bluuebunny/crossref_metadata_2025 huggingface dataset:
Vitamin B1 is an essential survival factor for naïve B cells but not IgA-secreting plasma cells (120.9)
Authors: Wake Taichi , Kunisawa Jun , Hashimoto Eri , Suzuki Yuji , Kiyono Hiroshi
Published: January 2023

It is generally accepted that dietary factors contribute in the regulation of various immunological functions. Among different kinds of dietary materials, vitamin families such as vitamin A, B6, B9, and D3 play critical regulatory roles for immune responses, but the immunological functions of the other vitamins remain largely unknown. Here, we demonstrate that vitamin B1, also known as thiamine, is an essential factor to keep naïve B cells but not IgA-secreting plasma cells. When mice were maintained with vitamin B1-deficient diet, IgM+ B220+ naïve B cells were significantly decreased in the spleen, mesenteric lymph nodes, and Peyer’s patches, while B220- CD138+ plasma cells secreting IgA antibodies were normally present in the small intestine. In agreement with this finding, despite the reduction of naïve B cells, levels of antigen-specific intestinal IgA responses were unchanged in mice receiving vitamin B1(-) diet after oral immunization with Choleratoxin. Similarly, IgA-secreting cells normally produced IgA even in the absence of vitamin B1 in vitro. These findings demonstrate a new immunological function of vitamin B1 at the different B cell stages.
ZEB2 plays a role in promoting T-bet expression and terminal differentiation of CD8+ T cells (121.10)
Authors: Dominguez Claudia
Published: January 2023

We are interested in deciphering the transcriptional programs that underlie the differentiation of the effector CD8 T cell response. Prior work has shown T-bet operates in a gradient fashion, with higher T-bet expression being critical for the development of terminal effector (TE) CD8 T cells. Expression profiling revealed that T-bet regulates ZEB2, a transcriptional repressor that is expressed 10 fold higher in TE cells compared to memory precursor (MP) effector CD8 T cells. To further investigate what role ZEB2 might play in promoting terminal differentiation of CD8 T cells we conditionally knocked out ZEB2 in activated CD8 T cells using Granzyme B Cre ZEB2 flox/flox mice (ZEB2 KO). Upon infection with LCMV, the CD8 T cell expansion in the ZEB2 KO was about half that of wild type mice. The ZEB2 KO CD8 T cells showed an accelerated maturation of the effector CD8 T cell compartment to a memory phenotype with increased IL-2 production and the acquisition of CD62L. Further analysis revealed that these phenotypes might be largely due to the reduction of the TE subset. In the absence of ZEB2, TE CD8 T cells adopt some MP characteristics such as CD27, CXCR3, and Eomes but reduced T-bet and Blimp-1. Our observations suggest that ZEB2 is critical for the maintenance of the transcriptional program that drives terminal differentiation including T-bet. Hence, we propose that ZEB2 and T-bet operate in a positive-feedback mechanism to drive CD8 T cells towards terminal differentiation.
Transcriptional regulator Id2 is required for the CD4 T cell immune response in the development of experimental autoimmune encephalomyelitis (121.12)
Authors: Lin Yen-Yu , Jones-Mason Mary , Inoue Makoto , Iavarone Antonio , Lasorella Anna , Shinohara Mari , Zhuang Yuan
Published: January 2023

Inhibitor of DNA binding 2 (Id2) is an important transcription regulator in lymphocytes. By forming heterodimers with E proteins, Id2 can inhibit E protein-mediated gene regulation, which is critical in controlling many steps of lymphocyte development. However, its role in mature CD4 T cell function is not well-characterized. Here we show that Id2 expression is higher in activated CD4 T cells. Utilizing a T cell-specific conditional Id2 knock-out mouse model, we find that Id2 is essential for the development of experimental autoimmune encephalomyelitis (EAE). Id2-deficient CD4 T cells fail to infiltrate the central nervous system, form a smaller pool of effector cells in the peripheral lymphoid organs and have increased cell death. These CD4 T cells express higher levels of Suppressor of cytokine signaling 3 (SOCS3), a negative regulator of T cell response and a potential target of E proteins. This is the first evidence showing an indispensible role of Id2 in the development of a Th17-mediated autoimmune disease.
Severe sepsis results in modulated chromatin modifying enzyme expression in bone marrow resident hematopoietic stem cells (121.15)
Authors: Carson William , Takahashi Sakuma , Allen Ronald , Kunkel Steven
Published: January 2023

Uncontrolled inflammatory responses drive the high mortality observed in severe sepsis and septic shock. Paradoxically, survivors of severe sepsis exhibit profound cellular immunosuppression, resulting in increased susceptibility to secondary, opportunistic and nosocomial infections. Recent studies have identified epigenetic repression of genes in peripheral immune cells post-sepsis as one possible mechanism underlying this immunosuppression. As post-septic immunosuppression can manifest long after recovery from sepsis, it was hypothesized that any observed epigenetic regulation of pro-inflammatory genes may also occur in hematopoietic stem cells, and in turn, these changes would be due to the up- or down-regulation of chromatin modifying enzymes (CMEs). Superarray analysis of mRNA isolated from CD117+ hematopoietic stem cells isolated from animals subjected to an experimental model of sepsis identified a distinct set of modulated mRNAs encoding distinct histone modifying enzymes - including methyltransferases (Dot1l, Prmt8 and Smyd1) and demethylases (Kdm5b), among others. These results indicate that severe sepsis can modulate the expression of CMEs in hematopoietic stem cells, suggesting distinct changes in the epigenetic signature of these cells. As these CMEs are associated with distinct histone modifications, these results can be used to guide further investigations into the genes targeted by these distinct epigenetic marks.
Multiphoton intravital microscopy analysis of signaling in T cells reveals persistent NFAT activation during dynamic APC scanning. (121.21)
Authors: Marangoni Francesco , Murooka Thomas , Manzo Teresa , Elpek Natalie , Mempel Thorsten
Published: January 2023

