bluuebunny/crossref_metadata_2025 huggingface dataset:
The splicing factor SF2/ASF is a novel regulator of IL-2 transcription and rescues IL-2 production in T lymphocytes from patients with SLE (P6288)
Authors: Moulton Vaishali , Grammatikos Alexandros , Fitzgerald Lisa , Tsokos George
Published: January 2023

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease, which afflicts mainly women in the childbearing 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 also are responsible for target organ infiltration. 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 splicing factor 2 / alternative splicing factor (SF2/ASF) enhances the expression of CD3ζ chain by limiting the production of an unstable mRNA splice variant. Here we show that SF2/ASF expression levels are decreased in T cells from SLE patients, more so in patients with higher SLE disease activity index (SLEDAI). Importantly, we show that overexpression of SF2/ASF in SLE T cells restores IL-2 production. In parallel, silencing SF2/ASF expression in normal T cells reduces their IL-2 secretion. Finally we show that SF2/ASF induces transcriptional activity of the IL-2 promoter and that SF2/ASF is recruited to the IL-2 promoter indicating its role in IL-2 transcription. These results identify SF2/ASF as a novel regulator of IL-2 expression in human T cells and a potential molecular mechanism underlying the T cell defect in SLE.
Two functional ligand binding sites on human CD30 separately located from the epitope of agonistic monoclonal antibodies (P6339)
Authors: Ise Tomoko , Abe Yasuhiro , Nagata Satoshi
Published: January 2023

Human CD30 is a member of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF8). CD30 is considered to exist as homotrimers on the cell surface without binding to the ligand. Several anti-CD30 agonistic antibodies have been isolated. One of them is currently clinically used as an antibody drug conjugate for therapy of CD30-positive lymphomas. Despite progress in clinical application, the binding mode required for the agonistic activity of anti-CD30 antibodies has not been fully understood. In particular, the presence of two duplicated sequences including the TNF receptor domain in human CD30 complicates the analysis of binding-function relationships. In this study, we produced a series of recombinant conformation-carrying CD30 mutants whose TNF receptor domains and epitopes are replaced with corresponding sequences of CD40, based on the structures. We tested their ability to transduce signals for NFkB activation in reporter cells upon binding to CD30-ligand or agonistic antibodies. We found that both of the predicted ligand binding sites activate NFkB pathways by the binding of CD30-ligand-Fc fusion protein or CD30-ligand expressed on 293T cells. In addition, a group of agonistic antibodies appeared to bind an N-terminus site of the non-ligand binding site mutants and activated NFkB. Our results suggest that agonistic antibodies induce the active complex formation of membrane CD30 for potent signaling through a different binding mode than the natural ligand.
C-C chemokine receptor regulates the antibody response to DNP-KLH (P6351)
Authors: Vines Charlotte , Welch T. , Fang Li , Kaiser Brian , Islam Shamima , Bill Charles , Sun Yuning , McBurney Tiffany
Published: January 2023

Functional loss of C-C chemokine receptor 7 (CCR7) and its ligands, CCL19 and CCL21, has been linked to development of autoimmune diseases. Therefore, we hypothesized that CCR7 antagonists could be used to promote an immune response that could be measured by tittering antibodies produced in animals injected with test antigens. To test this hypothesis we injected C57BL/6 mice on day 0 and boosted on day 20 with dinitrophenol (DNP)-Keyhole Limpet Hemocyanin (KLH) with a CCL19 antagonist, 8-83, or with the agonist solvent as a control. Similar to the CCR7-/- mouse, which has significantly elevated levels of IgG1, IgG2a, IgG2b and IgG3 by day 30, the animals treated with 8-83 generated significantly higher levels of IgG1 and IgG2a, than animals injected with the DNP-KLH/solvent alone. Previous studies by our laboratory have shown that CCL19 signals through ERK5. To determine if loss of signaling through ERK induced a similar increase in the levels of IgGs, we injected ERK5flox/flox control mice or ERK5flox/floxLck-Cre mice with DNP-KLH. Similar to animals treated with 8-83, the ERK5flox/flox Lck-Cre mice produced significantly higher levels of IgG1, IgG2a and IgG3 than control mice. From these results we conclude that CCR7/CCL19 signals through ERK5 to control the levels of antibodies made. Recent studies demonstrating a role for CCR7 in controlling the localization of regulatory T cells may provide insight into immune cells that are regulate observed increase in antibody titers.
IL-10-producing B cells limit CNS inflammation and infarct volume after experimental stroke in mice (P1090)
Authors: Offner Halina , Chen Yingxin , Murphy Stephanie , Bodhankar Sheetal
Published: January 2023

Clinical stroke induces inflammatory processes leading to cerebral injury. IL-10 expression is elevated during major CNS diseases and limits inflammation in the brain. Recent evidence demonstrated that absence of B cells led to larger infarct volumes and increased numbers of activated T cells, monocytes and microglial cells in the brain, thus implicating a regulatory role of B cell subpopulations in limiting CNS damage from stroke. The aim of this study was to determine whether the IL-10-producing regulatory B cell subset can limit CNS inflammation and reduce infarct volume following ischemic stroke in B-cell deficient (μMT-/-) mice. Five million IL-10-producing B cells were obtained from IL-10-GFP reporter mice and transferred i.v. to μMT-/- mice. After 24 h from this transfer, recipients were subjected to 60 min of middle cerebral artery occlusion (MCAO) followed by 48 h of reperfusion. Compared to vehicle-treated controls, the IL-10+ B-cell-replenished μMT-/- mice had reduced infarct volume and fewer infiltrating activated T cells and monocytes in the affected brain hemisphere. These effects in CNS were accompanied by significant increases in regulatory T cells and expression of the co-inhibitory receptor, PD-1, in the periphery. These novel observations demonstrate both immunoregulatory and protective functions of IL-10-secreting B cells in MCAO that potentially could impart significant benefit for stroke patients in the clinic.
Generation of mature activated regulatory myeloid cells: Differential effects of retinoic acid on myelopoiesis versus dendropoiesis (P1064)
Authors: VanGundy Zachary , Baker Julie , Stranger Heather , Papenfuss Tracey
Published: January 2023

