bluuebunny/crossref_metadata_2025 huggingface dataset:
Anti-inflammatory effects of WF10 in a mouse model of rheumatoid arthritis (P5185)
Authors: Leichsenring Anna , Lange Franziska , Isensee Thomas , Guntermann Henrich , Sack Ulrich
Published: January 2023

The purpose of our study was to investigate whether the immune modulator WF10 has an impact on clinical signs of experimentally induced rheumatoid arthritis in DBA/1 mice. Female DBA/1 mice were sensitized with a collagen II emulsion on day 0 and boosted on day 21. The animals were scored from day 17 for clinical signs of collagen induced arthritis (CIA). If the evaluated score exceeded a value of 5, CIA was considered to be established. From the day of CIA onset animals were treated with WF10 or saline in different doses and treatment regimens. Scoring was done for a total of 21 days after CIA onset. Animals which developed CIA within the first 35 days after the immunization were included into the study. Control animals as well as mice treated with WF10 exhibited CIA and showed an increasing score within the observation period, whereas animals treated with the compound showed a reduction of clinical symptoms. 21 days after CIA onset, animals were sacrificed and samples were preserved for further investigations like cytokine measurements, histology and mRNA expression analyses. In conclusion, WF10 had a reducing effect on the severity of CIA symptoms in mice and may therefore present a novel therapeutic approach for patients suffering from rheumatoid arthritis and other inflammatory autoimmune diseases.
Lovastatin in combination with metformin provides neuroprotection in experimental animal model of multiple sclerosis (P5195)
Authors: Singh Inderjit , Mohan Sarumathi , Paintlia Ajaib
Published: January 2023

Studies from our laboratory have previously reported that modulation of AMPK by metformin and isoprenylation of RhoA GTPase by statins attenuate inflammatory and autoimmune responses. Here, we evaluated the therapeutic efficacy of these drugs in combination to protect CNS in EAE, animal model of MS. Metformin and lovastatin are neuroprotective per se. Combination therapy using the suboptimal dose of the drugs following EAE induction provided synergistic effects in neuroprotection. Mechanistically, the neuroprotection offered was primarily through decreased infiltration of the immune cell (CD4, CD8 and CD68) and decreased axonal degeneration. Combination therapy also inhibited the EAE induced demyelination (LFB and MBP staining). The shattered homeostasis of phosphorylated form of neurofilament binding protein (SMI-31) during the EAE pathology was reverted back with the drug treatment. Further, CNPase, the marker of myelinating oligodendrotyes was increased by the metformin and lovastatin treatment. Combination treatment with lovastatin and metformin biased the class of elicited myelin basic protein antibody from IgG2a to IgG1 and IgG2b, suggesting a shift from Th1 to Th2 response. In summary, metformin and lovastatin treatment individually as well as in combination combats EAE disease. Since, metformin and lovastatin are FDA approved drugs; the present study suggests for a possible therapeutic application of their combination treatment for MS.
Removal of HMGB1 in inflammatory bowel disease using DNA-beads. (P5197)
Authors: Ju Zhongliang , Li Jianhua , Lu Ben , Dancho Meghan , Mahenda Ochani , Chavan Sangeeta , Tracey Kevin , Yang Huan
Published: January 2023

Inflammatory bowel disease (IBD) affects millions of Americans, and costs 1.7 billion annually. High mobility group box 1 (HMGB1) is a cytokine mediator of inflammation in the pathogenesis of IBD. Elevated levels of HMGB1 have been observed in human IBD and in mouse models of colitis (Vitali R, et al. Am J Gastroenterol. 2011. Yamasaki H, et al. Mol Med Report. 2009). Blocking HMGB1 induction by either anti-HMGB1 antibody or ethyl pyruvate (HMGB1 inhibitor) was able to ameliorate the inflammation in colitis mice. DNA with certain sequences or structures has been shown with high affinity binding to HMGB1, and has been reported to attenuate inflammatory responses by blocking HMGB1. In this study, we conjugated DNA sequences to sepharose beads and utilized them as novel reagents to remove HMGB1. We found that the DNA-beads were able to bind HMGB1 with a capacity of 7.6 μmol/L, and each immobilized DNA molecule was able to bind three HMGB1 molecules (DNA:HMGB1 ratio = 1:3). DNA-beads were able to capture HMGB1 from RAW 264.7 cell supernatant stimulated with LPS and from sera of septic mice induced by cecal ligation and puncture. When applied to stools of mice with dextran sulfate sodium (DSS) induced colitis, DNA-beads were able to capture HMGB1 from stools of these colitis mice. Hence, our data suggest a therapeutic potential for DNA-beads to remove HMGB1 in inflammatory diseases such as IBD.
An immuno-protective treatment strategy for autoimmune diseases (P5201)
Authors: McNally Jonathan , Elfers Eileen , Terrell Catherine , Grubblatt Eli , Jordan Michael , Hildeman David , Katz Jonathan
Published: January 2023

The current clinical approach for treating autoimmune diseases is to blunt all immune responses as a means of preventing autoimmune pathology. A major side effect of this strategy is depressed beneficial immunity, and increased rates of infections and tumors. Here we report the novel application of etoposide, a topoisomerase inhibitor, to treat experimental autoimmune encephalomyelitis (EAE), a model for human multiple sclerosis. The moderate and temporally-limited use of etoposide results in significant reduction and severity of EAE while sparing beneficial naïve and memory immunity. Our approach of eliminating activated encephalogenic effector T cells reduces clinical scores, pathogenic cytokine production, and overall pathology, while dramatically limiting the off-target effects on naïve and memory adaptive immunity. Etoposide-treated mice exhibited ameliorated or no EAE pathology and reduced antigenic spread, yet had normal T cell and T-dependent B cell responses to de novo antigenic challenges, as well as unimpaired memory T cells responses to viral rechallenge. Thus, dosage limiting etoposide therapy can selectively ablate effector T cells and limit pathology in an animal model of autoimmunity, while sparing protective immune responses. This strategy could lead to a broad new paradigm for the treatment of autoimmune diseases with both enhanced efficacy and decreased treatment-associated morbidities.
Inhibiting Th17 cells and reducing insulitis in diabetic NOD mice by GW9508, a GPR40 and GPR120 dual agonist (P5203)
Authors: Guo Zhiguang , Ansarullah A. , Lu Yan , Holstein Martha , Brittany Deruyter
Published: January 2023

