bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulatory dendritic cells suppress NK cell activation by promoting IL-10 secretion and activating an NKp46-mediated inhibitory circuit. (P1072)
Authors: Zwirner Norberto , Avila Damian , Spallanzani Raul , Rossi Lucas , Domaica Carolina , Ziblat Andrea , Fuertes Mercedes , Rabinovich Gabriel
Published: January 2023

Regulatory dendritic cells (regDC) constrain the immune response in physiological and pathological conditions including tumor settings. Cross-talk between mature dendritic cells (mDC) and NK cells leads to reciprocal activation of both cells, but the impact of regDC on NK cells remains unknown. Our aim was to investigate the functional outcome and mechanisms underlying the interactions between regDC and NK cells. Human regDC generated from monocyte-derived DC treated with LPS and dexamethasone, opposed to mDC, triggered very little amounts of IFN-γ secretion by NK cells. This effect was partially due to insufficient amounts of IL-18. Blocking experiments demonstrated that regDC have the potential ability to stimulate IFN-γ secretion by NK cells through NKp30, but that this effect was overridden by a suppressive circuit that involves IL-10, NK cell inhibitory receptors and, unexpectedly, engagement of the activating receptor NKp46. Surprisingly, the opposing role of NKp30 and NKp46 on IFN-γ secretion was also observed during the cross-talk between mDC and NK cells. Accordingly, similar amounts of NKp46 ligands were found to be expressed on mDC and regDC. Our findings unveil a previously unrecognized regulatory mechanism through which regDC regulate NK cell activation and identify a novel inhibitory role for NKp46 which counterbalances the stimulatory activity of NKp30. These results might be relevant in shutting-down NK cell-mediated anti-tumor immune responses.
B cell intrinsic and extrinsic roles of ADP-ribosyltransferase, PARP14 in regulation of IgA and IgE responses (P1078)
Authors: Cho Sung Hoon , Raybuck Ariel , Wei Mei , Erickson John , Williams John , Boothby Mark
Published: January 2023

Immunological memory, including both persistent and recall antibody (Ab) responses, enables more efficient and effective protection against infection, but key molecular mechanisms regulating emergence and persistence of memory B cells and plasma cells remain unclear. Poly-ADP-Ribose Polymerase14 (PARP14) is highly expressed in B cells and identified as a Stat6-interacting protein with intrinsic mono-ADP-ribosyltransferase (mART) activity. Although there are 17 PARP family members, the physiological function of most PARPs are not known well. Here we show that recall IgA and IgE production are impaired in PARP14-deficient mice. Further analysis revealed that, although PARP14 regulation of IgE involves B cell intrinsic function, IgA regulation by PARP14 was predominantly B cell extrinsic. Of note, PARP14 regulates the levels of CD103+ dendritic cells and T helper 17 (Th17) cells which might influence IgA generation in B cells. Moreover, PARP14 regulates the expression of RORα, Runx1 and Smad3 during Th17 differentiation, and mART activity of PARP plays a critical role in Th17 differentiation. Collectively, these results shed new light on differential mechanisms of Ig regulation, provide evidence that an endogenous mono ADP-ribosyltransferase regulates recall Ab production, and may have important implications for a new therapeutic target.
Targets of somatic hypermutation within IgH genes in zebrafish (P1080)
Authors: Caldwell Tomika , Zimmerman Anastasia
Published: January 2023

In mammals, somatic hypermutation (SHM) of IgH genes is critical for the generation of high affinity antibodies and effective immune responses. Knowledge of sequence specific biases in the targeting of mutations can be useful to understand antibody repertoires produced in response to infections, B cell neoplasms, or autoimmune disease. To evaluate potential nucleotide targets of SHM in zebrafish, we constructed a cDNA library of V(D)J_C segments from randomly selected clones which were sequenced and analyzed. Mutations were most prevalent in V regions with a bias towards single base transitions and increased mutation in complementarity determining regions. Mutations were overrepresented at WRCH/DGYW motifs suggestive of activation-induced cytidine deaminase (AID) targeting common in mice and humans. In contrast to mammals, AID hotspots were largely restricted to palindromic WRCH/DGYW motifs. Taken together, the data reveal specific nucleotide targets of SHM in zebrafish and suggest both AID and affinity maturation contributes to antibody diversification in this emerging immunological model.
Myeloid-derived suppressive cells enhance differentiation of Th17 cells in an IL-1β dependent manner (P1096)
Authors: Wang Xiang-Yang , Yi Huanfa , Guo Chunqing , Yu Xiaofei
Published: January 2023

Myeloid-derived suppressive cells (MDSCs) have been implicated in the pathogenesis of several diseases, including cancer and autoimmune disorders. We found that CD11b+Gr-1+ MDSCs from tumor- or experimental autoimmune encephalomyelitis (EAE)-bearing mice promote the differentiation of naïve CD4+ T cell precursors into Th17 cells in a highly efficient manner. The presence of MDSCs also results in elevation of IL-17A production and upregulation of the orphan nuclear receptor RORA and RORC in T cells. IL-1β-derived from MDSCs is identified as a major mediator of the enhanced Th17 differentiation, which involves the IL-1 receptor on CD4+ T cells. Depletion of MDSCs leads to significantly reduced Th17 response and decreased clinical scores of EAE as well as myelin injury. Our data reveal a new feature of MDSC action in the shaping of inflammatory Th17 cell responses and provide additional insights into the pleiotropic functions of this myeloid cell population. These new findings support the concept of targeting MDSCs for potentially effective intervention of Th17 cell/IL-17-mediated immunopathology in cancer and autoimmune diseases (e.g., multiple sclerosis).
Mesenchymal stem cells alleviate Propionibacterium acnes-primed mice liver injury by inducing differentiation of regulatory DCs (P1098)
Authors: Zhang Yi , Jin Min , Zhang Yanyun
Published: January 2023

