bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional and structural basis of T cell recognition in autoimmune gastritis: mapping of autoantigenic peptides of the α-chain of the H/K ATPase and X-ray structure of the I-Ad/peptide complexes (106.25)
Authors: Revilleza Maria Jamela , Levin Ditza , Mage Michael , Candon Sophie , Natarajan Kannan , Rui Wang , Teyton Luc , Robinson Howard , Shevach Ethan , Margulies David
Published: January 2023

We have previously reported the development of mouse models of autoimmune gastritis in which BALB/c mice expressing transgenic αβ TCR, designated A23 and A51, spontaneously develop acute Th1-mediated high-penetrance and chronic Th2-mediated low penetrance forms of the disease, respectively. Using an array of synthetic mutated peptides in functional and MHC II-binding assays, we now report the core peptides recognized by each of these TCR. A23 recognizes H/K ATPase residues 633-641 (AKAIAASVG) and A51 sees 892-900 (CVGLRPQWE), both exhibiting a canonical 1, 4, 6, 9 I-Ad -binding motif. To extend these findings, we determined the X-ray crystallographic structure of complexes of I-Ad with both covalent and non-covalent peptides that stimulate the A51 TCR, and used these structures to generate a molecular model of the A23-stimulating peptide/I-Ad complex. The structural identification of peptide anchor residues in individual I-Ad binding pockets and of solvent exposed side chains as T cell epitopic residues readily explains T cell proliferation data and I-Ad/peptide off-rate measurements of the large panel of truncated, chimeric, and alanine or glycine-substituted synthetic peptides. The first structure of I-Ad in complex with an autoimmune peptide offers new insight in the general question of the structural basis of responses to self antigens.
Crystal structure of the HLA-DM / DO complex and insight into the mechanism of DM-catalyzed peptide exchange. (106.28)
Authors: Stern Lawrence , Guce Abigail , Mortimer Sarah , Yoon Taejin , Painter Corrie , Jiang Wei , Mellins Elizabeth
Published: January 2023

Peptide loading onto class II MHC molecules, facilitated by the non-classical MHC II protein HLA-DM, is a key step in antigen presentation. The mechanism of HLA-DM action and its modulation by HLA-DO, another non-classical MHC II protein, are unknown. In this work we investigated that mechanism using kinetics, mutagenesis, and structural approaches. In the crystal structure, HLA-DM and HLA-DO bind in a side-by-side arrangement. HLA-DO adopts an MHCII-like structure with conformational alterations near the alpha subunit 3-10 helix. Kinetic analysis demonstrates that HLA-DO acts as a competitive inhibitor of HLA-DM, and analysis of the functional effects of a panel of HLA-DM mutants indicates that HLA-DO and MHC bind to the same site on HLA-DM. These results suggest a molecular mechanism for HLA-DM action, in which DM binding to MHC II facilitates peptide release by disrupting key MHC-peptide interactions and stabilizing a peptide-receptive conformation. Both peptide-MHCII H-bonding and P1 pocket interactions are disrupted.
A peptide/MHCII conformer generated in the presence of exchange peptide is the substrate for HLA-DM editing (106.30)
Authors: Ferrante Andrea , Gorski Jack
Published: January 2023

The mechanism of HLA-DM (DM) activity is still unclear. We have shown that peptide release from HLA-DR (DR) in the presence of DM is dependent on the presence of an exchange peptide. However, DM also promotes a small but measurable peptide release in the absence of exchange peptide. Here we show that non-denaturing SDS-PAGE separates purified peptide/DR1 (pDR1) complexes into two conformers whose ratio is dependent on peptide KD. In the absence of an exchange peptide, DM only releases peptide from the slower migrating conformer. Upon the addition of an exchange peptide, a portion of the DM-resistant conformer can be converted to the slower migrating conformer, which in turn is DM labile. Our results indicate that exchange peptide generates a conformer of the pDR1 complex, which constitutes the intermediate for peptide exchange and is also the substrate for DM activity. The resolution of the intermediate favors the peptide with the greatest ability to fold itself and the groove into a stable, low-energy complex. However, once folded into the DM-resistant conformer, even a low affinity peptide can be presented in the absence of free peptide, increasing the peptide repertoire available for presentation.
Equal Immune Rights For All Polypeptides (106.38)
Authors: Shastri Nilabh , Chen Keling
Published: January 2023

Effective immune surveillance requires that MHC class I molecules display a peptide repertoire on the surface representing all cellular proteins. How the peptide repertoire can be comprehensive despite large differences in abundance and stability of individual proteins is not known. We show here that peptide presentation by MHC I molecules is strongly influenced by the pioneer round of translation (PRT) associated with nonsense mediated decay (NMD) of mRNAs. The PRT allows cells to detect and eliminate aberrant mRNAs containing premature stop codons. Inhibition of PRT by knock-down of CBP80, a unique cap-binding protein reduced peptide presentation by MHC class I molecules on the cell surface without affecting global protein synthesis. On the other hand, introduction of premature stop codons changed mRNA stability without affecting peptide presentation by MHC I on the cell surface. Thus, the pioneer round of translation could be used to derive peptides presentation by MHC I molecules to generate a comprehensive display of virtually all endogenous proteins.
Neutrophils Control the Extent and Spread of the Immune Response in a Prostaglandin Mediated Process (106.46)
Authors: Yang ChiaoWen , Unanue Emil
Published: January 2023