Myeloid cells are increasingly recognized for their potent regulatory abilities. However, factors influencing regulatory myeloid cell (regMC) differentiation remain poorly understood. Retinoic acid (RA) is a steroid hormone important in regulating mucosal (gut) immunity. RA also promotes myeloid differentiation. We hypothesize that RA during differentiation will promote regMC formation (both regulatory DCs and macrophages). Using in vitro differentiation of myeloid cells with GM-CSF with or without RA, we found that day 6-7 RA BM-MCs were more mature/activated (i.e. increased expression of CD80, CD86, and MHCII) than control BM-MCs. Importantly, RA BM-MCs also had increased expression of the inhibitory marker PD-L1. Functionally, these RA BM-MCs expressed increased intracellular IL-10 and induced an increased percentage of T regulatory cells in vitro compared to controls. Notably, RA BM-MCs suppressed the proliferation of responder immune cells even after inflammatory (LPS) challenge. Surprisingly, CD11c+ (DCs) within the RA BM-MC population were not the suppressive population. Rather, the suppressive cells were CD11c-CD11b+Ly6Cint/low and likely represent a regulatory monocyte or other myeloid precursor population. Additional studies characterizing these cells are ongoing. These results suggest that RA has differential effects on myelopoiesis versus dendropoeisis which produce non-DC regMCs.
Cell hybridization technology produces monoclonal antibodies against MurA protein of Listeria monocytogenes (P3132)
Authors: Wen Yiming , Li YuChen , Li Zhiqing , Tong Jiyu , Yang Hongyu , Xiang Junjian
Published: January 2023

The research aims to develop and characterize monoclonal antibodies(MAbs) with high specificity and affinity for surface antigens of all major serotypes of Listeria monocytogenes(L. monocytogenes). Hybridomas were produced using extraction of L. monocytogenes surface protein as the immunogen. Detected by enzyme-linked-immunosorbent assay (ELISA), MAb1B10 was capable of binding to cell-surface antigens of live L. monocytogenes. MAb1B10, 2C10 and 1G3 reacted with both formalin-inactivated and heat-killed L. monocytogenes without any cross reactions with other Listeria spp.; MAb1C10 and 3G8 highly reacted with L. monocytogenes, L. innocua and L. welshimeri, but seldom reacted with non-Listeria spp.. Immunoprecipitation-mass spectrometry (IP-MS) and western blot were applied to convince that the reactive epitope was on the MurA protein of 72kDa. Recombinant MurA protein produced in Escherichia coli was confirmed to be the antigen which was recognized by MAb1B10, 2C10 and 1G3. To sum up, MAbs reacting with live L. monocytogenes, formalin-inactivated and heat-killed L. monocytogenes were produced. The results indicated that MurA could be exploited as a marker for specifically identifying pathogenic L. monocytogenes in suspect food samples.
Expression of two immunoglobulin binding proteins is necessary for S. aureus to avoid host mediated innate and adaptive immune systems (P3138)
Authors: Kim Hwan Keun , Falugi Fabiana , Missiakas Dominique , Schneewind Olaf
Published: January 2023

Human or animal infections with S. aureus do not elicit protective immunity against staphylococcal diseases. Staphylococci are notorious to deploy a wide spectrum of strategies to avoid host immune system. Previous work revealed that both innate and adaptive immune systems are hampered by the expression of two immunoglobulin binding proteins; staphylococcal protein A (SpA) and staphylococcal binder of immunoglobulins (Sbi). Staphylococcal protein A, a molecule that associates with both Fc and Fab portions of immunoglobulins, is necessary 1) to neutralize the antibody mediated opsonophagocytosis and 2) to trigger the expansion and apoptotic demise of B cell populations. The second immunoglobulin binding protein, Sbi, can associate with Fc portion of immunoglobulin and complement factor C3 to disable a critical juncture between the innate and adaptive immunity. Although much of biochemical analysis revealed how the two staphylococcal proteins engage with immunoglobulins for many years, their role as immuno modulatory virulence factors in vivo has not been fully understood. We have investigated the significance of immunoglobulin association with staphylococci during acute and chronic infection in animal model and characterized the molecular mechanisms to avert host immune defense system. Strategies are also discussed on how the two secreted products of staphylococci may be exploited for the development of vaccines and therapeutics.
Yersinia pseudotuberculosis persistence in the lungs is enabled by Ail and YadA targeting of Yops into neutrophils. (P3150)
Authors: Paczosa Michelle
Published: January 2023

