bluuebunny/crossref_metadata_2025 huggingface dataset:
Antigen-specific Treg and IL-10 influence DC immunogenicity hours following vaccination with a self versus a foreign antigen (P4232)
Authors: Marvel Douglas , Finn Olivera
Published: January 2023

Mice transgenic for the human tumor antigen MUC1 (MUC1.Tg) are hyporesponsive to MUC1 peptide relative to WT mice. A recently identified biomarker of this hyporesponsiveness is suppression in splenic dendritic cells (DC) of a family of pancreatic enzymes, represented by several trypsin isoforms and carboxypeptidase B1. These enzymes are upregulated following vaccination of WT mice with MUC1 peptide (foreign antigen) or MUC1.Tg mice with ovalbumin (foreign antigen), but are suppressed following vaccination of MUC1.Tg mice with MUC1 peptide (self antigen). This suppression can be alleviated by eliminating Tregs or by blocking signaling through the IL-10 receptor. This enzyme profile marking suppression or activation of DC could be identified as early as 4 hours post MUC1 peptide vaccination of WT and MUC1.Tg mice. Differences in enzyme expression also correlated with differential NK cell activation at the same early timepoint as measured by IFNγ and IL-10 secretion. This work shows that the decision for tolerance or immunity is established in multiple cell types no more than 4 hours post vaccination and that these enzymes are an early biomarker of this decision.
Pharmacological calcium mobilization synergizes with TLR signaling to enhance IL-12 production by murine DC (P4238)
Authors: Huang Chi , Berk Erik , Xu Min , Czerniecki Brian , Koski Gary
Published: January 2023

A critical DC-produced factor for polarizing Th1 immunity is the heterodimeric cytokine IL-12. Although IL-12 production can be triggered by stimulation of DC with single Toll-like receptor (TLR) ligands, optimized production requires that multiple signals be delivered to the DC. These include dual TLR ligands, single TLR ligands paired with the cytokine interferon-gamma or CD40L combined with these other signals. Although calcium mobilizing signals have been shown to induce phenotypical maturation of DCs, they have never been shown to synergize with TLR or cytokine signals to enhance IL-12 production. Murine bone marrow-derived DCs pretreated with calcium ionophore 4 hours prior to TLR ligand exposure significantly enhance IL-12 p70 production by DCs. Thapsigargin, a compound that enhances intracellular calcium levels by a different mechanism, had similar effects. The drugs cyclosporine A and ascomycin, which antagonize the activity of the calcium-dependent serine-threonine phosphatase, calcineurin, suppressed the IL-12-enhancing activity of calcium mobilization. When mouse T cells were co-cultured with allogeneic DCs activated with calcium ionophore, TLR ligands, or both, paired activation stimulation induced DCs with superior T cell stimulatory capacity as measured by IFN-gamma output (ELISA), and proliferation measured by CFSE dilution. Calcium mobilization may therefore provide a method for superior activation of Th1-polarizing DCs for clinical testing.
Antigen-specific tolerance induction by Tregitopes in vivo (P4202)
Authors: De Groot Anne , Cousens Leslie , Moise Leonard , Najafian Nader , Mingozzi Federico , Elyaman Wassim , Mazer Bruce , Khoury Samia , Su Yan , Scott David , Martin William
Published: January 2023

Modulation of T cell responses provide of new opportunities in the treatment of autoimmune and inflammatory diseases. Tregitopes are regulatory T cell (Treg) epitopes found in IgG that provide beneficial immunomodulatory effects, paralleling effects attributed to IVIG. In this presentation, we will provide evidence that Tregitopes derived from human IgG can reproduce immunomodulatory effects of IVIG in vitro and in vivo. Tregitopes cause CD4+CD25+FoxP3+ Treg to expand and produce IL-10 in vitro, and iTreg are induced in vivo. Induction and functions of Tregs have been examined in in vivo model systems such as D011.10 TCR transgenic mice, transplant of BM12 to C57BL/6, AAV-mediated gene transfer and EAE. Together, the data show that effector T cells, Th17, and Th9 cells are modified in the presence of Tregitopes. In OVA-induced allergic airway disease, we observed significant and reproducible expansion of Tregs in conjunction with decreased airway reactivity comparable to, if not greater than IVIG. We will provide additional unpublished evidence demonstrating the antigen specificity of tolerance induction using Tregitopes in conjunction with target antigens, and discuss the relevance of Tregitopes to the treatment of human immune-mediated diseases. Additionally, Treg epitopes (Tregitopes?) have been identified in other common serum proteins, suggesting that peripheral control of inflammatory signals may in part be due to intrinsic self-protein “off switches".
CD45RB ligation ameliorates experimental colitis by inducing Tregs and inhibiting inflammatory Teff cells (P4206)
Authors: Lin Yan , Froicu Monica , Rothstein David
Published: January 2023

In wt mice, α-CD45RB (MB23G2) treatment expands Tregs by both inducing iTregs and by enhancing homeostatic proliferation of nTregs. We examined the effect of α-CD45RB in a cell transfer model of colitis. Transfer of 3X105 CD4+Foxp3-CD45RBHi (Tconv) cells from Foxp3-reporter mice into Rag-/- mice resulted in severe colitis with 20% wt loss by 8 wks. α-CD45RB treatment (100ug; d0, 1) had no effect on number of Tconv but increased iTreg ≥ 2X in mesenteric LN (MLN), spleen, and colonic tissue. This completely prevented wt loss and decreased colitis score (at 8 wks) by 25% vs. Tconv cells alone (p<.05). While transfer of subtherapeutic (104) nTreg had no effect on colitis induced by Tconv, adding a-CD45RB prevented wt loss, further cut the colitis score by 50%, and increased Treg ~2.5X. To confirm the role of Tregs, Tconv were transferred from Foxp3-DTR mice and iTregs eliminated with diphtheria toxin. In this setting α-CD45RB still delayed onset of colitis, although all mice began losing wt by 3 wks (and there was no survival benefit). In this setting, delayed colitis by α-CD45RB was associated with a 2X decrease in IFNγ expression by Teff in MLN, whereas IL-17 was unchanged. These data demonstrate that α-CD45RB can uniquely ameliorate colitis by inducing iTreg or by expanding nTregs, and also inhibits colonic inflammation by Teff cells, possibly by inhibiting IFNγ.
Protection by a broadly neutralizing monoclonal antibody to pandemic Influenza requires selective Fc receptor engagement (P4216)
Authors: DiLillo David , Tan Gene , Palese Peter , Ravetch Jeffrey
Published: January 2023

