anti-inflammatory peptide, a H2A histone fragment, termed IIIM1 on MRL/lpr mice, an animal model of SLE. Oral administration of IIIM1 at early stage of disease caused reduction in proteinuria and serum anti-dsDNA antibodies. Starting the treatment at advanced stage of disease resulted in prolonged animal survival, decreased lymphadenosis and reduced levels of pathogenic or abnormal double negative CD4-CD8- cells and B220+ cells in lymph nodes and spleen. We discovered that IIIM1 induces the production of an additional peptide, a fragment of alpha-1-antitrypsin, termed UBE. Relatively low dose (1µg/kg) of UBE reduced proteinuria and hematuria in MRL/lpr mice. The beneficial effect of the peptide was corroborated by histological examination. Furthermore, a significant reduction in serum IL17, IL12 and anti dsDNA antibodies was observed in the UBE-treated mice. Isolated CD4 cells incubated with the peptide showed similar cytokine profile. Decreased levels of double negative CD4-CD8- and B220+ cells were determined in lymph organs of UBE-treated animals. The beneficial effects of both UBE and IIIM1 suggest these peptides as potential drugs for SLE.
Isolation of A Human GM-CSF Antibody with Potent Neutralizing Activity from a Subject with Idiopathic Pulmonary Alveolar Proteinosis (96.24)
Authors: Chan-hui Po-Ying , Cox Tom , Grondin Roxanne , Grandea Glen , Frey Steve , Branum Mark , Ward Courtney , Fox Ray , Olsen Ole , Hammond Phil , Cieplak Witold , Moyle Matthew
Published: January 2023

Background. GM-CSF has been implicated in the development of autoimmune disorders. The efficacy of neutralizing anti-GM-CSF has been demonstrated in several animal disease models rheumatoid arthritis, asthma and lung inflammation. While humanization of murine antibodies (Abs) and phage-display technology have generated potential therapeutic leads currently in development, these engineered leads face potential challenge of serum stability and immunoreactivity. We have isolated a fully human anti-GM-CSF from a patient of idiopathic pulmonary alveolar proteinosis (iPAP) who produces anti-GM-CSF as autoantibody. Method. To isolate natural human Abs, we used a short term memory B cell culture system to generate Ab-containing supernatants and screened for anti-GM-CSF that inhibited the proliferation of TF-1 cells in response to human GM-CSF. The specificity for GM-CSF was then confirmed via secondary binding screening in Alphascreen assays. Recombinant human Abs were isolated from the corresponding memory B cells using RT-PCR and subcloning. Results. By analyzing the serum from several iPAP patients for the neutralizing activity against TF-1 proliferation, we selected one peripheral blood sample for Ab isolation. TCN-350 was isolated with IC50 of 27 pM for TF-1 inhibition. It binds human GM-CSF with an affinity of 39 pM and cross-reacts with rabbit GM-CSF with an affinity of 1 nM. The high neutralization potency of TCN-350 warrants further evaluation as a therapeutic lead.
Revertant muscle fibers expressing dystrophin do not tolerize the immune system in Duchenne muscular dystrophy: lessons learned from a Phase I clinical trial (96.9)
Authors: Campbell Katherine , Rodino-Klapac Louise , Sahenk Zarife , Shilling Chris , Lewis Sarah , Bowles Dawn , Gray Steven , Li Chengwen , Galloway Gloria , Malik Vinod , Coley Brian , Clark Reed , Li Juan , Xiao Xiao , Samulski Jade , McPhee Scott , Samulski R.Jude , Mendell Jerry , Walker Christopher
Published: January 2023

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle weakness due to mutation of the dystrophin gene. Muscle inflammation is an unexplained feature of DMD. T lymphocytes dominate the inflammatory infiltrate but antigen(s) they target have not been identified. Here we evaluated T cell immunity to dystrophin in 6 humans with DMD before and after delivery of a gene therapy vector to skeletal muscle. The vector transgene encoded a miniaturized version of human dystrophin that restores muscle function in animal models of DMD. Sustained transgene expression was not observed in any of the subjects. Non-self epitopes encoded by the vector transgene, but deleted from the defective dystrophin gene, provoked CD4+ and CD8+ T cell responses in a subset of the subjects. Dystrophin-specific T cell immunity was unexpectedly detected in two subjects before gene therapy. Epitopes were mapped to revertant dystrophin protein spontaneously expressed in rare muscle fibers when alternative splicing restores the correct open reading frame. Delivery of the minidystrophin gene to skeletal muscle of one subject provoked a very rapid increase in the frequency of these self-reactive T cells. T cell priming by idiosyncratic expression of revertant dystrophin could explain muscle inflammation and contribute to the pathogenesis of DMD. Recall of auto-reactive T cells also has important clinical significance in the design and monitoring of experimental therapies for this disease.
Exercise increases regulatory T cell function and decreases Th2 and Th17 cytokine production in healthy and asthmatic mice (97.15)
Authors: Lowder Thomas , Dugger Kari , Estell Kim , Deshane Jessy , Schwiebert Lisa
Published: January 2023