The Ca2+ dependent activation and nuclear shuttling of the cytoplasmic transcription factor NFAT is a hallmark of productive T cell receptor (TCR) triggering. However, the kinetics of NFAT activation and the relationship to the Ca2+ dependent migratory stop signal have not been explored in vivo. We used Multiphoton Intravital Microscopy (MP-IVM) to visualize the subcellular dynamics of a fluorescent reporter of NFAT signaling (NFAT-eGFP) in cytotoxic T cells during their antigen-dependent interactions with various APC in lymph nodes (LNs) and in tumor tissue in vivo. During interactions with peptide-loaded B cells representing optimal APC in LNs, t1/2 of NFAT nuclear translocation was 1 minute. Relocation to the cytoplasm upon contact cessation occurred with a slower t1/2 of ~20 minutes. During its delayed nuclear export, NFAT continued to drive transcription of IFN-gamma. We therefore hypothesized that NFAT may facilitate the integration of TCR signals received through serial APC contacts. To test this in vivo, we performed MP-IVM to visualize contacts between cytotoxic T cells and tumor cells, representing suboptimal APCs. We observed that among T cells with continuously nuclear NFAT, ~30% remained motile while engaging in transient, serial contacts with multiple tumor cells. Hence, NFAT slow off-kinetics may constitute a mechanism of short-term biochemical memory enabling the integration of intermittent TCR signaling into a continuous transcriptional response.
A novel function of TRAF6 in TCR signaling (121.26)
Authors: Li Yingqiu , Xie Ji-Ji
Published: January 2023

As an essential ubiquitin E3 ligase in immune responses, the function and mechanism of TNF receptor-associated factor 6 (TRAF6) in adaptive immunity has not been illustrated as well as in innate immunity. Here we show that upon activation of T cells, TRAF6 is recruited to the peripheral ring of the T cells immunological synapse (IS), and is invovled in CD3/CD28-induced NFAT activation. The underlying mechanism is under study.
The SKAP55 dimer stabilizes TCR-induced SLP-76 microclusters (121.27)
Authors: Ophir Michael , Nguyen Ken , Bunnell Stephen
Published: January 2023

The adapter molecule SKAP55 enables the formation of T-cell:APC conjugates, facilitates T-cell cytokine production, and is required for optimal T cell proliferation in response to antigen. However, the molecular basis for the involvement of SKAP55 in these events remains unclear. Here, we demonstrate that both endogenous SKAP55 and exogenous SKAP55 chimeras enter T cell receptor (TCR)-induced signaling microclusters containing the adapter protein SLP-76. Using a SKAP55-deficient Jurkat T cell line, we demonstrate that SKAP55 is required for both the persistence and the movement of SLP-76 microclusters. Furthermore, the recruitment of SKAP55 into microclusters requires the presence of both SLP-76 and ADAP, an intermediary adapter protein capable of interacting with both SLP-76 and SKAP55. In addition, we show that SKAP55 homodimerizes via an N-terminal dimerization motif (DM). Our structure-function analyses indicate that the dimerization domain and the ADAP-interacting Src-homology domain 3 (SH3) domain of SKAP55 are both required for the recruitment of SKAP55 into SLP-76 microclusters, and for the stabilization of SLP-76 microclusters. Importantly, an artificial tandem dimer composed of two functional SH3 domains joined by a flexible linker was sufficient for entry into, and stabilization of these signaling complexes. This data indicates that SKAP55 could contribute to T-cell activation by increasing the overall avidity and stability of TCR-induced SLP-76 microclusters.
Distinct role of RIAM structural elements on IL-2 transcription (121.4)
Authors: Shaw Amy , Patsoukis Nikolaos , Boussiotis Vassiliki
Published: January 2023

RIAM is a novel adaptor that regulates actin polymerization, cell adhesion and PLC-γ1 activation. RIAM contains two coiled-coil regions in the N-terminus, a small proline-rich N-terminus region, a Ras-association (RA) domain, a pleckstrin homology (PH) domain and a large proline-rich C-terminus region. Using RIAM shRNA we determined previously that RIAM is mandatory for IL-2 transcription. The role of the distinct RIAM structural elements in this function remained unclear. In the present study we examined whether RIAM domains have distinct roles in regulating IL-2 transcription and whether this effect is mediated via specific transcription factor binding elements of the IL-2 promoter. Stable Jurkat T cells overexpressing RIAM displayed a dramatic increase in IL-2 transcription as determined by a lusiferase reporter construct driven by the entire IL-2 promoter. Expression of luciferase reporters driven either by AP-1, NFAT or NF-kB promoter elements revealed that NFAT was the dominant target of RIAM. By using various RIAM deletion mutants we found that the effects of RIAM on IL-2 transcription require the RA and PH domains in conjunction with the C-terminus region, while the N-terminus region of RIAM is dispensable for this function. Thus, RIAM promotes IL-2 production by regulating predominantly NFAT-mediated transcription and this effect requires the RA, PH and the C-terminus region of RIAM.
The Splicing Factor ASF/SF2 enhances Interleukin-2 production in human T lymphocytes (121.5)
Authors: Moulton Vaishali , Tsokos George
Published: January 2023