Neutralizing Abs against influenza viruses have traditionally been thought to provide protection by interfering with virus entry and/or fusion with host cells, events that rely solely on Ab variable region binding to viral spike proteins. However, the contributions of in vivo mechanisms conferred by Fc-Fc receptor (FcγR) interactions during protection by Ab remain unclear. We assessed the role of FcγRs during influenza virus neutralization in mice using mAbs directed to the HA antigen. We found that a broadly neutralizing mAb (bNAb) targeting the conserved stem region of HA confers protection to mice from lethal viral challenge, and required Fc-FcγR interactions for in vivo activity against both laboratory virus strains as well as the 2009 pandemic H1N1 virus. By contrast, a strain-specific mAb to the variable head domain of HA was equally protective both in the presence or absence of FcγR interactions. We then generated a chimeric human IgG1 Fc version of the anti-stem bNAb, modified to selectively enhance human activating FcγR engagement. We utilized FcγR-humanized mice, which express all human FcγRs on a genetic background lacking mouse FcγRs, to demonstrate augmented protection by the modified bNAb compared to wild type bNAb. Thus, Fc-FcγR interactions are required for bNAb-mediated protection and can be manipulated for optimal protection in vivo. These findings represent a general approach towards enhancing bNAb-mediated protection and warrant clinical investigation.
Monitoring the evolution of antigen-specific antibodies in circulation and their corresponding B cell clones in longitudinal immunization case studies through the use of NG-XMT (P4186)
Authors: Darce Jaime , Lutz Stephen , Schieferl Sandra , Moreno Elizabeth , Ramenani Ravi , Wieler James , Popova Lana , Beaudet Jason , Zhang Xiaowu , Sato Shuji , Beausoleil Sean , Polakiewicz Roberto , Cheung Wan
Published: January 2023

Previous studies aimed at determining the affinity maturation pathways of monoclonal antibodies during an immunization have relied on comparing germ-line with mature forms of antibodies present in either transgenic B cell receptor mouse models or by cloning circulating human B cells present in peripheral blood (PB) of pre and post-immunized individuals. These approaches have been insightful, but they do not identify in confidence the true B cell clone(s) producing the antigen-specific antibodies found in the sera of immunized animals or humans. We recently developed a technology, NG-XMTTM, encompassing tandem mass spectrometry and next generation sequencing1,2, which for the first time enables the 1) identification of the anatomical location of the B cell clone(s) corresponding to the antigen-specific serum antibodies 2) investigation of clonal diversity of antigen-specific B cell clones residing in primary and secondary lymphoid organs and circulating in PB and 3) analysis of the evolving affinity mature serum antibodies, in two longitudinal immunization studies of rabbits and humans. These results not only advance our understanding of the affinity maturation process and the dynamics of memory B cells and plasma cell development that occurs during an immunization response, they also allow us to use this information to generate useful recombinant antibodies that can be utilized in passive immunization strategies. 1Nat Biotechnol. 2012,30:447; 2Nat Biotechnol. 2012,30:1039
In vitro and in vivo pharmacological assessment of a targeted plasmid gene containing immunotoxin, DT390-IL-18-SRα on suppression of immunopathology in mice experimentally infected with Shistosoma mansoni (P4192)
Authors: Al harbi Naif , Bahashwan Saleh
Published: January 2023

Purpose: To evaluate both in vitro and in vivo effects of DT390-IL-18-SRα immunotoxoin on the development of liver immunopathology in murine schistosomiasis mansoni. Methods: The antiproliferative effect and in vitro granuloma formation (IVGF) inhibition were evaluated using methyl thaiazolyl tetrazolium and IVGF index respectively. In vivo evaluation, mice were divided into four groups of twelve mice each: Group1 normal non infected (-ve control), Group 2, 3 and 4 were infected percutaneously with 25 cercariae. After 6 weeks post infection, Group2 was treated with PBS (+ve control), Group3 was treated with plasmid DNA (50 µg) embedded with cationic liposome in 50 µl PBS, injected intramuscular in the hind limbs once daily for two weeks and Group4 was treated with DT390-SRα (mutant control). After animals sacrification, liver was removed for histopathological studies. Results: DT390-IL-18-SRα showed suppression of spleen lymphocytes proliferation and IVGF in a dose dependant manner as well as suppression of liver granulomas size by 80%, while mutant toxin had no significant suppression. Conclusion: The immunotoxin showed selective toxicity to antigen activated lymphocytes in vitro with reduced clinical and pathological severities of the disease.
Effects of sublingual immunotherapy on a murine model of Japanese cedar pollinosis (P4198)
Authors: Azuma Miyuki , Zhang Chenyang , Yanagisawa Shohei , Tomoda Taketo , Kang Siwen , Ohno Tatsukuni
Published: January 2023