Exercise training has been shown to significantly reduce Th2 cytokines, airway hyper-responsiveness (AHR), and lung pathology in asthmatic mice. Some of these changes have been postulated to be due to regulatory T cells (Tregs). In these experiments, mice were induced into an asthmatic state (OVA) and exercised (45min, 3d/wk, at 60-70% of VO2max) for a total of 10 bouts. Tregs isolated from the spleens, lungs, and mediastinal lymph nodes of exercise-trained mice and stimulated in vitro were able to suppress effector cells at a significantly greater rate than Tregs from sedentary mice irrespective of whether or not they were induced to an asthmatic state (p < 0.05). Blocking TGF-β did not reduce Treg-mediated suppression in the exercise-trained group, but suppression was decreased in control mice following anti-TGF-β administration. In vitro Treg:effector co-cultures demonstrated significant reductions in Th2 and Th17 cytokine production in exercise-trained mice compared to their sedentary counterparts. Surface expression of Tregs, as assessed by Foxp3EGFP, was significantly higher in exercise-trained asthmatic mice compared to sedentary controls. We show for the first time that exercise training increases Treg expression and function in asthmatic and non-asthmatic mice.
iNKT cells regulate plasmacytoid dendritic cell population in bone marrow (98.16)
Authors: Wen Xiangshu , Eriksson Anna , Kim Peter , Singh Ram
Published: January 2023

Plasmacytoid dendritic cells (pDC) develop and proliferate in bone marrow and migrate to peripheral organs. pDCs produce IFNα and play a role in viral infections and autoimmune diseases. Mechanisms that regulate the development of pDC in the bone marrow are not fully understood. Invariant natural killer T (iNKT) cells comprise of a small population of lymphocytes in the bone marrow. Here, we examined the role of iNKT cells on pDC population in the bone marrow in vivo and in vitro. Bone marrow cells from wild-type, CD1d-deficient, and iNKT cell transgenic (Vα14-Tg) BALB/c mice were cultured with Flt3 ligand (FL) and αGalCer. We found that although all DCs were activated, as determined by expression of MHCII and CD86, in the presence of activated iNKT cells, the proportion of pDCs (CD11c+PDCA1+B220+) was consistently reduced in the presence of activated iNKT cells. Similar results were found in vivo, where an injection of FL and αGalCer reduced the proportion of pDCs, whereas the proportion of myeloid DCs (CD11c+CD11b+) was unchanged or slightly increased, in the bone marrow and spleen. Interestingly, FL+αGalCer increased CCR7 expression on bone marrow pDCs, but not on myeloid DCs, suggesting that iNKT cells may contribute to the egress of pDCs from the bone marrow. Such effect of FL+αGalCer injection was not seen in CD1d-deficient mice. In summary, these data suggest a role of iNKT cells in the development and homeostasis of pDCs.
Regulation of CD8 T cell responses by follicular dendritic cell-bound antigen (98.17)
Authors: Erlebacher Adrian , Curotto de Lafaille Maria , Lafaille Juan , Carroll Michael , McCloskey Megan
Published: January 2023

Follicular dendritic cells (FDCs) are well known to retain the immune complexes that drive antibody affinity maturation. Here, we present evidence that FDC-bound antigens are also persistently sampled by bone marrow-derived DCs for presentation to CD8 T cells. These results are based upon our previous observation that ovalbumin (OVA) expressed in transmembrane form by the placenta is hematogenously released during pregnancy to accumulate on maternal FDCs throughout all secondary lymphoid organs. We now show that OVA loading onto FDCs is complement-dependent and that bound OVA is retained for several weeks after delivery. Strikingly, antigen retention correlated with a prolonged postpartum phase of OVA presentation to CD8 T cells that could be abrogated by the pharmacological ablation of FDC networks. Furthermore, bone marrow chimera experiments demonstrated that FDC-bound OVA could be acquired de novo by DCs arising in the postpartum period. The presentation of placental OVA acquired from FDC-associated depots was highly tolerogenic as it induced T cell deletion even in the presence of exogenous adjuvants. These results suggest an unanticipated level of control over peripheral tolerance induction potentially relevant to the pathogenesis of systemic lupus erythematosus, an autoimmune disease characterized by impaired tolerance to apoptosis-associated antigens opsonized by the complement system. Supported by NIH grant RO1-AI062980.
Regulatory function of myeloid-derived suppressor cells is restricted to inflammatory site. (98.25)
Authors: Haverkamp. Jessica , Ratliff Timothy
Published: January 2023