A murine model of Yersinia pseudotuberculosis (Yptb) pneumonia was previously developed using the IP2666 strain, which was found to robustly colonize the lungs but only sporadically colonize the liver and spleen (Fisher et al, 2007). Here we show that when the Yptb IP32953 strain was intranasally inoculated into mice, it colonized and persisted in the lungs at high levels, and had dramatically increased bacterial dissemination as compared to IP2666. As adhesins are important for infection and dissemination by Yersinia in other infection models, their role was investigated during IP32953 lung infection using isogenic ΔyadA, Δail, ΔpsaE and Δinv mutants in IP32953. Ail and YadA were critical and redundant for persistence in, but not colonization of, the lung. When a neutropenic mouse was infected with the ΔailΔyadA mutant, the mutant survived as well as wild-type, indicating that a major function of Ail and YadA is to circumvent killing by neutrophils. Further investigation indicated that these adhesins are necessary for selective translocation of Yops by the Type III Secretion System into neutrophils over other immune cell types. This enrichment of translocation into neutrophils correlates with altered binding by the ΔailΔyadA compared to the wild-type. In conclusion, Ail and YadA combined are critical for persistence in the lungs during Yptb infection, because they thwart the bactericidal effects of neutrophils by directing translocation of Yops into these cells.
Mycobacterium tuberculosis culture filtrate protein of 10 kDa inhibits B cell function (P3120)
Authors: Samten Buka , Wang Xisheng , Huang Fang-fang , Barnes Peter
Published: January 2023

Culture filtrate protein 10 kD (CFP10) and early secreted antigenic target of 6 kD (ESAT-6), are required in pathogenesis of tuberculosis in part by manipulation of host immunity. We reported the immune modulatory effects of ESAT-6, and the effect of CFP10 in this regard remains unexplored. We now demonstrate that recombinant CFP10 binds to peripheral blood B cells and monocytes, but not to T-cells. Stimulation of B cells through the B cell receptor BCR plus TLR9 induced IL-6, IL-8 and IL-10 production, and CFP10 but not rAg85 markedly reduced IL-10 secretion and mRNA expression, without affecting IL-6 and IL-8. CFP10 did not affect LPS-stimulated cytokine production by monocytes. CFP10 reduced BCR plusTLR9 stimulated B cell proliferation and IgG secretion. Using B cells as antigen-presenting cells, we found that CFP10 inhibited expression of CD80 and CD86, but not that of CD69 and MHC-II expression, resulting in reduced M. tuberculosis-specific T cell IFN-γ production. The effects of CFP10 on B cells were not due to changes in B-cell TLR9 expression, viability or apoptosis. However, CFP10 inhibited BCR plus TLR9 stimulated nuclear translocation of NF-κB, activation of AP-1 transcription factors and expression of BLIMP1, the master regulator of B cell maturation and activation. We conclude that CFP10 inhibits B functions by blocking TLR9 and BCR signaling, suggesting potential cell specific immune regulatory functions for CFP10 during tuberculosis infection.
Antigen specificity of tuberculosis-specific CD4+ T cells in IKEPLUS immunized mice (P3122)
Authors: Johnson Alison , Goldberg Michael , Lindestam Arlehamn Cecilia , Hegde Subray , Blanchard John , Sette Alessandro , Chan John , Jacobs William , Porcelli Steven
Published: January 2023

Mycobacterium tuberculosis (Mtb) infects one-third of the global population. A more efficacious vaccine is urgently needed for control and prevention of disease. We recently reported a genetically modified strain of M. smegmatis, IKEPLUS, which is a promising vaccine candidate due to its ability to generate an unprecedented level of bactericidal immunity against Mtb in mice. Since protective immunity induced by IKEPLUS is dependent on antigen-specific CD4+ T cell memory, we hypothesized that specificity of the CD4+ T cell response was critical for protection. To determine CD4+ T cell specificity directly, we generated MHC class II (I-Ab) restricted T cell hybridomas from IKEPLUS-immunized mice. Screening of T cell hybridomas against anion exchange-fractionated IKEPLUS and Mtb proteins provided evidence for multiple specific targets, one of which was identified as the AtpA protein, a product of the Mtb Rv1308 gene. In a complementary approach, using in vitro assays of IFNγ production by splenocytes from IKEPLUS-immunized mice, we identified an immunogenic peptide within the Mtb RplJ protein encoded by the Rv0651 gene. Our results demonstrate the CD4+ T cell response in IKEPLUS-immunized mice has multiple targets, among which AtpA and RplJ represent novel specificities for protective responses against Mtb. Further studies to more fully define the range of specificities of CD4+ T cells in IKEPLUS-immunized mice should help to inform the design of effective vaccines against Mtb.
Inhibition of B and T cell responses by Salmonella Pathogenicity Island II (P3124)
Authors: Nanton Minelva , Atif Shaikh , Lee Seung-Joo , Taylor Justin , Nuccio Sean-Paul , Way Sing Sing , McSorley Stephen
Published: January 2023

New vaccines against NTS and typhoid are urgently required and human and mouse infection models indicate a critical role for CD4+ T cells and B cells in protective immunity. Previous studies also suggest that live Salmonella can actively inhibit host protective immunity, but the mechanism remains poorly defined. Here, we utilized a novel OVA-B cell tetramer approach to examine the antigen-specific B cell response to live Salmonella-OVA vaccination. OVA-B cell expansion and germinal center formation were delayed until 45 days in vaccinated mice, suggesting active inhibition by bacteria. The Salmonella Pathogenicity Island II locus has been implicated in culling CD4+T cell responses during live vaccination. Thus, we examined the effect of the SPI2 locus on B cell responses by concurrently infecting mice with a ΔSPI2 mutant, immunizing with OVA protein and measuring the expansion and germinal center formation of OVA-B cells. Wildtype, and not ΔSPI2, Salmonella inhibited expansion of OVA-specific B cells 7 days after immunization. The results of this work suggest two dilemmas for Salmonella vaccination. 1. SPI2 effectors cause a sub-optimal Salmonella-specific B and T cell response during live vaccination. 2. Subunit vaccination in conjunction with active Salmonella infection, may elicit a weaker immune response to the administered vaccine. Studies are underway to identify the SPI2 effector protein responsible for dampening Salmonella-specific adaptive immune responses.
Ibrutinib (PCI-32765) administration modulates a Th1 dominated immune response during cutaneous leishmaniasis infection (P3070)
Authors: Natarajan Gayathri , Dubovsky Jason , Satoskar Abhay , Muthusamy Natarajan , Johnson Amy , Byrd John
Published: January 2023