Japanese cedar (Cryptomeria japonica) pollinosis (JCP) is one of the most common allergic diseases in Japan. Sublingual immunotherapy (SLIT) has been performed as a safer and more beneficial treatment to induce allergen-specific immune tolerance. However, immunological mechanisms and therapeutic biomarkers for SLIT remain unclear. In this study, we have established a new murine model of JCP that resembles human JCP with crude pollen grains and without the use of adjuvant and examined the effects of SLIT. SLIT was performed by the graded doses of cedar pollen extract painting onto the ventral surface of tongue for 28 days. [Results] The frequency of sneezing and the duration of rubbing for 10 min after the final challenge, serum IgE titer, pollen-specific IgG1, and the ratio of eosinophils in BALF and nasal mucosae were significantly elevated in the pollinosis mice. SLIT efficiently reduced the above manifestations. SLIT significantly decreased pollen-specific IL-4, IL-5, IL-13 and IL-10 production by splenic T cells and DC activation status such as MHC class II, CD86, and B7-H1 expression in the regional lymph nodes. Histological examination revealed that repeated pollen extract painting onto the sublingual mucosae replaced sublingual resident DCs with newly recruited CD11b+ DCs. These results suggest that our new pollinosis model is beneficial to evaluate and develop a new immunotherapy and therapeutic biomarkers.
CD4 T regulatory cells (Treg) control CD8 T cell responses to human herpesvirus 8 (HHV-8) lytic and latency proteins (P6056)
Authors: Lepone Lauren , Rappocciolo Giovanna , Knowlton Emilee , Jenkins Frank , Rinaldo Charles
Published: January 2023

Although CD8 T cells are considered important in controlling HHV-8 (aka KSHV) infection, the etiologic agent of Kaposi’s sarcoma (KS), there is little evidence to support this. Moreover, these T cell responses are relatively non-robust, suggesting that they are under tight regulatory control. We conducted a comprehensive, non-concurrent prospective study of the effect of Treg on CD8 T cell reactivity to HHV-8 lytic and latency proteins in HHV-8 monoinfected, healthy subjects compared to untreated, HHV-8/HIV-1 coinfected subjects who did and did not develop KS in the Multicenter AIDS Cohort Study. Direct staining of PBMC with MHCI multimers showed a relatively high frequency of HHV-8 antigen-specific CD8 T cells compared to low functional T cell activity (IFN-γ ELISPOT) in response to HHV-8 lytic and latency proteins and HIV-1 Gag beginning many years prior to development of KS. Removal of Treg enhanced HHV-8 specific, IFN-γ production by CD8 T cells, with or without HIV-1 coinfection and KS. These data show that beginning very early in HHV-8/HIV-1 coinfection, low antiviral T cell responses are related to eventual development of KS. Moreover, Treg suppress CD8 T cell responses to HHV-8 antigens, effectively masking more robust, underlying anti-HHV-8 T cell responses. The involvement of CD8 T cells and Treg in control of HHV-8 infection has important implications for understanding HHV-8 immunopathogenesis and development of vaccines to prevent KS.
HLA targeting efficiency correlates with human T cell response magnitude and with mortality from influenza A infection (P6082)
Authors: Hertz Tomer , Oshansky Christine , Roddam Phillipa , DeVincenzo John , Caniza Miguela , Jojic Nebojsa , Mallal Simon , Phillips Elizabeth , James Ian , Thomas Paul , Halloran M. , Corey Larry
Published: January 2023

Experimental and computational evidence suggests that human leukocyte antigens (HLAs) preferentially bind conserved regions of viral proteins, a concept we term “targeting efficiency,” and that this preference can provide improved clearance of infection in several viral systems. One potential advantage of targeting conserved regions of a virus is the higher likelihood of cross-reactive protection from other viral strains, including those newly introduced, such as pH1N1. We hypothesized that individuals that targeted less conserved regions of influenza- A viruses would have weaker T-cell responses following infection with pH1N1. To test this hypothesis, T cell responses to A/H1N1 (2009) were measured from PBMCs obtained from a household cohort study performed during the 2009-2010 influenza season. We found that HLA targeting efficiency scores significantly correlated with IFNγ ELISpot responses (p=0.042, multiple regression). A further population-based analysis found that the carriage frequencies of the alleles with the lowest targeting efficiencies, A*24, were associated with pH1N1 mortality (r=0.37, p=0.031). Interestingly, these are common in certain indigenous populations in which increased pH1N1 morbidity has been reported. The computational tools utilized in this study may be useful predictors of potential morbidity and identify immunologic differences of new variant influenza strains more accurately than evolutionary sequence comparisons.
Blockade of chronic type I interferon signaling to control persistent virus infection (P6048)
Authors: Wilson Elizabeth , Brooks David
Published: January 2023

While both immunostimulatory and inhibitory signals are triggered by acute viral infections, persistent infections are characterized by sustained expression of an immunosuppressive program that inhibits antiviral immunity, interferes with viral clearance, and poses a barrier to effective antiviral therapeutics. However, the signals that govern the immunosuppressive program are not understood. Viral infections elicit type I interferons (IFN-I) and these proteins have potent direct antiviral effects. However, IFN-I expression has also been associated with hyper immune activation and increased disease severity in persistent viral infections. We demonstrate that antibody blockade of IFN-I signaling diminished chronic immune activation, and the immunosuppressive program, while increasing multiple immune parameters associated with control of virus replication, ultimately allowing for clearance of the persistent infection. These data highlight the duality of the IFN-I response during viral infection-serving both as a direct extinguisher of viral replication and as a central rheostat and regulator of the immunosuppressive program, dampening T cell responses and preventing viral clearance. Thus, we identify IFN-I as a key factor in immuno-surveillance during viral infection and demonstrate that therapeutic ablation of chronic IFN-I signaling can enable control of persistent viral infection.
Th17 cells demonstrate stable cytokine production in allergic inflammation (P1143)
Authors: Glosson Nicole , Sehra Sarita , Yu Qing , Stritesky Gretta , Nguyen Evelyn , Kaplan Mark
Published: January 2023

