bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of hematopoietic stem cell quiescence by TXNIP (36.1)
Authors: Choi Inpyo , Jeong Mira , Yoon Suk Ran
Published: January 2023

Hematopoietic stem cells (HSCs) are distinct cells that are predominantly located in the bone marrow microenvironment, which is referred to as the niche. HSC quiescence, activation, and mobilization are coordinately regulated in their niche by intrinsic and extrinsic mechanisms. Several genes, including those that encode transcriptional regulators, cell cycle regulatory proteins, tumor suppressors, and proto-oncogenes, intrinsically regulate the balance between HSC quiescence and activation. The vitamin D3 up-regulated protein 1 (VDUP1) is a 397 amino acid residue, 50-kDa protein that belongs to the arrestin family. We hypothesized that VDUP1 plays a pivotal role in controlling HSC cell cycle, migration, and BM niche interactions. The expression of VDUP1 is decreased during HSC activation. In VDUP1-/- mice, the LT-HSC population is decreased and exhausted, and its capacity to repopulate is rapidly lost. The VDUP1 deficiency reduced the osteopontin/integrin β1-mediated interaction between HSCs and the bone marrow niche and impaired homing and retention in the osteoblastic niche, resulting in mobilized HSCs. Thus, we propose that VDUP1 is essential for maintaining HSC quiescence and interactions with the BM niche.
Analysis of human immunoglobulin gene repertoire in NOD/scid IL2Rγ null mice engrafted with human cord blood derived hematopoietic stem cells (36.14)
Authors: Chang Hong , Biswas Subhabrata , Tallarico Aimee , Sarkis Phuong , Marasco Wayne
Published: January 2023

The humanized NOD/scid IL2rg-/- (hNSG) and similar mouse models hold promise as testing platforms for preclinical studies. Here, we analyzed the human immunoglobulin (IG) variable heavy and light chain gene repertoire from sorted single B-cells of hNSG mice engrafted with cord blood derived hematopoietic stem cells. Unique IGHV (339) and IGLV (364, kappa and lambda) gene segments were derived from major B cell subsets identified in a largely normal B cell development pathway. However, the hNSG mice expressed elevated CD5+ mature B-1-like cells in the periphery. In mice and humans, B-1 cells are the major source of autoantibodies. Although the hNSG mice expressed a highly diverse repertoire comparable to humans, detailed genetic analysis of IG gene segments from the mature B cell subset revealed the presence of long IGH-CDR3 regions with positive charges—a hallmark for auto/polyreactivity. For functional tests, several in vitro expressed scFv-Fc IG candidates, derived from cloned heavy and light chain cognate pairs of single B cells, tested positive in an anti-nuclear antibody assay for auto/polyreactivity and/or bound to HIVgp120 in ELISA assays. The natural induction during HIV-1 infection of broadly neutralizing antibodies (BnAbs) which display autoantibody-like characteristics may be curtailed by the checkpoint control mechanisms during B cell ontogeny. We propose the hNSG mouse can be a model platform to test this hypothesis and interrogate how BnAbs are formed.
SHP2 protein-tyrosine phosphatase is a key regulator of mast cell signaling and development in mice (36.22)
Authors: Sharma Namit , Everingham Stephanie , Feng Gen-Sheng , Roers Axel , Craig Andrew
Published: January 2023

SH2 domain-containing phosphatase-2 (SHP2; also called PTPN11) is a positive effector of growth signaling pathways downstream of receptor tyrosine kinases and immune receptors. The development of conditional knockout mouse models for SHP2 has resulted in a better understanding of the role of SHP2 in adult tissues and cell types. In bone marrow-derived mast cells, recent studies have implicated SHP2 in functioning as a positive effector of growth signaling via Kit receptor tyrosine kinase, and TNFα production via the high affinity IgE receptor. In this study, we describe the use of transgenic mice expressing Cre recombinase in connective tissue mast cells (CTMCs) to generate a selective SHP2 knockout in CTMCs (MC-SHP2 KO). Compared to control mice lacking Cre, MC-SHP2 KO mice display a drastic reduction in mast cells within the peritoneum and skin. Previous studies have demonstrated the critical importance of Kit Y567 and Y719 signaling to Src family kinases/Gab2 and phosphatidylinositol 3’ kinase (PI3K) pathways for development of peritoneal and skin mast cells in mice. Thus, our results suggest that SHP2 plays a key role in signaling via the Src/Gab2/PI3K axis downstream of Kit during mast cell development in mice. In vitro differentiation assays for CTMCs are being investigated to further explore the molecular mechanisms by which SHP2 regulates mast cell development.
Identification of an IL-7 Responsive CD4+ Lymphoid Tissue Inducer Cell from Adult Human Blood (36.24)
Authors: Bekiaris Vasileios , Greenberg Brian , Sedy John , Ware Carl
Published: January 2023