Systemic lupus erythematosus (SLE) is a complex autoimmune disease which afflicts mainly women in the reproductive years, and causes painful arthritis, skin disease and complications in the kidneys and brain. Abnormal T lymphocytes in SLE not only regulate autoantibody-producing B cells, but are also responsible for target organ infiltration. Currently, no specific therapeutic strategies target T cell defects. Moreover, there are no known molecular markers to predict disease activity. Aberrant molecular mechanisms responsible for SLE T cell defects are thus promising targets for therapy, as well as potential biomarkers for disease. T cells from patients with SLE express decreased levels of the T cell receptor (TCR) - associated CD3 zeta (ζ) chain, a feature directly linked to their poor interleukin (IL)-2 production. We recently showed that the splicing regulator alternative splicing factor/ splicing factor 2 (ASF/SF2) enhances the expression of CD3ζ chain by limiting the production of an unstable mRNA splice variant. Here, we show that ASF/SF2 expression levels are decreased in T cells from SLE patients, and correlate with CD3ζ chain expression. Moreover, ASF/SF2 expression correlates inversely with SLE disease activity index (SLEDAI). Further, replenishment of ASF/SF2 in SLE T cells restores IL2 production. These results identify ASF/SF2 as a novel regulator of IL2 expression in human T cells and a potential molecular mechanism underlying the altered T cell defect in SLE.
Function of Syntaxin11 in cytotoxic T lymphocytes (121.9)
Authors: Halimani Mahantappa , Pattu Varsha , Junker Christian , Marshall Misty , Matti Ulf , Schwarz Eva , Krause Elmar , Hoth Markus , Rettig Jens
Published: January 2023

Cytotoxic T Lymphocytes (CTLs) form immunological synapses (IS) with antigen presenting cells to kill them. Killing requires the fusion of lytic granules at the IS to release the containing cytotoxic molecules (perforin and granzymes). The fusion process is mediated by SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins. Since mutations within the coding region of syntaxin11 lead to Familial Hemophagocytic Lymphohistiocytosis (FHL4), an immune disorder caused by impaired granule exocytosis. We have investigated the contribution of syntaxin11 to lytic granule release. Using CD3 specific antibodies as a marker for a functional IS, we show that syntaxin11 accumulates at the IS in CTLs. Syntaxin11 was also co-localized with Munc18-2, a docking factor in agreement with the role of Munc18-2 as a syntaxin chaperone in other cell types. Using high resolution nanoscopy (structured illumination microscopy) we determined the subcellular localization of syntaxin11 in CTLs with a syntaxin11 specific antibody. Syntaxin11 was associated with recycling endosomes and mannose 6-phosphate receptor (M6PR) positive compartments. Functional assays using Syntaxin11 specific siRNA (knockdown studies) show a reduction in degranulation and killing efficiency in contrast to control siRNA transfected CTLs. These results support the conclusion that syntaxin11 is required for lytic granule exocytosis.
Naïve anti-Id B cells and Id-specific CD4+ T cells collaborate efficiently to non infectious antigens in the absence of dendritic cells. (122.12)
Authors: Jacobsen Johanne , Schjetne Karoline , Munthe Ludvig , Bogen Bjarne
Published: January 2023

It is thought that activated dendritic cells are needed for induction of T cell responses. However, this makes it difficult to explain immune responses to self-like molecules such as allotypes and idiotypes where no activation of dendritic cells should be involved. We have here generated a double knock-in mouse that expresses prearranged H and L chain V regions of an anti-Id B cell receptor (BCR). By use of anti-Id B cells from this mouse, together with Id-specific CD4+ T cells from T cell receptor (TCR)-transgenic mice, we show that induction of isotype-switched anti-Id Abs depends on the presence of anti-Id B cells, Id-specific T cells, and Id+Ig. Naïve B and T cells were sufficient, and there was no need for MHC-matched dendritic cells. Responses were obtained with physiologically relevant concentrations of B and T cells, and non-adjuvanted Id+ Ig. These results demonstrate that conventional T-B collaboration explains induction of immune responses to non infectious, polymorphic self-like molecules and that dendritic cells are not required.
Repetitive peptide boosting induces functional memory cytotoxic T lymphocytes of a new phenotype (122.4)
Authors: Smyth Kendra , Garcia Karla , Sun Zhifeng , Tuo Wenbin , Xiao Zhengguo
Published: January 2023

Induction of functional memory cytotoxic T lymphocytes (CTLs) holds promise for fighting critical infectious diseases through vaccination, but so far, no effective regime has been identified. One round of immunization is normally not sufficient for generating functional memory CTLs, and subsequent boosting is required. We show here that repetitive intravenous boosting with peptide and adjuvant following priming progressively enhances memory CTLs and drives their differentiation to a unique and long-lasting effector memory phenotype characterized by decreased interferon-γ but increased granzyme B production. The resulting memory CTLs are clearly not immune senescent but are functional and able to provide protection against pathogen challenge. Furthermore, similar enhancement of functional memory CTLs can be achieved regardless of the adjuvant used. Overall, our results suggest that intravenous peptide boosting with adjuvant following priming may be a viable method for generating long-term functional memory CTLs through vaccination.
CD40 Engagement Ablates CTLA-4 expression on Th40 pathogenic cells: A Mechanism for Breach of Tolerance (122.5)
Authors: Wagner David
Published: January 2023