Th17 cells are critical for the clearance of extracellular bacteria and fungi, but also contribute to the pathology of autoimmune diseases and asthma. Th17 cells can acquire a Th1-like phenotype in vitro and in vivo, but less is known about their ability to adopt Th2 and Th9 effector programs. To explore this in more detail, we generated an IL-17F lineage tracer mouse strain that allows tracking of cells that formerly expressed IL-17F and can be used to test stability of the Th17 phenotype. Th17 cells cultured under polarizing conditions that promote Th1, Th2 or Th9 differentiation adopt the respective effector programs, expressing lineage associated transcription factors, and secreting IFN-γ, IL-4 or IL-9, while diminishing IL-17 production and expression of RORγt. To explore the stability of Th17 cells in an in vivo environment that is associated with the development of Th2 and Th9 cells, we used the adjuvant-dependent, OVA/Alum and the adjuvant-free, house dust mite models of allergic airway disease (AAD). In both models of AAD, Th17 cells from the lungs of diseased mice did not secrete alternative cytokines nor adopt Th1, Th2 or Th9 effector programs, but remained stable IL-17-secretors. Thus, although Th1-biased pro-inflammatory environments have been shown to induce alternative cytokine expression from Th17 cells in vivo, our data suggest that during allergic inflammatory disease, Th17 cells are remarkably stable, and retain the potential to produce IL-17.
Specialized microenvironment within the lymph node controls antigen-presentation properties of dendritic cells and Th2 differentiation. (P1119)
Authors: León Ruiz Beatriz , Ballesteros Tato André , Randall Troy , Lund Frances
Published: January 2023

T cells need to encounter antigen-bearing dendritic cells (DCs) to undergo clonal expansion and effector development. These interactions, within lymphoid tissues, are orchestrated by the expression of chemokines and chemokine receptors. Indeed, CCR7 expression by both T cells and DCs is thought to facilitate DC/T cell encounters within the T cell area of the lymph node. However, it is unknown whether co-localization of the DCs and T cells within the T cell area is obligate for effector generation. Here, we show that, following infection with an intestinal nematode, antigen-bearing DCs and the responding CD4+ T cells upregulate expression of CXCR5 and migrate in a CXCR5/CXCL-13 dependent fashion to the B cell area of the mesenteric lymph node. We further demonstrate that CXCR5-dependent localization of DCs and T cells near the CXCL13-expressing B cell follicles are required for optimal Th2 responses. Furthermore, we found that disrupting DCs from accumulating in the perifollicular region prevents the DCs from initiating Th2 differentiation. Finally, we demonstrated that DCs receive conditioning signals within the perifollicular microenvironment that change Class II Transactivator (CIITA) expression, maturation and antigen-presenting properties of DCs that allow the DCs to initiate Th2 priming. These results show that specialized microenvironments within the lymph node directly influence dendritic cell antigen presentation and function for effective priming of Th2 responses.
SIRT1 enhances Th17 cell generation by deacetylating RORγt (P1163)
Authors: Lim Hyung , Kang Seung Goo , Ryu Jae Kyu , Fei Mingjian , Lee Intelly , Kehasse Amanuel , Kasler Herbert , Kwon Hye-Sook , Shirakawa Kotaro , Schnölzer Martina , Gibson Bradford , Ott Melanie , Akassoglou Katrina , Xiao Changchun , Littman Dan , Verdin Eric
Published: January 2023

Although Sirtuin 1 (SIRT1), an NAD-dependent protein deacetylase, has emerged as a critical regulator of the cell cycle, replicative senescence, inflammation, and metabolism, its contribution to immune function remains to be fully investigated. Here we report that SIRT1 regulates the function of IL-17 producing CD4 T helper cells (Th17 cells) by modulating the activity of the Th17 master transcription factor RORγt. We found that Sirt1 interacts with RORγt and increases its transcriptional activity by deacetylating lysine residues K16 and K81. In mixed hematopoietic chimeras, cells from donors in which Sirt1 is deleted in ROR γ+ cells (SIRT1-cKO) exhibited compromised Th17 differentiation. Moreover, SIRT1-cKO mice were resistant to developing experimental autoimmune encephalomyelitis (EAE) upon MOG peptide immunization. Importantly, the SIRT1 specific inhibitor Ex-527 substantially inhibited trans-differentiation to Th17 cells in vitro, and injection of Ex-527 into mice strongly blocked EAE disease progression in vivo. These findings reveal a previously unappreciated proinflammatory role of SIRT1 in CD4 T cells, and provide significant evidence that pharmacological SIRT1 inhibitors might constitute a novel approach in the treatment of Th17-mediated autoimmune diseases such as multiple sclerosis.
Th2 differentiation during atopic asthma (P1167)
Authors: Pepper Marion , Hondowicz Brian , Garza Esteban , Moon James
Published: January 2023