G-protein coupled receptor 40 (GPR40) and G-protein coupled receptor (GPR120) are expressed on intestinal L cells, monocytes and/or macrophages. We investigated the effect of GW9508 (GW), a GPR40 and GPR120 dual agonist, on modulating autoimmunity in NOD mice. New-onset diabetic NOD mice were treated with GW for up to 8 weeks. Nonfasting blood glucose was monitored. After the treatment, CD4+CD25+FoxP3+ T (Treg) cells in thymus, spleen and pancreatic lymph nodes were measured. To detect IL-17+CD4+ T (Th17) cells, lymphocytes from these mice were cultured for 5 days. To determine in vitro effect of GW on Th17 cells, lymphocytes from NOD mice were cultured with or without GW. Cytokines and chemokines in supernatants were determined. Diabetes was reversed in 13% of vehicle (DMSO) treated mice, in 54% of mice treated with GW at 10mg/kg/day and in 69% of mice treated with GW at 20 mg/kg/day (P<0.01). The insulitis score was significantly lower in GW-treated mice (P<0.05). Although the percentage of Treg cells was not significant different, the percentage of Th17 cells and the ratio of Th17 cells to Treg cells in GW-treated mice were significantly lower (P<0.01). Th17 cells in GW-treated lymphocytes in culture were significantly reduced (P<0.05). IL-17, IFN-γ, IP-10, RANTES, and MIP-1α in supernatants of GW-treated lymphocytes were also significantly reduced (P<0.05). Our data indicate that targeting GPR40 /GPR120 is a new therapeutic approach for treating type 1 diabetes.
Lipoxin A4 decreases human memory B cell antibody production via an ALX/FPR2-dependent mechanism: A link between resolution signals and adaptive immunity (P5151)
Authors: Ramon Sesquile , Bancos Simona , Serhan Charles , Phipps Richard
Published: January 2023

Specialized proresolving mediators (SPMs) are endogenous bioactive lipid molecules that play a fundamental role in the regulation of inflammation and its resolution. SPMs are classified into lipoxins, resolvins, protectins and maresins. Lipoxins and other SPM have been identified in immunological tissues including bone marrow, spleen and blood. Lipoxins regulate functions of the innate immune system such as monocyte recruitment and inhibition of neutrophil infiltration to the site of inflammation. A major knowledge gap is whether lipoxins influence adaptive immune cells. Here, we discovered that B cells express the lipoxin A4 (LXA4) receptor, ALX/FPR2, and upregulate its expression following mitogen activation. Furthermore, LXA4 decreased IgM and IgG production on activated B cells through an ALX/FPR2-dependent mechanism. This caused a reduction in NF-κB p65 nuclear translocation. We found that memory B cells express higher levels of ALX/FPR2 compared to naïve B cells. Interestingly, LXA4 inhibited human memory B cell antibody production and proliferation, but not naïve B cell function. LXA4 also decreased antigen-specific antibody production in vivo. To our knowledge, this is the first description of the effects of lipoxins on B cells, which provides a link between resolution signals and adaptive immunity. The ability of lipoxins to decrease memory B cell antibody production could be particularly beneficial to threat chronic inflammatory diseases and autoimmune disorders.
A novel aza-anthrapyrazole modulates the development of CD4+ effector T cells (P5207)
Authors: Clark Matthew , Hacker Miles , Quinn Anthony
Published: January 2023

BBR3378 is a novel aza-anthrapyrazole that antagonizes autoimmunity and the progression of MOG-induced EAE without the complications noted with anthracycline-based (mitoxantrone) therapy in EAE and human multiple sclerosis. Mitoxantrone induces cell death in rapidly proliferating cells and significant lymphopenia in vivo; however, while most splenic CD4+ cells take up BBR3378 in vivo, only a few are deleted. Moreover, increased dosages of BBR3378 were associated with improved therapeutic impact without concomitant increases in lymphopenia. To determine if BBR3378-mediates regulation of EAE via lymphocyte deletion, splenic T cells were stimulated with anti-CD3 in the presence of low-dose BBR3378 or mitoxantrone. The CD3 blasts showed increased expression of CD25 and CD69, but the enhanced CD4 expression (CD4hi) typically associated with Th progressison to “effector” status was blocked on BBR3378 and mitoxantrone-treated cells, leading to enrichment of slowly proliferating CD4lo-CD69hi T cells. Cytokine production was confined to CD4hi-CD69hi blasts; however, in contrast to mitoxantrone, IL-17 and IFN-gamma production were significantly reduced in the BBR3378-treated cells in the absence of Th cell death. These findings suggest that BBR3378 can regulate specific genes in viable Th cells while impeding the progression of naïve Th cells to full effector status. The Th subsets associated with the induction of EAE were particularly sensitive to BBR3378-mediated suppression.
Dissecting fingolimod’s action: a novel immunomodulatory effect of the unphosphorylated compound on cytotoxic T cell function (P5159)
Authors: Ntranos Achilles , Grishkan Inna , Robinson Dionne , Hall Olivia , Klein Sabra , Calabresi Peter , Gocke Anne
Published: January 2023