Treatment for leishmaniasis, a neglected tropical disease with worldwide disease burden of 12 million, is ineffective due to rising drug resistance and toxicity. Hence, chemotherapies that modulate host immune response towards a leishmanicidal Th1 response are required. In this study we show that Ibrutinib (PCI-32765), a drug shown to be effective in Phase I/II lymphoma trials, is capable of modulating a Th1 dominated immune response during cutaneous leishmaniasis caused by Leishmania major. L.major infected BALB/c mice treated with Ibrutinib had similar lesion progression compared to vehicle treated L.major infected mice until six weeks of infection and subsequently developed smaller lesions from week-6 to week-9 post-infection. Draining lymph node cells from week-9 Ibrutinib treated mice stimulated with L.major antigen produced significantly lower concentrations of IL-4, IL-10 and IL-13 compared to vehicle treated mice while ratios of IFN-γ:IL-4, IFN-γ:IL-10 and IFN-γ:IL-13 were significantly higher in Ibrutinib treated mice compared to vehicle treated mice. Serum IgG1 antibody titers at week-8 post-infection were significantly lower in Ibrutinib treated mice compared to vehicle treated mice while IgG2a titers were similar between the two groups. Thus, our studies indicate that Ibrutinib (PCI-32765) administration leads to development of a Th1 dominated immune response in L.major infection. This drug requires exploration as a therapeutic agent against leishmaniasis.
IL-15 deficiency promotes accelerated colitis upon expansion of CD4+ T cells (P1231)
Authors: Tosiek Milena , Fiette Laurence , Freitas Antonio
Published: January 2023

Inflammatory bowel diseases (IBD) are idiopathic chronic inflammatory disorders affecting small intestine and/or colon. One of established animal models of IBD can be induced by adoptive transfer of naïve CD4+T lymphocytes from healthy wild type mice into syngeneic recipients lacking T and B cells (RAGko mice). IBD is becoming an attractive field of research for development of new therapeutic agents, including cytokines involved in intestinal inflammation. The clinical relevance of a pleiotropic cytokine IL-15, which shares some similarities with IL-2 and IL-21, was already demonstrated for rheumatoid arthritis, multiple sclerosis and suggested for IBD. In our study, we observed that adoptive transfers of wild type CD4+ T cells result in accelerated manifestation of IBD symptoms, i.e. remarkable body weight loss, rectal bleeding and diarrhoea when recipient mice are IL-15-deficient (IL-15koRAGko). Histologically, colitic IL-15koRAGko mice demonstrate marked infiltration of CD4+ T cells in the colon together with mucosal surface distortion and gland atrophy. Activation of CD4+ T cells transferred into IL-15-deficient mice positively correlates with enhanced IL-17 production and accumulation of neutrophils and lineage-negative cells at the site of inflammation. At the same time the frequency of regulatory T cells (CD4+CD25+Foxp3+) significantly decreases in colitic IL-15koRAGko, which addresses a question of unbalanced Treg function in colitis developed upon IL-15 deficiency.
Diacylglycerol kinase-ζ limits natural regulatory T cell generation. (P1213)
Authors: Schmidt Amanda , Zou Tao , Kambayashi Taku
Published: January 2023

Development of natural regulatory T cells (nTregs) is requisite to maintain central tolerance. nTregs are believed to develop after receiving strong T cell receptor (TCR) signals during T cell maturation in the thymus. This notion comes from experimental systems wherein TCR transgenic T cells adopt the Treg fate only if their cognate antigen is present in the thymus during development. However, whether strong TCR signals drive nTreg development in a polyclonal setting and the specific signaling pathways downstream of the TCR necessary for nTreg development remain elusive. In this study, we selectively perturbed the lipid second messenger diacylglycerol (DAG), which is a critical component of TCR signaling. Using mice that lack DAG kinase-ζ (DGKζ), a negative regulator of DAG, we demonstrate that enhanced DAG signals result in a significant increase in nTreg development in a cell-autonomous manner. DGKζ-deficient T cells exhibited increased c-Rel and ERK signaling, suggesting that these downstream pathways may contribute to enhanced Treg development in DGKζ-deficient mice. Indeed, the absence of c-Rel almost completely abrogated the increase in Treg development observed in DGKζ-deficient mice. Moreover, inhibition of ERK also prevented Treg development in the absence of DGKζ. Our study shows that (1) nTreg development is enhanced by increased TCR signaling in a polyclonal setting and (2) DGKζ limits nTreg development by attenuating signals leading to ERK and c-Rel activation.
The role of Discs Large Homolog 1 phosphorylation in translating TCR signal strength into discrete CTL functions (P1168)
Authors: Crocetti Jillian , Silva Oscar , Humphries Lisa , Miceli M.
Published: January 2023