Asthma is an inflammatory disease of the airways that affects more than 300 million people worldwide. Clinically, asthma is characterized by acute, intermittent and recurrent episodes of inflammation that can be induced by a specific allergen. CD4+ T cells contribute to this process by producing the Type 2 cytokines IL-4, IL-5, and IL-13 and inducing B cell production of IgE in response to T cell receptor (TCR) recognition of allergen peptides bound to MHCII molecules on host cells. In both murine models of disease and asthmatic patients, quiescent allergen peptide:MHCII (pMHCII)-specific CD4+ memory T cells can persist in lungs and lymphoid organs long after resolution of inflammation. Upon subsequent exposure to allergen, CD4+ memory T cells rapidly drive asthma-induced immunopathology making these cells attractive targets for allergen-specific immune modulation. Little is known, however, about how these cells orchestrate the asthmatic response due to the challenge of tracking small populations of CD4+ T cells that express allergen pMHCII-specific TCRs. To address this lack of knowledge, we have utilized a MHCII tetramer containing a peptide from the Der p1 protein of the house dust mite (HDM), Dermatophagoides pteronyssinus, the most common cause of atopic asthma. Using this tetramer and a novel magnetic bead-based cell enrichment method, we can assess the differentiation and function of Der p1 pMHCII-specific Th2 effector and memory cells in mice responding to HDM.
Serine antiprotease trappin-2 induces IL-9 in human peripheral blood mononuclear cells (P1171)
Authors: Alija Ana , Ambrosi Nella , Hernández Del Pino Rodrigo , Guerrieri Diego , Alvarez Belén , Tateosian Nancy , Garcia Verónica , Chuluyan Eduardo
Published: January 2023

Trappin-2 is a 9.9 kDa serine protease inhibitor produced by epithelial cells and immune cells such as macrophages and γδT cells. It has anti-inflammatory and microbicide activities. The aim of the present work was to evaluate the effect of Trappin-2 on peripheral blood mononuclear cells (PBMC) by analyzing proliferation response, apoptosis, cytokine production and transcription factors expression. After 5 days of culture, Trappin-2 decreased lymphocyte proliferation induced by IL-2 and diminished the early apoptosis of cells. Moreover, culture supernatants derived from PBMCs treated with Trappin-2 (48 h) showed a slight increase in IL-4 production, what was confirmed by intracellular staining of IL-4 on CD3 T cells. Besides, Trappin-2 significantly increased GATA-3 expression on PBMCs as determined by Western Blot. However, the most significant result was a clear increase in IL-9 expression on CD4 T cells, after 48 h of culture of cells with Trappin-2. Moreover, the expression of PU.1 was also increased on Trappin-treated cells. Finally, TGF-β was measured in the culture supernatants of PBMC treated with Trappin-2, given that naive CD4+ T cells differentiate in vitro into IL-9-secreting cells by effect of IL-4 and TGF-β. The results obtained showed that Trappin-2 in fact increased TGF-β at 48 h of culture. Overall, our findings suggest that Trappin-2 induces a cytokine microenvironment compatible with a potential Th9 pattern.
The role of TOX in peripheral T cell subsets (P1121)
Authors: Aliahmad Parinaz , Kadavallore Asha , Seehus Corey , de la Torre Brian , Kaye Jonathan
Published: January 2023

TOX (Thymocyte-selection associated HMG-box) is a DNA-binding protein identified in our laboratory that is required for development of the CD4 T cell lineage, including canonical CD4 T cells, NKT cells and T regulatory (Treg) cells. In addition, NK cell and lymphoid tissue inducer cell development requires TOX, the latter resulting in failure of lymph node development in TOX-deficient mice. Here we turn our attention to investigation of TOX in mature CD4 T cell subsets, facilitated by a knock-in Tox reporter (tdTomato) strain of mouse. The reporter faithfully mirrors TOX expression in the thymus and NK cells. We find that TOX (Tomato) is also upregulated in natural Treg and at highest levels in T follicular helper (Tfh) cells. Interestingly, both T cell subsets show heterogeneity in reporter expression, with cell populations of high, intermediate and low expression evident. In Treg, varied expression of TOX is independent of FOXP3 expression, as demonstrated by analysis of FOXP3 and TOX dual reporter mice. Among Tfh cells (CXCR5hiPD1hi), Toxhi cells are Bcl6hiCCR7-CD62Llo and likely represent mature follicular effector cells. To study the functional role of TOX, we expressed Cre in CD4 T cells from OTII Tox conditional knockout mice, and analyzed the development of phenotypically-defined Tfh in response to antigen upon adoptive transfer. Loss of TOX prevented Tfh cell development in this system, indicating a critical role for TOX in this peripheral T cell subset.
Foxp3+ regulatory T cells consist of rapidly dividing and quiescent subpopulations (P1179)
Authors: Punkosdy George , Shevach Ethan
Published: January 2023

While Treg exhibit an anergic phenotype in vitro, Treg proliferate in vivo at a rate similar to memory phenotype (MP; defined as CD44hi, CD62Llo) CD4+ T cells. In short-term BrdU labeling experiments, both populations uptake the label at a rate of 6% to 10% of cells in a 24-hour period. However, MP T cells are made up of multiple cell populations that proliferate at different rates and short-term labeling experiments only label the rapidly dividing population. To address whether Treg may also consist of multiple populations of cells dividing at different rates, we conducted long-term labeling experiments by providing BrdU in the drinking water. Adult thymectomized mice were used to control for new T cell development. In mice labeled for 20 days, only ~60% of the Treg were BrdU positive suggesting that a significant population of Treg is non-proliferative. BrdU incorporation was positively correlated with the level of CD44 expression on Treg and nondividing Treg uniquely expressed the cell surface marker Ly-6C. Ly-6C positivity represented a stable phenotype of Treg, as sorted Ly-6C negative and positive Treg retained their respective Ly-6C expression following adoptive transfer into a naïve host. These findings suggest that Treg are made up of rapidly dividing and quiescent subpopulations. The functional significance of these distinct populations is being investigated.
Mapping CD4+ T cell differentiation decision space: Flexibility and heterogeneity under mixed input conditions (P1181)
Authors: Eizenberg Inbal , Rimer Jacob , Friedman Nir
Published: January 2023

