bluuebunny/crossref_metadata_2025 huggingface dataset:
Genome wide mapping of epigenetic signatures identify novel enhancer elements that underpin virus-specific CD8+ T cell differentiation (IRM14P.445)
Authors: Turner Stephen , Olshansky Moshe , Li Jasmine , Nguyen Michelle , Chen Eddie , Nguyen T.H. , Rao Sudha , Russ Brendan
Published: January 2023

The molecular mechanisms that underpin acquisition and maintenance of lineage specific gene expression by virus-specific CD8+ T cells are not fully delineated. Post-translational modifications (PTMs) of genome associated histone proteins are a key mechanism for regulating gene expression. We mapped genome wide changes in histone PTM deposition during virus-specific CD8+ T cell differentiation. By comparing the location and level of deposition of a combination of H3K4me1, H3K4me2, H3K4me3, H3K27me3, H3K27Ac, chromatin accessibility and binding of the histone acetyltransferase, p300, we could map both known and putative enhancer elements that are differentially regulated between naive, effector and memory virus-specific CD8+ T cells. Using this epigenetic blueprint, we identified putative transcriptional enhancers located upstream of the Ccl5 gene, a key CD8+ T cell effector gene. We demonstrate that acquisition of Ccl5 transcription upon T cell activation is regulated via temporal and transcription factor (TF) dependent chromatin remodelling. Using chromatin conformation capture (3C) to measure enhancer-promoter interactions, we show that that TF dependent looping of these enhancer elements onto the Ccl5 promoter correlated with transcriptional activation and potential. These studies form a platform for more in depth analysis of key non-coding regulatory elements that regulate acquisition of virus-specific T cell gene expression and maintenance into memory.
The immunological clock: the role of cryptochrome in regulatory T cell function (IRM15P.602)
Authors: Mack Lauren , LeBlanc Mathias , Zheng Ye
Published: January 2023

Circadian rhythms are defined as 24 hour oscillations in physiological and behavioral processes. Clinical data suggests circadian involvement in the predisposition and progression of immune-related morbidities such as cancer and autoimmune disease. Recent studies have demonstrated the direct role of core molecular clock components in maintaining the immune system. A subset of CD4+ T cells known as regulatory T cells (Treg) are critical for preserving balance in the immune system and have been shown to be able to prevent a broad range of autoimmune disease. By studying the role of core circadian genes in Treg cells the connection between the circadian clock and autoimmune disease may be further elucidated. We have identified a core circadian clock gene, cryptochrome (Cry), to have a role in Treg cell function both in vitro and in vivo. The deficiency of Cry in Treg cells results in a breakdown in immune tolerance, which results in severe pathological autoimmunity in mice. This is characterized by immune cell infiltration into peripheral tissues as well as increased numbers of cytokine producing CD4 and CD8 T cells. It was also seen that ectopic expression of Cry in Treg cells results in increased suppression of T cell proliferation in vitro. Thus, we have demonstrated a direct role of Cry expression in the function of Treg cells.
Role of miR-27 in controlling T cell immunity (IRM15P.605)
Authors: Cruz Leilani , Cho Sunglim , Wu Chen-Jang , Yasuda Tomoharu , Lin Ling-Li , Rajewsky Klaus , Lu Li-Fan
Published: January 2023

miR-23~27~24 clusters are highly enriched in regulatory cells while expressed at low levels in conventional T cells. However, despite a selective expression pattern of miR-23~27~24 clusters in T cells, to date, studies of this miRNA family have primarily focused on their role in tumorigenesis. Here, we show miR-23~27~24 clusters play a pivotal role in regulating T cell immunity. Particularly, our results have demonstrated that the majority of miR-23~27~24 cluster-dependent phenotypes could be attributed to a single member of this miRNA family, miR-27. Mice with T cell-specific overexpression of miR-27 spontaneously developed autoimmune phenotypes. On the other hand, in vitro polarization studies revealed that T cells with miR-27 overexpression exhibited impaired differentiation and effector function of multiple T helper cell lineages. Finally, our mixed bone marrow chimeras studies further demonstrated that miR-27 could control different T cell responses through either a T cell-intrinsic or -extrinsic manner. Collectively, Our results identify a new "immune regulatory" miRNA that can play multi-faceted roles in regulating T cell immunity.
FOXP3 regulates RNA alternative splicing in regulatory T cells (IRM15P.461)
Authors: Zhou Baohua , Du Jianguang , Ziegler Steven
Published: January 2023

Alternative pre-mRNA splicing is an important mechanism for protein regulation and cellular function, affecting more than 95% of multi-exon genes in humans and contributing to a wide range of physiological and pathophysiological processes. It is well known that FOXP3, the master transcription factor for Treg differentiation and function, directly binds to its target genes to regulate their transcription. Here we report an interaction between FOXP3 and heterogeneous nuclear ribonucleoprotein F (hnRNPF), a protein that regulates pre-mRNA alternative splicing and/or 3’ polyadenylation. FOXP3 through its exon 2 region interacts with hnRNPF to modulate the alternative splicing pattern of hnRNPF targets like BCL-X. Interestingly, the alternatively spliced exon 2 of human FOXP3 contains hnRNPF putative binding sites and point mutations of these sites eliminate the alternative splicing of human FOXP3. These findings suggest human FOXP3 might autoregulate the alternative splicing of FOXP3 transcripts through its interaction with hnRNPF. Finally, overexpression of hnRNPF in mouse iTregs can reduce their ability to suppress CD4+ T cell proliferation. Taken together, our results reveal a novel molecular mechanism through which FOXP3 modulates pre-mRNA alternative splicing to regulate the differentiation and function of Tregs.
B cell differentiation governed by distinct microenvironments (LYM7P.622)
Authors: Cen Selena , Simard Nathalie , Paige Christopher
Published: January 2023