Myeloid-derived suppressor cells (MDSC) are identified in mice as Gr-1+CD11b+ cells able to suppress T cell proliferation. Suppressive function of MDSC is linked to expression of arginase I and inducible nitric oxide synthase (iNOS) and can be augmented by inflammation. While inflammation is linked to MDSC function, it is unknown if MDSC isolated from the inflammatory site possess equal regulatory function as those in distal sites such as the spleen. Using the POET-3 model of prostate inflammation, we show Gr-1+CD11b+ cells isolated from inflamed prostates express elevated levels of ARG I and iNOS. In contrast, cells from the spleens of mice with prostate inflammation do not express detectable levels of ARG I or iNOS. However, when function of Gr-1+CD11b+ cells from spleens or the inflammatory site were tested in conventional suppression assays, all cells demonstrated suppressive function. RT-PCR analysis revealed that arginase I and iNOS mRNA were induced in MDSC during suppression assays, in part through IFN-γ. Thus conventional suppression assays induce functional suppressor cells and do not test their actual activation state in vivo. To better evaluate the function of Gr-1+CD11b+ cells we developed a short term suppression assay designed to limit exposure to IFN-γ during culture. Only cells from the inflammatory site suppressed T cell proliferation. These data demonstrate the novel finding that only Gr-1+CD11b+ cells at the inflammatory site are functional MDSC.
Novel chalcone JSH 4-4 inhibit NF-kB-regulated inflammatory gene expression via targeting LPS-binding site of MD-2 (98.4)
Authors: Roh Eunmiri , Lee Heun-Sik , Kwak Jeong-Ah , Jung Sang Hun , Han Sang-Bae , Kim Youngsoo
Published: January 2023

Previous studies have shown that chalcone has beneficial effects with antioxidant and anti-inflammatory activities. However, molecular basis for these effects not yet fully understood. In this study, a chalcone derivative, 2’, 4’-dihydroxy-6’-isopentyloxy chalcone (JSH 4-4) was discovered as an inhibitor of NF-kB activation. To demonstrate molecular target affected by JSH 4-4, we examined a homotypic interaction of TLR4, using Ba/F3 cells stably expressing TLR4-Flag, TLR4-GFP CD14 and MD-2, seperately. As a result, JSH 4-4 inhibited the LPS-induced TLR4 oligomerization. Moreover, we demonstrated that JSH 4-4 inhibit LPS-induced NF-kB activation via targeting LPS-binding site of MD-2. This mechanism of action could contribute to suppressive effect of JSH 4-4 on NF-kB-regulated production of inflammatory mediators such as nitric oxide, COX-2 and cytokines. TLR4-mediated NF-kB signalling occurs following sequential activation of IRAK family and TAK1. JSH 4-4 inhibited LPS-induced phosphorylation of IRAK-1 and TAK1. In addition, JSH 4-4 significantly inhibited IKK activity and phosphorylation of IkBa, resulting in sequential prevention of downstream events, including degradation of IkBs, nuclear translocation of NF-kB p65. In conclusion, JSH 4-4 inhibit NF-kB-regulated inflammatory mediators via targeting the receptor complex MD2-TLR4 in macrophages, thus JSH 4-4 might be considered as a potential agent for the treatment of NF-kB-associated inflammatory diseases.
Abrogation of tolerance with Flt3L requires CD154 expression (98.9)
Authors: Gurung Prajwal , Griffith Thomas
Published: January 2023

Soluble proteins are poorly immunogenic, and will often lead to Ag-specific tolerance. Similarly, Ag associated with apoptotic cells will frequently induce tolerance. These types of Ag are not very “dangerous”, so that recruitment and stimulation of professional APC is minimal. Treatment with the hematopoietic growth factor Fms-like tyrosine kinase 3 ligand (Flt3L) results in tremendous DC expansion, and previous studies have shown that Flt3L treatment can prevent the establishment of tolerance. Yet, the exact mechanism by which Flt3L treatment enhances immune responses remains undefined. I.V. injection of apoptotic cells or soluble Ag into wt mice induces tolerance, but this tolerance is abrogated by treatment with Flt3L. Interestingly, similar abrogation of tolerance was not observed in CD154-/- mice, suggesting a role for CD40-CD40L interaction. Further analysis of Flt3L-treated mice found reduced expansion of CD11c+ cells in CD154-/- mice versus B6 mice. This reduction in CD11c+ cells correlated with reduced T proliferation, as determined by CFSE dilution, in Flt3L-treated CD154-/- mice compared to B6 mice. Phenotypic analysis of the CD11c+ DC from Flt3L-treated B6 and CD154-/- mice showed reduced costimulatory molecule expression, especially CD40, in the CD154-/- CD11c+ DC. Together, these data suggest that maximal DC mobilization by Flt3L requires CD40-CD154 binding, which primes the DC for stimulating immunity even against Ag that normally induce tolerance.
Roles of Bcl6 and Blimp1 in CD4 T follicular helper (Tfh) lineage commitment (99.11)
Authors: DiToro Daniel , Johnston Robert , Crotty Shane
Published: January 2023