T helper cell differentiation is influenced by the spectrum of cytokines in the cells' milieu. In-vivo, cells are expected to sense a complex cytokine environment. Therefore, we aimed to study differentiation under mixed input conditions. By exposing cells to varied combinations of cytokines, we mapped the "decision space" of the differentiation process. Naïve CD4+ T cells were exposed to all 64 binary combinations of 6 cytokines, which induce the differentiation of the main T helper subsets: Th1, Th2, Th17 and iTreg. We examined the differentiation outcome in terms of cytokine-production pattern and expression of lineage-specifying transcription factors, by multi-color flow cytometry. Our data indicate that mixed conditions result in mixed phenotypes with cells expressing markers of opposing subsets simultaneously. Clustering and principal component analysis revealed up to eight different Th subsets, some of which are in accordance with known states. We also identified a hierarchical organization of input signals in their influence on driving differentiation into those subsets. Our results demonstrate flexibility and heterogeneity in Th cell differentiation. Under uncertain conditions, rather than following a hard, binary decision, cells reflect uncertainty through a biased stochastic decision process. This strategy allows flexibility and plasticity of the response at the single-cell level, while maintaining an average, possibly optimized response at the population level.
Autoimmune Regulator (Aire) is critical for the thymic development of naturally occurring Treg specificities (P1185)
Authors: Leventhal Daniel , Malchow Sven , Nishi Saki , Fischer Benjamin , Paner Gladell , Savage Peter
Published: January 2023

While the transcription factor Autoimmune Regulator (AIRE) has been shown to play a critical role in establishing immune tolerance through the negative selection of T cells reactive to peripheral antigens, the role of AIRE in the thymic development of Foxp3+ regulatory T cells (Tregs) remains controversial. Previously in our lab, the naturally occurring Treg T-cell receptor (TCR) “MJ23”, isolated from the prostates of tumor-bearing TRAMP mice, was used to create a monospecific TCR transgenic (tg) mouse model. By generating MJ23 tg low frequency bone marrow chimeras (BMCs), we determined that the MJ23 TCR facilitates thymic Treg development in both male and female hosts, even though MJ23 T cells are reactive to a prostate-specific antigen. By generating MJ23 BMCs into Aire+/+ and Aire-/- hosts, we determined that the thymic development of MJ23 Tregs was Aire-dependent, regardless of host gender. These results were also observed for a second naturally occurring Treg specificity. In BMCs, we found no evidence of Aire-dependent MJ23 negative selection, suggesting that for this specificity, Aire plays an exclusive role in the thymic selection of Tregs. Finally, while autoimmune prostatitis was observed in male Aire-/- mice, we found no indications of Foxp3neg MJ23 T cell expansion in Aire-/- BMC hosts. In sum, our data provide mechanistic evidence that a subset of peripheral organ-specific Tregs are dependent on Aire for their selection into the Foxp3+ Treg lineage in the thymus.
Detection of AHR in in vitro-generated Th17 cells using novel monoclonal antibodies (P1191)
Authors: Funatake Castle , Araji Soha , Alvarez Miguel , Atteberry Brandi , Li Shourong , Huang Qiulong , Schrantz Nicolas , Kerkvliet Nancy , Just Peggy
Published: January 2023

Several studies have shown an increase in the mRNA levels of AHR in both mouse and human Th17 cells, however, protein levels in Th17 cells have been difficult to assess due to lack of reagents for flow cytometry. We developed novel monoclonal antibodies to enable detection of AHR by flow cytometry. The antibodies were characterized by western blot, flow cytometry, and immunocytochemistry. By western blot, both antibodies produced bands of approximately 95-100 kDa in lysates prepared from transiently transfected HEK-293T cells and HepG2 or Hepa1c1c7 cells (for human and mouse AHR, respectively). The anti-mouse AHR antibody also recognized a band of 95 kDa in liver lysates prepared from C57Bl/6 mice but not from AHR knockout mice. High levels of AHR protein were detected in cells polarized under Th17 conditions for 3 days, and these levels decreased on days 6 and 10, while AHR was not detected if Th17 were polarized from splenocytes from AHR knockout mice. Analysis of AHR expression by immunocytochemistry showed that these antibodies recognized AHR in the cytoplasm of untreated cells. Upon treatment of cells with FICZ, a high affinity ligand for AHR, AHR translocated to the nucleus. These antibodies will enable the examination of expression levels of AHR by flow cytometry and immunocytochemistry to provide insight into the role of AHR in many biological systems.
Epigenetic licensing of Th17 and Treg cell differentiation (P1197)
Authors: Antignano Frann , Burrows Kyle , Hughes Michael , Oudhoff Menno , Han Jonathan , Gold Matthew , McNagny Kelly , Levings Megan , Zaph Colby
Published: January 2023

Naïve CD4+ T helper (Th) cells acquire a range of fates depending on the cytokine milieu and anatomical location. For example, IL-17 producing Th17 and Foxp3+ regulatory (Treg) cells are dependent upon TGFβ1 and are critical for intestinal homeostasis. G9a is a histone lysine methyltransferase with di-methyl activity towards histone H3 lysine 9. H3K9me2 is a highly conserved histone modification and is commonly linked to transcriptional repression. In this study we examine the role of G9a in the control of Th17 and Treg cell differentiation. Using chromatin immunoprecipitation (ChIP) we found that there is a concomitant loss H3K9me2 and increase in H3K9Ac (an activating histone modification) at lineage specific genes of WT Th17 and Treg cells compared to naive. Meanwhile, naïve G9a-/- T cells have low levels H3K9me2 that results in enhanced sensitivity to TGFβ1 and an increase in the differentiation of both Th17 and Treg cells in vitro. Using the T cell transfer colitis model, we found that transfer of G9a-deficient T cells fails to cause the weight loss and colonic inflammation typical of the model. Furthermore, T effector-cell production of IFNγ was significantly reduced and IL-17 was enhanced when G9a-/- T cells were transferred. In addition, compared to WT controls, a higher proportion of G9a-deficient T cells express Foxp3. We conclude that G9a-dependent H3K9me2 is a homeostatic epigenetic checkpoint that controls the magnitude of Th17 and Treg cell responses.
MiR-17~92 family microRNAs are critical regulators of T follicular helper cell differentiation (P1133)
Authors: Kang Seung Goo , Liu Wen-Hsien , Teijaro John , Lim Hyung Wook , Shepherd Jovan , Verdin Eric , Qi Hai , Oldstone Michael , Xiao Changchun
Published: January 2023