In cytotoxic T lymphocytes (CTLs), engagement of the T cell receptor (TCR) triggers signaling pathways that culminate in cytokine production and cytotoxic killing. TCR signal strength is linked to CTL function with low signal triggering cytotoxicity while high signal leads to both cytotoxicity and cytokine production. Scaffold protein Dlg1 (Discs Large Homolog 1) is a key point of control downstream of the TCR that coordinates both cytokine production and cytotoxic functional output. However, the molecular mechanism by which Dlg1 regulates cytokine production and cytotoxicity in response to TCR signal strength remains elusive. We report that Dlgh1 is tyrosine phosphorylated in response to TCR stimulation, and that phosphorylation is a key step in Dlg1-mediated activation of the alternative p38 pathway and downstream cytokine gene expression. We demonstrate that Dlg1 phosphorylation is mediated by Dlg1-associated Lck, not Zap70; and that Dlg1 Tyr222 is a major site of TCR-induced phosphorylation. Importantly, T cells expressing Dlg1 with a tyrosine to phenylalanine mutation (Y222F) show a defect in TCR-induced activation of the alternative p38 pathway, activation of NFAT and transcriptional upregulation of TNFα and IFNγ genes, but not the transcriptional upregulation of the granzyme B gene or CTL degranulation. These data position Dlg1 phosphorylation as a molecular switch allowing selective activation of p38-dependent events in response to the strength of the TCR signal.
A novel mouse model for immune mediated skin dermatitis and fibrosis through targeted deletion of phosphoinositide dependent kinase-1 (P4132)
Authors: Yu Minjun , Ghosh Sankar , Farber Donna
Published: January 2023

We developed a novel conditional knockout mouse model in which the PDK1 is specifically ablated by OX40 Cre in a subset of CD4+ effector/memory T cells and Tregs , which are also concomitantly labeled with a fluorescent reporter YFP molecule, enabling tracking of the mutant PDK1-deficient cell populations in vivo. The resultant mice (designated PDK1-CKO) are born healthy similar to conditional heterozygous control mice (PDK1-CHET), but gradually and spontaneously develop symptoms and immune manifestations of human scleroderma- including skin dermatitis, fibrosis and thickening with increased collagen deposition, immune infiltration into the skin, an increase in Th2 effector cells, and elevations in Type 2 and pro-inflammatory cytokines in serum, including IL-4, IL-10, IL-13 and IL-6 also found in sera of human scleroderma. Moreover, the proportion of regulatory T cells is reduced compared to effector T cells, which are mostly activated Th2 cells in lymphoid organs. Transfer of wild type Tregs into PDK1-CKO mice ameliorates skin dermatitis, fibrosis and dampens the Th2 response, and transfer of effector CD4 T cells from PDK-CKO mice into immunodeficient hosts triggers skin pathology. Our results suggest a model for disease development in PDK1-CKO mice: Tregs are functionally impaired, enabling the development of pathogenic YFP+ Th2 effector CD4 T cells which migrate to skin and also promote systemic Th2-driven pathology.
IL-21 promotes autoreactive germinal center development in BXD2 mice partially by regulating TFR. (P4116)
Authors: Ding Yanna , Hsu Hui-Chen , Li Jun , Yang PingAr , Zajac Allan , Mountz John
Published: January 2023

Follicular regulatory T cells (TFR) are a regulatory CD4 (Treg) subset that suppress germinal center (GC) B cell differentiation. Interleukin 21 (IL-21) promotes T follicular helper (TFH) differentiation. In the present study we investigated the modulatory effects of IL-21 on TFR in autoimmune BXD2 mice that develop spontaneous GCs with accumulation of IL-21+TFH cells in the spleen. In BXD2-Il21-/- mice, GL-7+Fas+GC B cells and CXCR5+ICOS+TFHs were significantly reduced, but the frequency of TFR cells was 2-fold higher than that in wild-type BXD2 mice. Most CXCR5+ICOS+CD4 T cells in BXD2-Il21-/- mice highly expressed Treg markers including FoxP3, CTLA4, TGF-β1 and GITR. FoxP3+TFR cells were localized in spleen follicular areas of BXD2-Il21-/- mice. Surprisingly, transfer of TFRs from BXD2-Il21-/- mice into BXD2 mice reduced IgM but not IgG autoantibody producing B cells in the spleen. This may be due to the suppressive effects of IL-21 on TFRs as administration of AdIL-21 increased GC B cells and decreased the ratio of TFR/TFH in the spleen of BXD2-Il21-/- mice in vivo. In vitro, IL-21 increased TFH but decreased TFR cells and significantly reduced Tgfb expression by TFRs. IL-21 also counteracted TFR-induced B cells death and inhibition of TFHs. The present study suggests that IL-21 positively promotes autoreactive GC reactions in BXD2 mice at least partially by decreasing TFRs and compromising the suppressive function of TFRs on B cells and TFHs.
Highly suppressive CD39+ Tregs decrease during disease activity in patients with Systemic Lupus Erythematosus (P4124)
Authors: Syed Ahamed kabeer Basirudeen , Teo Yi Wei , Som Wen Chi , Foong Shu Fen Charlene , Thio Szu-Tien , Wang Linda , Salcido-Ochoa Francisco , Thumboo Julian , Ling Khoon Lin , Kok Yong Fong
Published: January 2023

Objective: Reports on changes in the number and function of Tregs in Systemic Lupus Erythematosus (SLE) have been contradictory, possibly because most of those studies focused on identifying the whole Treg population, without dissecting the Treg subsets. This study aimed to enumerate the number and to study the role of various Treg subsets such as CD39+ Tregs , Naïve Tregs and activated Tregs in SLE patients. Results: Thirty SLE patients, 8 with active disease and 22 in remission, were recruited. The proportion of Tregs (CD4+CD25+Foxp3+) did not differ between patients with active lupus and those in remission (8.2±2.4% vs 7.5±3.8%, respectively; p=0.83). Similarly, there were no differences in these two groups of SLE patients in respect to the numbers of the naïve (CD4+CD45RA+Foxp3+) and activated Tregs (CD4+CD45RA-Foxp3high). However, the proportion of CD39+ Tregs was found to be significantly lower SLE patients with a flare, compared to those on remission (21.6±8.1% vs 41.9±4.7%, respectively; p=0.01). FACS-sorted CD39+ Tregs were found to be significantly more suppressive than CD39- Tregs. At the cut-off point 15.3%, CD39+ Tregs differentiated patients with active lupus from those in remission with the sensitivity of 95% and specificity of 75%. Conclusions: Fewer CD39+ Tregs in patients with active lupus indicates that CD39+ Tregs may play a crucial role in the pathogenesis of SLE. Furthermore, peripheral blood CD39+ Treg may be a biomarker for flares in SLE patients.
An innovative method for assessing autophagy using the FlowSight imaging flow cytometer (P3253)
Authors: Kong Raymond , Friend Sherree , Hall Brian , Basiji David , Pugsley Haley
Published: January 2023