A distinct CD4 effector T cell subset, called T follicular helper cells (Tfh), is required for proper B cell responses in vivo. The mutually antagonistic transcription factors Bcl6 and Blimp1 play central but opposing roles in Tfh differentiation (Johnston et al., Science 2009). Bcl6 is both necessary and sufficient for in vivo Tfh differentiation, while Blimp1 specifically inhibits it. Expression of both Bcl6 and Blimp1, controlled temporally and spatially by cell-cell contact and multiple cytokines, is necessary for a normal CD4 T cell response. Naïve CD4 T cells express Bcl6 mRNA prior to, and independent of, commitment to the Tfh lineage. Bcl6 contains multiple functional domains and can affect different transcriptional outcomes depending on the local chromatin and co-repressor environment. Bcl6 repression of Blimp1 requires the co-repressor MTA3, which binds the Bcl6 RDII domain. We show that a loss of function mutation in the Bcl6 RDII domain results in a total failure of CD4 T cells to proliferate in vivo following infection with LCMV. We posit that this proliferation defect is a result of the failure to control expression of Blimp1, a strongly anti-proliferative protein, immediately following activation. Conversely, failure to attain adequate Blimp1 expression also results in an abnormal antiviral CD4 T cell response. Taken together, our findings suggest that the roles of Bcl6 and Blimp1 in CD4 T cells extend beyond Tfh lineage commitment.
SLAM-associated protein (SAP)-deficiency impedes the function of T follicular helper cells in germinal center formation (99.3)
Authors: Lu Kristina , Cannons Jennifer , Kanno Yuka , O'Shea John , Schwartzberg Pamela
Published: January 2023

Mutations affecting SAP lead to XLP, an immune disorder with impaired antibody responses. SAP-/- mice show defective formation of germinal centers (GC), due to a defect in T:B cell interactions. T follicular helper cells (Tfh) are important for providing help to B cells and GC formation. To investigate the role of SAP in Tfh cells for GC formation, we have developed a modified protocol to generate “Tfh-like” cells in vitro, resulting in IL-21 producing cells with elevated expression of hallmark Tfh markers CXCR5, ICOS, PD-1, BTLA, and SLAM family receptors CD84 and Ly108, along with enhanced levels of Bcl6 transcripts. Cell transfer of control Tfh-like cells into SAP-/- hosts can rescue GC formation upon immunization significantly better than polarized Th1, Th2 or Th17 cells, suggesting these cells act as functional Tfh cells in vivo. In vitro differentiation of SAP-/- cells is similar to control cells, yet, upon cell transfer, SAP-/- Tfh-like cells are unable to support GC formation. Interestingly, differentiation of Tfh-like cells is not terminal, as they can be repolarized to produce other distinct Th cell cytokines. Similarly, other Th cell populations can be repolarized to Tfh-like cells. Analyses of epigenetic modifications of transcription factors also supports plasticity between these Th cell lineages. These findings provide further insight into Tfh cell biology and suggest that SAP is critical for Tfh cell function in GC formation and immune homeostasis.
High-throughput characterization of MHC I-associated peptides presented by B lymphoblastoid cells (100.11)
Authors: Granados Diana Paola , Yahyaoui Wafaa , Côté Caroline , Muratore-Schroeder Tara , Thibault Pierre , Perreault Claude
Published: January 2023

Sensing of the MHC I-peptide (MHC I-p) repertoire allows elimination of infected or transformed cells. High-throughput strategies used to study MHC I-p include i) the use of secreted forms of MHC I transfected into model cell lines, or ii) chemical or metabolic labeling of MHC I, which is limited to certain MHCs and to cell culture model systems. Here, we used a quantitative proteomics approach to characterize and compare for the first time the MHC I-p repertoire associated to unlabeled HLA-A,B molecules of untransfected B lymphoblastoid cells (B-LCLs). Peptides were obtained from B-LCLs from 3 subjects by mild acid elution, fractionated by liquid chromatography and analyzed by nanoLC-MS/MS on a LTQ-Orbitrap mass spectrometer. We have identified more than 1000 unique 8-11mers associated to 10 HLA-A,B alleles: HLA-A*0101,0201,0205,0301,2902; B*0801,1501,4403,5001,5701. We found MHC-dependent differences in the predicted binding affinity of identified peptides. We used bioinformatic tools to identify the genes source of peptides and to characterize and compare the proteins encoding the MHC I-p repertoire. Peptides were encoded by genes located in all chromosomes and the mitochondria, and were significantly enriched for genes located in chromosomes 17 and 6. Our analysis revealed an enrichment of genes implicated in cell cycle, transcription and protein synthesis, as well as in immune and viral infection-related pathways, reflecting a B cell-specific signature.
The mechanism of resistance to parasite infection. (100.14)
Authors: Grover Harshita , Reese Michael , Blanchard Nicolas , Gonzalez Federico , Chan Shiao , Robey Ellen , Shastri Nilabh
Published: January 2023