Biomarkers defining pathogenic effector T cells slowly have been forthcoming. Towards this we identified CD4+ cells that express CD40 (CD4CD40), defining these cells as pathogenic in the NOD type 1 diabetes (T1D) model where these cells are unique in their ability to adoptively transfer T1D. To study the origin of CD4CD40 cells and disease pathogenesis we employed a dual transgenic model expressing OVA323-339 peptide as a neo-self antigen on islet beta cells and medullary thymic epithelial cells (mTEC) and a transgenic TCR recognizing the OVA323-339 peptide. CD4CD40 cells and Tregs each recognizing the cognate neo-antigen, rather than being deleted through central tolerance drastically expand in the thymus. DO11.RIPmOVA mice develop autoimmune-type diabetes. In pancreatic lymph nodes CD4CD40 cells are greatly expanded while Tregs remain equivalent to controls and importantly remain functional throughout the disease process, suggesting another breach of tolerance mechanism. DO11.RIPmOVA CD4CD40 cells do not express CTLA-4, a molecule for cell auto-regulation, while DO11 Th40 cells do. CD3/CD28 engagement induces CTLA-4 cell surface expression on DO11 CD4CD40 cells while CD40 co-engagement ablates that induction. Injected antigen (OVA) challenge induces CTLA-4 on CD4CD40 cells while the same antigen expressed endogenously does not. Thus the mechanism of antigen exposure dictates potential tolerance. These data suggest a unique means for CD4CD40 cells to thwart tolerance.
Effects of depletion of CD4+FoxP3+ Treg on development of spontaneous autoimmune thyroiditis (SAT) in WT and B cell deficient (B-/-) NOD.H-2h4 mice (123.10)
Authors: Ellis Jason , Braley-Mullen Helen
Published: January 2023

NOD.H-2h4 mice given NaI in their water develop SAT with chronic inflammation of the thyroid by T and B cells. B-/- mice are resistant to SAT, but develop SAT after transient depletion of Treg by anti-CD25. However, NOD.H-2h4 mice have significant numbers of CD4+FoxP3+ Treg that are CD25-. Generation of mice expressing the human diphtheria toxin receptor (DTR) under control of the FoxP3 promoter allows for selective depletion of Foxp3+ cells by diphtheria toxin (DT). Anti-CD25 depletion of Treg lasts 3-4 weeks, whereas most Treg return 4-5 days after DT treatment. In WT and B-/- FoxP3-DTR NOD.H-2h4 mice, injection of DT eliminates essentially all Treg in blood and spleen within 2 days. As with anti-CD25 depletion, DT treatment allows for SAT development in B-/- mice even though Treg are depleted for only a few days, and SAT in DT-treated WT mice is slightly increased. We established a transfer model in which splenocytes from CD28-/-B-/- mice (effector cells) were cultured with or without sorted Treg from FoxP3-DTR WT or B-/- mice. After transfer to TCRα-/- mice, recipients of Treg from B-/- mice developed significantly less severe SAT than did mice receiving Treg from WT mice. As there is no source of new FoxP3-DTR Treg, DT treatment 2 days after transfer to recipient mice resulted in permanent depletion of Treg. This abrogated the suppression, suggesting that suppression of SAT requires continued inhibition of effectors by Treg during disease development.
Spontaneous development of encephalitogenic T cells is regulated by age-dependent induction of tolerance (123.11)
Authors: Ito Naoko , Boppana Sridhar , Mindur John , Dhib-Jalbut Suhayl , Ito Kouichi
Published: January 2023

Multiple Sclerosis (MS) is an autoimmune disease against CNS and disease onset most often occurs in young adults, suggesting that aging is a critical factor for the development of MS. We examined the effect of aging on the induction of tolerance in the CNS-specific T cells and their differentiation into pathogenic Th1 and Th17 cells in the transgenic (Tg) mice that express HLA-DRB5*0101 and MBP-specific human TCR (3A6) genes isolated from an MS patient. The 3A6-TCR Tg CD4 T cells undergo thymic and peripheral deletion with aging and the surviving CD4 T cells undergo anergy in the periphery. In spite of induction of age-dependent tolerance induction in the MBP-specific CD4 T cells, 3A6 Tg CD4 T cells differentiate into Th1 and Th17 cells in the thymus, migrate into the periphery, and further differentiate into Th1 and Th17 encephalitogenic T cells spontaneously in 5-10-week-old young adult mice. However, increase in age-dependent tolerance reduces the spontaneous development of encephalitogenic T cells. These data suggest that spontaneous development of encephalitogenic T cells is dependent on the balance between age-dependent tolerance induction and spontaneous development of CNS-specific Th1 and Th17 cells.
HLA-DQ trans heterodimer association to type 1 diabetes in East Asian population (123.15)
Authors: Miyadera Hiroko , Ohashi Jun , Tokunaga Katsushi , Japan Diabetes Society Japan Committee on Type Diabetes,
Published: January 2023

HLA-DQ provide the strongest genetic contributions to susceptibility and resistance to type 1 diabetes (T1D). In Europeans, haplotypes carrying non-Asp57β polymorphism in DQB1 (DQA1*03-DQB1*03:02, DQA1*05-DQB1*02) associate strongly to the susceptibility to T1D, while T1D-risk haplotypes in East Asians encode Asp57β (DQA1*03-DQB1*03:03/*04:01). The distinct haplotype associations are ascribed to differences in HLA-DQ-DR haplotype composition and distribution across populations. To understand the mechanistic basis of T1D pathogenesis in East Asian population, we evaluated the disease association of DQA1-DQB1 combinations in trans in T1D case/control samples in Japanese population, which was collected and genotyped by the Committee on Type 1 Diabetes, Japan Diabetes Society. The DQA1 alleles were estimated from DRB1-DQB1 haplotype distributions in general populations, and trans HLA-DQ dimer formation was estimated from the dimerization profile for the entire HLA-DQ alleles (data not shown). Homozygous individuals were also assumed to form trans dimers (dimers identical to cis haplotype) and counted as "trans", in evaluating the associations to T1D. The preliminary result do not suggest the contributions of particular trans DQA1-DQB1 combinations to T1D. The association analyses using the larger sample size will be presented and discussed with the potential mechanistic basis of T1D in Asians.
DEC-205-mediated antigen targeting to steady-state dendritic cells induces deletion of cognate CD8+ T cells independently of the PD-1 pathway (123.19)
Authors: Mukherjee Gayatri , Geliebter Ari , Sharpe Arlene , Freeman Gordon , DiLorenzo Teresa
Published: January 2023