Germinal center (GC) reaction is a key event in B cell-mediated immune responses and CD4+ T cell help plays an essential role in these process. A distinct CD4+ effector T cell subset, T follicular helper cells (TFH), provides this help to B cells. However, molecular mechanisms underlying TFH differentiation are still largely unknown. A recent study revealed that Bcl-6 is a signature transcription factor regulating TFH differentiation and it represses the expression of miR-17~92 in CD4+ T cells. MiR-17~92 in turn suppresses the expression of CXCR5, a critical chemokine receptor controlling CD4+ T cell migration into B cell follicles. The authors therefore concluded that miR-17~92 functions as a negative regulator of TFH differentiation. Here we report the surprising finding that mice with miR-17~92 family microRNAs (miRNAs) deleted specifically in T cells exhibited severely compromised TFH differentiation, germinal center formation, and reduced antibody responses upon protein antigen immunization. Those mutant mice were also defective in TFH differentiation and germinal center B (GCB) cell formation during lymphocytic choriomeningitis virus (LCMV) clone-13 infection and failed to control the virus. Conversely, T cell-specific miR-17~92 transgenic mice spontaneously accumulated TFH cells. Novel cellular and molecular mechanisms underlying miR-17~92 family miRNA-mediated regulation of TFH differentiation will be presented at this meeting.
Modulation of autoimmunity associated with blocked nuclear export of histone Deacetylase 7 in thymocytes by CCR9 and the NOD genetic background. (P4088)
Authors: Kasler Herbert , Lim Hyung , Lee Intelly , Verdin Eric
Published: January 2023

Histone Deacetylase 7 (HDAC7) is a chromatin-modifier that, like other Class IIa HDACs, regulates cell differentiation through extracellular signal-dependent nucleo-cytoplasmic shuttling. We recently showed that nuclear export of HDAC7 in response to TCR signaling in thymocytes is required for negative thymic selection, and that thymic expression of a constitutively nuclear mutant of HDAC7 (HDAC7-ΔP) consequently causes lethal autoimmunity. Although the block in negative selection appears broad, in C57bl/6 mice autoimmunity is largely restricted to the abdominal viscera, with lethality due to exocrine pancreatitis. Investigating the cause of this restricted autoimmunity, we noted abnormally high expression of CCR9, a receptor associated with abdominal homing, in the CD8 T cells of HDAC7-ΔP mice. While this suggested that the type of disease caused by HDAC7-ΔP was due to its effects on both negative selection and chemokine receptor expression, we found that expression of HDAC7-ΔP in a CCR9-deficient background lengthened survival but did not alter the tissue distribution of autoimmune attack. In contrast to this result, we found that expression of HDAC7-ΔP in the NOD genetic background extensively altered the tissue distribution of autoimmunity and also greatly reduced mean survival time. These results shed significant new light on how a specific molecular lesion affecting thymic selection can interact with different genetic backgrounds to produce distinct disease outcomes.
CASPASE-12 genotype influences the expression of systemic lupus erythematosus in African-Americans (P4090)
Authors: Hermel Evan , Fuchs Trista , Kelly Jennifer , Moser Sivils Kathy , Velasco H. Eduardo
Published: January 2023

CASPASE-12 (CASP12) modulates inflammation by down-regulating inflammatory cytokines. CASP12 is a risk factor for sepsis in persons of African ancestry, as the functional CASP12 allele is present in about 20% of persons of African descent. Nearly all other human populations lack a functional CASP12 gene. Systemic lupus erythematosus (SLE) is a serious and complex autoimmune disease affecting multiple organ systems and is most common in African-American (AA) women. To determine if CASP12 may be protective against SLE, 596 AA SLE patients and 296 healthy controls from the Lupus Family Registry and Repository were genotyped for CASP12. Serum from 30 patients and controls was assayed by antibody arrays for 23 inflammatory cytokines and chemokines. There was a significant association between CASP12 and protection against serositis. CASP12 homozygosity was seen only in controls, and CASP12 was only in female, but not male patients without serositis compared to those with serositis. In male patients, CASP12 was protective against the development of arthritis. Expression of the Mig chemokine was statistically different between patients and controls, independent of genotype. No influence of CASP12 genotype was seen upon serum cytokine levels in patients vs controls. The results indicate that CASP12 is protective against lupus serositis and arthritis in AA patients, with possible gender influences for both. In addition, chemokines may contribute to the pathogenesis of SLE.
A short murine lupus-susceptibility Sle2c1rec1sublocus functions as both contributor and modifier of autoimmune disease in CD95 deficiency (P4098)
Authors: Xu Zhiwei , Morel Laurence
Published: January 2023