We have studied the influence of neutrophils on immune responses to protein antigens in adjuvants. Neutrophils enter lymph nodes shortly after immunization starting within the first hour and continuing for several days. By using neutrophil depleting Abs or genetically neutropenic mice we found suppressive effects of neutrophils on CD4 T cell as well as Ab responses. One of the mechanisms by which neutrophils modulated the extent of T cell response was by reducing the amount of antigen available to the APC. Neutrophil-DC interaction also affected the interaction of DC with T cells. We now found that neutrophils control the spread of T cell responses to distant lymph nodes. In the presence of neutrophils ~75% of the response is restricted to the draining node but in their absence most of the response is found in distal nodes. A critical component is the production of prostaglandin made by the entering neutrophils. Prostaglandin deficient mice or in vivo inhibition of prostaglandin production by indomethacin in the presence of neutrophil entry showed unrestricted response similar to neutropenic mice. Adoptive transfer of neutrophils treated with indomethacin or transferring neutrophils from prostaglandin deficient mice into genetically neutropenic recipients supported a role for neutrophil derived prostaglandins in restricting T cell responses in popliteal nodes. In brief neutrophils modulate the extent and the spread of T cell responses in a prostaglandin mediated process.
Initiation of TCR Phosphorylation and Signal Transduction. (106.50)
Authors: Casas Javier , Brzostek Joanna , Hoerter John , Gascoigne Nicholas
Published: January 2023

Recognition of antigen by the T cell receptor (TCR) is the key event for the T cell activation. The earliest molecular events in T cell recognition have not yet been fully described, and the TCR triggering mechanism remains a subject of controversy. By using supported lipid bilayers to present peptide MHC class I complexes to CD8+ cells, we can monitor the very early molecular events occurring at the immune synapse. The lipid bilayer technology mimics the signaling environment for T cell activation as determined by Ca2+ flux, total phospho-Tyr levels, and IL-2 secretion. Using TIRF/FRET microscopy we have observed an early (> 1 min) interaction between CD3ζ and the coreceptor CD8 that is independent of the MHC-CD8 binding, but requires CD8 association with Lck. Later (< 10 min) CD3ζ-CD8 interactions require CD8-MHC binding. This suggests that, upon antigen recognition, TCR may be initially phosphorylated by Lck not associated with coreceptor, followed by MHC dependent recruitment of CD8-Lck complexes. We are currently working on elucidating the proposed different roles of unbound and coreceptor associated Lck during the early stages of antigen recognition.
human metapneumovirus attachment protein G targets RIG-I to inhibit innate immune responses (108.11)
Authors: Bao Xiaoyong , Kolli Deepthi , Liu Tianshuang , Garofalo Roberto , Casola Antonella
Published: January 2023

hMPV is a recently identified paramyxovirus. It is associated with respiratory illnesses in children, adults, and immunocompromised patients. Evidence suggests that the virus may also cause repeated infection throughout life. Recent investigations in our lab identified hMPV attachment glycoprotein G as an important virulence factor, as a recombinant hMPV with G protein deleted (rhMPV-ΔG) exhibited enhanced production of important immune and antiviral mediators in airway epithelial cells as well as in primary immune cells. In this study we have identified RIG-I, a major intracellular viral RNA sensor, as the target of hMPV inhibition of cellular signaling in airway epithelial cells. G protein interacts with RIG-I and prevents its recruitment to mitochondria and interaction with the adaptor protein MAVS, necessary to activate the downstream signaling pathways leading to activation of cytokine and chemokine gene expression. Site-directed mutagenesis indicates that hMPV G cytoplasmic domain is required for the inhibitory activity, as a rhMPV in which Glu 2 or Val 3 of G is substituted with Ala recapitulates the phenotype observed with G protein deleted mutant. As rhMPV-ΔG is under consideration as potential vaccine candidate, it would be important to have a mutant virus which has no or reduced inhibitory activity and contains the fewest changes, while retaining full G protein expression, which could be important for generation of fully protective immune responses.
Asymmetric division of germinal center B cells (109.1)
Authors: Barnett Burton , Ciocca Maria , Goenka Radhika , Barnett Lisa , Wu Junmin , Laufer Terri , Burkhardt Janis , Cancro Michael , Reiner Steven
Published: January 2023

B cells that encode high-affinity, protective antibodies are generated in the germinal center (GC) reaction, a microanatomical structure that includes GC B cells and follicular helper T cells (TFH). The selection of GC B cells to proliferate and differentiate into plasma cells and memory B cells relies on contacts with TFH. In other instances where cells undergo external contacts, polarity cues are imparted that lead to asymmetric division. We hypothesized TFH may provide polarity cues during these interactions, in addition to mitogenic and differentiative signals, so that GC B cells may divide asymmetrically to generate diversity. Using confocal microscopy we observe that GC B cells asymmetrically segregate and unequally inherit the ancestral polarity regulator PKCζ, and the receptor for interleukin 21 (IL-21R) and Bcl6, which are responsible for initiating and maintaining the GC B cell fate, respectively. Homeostatic proliferation does not involve asymmetric divisions, while ICAM-1 and CD40 both contribute to asymmetric division of B cells in vivo and in vitro, respectively. In the absence of asymmetric division, we observe a failure to generate antibody secreting cells from GCs. Together, these data support a model where, in addition to canonical signals, GC B cells receive polarity cues from TFH that result in the unequal inheritance of fate determinants by daughter B cells, leading to divergent differentiation.
Heterogeneity of the bone marrow plasma cell pool (109.14)
Authors: Chernova Irene , Bortnick Alexandra , Allman David
Published: January 2023