Fulminant hepatic failure is a clinical syndrome characterized by sudden and severe impairment of liver function. Mesenchymal stem cells have been proposed as a promising therapeutic approach for fulminant hepatic failure. In this study, we have used Propionibacterium acnes-primed, LPS-induced liver injury in mice as an animal model of human fulminant hepatic failure. We demonstrated that administration of mesenchymal stem cells significantly ameliorated liver injury and improved the survival rates of mice subjected to fulminant hepatic failure. Therapeutic efficacy of mesenchymal stem cells could be attributed to decreased infiltration of CD4+ T cells in the liver, inhibition of Th1 cells, and induction of regulatory T cells. Intriguingly, a distinct liver population of CD11c+MHCIIhiCD80loCD86lo regulatory DCs was induced. Further mechanistic studies demonstrated that mesenchymal stem cell-derived prostaglandin E2 and one of its receptors, EP4, played essential roles in the differentiation of CD11c+B220- DC precursors into regulatory DCs in a PI3K-dependent manner. Our findings demonstrated, for the first time, critical regulatory properties of mesenchymal stem cells for induction of regulatory DCs from CD11c+B220- DC precursors. This study further lays a solid foundation for application of mesenchymal stem cells in fulminant hepatic failure therapy.
Human B cells are targets for Brucella abortus infection. (P3130)
Authors: Ramírez Saldaña Maricela , Aguilar Santelises Miguel , Moreno Lafont Martha , Santos Argumedo Leopoldo , Paredes Cervantes Vladimir , López Santiago Rubén
Published: January 2023

It has been demonstrated that Brucella abortus is capable to multiply into professional antigen presenting cells such as macrophages and dendritic cells. However, there are a few reports about Brucella infection into B cells. In this work, we evaluated the ability of B. abortus 2308 for infect the human B cell line, NALM6 R. B cells cultured in RPMI medium supplied with L-glutamine and 10% of fetal calf serum were infected with several MOIs of Brucella abortus 2308 transformed by electroporation with the pBBR1MCS-2-gfp-mut3 plasmid to evaluate the entry of green fluorescent Brucella. The percentage of cells containing bacteria was evaluated by flow cytometry at three time points. To evaluate whether Brucella was on the surface or into the B cells, kinetics of infection was performed and analyzed by transmission electron microscopy. Association of Brucella abortus 2308-gfp-mut3 with NALM6 R cells increased in a dose-time dependent manner to reach a maximum of 50% of NALM6 R infected cells. Electronic microscopy revealed that NALM 6R B cells produce filopodia over B. abortus. Once inside, the bacteria resided into small vesicles. Apparently bacteria underwent some bactericidal mechanism, because the electron microscopy showed bacteria degraded into vesicles. It was not observed evidence of replication of bacteria inside B cells. Brucella abortus 2308 is capable to interact with NALM6 R membrane, to induce filopodia formation and to entry into B cells inside vesicles.
Adhesins and host serum factors drive Yop translocation by Yersinia pseudotuberculosis into professional phagocytes during animal infection (P3142)
Authors: Maldonado-Arocho Francisco , Green Carlos , Fisher Michael , Paczosa Michelle , Mecsas Joan
Published: January 2023

Yersinia delivers Yops into numerous types of cultured cells, but predominantly to professional phagocytes during animal infection. The basis for this cellular tropism during animal infection is not understood. This work demonstrates that Yop translocation into specific cell types by Yersinia pseudotuberculosis (Yptb) is a multi-factorial process where several adhesins and host factors are employed for efficient Yop delivery into phagocytes. Strikingly, when WT or a multiple adhesin mutant strain, ΔailΔinvΔyadA, colonized tissues to comparable levels, ΔailΔinvΔyadA translocated Yops into significantly fewer cells, indicating that adhesins influence the number of cells successfully translocated into during infection. However phagocytes were still selectively targeted for translocation, indicating that other bacterial and/or host factors contribute to this function. Complement depletion showed that complement both limits infection by ΔailΔinvΔyadA but not WT and that adhesins enlist complement to directs Yop translocation into professional phagocytes during animal infection.
Brucella induces an unfolded protein response via TcpB in macrophages (P3116)
Authors: Khan Mike , Magnani Diogo , Harms Jerome , Radhakrishnan Girish , Durward Marina , Liu Yi-Ping , Splitter Gary , Smith Judith
Published: January 2023

Brucella melitensis is a facultative intracellular bacterium that invades and replicates within macrophages and dendritic cells. With greater than 500,000 new infections per year, brucellosis is the most prevalent zoonosis worldwide. Currently, a safe and effective human vaccine does not exist and Brucella’s intracellular replicative niche renders the organism resistant to antibiotics. A better understanding of host-pathogen interactions supporting Brucella replication is necessary for vaccine and therapeutic development. Brucella fuses with the endoplasmic reticulum (ER) in order to replicate and may provoke a conserved ER stress response called the Unfolded Protein Response (UPR). B. melitensis infection in RAW 264.7 murine macrophages and bone marrow-derived macrophages upregulated the UPR target genes BiP, CHOP, and ERdj4, and induced XBP1 mRNA splicing. Upregulation of CHOP and ERdj4 was MyD88-independent whereas XBP1 splicing was MyD88-dependent. A Brucella TcpB mutant showed significantly reduced expression of BiP, CHOP, and ERdj4. Purified TcpB, a microtubule-modulating protein, induced UPR target genes. Also, purified TcpB caused ER restructuring similar to infection with B. melitensis. Finally, Brucella replication was significantly impaired by tauroursodeoxycholic acid, a pharmacologic UPR inhibitor. These results suggest that Brucella induces the UPR to enable its intracellular replication and thus provides a novel therapeutic target for brucellosis treatment.
B cells shape T cell responses during Pneumocystis infection (P3053)
Authors: Elsegeiny Waleed , Zheng Mingquan , Ricks David , Chen Kong , Kolls Jay
Published: January 2023

