bluuebunny/crossref_metadata_2025 huggingface dataset:
Scaffold functions of 14-3-3 adaptors in B cell immunoglobulin class switch DNA recombination (IRC3P.472)
Authors: Lam Tonika , Thomas Lisa , White Clayton , Li Guideng , Pone Egest , Xu Zhenming , Casali Paolo
Published: January 2023

Immunoglobulin (Ig) class switch DNA recombination (CSR) crucially diversifies antibody biological effector functions. CSR involves the induction of activation-induced cytidine deaminase (AID) expression and targeting to switch (S) regions by 14-3-3 adaptors. We hypothesized that 14-3-3 adaptors function as scaffolds to stabilize CSR elements on S regions. Here, we demonstrate that all seven 14-3-3 adaptors (β, ε, γ, η, σ, τ and ζ), directly interacted with AID, PKA-Cα (catalytic subunit) and PKA-RIα (regulatory inhibitory subunit) and uracil DNA glycosylase (Ung). 14-3-3 adaptors, however, did not interact with AIDΔ(180-198) C-terminal truncation-mutant or AIDF193A and AIDL196A point-mutants (which have been shown not to bind to S region DNA and fail to mediate CSR). 14-3-3 adaptors colocalized with AID and replication protein A (RPA) in B cells undergoing CSR. 14-3-3 and AID binding to S region DNA was disrupted by HIV-1 viral protein R (Vpr), which inhibited CSR without altering AID expression. Accordingly, we demonstrate that 14-3-3 directly interacted with Vpr, which in turn, also interact with AID, PKA-Cα and Ung. Thus, we describe that 14-3-3 adaptors play important scaffold functions and nucleate the assembly of CSR factors on S regions. Such assembly can be disrupted by a viral protein, thereby allowing us to hypothesize that small molecule compounds that specifically block 14-3-3 interactions with AID, PKA and/or Ung can be used to inhibit unwanted CSR.
Mechanism of B cell membrane repair and the effects of repair on B cell receptor activation (IRC3P.460)
Authors: Miller Heather , Tam Christina , Corrotte Matthias , Castro-Gomes Thiago , Andrews Norma , Song Wenxia
Published: January 2023

The plasma membrane of B cells is at risk for injury by bacterial toxins, viruses, and possible mechanical damage induced by interactions with other cells during antigen presentation. Whether and how B cells repair membrane damage and its impact on B-cell functions are unknown. Here we show that upon injury by the pore forming toxin streptolysin O (SLO), primary mouse splenic B cells can repair their membrane in a Ca2+-dependent manner, like muscle and epithelial cells. B cells exocytose their lysosomes in response to Ca2+ influx and release acid sphingomyelinase from lysosomes, which induces endocytosis. In B cells activated by cross-linking B-cell receptors (BCRs), repairing membrane damage by SLO inhibits BCR clustering and internalization, consequently reducing BCR-induced tyrosine phosphorylation. Further analyses show that membrane repair inhibits the colocalization of BCRs with lipid rafts on the cell surface and during their internalization, indicating that membrane repair-induced endocytosis competes with BCRs for lipid rafts and interferes with the interaction of BCRs with lipid raft-associated kinases. Our results reveal the ability and the mechanism for B cells to repair membrane damage, and discover a negative regulatory role of membrane repair on B cell signaling and antigen uptake functions.
Integrin β8 is a marker of mouse regulatory T-cells (Treg) and is required for processing of latent TGF-β1 from the GARP/latent TGF-β1 complex. (IRC4P.483)
Authors: Edwards Justin , Thornton Angela , Shevach Ethan
Published: January 2023

Previous studies have demonstrated that activated Tregs express latent TGF-β1 on their cell surface bound to the tethering molecule GARP. Although integrins have been implicated in mediating the release of biologically active TGF-β1 from the complex of latent TGF-β1 and latent TGF-β1 binding protein, their role in processing latent TGF-β1 from the latent TGF-β1/GARP complex is unclear. We detected integrin β8 expression by CD4+Foxp3+ Treg, but not by CD4+Foxp3- T cells, by qRT-PCR. Furthermore, integrin β8 expression appeared to be a marker of thymically-derived Treg (tTreg), as its could not be detected on Foxp3+ T cells induced in vitro nor in vivo on peripherally induced Foxp3+Helios- Tregs. While Tregs from integrin β8 conditional knockouts suppressed T effector cell proliferation normally in vitro and in vivo in a model of colitis, integrin β8 deficient Tregs failed to provide a source of TGF-β1 to drive Th17 or iTreg differentiation in vitro. In addition, the knockout Tregs expressed higher levels of latent TGF-β1 on their cell surface consistent with defective processing. Taken together, integrin β8 is not only a marker of tTregs, but also functions in a cell intrinsic manner as the major pathway for processing latent TGF-β1 from the latent TGF-β1/GARP complex on the surface tTregs.
Type I IFNs modulate T effector (Teff) and T regulatory (Treg) cell function during experimental autoimmune encephalomyelitis (EAE) (IRC4P.485)
Authors: Metidji Amina , Shevach Ethan
Published: January 2023