B cell differentiation is regulated by the surrounding microenvironment, which produces cytokines, chemokines, and adhesion molecules that guide B cell commitment and differentiation. The gastrointestinal tract, in constant interaction with microbiota, constitutes a distinctive microenvironment for B cell differentiation. While the bone marrow and spleen contain few IgA+ cells, the gut lamina propria is largely colonized by IgA+ cells, which contain a subset of myeloid-like plasma cells expressing IgA, TNFα, iNOS, CD11c, Ly6C and Ly6G. Whether the bone marrow stroma produces inhibitory factors for IgA+ cell generation or the gut lamina propria stroma produces inducing factors needs further investigation. We developed an in vitro system to co-culture stromal cells with B cells to characterize the signals necessary to generate IgA+ cells. It was found that intestinal microbial components along with the intestinal lamina propria stromal cells played an important role in the emergence of both IgA+ and myeloid-like plasma cells. Interestingly, bone marrow and spleen-derived stroma showed an inhibitory effect on the generation of IgA+ cells. Stromal soluble factors (e.g. IL-1, IL-6, and MCP-1) might play a role in the guidance of B cell IgA switching, proliferation, survival, and differentiation. These experiments will provide a better understanding of the impact of microenvironments in shaping B cell development, and we will identify the molecular mechanisms they use.
Gut microbial dysbiosis due to Helicobacter drives marginal zone B cell development in the absence of IL-10 signaling in macrophages (LYM7P.623)
Authors: Dittel Bonnie , Basu Sreemanti , Gharaibeh Raad , Fordor Anthony , Ray Avijit
Published: January 2023

IL-10 plays an essential role in maintaining homeostasis in the gut in response to the microbiome. However, it is unknown whether IL-10 also facilitates immune homeostasis at distal sites. To address this, we asked whether splenic immune populations were altered in IL-10-deficient mice with differential susceptibility to microbiome-dependent spontaneous enterocolitis. Susceptible mice exhibited a significant increase in splenic macrophages, neutrophils and marginal zone (MZ) B cells that was inhibited by IL-10 signaling in macrophages, but not B cells. The increase in myeloid cells was due to increased proliferation that correlated with increased MZ B cell differentiation. Cohousing and antibiotic treatment suggested that the alteration in splenic immune homeostasis was microbiome-dependent. 16S rRNA sequencing reveled that susceptible mice harbored a more diverse microbiome with a significant increase in the abundance of the bacterial genus Helicobacter. The introduction of H. hepaticus to the gut of nonsusceptible mice was sufficient to drive myeloid cell expansion and MZ B cell development. Given that MZ B cells are part of the first line of defense against blood-borne-pathogens, their increase following a breach in the gut epithelial barrier would be protective. Thus IL-10 is an essential gatekeeper that maintains immune homeostasis at distal sites that can become functionally imbalanced upon the introduction of specific bacteria to the intestinal track.
Tumor progression locus 2 inhibits regulatory T cell development and immunosuppressive functions (LYM8P.640)
Authors: Li Xin , Acuff Nicole , Peeks Angela , Kirkland Rebecca , Hardwick Kara , Nagy Tamas , Watford Wendy
Published: January 2023

Regulatory T cells (Tregs) are a specialized subset of immunosuppressive T cells that function to maintain peripheral tolerance. We previously demonstrated that the serine-threonine kinase, tumor progression locus 2 (Tpl2, also designated Cot/Map3k8), regulates TCR signaling and inflammatory cytokine secretion in CD4+ T cells. Herein, we demonstrate that Tpl2 is preferentially expressed by Tregs and regulates their development and functions. Tpl2-/- mice exhibited increased proportions of thymic natural Tregs (nTregs) as well as peripheral splenic Tregs, in vivo. Enhanced Treg development was due to a T cell autonomous defect, since Treg development from tpl2-/- naïve T cells cultured in vitro was similarly enhanced, and peripheral Treg proportions were also increased within the tpl2-/- donor compartment of mixed bone marrow chimeras. This defect depended upon TCR signal strength and was enhanced at low antigen concentrations. Importantly, tpl2-/- Tregs had increased expression of the immunosupressive cytokines, IL-10 and IL-35, and provided better protection than wild-type Tregs in vivo in a T cell transfer model of colitis. These results demonstrate that Tpl2 has an important physiological role in limiting Foxp3 expression, Treg development and functions. Therefore, Tpl2 inhibition could potentially be used to deviate pathological immune responses in a variety of autoimmune diseases towards a protective, tolerogenic response through preferential Treg induction.
Blood transcriptional signatures of early tuberculosis infection and disease in cynomolgus macaque model (MPF6P.658)
Authors: Gideon Hannah , Skinner Jason , Flynn JoAnne , Lin Philana
Published: January 2023