Pneumocystis pneumonia (PCP) remains one of the leading causes of death in immunocompromised patients. Disease is attributed to the loss of CD4 T cells; however recent studies suggest that B cells also play a protective role in clearing PC in an Ig-independent manner through antigen presentation. Several patients treated with anti-CD20 have developed PCP and thus we investigated the effect of anti-CD20 on primary PC infection. We hypothesized that B cells in part function as a critical antigen-presenting cell to helper T cells. To test this, C57BL/6 WT mice were administered anti-CD20, anti-CD4 (as a pos. control) or both, and then infected with a 105 inoculum of Pneumocystis murina. Lung mononuclear cells were harvested two weeks after infection, assessed for cellularity and then stimulated in vitro with PC antigen for cytokine profiling. Even though CD20-depleted animals had unaltered T cells numbers, there were diminished cytokine responses for IL-5, IL-17, and IL-10. These data suggest that B cells are involved in mediating optimal T cell cytokine responses against PC. We are currently testing if B cells are required in the recall responses from memory T cells by performing B cell depletion during the re-challenge of mice that have recovered from primary PC infection. We are also investigating if B cells modify T regulatory cells during Immune Reconstitution Inflammatory Syndrome (IRIS), a disease that affects a subset of patients upon reconstitution of CD4+ cells.
Regulatory T cells mediate immunosupression induced by experimental hypothyroidism. (P1215)
Authors: Valli Eduardo , Sterle Helena A , Méndez Huergo Santiago , Mascafroni Ivan , Klecha Alicia J , Barreiro Arcos María L , Rabinovich Gabriel A , Cremaschi Graciela A
Published: January 2023

Thyroid status modulates immunity, being the circulating levels of thyroid hormones (THs) responsible for the regulation of lymphocyte activity. Formerly we observed that experimental hypothyroidism (Exp-hypo) decreases Th1 cytokine production and T cell-mediated immunity. Here we aimed to elucidate the mechanisms underlying immunosuppression triggered by Exp-hypo. Lymphocyte subsets were assessed in spleens and lymph nodes of hypothyroid (hypo-WT) and control (C) mice. Percentage of Treg (CD4+CD25+FoxP3+) cells (Tregs %) was significantly increased in the hypo-WT group without differences in CD3/CD19 or CD4/CD8 ratios. When naïve T cells were differentiated to inducible Treg cells in vitro, similar results were obtained. Treg cells overexpress galectin-1 (Gal-1), an immunoregulatory β-galactoside-binding protein, and mice lacking this lectin (Lgals1-/-) showed reduced Treg cell activity. To evaluate the role of Gal-1 in the regulation of Tregs in Exp-hypo we also analyzed lymphocyte reactivity and the percentage of Tregs % in eu- and hypothyroid Lgals1-/- mice (hypo-KO). We found an increased T cell proliferation and a reduction in the Tregs % in hypo-KO mice respect to the hypo-WT group. We conclude that modulation of immunity in the Exp-hypo with low circulating levels of THs is associated to a significant rise in the percentage of spontaneous and inducible Tregs. This increase relies on the expression of Gal-1 and may be related to a functional deficit of T cells.
CD39-mediated Effect of human mesenchymal stem cells on the human Th17 cells (P1221)
Authors: Kim Mee Kum , Jeong Hyun Jeong , Kim Dong-Hyun , Wee Won Ryang , Lee Won Woo
Published: January 2023

Mesenchymal stem cells (MSCs) have been known to have anti-inflammatory properties in various inflammatory diseases. However, the reported effects of human MSCs on human Th17 cells have not been confirmed. This study was aimed to investigate inhibitory effects of human mesenchymal stem cells (hMSCs) on human CD4+ T cells through CD39 mediated pathway. The study was performed in accordance with the guidelines of the Declaration of Helsinki and was approved from the Institutional Review Board of the Seoul National University Hospital. Human peripheral blood mononuclear cells was obtained from the healthy voluntary donors with informed consents(n=6). Bone marrow-derived hMSCs were co-cultured with human CD4+ T cells at a MSC:T cell ratio of 1:10. The suppressive effects of MSCs were evaluated by assessing their effects on the proliferation of CD4+ T cells by carboxyfluorescein diacetate succinimidyl ester (CFSE) staining and the secretion of IFN-γ and IL-17A by CD 4+ memory T cells with or without anti-CD39 treatment. hMSCs effectively suppressed the proliferation and both IFN-γ and IL-17A secretion of CD4+ CD45ROhi T cells, while, anti-CD39 treatment significantly reduced the inhibitory function of hMSCs on the CD4 T cells. The expression of CD73 on the T cells was highly induced with co-culture of hMSCs. It suggests that hMSCs effectively suppressed the function of CD4+ T cells which was mediated by the CD39 and CD73.
Mechanism of combinatorial cytokine effects on dendritic cell state (P1200)
Authors: Hartmann Boris , Zaslavsky Elena , Marjanovic Nada , Moran Thomas , Sealfon Stuart
Published: January 2023