The parasite, Toxoplasma gondii (T. gondii) can cause severe disease in immunocompromised humans. Susceptibility to T. gondii is also linked to the MHC haplotype: B10.D2 (H-2d) mice are relatively resistant to T. gondii infection than C57Bl/6 mice (B6, H-2b). CD8 T cells, activated by MHC molecules, are known to be that primary mediators of protection from T. gondii, and it is possible that differences in their responses might determine susceptibility to infection. Indeed, in H-2d mice, we recently identified the key immunodominant peptide, HF10, contained in the GRA6 protein that elicited protective CD8 T cell responses. In contrast, we show here that the CD8 T cell response to T. gondii in susceptible B6 mice is weaker and specific for a distinct peptide derived from the ROP5 protein. Furthermore, unlike the HF10 peptide that generated protective CD8 T cells in H-2d mice, immunization with the ROP5 peptide failed to diminish the parasite burden in chronically infected H-2b mice. Thus, susceptibility to T. gondii correlates with MHC-linked processing of specific parasite proteins that elicit differential CD8 T cell responses in mice.
The role of ERAAP polymorphisms in the autoimmune disease Ankylosing Spondylitis (100.17)
Authors: Bakker Arne , Shastri Nilabh
Published: January 2023

Ankylosing spondylitis (AS) is a chronic inflammatory joint disease of the axial skeleton, ultimately leading to fusion of the spine. For over three decades it has been known that the MHC class I molecule HLA-B27 is strongly associated with AS. In addition, the ER-resident aminopeptidase ERAAP (or ERAP1) was recently shown to be associated with as much as 26% of the risk for developing AS in HLA-B27 positive individuals. Multiple non-synonymous SNPs have been identified in the ERAAP gene that increase the risk of AS, but the functional significance of these polymorphisms is not known. The ERAAP protease is an essential component of the MHC class I pathway where it trims antigenic peptides in the ER. The significant association of both HLA-B27 and ERAAP with AS suggests that an altered MHC class I antigen processing pathway could play an important role in this partially T cell-mediated disease. To test this hypothesis we have analyzed the antigen processing capacity of several polymorphic forms of human ERAAP in ERAAP-deficient cells. All the polymorphic ERAAP constructs tested were enzymatically active and able to trim model antigenic peptides in different cell lines, with either no or minor differences in the quantity of total epitopes generated. We will discuss the implications of these findings.
Presentation of citrullinated peptides by class II histocompatibility molecules is associated with autophagy. (100.19)
Authors: Rimer Jamie , Unanue Emil
Published: January 2023

Autoimmune responses to citrullinated proteins are associated with rheumatoid arthritis; however, little is known of the mechanism of citrullination by the antigen presenting cells (APC) of the immune system. Using T cell hybridomas as probes, we found constitutive presentation by class II-Major Histocompatibility Complex (MHC) molecules of a dominant citrullinated peptide from hen egg-white lysozyme (HEL) by either macrophages or dendritic cells. Treatment of APC with 3-Methyladenine (3MA) blocked presentation of the citrullinated peptides after processing unmodified HEL, but presentation of unmodified peptides was not at all affected. In contrast presentation of citrullinated peptides by splenic B cells or B lymphoma cells was not detected under normal culture conditions. However, B cell lines presented citrullinated peptides after serum starvation in a process affected by 3MA treatment or by reducing the expression of the autophagy protein Atg5. Higher levels of citrulline in naturally processed peptides from serum starved B cell lines were detected biochemically. Primary B cells presented citrullinated HEL peptides after engagement of their B cell receptor by antigen or by using anti-immunoglobulin antibodies. Our findings demonstrate a role for autophagy in citrullination of antigen during processing by APC and indicate a link in B cells between engagement of their antigen receptor and presentation of citrullinated peptides.
Flow cytometry based analysis for subsets of Plasmacytoid Dendritic Cells (pDC) from human PBMC (100.31)
Authors: Chitta Sriram , Quintel Payton , Lee Hyun-Ku , Wardle Lauren , Stampfl Jason , Singh Gita , Rosenberg Jonathan , Singh Sujay
Published: January 2023

Dendritic cells are a heterogeneous group of antigen presenting cells that link innate and adaptive immunity. In mice and humans, subsets of DC have been identified, based on origin, location, differentiation, phenotype and functions. Newly identified markers, availability of mAb(s) and advanced flow-cytometry techniques have helped in enumerating existence of such subsets. Plasmacytoid dendritic cells (pDC) are peripheral blood DC subsets, which produce IFN (Type I) and are vital for anti-viral immunity. pDC exist in small numbers (~0.1-0.5%) in peripheral blood and their phenotype analysis by flow, includes CD123 and HLADR expression on lineage negative populations of PBMC. Recently, pDC were shown to play important roles (besides anti-viral) in other human diseases such as lupus and psoriasis but it is not known if distinct subsets are associated. In the present study we used three markers viz. CD123, BDCA-2 and ILT7 (newly identified) to analyze potential subsets of pDC. As Type I IFN producers, pDC express intracellular toll-like receptors TLR7 and TLR9 and our analysis extends to include whether these subsets differ in TLR expression, particularly focusing on TLRs playing an important role in innate-IFN (Type I) production. In order to understand the role of pDC (subsets) in induction of anti-immune responses towards other microbes, expression of non-specific markers such as Dectin-2 and TLR 10 were also tested using newly available mAb(s).
Uptake and disposition of recombinant chimeric immunogens constructed from Streptococcus mutans SBR-CTA2/B at mucosal immune inductive sites (100.35)
Authors: Zhao Weiwei , Minderman Hans , O'Loughlin Kieran , Russell Michael
Published: January 2023