Long-lived plasma cells (PCs) are responsible for maintaining antibody titers and are believed to populate unique survival niches in the bone marrow (BM). Current models predict that BM PCs consist chiefly of long-lived, slowly renewing cells. However, we find that more than 50% of BM PCs exhibit characteristics of recently formed PCs. These characteristics include surface expression of the canonical naïve B cell surface proteins B220 and CD19, and a 50% renewal rate of less than 3 days. Surprisingly, despite the rapid turnover rate exhibited by B220+ BM PCs, antigen-induced antibody secreting cells are found within this population for more than 100 days post-immunization. Together these data offer new insights into the cellular basis of antibody titer maintenance, and suggest that BM niches are continuously repopulated by newly generated plasma cells well after antigenic exposure.
Direct detection of IgE-switched B cells in an IgE-GFP reporter mouse reveals a germinal center pathway for the generation of IgE memory B cells and plasma cells (109.3)
Authors: Talay Oezcan , Yan Donghong , Brightbill Hans , Straney Elizabeth , Zhou Meijuan , Ladi Ena , Lee Wyne , Xu Min , Egen Jackson , Austin Cary , Wu Lawren
Published: January 2023

IgE antibodies are pathogenic in asthma and allergic diseases, but the in vivo biology of IgE cells is poorly understood, largely due to their low frequency and a lack of good reagents to detect them specifically. It has been proposed that IgE cells develop through a germinal center IgG1 intermediate and that IgE memory resides in the IgG1 memory B cell compartment. Here we have utilized a recently generated IgE-GFP reporter mouse that enables the direct detection of IgE-switched cells to assess in vivo IgE responses. In contrast to the IgG1-centered model of IgE switching and memory, we find that IgE cells develop through a germinal center IgE intermediate to form IgE memory B cells and plasma cells. Moreover, IgE memory B cells, as opposed to IgG1 memory B cells, constitute cellular IgE memory and respond quickly upon rechallenge to produce serum IgE. Our studies provide a new model for the in vivo biology of IgE switching and memory.
A novel role for blood endothelial cells in the development and maintenance of immune memory (110.17)
Authors: Swift Stephanie , Bassett Jennifer , Bramson Jonathan
Published: January 2023

Vaccination with recombinant human adenovirus serotype 5 (rHuAd5) drives a CD8+ T cell response that is dominated by effector and effector memory cells. In investigating the immunobiological mechanisms that result in this effector-skewed response, we previously showed that non-haematopoietic cells play a key role in defining the rHuAd5-elicited CD8+ T cell memory population. Furthermore, maintenance of this memory population relies upon sustained transgene expression from the adenovirus vector. While we know that following vaccination rHuAd5 drains to local lymph nodes, long-term antigen persistence occurs outside this location, since surgical removal of these nodes does not impact the nature of the memory response. Here, we show that following intramuscular vaccination, the adenovirus transgene is predominantly detected in the perinodal adipose tissue, in addition to draining lymph nodes and the muscle injection site itself. In all three locations, blood endothelial cells are the primary cell type that express the adenovirus-encoded antigen, strongly indicating that this radio-resistant cell population may have a role in presenting antigen to CD8+ T cells and ultimately in defining the memory response. Long-term antigen presentation from this cell compartment may represent a mechanism to sustain CD8+ T cell memory within the circulation and to ensure adequate protective immunity is maintained in multiple organs.
Blimp-1 regulates CD8 T cell effector/memory development via regulation of cytokine receptor expression (110.20)
Authors: Shin Hyun Mu , Kapoor Varun , Welsh Raymond , Berg Leslie
Published: January 2023

During infections, the pathogen-induced cytokine milieu has been shown to determine T cell fate decisions between effector cells and memory precursor cells. However, how changes in cytokine receptor expression contribute to this process is poorly understood. Blimp-1 is known to be an important transcriptional repressor involved in the differentiation of short-lived CD8+ effector T cells following LCMV infection. Using a genome-wide ChIP-sequencing technique, we found that Blimp-1 directly controls cytokine receptor expression during the contraction phase of CD8+ T cells. During the expansion phase at day 5 post-infection (p.i.), CD8+ T cells are highly responsive to several cytokines, whereas at day 7 or 9 p.i. their responses are significantly decreased. In contrast, Blimp-1-/- CD8+ T cells maintain their cytokine responsiveness, and have increased cytokine receptor expression and increased formation of memory CD8+ T cells. Furthermore, Blimp-1 recruits histone modifying enzymes, HDAC2 and G9a to the genomic loci of cytokine receptors IL-2Rα and CD27. This study defines the mechanism by which Blimp-1 association with histone modifying enzymes controls cytokine receptor expression during the expansion and contraction phases of CD8+ T cells following LCMV infection, and thereby contributes to the generation of CD8+ effector and memory T cells.
Enhanced CD8 T cell expansion and protective immunity during VSV infection by agonistic anti-CD40 is dependent on CD70 and accessory signals (110.7)
Authors: Zickovich Julianne , Yagita Hideo , Obar Joshua
Published: January 2023

Listeria monocytogenes and VSV infection result in the generation of large memory CD8 T cell populations that are capable of rapid secondary expansion. However, only memory cells induced by LM are protective against secondary infection. Optimal activation, expansion, and programing of CD8 T cells are dependent on at least three signals: 1) antigenic stimuation, 2) co-stimulation, and 3) cytokines. Here we found that treatment with agonistic anti-CD40 during VSV infection enhances CD8 T cell expansion and protective immunity. Enhancement of the CD8 T cells by agonistic anti-CD40 treatment is dependent on CD70 signaling. However, treatment with agonistic anti-CD27 did not enhance CD8 T cell expansion or protective immunity, suggesting a role for accessory signals working in conjugation with CD70. Agonistic anti-CD40 treatment induced IL-12p70 and IL-6 in a CD70-dependent manner. Blockade of IL-12p40, but not IL-6, after agonistic anti-CD40 treatment impaired CD8 T cell expansion. In conclusion, our data demonstrate that enhancing signaling through CD40 during VSV infection enhances CD8 T cell expansion in a CD70 and IL-12p40-dependent manner. Thus, understanding how the CD40 signaling pathway regulates the generation of highly protective memory CD8 T cells will provide key insights into memory CD8 T cell biology and vaccine development.
p21 and Paf as targets of E47 in regulating hematopoietic stem cell (HSC) function (111.10)
Authors: Santos Patricia , Borghesi Lisa
Published: January 2023