The effects of cytokine microenvironment on cellular response state are potentially important and relatively unexplored in the immune system. We focus on elucidating the combinatorial effects of extracellular signaling in the dendritic cells (Dc) and the mechanisms responsible for decoding these signals. Newcastle disease virus infected human dendritic cells in culture secret 21 extracellular factors for which they express receptors. Previously we showed that paracrine cytokines involving more than type I interferon released by virus-infected human Dcs dictate an antiviral maturation state of naïve Dcs. Microarray studies identified 260 genes that are differentially regulated (p≤0.05; >2-fold) by infected Dc supernatant compared to cells treated with IFNβ alone. Combinatorial cytokine studies indicate that IFNβ and TNFα are major factors that together are responsible for stimulating this state in Dcs. Linear regression modeling of microarray data identified 727 synergistically regulated genes and gene set enrichment analysis implicated STAT-5, STAT-6 and IRF-1. The combinatorial activation of these transcription factors was confirmed by imaging flow cytometry. These results suggest that the cytokine microenvironment generate Dc response states via the activation of combinatorial-responsive transcription factors. Combinatorial regulation mechanisms may provide the ability for localized control of the activation state of immune cells to spatially tune the response to infection.
Human metapneumovirus infection induces expression of thymic stromal lymphopoetin and IL-33 in airway epithelial cells in vitro and lung infiltration of OX40L+ DCs in vivo (P6200)
Authors: Lay Margarita , Cespedes Pablo , Palavecino Christian , Leon Miguel , Bueno Susan , Kalergis Alexis
Published: January 2023

Human metapneumovirus (hMPV) is a common virus known to induce acute respiratory tract infection. The specific mechanism by which hMPV modulates the immune response in infected airway epithelial cells (AECs) leading to tissue damage, still remains unknown. Thymic stromal lymphopoetin (TSLP) and IL-33, epithelial cell derived cytokines, induce high expression levels of OX40L on DCs, leading to Th2 responses and allergic inflammation. Recent reports have shown that Th2 inflammatory responses in RSV infections are mediated by TSLP-dependent OX40L expression in DCs. We hypothesize that the TSLP, IL-33 and OX40L pathway mediates lung inflammation in hMPV-infected individuals. We tested the expression of TSLP and IL-33 in human AECs after hMPV infection and compared them with RSV-infected AECs by qRT-PCR. We also evaluated lung infiltration of GR1+CD11b+ and CD11c+CD11b+OX40L+ cells in hMPV-infected mice by flow cytometry. We found an induction of TSLP mRNA levels in hMPV-infected AECs in a MOI dependent-manner. We also proved that hMPV induces higher TSLP mRNA levels than RSV in AECs, and hMPV but not RSV up-regulates IL-33 levels in AECs. Furthermore, we showed that hMPV infection in mice induces an increased lung infiltration of neutrophiles and OX40L+ DCs than the mock control group. These results suggest that hMPV infection in lungs may be inducing an augmented Th2 inflammatory response, through TSLP and IL-33 produced by hMPV-infected AECs and activated DCs expressing OX40L.
Narrowing of influenza A virus specific T-cell receptor alpha and beta repertoire with increasing age (P6176)
Authors: Gil Anna , Yassai Maryam , Naumov Yuri , Selin Liisa
Published: January 2023

Alterations in memory CD8 T cell responses may contribute to the high morbidity and mortality caused by seasonal influenza A virus (IAV) infections in older individuals. We questioned whether memory CD8 responses changed overtime with increasing age to this non-persistent virus, to which recurrent exposure with new strains is common. Here, for the first time we show that with age the HLA-A2-restricted IAV M158-66-specific Vα T cell repertoire was significantly narrowed leading to oligoclonal expansions including the usage of a single identical VA12 clonotype in all 8 older donors. The VA repertoire of older individuals also had longer CDR3 regions with increased usage of alanine/glycine runs which may enhance TcR promiscuity. Collectively these results suggest that CD8 memory responses in humans to non-persistent viruses like IAV are dynamic, and with aging become narrower with preferential retention of T cell repertoires with features of enhanced promiscuity.
B cell activation is regulated by the stiffness properties of the substrate presenting the antigens (P1152)
Authors: Liu Wanli , Wan ZhengPeng
Published: January 2023

B lymphocytes are activated upon the sensing of antigens by BCRs. The substrate presenting the antigens can show different degree of stiffness. However it is not clear if B cells can respond to substrate stiffness changes. In this report, we use high resolution, high speed live cell imaging techniques to capture the early events in B cell activation shortly after the recognition of antigens tethered to polyacrylamide gel substrates with variable degrees of stiffness as quantified by Young’s modulus (2.6-22.1 kPa). We show that the initiation of B cell activation is extremely sensitive to substrate stiffness. B cells exhibit much stronger activation responses when encountering antigens tethered to substrates with a high degree of stiffness as measured by the accumulation of BCRs, pSyk and pTyr molecules into B cell immunological synapse. Moreover, BCR and pSyk microclusters induced by antigens on stiffer substrate are more prominent and show significantly enhanced colocalization compared to activation with antigens on softer substrate. As a result, the expression of the B cell activation marker CD69 is greatly enhanced in B cells encountering antigens on stiffer substrate. Using a series of cytoskeleton inhibitors, we determine that the mechanosensing capability of B cell is strictly dependent on microtubules, and only mildly linked to actin cytoskeleton. These results suggest the importance of mechanical properties mediated by substrate stiffness in B cell activation.
TRAF6 regulates TCR signaling via interaction with and modification of LAT adapter (P1178)
Authors: Yingqiu Li , Xie Ji-Ji , Liang Jia-Qi , Altman Amnon
Published: January 2023