In mice, the lymphoid tissue inducer (LTi) cell forms a distinct lineage of hematopoietic cells required for the development of lymphoid tissues, and in the adult may be important for regeneration of lymphoid tissues following infection. LTi cells are rare and constitute a phenotypically mixed population of cells that may differentiate into NK-like cells, which has hindered their identification and characterization. Here, we isolated cells from adult human blood with phenotype and functional responses conserved with mouse CD4+ LTi cells. Human LTi-like cells expressed CD4 and IL-7 receptor-α, Rorc and Tnfsf8, signature markers for mouse CD4+ LTi cells, but lacked lineage markers for T and B lymphocytes, NK and myeloid cells. A culture system that allowed survival of human and mouse LTi cells was independent of IL-7, yet human LTi cells retained responsiveness to IL-7 by induction of lymphotoxin (LT)-α and LTβ, the key signaling system for lymphoid organogenesis. Human CD4+ LTi cells, like T cells, expressed the lymphoid tissue homing chemokine receptor, Ccr7, and the coreceptor for HIV, CXCR4. The identification of adult human CD4+ LTi cells should expand the investigation of these cells in human immunobiology and pathogenesis.
What goes up must come down: The consequences of not turning down pre-TCR induced β-catenin (36.29)
Authors: Sharma Archna , Yu Qing , Misra Sen Jyoti
Published: January 2023

Pre-T cell receptor (pre-TCR) signals regulate β-selection during the maturation of CD4- CD8- double negative (DN) thymocytes to the CD4+ CD8+ double positive (DP) stage. T-cell specific deletion of β-catenin, a central mediator of Wnt- β-catenin-TCF/LEF signaling pathway, impairs T cell development at the β-selection checkpoint. We demonstrate that pre-TCR signals specifically stabilize β-catenin in DN thymocytes during β-selection. Pre-TCR induced β-catenin regulates expression of IL-7 receptor (IL-7R), early growth response (Egr) and Ror-γ-T genes that are essential for traversal through β-selection checkpoint. However, expression of pre-TCR induced β-catenin must be transient and must return to background levels for efficient transition to the DP stage. Sustained expression of β-catenin results in the development of IL-7R and Egr expressing pre-DP thymocytes that fail to up-regulate the expression of Ror-γ-T, which is essential for development to the DP stage. The stalled DN cells, expressing β-catenin, exhibit DNA damage, growth arrest, oncogene-induced-senescence (OIS) and p53 dependent apoptosis. In p53-deficient mice, expression of β-catenin still results in DNA damage and OIS, but the thymocytes survive and eventually progress to thymic lymphoma. We suggest that oncogenic β-catenin is a part of normal developmental signaling event but is prevented from transforming developing thymocytes by p53-independent growth arrest and OIS and p53-dependent apoptosis.
Dkk1 mediated inhibition of Wnt Signaling in postnatal mice leads to loss of TEC progenitors and thymic degeneration (36.32)
Authors: Pezzano Mark , Osada Masako , Jardine Logan , Misir Ruth , Andl Thomas , Millar Sarah
Published: January 2023

The molecular mechanisms, which control both the initial development and subsequent maintenance of TEC microenvironments, are poorly defined. Regulation of Wnt signaling has been shown to impact both early thymocyte and thymic epithelial development. Early blocks in thymic organogenesis or death of the mice have prevented analysis of a role of Wnt signaling in the maintenance of TECs in the postnatal thymus. Tetracycline-regulated expression of the Wnt inhibitor DKK1 in cTECs and mTECs of adult mice, results in rapid thymic degeneration characterized by a reduced Foxn1 expression, loss of K5K8DP putative TEC progenitors, decreased proliferation and the development of cystic structures, similar to an aged thymus. Removal of DKK1 from DKK1-involuted mice results in full recovery, suggesting that canonical Wnt signaling is required for the differentiation or proliferation of TEC populations needed for maintenance of properly organized adult thymic epithelial microenvironments. The results of this study demonstrate that canonical Wnt signaling within TECs is required for the maintenance of epithelial microenvironments in the postnatal thymus, possibly through effects on TEC progenitor/stem cell populations. Downstream targets of Wnt signaling, which are responsible for maintenance of these TEC progenitors may provide useful targets for therapies aimed at counteracting age associated thymic involution or the premature thymic degeneration associated bone marrow transplants.
Impaired thymic selection and abnormal antigen-specific T cell responses in Foxn1Δ/Δ mutant mice (36.36)
Authors: Xiao Shiyun , Manley Nancy
Published: January 2023