HSCs replenish all blood lineages maintaining a functional immune system throughout adult life. It remains unclear how E47, a major transcriptional regulator of stem cell integrity, controls HSC function and maintenance. Here, we focus on p21 and Paf, two candidate E47 targets known to be important for HSC proliferation. E47 deficiency results in downregulation of p21 in uncommitted progenitors. Using compound heterozgyotes to examine the in vivo interaction of E47 and p21, we show an increase in BrdU+LSKs in E47hetp21het mice. More importantly, ~15% of E47hetp21het LT-HSCs are BrdU+ compared to ~10% LT-HSCs from WT or single HET controls. Consistent with the increase in proliferation, uncommitted progenitors from these mice are more sensitive to 5-FU treatment. At 16 weeks after adoptive transfer, E47hetp21het BM recipients had lower frequency of peripheral blood lymphocytes suggesting a partial defect in lymphopoiesis. In addition to p21, we show that Paf, a 15kDa intracellular protein expressed mostly in proliferating cells, is a direct target of E47. We observed a 2-fold increase in Paf mRNA levels in E47-/- flk2- and flk2+ LSKs compared to E47 WT mice, and a 1.5-fold increase in Paf mRNA levels in E47-/- LT-HSCs consistent with the increased proliferation of uncommitted progenitors observed in these mice. Together, these data define a molecular pathway in which E47 can differentially regulate the expression of both p21 and Paf to control HSC proliferation.
Fate Mapping Mononuclear Phagocytes using the CX3CR1 promoter (111.13)
Authors: Kim Kiwook , Yona Simon , Wolf Yochai , Mildner Alexander , Jung Steffen
Published: January 2023

The mononuclear phagocyte system comprises monocytes, dendritic cells (DCs) and macrophages. Collectively these cells play central roles in the maintenance of tissue integrity during development and after injury, as well as the initiation and resolution of innate and adaptive immunity. The chemokine receptor CX3CR1 is widely expressed in the mononuclear phagocyte system. Indeed mice harboring a GFP reporter in the CX3CR1 locus have been instrumental in identifying and defining clonotypic lineage precursors, dedicated classical DC precursors, as well as tissue-resident mononuclear phagocyte populations. Moreover, discrete expression levels of CX3CR1/GFP in CX3CR1gfp animals led to the identification of two monocyte subsets in mice as CX3CR1int Ly6C+ and CX3CR1hi Ly6C- cells. Here, we report a fate mapping study of the murine mononuclear phagocyte compartment using mice that harbour conditional and constitutive active Cre recombinase genes in their CX3CR1 loci. Our data obtained by side-by-side comparison of CX3CR1Cre and CX3CR1CreERT2 mice, as well as CX3CR1gfp mice suggest the existence of three distinct pathways yielding peripheral mononuclear phagocyte populations in the adult organism: (1) macrophages seeded by primitive macrophages and maintaining themselves through longevity and limited self-renewal; (2) macrophages seeded during embryonic development from definitive precursors and (3) macrophages that are continuously renewed from blood monocytes.
Zinc Finger Protein 521 (ZFP521) is required for the development of mast cells in mice (111.16)
Authors: Lukin Kara , Lei Hong , Possada Jessica , Rodriguez Valerie , Straign Desiree , Roers Axel , Warming Søren , Hagman James
Published: January 2023

Mast cells play crucial roles in both innate and adaptive immune responses. Evidence in our laboratory suggests that the ZFP521 (Evi3/ZNF521/EHZF) transcription factor orchestrates key pathways required for proper development and function of mast cells. ZFP521 is a nuclear protein that contains 30 zinc fingers. ZFP521 is important for the renewal and development of hematopoietic cells. Here, we demonstrate that ZFP521 is expressed highly in murine mast cells. To address the intrinsic roles of ZFP521 in mast cells, we developed a model system in which Zfp521 gene expression is ablated conditionally in mast cells of mice with floxed alleles (Zfp521 cKO mice) by mast cell-specific Cre (Mcpt5-Cre transgene). Zfp521 is required for normal numbers of mast cells in the peritoneal cavity (PMC). In part, the reduction of PMC may be due to the reduced expression of CD117 (c-Kit) on Zfp521 cKO-derived mast cells. Both CD117 and its ligand, Stem Cell Factor (SCF), are essential for mast cell development in the bone marrow. Additionally, the expression of several transcription factors, including GATA-1, GATA-2 and GATA-3, which regulate mast cell development and function is reduced in bone marrow-derived Zfp521 cKO mast cells. Together, our data provide insights into 1) a novel role for ZFP521 in hematopoiesis, and 2) the transcriptional regulatory network that governs the development and function of mast cells.
IFN-gamma is the primary cause of aplastic anemia not autoreactive T cells (111.2)
Authors: Lin Fanching , Macfarlane Michael , Saleh Bahara , Hodge Deborah , Young Howard
Published: January 2023