Mycobacterium tuberculosis (Mtb) remains one of the successful human bacterial pathogen, yet little is known about the factors that influence infection outcome. The Cynomolgus macaque model of tuberculosis (TB) recapitulates many of the manifestations of human TB and provides an excellent platform to study early disease progesssion, which is not tractable in the humans. Macaques (n=38) were infected with low dose of Mtb (Erdman strain, ~25CFU) and whole blood samples were obtained for transcriptional analysis at serial time-points (pre infection to 6 months post infection) when the animals were declared to have clinically active TB or latent infection. Linear Mixed Modeling Analysis (LMMA) revealed increased activity of transcript clusters associated with interferon response, inflammation, hematopoiesis, early immune regulation, innate, and acquired immune responses between 20-56 days post Mtb infection in all animals. Despite considerable overlap in the transcriptional signatures of animals that developed active TB (n=16) or remained with latent infection (n=22), cross correlation of transcript cluster activity and disease activity (scan score) revealed significant immunologically relevant associations especially with interferon and lymphocyte responses that differed in composition and timing between active and latent cohorts. In summary, this study elucidates novel associations of infection outcome and the timing and composition of transcriptional signatures in TB.
IL2RA variants that reduce IL-2 signaling through its receptor aggravate cutaneous leishmaniasis (MPF6P.700)
Authors: Dessein Alain , Oliveira Pablo , Dessein Helia , Romano Audrey , Cabantous Sandrine , pereira Valeria , de Brito Maria , Rodrigues Virmondes , Rafati Sima
Published: January 2023

Leishmania infection may be asymptomatic, cause mild or severe skin ulcers (cutaneous leishmaniasis, CL), limited or disseminated lesions, or lethal visceral disease. We previously reported an association between IL-2 receptor mutations and susceptibility to visceral leishmaniasis in children infected with Leishmania donovani. Here, we evaluated the possible role of IL-2 signaling in human CL. We first showed that transcript abundance was high for several genes of the IL-2 pathway in skin lesions caused by Leishmania braziliensis. We then carried out a genetic analysis, focusing on major genes of the IL-2 pathway. We used a family-based approach and found that polymorphisms of several genes appeared to be associated with CL in a Brazilian population. Moreover, two polymorphisms of the IL2RA gene were significantly and independently associated with CL. We confirmed this result in a second Brazilian sample and in Iranians infected with Leishmania tropica: IL2RA rs10905669 T (Pcombined = 6 x 10-7) and IL2RA rs706778 T (Pcombined = 2 x 10-9) were associated with greater susceptibility to lesion development. These alleles were also correlated with a poor IFN-g response and poor FoxP3+ Treg cell activation. Thus, IL-2 plays a crucial role in protection against the cutaneous ulcers caused by Leishmania, and the IL-2 pathway is a potential target for strategies aiming to control Leishmania-related diseases.
Type I IFN enhances Coxiella burnetii infection (MPF6P.646)
Authors: Hedges Jodi , Robison Amanda , Christensen Kelly , Kimmel Emily , Lucas Erin , Ramstead Andrew , Jutila Mark
Published: January 2023

Coxiella burnetii is an intracellular pathogen and the cause of Q fever. A role for type I IFN in C. burnetii pathogenesis has not been determined but this pathway supports host protection from a related pathogen, Legionella pneumophila. Spleen cells from type I IFN receptor (IFNAR)-deficient mice supported diminished replication of the avirulent Nine Mile phase II strain (NMII) in vitro compared to spleen cells from wild type mice, and addition or induction of type I IFN enhanced replication, suggesting that type I IFN promoted C. burnetii infection. The inverse relationship between type I IFN and inflammasome activation was explored as a potential mechanism of type I IFN-dependent enhanced C. burnetii infection. When human cells were infected with NMII in vitro inflammatory transcripts, such as IL-1β, were strongly induced. This transcript expression was reversed by exogenous induction of the type I IFN pathway. In vivo infection with the virulent phase I strain (NMI) of C. burnetii confirmed in vitro predictions; IFNAR-deficient mice were significantly protected from weight loss compared to wild-type mice, and treatment with rIFNa exacerbated disease. In contrast, IL-1β- and ASC-deficient mice had enhanced bacterial replication in the lungs. The data suggest that type I IFN signaling may dampen IL-1β expression, which is important for protection from C. burnetii.
Elucidating a role for eosinophils and discovering novel vaccine targets in Pneumocystis pneumonia using transcriptomics (MPF6P.651)
Authors: Eddens Taylor , Elsegeiny Waleed , Horne William , Zheng Mingquan
Published: January 2023

Pneumocystis (PC) pneumonia remains the most common serious opportunistic infection in patients with HIV/AIDS and is re-emerging in non-HIV infected immunosuppressed patients. Clearance of PC occurs through CD4+ T-cells with antibodies acting as one documented mechanism of clearance, although protective antibody epitopes have yet to be elucidated. To address the role of CD4+ T-cells, we used transcriptomics in mice over the course of PC infection and we observed a strong eosinophil signature in CD4-replete animals by RNA sequencing and by BAL analysis. Hypereosinophilic CD4-depleted mice receiving pIL5 via hydrodynamic injection had increased eosinophils in the lung and decreased PC burden. Finally, mice deficient in eosinophilopoiesis, had no change in burden with pIL5 treatment following CD4-depletion, further implicating eosinophils as novel mediators of immunity against PC. To identify protective antibody epitopes as potential vaccine candidates, we used a life-cycle based approach and sequenced RNA from the ascus and troph life forms of PC and found GSC-1 (ascus) and Meu10 (troph) were differentially regulated. Both GSC-1 and Meu10 generate strong humoral immune responses. Meu10 peptide vaccination decreases PC burden in CD4+ T-cell depleted animals, while immunization with recombinant GSC-1 failed to provide complete protection in a challenge model. Future studies will evaluate a multivalent life-cycle based vaccine in both a PC challenge model and a co-housing model.
Rescuing cells from apoptosis after induction in cells protected by Toxoplasma infection (MPF7P.720)
Authors: Crouse Elle , Nash Paul
Published: January 2023