Foxn1Δ/Δ mutant mice have a specific defect in thymic development, characterized by a block in TEC differentiation at an intermediate progenitor stage, and blocks in thymocyte development at both the DN1 and DP cell stages, resulting in the production of abnormally functioning T cells that develop from a atypical progenitor. To test the effects of deficient TEC on thymic selection, we used Foxn1Δ/Δ;DO11 Tg and Foxn1Δ/Δ;OT1 Tg mice as positive selection and Foxn1Δ/Δ;MHCII I-E mice as negative selection models. An in vivo system of antigen-specific reactivity was used to test the function of peripheral T cells. Our data show that the capacity for positive and negative selection was reduced in Foxn1Δ/Δ mutants compared to Foxn1+/Δ control mice. These defects were due in part to reduction of MHC I, II expression, although the resulting peripheral T cells have a broad TCR Vβ repertoire. In this deficient thymic environment, immature CD4 and CD8 SP thymocytes emigrate from the thymus into the periphery. These T cells had an incompletely activated profile under stimulation of the TCR signal in vitro, and were either hypersensitive or hyporesponsive to antigen-specific stimulation in vivo. These cell-autonomous defects were compounded by the hypocellular peripheral environment caused by low thymic output, showing that a primary defect in the thymic microenvironment can cause indirect effects on the periphery that can in turn exacerbate functional defects in T cells.
Spontaneous development of IL-17-producing γδ T cells in the thymus occurs via a TGFβ1-dependent mechanism (36.37)
Authors: Do Jeongsu , Fink Pamela , Li Lily , Spolski Rosanne , Robinson Janet , Leonard Warren , Letterio John , Min Booki
Published: January 2023

In naive animals γδ T cells are innate sources of IL17. However, mechanism(s) involved in the generation of these cells remains unexplored. We show here that some γδ T cells spontaneously differentiate into IL17+ cells in vivo and IL17-competent γδ T cells were preferentially found in the peripheral LN but not in the mesenteric LN. Ontogeny study revealed that γδ T cell IL17 differentiation mainly occurs in the thymus particularly during the postnatal period, e.g. prior to 7 days of age; ~40% of γδ TCR+ thymocytes in 5-day old mice expressed IL17 upon stimulation. IL17+ γδ TCR thymocytes were mainly found to be CD44highCD25low, ‘DN1 phenotype’ cells. Because γδ TCR is expressed after the DN2/3 stage of the thymic development pathway, this finding strongly suggest that IL17+ CD44highCD25low cells may be the DN4 γδ TCR+ thymocytes that have upregulated CD44 expression and acquired IL17 expression. In deed, in vitro culture of FACS sorted DN4 γδ TCR+ cells with OP9-DL4 stromal cells confirmed this possibility. Using gene-deficient mice, we also found that TGFβ is the primary factor mediating IL17 differentiation of γδ T cells in the thymus. However, other cytokines involved in Th17 differentiation including IL23, IL6, and IL21 played little or no roles as IL17+ γδ T cells were unaffected in these strains of mice. Taken together, our results identify a novel developmental pathway during which IL17-competent γδ T cells arise in the thymus by a TGFβ1-dependent mechanism
Dynamic regulation of microRNAs in the thymus during stress response (36.41)
Authors: Silge Robert , Belkaya Serkan , de la Morena Maite , van Oers Nicolai
Published: January 2023

Immune responses are dramatically affected by stress, with developing thymocytes undergoing marked atrophy following bacterial infections or steroid injections. MicroRNAs (miRs) have been implicated in stress responses in many different organ systems. We performed miR expression profiling to identify miRs affected by lipopolysaccharide (LPS) or dexamethasone induced thymic atropy. Both treatments caused significant reductions in overall thymic cellularity, with the CD4+CD8+ subset primarily affected. Seven and ten distinct miRs were significantly up- and down- regulated, respectively, in the thymic tissue, compared to controls. Synthetic mimics or inhibitors of several of these miRs, including miR-181a, differentially affected T cell development, as assessed in fetal thymic organ culture assays. These findings indicate that many thymic miRs are stress responsive and can influence the efficiency of T cell development.
The effect of forcibly expressed “immunoproteasome” subunit β5i in cortical thymic epithelial cells (36.46)
Authors: Xing Yan , Hogquist Kristin
Published: January 2023