TNF receptor-associated factor 6 (TRAF6) is an essential ubiquitin E3 ligase in immune responses, but its function in adaptive immunity is not well understood. Here we show that TRAF6 is recruited to the peripheral ring of the T cell immunological synapse in Jurkat T cells or human primary CD4+ T cells conjugated with SEE-pulsed B cells. This recruitment depends on TRAF6 interacting with linker for activation of T cells (LAT) via its TRAF domain. Although LAT was indispensable for TCR/CD28-induced TRAF6 ubiquitination and its ligase activity, RNA interference-induced TRAF6 knockdown in T cells decreased TCR/CD28-induced LAT ubiquitination, tyrosine-phosphorylation. Overexpression of TRAF6 or its catalytically inactive form C70A promoted and decreased, respectively, LAT tyrosine-191 phosphorylation upon stimulation. Moreover, LAT was ubiquitinated at Lysine-88 by TRAF6 via K63-linked chain. In addition, TRAF6 was required for and synergized with LAT to promote the TCR/CD28-induced activation of NFAT. These results reveal a novel function and mechanism of TRAF6 action in the TCR-LAT signaling pathway distinct from its role in TCR-induced NF-κB activation, and indicate LAT also play an adapter role in TCR/CD28-induced activation of TRAF6.
Effects of sitagliptin on immune function of CD26/DPP-IV in T helper cells (P1190)
Authors: Yoon Hyunyee , Yang Man Gil , Seo Yu Rim , Sung Ji Hyun , Cho Su Hee , Lee Jae Eun , Song Moon Jung
Published: January 2023

CD26 is a multifunctional protein containing an ecto-domain with dipeptidyl peptidase (DPP-IV) activity and known to involve in T cell immune response. A membrane form of DPP-IV (mDPP-IV) is mainly expressed on the surface of T cells, while soluble DPP-IV (sDPP-IV) exists at a high level in blood. Sitagliptin, an sDPP-IV inhibitor is currently available for the treatment of the Type 2 Diabetes. However, whether or how the mDPP-IV activity of T helper cells contribute to their immune functions is not known. In this study, we investigated whether sitagliptin inhibits the mDPP-IV of T helper cells and consequently affects their immune functions when stimulated by pokeweed mitogen (PWM). Our results showed that PWM treatment alone strongly stimulated the expression of mDPP-IV and cytokines such as IL-2, IL-10, TNF-α, IFN-γ, IL-13, GM-CSF and MMP9 in CD4+CD26+ H9 cells. Although apoptosis was induced, the overall cell viability was marginally affected in PWM-treated H9 cells. On the other hand, sitagliptin co-treatment potently inhibited the mDPP-IV activity in a dose-dependent manner, but did not affect either the cell viability or cytokine profiling in PWM-treated H9 cells. The results suggest that T cell signaling pathways for cytokine expression function normally upon immune stimulation although sitagliptin effectively inhibits the mDPP-IV of T helper cell.
Modification of glycan chains as a mechanism to regulate SOCE (P1156)
Authors: Kilch Tatiana , Alansary Dalia , Dörr Kathrin , Peinelt Christine , Niemeyer Barbara
Published: January 2023

T cell receptor mediated activation requires the influx of calcium from the extracellular medium. Here, the endoplasmic reticulum (ER) Ca2+ sensor STIM1 reports the decrease in luminal [Ca2+] caused by IP3 receptor activity, clusters and directly binds and activates Orai1 channels, providing Ca2+ influx (store-operated, SOCE) essential for immune cell activation. We are interested in the regulation and modulation of this pathway. Because alterations of glycosylation of several T cell epitopes are implicated in immunsenescence and cancer, we investigated N-glycosylation site mutants of STIM1 and Orai1 and describe a STIM1 mutant (NN/DQ) which results in a strong gain-of-function. The mutant leads to a significant increase in the number of active channels despite a concomitant decrease in Orai1 protein, uncovering a novel CRAC channel regulatory mechanism. While we have already shown that the STIM1 mutant phenotype is mainly due to an increased rate of STIM1 oligomerization, we now investigate the specificity of this effect by analysing Ca2+ influx mediated by Orai2 and Orai3 and also started delineating the mechanism of Orai1 protein reduction. In addition, we are investigating the complex glycosylation pattern of primary human T cells and its effect on SOCE. Our findings indicate that the cell type specific repertoire of N-glycoslytransferases modifies the influence of the Orai1 glycan chains on Ca2+ influx.
Genome-wide analysis highlights the involvement of calcineurin/NFAT pathway signaling in transcriptional elongation in restimulated memory CD8 T cells (P1166)
Authors: Martinez Gustavo , Aijo Tarmo , Cruz-Guilloty Fernando , Ohora Masatsugu , Greenbaum Jason , Peteres Bjoern , Lähdesmäki Harri , Pipkin Matthew , Rao Anjana
Published: January 2023

Memory T cells respond to antigen encounter more robustly than naïve T cells, but the cell-intrinsic molecular mechanisms underlying this accentuated responsiveness are unknown. Here we show that in in vitro-generated memory-like CD8+ T cells, perforin (Prf1) gene transcription in response to secondary stimulation requires all NFAT transcription factors present in immune cells (NFAT1, NFAT2 and NFAT4), activation of the phosphatase calcineurin, and store-operated Ca2+ entry mediated by the ER Ca2+ sensors Stim1 and Stim2. The calcineurin inhibitor cyclosporine A (CsA) blocked transcriptional elongation through the Prf1 gene, with much less effect on recruitment of RNA polymerase II (Pol II) and transcriptional initiation. The generality of this effect was confirmed by genome-wide RNA-sequencing and chromatin immunoprecipitation for Pol II, NFAT1, H3K4me3 and H3K36me3 histone modifications, and cyclinT1, a component of positive transcription elongation factor b (P-TEFb). Of 1340 genes upregulated upon restimulation, 199 genes were CsA-sensitive, with the majority of these genes affected by transcription elongation rather than Pol II recruitment. CyclinT1 binding to the transcription start sites of restimulation-induced genes overlapped with NFAT1 occupancy and was CsA-sensitive. Thus, a substantial fraction of the rapid TCR-induced memory CD8 T cell responses is regulated by transcription elongation via calcineurin/NFAT signaling and recruitment of P-TEFb.
Autoregulatory CD8 T cells modulate CNS autoimmunity by targeting encephalitogenic CD4 T cells (P4130)
Authors: Ortega Sterling , Kashi Venkatesh , Tyler Andrew , Mendoza Jason , Cunnusamy Khrishen , Karandikar Nitin
Published: January 2023