Aplastic anemia (AA) is characterized by hypocellular marrow and peripheral pancytopenia. Although cases of AA caused by environmental factors have been reported, the etiology of AA is largely unknown. Since IFN-γ and T-bet can be detected in AA patients' T cells, it was believed that autoreactive T lymphocytes played a major role in destroying the hematopoietic stem cells (HSCs) in bone marrow (BM). Thus, AA has been treated as an autoimmune disease. We have observed AA-like symptoms in our IFN-γ AU-rich element (ARE) knock-out (KO) mice. These mice have a targeted deletion in the 3'untranslated region of the IFN-γ mRNA. The deletion increases the half-life of IFN-γ mRNA. Thus, these mice constitutively express low level of IFN-γ under normal condition. In this study, we used IFN-γ ARE KO mice as an animal model to understand the etiology of AA. These mice exhibited signs of immunodeficiency when their immune organs were examined. Also, T cells from these mice failed to produce cytokines when stimulated with anti-CD3 and anti-CD28 antibodies. Upon examining the cell population in BM, we did not detect any infiltration of T cells. In addition, we observed an increase in long term HSCs (LT-HSCs) and a decrease in short term HSCs (ST-HSC). We also observed the same phenomena in WT recipients of IFN-γ ARE KO BM. The results of this study suggest AA occurs when IFN-γ inhibits the generation of ST-HSCs from LT-HSCs, as opposed to infiltration of autoreactive T cells.
DNA damage in hematopoietic stem cells as a mechanism for lymphoid progenitor exhaustion in E47 deficient mice (111.20)
Authors: Cruz J. Agustin , Borghesi Lisa
Published: January 2023

Aging and chronic infection are known to cause decreases in lymphopoiesis and in early hematopoietic progenitors, ultimately manifesting as poor responsiveness to new pathogens and reduced efficacy of vaccination. However, the mechanisms involved in these manifestations are still not completely understood. E47 is a transcription factor that regulates the proliferative integrity of early hematopoietic progenitors, and chronic TLR stimulation decreases the levels of E47 in mice. Additionally, E47 KO mice develop an exhaustion of lymphoid progenitors as they age. Consequently, we hypothesize that the responsive pathway to the aging process of the immune system involves a decreased expression of E47, and increases in both cell cycling and reactive oxygen species exposure, which lead to an increase of DNA damage in hematopoietic progenitors and subsequent exhaustion. In order to test our hypothesis, we assessed the DNA damage repair response in E47 KO mice using flow cytometry by measuring the expression level of the γ-pH2A.X. Although we found normal numbers of phenotypic HSCs under steady-state homeostasis, the γ-pH2A.X response in E47 null mice is increased respect to the E47 WT control group. This is the first evidence showing that deficiency of E47 leads to increased DNA damage response in HSCs and other early progenitors subset. Further investigations on the role of E47 in the response to DNA damage may provide novel insights into aging and exhaustion of HSCs.
Molecular targets of Notch signaling, HES1 and c-Myc, oppose a PTEN-dependent check on survival, differentiation and proliferation of TCR-beta-selected thymocytes (111.21)
Authors: Wong Gladys , Zuniga-Pflucker Juan Carlos , Knowles Gisele , Mak Tak , Ferrando Adolfo
Published: January 2023

The developmental progression of immature thymocytes requires cooperative input from several pathways. Notch signals playing an indispensible role at the T cell receptor (TCR)-beta selection checkpoint, by activating the PI3K/Akt pathway, which is required for pT-alpha/TCR-beta (preTCR)-induced survival, differentiation and proliferation of developing alphabeta-lineage thymocytes. However, the molecular players responsible for this interaction between the Notch and PI3K pathways are unknown. Here, we show that Notch induction of Hes1 is necessary to repress the PI3K/Akt pathway inhibitor, PTEN, which in turn facilitates preTCR-induced differentiation. In support of this mechanism, deletion or down-regulation of Pten overcomes the Notch signaling requirement for survival and differentiation during beta-selection. Additionally, c-Myc is a critical target of Notch at this stage, as c-Myc expression overcomes the Notch signaling requirement for proliferation during beta-selection. Collectively, our results point to HES1, via repression of PTEN, and c-Myc as critical mediators of Notch function at the beta-selection checkpoint.
Adipocytes inhibit B lymphopoiesis at the common lymphoid progenitor (CLP) to pre-proB cell stage (111.30)
Authors: Bilwani Fareena , Knight Katherine
Published: January 2023

Like in humans, B lymphopoiesis declines in rabbits with age, and we previously showed that adult rabbit BM stroma is unable to support generation of proB cells. We investigated changes in BM stroma of adult rabbits and found an increase in adipocytes similar to that in aged humans. We also found that both rabbit and human adipocytes secrete a soluble factor(s) that inhibits B lymphopoiesis. We investigated the stage at which the inhibitory molecule blocks B lymphopoiesis by co-culturing cord blood-derived human HSC and OP9 stromal cells with rabbit adipocyte-conditioned media (CM) and found that the inhibitory factor does not impair the generation of CLP but inhibits generation of pre-proB cells. We tested if the inhibitory molecule acts via stromal cells by treating OP9 stromal cells with adipocyte CM and then co-culturing them with rabbit BM mononuclear cells (MNC). The capacity of pre-treated OP9 stromal cells to support B lymphopoiesis was unchanged. In contrast, treatment of BM MNC of adult rabbit with the adipocyte CM and subsequent co-culture with OP9 stromal cells without adipocyte CM, showed that pre-treated cells were unable to differentiate into proB cells. We conclude that the adipocyte-derived soluble factor acts on early lymphoid precursors to inhibit B lymphopoiesis from the CLP to pre-proB cell stage. Such inhibition likely contributes to the decline of B lymphopoiesis in aged rabbits and humans and can be exploited to enhance immunity in elderly.
Notch ligand Jagged-1 negatively regulates dendritic cell differentiation during emergency myelopoiesis by inhibiting Wingless signaling (111.4)
Authors: Liu Hao , Zhou Jie , Cheng Pingyan , Gabrilovich Dmitry
Published: January 2023