Fingolimod (FTY), a sphingosine-1-phosphate receptor modulator, is a multiple sclerosis (MS) therapeutic that upon phosphorylation traps CCR7+ T cells in lymph nodes (LN). CCR7- effector T cells are spared, thus allowing effective infection clearance. Nonetheless, FTY-treated patients are more susceptible to viral infections, indicating a cytotoxic T (Tc) cell defect. Yet, FTY’s effect on Tc cells remains unclear. To address this question, we utilized experimental autoimmune encephalomyelitis (EAE) and a murine influenza model. FTY ameliorated EAE by sequestering T cells, but also inhibited splenic Tc cell IFNγ and Granzyme B(GrB) expression. Influenza was exacerbated and mortality was increased, as FTY inhibited Tc cell activation and lung infiltration. To elucidate its mechanism of action, murine splenocyte cultures were treated with phosphorylated and unphosphorylated FTY, as both exist in vivo. Remarkably, only the latter reduced IFNγ and GrB levels in Tc cells and inhibited their function in an LDH cytotoxicity assay. The addition of arachidonic acid rescued Tc cell function, suggesting that FTY’s effect is due to inhibition of cytosolic phospholipase A2. Herein, we demonstrate that FTY suppresses Tc cell function independently of its cell trafficking effects. This provides a novel explanation not only for the increased rate of viral infections in FTY-treated patients, but also for its efficacy in MS, as Tc cells have emerged as crucial mediators of MS pathogenesis.
Histone deacetylase inhibition by ITF2357 reduces SLE pathogenesis in vivo (P5161)
Authors: Regna Nicole , Chafin Cristen , Hammond Sarah , Caudell David , Reilly Christopher
Published: January 2023

Histone deacetylase (HDAC) inhibitors are important modifiers of gene and protein expression. In our studies, we sought to determine if a specific HDAC I and II inhibitor (ITF2357) was able to decrease disease in lupus-prone New Zealand Black/White (NZB/W) F1 female mice through regulation of T cell profiles. From 22 - 38 weeks-of-age, NZB/W and non-lupus New Zealand White (NZW) mice were injected intraperitoneally with 10mg/kg of ITF2357 (high dose), 5mg/kg of ITF2357 (low-dose), or the vehicle control. Body weight and proteinuria were measured every 2 weeks, while sera anti-dsDNA and cytokine levels were measured every 4 weeks. Kidney disease was determined by sera BUN and creatinine, immune complex deposition, and renal pathology. T lymphocyte profiles were assessed using flow cytometric analyses and T regulatory cell function was determined by CFSE assay. Our results showed NZB/W mice treated with the high-dose of ITF2357 had increased histone acetylation resulting in a decrease in overall renal disease and inflammatory cytokines in the sera. Treatment with ITF2357 at 10 mg/kg decreased the Th17 phenotype while increasing the percentage and function of Treg cells as well as Foxp3 acetylation. These results suggest that specific HDAC inhibition may decrease disease by altering T cell differentiation and function.
CD19+CD24highCD38high B cells exhibit higher ability in TGF-β and IL-10 secretion and suppressing of CD4+ T cell proliferation but are quantitatively decreased in G-BM of healthy donors (P2146)
Authors: Chang Ying-Jun , Zhao Xiao-Su , Lv Meng , Zhao Xiang-Yu , Han Ting-Ting , Wang Hong-Tao , Huang Xiao-Jun
Published: January 2023

In this study, we investigated the alteration of B cell compartment before and after treating healthy donors with granulocyte colony-stimulating factor (G-CSF) and the principal role of CD19+CD24highCD38high B cells in suppressing T cell proliferation. Our studies revealed that G-CSF-primed bone marrow (G-BM) contains significantly lower numbers of CD19+CD24highCD38high B cells than that of steady-state bone marrow (SS-BM) (P=0.001). Compared to SS-BM, the expression of L-selectin, LFA-1, and ICAM-1 on CD19+CD24highCD38high B cells was significantly lower than that of SS-BM (P=0.019, 0.001, 0.015, respectively). The percentages of interleukin-10 (IL-10), and transform growth factor-β (TGF-β) producing CD19+CD24highCD38high B cells were significantly higher in G-BM than those in SS-BM (P=0.011, and 0.001, respectively). The concentration of IL-10 and TGF-β in cultural supernatant of purified bone marrow B cells in G-BM was also significantly higher than those of SS-BM (P<0.05 for all). Ability in suppression of CD4+CD25- T cell proliferation by activated bone marrow B cells in G-BM was significantly higher than that of SS-BM (P=0.001). Our results suggest that CD19+CD24highCD38high B cells in G-BM of healthy donors exhibit higher ability in TGF-β and IL-10 secretion and suppressing of CD4+ T cell proliferation but are quantitatively decreased. ** Address for Correspondence: Xiao-Jun. Huang, MD E-mail: huangxiaojun@bjmu.edu.cn
Transplant tolerance depends on regulatory T cells induced by TGF-β-producing regulatory B cells (P2168)
Authors: Lee Kang Mi , Kim James , Yeh Heidi , Stott Ryan , Shi Shuai , Deng Shaoping , Markmann James
Published: January 2023

Regulatory B cells (Bregs) have been shown to play a critical role in immune homeostasis and in autoimmunity models. We have recently demonstrated that combined anti-TIM-1 and anti-CD45RB antibody treatment results in tolerance to full MHC mismatch islet allografts in mice, generating Bregs that are necessary for tolerance, and capable of transferring tolerance to untreated animals. Herein, we demonstrate that adoptively transferred Bregs require the presence of Tregs to establish tolerance, and that adoptive transfer of Bregs increases the number of Tregs. Bregs induce significantly more Foxp3 expression in CD4+CD25- T cells, in vivo, compared to naive B cells. We also show that Bregs express the TGF-β associated latency-associated peptide (LAP). Furthermore, Breg-mediated graft prolongation post-adoptive transfer is abrogated by neutralization of TGF-β activity. Collectively, these findings suggest that in this model of antibody-induced transplantation tolerance, Bregs promote graft survival by promoting Treg development, possibly via TGF-β production.
Characterization of lymphocytes emerging early after nonmyeloablative conditioning and hematopoietic stem cell transplant supported with sirolimus (P2186)
Authors: Weitzel R. , Biancotto Angelique , Fitzhugh Courtney , McCoy Philip , Hsieh Matthew , Tisdale John
Published: January 2023