CTLL-2, a cell line commonly used to quantify IL-2 levels, undergo apoptosis when IL-2 is not present. We previously demonstrated that infection with Toxoplasma gondii prevents this programmed cell death. We also demonstrated that Toxoplasma infection prevents apoptosis induced by beauvericin. In this study, we undertook to determine whether the protection from apoptosis provided by Toxoplasma infection is partial and only protects to the level of our assay determination, or whether it is complete and the cells could continue to grow once the apoptotic inducer is removed. Of course this is an artificial system since the parasite would normally quickly kill the cell. However, the results could be relevant when cytotoxic T lymphocytes are killing infected cells when at the same time Toxoplasma treatment drugs are employed. More importantly, the information gained from this study will help us better understand inhibition of apoptosis by Toxoplasma and of apoptotic mechanisms. Since apoptosis is involved in so many processes, understanding and potentially being able to modulate its occurrence could be valuable. Here we present the fate of cells that had apoptosis induced, were treated to prevent Toxoplasma from killing the cells, and then maintained under normal conditions.
Exacerbated endothelial activation and type 1 immune responses, but impaired type 2 responses, contribute to severe scrub typhus (MPF7P.706)
Authors: Soong Lynn , Wang Hui , Shelite Thomas R , Liang Yuejin , Saito Tais , Mendell Nicole L , Zhang Wenbo , Sun Jiaren , Gong Bin , Walker David
Published: January 2023

Dysregulated immune responses can contribute to pathogenesis of severe scrub typhus, a neglected tropical disease caused by Gram/LPS-negative Orientia tsutsugamushi bacteria. However, the molecular basis of immune alterations is poorly defined. We have developed a murine model of severe scrub typhus and examined innate regulators in O. tsutsugamushi-infected mice. We found a selective impairment in type 2 cytokines (IL-4, -7, -13), but a significant activation of IL-1β, IL-6, IL-10, TNF-α, IFN-γ, as well as CXCR3- and CXCR1-related chemokines at 2, 6, and 10 days in the lungs, liver and spleen. The elevated angiopoietin (Ang)-2 expression and Ang2/Ang1 ratios suggested the presence of excessive inflammation and the loss of endothelial integrity. These alterations, together with extensive recruitment of myeloperoxidase (MPO)-expressing neutrophils and an influx of T cells, contributed to pronounced apoptosis and acute tissue damage and death. However, targeted depletion of CXCR3 by itself was insufficient to offset progressive disease, although lung edema and vascular leakage were much attenuated in CXCR3-/- mice. We believe that this is the first report of endothelial dysregulation and selective alterations in a panel of immune regulators during early O. tsutsugamushi infection in intravenously inoculated C57BL/6 mice. Our findings shed new light on the pathogenic mechanisms associated with severe scrub typhus and suggest potential targets for therapeutic investigation.
Rac GTPases have opposite roles in T cells and dendritic cells in mucosal immune function (MUC8P.730)
Authors: Cruz Morales Elisa , Song Yan , Marin Romela , Khor Bernard , Nguyen Deanna
Published: January 2023

Variants in RAC1 and RAC2, which encode small Rho-family GTPases, are associated with risk for inflammatory bowel disease. We have shown reduced regulatory T-cell (Treg) proportions in Rac2 knockout (KO) mice with additional dendritic cells (DC)-specific deletion of Rac1 compared to WT. Since Rac is crucial to TCR signaling, we hypothesized that a T-cell specific deletion of Rac1 would also result in defects in Treg development. Treg proportions and susceptibility to dextran sulfate sodium(DSS)-induced intestinal inflammation were assessed in mice deficient in Rac2 in addition to either T cell- or DC-specific deletion of Rac1 (cDKO). Surprisingly, elevated Treg proportions were observed in T-cell specific cDKO mice compared to WT as well as higher Treg proportions within the cDKO compared to the WT compartment in mixed bone marrow chimera. Furthermore, cDKO naïve CD4+ T cells were able to differentiate into Tregs, unlike cDKO DCs, which led to defective Treg generation of WT naïve CD4+ T cells in in vitro Treg generation assays. In vivo Treg generation was reduced in DC-specific cDKO mice compared to WT controls with ovalbumin (OVA) feeding following transfer of CD4+ T cells transgenic for OVA. Lastly, exacerbated DSS colitis was observed in DC-specific, but not T-cell specific cDKO mice, compared to WT. In conclusion, deficiency of Rac proteins result in contrasting effects on mucosal immune function in DCs compared to T cells. The precise mechanism is under investigation.
An NK1.1-expressing regulatory leukocyte in gut-associated lymphoid tissue controls IgA production (MUC8P.733)
Authors: Karmele Erik , Waggoner Stephen
Published: January 2023

Immunoglobulin A (IgA) is the most abundant immunoglobulin in the body and is responsible for maintaining homeostasis at mucosal surfaces. Gut microbes elicit continuous germinal center (GC) reactions within gut associated lymphoid tissues (GALT), which in turn contribute to anti-microbial IgA production by promoting maturation and IgA class switching of B cells that populate the gut lamina propria. Dysregulation of IgA levels is a common disease of unknown etiology. Thus, a better understanding of the mechanisms regulating IgA is critical. We previously found that NK1.1+ cells suppress GC responses and generation of long-lived humoral immunity during systemic viral infection of mice. In the GALT, these NK1.1+ cells may be conventional natural killer cells, group 1 innate lymphoid cells, or natural killer T cells. Surprisingly, systemic depletion of NK1.1+ cells in uninfected specific pathogen free (SPF) mice resulted in an increase in the numbers of GC B cells and T follicular helper cells within GALT (e.g. mesenteric lymph nodes) but not peripheral lymph nodes. In addition, the concentration of IgA in fecal pellets was increased following depletion of NK1.1+ cells when compared to non-depleted control mice. Together our data suggests that an NK1.1-expressing leukocyte inhibits IgA production by impairing GALT GC responses against the gut microbiota. This novel immunoregulatory mechanism may control intestinal homeostasis and prevent development of IgA nephropathy.
An inhibitory receptor leukocyte mono-immunoglobulin-like receptor 3/CD300f deficiency aggravates DSS-induced colitis (MUC8P.737)
Authors: Matsukawa Toshihiro , Teshima Takanori , Okumura Ko , Kitaura Jiro
Published: January 2023