Purpose: To investigate cellular pathways of uptake and processing of the recombinant chimeric immunogen SBR-CTA2/B consisting of the saliva-binding region (SBR) of S. mutans AgI/II coupled to cholera toxin A2/B subunits, in comparison with SBR alone. Methods: Amnis ImageStream technology was used to characterize cells from lamina propria (LP), Peyer’s patch (PP), and mesenteric lymph nodes (MLN) of mice before and 2, 4, 16, and 48h after intragastric immunization with FITC-labeled proteins. Dendritic cells (DC) were stained for phenotypic markers and the migration receptor CCR7 using contrasting fluorochromes. Results: LP CD103+, CD11c+, and especially CD103/CD11c double+ DC took up SBR-CTA2/B through 4h, after which the proportion of antigen-containing DC stayed level until 16h, although the number of DCs continued to increase. DCs in PP captured SBR-CTA2/B through 16h. The proportion of LP DCs containing SBR peaked at 2h and declined by 16h. CCR7 expression by CD11c+ LP DCs taking up SBR-CTA2/B rose at 2h and dropped at 4h, whereas CCR7 expression by DC taking up SBR declined from 0h levels. In MLN the number of CD11c+/CD4+ cell doublets dramatically increased at 48 h after SBR-CTA2/B immunization. Conclusions: Compared to SBR alone, the chimeric protein SBR-CTA2/B enhanced the functional activation of DCs in LP and PP, in which most antigen uptake occurred within the first few hours. DCs loaded with protein may then migrate to MLN and intercommunicate with T cells.
Immunoproteasomes regulate gene expression in dendritic cells (100.36)
Authors: de Verteuil Danielle , Laperrière David , Mader Sylvie , Thibault Pierre , Perreault Claude
Published: January 2023

A constitutive form of the proteasome (CP) is expressed in all eukaryotic cells and is involved in many cellular processes. Immune and cytokine-activated cells also express a second type of proteasome named the immunoproteasome (IP). Since its discovery, it has been thought that the main role of IPs is to produce MHC I-associated peptides. However, an increasing number of studies report peptide-independent effects of IPs. In line with this, we recently showed using microarrays that knocking-out two IP-subunits (LMP7 and MECL1) in bone marrow-derived dendritic cells alters the expression of 171 genes mostly associated with immune functions, with enriched clusters on chromosomes. We now show by qPCR experiments that the disruption of both LMP7 and MECL1 is necessary to alter gene expression. Bioinformatic predictions of transcription factor binding sites suggest that this effect of IPs hinges mainly on transcriptional regulation. Comparing the abundance of unspliced and mature mRNAs in WT and IP knock-out cells shows that IPs also influence the maturation of certain transcripts. However, IPs do not affect mRNA stability overtime when blocking mRNA synthesis. The control of gene expression by IPs might explain aberrant T and B cell phenotypes previously described in IP-subunit knock-out mice. A better comprehension of how IPs regulate gene expression could help us understand the selective pressure that allowed the conservation of more than one type of proteasome in vertebrates.
Targeting Antigens to Human Dendritic Cells via Dectin-1 Elicits Potent Antigen-specific Th17 CD4+ T Cells That Express Mucosal Homing Receptors (100.39)
Authors: Ni Ling , Li Xiaohua , Duluc Dorothee , Joo HyeMee , Kluca Peter , Agouna-Deciat Olivier , Zurawski Sandy , Banchereau Jacques , Zurawski Gerard , Oh SangKon
Published: January 2023