Type 1 diabetes (T1D) is an autoimmune disease characterized by T cell-mediated destruction of the pancreatic islet beta cells. Cytotoxic CD8+ T cells specific for beta cell antigens are a main contributor to T1D development. CD8+ T cells specific for insulin and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) epitopes have been implicated in T1D development in both patients and NOD mice. Targeted delivery to steady-state dendritic cells (DCs) of a beta cell peptide mimotope linked to anti-DEC-205 leads to deletional tolerance of transferred cognate CD8+ T cells in NOD mice. Treatment with anti-DEC-205 linked to NRP-V7, a superagonist mimotope of IGRP206-214, selectively deleted endogenous IGRP206-214-specific T cells, a highly prevalent specificity in NOD mice, and showed promise in T1D prevention. Simultaneous treatments with anti-DEC-205/InsI9, a mimotope peptide of the CD8+ T cell insulin epitope InsB15-23, and anti DEC-205/NRP-V7 are being held to study the effect of targeting multiple CD8+ T cell specificities on T1D prevention. Investigation of the operative tolerance mechanisms revealed that antibody blockade of the PD-1 pathway, often implicated in DC- mediated induction of peripheral tolerance, could not inhibit the deletion of transferred T cells by DEC-205-mediated targeting of their antigen to steady- state DC. Involvement of the CTLA-4 or Fas pathways will be probed to further study the molecular mechanism of tolerance induction. .
Pre-B Cell Leukemia Homeobox 1is a novel susceptibility gene that regulate T cell functions. (123.2)
Authors: Sengupta Mayami , Liang Shujuan , Morel Laurence
Published: January 2023

Pre-leukemia homeobox (Pbx) 1 is a novel lupus susceptibility gene that regulates T cell activation and tolerance in both the NZM2410 lupus-prone mouse model and lupus patients. Lupus susceptibility is associated with the expression of a novel splice isoform Pbx1-d, which, when over expressed leads to an increased CD4+ T cell activation and a reduced induction of Tregs by retinoic acid. The exons encoding for the DNA binding domain and the Hox binding domain are deleted in Pbx-1d, predicting that this isoform functions as a dominant negative. To test this hypothesis, we used an osteogenesis model in which Pbx1 expression prevents differentiation. Pbx1-d expression in MC3T3 osteoblast progenitors mimicked the effect of shRNA Pbx1 in accelerating osteoblast differentiation. Furthermore, Pbx-1d binding to the SOX3 promoter was weaker than that of the full-length Pbx1-b isoform, and SOX3 expression was attenuated in Pbx1-d transfected cells. These results demonstrated that Pbx1-d functions as a dominant negative. C57BL/6 transgenic mice expressing Pbx1-d in their CD4+ T cells show an age-dependent increased in CD4+ T cell activation, with an expansion of CD44+ CD4+ memory cells, and the production of anti-nuclear autoantibodies. These results demonstrate that Pbx1 represents a novel regulator of T cell activation, whose impaired function leads to systemic autoimmunity.
T-regulatory cell (Treg)-extrinsic role of IL-2 in protection against Autoimmune Pancreatitis (123.31)
Authors: Sharma Rahul , Ju Shyr-Te
Published: January 2023

Autoimmune pancreatitis (AIP) is a chronic inflammation of pancreas marked by high serum immunoglobulins and leukocytic infiltration. While the Il2-/- mice, which have reduced Treg levels, develop severe pancreatitis, the Foxp3-mutant Scurfy (Sf) mice, with a complete Treg-deficiency, are resistant to pancreatitis though they have a fatal multiorgan inflammation. Rendering Sf mice deficient for IL-2 (Sf.Il2-/-) abolished this resistance to AIP. We have shown that IL-2 is critical for the Th2 and not the Th1 response in the Sf mice. Thus, we hypothesized that a strong Th2 response in the Sf mice protects against AIP while a poor Th2 response due to IL-2 deficiency fails to suppress the disease. Indeed, Sf mice lacking IL-4 (Sf.Il4-/-) or STAT6 (Sf.Stat6-/-) developed severe AIP, while IFN-γ -deficient Sf mice (Sf.Ifng-/-) remained free from pancreatitis. Interestingly, all these mice had hyper-gammaglobulinemia and serum reactivity to carbonic anhydrase II. Our data further shows that lack of IL-2 or inadequate Th2 expression in the Sf mice causes an increase in the IFN-γ levels leading to upregulation of CXCR5, CXCR3 and CCR5 on the CD4+ T-cells and an increase in IP-10 and RANTES levels locally in the pancreas. The CD4+ T-cells then infiltrate the pancreas and form tertiary lymphoid follicles leading to AIP. We propose that although Treg-deficiency contributes to AIP, IL-2 regulates clinical manifestation of the disease in a Th2 dependent manner.
Cell mediated injury in autoimmune anti-glomerular basement membrane disease is defined by a critical T cell epitope in humanized mice (123.33)
Authors: Ooi Joshua , Chang Janet , Hudson Billy , Fugger Lars , Holdsworth Stephen , Kitching A Richard
Published: January 2023