Autophagy is a process in which normal cellular components that accumulate during growth and differentiation are degraded via the lysosome; it is a survival mechanism that reallocates nutrients from unnecessary processes to more vital processes in the cell. Basal levels of autophagy are usually low but can be up-regulated by numerous stimuli including starvation, physiological stress, pharmacological agents and infections. In addition, suppression of autophagy has been associated with cancer, neurodegenerative disorders, infectious diseases and inflammation. During autophagy, cytoplasmic LC3 is processed and recruited to the autophagosomal membranes; therefore, cells undergoing autophagy can be identified by visualizing LC3 puncta using immunofluorescence microscopy. While manual microscopy allows visual identification of autophagy on a per-cell basis, an objective and statistically rigorous assessment is difficult to obtain. To overcome these problems, we used the FlowSight imaging cytometry platform collect imagery of large numbers of cells to assess autophagy in an objective, quantitative, and statistically robust manner. In this study, we demonstrate a method for determining the best image-based parameter for assess the LC3 puncta in starved and non-starved U2OS RFP-LC3 human osteosarcoma reporter cells.
Innovative nanotherapy for the treatment of the chronic skin condition, rosacea. (P3259)
Authors: Mahajan Supriya , Aalinkeel Ravikumar , Reynolds Jessica , Nair Bindukumar , Sykes Donald , Law Wing-Cheung , Prasad Paras , Schwartz Stanley
Published: January 2023

A chronic skin disorder called Rosacea is primarily characterized by inflammation associated with abnormal innate immune response and the current treatments include oral and topical antibiotics and antimicrobials. CD163 plays a key role in etiology of Rosacea and a combination targeted nanotherapy which includes an topical antibiotic such as doxycycline, an anti-microbial such as Azelaic acid and the anti CD163 antibody would be an effective therapy for Rosacea. We developed a nanoemulsion for the treatment of Rosacea using fluorophores called nanophosphors which are upconversion nanoparticles (UCNPs), that have the ability to convert near infrared radiations with lower energy into visible radiations with higher energy via a nonlinear optical process and exhibit unique luminescent properties, including high penetration depth into tissues. Our goal was to evaluate the transdermal delivery of this nanoemulsion using an in-vitro skin barrier model (EpiDerm™). Dermal permeability coefficient is calculated and drug concentrations are determined by HPLC. Our results show a significantly higher 72% increase (p<0.01) in uptake of this nanoemulsion by dermal keratinocytes and fibroblasts and deeper penetration into the skin layers as compared to topical application of the antibiotic and/or antimicrobial alone. Although, this nanoformulation is specific to the treatment of Rosacea, this nano-therapeutic approach can be applied to other skin disorders.
A simple method for assessment of human anti-Neu5Gc antibodies applied to Kawasaki disease (P3263)
Authors: Padler-Karavani Vered , Tremoulet Adriana , Yu Hai , Chen Xi , Burns Jane , Varki Ajit
Published: January 2023

N-Glycolylneuraminic acid (Neu5Gc) is a dietary immunogenic sugar that metabolically incorporates into diverse glycoconjugates in humans. Anti-Neu5Gc antibodies are detected in all human sera, play a role in several inflammation-mediated pathologies and are potential diagnostics of such diseases. Currently, it is difficult to determine their levels in individual human samples due to diverse Neu5Gc-epitopes recognition. Here we describe a simple method for detection of human anti-Neu5Gc antibodies. We exploit the difference between two mouse models that differ only by Neu5Gc-presence (wild-type) or Neu5Gc-absence (Cmah-/- knockout). We characterize mouse serum from both strains by HPLC, lectin and mass-spectrometry. We then use Cmah-/- knockout sera to inhibit all non-Neu5Gc-reactivity followed by binding to wild-type sera to detect overall anti-Neu5Gc response in a single assay. We applied this methodology to characterize anti-Neu5Gc IgG and IgA in sera of Kawasaki disease (KD) patients at various stages compared to controls. KD is an acute childhood febrile disease characterized by inflammation of coronary arteries that untreated may lead to coronary artery aneurysms, thrombosis and myocardial infarction. This estimated response is comparable to the average of detailed anti-Neu5Gc IgG profile analyzed by a sialoglycan microarray. Both assays revealed an elevated response in acute KD patients with normal coronaries compared to patients with aneurysm or dilated coronaries.
Real-time impedance monitoring of pathogen induced apoptosis as a screening method for antiviral drug discovery (P3279)
Authors: Campbell Carmen , Motzfeldt-Laane Carl , Moses Ashlee
Published: January 2023