Type I IFNs (IFNα and IFNβ) are a family of cytokines that mediate multiple roles in inflammation and autoimmunity. Previous studies have demonstrated that Type I IFNs primarily act on myeloid cells during the course of EAE. We have re-examined the role of Type I IFNs in EAE by generating mixed bone marrow chimeras between wild type (WT) mice and mice with a deficiency in the IFN receptor (IFNAR-/-). IFN plays a role in the T lymphocyte homeostasis as the ratio of WT to IFNAR-/- CD4+ T cells is skewed toward WT and the skewing is more marked in the Treg compartment. Surprisingly, after induction of EAE, the ratio of WT to IFNAR-/- T cells is reversed in the periphery and the mixed chimeras develop more severe disease than the WT/WT chimeras. Almost all of the CD4+Foxp3- and CD4+Foxp3+ T cells in the CNS of chimeric mice are derived from the IFNAR-/- donor, but the percentage of Foxp3+ T cells was similar in WT/WT and WT/IFNAR-/- chimeras. Both CD4+Foxp3- and CD4+Foxp3+ T cells from the IFNAR-/- donor expressed higher levels of α4 integrins, which may account for their increased numbers in the CNS during EAE. Mice with a conditional deletion of the IFNAR in Treg rapidly developed a very severe form of EAE. Taken together, these results demonstrate that signaling via the IFNAR is required for Treg suppressor function in EAE and that IFNAR signaling may also modulate homing of Teff cells to the CNS and/or their susceptibility to suppression by Treg.
PCAF promotes TGF-β-induced Foxp3+ regulatory T cell production (IRC4P.486)
Authors: Liu Yujie , Wang Liqing , Han Rongxiang , Beier Ulf , Dent Sharon , Hancock Wayne
Published: January 2023

We recently showed that 2 histone/protein acetyltransferases (HATs), p300 and CBP, are important for Foxp3+ Treg function, and that their targeting promotes anti-tumor immunity in immunocompetent mice. We now report on functions of another HAT, PCAF, in Tregs. Like CBPflflp300flflFoxp3YFP-cre mice, PCAF-/-GCN5flflFoxp3YFP-cre mice develop severe autoimmunity and die in early life, indicating their importance for Treg development. While mice with a single deletion (PCAF-/- or GCN5flflFoxp3YFP-cre) are born at expected Mendelian ratios and do not develop obvious autoimmunity, T cells from PCAF-/- mice are activated, with more CD4+CD44hiCD62Llow, CD4+CD69+ and CD8+IFN-γ+ cells than WT mice. PCAF-/- mice have normal CD4+Foxp3+ percentages in thymus and peripheral lymphoid tissues, but these undergo increased apoptosis upon CD3/CD28 stimulation. Furthermore, compared to control naïve CD4+ T cells (CD4+CD25-CD44lowCD62Lhigh), PCAF naïve CD4 T cells show impaired conversion into Foxp3+ Tregs when cultured with TGF-β in vitro. Adoptive transfer of naïve PCAF-/- vs. WT CD4 cells into Rag1-/- mice also resulted in fewer PCAF-/- CD4 T cells converting into Foxp3+ Tregs than with WT controls, and was accompanied by lower pSmad2 levels in PCAF-/- CD4 T cells. Hence, PCAF regulates iTreg production in vivo and in vitro. Our studies suggest that PCAF is a promising target for development of pharmacologic inhibitors to modulate iTreg production and promote anti-tumor responses.
R-Ras promotes autoimmunity through negative regulation of natural T regulatory cell numbers by inhibiting tolerogenic dendritic cell development (IRC4P.488)
Authors: Ray Avijit , Basu Sreemanti , Chan Andrew , Dittel Bonnie
Published: January 2023

R-Ras is a member of the Ras family of small GTPases, which regulate various cellular processes including adhesion, survival, proliferation, trafficking, and cytokine production. R-Ras is expressed by immune cells and has been shown to modulate dendritic cell (DC) function in vitro. Although, higher expression of R-Ras is associated with liver autoimmunity in humans, its pathogenic mechanism is unknown. We used Rras-/- mice to identify the mechanism whereby R-Ras contributes to autoimmunity using experimental autoimmune encephalomyelitis (EAE), a mouse model of the CNS autoimmune disease multiple sclerosis. We found that a lack of R-Ras in peripheral immune cells resulted in attenuated EAE disease. In accordance with reduced EAE disease, numbers of inflammatory immune cells in the CNS of Rras-/- mice at the peak of EAE were significantly reduced. Further investigation revealed that during EAE, absence of R-Ras promoted the formation of MHC IIlo tolerogenic DC concomitant with a significant increase in Neuropilin-1hiCD4+Foxp3+ natural T regulatory cell (nTreg) numbers in the periphery. Mechanistically, we found that Rras-/- tolerogenic DCs induced proliferation of nTregs thereby enhancing their numbers and maintaining them in vivo. Our study suggests a novel role for R-Ras in promoting autoimmunity through negative regulation of nTreg numbers by inhibiting tolerogenic DC development.
WRNIP1 is a key regulator of antigen-specific Treg cells. (IRC4P.480)
Authors: Du Weiting , Wang Ding , Shen Yueh-Wei , Lee Wen-Hui , Riggs Art , Liu Chih-Pin
Published: January 2023

It is well documented that, both Foxp3+ regulatory T cells (Treg) and antigen-expanded Foxp3lo/- Treg play critical roles in regulating immunity and autoimmunity, as well as in preventing autoimmune diseases. However, the molecular mechanisms and genes responsible for modulating function of these Treg still remain incompletely understood. WRNIP1 (Werner helicase-interacting protein 1) was identified as a protein that interacts with WRN, the Werner syndrome responsible gene product, and may participate in DNA synthesis, damage and repair. Here, we report an unexpected finding that WRNIP1 plays an important role in regulating the function of autoantigen-expanded Foxp3lo/- Treg. Down regulation of WRNIP1 in these Treg significantly increased their proliferation and reduced activation-induced cell death. More importantly, WRNIP1-downregulated Treg showed markedly enhanced suppressive function in vitro, partly due to an increased production of IL-10 and TGF-β. These Treg also significantly improved their ability in preventing diabetes development in NOD mice. To understand how WRNIP1 regulates Treg function, we found that elevated Foxo1 expression may contribute to the enhanced Treg function through promoting IL-10 and TGF-β production. These studies demonstrate that WRNIP1 negatively regulates Treg function and suggest previously undisclosed molecular pathways and targets for improving Treg-mediated immune tolerance to treat autoimmune diseases like type 1 diabetes.
Chronic viral infection: the perfidious role of IL-2 and IL-15 on CD8 T cell exhaustion (IRM4P.500)
Authors: Beltra Jean-Christophe , Charpentier Tania , Michaud Eva , Lamarre Alain , Decaluwe Hélène
Published: January 2023