Leukocyte mono-immunoglobulin-like receptor 3 (LMIR3) (also called CD300f) is an inhibitory receptor. LMIR3 is mainly expressed in myeloid cells, including mast cells. The binding of LMIR3 to its ligand ceramide inhibits IgE- and mast cell-dependent allergic responses. It was recently reported that ATP-induced activation of mast cells via P2X7 purinoceptor contributes to the initiation and progression of intestinal inflammation. Here we show that LMIR3 is a negative regulator of dextran sodium sulfate (DSS)-induced colitis, a murine model of ulcerative colitis (UC). Notably, Lmir3-/- mice were highly susceptible to DSS-induced colitis. We found a prominent increase in inflammatory cells, including degranulated mast cells, in the colonic lamina propria of DSS-treated Lmir3-/- mice. Mast cell-deficient KitWsh/Wsh mice engrafted with LMIR3 deficient bone-marrow-derived mast cells (BMMCs) exhibited increased susceptibility to DSS-induced colitis as compared with those with wild-type (WT) BMMCs. Importantly, ceramide-LMIR3 binding suppressed activation of ATP-stimulated BMMCs. Consistently, treatment with ceramide liposomes improved DSS-induced colitis in WT mice, but not in Lmir3-/- mice. Taken together, these results indicated that LMIR3 negatively regulated DSS-induced colitis by inhibiting ATP-mediated mast cell activation through ceramide-LMIR3 binding. Accordingly, LMIR3-targeted ceramide liposomes would provide novel therapeutic strategies for UC.
CCR9 regulates colitis development by inhibiting regulatory T cell development (MUC8P.725)
Authors: Evans-Marin Heather , Cao Anthony , Yao Suxia , Chen Feidi , Gonzalez Maria , He Chong , Liu Han , Wu Wei , Dann Sara , Cong Yingzi
Published: January 2023

T cells reactive to microbiota regulate the pathogenesis of inflammatory bowel disease (IBD). As T cell trafficking to intestines is regulated through interactions between highly specific chemokine-chemokine receptors, great efforts have been made to develop intestinal specific immunosuppression based on blocking these key processes. CCR9, a gut-trophic chemokine receptor expressed by lymphocytes and dendritic cells, has been implicated in regulation of IBD through mediating recruitment of T cells to inflamed sites. However, the role of CCR9 in inducing and sustaining inflammation in the context of IBD is poorly understood. In the current study, we demonstrate that CCR9 inhibits Treg cell development, which contributes to its regulation of intestinal inflammation. While CCR9-/- mice are more resistant to disease compared to wild type (WT) mice upon DSS insults, CCR9 deficiency does not affect effector T cell induction of colitis in a microbiota antigen specific T cell-mediated model. Interestingly, CCR9-/- mice demonstrate a high level of Foxp3+ Tregs, and ligation of CCR9 by its ligand CCL25 inhibited Treg cell differentiation in vitro. Furthermore, partial depletion of Tregs in CCR9-/- mice increased susceptibility to DSS insults to a level similar to WT mice. Collectively, our data indicates that CCR9 signaling inhibits Treg cell development, which contributes to its regulation of colitis development, in addition to acting as a gut-homing molecule.
IL-10 engages macrophages to shift Th17 cytokine dependency and pathogenicity during T cell-mediated colitis (MUC8P.729)
Authors: Geiger Terrence , Li Bofeng , Gurung Prajwal , Malireddi RK , Vogel Peter , Kanneganti Thirumala-Devi
Published: January 2023

Polymorphisms attenuating IL-10 signaling confer genetic risk for inflammatory bowel disease. Yet how IL-10 prevents mucosal autoinflammation is incompletely understood. We demonstrate using lineage-specific deletions of IL-10Rα that IL-10 acts primarily through macrophages to limit colitis. A macrophage-selective deficiency of IL-10Rα increases the severity of colitis induced by T cell transfer into Rag1-/- mice to the same extent as germline deletion of the gene. Colitis mediated by T cell transfer is Th17 cell dependent regardless of the expression of IL-10Rα. Disease development requires IL-6 to support pathologic Th17 cell generation in wild type mice. However, specific ablation of macrophage IL-10Rα provokes excessive IL-1β production that overrides Th17 IL-6 dependence, amplifying the colonic Th17 response and disease severity. IL-10 not only inhibits pro-IL-1β production transcriptionally in macrophages, but suppresses caspase-1 activation and caspase-1 dependent maturation of pro-IL-1β to IL-1β. Therefore lineage-specific effects of IL-10 skew the cytokine dependency of Th17 development required for colitis pathogenesis. Coordinated interventions may be needed to fully suppress Th17-mediated immunopathology.
Dynamics of intestinal IgA expression after early-life antibiotic treatment. (MUC9P.738)
Authors: Ruiz Victoria , Teitler Isabel , Ou Amy , Weber Laura , Chess Edith , Battaglia Thomas , Cadwell Ken , Blaser Martin
Published: January 2023