Hematopoietic stem cell transplant is a curative option for patients with severe congenital anemias such as Sickle Cell Disease. Due to the higher transplant-related mortality associated with myeloablative conditioning and the reversal of the SCD phenotype achieved with as low as 11% stable donor chimerism, we developed a successful non-myeloablative regimen for patients with matched-sibling donors consisting of pre-transplant alemtuzumab, 300cGy TBI, and post-transplant sirolimus. However, mechanisms that promote tolerance in these patients remain unclear. We hypothesized that lymphocytes emerging early post-transplant would display a toleragenic phenotype. Samples were thawed from time points near day 60 or day 100 post-transplant. 17-parameter flow cytometry was performed, revealing dramatically high proportion of Tregs (6.3%-32.8%) and T-helper-17 (Th17) (8.2-21.2%) cells, elevated markers of proliferation, activation, and B cell maturity in engrafting patients. Using multiplex cytokine immunoassay, 67 analytes were measured, revealing high expression of IL-17A in the engrafting patients, MCP-1 in the rejecting patients, but little difference in Treg/Th1/Th2-associated cytokines. These unexpected observations have provided insight into reconstitution following nonmyeloablative conditioning and sirolimus-based immunosupporession and suggest targets for further investigation into both the long-term, stable mixed chimerism and the lack of GvHD observed in our patients.
Varying frequencies of T cells exhibiting CMV peptide-specific reactivity to allo-MHC are present in cord blood transplant units (P2228)
Authors: White Jason , Kurche Jonathan , Gutman Jonathan , Kedl Ross
Published: January 2023

Cord blood transplants are a desirable treatment option for a number of hematopoietic diseases due partly to the increased tolerance of HLA mismatches between transplant donor and recipient, manifest in the decreased occurrence of GVHD. Unfortunately, these patients also have an increased risk of reactivating latent infections (e.g. CMV) post-transplant which will ultimately be fatal; likely due to the same decreased T cell functioning that results in HLA mismatch tolerance. Viral reactivation is by no means universal after transplant, however, and while certain factors are known to increase a patient’s risk (e.g. GVHD or in-vivo t cell depletion), there remains no good method for predicting susceptibility. Using MHC tetramers, our lab has found a population of T cells specific to the HLA-A2 CMV peptide in not only A2, but also non-A2 cord blood samples. The frequency of these cells varies widely between cord blood units, which could be a potential signifier of a unit’s efficacy in fighting viral reactivation. Further, preliminary studies suggest that these cells have a specificity and functionality that would make them therapeutically useful.
Retention of autoreactive CD4+ recent thymic emigrants in the liver (BA8P.127)
Authors: Xu Xi , Ge Qing
Published: January 2023

T cells escape from negative selection in the thymus require peripheral tolerance enforcement to prevent them from triggering T cell-mediated autoimmunity. Using adoptive transfer model and RAG2p-GFP transgenic mouse system, we demonstrated that autoreactive CD4+ recent thymic emigrants (RTEs) were retained and some were activated in the liver directly after their egress from the thymus. Educations from secondary lymphoid organs were not required for this hepatic retention but TCR-MHC interaction in the liver was important for the activation of CD4+ liver RTEs. Compared to RTEs in the lymph nodes, liver RTEs exhibited higher TCR reactivity. CD4+ liver RTEs preferentially repopulated the liver and peyer’s patches when adoptively transferred into lymphoreplete mice. When transferred into RAG1-/- mice, liver but not lymph node RTEs caused T cell infiltration and severe inflammation in the lung. These data suggest that the liver may be the first checkpoint in the periphery to filter and retain autoreactive CD4+ thymic emigrants that escape from negative selection. Once unchecked in the absence of regulatory T cells in lymphopenic hosts, these cells could induce Th1 type autoimmunity primarily in the lung.
Cytokines secreted by Th2 cells negatively regulate lymphangiogenesis (CAM3P.202)
Authors: Kim Dong-Eon , Shin Kihyuk , Lee Seung-Hyo
Published: January 2023

Abstract Lymphatic vessels (LVs) consist of a unidirectional network that absorbs protein-rich fluid from peripheral tissues and drains it into the venous circulation through a conduit system of collecting vessels, lymph nodes (LNs), and ducts. In addition to fluid homeostasis, LVs also play an important role in immune surveillance by regulating immune cell migration and antigen delivery to draining LNs. Previous study showed IFN-g, a major Th1 cell-associated cytokine, negatively regulates lymphangiogenesis. However, the effects of the Th2 cell cytokines, IL-4 and IL-13, are still unclear. To reveal this, by using mouse lymphatic endothelial cells (mLECs), human LECs (hLECs), and Prox-1-GFP transgenic mice, we investigated the effects of IL-4 and IL-13 on lymphangiogenesis. Here we show that treatment of mLECs and hLECs with IL-4 and Il-13 down-regulated expression of Prox-1, essential transcription factor expressed by LECs. IL-4 and IL-13 also inhibited tube formation by LECs in vitro. Similarly, LECs co-cultured with Th2 cells showed decreased tube formation, and this effect was fully restored by cytokine neutralization with anti-IL-4 and anti-IL-13 antibodies. Moreover, in vivo blockade of IL-4 and IL-13 increased not only the density of LVs, but also the function of LVs in the airways. Therefore, our findings indicate a novel anti-lymphangiogenic function for Th2 cells via production of IL-4 and IL-13.
LFA-1 and CD62L dependent transient surface CCR5 expression in naïve CD8+ T cells enhances T cell immune memory (CCR3P.214)
Authors: Huang Alex , Askew David , Su Charles , Liou Rachel , Nthale Joseph , Barkauskas Deborah
Published: January 2023