CD8 T cell exhaustion is a major immuno-regulatory mechanism by which chronic viruses escape the immune response. However, little is known on the factors regulating the unresponsiveness of CD8 T cells is this context. Here, we investigated the combined role of IL-2 and IL-15, two common gamma chain-dependent cytokines, on CD8 T cell differentiation and exhaustion during a chronic viral infection. We performed adoptive transfer of WT or IL-2RβKO P14 CD8 T cells (unresponsive to both IL-2 and IL-15 signals) in mice challenged with LCMV clone 13. Initially, IL-2Rβ-signals were critical for short-lived effector cell differentiation. IL-2 and IL-15 conditioned optimal proliferation of early effector CD8 T cells and promoted their cytotoxic functions. Interestingly, over the chronic phase, IL-2Rβ-signals dramatically worsened CD8 T cell exhaustion. IL-2 and IL-15 sustained the expression of several inhibitory receptors and fostered the development of the highly exhausted PD-1hi progeny population. Finally, IL-2Rβ-signals precluded central memory CD8 T cell differentiation capable of homeostatic proliferation and robust secondary expansion. Altogether, these data demonstrated for the first time an important deleterious effect of IL-2 and IL-15 on the CD8 T cell response to a chronic viral infection. Reconsidering their role in this context may provide new insights for therapeutic regimens against chronic viruses.
Pregnane X receptor negatively regulates inflammation by directly modulating the Toll-like receptor 4 pathway (IRM5P.701)
Authors: Qiu Zhijuan , Cicek Basak , Cervantes Jorge , Salazar Juan , Mani Sridhar , Khanna Kamal
Published: January 2023

Pregnane X receptor (PXR) is a nuclear receptor that plays a critical role in regulating the metabolism of xenobiotics. Here we report a new role for PXR as a negative regulator of the inflammatory pathway. Quiescent Pxr-/- BMDMs exhibited a pro-inflammatory state as transcripts of inflammatory genes were significantly elevated. Upon stimulation with Listeria monocytogenes (Lm), Pxr-/- BMDMs produced significantly higher protein levels of inflammatory cytokines. Moreover, enhanced inflammation was also observed in Pxr-/- mice challenged with Lm. Furthermore, disruption of the toll-like receptor 4 (TLR4) gene reversed the pro-inflammatory state in Pxr-/- BMDMs suggesting that PXR negatively regulated inflammation through the TLR4 pathway. In fact, both Pxr-/-Tlr4-/- BMDMs and mice exhibited normal inflammation in response to Lm infection. Transfection of HEK-Blue™-mTLR4 cells with DNA vector expressing PXR inhibited the response to LPS in a dose-dependent manner, which suggested that PXR can directly modulate the TLR4 pathway. In conclusion, our study demonstrates that PXR negatively regulates the inflammatory pathway and the regulation is through directly modulating the TLR4 pathway. Thus, our results suggest that PXR can be targeted as a therapeutic strategy to treat inflammatory diseases.
Differential phosphorylation of T-bet in the presence and absence of mTORC1 signaling (IRM6P.722)
Authors: Chornoguz Olesya , Powell Jonathan
Published: January 2023

Previously we have demonstrated that T cells lacking the mTORC1 activator Rheb fail to differentiate in Th1 cells upon appropriate skewing conditions. T-box transcription factor T-bet plays a critical role in Th1 differentiation. We wondered whether mTORC1 regulates either T-bet expression or activity. Interestingly, T-bet contains several motifs of sites typically phosphorylated by AGC kinases. This raised the possibility that mTORC1 activation might play a role in the phosphorylation and control of T-bet function. To test this hypothesis we employed targeted mass-spectrometry proteomic analysis (MRM-MS) to identify novel T-bet phosphorylation sites that might be controlled by mTORC1. By analyzing activated WT and Rheb-/- T cells as well as T cells stimulated in the presence of rapamycin we were able to detect 6 T-bet phosphorylation sites. Specifically S52, T54, S224 are novel and Y77, Y118, S508 are previously published phosphorylations. T54, Y77 and S508 were either absent or downregulated in Rheb deficient T-cells and T-cells treated with Rapamycin suggesting that they are controlled by mTORC1 signaling. Studies are underway to determine the precise functional importance of these sites. Thus using MRM-MS we have identified a potentially novel mechanism by which mTORC1 signaling controls T-bet.
CD4 T Cell-autonomous TNF receptor type 2 promotes interleukin-2 transcription (IRM6P.723)
Authors: McKarns Susan , Miller Patrick
Published: January 2023