Class IgA secretory antibodies (SigA) are an early line of antigen-specific immune defenses that are influenced by commensal microbial populations. Perinatal antibiotic exposures may affect the host microbiota and have long-term consequences on host immunity. We hypothesize that early-life pulsed antibiotic treatment (PAT) perturbs the intestinal microbiota leading to alterations in sIgA expression. To test this hypothesis, C57BL/6 mice were exposed to 5 therapeutic doses of the β-lactam, amoxicillin, or the macrolide, tylosin. Exposure to either the tylosin or amoxicillin delayed sIgA expression. sIgA expression recovered in β-lactam-exposed mice but was abrogated with macrolide exposure. Compared to age-matched controls, 1 or 3 tylosin pulses decreased sIgA levels by 40% and 75%, respectively. Alterations in sIgA production were associated with decreased bacterial richness (α-diversity), altered community structure (β-diversity), and changes in microbial compositional, including increased abundance of Enterobacteriaceae and Bacteroides uniformis. Whereas tylosin had multiple immunological effects on conventional mice, essentially none were detected in germ-free animals; indicating that an antibiotic-perturbed microbiota influences developing immunity. These results provide evidence that early-life alterations of the intestinal microbiota by PAT modulates sIgA production, phenotypes that may persist after microbial recovery, with long-term consequences to host immunity.
Comparison of flow cytometers using the side population technique to identify stem cells (TECH2P.900)
Authors: Guenther Garret , Telford William
Published: January 2023

Traditional methods using the side population analysis to identify stem cells involved the use of a flow cytometer with an ultra violet laser excited dye. This presents some challenges for researchers in relation to costs, and even though other dyes such as DyeCycle Violet (DCV) are now available that use the more economical violet laser, the cost of a flow cytometer to provide this type of analysis is still an obstacle. More recently, new benchtop flow cytometers have become available that can provide the quality of a high-end flow cytometer at a much more affordable cost. In this analysis, we sought out to identify stem cells and progenitors using the side population technique in murine bone marrow using the ACEA NovoCyte flow cytometer which maximizes optical design through its use of shared PMTs. Samples were stained and evaluated with (DCV), Lineage markers, Sca, and c-kit. To identify stem cells using the side population technique, we first gated on Lineage- cells and subsequently on Sca+, c-kit+. To confirm the correct identification of these cells, this population was back gated comparing DCV red fluorescence vs DCV blue fluorescence and verified the cells are localized to the expected tail-region. This study is evidence that quality data can be generated on a budget-friendly flow cytometer and allows researchers with limited resources to use flow cytometry.
Novel targeting methods for pancreatic cancer. (TECH2P.914)
Authors: Knowlton Andrea , Restuccia Antonietta , Delitto Daniel , Trevino Jose , Hudalla Greg , Wallet Shannon
Published: January 2023

Pancreatic cancer is the fourth leading cause of cancer deaths in the United States and one of the most difficult-to-treat cancers. Despite advances in chemotherapy, 5-year survival following surgical resection is only 23% in specialized pancreatic surgery centers. A major stumbling block for these and other cancer treatments is a way to efficiently deliver therapeutics to the target cells. Thus, we have identified high affinity molecular targeting molecules unique to an individual pancreatic cancer, which were generated utilizing a high throughput system using fresh surgical specimens along with phage display to develop novel small chain variable fragments (scFv). Here we present the characterization of two scFv molecules identified, through which we demonstrate by ELISA and BioLayer Interferometry that our scFv bind with high specificity to the neoplasm from which they were derived, while remaining minimally reactive to healthy pancreatic tissue. In addition, we have engineered these scFV to be assembled into a homotrimeric alpha-helical coiled coil using a novel platform in order to retain its multimeric nature. Future studies will assess the ability of these scFv to home to the original neoplasm using our established patient-derived tumor xenograft models whereby the human tumor is grown in NOD/SCID mice. Together these data will determine whether screening individual neoplasms using phage display can be used to develop scFv as personalized targeting molecules.
Rapid quantification of maleimide in bioconjuated samples using a novel colorimetric method (TECH2P.761)
Authors: Liao Jinfang , Guo Haitao , Luo Zhen , Zhao Qin , Diwu Jack
Published: January 2023

A variety of crosslinking reagents with a maleimide group are widely used for crosslinking proteins to proteins, or proteins to other macromolecules. A key challenge in maleimide crosslinking technology is the lack of rapid and sensitive methods for quantifying the amount of maleimide groups in a biopolymer. Maleimides can be directly measured spectrophotometrically at 302 nm. However, the small extinction coefficient of maleimide renders this approach insensitive. In addition, the measurement is non-specific due to the fact that proteins have absorbance at the same wavelength. To address this unmet need, a rapid colorimetric method was developed to quantify maleimide group by using a novel maleimide sensor with the maximum absorbance at ~780 nm. The maleimide sensor can react with maleimide groups with high selectivity and sensitivity. After reaction with the maleimide sensor, the maleimide-linked sample was purified through a single spin column to remove the excess sensor, and the absorption spectrum was further measured. The ratio of maleimide to protein in the maleimide-linked samples was calculated by the ratio of absorbance at 780 nm to the absorbance at 280 nm. Based on this newly developed method, number of maleimide groups can be measured rapidly and precisely for a biopolymer (such as antibodies).
Metal-conjugated neutravidin for MHC multimer assays using mass cytometry (TECH3P.939)
Authors: Ornatsky Olga , Wong Gladys , Majonis Daniel , Delcommenne Marc , Narumiya Kohei
Published: January 2023