It is widely recognized that genetic susceptibility plays a significant role in lupus-like autoimmune diseases. The Sle2c1 sublocus belongs to the lupus susceptibility Sle2 locus identified on the chromosome 4 of lupus-prone NZM2410 mouse, and resulted in T cell-mediated autoimmune pathology in the bicongenic Sle2c1.lpr strain. Subsequently, a new recombinant congenic Sle2c1rec1.lpr strain carrying an about 10 Mbp-long Sle2c1rec1 sublocus produced from Sle2c1.lpr mouse was validated with the same autoimmune phenotypes as does Sle2c1.lpr mouse. In current study, we generated four new congenic recombinant stains, Sle2c1rec1a.lpr, Sle2c1rec1b.lpr, Sle2c1rec1c.lpr and Sle2c1rec1d.lpr, and performed a fine mapping of their recombinant intervals and a detection of their phenotypic alterations. The overlapping region between the Sle2c1rec1a and Sle2c1rec1d intervals mapped to the middle part of Sle2c1rec1 strongly promoted T cell expansion, incidence increase in inflammatory dermatitis and production of IgG autoantibodies. In contrast, Sle2c1rec1b in the proximal part of Sle2c1rec1 led to significant decrease in incidence of inflammatory dermatitis. Besides, Sle2c1rec1c in the distal end of Sle2c1rec1 obviously interacted with the mid part of Sle2c1rec1 sublocus to enhance T cell expansion. Overall, our results showed that Sle2c1rec1 harbors at least three functional domains, which are involved in regulating T cell mediate autoimmune pathology in epistasis with Fas deficiency.
Expression of Ikaros family member molecules in lupus-prone mice: a preliminary analysis (P4102)
Authors: Divekar Anagha , Lee Michael , Singh Ram , Halder Ramesh , Guiterrez Miguel-Angel , Yang Xifeng , Ransom John
Published: January 2023

Ikaros family of zinc-finger proteins, namely Aiolos, Helios and Ikaros, are hematopoietic-specific transcription factors involved in lymphocyte development. Aiolos-deficient mice develop SLE-like disease, and Ikaros family members have been linked to the balance between T cell activation and tolerance. To begin to investigate the role of Ikaros proteins in SLE, we first determined the expression of these proteins in spleen cells of NZM.2328 mice that develop disease that mimics human SLE in many ways, including a profound female bias. Results show that Aiolos expression in T cells is significantly lower than that in B cells from NZM.2328 mice. Similar reduction in Aiolos expressing T cells was observed in old lupus-prone MRL-MpJ-Fas+/+ compared to age-matched control B6 mice. Almost all B cells expressed Aiolos in all mice tested, but the expression level was much lower in MRL-MpJ+/+ B cells than in control B6 B cells. In contrast to Aiolos expression, T cells expressing Helios were 3-4-fold higher in old NZM.2328 females than in age-matched male and younger female mice. Ikaros expression was also higher on both T and B cells in older NZM.2328 females than in age-matched male and younger female mice. Ongoing studies will investigate the relationship of dysregulated Ikaros family molecules with lupus disease and examine the role of this dysregulation in female bias and lupus pathogenesis.
Defining epigenome responses in T-cell arthritogenicity: in search for effectors of autoimmunity. (P4076)
Authors: Backdahl Liselotte , Holmdahl Rikard
Published: January 2023

Recent successes of therapeutic intervention of chronic inflammatory diseases using epigenetic modifiers such as histone deacetylase inhibitors and inhibitors of DNA methylation suggest that epigenetic reprogramming plays a role in the aetiology of these diseases. However, the disease mechanisms driving epigenetic modulation in chronic inflammation are poorly understood. One of the major problems with assessing epigenetic regulation is that it is cell specific. DNA methylation is here studied in a population of arthritogenic CD4 T cells that can transmit pristane-oil induced arthritis, a model for Rheumatoid arthritis, after injection into healthy rats. Pristane primed spleenocyte derived T cells subjected to 24 hrs treatment with 5-AZA Cytidine completely lose the ability to transfer arthritis. Demonstrating that arthritogenic T cells have an altered DNA methylation profile that can be reversed. On the transcription level, treated CD4 T cells showed significant increase in IL-10, TGFb and Foxp3 indicating that regulatory T cells to be important for the protective effect. Assessing cellular phenotypes, MHCII is decreased and CD8+ is increased, indicating that activated CD4 cells but not naïve, are being killed by the regulatory T cells. Furthermore 3 CpG sites in the IL10 promoter were differentially methylated after 5-AZA. Identifying specific epigenetic marks in the genome would provide excellent clues to disease pathways as well as serve as potential therapeutic targets.
Tregs from T1D subjects with a susceptible IL-2R gene SNP, respond aberrantly to IL-2 stimulation. (P4080)
Authors: Marwaha Ashish , Staiger Spencer , Hirschfield Aaron , Panagiotopoulos Constadina , Turvey Stuart , Buckner Jane , Tan Rusung
Published: January 2023

Type 1 diabetes (T1D) results from the destruction of beta cells by autoreactive lymphocytes. Regulatory T cells (Tregs) maintain self-tolerance in part by suppressing the effector functions of self-reactive lymphocytes. Tregs require the cytokine interleukin-2 (IL-2) for survival and maintenance of their suppressive function. IL-2 binds to the high-affinity IL-2 receptor (IL-2R), leading to phosphorylation of signal transducer and activator of transcription 5 (STAT5) and downstream transcription of effector proteins.The IL-2 receptor gene has single nucleotide polymorphisms (SNPs), which are associated with T1D incidence. We hypothesized that T1D subjects with SNPs in the IL-2R gene possess Tregs that respond aberrantly to IL-2 and do not optimally induce signaling in the STAT5 pathway. PBMC from T1D subjects with a homozygous susceptible (GG), heterozygous (AG) and homozygous wild-type (AA) genotype for a SNP located in the rs3118470 locus of the IL-2R gene were stimulated with IL-2. Our results indicate that Tregs from individuals with the homozygous susceptible GG genotype have reduced levels of pSTAT5 compared to those with the AA genotype (p=0.0133). This provides evidence that Tregs from T1D subjects with SNPs in the rs3118470 locus of the IL-2R gene have diminished STAT5 phosphorylation in response to IL-2. This is a potential mechanism of defective Treg function in a genetically identifiable subset of children with T1D.