Human anti-glomerular basement membrane (GBM) disease has strong associations with HLA DRB1*1501. The target autoantigen is the non-collagenous domain of the α3 chain of Type IV collagen, α3(IV)NC1. T cell epitopes of α3(IV)NC1 were identified by immunizing mouse MHC-/-, HLA DRB1*1501 transgenic (1501tg) mice with overlapping 20mers. They defined a HLA DRB1*1501 restricted epitope (aa128-147) not reactive in HLA DRB1*0101 transgenic (0101tg) or C57BL/6 mice. 1501tg mice were immunized with α1(IV)NC1, α3(IV)NC1 or α1/α3 chimeric molecules, made by inserting the α3 aa17-31 (C2) or aa127-141 (C6) into the non-immunogenic α1 backbone. 1501tg mice immunized with α3(IV)NC1 or C6 responded to aa128-147 but not to aa8-27, but α1(IV)NC1 or C2-immunized mice did not respond to either peptide. CD4+ T cell clones, made from 1501tg mice and transferred into naive 1501tg mice, induced disease with necrotizing and crescentic glomeruli, albuminuria and renal failure, and glomerular CD4+ T cell and macrophage infiltrates. To determine if immunization with the restricted epitope causes disease, 1501tg or 0101tg mice were crossed with FcγRIIB-/- mice (Fcγ receptors are implicated in disease susceptibility). Immunization with either whole α3(IV)NC1 or the identified restricted epitope induced glomerulonephritis in 1501tg.FcγRIIB-/- mice but not in 0101tg.FcγRIIB-/- mice. These studies demonstrate that T cell responses in anti-GBM disease are defined by a HLA DRB1*1501 restricted T cell epitope.
CD28-ITK signals regulate autoreactive T cell trafficking into tissues (123.41)
Authors: Jain Nitya , Miu Bing , Jiang Jian-kang , Prince Amanda , Greiner Dale , Thomas Craig , Sanderson Michael , Berg Leslie , Kang Joonsoo
Published: January 2023

Autoimmune diseases such as Type 1 diabetes arise from the activation of self- reactive T cells and their trafficking to target tissues, ultimately leading to impaired organ function. Immune-based therapies for autoimmunity including corticosteroid regimens, modulation of inflammatory effectors and tolerance induction by inhibition of T cell activation fail to target self-reactive T cells specifically, resulting in compromised general immunity. A recent focused approach aims at regulating self-reactive T cell migration to organs to limit immune pathology. The signaling pathways regulating migration of activated tissue-antigen specific T cells versus activated foreign-antigen specific T cells are unknown. Here we demonstrate that the B7-CD28 costimulatory pathway of T cell activation is an important regulator of the trafficking of self-reactive T cells. The Tec-kinase ITK is a downstream mediator of the CD28 signal controlling autoreactive T cell migration. Using the Ctla4-/- mouse as a model of multi-organ autoimmunity, we show that genetic and pharmacological inhibition of ITK prevents the migration of activated T cells into target tissues. Furthermore, small molecule ITK inhibitors prevent pancreatic islet infiltration by diabetogenic T cells in mouse models of type I diabetes. Our results suggest that pharmacological disruption of ITK function should be considered as a treatment of human T cell mediated organ-specific autoimmune diseases.
IL27p28 inhibits induction and expression of experimental autoimmune uveitis by concurrently antagonizing autoaggressive Th1 and Th17 responses. (123.43)
Authors: Chong Wai Po , Horai Reiko , Silver Phyllis , Mattapallil Mary , Chen Jun , Caspi Rachel
Published: January 2023

Human autoimmune uveitis is accompanied by elevated Th1 and Th17 responses. In experimental autoimmune uveitis (EAU) induced in mice with the retinal antigen IRBP, both responses are present and each effector lineage can mediate disease independently. A therapeutic approach that targets both Th1 and Th17 would therefore be attractive. IL27p28 is an antagonist of gp130, which is required for IL-6 and for IL-27 signaling. Since IL-6 and IL 27 promote Th17 and Th1 responses respectively, we hypothesized that IL27p28 would regulate EAU by affecting both responses concurrently. Overexpression of IL27p28 reduced IFN-γ and IL-17A production from eye-infiltrating CD4+ T cells and ameliorated EAU induced by active immunization. Mechanistic studies revealed that IL27p28 suppressed IL6-mediated STAT3 and IL27-mediated STAT1 phosphorylation during Th17 and Th1 polarization, respectively. Furthermore, overexpression of IL27p28 ameliorated EAU induced by passive transfer of uveitogenic effector Th1 or Th17 cells obtained from donors expressing an IRBP-specific TCR. Donor TCR-transgenic T cells recovered from these mice expressed reduced levels of Th17 and Th1 signature cytokines, IL-17A and IFN-γ, and of their corresponding transcription factors, RORγt and Tbet. The ability of IL27p28 to inhibit both Th1 and Th17 responses and to ameliorate not only the induction, but also the effector phase of EAU, bodes well for clinical usefulness of IL27p28 augmentation to control autoimmunity.
CDK2 regulates Foxp3 activity through protein destabilization (123.7)
Authors: Morawski Peter , Mehra Parul , Chunder Neelanjana , Wells Andrew
Published: January 2023

Foxp3 is a transcription factor that directs development of the regulatory T cell (Treg) lineage. While the signals that regulate expression of the foxp3 gene are established, it remains unclear how the activity of the Foxp3 protein is controlled. Examination of the primary structure of Foxp3 revealed four putative cyclin-dependent kinase (CDK) substrate motifs: S/T-P. In vitro kinase and mass spectrometry analyses showed that Foxp3 can be phosphorylated on at least three of these motifs by the E-type cyclin-dependent kinase, CDK2. Ser/Ala and Thr/Ala mutations within these motifs (S/T>A, or STAble) resulted in an increase in the stability, measured through a CHX assay, and steady-state level of Foxp3 protein within the cell. Conventional CD4 cells transduced with the STAble mutant of Foxp3 exhibited increased CD25 expression, repression of IL-2, and suppressive function compared to CD4 cells transduced with wild type Foxp3, suggesting that phosphorylation of Foxp3 by CDK2 may negatively regulate its function. Consistent with this, natural Foxp3+ Tregs from mice genetically deficient for CDK2 likewise exhibited a gain of suppressive function compared to wild type Tregs. Based on these data, we hypothesize that mitogenic signaling through CDK2 negatively regulates Foxp3 activity and Treg function through phosphorylation-dependent protein destabilization.
Direct Effects of Increased Sugar Concentration on Proliferation and Activation of Jurkat T-cells. (123.8)
Authors: Snitsarev Vladislav
Published: January 2023