The initiation of an effective adaptive immune response requires choreographed interaction among rare antigen-bearing dendritic cells (DCs) with antigen-specific CD4+ and CD8+ T cells in the lymph node (LN). This interaction is especially critical for the magnitude of memory T cell generation. We and others have previously demonstrated that CCL3/CCL4 secretion from productive interaction between antigen-specific T cell and antigen-bearing DCs promote local recruitment of naïve CD8+ T cells in the inflamed LNs, implying that naïve CD8+ T cells can express CCR5 in the inflamed LN microenvironment. Here, we examined the kinetics of CCR5 expression on naïve T cells upon entering an inflamed LN. Maximum surface CCR5 expression occurs in 10-30% of newly arrived naive T cells within the first 6 hours via mobilization of pre-existing CCR5 protein found in intracellular vesicles, and this transient up-regulation of surface CCR5 expression in CD8+ T cells was dependent on direct physical contact with LFA and CD62L, as ligation of CD8+ T cells with anti-CD11a and/or anti-CD62L antibodies enhances surface CCR5 expression within 1 hour in the absence of TCR stimulation. More importantly, CCR5+ fraction of naïve CD8+ T cells exhibit enhanced ability to become memory T cells. Our study implicates an important functional role for CCR5 expression by naïve lymphocytes prior to cognate antigen recognition for enhanced memory T cell development.
IL-36 contributes to robust effector function of CD8+ T cells co-stimulated with 4-1BB (CD137) and OX40 (CD134) through a TCR independent mechanism (CCR3P.216)
Authors: Tsurutani Naomi , St. Rose Marie-Clare , Adler Adam , Vella Anthony
Published: January 2023

IL-36 cytokines are members of the IL-1 family of cytokines. It has been reported that murine bone barrow-derived dendritic cells and CD4+ T lymphocytes constitutively express IL-36R and respond to IL-36. Also, during CD4+ T cell stimulation, inclusion of IL-36 is known to alter cytokine potential. However, the role of IL-36 in CD8+ T cells is not well understood. In this study, we investigated whether IL-36R is expressed on CD8+ T cells and whether IL-36 exerts effects on CD8+ effector T cell function and phenotype. We found that by quantitative real-time RT-PCR IL-36R is highly expressed on CD8+ T cells after 4-1BB (CD137) and OX40 (CD134) dual co-stimulation. Importantly, these effector CD8+ T cells responded to IL-36β and IL-36γ. We found enhanced IFN-γ production when IL-36 was combined with IL-12, or IL-2. Surprisingly, IFN-γ production was induced in resting effector CD8+ T cells even in the absence of any known TCR trigger. Secondly, the synergy of IL-36 with IL-2 was time dependent and evident at IFN-γ mRNA and protein levels. Together, these data suggest that IL-36 can enhance the effector function of CD8+ T cells and may be a promising immunotherapy candidate.
TL1A level (low versus high) differentially affects expansion and function of regulatory and effector T cells. (CCR3P.218)
Authors: Sidhu Maninder , Shih David , Targan Stephan
Published: January 2023

Patients with Crohn’s disease have increased mucosal expression of TL1A. Mice generated with sustained and elevated T cell expression of Tl1a (L-Tg) have increased Foxp3+ Treg cells yet develop worsened ileitis, proximal migration of colitis and intestinal fibrosis. The aim of this study was to investigate the role of Tl1a in the differentiation and function of Tregs. In the L-Tg mice, the CD4+ T cell population was comprised of GFPhigh and GFPlow cells that correlated with the level of Tl1a expression. We found that Foxp3 expression was significantly higher in the GFPlow population of CD4+ T cells compared to the GFPhigh cells. The GFPlowFoxp3+ T cells expressed high Nrp-1, GARP, and LAG-3 surface markers and were more suppressive in function whereas the GFPhigh Foxp3+ T cells had an activated effector memory phenotype and were highly proliferative. Induced Tregs differentiated in-vitro from naïve GFPlow CD4+ T cells expanded into TGF-β producing Foxp3 T cells whereas naïve T cells from the GFPhigh fraction expanded into cells with a Th2 phenotype, producing IL-4, IL-13 and IL-10. Preliminary data from a T cell co-transfer study suggested that mice cotransferred with the GFPlowFoxp3+ T cells were better protected from weight loss compared to mice cotransferred with GFPhighFoxp3+ T cells. These data suggest that level of Tl1a (high v. low) differentially influenced the expansion of effector and Treg cells leading to either a pro-inflammatory or protective mucosal environment.
Expression of secreted osteoclastogenic factor of activated T cells (SOFAT) by T- and B-lineage cells may contribute significantly to bone loss in periodontitis. (CCR3P.221)
Authors: Napimoga Marcelo , Martinez Elizabeth , Peruzzo Daiane , Carregaro Vanessa , de Araújo Vera , Weitzmann M.
Published: January 2023