Many intracellular bacteria and viruses cause host cell death by apoptosis. Antimicrobial agents that block host cell apoptosis are lead drug candidates. To facilitate this, one requires a real-time, automated detection method that monitors pathogen-induced apoptosis to screen for anti-apoptotic drugs. In the current work, we demonstrate that Electric Cell-Substrate Impedance Sensing is a powerful tool for real-time monitoring of pathogen induced cell death in a cell culture system. The impedance biosensor is sensitive enough to detect micro-motion of single cells on an electrode and measures changes in impedance over time. As cells adhere to and spread out across the electrodes, the impedance of the electrode increases relative to the amount of electrode area covered with adhered cells. As virus replicate inside the cells and induce apoptosis, the cell membranes begin to disintegrate and detach from the surface effectively restoring the flow of current resulting in decreased impedance. The method has proven to be remarkably accurate for measuring viral induced cell death in a time and dose dependent manner in both fish and human cell models. Results of the experiments and future applications of this method for antiviral drug discovery will be presented. Reference: Campbell, C.E., Motzfeldt-Laane, M., Haugarvoll, E., and Giaever, I., "Monitoring Viral Induced Cell Death using Electric Cell-Substrate Impedance Sensing." Biosensors and Bioelectronics: 23(4): 536-542 (2007).
Systematically defining murine immunity at the phenotypic and functional levels via mass cytometry (P3283)
Authors: Spitzer Matthew , Hotson Andrew , Bendall Sean , Engleman Edgar , Nolan Garry
Published: January 2023

Despite decades of immunology research in the mouse, no systematic and comprehensive characterization of murine immunity at the proteomic level has been reported. While flow cytometry has been utilized to interrogate immune cells under many circumstances, overlap in fluorochrome emission spectra limits the number of simultaneous parameters available, impeding a thorough analysis of all immune cells and their behavior in complex tissues. Mass cytometry, which couples flow cytometry to time-of-flight mass spectrometry, circumvents this limitation. Here we report the application of single-cell mass cytometry to characterize wild-type murine immunity in three common mouse strains. Using a 40-dimensional panel of immunophenotypic markers, we have assessed the cellular frequency and phenotype of immune cells in the peripheral blood, spleen, lymph nodes, bone marrow, and thymus in an unsupervised manner. Moreover, by combining phenotypic analysis with phospho-specific flow cytometry, we have determined the cell signaling response of immune cells in these tissues to over 20 different stimuli. By characterizing 10 mice per strain, we have defined the ranges of intra-strain and inter-strain variability in immune cell phenotype and behavior. Thus, we report a wild-type murine immune reference map as a resource for the field. We believe this tool will greatly facilitate thorough characterization of pathology, genetic mutations and therapeutic interventions in mouse models.
A novel haploid genetics approach to dissect immune pathways (P3235)
Authors: Shen Jingshi , Dinarello Charles
Published: January 2023

Unbiased loss-of-function genetic screens in model organisms have been major driving forces for modern biological research. However, many biological functions of humans such as adaptive immunity are not found in model organisms amenable to classic genetic screens. Despite tremendous efforts, human somatic cell genetics remains unattainable due to the inability to efficiently induce homozygous mutations in diploid human cells. Our group developed a novel haploid genetics approach to dissect immune pathways in human cells. We take advantage of human myeloid cells lines (e.g. KBM7) that exhibit a haploid karyotype. Retroviral insertional mutagenesis of the haploid cells generates a library of mutant cells with individual genes completely disrupted. First, we validated the haploid mutant library by screening for mutants resistant to the cytotoxicity of diphtheria toxin. After selection, the retroviral insertion sites of the resistant clones were mapped by deep sequencing. We identified HBEGF, the known cell surface receptor for diphtheria toxin. We also isolated multiple endocytic membrane proteins, which likely mediate the internalization of diphtheria toxin. Next, we demonstrated that the haploid mutant library is also suitable for studying complex immune pathways such as the elimination of tumor cells by cytotoxic T lymphocytes. This haploid genetics approach provides a novel and powerful platform for dissecting immune pathways on a genome-wide scale.
Repertoire-wide single-cell linked immunoglobulin DNA libraries (P3247)
Authors: Johnson David , Conant Carolyn , Withey Gary , Löhr Andrea , Hsu Andro , Meyer Everett
Published: January 2023

Deep sequencing is used to broadly characterize mammalian B cell repertoires. DNA from these libraries might be used to produce monoclonal antibodies in vitro, but current methods do not amplify paired heavy and light chain immunoglobulin (Ig). We have developed a method that combines microfluidics and DNA amplification to produce repertoire-wide single-cell linked Ig DNA libraries. We encapsulate hundreds of thousands of single B cells into aqueous-in-oil picoliter droplets. Inside droplets, we perform overlap extension reverse transcriptase PCR, which reverse transcribes and amplifies heavy and light chain Ig separately in early cycles. In later cycles, a linker DNA sequence drives synthesis of a fusion product containing both heavy and light chain Ig. The fusion products encode fragments of monoclonal antibodies (mAbs). We spiked immortal B cell lymphocytes into peripheral blood at several concentrations. Previously, we sequenced the immunoglobulin kappa (IgK) and heavy (IgH) chain variable regions of each cell line. Then, we produced linked Ig repertoire DNA libraries using a set of primers targeting the full variable regions (CDR1, CDR2, and CDR3) for the full IgK and IgH repertoires. We were able to recover expected IgK and IgH pairings for each of the spike-ins, with <5% background. Affinity-selected mAbs from linked repertoire libraries could eventually be used as therapeutics in autoimmunity or cancer, and may have higher efficacy than artificially derived mAbs.
Plasticity in the differentiation of IL-22 producing cells (P6265)
Authors: Shen Wei , Li Wenqing , Feigenbaum Lionel , Hixon Julie , Durum Scott
Published: January 2023