Emerging evidence indicates important roles of IL-17-producing (Th17) CD4+ T cells in protective immunity against microbial pathogens, including influenza viruses. Thus we tested the magnitudes and spectrum of influenza viral hemagglutinin subunit 1 (HA1)-specific Th17 CD4+ T cells in healthy donors by targeting HA1 to dendritic cells (DCs) via Dectin-1 using recombinant fusion proteins of agonistic anti-Dectin-1 fused to HA1 (anti-Dectin-1-HA1). Our data show that healthy individuals maintain broad ranges of influenza HA1-specific Th17 CD4+ T cells expressing CCR4 and CCR6. Interestingly, HA1-specific Th17 CD4+ T cells were distinguished from other Th17 CD4+ T cells by expressing increased levels of CCR9 and decreased levels of CD161. We further found that DCs activated through hDectin-1 upregulated other mucosal homing receptors, beta7 integrin, CCR4, and CCR9 on the surface of HA1-specific Th17 CD4+ T cells. Such HA1-specific Th17 CD4+ T cell responses were greatly enhanced by targeting DCs with anti-Dectin-1-HA1 in the presence of TLR2 ligands. This enhancement was mainly due to the expansion of pre-existing HA1-specific memory Th17 CD4+ T cells that express IL-1R1, but not naïve CD4+ T cells which do not. Induction of IL-1R1 on naïve CD4+ T cells required for priming HA1-specific Th17 CD4+ T cells. Taken together, we conclude that antigen targeting to DCs via Dectin-1 could mount potent protective immunity against microbial pathogens, including influenza viruses.
Insulin peptide specific CD4+ T cells escape negative selection and cause diabetes in NOD mice (100.44)
Authors: Mohan James , Calderon Boris , Petzold Shirley , Herzog Jeremy , Unanue Emil
Published: January 2023

The importance of insulin as an autoantigen in type 1 diabetes pathogenesis has been extensively associated with the development of disease. Within the NOD mouse the majority but not all of autoreactive T cells that recognize insulin are deleted during development in the thymus. We found a set of autoreactive CD4+ T cells specific for an insulin peptide, incapable of recognizing the insulin protein when processed by the antigen presenting cells (APC). These unique autoreactive cells escape thymic control, infiltrate the islets of Langerhans in pre-diabetic mice and are pathogenic. The mechanism by which these peptide specific T cells recognize antigen results from the formation of a unique state of the peptide-MHC complex (pMHC) that differs from the pMHC generated from the processed protein. Differences in pMHC states of the same peptide bound to the MHC molecule may arise from the particular vesicle within the APC were exogenous peptide vs. processed protein is loaded. We have also found that APC situated in close contact with the beta cells in the islets of Langerhans bear vesicles with the antigenic insulin peptides and activate the peptide-specific T cells. The importance of these T cells is that they readily escaped negative selection when the insulin protein was expressed by the thymic APC even though they can be activated in the periphery by islet APC in the absence of inflammation.
Analysis of several CD4+ T cell clones reveals a >10,000 fold range in 2D affinity of antigen recognition (100.45)
Authors: Hood Jennifer , Sabatino Joseph , Rosenthal Kristen , Zhu Cheng , Evavold Brian
Published: January 2023

Using a micropipette adhesion frequency assay, we are able to determine the affinity of polyclonal and monoclonal T cells without needing to isolate the TCR. This assay allows for the direct measuring of adhesion between a T cell and a surrogate APC, which is a human red blood cell that has been coated with peptide:MHC monomers. Utilizing this two dimensional measure of affinity, we determined the effective affinities for the following CD4+ TCR transgenic clones: 2D2, BDC2.5, and SMARTA for their cognate antigens. For the 2D2 cells, we assessed both MOG38-49:IAb and NF-M18-30:IAb monomers since it has been shown that 2D2 cells can cross-react with both of these CNS antigens. Our results demonstrate a wide range of affinities for these various transgenic cells. The hierarchy of the affinities from highest to lowest for the clones (clone:peptide) was; BDC2.5:1040-63, SMARTA:GP-61, 2D2:NFM18-30 and 2D2:MOG38-49 and roughly correlated with their responsiveness to antigen. The affinities ranged from 10-4 µm4 to less than 10-8 µm4, which is below the limits of detection for this technique. These data demonstrate that there is a wide range of affinities for CD4+ T cells that are capable of responding to antigen.
Modulation of MHC class I antigen presentation by the adenovirus E3-19K protein (100.8)
Authors: Fu Jie , Li Lenong , Bouvier Marlene
Published: January 2023

The presentation of viral antigens by MHC I to CTLs is vital for elimination of infected cells. In turn, viruses have evolved various strategies to abrogate antiviral cellular immune responses. For example, the adenovirus (Ad) E3-19K protein retains MHC I in the endoplasmic reticulum, dramatically suppressing the cell-surface presentation of viral antigens. Many aspects of how E3-19K exerts its function towards MHC I are poorly understood. This knowledge is important as the E3-19K/MHC I interaction is thought to play a role in enabling Ads to cause persistent infections. We characterized biophysically and cellularly interaction between E3-19K proteins of different Ad serotypes (Ad 7 and 35, subgroup B; Ad 2 and Ad 5, subgroup C; Ad 37, subgroup D; and Ad 4, subgroup E) and HLA-A, -B, and -C molecules. Our results show that E3-19K proteins from Ad serotypes of the same subgroup display more similar binding properties for a given MHC I than those of different subgroups. Furthermore, E3-19K proteins exhibited allele- and locus-specificity; higher avidity for HLA-A relative to -B molecules, and no interaction with HLA-C molecules. FACS analysis showed that binding affinities correlated (in a negative way) with levels of MHC I expression on infected cells. Finally, we propose the first model of interaction between E3-19K and MHC I. Overall, our studies provide novel insights into the structure/function relationship of E3-19K and the molecular cell biology of antigen presentation.
Serologic analysis of MHC class I folding intermediates (100.9)
Authors: Simone Laura , Tuli Amit , Wang Xiaojian , Sharma Mahak , Simone Peter , Naslavsky Naava , Caplan Steve , Solheim Joyce
Published: January 2023