A novel T cell-secreted cytokine, termed secreted osteoclastogenic factor of activated T cells (SOFAT), that induces osteoclastic bone resorption in a RANKL-independent manner, has recently been described. We have previously reported that SOFAT is highly expressed in gingival tissues of patients with chronic periodontitis (CP) suggesting a putative role in the bone loss associated with CP. Here we characterized the cell types producing SOFAT in CP. Gingival tissues were biopsied from subjects without periodontal disease (n=5) and patients with CP (n=5) and SOFAT quantified by immunohistochemistry and immunofluorescence staining. Our data reveal marked SOFAT staining in CP that was predominantly associated with the lymphocytic infiltration of gingival tissues. Interestingly, besides CD3+ T cells, B-lineage cells including plasma cells also exhibited strong staining for SOFAT. Because SOFAT has not previously been reported in B-linage cells, we further purified splenic T cells and B cells from Balb/c mice and activated them using LPS or CD3/CD28 antibodies respectively. SOFAT was quantified by RT-qPCR and revealed significant induction of SOFAT expression in both activated T cells and B cells in comparison to unstimulated cells. Our data support a putative role of SOFAT in the bone loss associated with chronic periodontitis and further demonstrates for the first time that in addition to T cells, B-linage cells may also be a significant source of SOFAT in inflammatory states.
The biological function of membrane versus soluble TL1A (CCR3P.205)
Authors: Ferdinand John , Meylan Francoise , Siegel Richard , Al-Shamkhani Aymen
Published: January 2023

The Tumour Necrosis Factor Superfamily (TNFSF) are an important source of costimulatory signals required for activation of T cells. The TNFSF is composed of 29 receptors and 18 ligands; one such pair is Death receptor 3 (DR3) and TNF-like ligand 1A (TL1A). Signalling via DR3 costimulates lymphocytes and polymorphysims in TL1A and DR3 have been associated with Crohn's Disease. In mice, signalling via DR3 has been shown to be required for maximal pathology in models of asthma and lupus and transgenic constitutive expression of TL1A results in an IL-13 and IL-17a driven intestinal pathology. Several TNFSF ligands, including TL1A, can be expressed on the membrane and cleaved to yield a soluble product. Some cytokines, such as TNF are active in soluble form whereas others, such as FasL, are not. The biological roles of these distinct forms of TL1A in vivo are not known. To investigate the biological function of membrane vs. soluble TL1A we used retrotransgenic technology to express cleavable vs. membrane bound TL1A throughout the murine hematopoietic system. Membrane restricted TL1A promoted a strong inflammatory pathology in both the ileum and lung. Full length TL1A expression yielded little membrane TL1A. These mice failed to develop pathology despite a greater than physiological level of TL1A in the serum. This work demonstrated that membrane restricted TL1A is a more potent ligand for DR3. We have generated membrane-restricted TL1A transgenic mice to investigate this further.
Inhibiting TWEAK (TNF-like weak inducer of apoptosis) signaling ameliorates blood brain barrier integrity and neuronal damage in neuropsychiatric lupus prone MRL/lpr mice (CCR3P.212)
Authors: Wen Jing , Doerner Jessica , Stock Ariel , Sellers Rani , Burkly Linda , Gulinello Maria , Putterman Chaim
Published: January 2023

Neuropsychiatric disease (NPSLE) is one of the most common manifestations of human lupus, but remains poorly understood. TWEAK is a cytokine member of the TNF superfamily; its sole receptor, Fn14, is expressed in brain endothelial cells, astrocytes, microglia and neurons. In vitro, TWEAK signaling promotes expression of MCP-1, IL-6 and IL-8, decreases ZO-1 expression, and increases the permeability of human brain microvascular endothelial cells. Our previous study showed that Fn14 knockout (KO) in the MRL/lpr lupus strain led to markedly attenuated neuropsychiatric disease, as revealed by significant reduction in depressive-like behavior and improved cognitive function. The purpose of the present study was to determine the mechanisms by which TWEAK signaling is instrumental in the pathogenesis of NPSLE. We found that Fn14KO mice had improved BBB integrity as shown by decreased fibronectin and IgG deposition in the brain. Further, there were fewer cellular infiltrates in Fn14KO mice. Additionally, Fn14KO mice displayed reduced neuron apoptosis and hippocampal gliosis. Interestingly, there were no differences in neurogenesis and microglia activation, or in circulating autoantibody titers, between Fn14KO and Fn14WT mice. Our studies indicate that TWEAK/Fn14 interactions play a central role in the pathogenesis of NPSLE by improving BBB integrity and reducing neuronal damage, suggesting a novel target for therapeutic intervention for this major disease manifestation.
Recruitment of adult thymic progenitors is regulated by lymphotoxin beta receptor (HEM4P.240)
Authors: Shi Yaoyao , Wu Weiwei , Fu Yang-Xin , Zhu Mingzhao
Published: January 2023

Continuous homing of hematopoietic progenitors from peripheral blood to thymus is essential for the functional maintenance of thymus, especially upon thymic injury. Here we identified the signaling of lymphotoxin beta receptor (LTβR), is essential for the thymic progenitor settling. Dramatically reduced frequency and number of early T-lineage progenitors (ETP) were found in the steady thymus of LTβR knockout (KO) mice. This is probably not due to impaired development of hematopoietic progenitors, since normal frequencies of T-cell precursor were found in the bone marrow of LTβR KO mice. Further experiment by tracing adoptively transferred bone marrow cells directly confirmed a thymic homing defect in LTβR KO mice. Progenitor thymic settling is mediated by a cascade of adhesion and signal events, which involves P-selectin, CCL25, VCAM-1/ICAM-1, etc. We identified P-selectin is down-regulated in the thymi of LTβR KO mice while the expression levels of CCL25, VCAM-1, VCAM-1 are normal. Data from bone marrow chimeric mice suggest that LTβR on radio-resistant, likely stromal cells, regulates thymic progenitor receptivity. Finally, LTβR KO mice demonstrated severely impaired thymus regeneration upon thymus-targeted injury. Taken together, our data reveal a novel role of LTβR on the regulation of thymic progenitor homing. Manipulation of this signaling pathway may boost thymic regeneration and immune reconstitution during chemotherapy, radiotherapy or AIDS HAART therapy.
Thymic aging in the absence of klotho (HEM4P.243)
Authors: Smith Michelle , Goetz Christine , Min Dullei , Stefanski Heather , Hollander Georg , Weinberg Kenneth , Blazar Bruce
Published: January 2023