Interleukin (IL)-2 plays a key role in adjusting the dynamic state of equilibrium between effector T cells (Teffs) and regulatory T cells (Tregs) as well as channeling the outcome of immune responses. IL-2 is mainly produced by activated CD4 T cells and the absence of IL-2 signaling results in lethal autoimmunity. In this study we investigated the role of tumor necrosis factor alpha (TNF) receptors type 1 (TNFR1) and type 2 (TNFR2) on the regulation of IL-2 at the level of transcription during the primary activation of CD4 T cells. We generated TNFR1- and/or TNFR2-deficient-IL-2-GFF knock-in reporter mice to examine Il2 promoter activity at the single cell level. We found that the deficiency of TNFR2, but not TNFR1, markedly diminished the number of CD4 T cells that produced IL-2. We further correlated decreased Il2 transcription with impaired intracellular c-Rel expression in TNFR2 KO CD4 T cells. IL-2 production was augmented in response to the addition of soluble TNF implicating a role for transmembrane TNF (tmTNF). Collectively, these data suggest that CD4 T cell autonomous tmTNF/TNFR2 signaling contributes to the number of IL-2 producers by providing a critical third signal necessary for threshold Il2 promoter activity. These results have important implications for understanding the contribution of IL-2 signaling on the dynamic equilibrium between Teff cells and Treg cells and the outcome of inflammatory immune responses.
Peroxisome proliferator-activated receptor-alpha (PPARα) suppresses T helper 1 (Th1) cytokine production in male mice by inducing post-translational histone modifications at the interferon-gamma (IFNγ) locus (IRM6P.724)
Authors: Zhang Monan Angela , Dunn Shannon
Published: January 2023

Women develop more robust Th1 inflammatory responses than men. Previously, we showed that lower Th1 cytokine responses in males correlated with higher expression of the androgen-sensitive nuclear receptor PPARα, which represses IFNγ production by T cells (Zhang et al. PNAS 2012). The current study aimed to elucidate the molecular mechanism of repression of IFNγ by PPARα in male mouse CD4+ T cells. We first contrasted the expression of various Th-associated genes between anti-CD3 and anti-CD28-stimulated PPARα+/+ (WT) and PPARα-/- (KO) T cells. Of these genes, only T-bet, Eomes, and IFNγ mRNAs were expressed at higher levels in KO vs. WT T cells. Since these genes can reinforce each others’ expression, we further examined their expressions under conditions of either T-bet deficiency or IFNγ neutralization. We found that the higher expressions of T-bet and Eomes were lost, but IFNγ mRNAs remained elevated in KO cells, indicating IFNγ as the likely gene repressed by PPARα. We then performed chromatin immunoprecipitation experiments to examine the abundance of a permissive histone mark (acetylated-H4) and a co-repressor protein (NcoR) at conserved non-coding sequence sites across the ifng locus. We detected global de-repression of ifng in KO vs. WT cells while treating WT cells with a specific agonist induced PPARα recruitment and decreased H4 acetylation at certain sites. Thus, our data suggest that PPARα suppresses IFNγ in males by keeping ifng less poised for transcription.
The RNA-binding protein HuR is required for normal homeostatic control of IL-2 expression in activated CD4 T cells (IRM6P.713)
Authors: Techasintana Patsharaporn , Magee Joseph , Atasoy Ulus
Published: January 2023

Upon T cell activation approximately 50% of genes are regulated at the posttranscriptional level by RNA-binding proteins (RBPs). HuR is a stabilizing RBP which regulates mRNA via interactions with AU-rich elements (ARE) in mRNA 3’UTRs. HuR has been shown to be crucial for T cell development, thymic selection and egression. However, it is unclear how HuR regulates T cell differentiation and cytokine expression. To further investigate these questions, we generated a novel conditional HuR KO mouse, distal-Lck Cre ROSA HuRfl/fl which ablates HuR in mature T cells prior to activation. HuR KO T cells develop and egress from thymus normally; however, they display aberrant cytokine and receptor expression upon activation. Activated HuR KO T cells show striking increases in IL-2 (20-30 fold) and display an inability to turn off IL-2 expression, but significant decreases in IL-4, IL-5 and IL-13 with no change in IFN-γ. Over 90% of HuR KO CD4 cells still make IL-2 five days after activation but display impaired IL-4, IL-5 and IL-13 secretion. Increases in IL-2 in HuR KO cells are due to augmentation of IL-2 transcription and mRNA stability. Moreover, there are significant decreases in proliferation and CD25 (IL-2Rα) expression in HuR KO cells as a result of reductions in CD25 transcription. Thus, HuR is required for cytokine homeostasis in CD4 cells and may well function as a critical off-switch for IL-2 expression during T cell activation.
β-catenin promotes colitis and colon cancer through imprinting of pro-inflammatory properties in T-cells. (LYM3P.727)
Authors: Keerthivasan Shilpa , Aghajani Katayoun , Dose Marei , Khazaie Khashayarsha , Gounari Fotini
Published: January 2023

The density and type of lymphocytes infiltrating colon tumors are predictive of the clinical outcome of colon cancer. High densities of TH17 cells and inflammation predict poor outcome, while infiltration by T regulatory cells (Tregs) that naturally suppress inflammation is associated with longer patient survival. However, the role of Tregs in cancer remains controversial. We previously reported that Tregs in colon cancer patients can become pro-inflammatory and tumor promoting. These properties were directly linked with their expression of RORγt, the signature transcription factor of TH17 cells. Here, we report that Wnt/β-catenin signaling in T-cells promotes expression of RORγt. Expression of β-catenin was elevated in T-cells, including Tregs, of patients with colon cancer. Genetically engineered activation of β-catenin in mouse T-cells resulted in enhanced chromatin accessibility in the proximity of Tcf-1 binding sites genome-wide, induced expression of TH17 signature genes including RORγt, and promoted TH17-mediated inflammation. Strikingly, the mice had inflammation of small-intestine and colon and developed lesions indistinguishable from colitis-induced cancer. Activation of β-catenin only in Tregs was sufficient to produce inflammation and initiate cancer. Based on these findings we conclude that activation of Wnt/β-catenin signaling in effector T-cells and/or Tregs is causatively linked with the imprinting of pro-inflammatory properties and promotion of colon cancer.
Deep sequencing of the gag-specific CD4+ T cell receptor repertoire and CD4+ memory subsets of HIV viral controllers reveals significant sharing of unique gag-specific TCRs in central and effector memory TCR repertoires (LYM3P.742)
Authors: Pilkinton Mark , Barnett Louise , Kalams Spyros
Published: January 2023