Antigen-specific T cells in blood can be detected through the use of soluble MHC-peptide ligands that engage αβTCR. The fluorescent tetramer assay, developed in part by John Altman et al. (1) of the Vaccine Research Center, has become a standard tool for immunologists. Evan Newell et al. ( 2) adapted this peptide-MHC tetramer technology to mass cytometry for the purpose of screening of up to 109 different peptide-MHC tetramers in a single human blood sample, as well as analyzing another 23 markers of T-cell phenotype and function using a recombinant form of streptavidin conjugated to metal tags (Maxpar® kits, Fluidigm CA). We will describe the workflow for enumeration and identification of CMV-specific CD8+ T-cells with Neutravidin-metal reagent complexed with HLA-A*0201 CMV pp65 biotinylated monomer (MBL International, MA) simultaneously with metal-labeled surface markers, cisplatin dead-cell identifier, and the use of metal barcoding of several samples into one. There are 198 different biotinylated monomers, commercially available from MBL International, which can be combined with up to 35 isotope-tagged Neutravidin reagents to design a highly multiparametric assay. 1. Altman, J. D. et al. Phenotypic analysis of antigen-specific T lymphocytes. Science 274: 94-96 (1996) 2. Newell, E. W. et al. Combinatorial tetramer staining and mass cytometry analysis facilitate t-cell epitope mapping and characterization. Nat. Biotechnol. 31, 623-629 (2013).
Manipulating DNA damage response pathways for the treatment of experimental autoimmune encephalitis (THER7P.952)
Authors: McNally Jonathan , Millen Scott , Terrell Catherine , Carroll Katelyn , Hildeman David , Katz Jonathan , Jordan Michael
Published: January 2023

The race between pathogens and the immune response drives rapid expansion of lymphocyte populations, suggesting genomic stress in responding lymphocytes. We have previously reported the unique apoptotic sensitivity of activated T cells to DNA damaging drugs in diverse therapeutic contexts. We now report that activated T cells display a pronounced DNA damage response (DDR) in vitro and in vivo. We hypothesized that this intrinsic DDR could be manipulated with targeted small molecules for the elimination of pathologic T cells in a therapeutic context. We found that potentiation of p53 (via inhibition of MDM2)) or abrogation of the G2/M cell-cycle checkpoint (via inhibition of WEE1) synergized with DNA damaging drugs for the elimination of acutely activated, pathologic T cells. Moreover, a combination of p53 potentiation and checkpoint abrogation displayed therapeutic benefits in Experimental Autoimmune Encephalitis a preclinical disease model of Multiple Sclerosis, without the concurrent use of genotoxic chemotherapeutics. Additionally, combination inhibitor therapy showed minimal off target toxicities on other immune populations. Thus, targeted manipulation of p53 and cell cycle checkpoints may represent a new therapeutic modality with significant translational potential for diverse immune mediated diseases.
Engineered MBP antigen-specific human regulatory T cells: direct and bystander immunosuppressive function (THER7P.954)
Authors: Kim Yong , Zhang Ai-Hong , Wucherpfennig Kai , Scott David
Published: January 2023

Clinical application of expanded T regulatory cells (Tregs) offers great promise for the treatment of undesirable immune responses. However, treatment with polyclonal Tregs, which are not specific, could potentially cause global immunosuppression. To avoid this problem, we propose that antigen-specific Tregs, based on chimeric antigen receptor (CAR) therapy in leukemia, would be desirable to control autoimmune responses. We engineered a myelin basic protein-specific TCR in a retroviral vector and transduced it to human naïve CD4+ T cells and Tregs. The TCR V genes came from an MBP-specific T-cell clone derived from an MS patient (Ob2F3, Ota et al., Nature 346, 183-7, 1990). Transduced Tregs proliferated in vitro in response to MBP peptide on DR2 APC. Foxp3 and Helios expression were preserved in transduced Tregs during long-term expansion and these specific Tregs did not produce any inflammatory cytokines. Treg-unique activation markers, Foxp3 and LAP, were upregulated by MBP. Importantly, MBP-specific Tregs suppressed proliferation and cytokine production by MBP-specific T effector cells in the presence of MBP peptide. We also observed bystander suppression of MBP responses in vitro with Tregs specific for a different epitope when both peptides were present. We propose that MBP-specific Tregs can control pathologic inflammation as a future therapy in MS patients.
Unique gut commensal bacteria in CD44 deficient mice can ameliorate experimental autoimmune encephalomyelitis (THER7P.943)
Authors: Guan Hongbing , Singh Udai , Singh Narendra , Hao Haiping , Nagarkatti Prakash , Nagarkatti Mitzi
Published: January 2023

Gut commensal microbiota synergize diet, metabolites, and innate immunity to maintain homeostasis of biological system. There are now emerging evidences implicating the role of gut microbiota in elicitation of human autoimmune diseases. It is has been reported that gut commensal microbiota and myelin autoantigen cooperate to trigger autoimmune demyelination and experimental autoimmune encephalomyelitis (EAE). In the current study, we aimed to identify specific microbiota associated with the pathogenesis of EAE. We used EAE-susceptible CD44+/+ (WT) mice and EAE-resistant CD44-/- (KO) mice for the analysis of gut commensal bacteria. We found distinct bacteria profiles in naïve WT and naïve KO mice. After immunization with MOG35-55 peptide, the profiles dramatically changed on the bacterial classification levels. We identified several species as putative probiotics against the disease. We also observed these probiotics can prevent the development of EAE, and significantly ameliorate the disease. Upon analysis of the metabolites in situ in the gut, we identified probiotic-composites that suppressed encephalitogenesis of CD4+ effector T cells. Our results may offer novel strategies for the prevention and treatment of multiple sclerosis.
Plant-based product Apigenin attenuates EAE severity through the modulation of immune cell function (THER7P.948)
Authors: Ginwala Rashida , McTish Emily , Sagar Divya , Raman Chander , Jain Pooja , Khan Zafar
Published: January 2023