It has been known that the terminal differentiation of dendritic cells (DCs) takes place outside bone marrow (BM), although accumulation of DC precursors occurs in BM. However, cell signaling regulating this process, remains poorly understood. We demonstrated that this process could be differentially regulated by Notch ligands: Jagged-1 (Jag1) and Delta-like ligand 1 (Dll1). In contrast to Dll1, Jag1 in vitro and during emergency myelopoiesis in vivo prevented DC differentiation by promoting accumulation of their precursors. Although both ligands activated Notch in hematopoietic progenitor cells (HPC), they had an opposite effect on Wnt signaling. Dll1 activated Wnt pathways; whereas, Jag1 inhibited it via down-regulation of the expression of Wnt receptors Frizzled (Fzd). Jag1 suppressed fzd expression by retaining histone deacetylase 1 in the complex with the transcription factor CSL/CBF-1 on fzd promoter. Our results suggest that DC differentiation, during emergency myelopoiesis, can be regulated by the nature of the Notch ligand expressed on adjacent stroma cells.
Genome-wide genetic and transcriptome analyses of early T-cell developmental checkpoint failure and lymphocytic leukemia initiation in Rag1-deficient non-obese diabetic (NOD) mice (111.40)
Authors: Yui Mary , Feng Ni , Zhang Jingli , Liaw Chen Yee , Rothenberg Ellen , Longmate Jeffrey
Published: January 2023

All T-lineages arise from multipotent progenitors that proliferate and differentiate under the influence of the thymic environment. We previously reported defects around the earliest T-cell developmental checkpoints in both Rag1-deficient and wild type NOD mice. To investigate the genetic and molecular source of the NOD early T-cell defects and possible linkage to T-cell mediated autoimmunity, we carried out genome-wide genetic and transcriptome studies on the checkpoint violation in NOD.Rag1-/- mice. In a QTL analysis, the checkpoint defect mapped to the diabetes susceptibility Idd9/11 region, which was confirmed using congenic mice. Transcriptome analysis, comparing Rag1-deficient NOD and B6 cells, showed a significant enrichment of signal transduction genes among those differentially expressed, implicating signaling in the checkpoint defect. Furthermore, emerging NOD.Rag1-/- breakthrough cells aberrantly co-expressed legacy stem/progenitor genes, including Lmo2 and Kit, which are normally extinguished at the earliest T-cell commitment checkpoint, along with post-β-selection T-cell genes, including Cd4 and Cd5. The gene expression profile resembles early subtypes of human acute T-cell lymphoblastic leukemia (T-ALL), suggesting that the breakthrough cells are the source of T-cell lymphoma/leukemia found in these mice. Our results show that the earliest checkpoints are defective in NOD T-progenitors, with possible consequences for both T-cell leukemia and autoimmunity.
Tcf-1 protects thymocytes from malignant transformation. (111.43)
Authors: Dose Marei , Konstantinou Tassos , Zhang Jiangwen , Aghajani Katayoun , Le Beau Michelle , Rosen Steven , Georgopoulos Katia , Gounari Fotini
Published: January 2023

The DNA binding protein Tcf-1 is a gatekeeper of lymphocyte fate, yet its molecular functions remain largely elusive. Tcf factors are mediators of Wnt/ β-catenin signaling, a pathway that is linked with many genomically unstable cancers. Here we provide evidence that Tcf-1 may play a crucial role in protecting the genome from damage and genomic instability. This is particularly important during TCR rearrangement when developing T-cells introduce damage to their DNA as a consequence of Rag activity. Interestingly, the Tcf-1 binding pattern on DNA as determined by ChIP-seq is highly correlated with that of Rag2. We further show that Tcf-1 binds several components of the DNA repair and remodeling machineries. Importantly, β-catenin outcompetes some of these interactions, with fatal consequences, as overt activation of β-catenin in murine thymocytes (CD4Cre;Ctnnb1ex3) causes Rag-dependent lymphomas that carry recurring TCRα ;Myc translocations. CD4Cre; Ctnnb1ex3 thymocytes also display increased chromatin accessibility (H3K4me3 and H3K9/14Ac), a risk-factor for translocations. In summary, our data point to novel roles for components of the Wnt/ β-catenin signaling pathway in defining chromatin structure, recombination and repair in general, and for the generation of recurring t(14;15)(C2;D1) translocations in particular, with implications for human malignancies in which analogous translocations have been described.
The role of p38 MAPK/GSK3β signaling in T and B lymphocytes undergoing programmed DNA recombination (111.47)
Authors: Thornton Tina , Rincon Mercedes
Published: January 2023

In addition to environmentally induced DNA double strand breaks (DSBs), developmentally programmed DSBs are also generated in T and B cells during V(D)J recombination. We have shown that in response to DSBs, p38 MAPK is activated and translocated to the nucleus. Although typically associated with cell death, we found that p38 MAPK promoted survival of cells following DNA damage by phosphorylating GSK3β at a novel residue (S389). GSK3β is a constitutively active kinase that can promote death by phosphorylating and targeting survival factors for destruction. GSK3β has been shown to be inhibited by the phosphorylation of S9 by Akt. Our biochemical analysis showed that phosphorylation of GSK3β at S389 resulted in inhibition of GSK3β similar to the S9 phosphorylation of GSK3β. Here we show that in response to DSBs in the development of T and B cells, activation of p38 MAPK leads to S389 phosphorylation and inactivation of GSK3β. We also show that unlike S9 phosphorylation that occurs in the cytosol, S389 phosphorylation occurs within the nucleus. To investigate the role of this pathway in vivo, we generated knockin mice (KI) where the S389 has been mutated to alanine. Lymphocytes for our KI mice appear to be more sensitive to death in response to DSBs. Our data indicate that GSK3β inactivation by p38 MAPK is selectively induced by DSBs as a mechanism to inhibit GSK3β activity within the nucleus allowing for the accumulation of survival factors.
TAK1-dependent checkpoint in dendritic cell survival promotes immune homeostasis and function (111.53)
Authors: Huang Gonghua , Wang Yanyan , Chi Hongbo
Published: January 2023