HIV viral controllers maintain preserved CD4+ T cells counts and undetectable viral levels off antiretroviral therapy. The diversity of the HIV-specific T cell receptor (TCR) repertoire within memory may be important for maintaining viral control, however, sequencing the entire CD4+ memory repertoire to evaluate for HIV specific TCRs would be prohibitive by conventional methods. In this study, the TCR repertoire of two HIV controllers was analyzed with deep sequencing. Peripheral blood mononuclear cells (PBMC) of HIV+ individuals were stimulated by Gag peptides and the Gag-specific CD4+ T cells were sorted by expression of the activation markers. Unstimulated PBMC were sorted for CD4+ memory subsets based on central memory (CD45RO+CCR7+), effector memory (CD45RO+CCR7-), and naïve (CD45RO-CCR7+). Subject 1 had 46 Gag-specific CD4+ TCR. 18 were in the Tcm population and 24 in the Tem population, though 15 of these sequences were identified in both subsets. In Subject 2, there were 29 Gag-specific TCR with 27 in the Tcm population and 20 in the Tem repertoire with 20 Gag-specific TCR identified in both populations. Deep sequencing of the HIV specific TCR repertoire and unbiased memory subsets revealed a high degree of overlap of CD4+ HIV-specific cells in both the Tcm and Tem populations. Further studies will need to be performed to determine if this provides an advantage for virologic control.
Harnessing the heterogeneity of T cell differentiation fate to fine-tune generation of effector and memory T cells (LYM4P.759)
Authors: Gong Chang , Linderman Jennifer , Kirschner Denise
Published: January 2023

Recent studies show that naïve T cells bearing identical T cell receptors experience heterogeneous differentiation and clonal expansion processes. The factors controlling this outcome are not well characterized, and their contributions to immune cell dynamics are similarly poorly understood. In this study, we develop a computational model to elaborate mechanisms occurring within and between two important physiological compartments, lymph nodes and blood, to determine how immune cell dynamics are controlled. Our multi-organ (multi-compartment) model integrates cellular and tissue level events and allows us to examine the heterogeneous differentiation of individual precursor cognate naïve T cells to generate both effector and memory T lymphocytes. Using this model, we simulate a hypothetical immune response and reproduce both primary and recall responses to infection. Increased numbers of antigen-bearing dendritic cells are predicted to raise production of both effector and memory T cells, and distinct “sweet spots” of peptide-MHC levels on those dendritic cells exist that favor CD4+ or CD8+ T cell differentiation towards either effector or memory cell phenotypes. This has important implications for vaccine development and immunotherapy.
Probiotic lactobacillus ameliorates heightened colonic inflammatory responses in infected stressor-exposed C57BL/6 mice and inhibits dysbiosis (MPF1P.771)
Authors: Galley Jeff , Mackos Amy , Parry Nicola , Yu Zhongtang , Ahmer Brian , Bailey Michael
Published: January 2023

The mammalian colon is a primary niche for the microbiota, a community of bacteria in part involved in immune maintenance. Alterations in these microbial populations, referred to as dysbiosis, can have deleterious effects on host health and previous studies show psychological stress can lead to dysbiosis. We investigated if stressor exposure could increase the severity of colonic infection with the enteric pathogen, Citrobacter rodentium. Mice that were subjected to social stressor exposure had increases in pathogen burden and the inflammation-related gene transcripts TNF-alpha, iNOS, and CCL2. The stressor-exposed mice also had increased colitic pathology. When mice were treated with a probiotic strain of Lactobacillus reuteri, the colitic pathology and resultant inflammatory markers were abrogated. The mechanism by which L. reuteri reduces colitis is unknown. Probiotic treatment of germ-free mice mono-associated with C. rodentium did not reduce inflammation, indicating that direct immune intervention is unlikely. However, quantitative PCR of conventional mice infected with C. rodentium revealed that stressor-exposed mice had altered levels of commensal microbes, and probiotic treatment caused these populations to rebound. Thus, stressor exposure exacerbated infection-induced dysbiosis and colonic inflammation. It is proposed that probiotic treatment prevents stressor-induced exacerbation of colonic inflammation by preventing dysbiosis.
IL-1α is critical for leukocyte recruitment during pulmonary Aspergillus fumigatus infection (MPF1P.776)
Authors: Caffrey Alayna , Lehmann Margaret , Shepardson Kelly , Watschke Christopher , Hilmer Kimberly , Zickovich Julianne , Barker Bridget , Cramer Robert , Obar Joshua
Published: January 2023

Aspergillus fumigatus is a mold that causes severe pulmonary infections. Currently, our knowledge of how A. fumigatus growth is controlled in the respiratory tract is limited. Alveolar macrophages and the airway epithelial cells constitute the first lines of defense against inhaled A. fumigatus conidia; subsequently, neutrophils and macrophages are sequentially recruited to the respiratory tract to control fungal growth and germination. How neutrophils and macrophages are recruited to the respiratory tract after A. fumigatus infection remains ill defined. A. fumigatus instillation induced biphasic expression of the inflammasome-dependent cytokines IL-1β and IL-18 during the first 48 hours, while IL-1α expression linearly grew over the same period. Il1r1-deficient mice are highly susceptible to pulmonary A. fumigatus infection. Enhanced susceptibility of Il1r1-deficient mice correlated with defects in leukocyte recruitment and anti-fungal activity. Interestingly, IL-1α rather than IL-1β was crucial for optimal leukocyte recruitment. IL-1α signaling enhanced the production of CXCL1, which was responsible for pulmonary leukocyte recruitment. In contrast, the inflammasome and IL-1β was only essential for optimal activation of anti-fungal activity of leukocytes. As such, Pycard-deficient mice were only mildly susceptible to A. fumigatus infection. Taken together, our data reveal central, non-redundant roles for IL-1α and IL-1β in controlling A. fumigatus infection in the lung.
Basic leucine zipper transcription factor, ATF-like (BATF) is essential for the development of protective immunity against parasitic helminth infection (MPF1P.779)
Authors: Bao Katherine , Reinhardt Richard
Published: January 2023