Multiple Sclerosis (MS) is an inflammatory demyelinating disease with a preponderance of CD8 T-cells in central nervous system (CNS) lesions. We have previously shown that myelin-specific autoreactive CD8 T-cells are capable of suppressing MOG35-55 peptide-induced experimental autoimmune encephalomyelitis (EAE), a murine model of MS. We now report that myelin-specific CD8 T-cells can suppress CD4 T-cell-mediated EAE disease. Whereas in CD8-/- mice, increased EAE severity was associated with increased autoreactivity and inflammatory cytokine production by myelin-specific CD4 T-cells, the adoptive transfer of MOG35-55-specific CD8 T-cells was found to be sufficient for disease reversal. These autoregulatory CD8 T-cells required MHC Class Ia (KbDb) presentation both for in vitro activation and in vivo disease suppression. Interestingly, MOG35-55-specific CD8 T-cells could suppress adoptive EAE using wild type MOG35-55-specific CD4 T-cells induced in KbDb-/- recipients mice, suggesting direct targeting of encephalitogenic CD4 T-cells. Mechanistically, autoregulatory CD8 T-cell-mediated suppression is IFN-γ and perforin dependent and can be clinically augmented with in vitro IL-12 modulation. These studies define a novel and unexpected immune regulatory function for tissue-reactive “autoregulatory” CD8+ T-cells that can be harnessed for the development of immunotherapeutic intervention in MS and other autoimmune diseases.
Higher susceptibility to experimental autoimmune encephalomyelitis in Muc1-deficient mice is associated with increased Th1/Th17 responses (P4134)
Authors: YEN Jui-Hung , Xu Shuyun , Ganea Doina , Kim Kwang Chul
Published: January 2023

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system in which dendritic cells (DC) play an important role in the development of inflammatory responses. Recently it has been shown that Muc1, a membrane tethered glycoprotein, has an ability to suppress inflammatory responses in cultured DC. The objective of this study was to investigate the possible involvement of Muc1 in the development of MS using experimental autoimmune encephalomyelitis (EAE) in mice, a widely used animal model of MS. Our results showed that: (1) Muc1-/- mice developed greater EAE severity compared with wild type (wt) mice, which correlated with increased numbers of Th1 and Th17 cells infiltrating into the CNS; (2) Upon stimulation, splenic DC from Muc1-/- mice produced greater amounts of IL-1β, IL-6, and IL-12 but less amounts of IL-10 compared with those from wt mice; and (3) The ability of splenic DC to antigen-specific CD4+ T cells into Th1 and Th17 cells was greater in Muc1-/- mice compared with wt mice. We conclude that Muc1 plays an anti-inflammatory role in EAE. This is the first report demonstrating the possible involvement of Muc1 in the development of MS and might provide a potential target for immunotherapy.
Alternative splicing of FOXP3 controls regulatory T cell function and Th17 differentiation (P4118)
Authors: Mailer Reiner , Joly Ann-Laure , Liu Sang , Andersson John
Published: January 2023

Eukaryotic cells diversify gene function through alternative splicing, i.e. differential assembly of exons to create distinct protein isoforms. Alternative splicing is common for human FOXP3, the key transcription factor of regulatory T (Treg) cells that is indispensable for maintaining immunological tolerance. Previous studies have demonstrated that Treg cells can exclude exon 7 of FOXP3, which encodes for a leucine zipper region that sustains the Treg cell phenotype. Here we demonstrate that patients suffering from Crohn’s disease (CD) have elevated levels of FOXP3 lacking exon 7, which correlates with disease severity. Moreover, by modulating the pattern of alternative splicing towards exclusion of FOXP3's seventh exon we were able to generate interleukin-2 (IL-2) producing Treg cells. FOXP3 is transiently expressed during Th17 differentiation and we found that exclusion of FOXP3's seventh exon promoted Th17 differentiation. Finally, we demonstrate that expression of FOXP3 lacking exon 7 correlates with increased expression of IL-17 in CD patients. In summary, alternative splicing of FOXP3 regulates both the regulatory T cell phenotype and Th17 differentiation.
iVAX: a sophisticated suite of online vaccine design tools (P3269)
Authors: De Groot Anne , Terry Frances , Spero Denise , Moise Leonard , Martin William
Published: January 2023

Emerging and re-emerging infectious diseases represent a significant challenge for next-generation vaccine design and bioterror preparedness. We have composed a suite of online immunoinformatics tools for accelerated design of genome-derived, epitope-driven vaccines generated from protein sequences. Using the Conservatrix algorithm, even the most mutable pathogenic genomes may be probed for highly conserved segments, which are then mapped for T cell epitopes and regions of high epitope density using EpiMatrix and ClustiMer. Pathogen sequences which could potentially elicit an undesired autoimmune or T-regulatory response due to homology with sequences encoded by the human genome are screened out using BlastiMer and JanusMatrix, an improved homology analysis tool examining pathogen/host sequence similarity with respect to the HLA and TCR faces of an epitope. Immunogenic Consensus Sequences are created by EpiAssembler, a tool which optimizes the balance between pathogen and population coverage. VaccineCAD links potential vaccine candidate epitopes into a string-of-beads design while minimizing non-specific junctional epitopes that may be created in the linking process. With proof of principle established in animal models for vaccines against Tularemia, Vaccinia and H. pylori, the iVAX toolkit exemplifies a rapid, efficient, easily accessible and broadly applicable solution to accelerate the development of critically important vaccines for human health and biodefense.
Production of fully human monoclonal antibodies to therapeutic self-antigenic targets (P3285)
Authors: Sutkowski Natalie , Sun Wei , Fernandes Daniel , Rubinchik Semyon
Published: January 2023