Age-related thymic involution, characterized by a decrease in thymic epithelial cell (TEC) proliferation, absolute number, and organization, results in overall defective support of T cell development and proliferation and is a major cause of thymic dysfunction. Using a genetic model of advanced aging caused by deficiency of Klotho, an anti-aging gene and modifier of FGF signaling, we investigated TEC-intrinsic aging in the absence of a globally aging environment. By one month of age, Klotho deficient (Kl-/-) mice display a disrupted thymic architecture, a 4-6 fold decrease in TEC numbers, as well as significant decreases in all thymocyte populations and also a 3-8 fold decrease in peripheral T cell numbers. To separate thymus-intrinsic aging from the effects of organismal aging, we implanted fetal thymi from Kl-/- mice under the kidney capsule of mice lacking thymi. Kl-/- and WT fetal thymi supported comparable levels of thymopoiesis as measured by the numbers of T cells in peripheral blood and lymphoid organs up to 4 months post-implantation, with both cohorts of implants not detectable past 5 months. In this aging model, isolation from the systemic effects of Klotho deficiency attenuates age-related thymic dysfunction, suggesting that extrathymic factors affect thymic aging.
Human T cell development in HSC-engrafted NSG mice is dependent on age of the recipient (HEM4P.247)
Authors: Hasgur Suheyla , Shultz Leonard , Brehm Michael
Published: January 2023

Humanized mouse models are an exciting alternative for the investigation of human hematopoiesis, immunobiology, autoimmunity, infectious diseases, cancer biology, transplantation biology and regenerative medicine. However the efficient generation of humanized mice is complicated by the availability of numerous mouse strains and the large diversity of engraftment protocols. NOD-scid IL2rγnull (NSG) mice injected with human HSC support high levels of engraftment and develop a functional human immune system including both innate and adaptive immune cells. Previous studies have shown that injection of human HSC into newborn NSG mice results in more efficient and rapid T cell development as compared to adult NSG (>6 wk) mice. Here we compared engraftment of human HSC and immune system development following intracardiac injection into irradiated newborn NSG mice or intravenous injection into irradiated 3-wk old NSG mice. Total human cell chimerism was monitored in the blood between 6 and 18 wk after transplantation. We also evaluated the development of human T cells, B cells, monocytes, NK cells, NKT cells, conventional and plasmacytoid DC. Our results show that newborn NSG mice have higher levels of T cell reconstitution at earlier time points as compared to 3-wk old NSG recipients but that the older mice achieved comparable T cell levels at later times. Together these findings show that T cell development in humanized mice occurs with slower kinetics in older NSG mice.
Alteration of TCR Dβ sequence content alters Treg development (HEM4P.248)
Authors: Levinson Michael , Sanchez-Silva Aaron , Zhuang Yingxin , Sleckman Barry , Schroeder Harry
Published: January 2023

Diversity of the T cell receptor is mainly developed through V(D)J rearrangement and N addition during TCR development. The product of V(D)J rearrangement is the CDRβ3, a region of high variability that recognizes antigen and includes all of the D gene. The Dβ sequence is highly conserved across various species, from trout to mouse to humans. This suggests that there are some natural constraints on the TCR; these constraints are thought to limit deleterious T cells from reaching the periphery. We hypothesize that altering the D region will have an effect on the development of thymocytes. To do this we have created Dβ altered mice. These mutants are a Dβ2 KO (Dβ1); a replacement of the Dβ locus with a charged Dβ (DβDKRQ); and a replacement of the Dβ locus with a hydrophobic DH (DβYTL). When compared to WT mice, the mutant Dβ mice have an altered T cell repertoire in both CDRβ3 amino acid composition and length and these differences can be attributed to the changes in the germline sequence. Changing the Dβ also changes the total T cell number in both developing and mature T cells, with an altered Dβ being selected against. However, Treg numbers from the DβDKRQ mice are increased in the mucosa, suggesting a link between charge, self-reactivity and nTreg induction. Ongoing experiments will elucidate the role of the Dβ sequence on the development of T cells and the role it plays in infection and inflammation.
Ikaros and aiolos undergo different post-transcriptional regulation in mice and humans (HEM4P.249)
Authors: Mitchell Julie , Szarejko John , Yankee Thomas
Published: January 2023

The Ikaros family of transcription factors is important for T cell development and leukemogenesis. In mice, homozygous expression of an Ikaros isoform that is unable to bind DNA leads to a complete block in T cell development, while heterozygous expression of the same Ikaros isoform results in leukemogenesis. To better understand the roles of Ikaros family members in human and murine T cell development, we used quantitative RT-PCR and Western blot analysis to determine the mRNA and protein levels of Ikaros family members in FACS-purified murine and human thymocytes. As thymocytes mature from the double negative (DN) to double positive (DP) stages, Ikaros mRNA levels increase 3.0±1.8 fold (p<0.01, n=6) in mice and 4.4±1.7 fold (p<0.001, n=5) in humans. Similarly, Aiolos mRNA levels increase 27±2.3 fold (p<0.001, n=6) in mice and 18±2.8 fold (p<0.001, n=5) in humans. However, during the same DN to DP transition, protein levels only increase significantly in mice for both Ikaros (2.5±0.79 fold, p<0.001, n=6) and Aiolos (11±5.4 fold, p<0.01, n=5). These results suggest that Ikaros and Aiolos undergo different post-transcriptional control in murine and human thymocytes. Further studies are needed to understand the method of this post-transcriptional control, and how it regulates human T cell development and leukemogenesis.
Clinical isolates of Mycobacterium tuberculosis associated with different transmission phenotypes induce differential activation of the host immune response (HEM4P.251)
Authors: Verma Sheetal , Rodrigues Rodrigo , Jones-Lopez Edward , Palaci Moises , Alland David , Dietze Reynaldo , Ellner Jerrold , Salgame Padmini
Published: January 2023