Parasitic helminth infections afflict roughly a quarter of the world’s population. Immunity against parasitic helminth infection relies on the induction of allergic inflammation. Major hallmarks of allergic inflammation, such as IgE, eosinophilia, and mucus production, depend on the cytokines interleukin-4 (IL-4) and IL-13. In mice, IL-4 from CD4+ follicular T helper (Tfh) cells is required for IgE production, while IL-13 from CD4+ T-helper 2 (Th2) cells is required for worm clearance and mucus production. The basic leucine zipper transcription factor, ATF-like (BATF) is critical for Tfh function, however its necessity in Th2 cell development and IL-4 production remains unclear. To investigate the role of BATF in the context of allergic inflammation, we infected wild-type and BATF-deficient mice with the helminth Nippostrongylus brasiliensis to compare the resultant immune responses. In wild-type mice, N. brasiliensis infection induced a robust response as indicated by lung eosinophilia, high serum IgE, and worm expulsion. In contrast, BATF-deficient mice exhibited dramatically reduced eosinophilia, no IgE, and were unable to clear parasites from the intestine. Studies with IL-4-reporter mice revealed that these defects in allergic immunity were attributed to impaired Tfh and Th2 cell development. These findings suggest that BATF plays a central role in protection against helminths, and BATF is critical for the development of both Th2 and Tfh cells during allergic responses.
Interleukin-3-deficient mice have increased resistance to blood-stage malaria (MPF1P.764)
Authors: Auclair Sarah , Roth Kenneth , Saunders Bryan , Ogborn Kathryn , Sheikh Abdalla , Naples Julianne , Young Anna Marie , Boisen Dorottya , Tavangar Amelia , Welch Jane , Lantz Chris
Published: January 2023

The contribution of interleukin-3 (IL-3), a hematopoietic growth factor and immunoregulatory cytokine, to resistance to blood-stage malaria was investigated by infecting IL-3 deficient (knockout [KO]) mice with Plasmodium berghei NK65. Male IL-3 KO mice, but not female mice, were more resistant to infection than wild-type (WT) mice, evidenced by lower peak parasitemia and prolonged survival. Both male and female IL-3 KO mice had increased splenomegaly and were more anemic than corresponding WT mice. Anemia was compensated for by an increase in bone marrow and splenic erythropoiesis in IL-3 KO mice, as evidenced by higher levels of erythroid progenitors. Plasma levels of gamma interferon (IFN-γ) and CXCL9 (Monokine Induced by IFN-γ; [MIG]) were found to be significantly reduced in IL-3 KO mice during early stages of infection whereas G-CSF levels were significantly greater than in WT counterparts. Overall, our results indicate that IL-3 plays a critical role in suppressing protective immunity to P. berghei NK65 infection and that it is involved in inhibiting the development of splenomegaly, anemia, and erythropoiesis. IL-3 also influences IFN-γ, CXCL9, and G-CSF production in response to infection. The abnormal responses seen in infected IL-3 KO mice may be due to the lack of IL-3 during development, to the lack of IL-3 in the infected mature mice, or to both.
Influenza infection suppresses NADPH oxidase-dependent phagocytic bacterial clearance and enhances susceptibility to secondary MRSA infection (MPF2P.811)
Authors: Sun Keer , Metzger Dennis
Published: January 2023

Methicillin-resistant S. aureus (MRSA) has emerged as a leading contributor to mortality during recent influenza pandemics. The mechanism for this influenza-induced susceptibility to secondary S. aureus infection is poorly understood. Here we show that innate antibacterial immunity was significantly suppressed during the recovery stage of influenza infection, despite the fact that MRSA super-infection had no significant effect on viral burdens. Compared to mice infected with bacteria alone, post-influenza MRSA infected mice exhibited impaired bacterial clearance, which was not due to defective phagocyte recruitment, but rather coincided with reduced intracellular reactive oxygen species (ROS) levels in alveolar macrophages and neutrophils. NADPH oxidase is responsible for ROS production during phagocytic bacterial killing, a process also known as oxidative burst. gp91phox-containing NADPH oxidase activity in macrophages and neutrophils is essential for optimal bacterial clearance during respiratory MRSA infections. In contrast to WT animals, gp91phox-/- mice exhibited similar defects in MRSA clearance before and after influenza infection. Using gp91phox+/- mosaic mice, we further demonstrate that influenza infection inhibits a cell-intrinsic contribution of NADPH oxidase to phagocyte bactericidal activity. Together, our results establish that influenza infection suppresses NADPH oxidase-dependent bacterial clearance and leads to susceptibility to secondary MRSA infection.
Increased CD4 and CD206 expression in a rich environment of IL-4 and IFN-γ in fatal cases of coccidiodomycosis (MPF2P.813)
Authors: Rosas-Taraco Adrian , Rodriguez-Ramirez Heidi , Barboza-Quintana Oralia , Ceceñas-Falcon Luis , Gonzalez Gloria , Salinas-Carmona Mario
Published: January 2023