Cortical thymic epithelial cells (cTECs) provide an essential microenvironment for the positive selection of MHC-restricted T cells. Data in recent years suggests that a novel proteasome subunit—β5t, exclusively expressed in cTECs in the thymus, plays a key role in generating the MHC Class I-restricted CD8+ T cell repertoire during thymic selection. In contrast, the “immunoproteasome” subunit β5i is normally expressed in dendritic cells and medullary thymic epithelial cells, and can be upregulated in other MHC Class I positive cells by IFNγ. To study how positive selection is regulated by a unique peptide repertoire expressed in cTEC, we generated K14-β5i transgenic (Tg) mice in which β5i was expressed in cTECs under the control of the human keratin 14 (K14) promoter. Forced expression of β5i in cTECs did not result in a reduced percentage or number of CD8 T cells. However, there was an increase in the percentage and number of CD44high “memory phenotype” CD8 T cells in the periphery of K14- β5i Tg mice, similar to that observed in β5t deficient mice. This suggests that the expression of β5i in cTECs could induce a distinct peptide repertoire and affect the selection of CD8 T cells in thymus. In addition, these mice exhibit hyperkeratosis of the skin. RAG-deficiency did not rescue the skin disease of K14-β5i Tg mice, indicating that it might be promoted by the over-expression of β5i in keratinocytes.
Importance of TCR ITAM multiplicity in the establishment of central tolerance (36.48)
Authors: Hwang SuJin , Song Ki-Duk , Chu Hamlet , Lee Jan , Lesourne Renaud , Li LiQi , Pinkhasov Julia , Jenkins Marc , Love Paul
Published: January 2023

Thymocyte selection is controlled by signaling motifs (ITAMs) contained within the invariant T cell receptor (TCR) subunits. The current model of thymocyte selection posits that differences in the affinity of the TCR for self-peptides results in differences in the TCR signaling response, with low affinity interactions promoting positive selection and high affinity interactions triggering negative selection. We and others previously demonstrated that attenuation of TCR signaling potential (by mutation of the zeta chain ITAMs) leads to the generation of T cells that express auto-reactive TCRs. However, these T cells did not cause autoimmune disease, presumably because TCR/self-peptide interactions do not induce strong effector responses in the absence of zeta ITAMs. This paradigm was recently challenged in a study reporting that retroviral reconstitution of zeta deficient bone marrow with ITAM mutant zeta chains results in fatal autoimmune disease. In the current study, we generated a knock-in (KI) mutation at the zeta locus that results in expression of a zeta chain where each of the ITAM tyrosines is mutated to phenylalanine (6YF). The phenotype of zeta-6YF mice demonstrates that attenuation of TCR signaling potential is not, by itself, sufficient to abrogate central tolerance. In addition, our results confirm that the threshold for T cell activation is determined by both the affinity of the TCR and by the TCR signaling potential
Age related dynamics of committed T cell progenitors in mice (36.57)
Authors: Peiris Tanuja , Ciriza Jesús , Al-Kuhlani Mufadhal , Graham Ashley , García-Ojeda Marcos
Published: January 2023

Immunosenescence is the reduced ability of the body to mediate an adaptive immune response. Multiple factors contribute to immunosenescence, including age-related changes in hematopoietic stem cell (HSC) phenotype, its niche, and the tissues responsible for lymphocyte development, such as the bone marrow and the thymus. Therefore, understanding lymphocyte development in the aged is fundamental to enhance the immune response capacity of the aging immune system. Here, we investigate the age-related dynamics of committed T cell progenitors (CTP), an HSC-derived bone marrow population capable of generating functional T cells via both thymic and extrathymic pathways. Specifically, we examined the CTP’s frequency and absolute numbers in the bone marrow of young (1 month) and old (12 and 18 months) C57BL/6 mice using flow cytometry. Moreover, we compared the capability of young and aged CTP to generate T cells in vitro. Understanding the biology of CTP in relationship to aging could have important clinical applications in the treatment of diseases related to immunodeficiencies and cancer by hematopoietic stem cell transplantation, as CTP-derived T cells protect against viral infections without inducing graft vs. host disease. This work could pave the way to studies related to human CTP populations and their therapeutic potential.
A simple and fast method for the isolation of mouse lymphoid progenitors from bone marrow (36.6)
Authors: Tabatabaei-Zavareh Nooshin , Fairhurst Maureen , Thomas Terry
Published: January 2023