T helper type 22 (Th22) cells are a subset of CD4+ effector T cell that primarily produces interleukin 22 (IL-22), IL-13, and TNF-alpha and have an indispensable role in host defense, barrier function, tissue repair and inflammation. However, the stability and plasticity of Th22 cells have not been elucidated. In this study we developed an IL-22-tdTomato reporter mouse to examine the plasticity of Th22 cells in vitro and in vivo. Briefly, a bacterial artificial chromosome (BAC) was modified to introduce the fluorescent reporter gene into the Il-22 locus. By homologous recombination, the synthesis of the signal peptide of Il-22 in the BAC was disrupted and the tdTomato gene with poly A was inserted immediately after the ATG start site of Il-22, replacing exon 1. Mice were generated that harbor the BAC construct. We found that in vitro generated Th22 cells still maintained their differentiation program in vitro in the early stage of development and could be reprogrammed into Th17 and Th1 cell lineages, but not Th2 cells, in the later stage. To observe Th22 plasticity in vivo, we sorted IL-22 producing cells from mouse T cell transferred colitis model and injected them back to the Rag-/- mouse. It demonstrated that Th22 cells slowly lost their IL-22 expression and converted into Th17 cells in a TGF-beta dependent manner. These results, although suggesting the flexibility of Th22 cells, also indicate active maintenance of their program in vivo.
Dose-dependent suppression of cytokine production from T cells by a novel phosphoinositide 3-kinase delta inhibitor (P6275)
Authors: Way Emily , Chen Kong , Puri Kamal , Kolls Jay
Published: January 2023

The use of specific cytokine inhibitors to treat inflammatory conditions is common but typically utilizes monoclonal antibodies, fusion proteins, and other molecules whose large size necessitates parenteral administration. There remains a significant need for development of effective small molecule cytokine inhibitors for inflammatory conditions. Phosphoinositide 3-kinase (PI3K) plays a critical role in multiple cell signaling pathways, cell cycle progression, and cell growth, and is thus a potential target for drug development. We examined the effect of a novel PI3Kδ inhibitor on CD3/CD28 stimulated T-cell cytokine production. When added to culture after allowing naïve T-cells to polarize into effector T-cells, we observed dose-dependent suppression of multiple cytokines by Luminex and ELISA, including IL-17, IFNγ, and IL-4 from Th17, Th1, and Th2 cells, respectively. Real time PCR confirmed the suppression of cytokine gene expression suggesting the inhibition is mediated at the transcriptional level. However, we do not observe suppressed gene expression of Tbet, GATA3, or RORc, the critical transcription factors for production of those cytokines. A potential mechanism is that the PI3Kδ inhibitor alters nuclear localization of these transcription factors post-translationally. These experiments show effectiveness of this small molecule inhibitor of PI3Kδ activity to modulate inflammation in vitro and exhibit promise as a treatment for in vivo models of inflammation.
Activation of Complement5a receptor on macrophages inhibits Type I Interferon mediated suppression of Th17 cells in lupus (P6277)
Authors: Biswas Partha , Pawaria Sudesh , Maers Kelly
Published: January 2023

Systemic lupus erythematosus is a type I Interferon (type I IFN) driven systemic autoimmune disorder with exaggerated B and T-helper (Th) cells response. Th17 cells, a recently identified T-helper cell known for its ability to produce inflammatory cytokine Interleukin (IL)-17 have been implicated in the pathogenesis of lupus. Since, type I IFN can suppress the generation Th17 cells in an IL-27 dependent manner, it is unclear how pathogenic Th17 cells are generated in the presence of type I IFN in lupus prone mice and patients. Here we show that, activation of Complement5a-receptor (C5aR) on lupus prone macrophages blocked type I IFN mediated IL-27 production and facilitated the development of Th17 cells. C5aR activation on macrophages inhibited IRF1 mediated upregulation of IL-27 gene expression. Consequently, in a pristane-induced lupus model, C5aR-deficient mice had fewer Th17 cells and reduced disease severity in comparison to wild type control mice. Finally, we are able to demonstrate that C5aR activation on macrophages from lupus patients produced diminished level of IL-27 in response to type I IFN stimulation. Collectively, these data clearly indicate an essential role for innate immune signals in the generation of pathogenic Th17 response in lupus. In the long run knowledge gained from this study will be beneficial in designing potential therapeutic strategies to treat lupus patients.
PD-1 interaction with PD-L1 but not PD-L2 on B cells mediates protective effects of estrogen against EAE (P5176)
Authors: Bodhankar Sheetal , Offner Halina
Published: January 2023

Increased remissions in multiple sclerosis (MS) during late pregnancy may result from high levels of sex steroids such as estrogen (E2) and estriol (E3). In fact, treatment with relatively low doses of E2 can protect against clinical and histological signs of MOG-35-55 induced experimental autoimmune encephalomyelitis (EAE) through mechanisms involving PD-1, PD-L1 and B cells. The current study sought to determine more specifically the contribution of PD-L1 and PD-L2 on B cells in E2-mediated protection. Thus, WT, PD-L1 knockout (KO) and PD-L2 KO mice were implanted with E2 pellets prior to EAE induction and the disease course was followed. Unlike PD-L2 KO mice that were fully protected against EAE after E2 treatment, E2-implanted PD-L1 KO mice were fully susceptible to EAE, with increased numbers of proliferating Th1/Th17 cells in the periphery and severe cellular infiltration and active demyelination in the CNS. Moreover, transfer of B cells from MOG-immunized PD-L1 KO or PD-L2 KO donors into E2-preconditioned B cell deficient μMT-/- recipient mice revealed significantly reduced E2-mediated protection against EAE in recipients of PD-L1 KO B cells, but complete protection in recipients of PD-L2 KO B cells. We conclude that PD-1 interaction with PD-L1 but not PD-L2 on B cells is crucial for E2-mediated protection in EAE and that strategies that enhance PD-1/PD-L1 interactions might potentiate E2 treatment effects in EAE and MS.