Not all autoreactive antibodies are harmful, despite their role in autoimmune disease. Autoantibodies targeting tumor antigens, growth factors and their receptors are currently used therapeutically for treatment of cancer and autoimmunity. Ordinarily, healthy subjects do not exhibit strong secondary antibody responses to self-proteins, because most autoreactive B cells do not receive sufficient T cell help required for the germinal center reaction and for differentiation into IgG secreting cells. Our data demonstrate that rare, potentially therapeutic autoreactive B cells can be efficiently rescued from tonsil with EBV immortalization, and manipulated in vitro to secrete IgG, by supplying factors that mimic antigen stimulation and T cell help. Because this process is performed wholly in vitro, it constitutes a rapid and cost effective technology, resulting in the isolation of fully human antibodies. In addition, antibodies can be generated against highly toxic antigens, since immunization is unnecessary. As proof of concept, we have developed a panel of human monoclonal antibodies targeting the self-tumor antigen nucleolin for use in cancer immunotherapy. Nucleolin is normally expressed in the nucleus of all cells, but it is over-expressed in both the cytoplasm and on the cell surface of tumor cells in the most common forms of breast cancer, making nucleolin a new type of tumor antigen. The antibodies are currently undergoing pre-clinical testing against breast cancer.
Deep-sequencing reveals WHO class-specific urinary microRNAs in human lupus nephritis (P3245)
Authors: Goilav Beatrice , Ben-Dov Iddo , Loudig Olivier , Blanco Irene , Wahezi Dawn , Putterman Chaim
Published: January 2023

Lupus nephritis (LN) is associated with significant morbidity and mortality. microRNAs (miRs) are small, non-coding RNAs that regulate translation. Previous, studies report changes in miR expression in kidney tissue, urine and PBMC that correlate with LN disease activity. However, LN WHO class-specific miRs have not been described. Using deep-sequencing, we aimed to identify WHO class-specific miRs in urine from adult and pediatric patients with biopsy-proven LN. Cell-free urine from adult (n=25) and pediatric (n=8) female patients with WHO class IV and V LN were obtained at time of active disease and during remission. Total RNA was used to prepare small RNA cDNA libraries for sequencing. Multiplexing through sample-specific 3' adapters was applied. Sequence reads were mapped to the human genome and small RNA databases. miRs were quantified by relative read abundance. We obtained reproducible profiles of miRs. In a paired-sample analysis comparing miR abundance in urine of adult and pediatric patients with active class IV versus class V LN, we found significant changes in 6 miRs, including up-regulation of miR-193a-5p, -423, 501-3p, and -874 by 400-1,000x in WHO class IV LN. In conclusion, we detected higher counts of specific miRs in urine of adult and pediatric patients with LN class IV, versus class V. Given the worse prognosis of class IV LN identifying miRs associated with this LN class is an important step in biomarker discovery of this particulary aggressive disease.
IL-22 is a target for the treatment of anaplastic lymphoma kinase-positive anaplastic large cell lymphoma (P6296)
Authors: Lim Chrissie , McFarland Adelle , Savan Ram
Published: January 2023

We have shown that leukemic T cells from anaplastic lymphoma kinase-positive anaplastic large cell lymphoma (ALK+ALCL) patients aberrantly express IL-22R1, the private receptor for IL-22, on their surface. This causes these cells to resemble a Th17 inflammatory cell type. The expression of IL-22R1 leads to IL-22 production, which then feeds back in an autocrine manner. This signaling also stimulates IL-17 in these cells that results in damaging systemic inflammation, a common complication seen in ALK+ALCL patients. We have shown that IL-22 may be a potential prognostic marker as it is likely a survival factor for the leukemic cells. The objective of the current study is to identify the role of the IL-22/IL-22R1 signaling pathway in ALK+ALCL development and during treatment. Since forced expression of IL-22R1 is sufficient to skew T cells to a Th17 phenotype and induce neutrophilia in vivo, targeting IL-22 may reduce inflammatory disease in ALK+ALCL patients. As IL-22 has proliferative effects, we hypothesized that IL-22 contributes to persistence of ALK+ALCL. We show that neutralizing anti-IL-22 antibody has a cytotoxic effect against an ALK+ALCL cell line when combined with a monoclonal anti-CD30 antibody. If neutralization of IL-22 shows therapeutic promise in our ALK+ALCL model, it could have rapid translational turnover.
E. coli fimbriae serve as an IL-35 agonist stimulating indoleamine 2,3 dioxygenase for protection against collagen-induced arthritis (P5154)
Authors: Kochetkova Irina , Maddaloni Massimo , Thornburg Theresa , Hoyt Teri , Pascual David
Published: January 2023