Homeostatic control of dendritic cell (DC) survival is crucial for adaptive immunity, but the molecular mechanism is not well defined. Moreover, how DCs influence immune homeostasis under steady state remains unclear. Combining DC-specific and inducible deletion systems, we report that the kinase TAK1 is an essential regulator of DC survival and immune system homeostasis and function. Deficiency of TAK1 in CD11c+ cells induced markedly elevated apoptosis, leading to the depletion of DC populations, especially the CD8+ and CD103+ DC subsets in lymphoid and non-lymphoid tissues, respectively. TAK1 also contributed to DC development by promoting the generation of DC precursors. Pro-survival signals from Toll-like receptors, CD40 and RANK are integrated by TAK1 in DCs, which in turn mediated activation of downstream NFκ-B and AKT-Foxo pathways and established a gene-expression program. TAK1 deficiency in DCs caused a myeloid proliferative disorder characterized by expansion of neutrophils and inflammatory monocytes, disrupted T cell homeostasis, and prevented effective T cell priming and generation of regulatory T cells. Moreover, TAK1 signaling in DCs was required to prevent myeloid proliferation even in the absence of lymphocytes, indicating a previously unappreciated regulatory mechanism of DC-mediated control of myeloid cell-dependent inflammation. Therefore, TAK1 orchestrates a pro-survival checkpoint in DCs that affects the homeostasis and function of the immune system.
GM-CSF induces podoplanin expression during dendritic cell differentiation (111.56)
Authors: Zhang Hong , Lin Xiao , Li Sky , Pahuja Anil , Ransom John , Yang Xifeng
Published: January 2023

Podoplanin is a mucin-type transmembrane glycoprotein containing extensive Oglycosylation in extracellular domain and two phosphorylation sites in a short cytoplasmic tail. It is expressed in kidney glomerular epithelial cells, lymphatic endothelium and subcapsular epithelial cells in murine thymus. Recently, podoplanin was found on murine F4/80 positive peritoneal macrophages and human follicular dendritic cells (FDCs) indicating that podoplanin is involved in immune response. We have reported that podoplanin is induced during Th9 and Th17 polarization. Here, we studied podoplanin expression on different types of murine myeloid cells. We found that podoplanin was not detectable on circulating and bone marrow derived neutrophils, monocytes, basophils and esinophils, its expression level on neutrophil and monocytes was not inducible followed by fMLP or TNF-a stimulation. Podoplanin was also not detectable on spleen and lymph node resided CD11b+macrophages, CD11c+ dendritic cells (cDCs) and CD8+ plasmacytoid dendritic cells (pDCs). However, podoplanin expression level has been dramatically increased on GM-CSF driven bone marrow-derived dendritic cells. These data suggest that podoplanin can be induced during GM-CSF driven dendritic cell differentiation, indicating that podoplanin may be a transient marker during dendritic cells differentiation and maturation. The mechanism of GM-CSF induced podoplanin and it significance need to be further investigated.
Interleukin-8 promoter polymorphism rs4073 is not associated with its expression level or tuberculosis susceptibility in China. (112.17)
Authors: Li Chuanyou , Che Nanying
Published: January 2023

Interleukin-8 (IL-8) is an important chemokine eliciting host immune response against tuberculosis (TB). However, whether IL-8 gene polymorphism is associated with TB susceptibility and its biological function remains to be answered. Ma et al. reported that rs4073 polymorphism in IL-8 promoter region was associated with TB susceptibility in the two ethnic groups in the United States. On the contrary, Cooke et al. reported that this polymorphism was not a true factor affecting TB susceptibility in Gambia. So in this study, the association between rs4073 polymorphism and TB susceptibility was analyzed on 433 TB patients and 505 healthy controls in China. Allelic and genotypic analysis showed that rs4073 polymorphism distribution between TB patients and healthy controls was not significantly different (p>0.05). The effect of IL-8 rs4073 polymorphism on IL-8 expression level was also determined. ELISA analysis showed that IL-8 expression level in the peripheral blood among different genotypic doners (A/A, A/T, T/T) was not significantly different (p>0.05). These results demonstrate that rs4073 polymorphism is not associated with TB susceptibility or with IL-8 expression level in Chinese population. Genetic susceptibility to TB in different ethnic groups needs to be studied carefully.
Functional role of Thioredoxin peroxidase from Clonorchis sinensis in host immune reactions (112.3)
Authors: Wi Hae Joo , Jin Yan , Bae Young Mee
Published: January 2023