Coccidioidomycosis is a fungal disease caused by Coccidioides immitis or Coccidioides posadasii. Little is known about the immunological environment in human lung of fatal cases. The aim of this study was to analyze inflammatory and anti-inflammatory cytokine expression (IL-4, IL-10, IL-17, and IFN-γ), as well as, CD4 (T helper, Th), CD8 (T cytotoxic, Tc), FOXP3 (Regulatory T cell), and CD206 (alternatively activated macrophage, AAM) markers in human lung tissue from fatal cases of coccidioidomycosis. A total of five biological samples from fatal cases and five controls were included in this study. Paraffin embedded blocks were cut in sections and prepared for immunohistochemistry (IHC) for each cytokine or cell marker. No differences were found in IL-10 and IL-17 expression, however, high levels of IL-4 and IFN-γ were found in lung of fatal cases of coccidioidomycosis (P<0.05). CD4+ and CD206+ cell markers were elevated in coccidioidomycosis samples when compared with control (P<0.05). In conclusion, our data suggest that AAM/Th2 and Th1 environment co-exists in fatal cases of coccidioidomycosis.
Chlamydia muridarum used in the mouse model of C. trachomatis, contains multiple variants that lead to differential immune responses and disease development. (MPF2P.807)
Authors: Sullivan Jeanne , Frazer Lauren , Darville Toni , O'Connell Catherine
Published: January 2023

C. muridarum Nigg is commonly used in the mouse model of C. trachomatis genital tract infection. Plaque-purified isolates derived from a C. muridarum Nigg stock were compared in the murine model of genital tract infection. C57BL/6 mice were intravaginally infected with strains CM001, CM002, CM005, CM006, CM012, or the parental AR Nigg and differences in infection course and disease outcome were observed. We identified CM001 as hyper-virulent because infection course in the lower tract was extended, bacterial load was elevated (P=0.02) and we observed increased frequency of hydrosalpinx (P<0.01) and oviduct dilatation scores when compared to AR Nigg. CM012 was attenuated, with reduced lower genital tract course of infection (P=0.05), decreased pyosalpinx early, and decreased hydrosalpinx (P<0.001) and oviduct dilatation (P<0.01) at resolution. Infection with CM002, CM005, and CM006 yielded similar bacterial burden and oviduct pathology to the parental stock. Inoculation of immune deficient mice revealed that infection with CM001 resulted in the death of rag1-/- mice in contrast to both CM012 and AR Nigg, which established chronic infections. Furthermore, CM001 caused enhanced oviduct pathology in tlr2-/- mice compared with AR Nigg while CM012 infection resulted in even milder pathology than the parent. Importantly, differences between chlamydial strains used for animal modeling of human disease should be considered as a cause for observed differences in disease phenotype.
Vaccine mediates protection against penicilliosis in BALB/c mice (MPF2P.810)
Authors: Yang Fengjuan , Yuen Kwok-Yung , Lau Susanna KP , Woo Patrick CY
Published: January 2023

Dimorphic pathogenic fungus Penicillium marneffei can cause penicilliosis which is a common infectious disease in AIDS patients in Southeast Asia and southern parts of China. Mp1p is a highly antigenic cell wall mannoprotein encoded by MP1 gene of P. marneffei and it is a pivotal factor for the pathogen infection and survival. Recombinant protein Mp1p produced in E.coli and Pichia pastors was used as an antigen vaccine against penicilliosis. While administered with different adjuvants and injection routes to BALB/c mice, this vaccine protein combination with Sigma Adjuvant System by intraperitoneal injection elicited a strong immunoglobulin G antibody response and conferred protection against a lethal P. marneffei challenge. These results showed that protein Mp1p could protect BALB/c mice in a dose, adjuvant and injection dependent manner. Protein vaccine Mp1p induced resistance was assessed in terms of survival, fungal growth (CFU) and histopathological examination. Immunised BALB/c mice could be protected against P. marneffei with 100% survival. CFU and histopathological analysis implied that fungal growth was systemically restricted at target organ of vaccinated mice, especially in liver where there was a significant lower value of CFU than non-vaccinated mice. Our results support the further development of an effective vaccine against penicilliosis.
CCR10 regulates intestinal immune homeostasis through regulating both Treg and Teff cells in the intestine (MUC2P.832)
Authors: Hu Shaomin , Yang Jie , Xiong Na
Published: January 2023

Intestinal immune homeostasis is an active process of balanced tolerant responses towards the gut-resident commensals and other food-born stimulations. Failure of the balanced response could lead to inflammatory diseases and cancers. Understanding molecular mechanisms that differentially regulate the homeostatic vs. inflammatory activation of T cells are fundamentally important in the intestinal immunity. CCR10, through interaction with its mucosa-specific ligand CCL28, was previously found important in regulation of IgA antibody-producing B cells in intestines. On the other hand, CCR10 was suggested to be a skin- but not gut- homing molecule for T cells. Using a novel strain of CCR10-knockout/EGFP-knockin mouse, we found that CCR10 is specifically induced on activating T cells in healthy intestines in situ and is critically important in balanced maintenance and functions of Treg and Teff cells to promote the intestinal homeostasis. On the other hand, during intestinal immune activations associated with infection or other inflammatory stimulations, the induction of CCR10 on activated intestinal T cells were suppressed that allows the T cell activation along different lineages. Our findings represent the first demonstration that a chemokine receptor is specifically associated with the homeostatic status of the intestine and reveal that there exists an auto-promoting feedback circuit of intestinal homeostasis in which the CCR10/ligand axis are a critical molecular link.
Janus-faced immunity to an enteric pathogen: how Campylobacter mediates colitis and autoimmunity (MUC2P.820)
Authors: Malik Ankit , Gadsden Barbie , Sharma Deepika , A. Dybas Leslie , Mansfield Linda
Published: January 2023