During the process of peptide selection, MHC class I molecules transition from an open, peptide-free conformer to a folded, peptide-bound state. Thus, MHC class I molecules have been proposed to adopt an intermediate conformation during peptide binding; however, the molecular nature of this transition state is unknown. Here, we report evidence that the 34-1-2 antibody, which recognizes several murine MHC class I molecules, is able to distinguish an intermediately folded conformer. The majority of 34-1-2+ Ld molecules were found to represent a distinct pool of MHC class I molecules that were not recognized by the anti-folded Ld antibody 30-5-7. Moreover, 34-1-2+ Ld molecules were found to display characteristics indicative of incomplete folding, including increased tapasin association and endoplasmic reticulum (ER) retention. Additionally, our data suggest that tapasin associates with sub-optimally loaded 34-1-2+ Kd molecules and may facilitate the retrieval of these molecules to the ER. We have designated this MHC class I conformation as 34-1-2-INT (34-1-2+, intermediately folded). Together with prior knowledge regarding the 34-1-2 epitope, our analysis supports a model whereby binding of the peptide N-terminus permits MHC class I molecules to adopt an intermediately folded conformation which subsequently converts to the fully folded form upon optimization of C-terminal interactions. Thus, the 34-1-2 antibody is an important tool in understanding MHC class I peptide loading.
TCR tuning by homeostatic cytokines — implication for autoimmunity (101.16)
Authors: Deshpande Pratima , Singh Karnail , Weyand Cornelia , Goronzy Jorg
Published: January 2023

In animal models, lymphopenia and the associated function of homeostatic cytokines (HC) set the stage for autoimmunity. This model is relevant for rheumatoid arthritis (RA) which has defects in T cell homeostasis due to a failure in DNA repair and which responds to JAK inhibition. We have previously shown that RA T cells have a hyperreactive ERK response which impairs the induction of anergy. Here, we examined whether HC tune TCR activation thresholds by calibrating the ERK rheostat. RA T cells exhibited increased levels of phosphorylated STAT3 and STAT5 indicating in vivo activity of IL-21/IL6 and IL-7/IL-15. Incubation of healthy donor T cells with IL-7, IL15 or IL21 prior to anti-CD3/CD28 stimulation augmented ERK activation and expression of activation markers (CD69, CD40L) and cytokines (TNFα, IFNγ and IL-17). Preincubation with IL-1β or TNFα did not exert an effect. In DR4 healthy donors, HC priming facilitated responses to self-antigens such as citrullinated vimentin peptide. The effect of HC lasted for less than 3 hours and was sensitive to PI3K inhibition suggesting a non-transcriptional PI3K mediated priming of the ERK pathway. HC dose titration showed a bimodal distribution in pERK consistent with an on-off switch characteristic of a positive feedback loop. Allosteric binding of RAS-GTP to SOS is known to induce such an on-off switch. Indeed, HC incubation increased active RAS. We propose that HC lower the threshold for autoimmune responses by priming of SOS.
Expansion of IL-7Ralow memory CD8 T cells in human lupus (101.19)
Authors: Lee Naeun , Shah Kamini , Shin Min Sun , Kim Sang Hyun , Craft Joe , Kang Insoo
Published: January 2023

Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disorder that is characterized by production of autoantibodies and immune complex deposition. In the immunopathogenesis of SLE, CD4+ T cells play as critical drivers of B cell dependent autoantibody response, but little is known about the role of CD8+ T cells in the development of SLE. Recent studies have shown that the expansion of memory CD8+ T cells occurs in SLE patients. We have measured the expression of IL-7 receptor alpha (IL-7Ra, CD127) on memory CD8+ T cells in SLE patients, since IL-7 and IL-7R are involved in homeostasis of CD8+ T cells. As previous reported, we found two subsets expressing high and low levels of IL-7Ra on memory CD8+ T cells in SLE patients and healthy controls. In this study, we showed that SLE patients have an increased frequency of IL-7Ralow memory CD8+ T cells compared to healthy controls. This cell subset expresses high levels of cytotoxic molecule perforin with the capacity to damage tissue. Furthermore, the frequency of IL-7Ralow memory CD8+ T cells correlated with disease activity as measured by SLE disease activity index (SLEDAI). Although T cell receptor triggering barely induced IL-7Ralow cell proliferation, the homeostatic cytokine IL-15 substantially induced the proliferation of this cell subset. Our findings suggest a potential role for IL-7Ralow memory CD8+ T cells in the development of SLE.