Orally delivered colonization factor antigen I (CFA/I) fimbriae stimulate IL-35 producing Foxp3+CD39+CD4+ regulatory T cells (Tregs) essential against CIA in C57BL/6 mice. To begin to understand how CFA/I-induced IL-35 mediates protection, recent studies have begun to examine immunosuppressive mediators by dendritic cells (DCs). It was found that IDO was induced in splenic, mesenteric lymph nodes (MLNs), and inguinal, axillary, and iliac lymph nodes (LNs) DCs in protected mice. We hypothesize that CFA/I-induced IL-35 could directly regulate development of IDO+ DCs to sustain anti-inflammatory responses. By flow cytometry, recombinant IL-35 was found capable of binding DCs in vitro suggesting direct action of IL-35 upon DCs. Mice deficient in IL-35 (EBI3-/-) were unresponsive to CFA/I fimbriae and unable to induce IDO. Similar results were observed in CD39-/- mice correlating with the lack of clinical interventions. To inhibit IL-35's impact, CFA/I- or PBS-dosed C57BL/6 mice were treated in vivo with rabbit IgG anti-mouse IL-35 during arthritis manifestation resulting in 50% suppression of intracellular IDO expression along with significantly enhanced CIA severity when compared to nonimmune IgG-treated mice. Adoptive transfer of CFA/I-induced CD39+CD4+ Tregs to mice induced with CIA elevated IDO expression by DCs. Thus, our findings demonstrate the effect of induced IL-35 by Tregs upon IDO+ DCs in conferring protection against CIA. Supported by NIH P01 AT-04986.
Dissecting the molecular mechanisms for the increased risk of T1D in HLA-DQ8/DQ2 heterozygous individuals (P5182)
Authors: Chow I-Ting , James Eddie , Gates Theresa , Yang Junbao , Torres-Chinn Nadia , Kwok William
Published: January 2023

HLA-DQ8trans is the HLA molecule that confers the highest risk for human type 1 diabetes (T1D), yet the reason for this risk remains elusive. Using tetramer-guided epitope mapping, we discovered a high degree of peptide overlap between DQ8 and DQ8trans for antigens from influenza A viruses, with only one peptide exclusively presented by DQ8trans. Among influenza epitopes shared by DQ8, DQ2 and DQ8trans, cross-reactivity was observed between DQ8 and DQ8trans, but not between DQ8 and DQ2 or between DQ8trans and DQ2. In agreement with foreign antigen observations, glutamic acid decarboxylase 65 (GAD65)-, and islet tyrosine phosphatase (IA-2)-specific T cell clones isolated from DQ8 or DQ8/DQ2 patients responded to peptides loaded on DQ8 or DQ8trans molecules. The autoantigenic peptides bound to DQ8 and DQ8trans in the same binding registry, but peptides presented by DQ8trans bound with higher affinity to both T cell clones and in vitro expanded T cells. Proliferation assays and cytokine ELISA revealed that DQ8trans elicited stronger functional responses from cross-reactive autoantigenic T cells and created an environment that favored the expansion of self-reactive T cells by preferentially promoting the secretion of IFN-gamma from cross-reactive T cells. Overall, our data suggest that the presence of higher affinity HLA-DQ8trans molecules enhances self-reactive DQ8 and DQ8trans cross-reactive T cell responses, leading to increased susceptibility to T1D in DQ8/DQ2 individuals.
NADPH oxidase 2 activity modulates antigen processing and epitope repertoires (P5186)
Authors: Lemmon Jason , Balce Dale , Rybicka Joanna , Yates Robin
Published: January 2023

Phagocytes such as macrophages and dendritic cells are essential to both the innate and acquired immune systems of mammals. To produce effective immune responses, material antigens must first be processed and presented by phagocytes. The importance of antigen processing is often overlooked, which is exemplified by current therapies focusing on downstream immune processes in T cells and B cells. However, these therapies often result in modulation of the immune system as a whole. Instead, it would be ideal to modulate antigen processing to prevent the generation of epitopes able to stimulate and re-stimulate specific T cells. Antigen processing within phagosomes is a dynamic process influenced by several environmental factors such as pH and oxidative burst. Of particular importance to our lab is the oxidative burst generated by the protein complex NADPH oxidase 2. Our lab has previously published data showing that regulation of NADPH oxidase 2 activity results in modulation of phagosomal proteolysis through modulation of cysteine cathepsin activity. We further hypothesize that protein antigen processing and epitope repertoire generation can be predicted and more importantly influenced by changing the activity of NADPH oxidase 2. Using a combination of fluorometric data, HPLC, and mass spectrometry we have begun to show, through preliminary data, that epitope repertoires can indeed be influenced without knocking down antigen processing and presentation as a whole.
Modeling the effect of PKCθ on T cell motility in lymph nodes (P5192)
Authors: Cannon Judy , Asperti-Boursin Francois , Oruganti Sreenivasa , Letendre Kenneth , Moses Melanie
Published: January 2023

The adaptive immune response is an integrated process that requires the interaction of T lymphocytes and antigen-presenting cells (dendritic cells) within the context of lymph nodes. Under homeostatic conditions, T lymphocytes actively “scan” the lymph node cortex, interacting with multiple dendritic cells (DCs) in search of cognate antigen. T cell motility is driven by CCR7-CCL21/19 and this chemokine-driven motility dictates the rate of interaction between T cells and DCs in lymph nodes and sets the properties of T cell - dendritic cell encounters. We recently found that CCR7 signaling alone without T cell receptor stimulation can activate PKCθ in naïve T cells. Using 2-photon imaging as well as in vitro migration assays, we show that PKCθ is required for optimal T cell migration. We have now generated a computer model to recapitulate the motility of both wild type and PKCθ-deficient T cells in the lymph node. By comparing the behavior of WT versus PKCθ-deficient T cells in our model, we will be able to determine whether the absence of PKCθ compromises interactions between T cells and dendritic cells in the lymph nodes, and therefore the initiation of an adaptive immune response. Our model will allow us to test not only the role for PKCθ in regulating T cell search for antigen, but also the role for other signaling molecules that may regulate T cell motility in vivo.