Clonorchis sinensis (C. sinensis), one of liver flukes, can cause chronic infection while they reside in the bile duct and leads to cholangiocarcinoma in humans. We investigated interaction between live C. sinensis worm and murine macrophage cell line, RAW264.7, using a non contact co-culture transwell plate. The results shown that the worms are able to activate RAW cells to express surface TLR4 and to promote IL-10 and TGF-β secretion. There are minor differences between direct and indirect exposure of the worms. As we hypothesized that anti-oxidant enzyme produced by C. sinensis might stimulate host immune reactions, we cloned antioxidant enzyme, Thioredoxin peroxidase (TPX, 2-Cys peroxidase), and the recombinant protein (rTPX) has been produced by using the bacterial system. Mouse macrophage cells, RAW264.7 cells, were treated with rTPX and checked the level of cell surface markers. We found that expression of TLR4 and CD40 molecules were augmented at 3 and 6 hours time points, respectively. Surface expression of TLR4 was prolonged up to 48 hrs, while CD40 was temporarily expressed. ELISA results showed that IL-4 level was slightly down-regulated by TPX treatment, however, IL-10 secretion was increased and even doubled when IL-4 and TPX were treated together. Although, more than one molecule could be involved in antigenicity of C. sinensis, we report here that the TPX from C. sinensis is a candidate that acts as “infection alarm” to awake the host innate immune cells.
C-reactive protein-mediated initial protection of mice from pneumococcal infection (112.4)
Authors: Gang Toh , Hammond David , Singh Sanjay , Ferguson Donald , Agrawal Alok
Published: January 2023

Human C-reactive protein (CRP) protects mice from lethal Streptococcus pneumoniae infection when injected into mice 6 h before to 2 h after the administration of pneumococci. Because CRP binds to phosphocholine, it has been proposed that the protection begins with the binding of CRP to phosphocholine residues present on pneumococcal C-polysaccharide. To test this proposal, in this study, we utilized a CRP mutant incapable of binding to phosphocholine. Based on the crystal structure of CRP-phosphocholine complexes which showed that amino acids Phe66, Thr76 and Glu81 form the phosphocholine-binding site of CRP, we constructed a CRP triple mutant F66A-T76Y-E81A. We purified the mutant CRP by affinity chromatography on a phosphoethanolamine-conjugated Sepharose column. Compared to wild-type CRP, the mutant CRP bound more avidly to phosphoethanolamine but did not bind to phosphocholine-BSA, pneumococcal C-polysaccharide and whole pneumococci. In C57BL/6 mice, the half-life of the mutant CRP was the same as that of wild-type CRP. Unlike wild-type CRP, passively administered mutant CRP did not protect mice from lethal pneumococcal infection. Mice injected with mutant CRP had higher mortality rates than mice that received wild-type CRP. Decreased survival was due to the increased bacteremia in mice treated with the mutant CRP. We conclude that the phosphocholine-binding function of CRP is required for CRP-mediated initial protection of mice from pneumococcal infection.
Identification of “Asymptomatic” HLA-A*0201-Restricted CD8+ Cytotoxic T-Lymphocyte Epitopes from the Herpes Simplex Virus Glycoprotein B (113.13)
Authors: Benmohamed Lbachir , Chentoufi Aziz , Dervillez Xavier , Dasgupta Gargi , Kabbara Khaled , Villacres Maria , Nguyen Chelsea , Wechsler Steven , Sidney John , Nesburn Anthony
Published: January 2023

Protective “asymptomatic” human CD8+ T-cell epitopes from herpes simplex virus type 1 and type 2 (HSV1 & HSV-2) protein antigens remain largely undefined. Here, we have identified immunodominant protective "asymptomatic" human CD8+ T cell epitopes on the HSV-1 gB that are restricted to HLA-A*0201, an haplotype that covers over 50% of the human population. The 904-amino acid HSV-1 (strain 17) gB sequence was screened for potential CD8+ T cell epitopes. Ten epitopes were identified, synthesized and tested for affinity to HLA-A*0201 molecule. Two of these peptide epitopes, gB441-449 and gB561-569, exhibited high affinity for soluble HLA-A*0201 molecules. Peptide-specific CD8+ T cell proliferation (CFSE dilution) and CD107a/b cytotoxic degranulation were demonstrated against the same two epitopes in 30 sequentially studied HLA-A*0201-positive, HSV-1 seropositive asymptomatic individuals (i.e. during control of herpetic disease), but not in symptomatic individuals (i.e. with frequent herpetic disease). A heterologous lipopeptide/rAdv5 prime/boost vaccine expressing these two asymptomatic gB CD8+ epitopes induced a strong and long lasting CD8+ T-cell protective immunity against ocular and genital herpes in a susceptible HLA-A*0201 transgenic mice. Human protective asymptomatic CD8+ T-cell epitopes on HSV-1 proteins should continue to provide new insight into immune correlates of protection and aid in the development of a safe and efficient immunotherapeutic vaccine for herpes.
Development of goose monoclonal IgY antibodies for use as a passive therapeutic. (113.6)
Authors: Haese Nicole , Bradley David
Published: January 2023

When vaccination is not cost effective or possible, one of the primary alternatives is passive immunotherapies. Passive therapeutic antibodies have been shown to be effective in the treatment, not only of infectious agents, but also for toxins, venoms, and cancer targets. Recent research in our laboratory as well as others, has demonstrated the potential of avian-derived passive therapeutic antibodies. We have demonstrated goose-derived polyclonal antibodies, from either sera or eggs, are therapeutic for several viral diseases. The avian immunoglobulin IgY is the primary avian antibody isotype. IgY is similar to mammalian IgG, but lacks the ability to interact with mammalian Fc receptors, complement, and other factors that promote inflammation. IgY’s potential as a therapeutic agent is boosted by its higher avidity than IgG and recognizes more epitopes than mammalian antibodies. These advantages are due to the evolutionary distance between mammalian and avian species. In this work we present initial studies to develop goose mIgY, utilizing traditional mammalian monoclonal protocol that will be commercialized against viral or other targets responsive to single epitope treatment, with the product displaying the inherent advantages of avian antibodies.