High-fructose corn syrup has been used with increasing commonality in recent decades. Parallel with its use, an increased occurrence of autoimmune disease has been reported suggesting a potential correlation. We hypothesized that increased sugar concentration may activate T-cells. Using Jurkat T-cells as a model, we found that increased glucose and fructose concentration decreased Jurkat T-cell proliferation and death rates. This effect was significant at pH 7.4, while at pH 7.2 the above effects did not reach significance. Cells grown in high glucose and fructose concentration formed clusters that were unbreakable by standard trituration techniques indicating possible expression of high-affinity adhesion molecules characteristic of T-cell activation; this effect was more prominent in fructose than glucose formulations. We conclude that exposure of T-cells to sustained higher sugar concentration can lead to cells aggregation indicating possible activation in an antigen-independent manner, and can potentially contribute to the development of autoimmune disease.
Rapid Methods for Cell Health Assessment of Peripheral Blood Mononuclear Cells (124.15)
Authors: Clor Julie , Gillis Katherine , Khan Asima , Tyagarajan Kamala
Published: January 2023

Immunological studies evaluating phenotypic, mechanistic and activation characteristics of peripheral blood mononuclear cells (PBMCs) involve multiple samples, long treatment times, and laborious methods. The initial screening and assessment of PBMC quality for parameters such as count, viability and apoptosis is critical in determining the success of downstream immunological studies. Traditionally, such screening has been limited since it has required access to complicated and expensive platforms. In this study, we present results from a simplified method for the characterization of fresh or cryopreserved PBMC’s on the novel Muse Cell Analyzer. The methods utilize simple mix and read assays, provide quick assessment of count and viability measurements and the apoptotic status of PBMC’s. Our results indicate that good accuracy and linearity data on count and viability can be obtained for PBMC’s in a wide concentration range can be obtained using sample sizes as low as 20 µL. Precise results were obtained for all assays tested as shown by the low coefficient of variation (CV<10%). The simplified assay format allows for low operator to operator variability with inter-operator CV’s of <10%. Data on impact of PBMC viability and apoptosis and correlation with downstream performance will be presented. The combination of rapid methods with a novel accessible platform will empower researchers to easily select optimal PBMC samples for downstream experiments.
Cytokine responses and ADCC activity in cryopreserved PBMC (124.6)
Authors: Bahunde Faith , Awoyode Rotimi , Przybylek Andrew , Harwood William , Meixell Ron , Anekella Bharathi
Published: January 2023

Cryopreserved peripheral blood mononuclear cells (PBMC) are a good alternative to fresh PBMC in clinical trials when samples are collected at multiple sites and processed later. Optimal retention of cryopreserved PBMC functionality depends on cell isolation, freezing and storage. SeraCare developed validated PBMC cryopreservation procedures to ensure functionality is maintained on cryopreservation. Two assays were performed to demonstrate the equivalence of cryopreserved vs. fresh PBMCs. Intracellular cytokine staining (ICS) quantitatively measures antigen specific T cell cytokine production at the single cell level. Using flow cytometry ICS analyzes multiple parameters on a single sample to obtain the precise cytokine producing T cell phenotype. Our data demonstrates production of IFN-γ, IL-2, TNF-α and IL-4 from cryopreserved PBMC suggesting full retention of PBMC functionality after cryopreservation. The antibody dependent cellular cytotoxicity (ADCC) assay measures natural killer cell mediated destruction of antibody targeted cells. ADCC is a powerful tool to measure immune responses in HIV and cancer immunotherapy. Most studies used fresh PBMC in ADCC assays, noting that cryopreserved PBMC result in lower activity. We show cryopreserved PBMCs have comparable ADCC activity to fresh PBMC. ADCC activity appeared donor specific and was not affected by cryopreservation. Our data suggest that cryopreservation has no adverse affect on PBMC functionality in immunological assays.
Analysis of T and B cell phenotype and effector memory T cell response in a mouse model of tree nut allergy (125.10)
Authors: Gangur Venugopal , Gonipeta Babu , Parvataneni Sitaram , Duriancik David , Kim Eunjung , Gardner Elizabeth
Published: January 2023

Tree nuts cause potentially fatal anaphylactic reactions for reasons that are not well understood. We previously reported an adjuvant free mouse model of hazelnut allergy. Here we analyzed the T & B cell phenotype and effector memory responses. Balb/c mice were transdermally sensitized with hazelnut. Hazelnut allergy was confirmed by measuring hazelnut specific IgE antibodies and systemic anaphylaxis (hypothermia) upon challenge. Using flow cytometry T cell and B cell phenotypes were studied. Hazelnut allergic mice exhibited a significant increase in the number of CD4+ CD62L - T cells in the bone marrow and the spleen. Among B cells, there was increase in CD80+ cells in BM, spleen and MLN and CD62L- cells in BM and spleen of allergic mice. Among CD80+ B cells a significant proportion were CD73- in the MLN of allergic mice. Effector memory Type-2 cytokine responses were analyzed using spleen cell cultures. Allergic mice exhibited a robust IL-4, IL-5 and IL-13 response that was blocked by anti-CD4 mAb. The STAT6 KO mice exhibited IL-5 and IL-13 but not IL-4 memory response. These data demonstrate that hazelnut elicits: (i) significant expansion of CD4+CD62L- T cells in both the BM and the periphery; (ii) a modest increase in CD80+ and CD62L- B cells in BM and the periphery; and a significant down regulation of CD73 expression on a subset of CD80+ B cells in the MLN; and (iii) STAT6 and CD4 dependent IL-4 memory; and STAT6 independent, CD4 dependent IL-5/IL-13 memory.
LTB4 driven neutrophil recruitment to the skin is essential for allergic skin inflammation (125.17)
Authors: Oyoshi Michiko , He Rui , Li Yitang , Yoon Juhan , Afshar Roshi , Chen Mei , Lee David , Luo Hongbo , Luster Andrew , Larson Allison , Murphy George , Geha Raif
Published: January 2023