The use of Apigenin, a naturally occurring plant flavone, for centuries to treat asthma, Parkinson's disease, neuralgia, and shingles, indicates its importance in the regulation of inflammation. However, its effect on dendritic cells (DC) that maintain the critical balance between an immunogenic and tolerogenic immune response especially in neuroinflammation is relatively unknown. To test if Apigenin treatment ameliorates disease after onset of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, C57BL/6 mice immunized with MOG35-55 were treated with Apigenin. A significant reduction in severity of EAE progression was observed in the treated mice with disease peak lasting only a day. Splenocytes isolated from Apigenin treated EAE mice show decreased expression of α4 integrin and increased DCs and macrophages compared to untreated EAE mice. This correlated with immunohistochemistry findings of decreased immune cell infiltration and reduced demyelination in the CNS. In vitro, Apigenin inhibited TNF-α and IL-6 secretion in mice splenic DCs stimulated with LPS and the cell surface expression of MHC II and CD86 molecules on bone marrow derived DCs. These results indicate a protective role of Apigenin against the neurodegenerative effects resulting from the entry of DC stimulated pathogenic T cells into the CNS. Apigenin can thus serve as a potential therapy for neuroinflammatory disease through its regulation of immunogenic T cell response.
Cross reactivity of allo and xenoimmune response in transplantation (TRAN2P.964)
Authors: Byun Nari , Kim Hyun-Je , Yoon Il-Hee , Kwon Oh-Sang
Published: January 2023

Many type I diabetes patients are suffering from hypoglycaemia unawareness. And porcine islet transplantation is one of the promising modalities to treat the problematic condition in a physiologic manner. But by transplanting porcine islet, xenoantigen sensitization is inevitable. For clinical application of porcine islet transplantation, the knowledge of the outcome and consequent effects of xenoantigen sensitization needs to be fully established. Our focus was whether xenoantigen sensitization would influence subsequent allotransplantation in any way. We confirmed that xenoantigen sensitization does not induce alloreactive antibody. And by ELISpot assay we demonstrated that xenoreactive T cells are not responsive to alloantigen. Lastly, we confirmed that xenoantigen sensitized mice have the same graft survival day compared with non sensitized mice in allogeneic islet transplantation setting. With these results put together, we conclude that porcine antigen sensitization can not induce alloantigen sensitization in mice.
Induction of antigen specific transplantation tolerance using chimeric antigen receptor type T cells engineered to kill allospecific T cells by a gene therapy immunotherapeutic approach (TRAN2P.968)
Authors: Rajawat Yogendra , Berkson Julia , McFarland Hugh , Morgan Richard , Zhao Yangbing , Rosenberg Amy
Published: January 2023

Development of specific immunotherapies to prevent graft rejection and graft-versus-host disease offers the promise of selectively deleting only allospecific immune responses. Veto activity is the ability of a cell to specifically suppress/delete only T cells directed against antigens of the veto cells themselves, but not against third-party antigens. In the present study, we reasoned that providing TCR-like signaling to allogeneic major histocompatibility complex (MHC) molecules on the surface of CD8+CTLs might result in enhanced veto function. To generate the donor antigen specific veto cells, we have utilized the Chimeric Antigen Receptor (CAR) T cell approach, in which CD8+CTLs express an MHC alloantigen fused to signaling molecule apparatuses. We fused mouse MHC class I alloantigen (H-2Dd) to the hinge and trans-membrane domains of the mouse CD8 chain plus intracellular signaling sequences derived from mouse 4-1BB, and CD3-ζ chain creating various CAR constructs. These CARs were cloned into retroviral vectors. Ex-vivo transduced T cells (FVB-Dd) were adoptively transferred in the recipient parental mice strain (FVB-H-2q) which do not express Dd. Two weeks post-adoptive transfer, we detected low levels of Dd+CD8+ T cells in lymph node and spleen, ranging from 0.2-1.2% of total CD8+ T cells. Veto activity of these Dd+CD8+T cells will be examined by in vitro Dd specific CTL assays, proliferation assays, and rejection of Dd skin grafts in parental strain (FVB) mice.
Accelerated progression of Hodgkin’s-like lymphoma in golli-deficient mice is associated with higher induction of CD4+FoxP3+ regulatory T cells (TUM9P.1018)
Authors: Feng Ji-Ming , Ouyang Suidong , Han Guangming
Published: January 2023

The spontaneously occurred lymphomas in SJL/J mice have many pathological features similar to Hodgkin’s lymphoma in human. The malignant growth of the tumor cells is dependent on the support of host FoxP3+ CD4+ regulatory T cells (Tregs). In this study, we report that the golli protein, a negative regulator of CRAC (calcium release activated calcium) channel in T cells, plays a critical role in the tumorigenesis of Hodgkin’s-like lymphoma. The ablation of golli protein in SJL/J mice results in an accelerated progression of Hodgkin’s-like lymphoma which is associated with a facilitated induction of FoxP3+ Treg cells. Our results suggest that through regulating the induction of Treg cells, golli protein affect the progression of Hodgkin’s-like lymphoma.