In a Household contact (HHC) study conducted in Brazil, we have categorized “index” cases into High (HT) and Low (LT) transmission groups based on the number of their household contacts testing positive for the tuberculin skin test. A balanced induction of lipid mediators (Leukotriene B4, Prostaglandin E2 and Lipoxin A4) and Tumor necrosis factor (TNF) is essential for host defenses. Virulent Mycobacterium tuberculosis (Mtb) strains induce an imbalance in host lipid mediators to result in increased TNF production and activation of pro-necrotic pathway in macrophages. As necrosis is associated with increased growth of extracellular bacilli, we hypothesize that Mtb isolates derived from HT index cases exploit the TNF/necrotic axis in macrophages to increase the infectiousness in the index case, while LT isolates reduced necrosis but enhanced intracellular replication. Consistent with this, preliminary analysis of eight isolates confirms significantly higher TNF expression in human monocyte-derived macrophages infected with HT isolates in comparison to LT isolates. All isolates induced similar expression of IL-1β, IL-8 and IL-10. Ongoing experiments are evaluating the lipid mediator response, induction of pro-necrotic pathway, and bacterial growth in cells infected with HT and LT Mtb isolates. Together, the findings will provide insight into the role of Mtb strain variation and the resultant differing host immune response that contributes to their transmission phenotype.
TSLP signaling in CD4 T cells is required for T helper 2 memory (HYP6P.259)
Authors: Zhou Baohua , Wang Qun , Du Jianguang , Zhu Jingjing
Published: January 2023

Thymic stromal lymphopoietin (TSLP) plays important roles in the differentiation of T helper 2 (Th2) cells and thus the pathogenesis of allergic diseases. Whether TSLP is required for the generation and maintenance of Th2 memory cells has not been clearly defined. In this study, we show that TSLP signaling in CD4 T cells is required for the generation of memory cells following OVA+Alum priming. TSLPR-deficient CD4 T cells have no defects in proliferation but fail to survive one week after sensitization, while elevated TSLP expression during sensitization significantly increased the frequency of memory cells. Although in vitro differentiated TSLPR-deficient Th2 cells develop into memory cells with equal efficiency to wild type cells, the recall response to airway antigen challenge is impaired. Moreover, after antigen challenge of mice receiving in vitro primed Th2 cells, TSLP signaling in CD4 T cells significantly affects memory cell generation from the secondary effector cells. Therefore, TSLP plays a critical role not only in Th2 memory generation in vivo, but also in recall response of the memory cells to local antigen challenge.
Visualization of allergic immunity reveals non-coordinate expression of IL-4 and IL-13 in the innate immune system (HYP6P.260)
Authors: O'Brien Thomas , Bao Katherine , Ang Wei Xin Gladys , Abraham Soman , Reinhardt Richard
Published: January 2023

Allergic disease affects more than 3 billion people globally. In developed countries, allergic disease manifests itself as allergy and asthma, while in developing countries it takes the form of parasitic infections. The pathogenesis that develops as a consequence of allergic inflammation is mediated by the cytokines interleukin-4 (IL-4) and IL-13. Re-stimulation studies have demonstrated that a variety of cells in the lung are competent to produce IL-4 and IL-13 in vitro. At the present time, however, we have no clear understanding of which cells are responsible for producing these cytokines in vivo. In order to examine the cellular sources of IL-4 and IL-13 during allergic inflammation, we used IL-4 and IL-13 cytokine reporter mice to directly visualize cytokine production in vivo. These studies revealed that CD4+ T cells were the primary source of both IL-4 and IL-13 in allergic inflammation. Additionally, these studies revealed that CD4+ T cells produce IL-4 and IL-13 equivalently on a per cell basis. This is in stark contrast to innate cells, which preferentially produce either IL-4 or IL-13. NKT cells, eosinophils, and basophils produce IL-4, while ILC2 cells produce IL-13. Interestingly, IL-13 production was restricted to cells expressing high GATA-3. No such relationship was observed in IL-4 producing innate cells. In summary, these findings challenge the current dogma of coordinate regulation of IL-4 and IL-13 during allergic inflammation.
Proinflammatory role of natural killer cells in allergic airway disease (HYP6P.264)
Authors: Mathias Clinton , Guernsey Linda , Zammit David , Brammer Clair , Thrall Roger , Aguila Hector
Published: January 2023

Natural Killer (NK) cells have been implicated in the development of allergic airway inflammation. However the in vivo role of NK cells has not been firmly established due to the lack of animal models with selective NK cell deficiencies. We studied the role of NK cells in a model of ovalbumin-induced allergic airway disease (OVA-AAD) using NK-deficient (NKD) mice, perforin-/- mice, and mice depleted of Ly49A/D/G+ NK cell subsets. Induction of OVA-AAD in wild-type (WT) mice resulted in the expansion of airway NK cells and the development of pronounced airway eosinophilia. In the absence of NK cells or specific NK cell subsets, either in NKD mice, or after depletion of Ly49A/D/G+ NK cells, the development of OVA-AAD was significantly impaired as seen by decreased airway inflammation, decreased secretion of IL-4, IL-5 and IL-13 and diminished OVA-specific antibody production. Furthermore, while OVA-exposure induced a dramatic expansion of dendritic cells in WT mice, their induction was significantly attenuated in NKD mice. Development of OVA-AAD in perforin-/- mice suggested that the proinflammatory role of NK cells is not dependent on perforin-mediated cytotoxicity. Lastly, induction of allergic disease by OVA-specific CD4 T cells from WT but not NK-depleted or NKD mice in RAG-/- recipients, demonstrates that NK cells are essential for T cell priming. In summary, our data demonstrate that NK cells play an important and distinct role in the development of AAD.