Scratching triggers skin flares in atopic dermatitis (AD), a disease marked by itching and allergic skin inflammation. Scratching of human skin, and tape stripping of mouse skin, caused a neutrophil influx. This influx in mice was largely dependent on the generation of leukotriene B4 (LTB4) by neutrophils and their expression of its receptor BLT1. Allergic skin inflammation in response to epicutaneous (EC) application of ovalbumin to tape-stripped skin was severely impaired in BLT1-/- mice, and required the expression of BLT1 on both T cells and non-T cells. Co-transfer of WT neutrophils, but not neutrophils deficient in BLT1 or the LTB4 synthesizing enzyme LTA4H, restored the ability of WT CD4+ effector T cells to transfer allergic skin inflammation to BLT1-/- recipients. Pharmacologic blockade of LTB4 synthesis inhibited allergic skin inflammation elicited by cutaneous antigen challenge in previously EC-sensitized mice. Blockade of the LTB4-BLT1 axis might provide a potential therapy for AD.
Role of carbohydrate moieties in recognition and uptake of allergens by innate immune cells (125.2)
Authors: Ghaem Maghami Amir , Al-Ghouleh Abeer , Harrington Helen , Shakib Farouk
Published: January 2023

Allergens are initiators of both innate and adaptive immune responses. They are recognised at the site of entry by epithelial and dendritic cells (DCs), both of which activate innate inflammatory circuits that can collectively induce Th2 immune responses. In an attempt to have a better understanding of the role of carbohydrates in the recognition and uptake of allergens by the innate immune system, we defined common glycosylation patterns in major allergens like Der p 1, Fel d 1, Ara h 1, Ber e 1, Der p 2, Bla g 2 and Can f 1. Our data showed that all these allergens are glycosylated and that the main dominant sugars on allergens are 1-2, 1-3 and 1-6 mannose. We then investigated uptake and recognition of different allergen glycofroms (i.e. natural, hyper- and a-glycosylated) by DCs and lung epithelia. The deglycosylated preparation of Der p 1 exhibited minimal uptake by DCs compared to the natural and hyperglycosylated recombinant counterparts, with the latter being taken up more readily than the other preparations. Interestingly, our data also showed an increase in Thymic stromal lymphopoietin (TSLP) (known to drive DC maturation in support of Th2 responses) secretion by lung epithelia upon stimulation with natural Der p 1 which was carbohydrate dependent. Collectively, our data indicate that carbohydrate moieties on allergens play a vital role in their recognition by innate immune cells, implicating them in downstream deleterious Th2 cell activation and IgE production.
Tolerance induction using novel antigen-coupled carriers in a model of allergic asthma (125.5)
Authors: Smarr Charles , Yap Woon Teck , McCarthy Derrick , Bryce Paul , Shea Lonnie , Miller Stephen
Published: January 2023

Current immunotherapy for Th2-associated allergic disease is inefficient and has a significant risk of inducing anaphylaxis. There is significant need for new treatments to safely, efficiently induce antigen-specific tolerance in allergic disease. Antigen-coupled cells (Ag-SP) have been used by our lab to prevent and treat Th1/Th17-mediated models of autoimmune disease and, more recently, Th2-mediated models of allergic asthma and food allergy. Ag-SP treatment is currently entering a Phase II clinical trial in multiple sclerosis patients in Germany. Due to the technical complications of such treatment, however, we have begun to explore new antigen carriers for the induction of tolerance in autoimmune and allergic diseases. Initial work with antigen coupled to inert polystyrene and biodegradable PLGA microparticles (Ag-MP) identified these carriers as effective at inducing tolerance in EAE. We have extended these findings to a common ovalbumin (OVA)-alum induced mouse model of allergic asthma. Prophylactically, OVA- MP induce protective tolerance - inhibiting production of OVA-specific IgE antibodies and lung inflammation and preventing OVA-specific proliferation and Th2 cytokine production by restimulated lymphocytes. Given post-sensitization, OVA-MP inhibit Th2 responses in restimulation cultures but do not, in the short frame examined, inhibit other parameters of disease. This work identifies Ag-MP as promising inducers of tolerance for allergic disease.
Retinoic acid downregulates IL-4-induced IgE production through retinoic acid receptor (125.8)
Authors: Seo Goo-Young , Lee Jeong-Min , Yoo Hana , Nagler Cathryn , Kim Pyeung-Hyeun
Published: January 2023

Retinoic acid (RA) has been reported to downregulate IgE Ab synthesis. In the present study, we assessed the mechanisms by which RA suppresses IL-4-induced IgE expression by murine splenic B cells. We found that AM80, a RA receptor (RAR) agonist, inhibits the production of IL-4-induced IgE but not IgA or IgM. Addition of the RAR antagonist LE540 to these cultures abrogated the RA mediated inhibition of IgE, as well as the expression of germ-line IgE transcripts (GLTϵ). In addition RA decreased IL-4/αCD40-induced GLϵ promoter reporter activity while increasing GLα promoter activity. Finally, serum IgE was substantially elevated in vitamin A-deficient (VAD) mice. Taken together, these results suggest that RA downregulates IgE isotype switching through a RAR-dependent pathway in vivo.