Campylobacter jejuni is a leading cause of foodborne enteritis and has also been linked to the autoimmune neuropathy, Guillain Barré syndrome. IL-10−/− mice serve as C. jejuni colitis model, but a mouse model for GBS is lacking. Immune mechanisms responsible for C. jejuni mediated colitis, protection from colitis or initiation of autoimmune sequelae are also largely unknown. We demonstrate that IL-10−/− mice infected with colitogenic human isolates of C. jejuni had significantly upregulated Type1/17 but not Type2 cytokines in the colon coincident with infiltration of phagocytes, T cells and Innate Lymphoid Cells. Both ILCs and T cells participated in IFN-γ, IL-17 and IL-22 upregulation but in a time- and organ-specific manner. However, T cells were necessary for colitis. Furthermore, depleting IFN-γ, IL-17 or both significantly ameliorated colitis and drove the colonic responses towards Type2 cytokine and antibody induction. In contrast, C. jejuni GBS patient strains induced mild colitis associated with blunted Type 1/17 but enhanced Type2 responses. Moreover, IL-4 and CD4+ T cell dependent Type2 antibodies (but not the Type1/17 antibodies) elicited exclusively by the GBS strains cross-reacted with peripheral nerve antigens. This lead to macrophage mediated demyelination of sciatic nerves that manifested with gait ataxia and hind limb paraparesis in the IL-10-/- mice. Therefore, this is the first mouse model of an autoimmune disease induced directly by an enteric bacterium.
A next-gen pipeline for generation, error correction and annotation of high-throughput immunosequencing data (TECH1P.842)
Authors: Emerson Ryan , Sherwood Anna , DeWitt William , Howie Bryan , Rieder Mark , Robins Harlan
Published: January 2023

Immunosequencing is emerging as a new technology with substantial scientific and clinical relevance in immunology, hematology and oncology. Thanks to the highly-randomized somatic rearrangement of antigen receptor genes in lymphocytes, each clonal population of lymphocytes bears a receptor rearrangement that is essentially unique. Thus, sequencing of rearranged antigen receptors in B and T cells can provide a thorough characterization of the diversity and clonal structure of the adaptive immune system. Despite the significant promise of immunosequencing, accurate and consistent methods for data generation and cleaning have proven elusive in practice. Outstanding issues include biased amplification of rearranged antigen receptor genes; computational correction of PCR and sequencing errors in raw sequencing data; alignment of receptor sequences to germline gene receptor loci for accurate reconstruction of rearrangement events; and methods for extrapolation from sequencing reads attributed to each unique rearrangement to absolute input cell numbers. Using a set of robust experiments in which carefully-controlled conditions and synthetic molecules are utilized to precisely determine correct output, we have developed an experimental paradigm and data analysis pipeline that address these concerns and set a new standard for accuracy and data quality in the field of immunosequencing.
Annotation of pseudogenous gene segments by high-throughput sequencing of rearranged lymphocyte receptor loci (TECH1P.844)
Authors: Sherwood Anna , Emerson Ryan , Williamson David , Chung Moon-Wook , Robins Harlan , Rieder Mark , Carlson Christopher
Published: January 2023

The adaptive immune system generates a huge breadth of antigen-specific T (TCRs) and B cell receptors (BCRs) by combinatoric shuffling of gene segments. Lymphocytes undergo somatic recombination of variable (V), diversity (D), and joining (J) region gene segments, with deletions and non-templated insertions at the junctions, collectively encoding the CDR3 region. Many of the gene segments are annotated as non-functional; however, full annotation of genes as functional, pseudogene or ORF has proven elusive. We used high-throughput sequencing to identify 100K’s of rearranged TCRB and IGH receptors from a large cohort of healthy adults. We then annotated V and J gene segments as functional or pseudogenes based on selection. Random chance predicts that less than one-third of somatic rearrangements will lead to in-frame transcripts, but only lymphocytes with a productive receptor are selected. Non-functional rearrangements (out-of-frame or pseudogene) persist only as a second allele in mature T and B cells. Based on these data we annotated the functional status of each gene segment based on the realized ratio of productive to non-productive rearrangements. Some gene segments are currently mis-annotated: in TCRB 2 presumed-functional gene segments are pseudogenes, in IgH 3 presumed-functional genes are pseudogenes and 1 annotated pseudogene is functional. Additionally, in both loci we identified cases of gene segment functional/pseudogene status segregating among healthy subjects.
Improved GAA activity in tregitope-treated pompe disease: preclinical studies (TECH1P.864)
Authors: Cousens Leslie , Koeberl Dwight , Mingozzi Federico , Terry Frances , Martin Bill , De Groot Anne
Published: January 2023

“Tregitopes” are immunoglobulin-derived regulatory T-cell (Treg) epitopes that lead to suppression of inflammation and, when administered with a target antigen, adaptive tolerance. One potential application of Tregitopes is mitigating immune responses to therapeutic proteins, manifest when a protein appears foreign either because it is derived from a different species or because patients express a genetic deletion or modification. In Pompe disease, genetic deficiencies in acid alpha-glucosidase (GAA) result in lysosomal glycogen accumulation throughout the body. Life-saving treatment with fully human recombinant GAA can trigger high titer anti-drug-antibodies (ADA) that interfere with drug efficacy. We report here our efforts to apply Tregitopes to provide GAA-specific tolerance induction. Therapeutic GAA protein was expressed in GAA-deficient mice by administering AAV-GAA under conditions known to induce ADA +/- Tregitopes, also delivered by AAV expression vectors. Tregitope administration inhibited ADA formation compared to vehicle-treated controls. In addition, a significant increase in GAA activity was observed for Tregitope, consistent with Tregitope inhibition of glycogen buildup in muscle. Current experiments are designed to refine the Tregitope treatment regimen to achieve clinically-relevant ADA inhibition and to evaluate cellular immune responses towards the goal of providing effective and lasting Tregitope-mediated tolerance to GAA for Pompe patients.