Lymphocytes are derived from hematopoietic stem cells through an important developmental intermediate called a common lymphoid progenitor (CLP). CLPs are defined as Lin−IL-7Rα+c-KitloSca-1lo. They can differentiate into T, B, and natural killer (NK) cells but lack myeloid and erythroid potential. Study of lymphocyte development largely relies on access to this rare cell population. Fluorescence-activated cell sorting (FACS) commonly used to isolate CLPs is costly, time-consuming and perhaps most importantly detrimental to the cell viability and function. We describe a fast and efficient method for the isolation of CLPs from mouse bone marrow (BM). This method is based on immunomagnetic, column-free cell separation technology (EasySep). Using this method, lineage positive cells were first depleted by cross-linking them to magnetic particles using biotinylated antibodies. Next, IL-7Rα+ cells were positively selected from the Lin−/lo population. Purity of Lin−IL-7Rα+c-Kit+ lymphoid progenitors as assessed by flow cytometry ranged from 18-41%. Limiting dilution analysis of the EasySep enriched cells showed increased frequencies of B cell (1:25), T cell (1:6) and NK cell (1:49) progenitors as compared to non-depleted control BM. This system introduces a rapid and easy method for the enrichment of rare CLPs with greater yield than cell sorting and good viability. This will enable research in the field of cellular, molecular and developmental immunology.
TGF-beta is an essential factor for IL-9 production (36.60)
Authors: Wu Xiao-wei , Rabenstein Jacob , Sepulveda Homero , Wei Ai-Li , Hingorani Ravi , Ernst David , Rohrer Jurg
Published: January 2023

Interleukin-9 (IL-9) is a multifunctional cytokine involved in proliferation, differentiation, and effector functions of various cell types that include T cells, B cells, mast cells, eosinophiles neutrophils, and epithelial cells. In previous studies, Th2 cells were shown to produce IL-9. Recent studies suggest that IL-9 can also be produced by so-called Th9 cells. In this study, we report that TGF-beta is an essential factor for in vitro cultured human peripheral blood mononuclear cells (PBMC) into subsets that secrete IL-9. PBMC were activated in the presence of anti-CD3/CD28 antibodies and polarizing cytokines with or without the addition of TGF-beta. Using flow cytometry to detect both intracellular IL-9 expression and soluble IL-9 secretion, we found that IL-9 was only produced in the presence of TGF-beta. Addition of other cytokines, especially IL-4 further increased IL-9 production. Interestingly, a subset of CD4+IL-9+ T cells was seen to also express IL-17, but not IL-4, IL-10 or IL-12. Additional studies will address the plasticity between the Th9 and Th17 CD4 T cell subsets.
Three functional regions in human tonsil distinguished by in situ gene expression (37.1)
Authors: Zhao Zeng , Rabin Ronald
Published: January 2023

We stained tonsil sections for T cell, B cell, and DC markers and found that T cell zones could be subdivided according to distribution of CD303+ plasmacytoid DC into pDC rich and pDC poor regions; pDC rich regions surrounded the high endothelial venules and pDC poor regions were relatively well populated with CD1c+ myeloid DC. We then modified our immunuhistochemistry technique to preserve RNA integrity and used laser capture microdissection (LCM) to dissect three discrete target regions and characterize differential patterns of gene expression among them. Fifty of the 80 genes probed were differentially expressed among the three regions. Thirty genes in the pDC rich region represented T cell activation and thirteen genes in the pDC-poor region included T cell regulation. Not surprisingly, the seven genes most highly expressed in the GC were primarily related to B cell activation. Expression patterns of master transcription factors of adaptive immune function, IFN-g, and subtypes of types I and III IFN validated a model whereby T cells. Three distinctive regions and genomic functions in situ of tonsil were characterized corresponding to distribution of DC subtypes. The gene profile provides basis of immune responses in vivo for administration of surface vaccines.
A pathogenic role of macrophages in mice intradermally infected with methicillin-resistant Staphylococcus aureus (37.18)
Authors: Asai Akira , Tsuda Yasuhiro , Kobayashi Makiko , Herndon David , Suzuki Fujio
Published: January 2023

In this study, intradermal MRSA infection in burn mice was pathogenically analyzed. Burn mice (3rd degree, 25% TBSA flame burn) and normal mice were infected i.d. with 108 CFU/mouse of MRSA, and abscesses formed in the infection sites and mortality rates of these mice were examined. Also, the abscess formation was examined in burn and normal mice after inoculation with F4/80+Mϕ isolated from tissues surrounding the infection sites of normal (N-F4/80 Mϕ) or burn mice (B-F4/80 Mϕ). Finally, these Mϕ were biophysically characterized. In the results, the abscess was formed in normal mice infected with MRSA and all of them survived. However, the abscess was not formed in burn mice infected with MRSA, and all of them died. The abscess was formed and sepsis was not developed in burn mice inoculated with N-F4/80 Mϕ. However, the abscess was not formed and sepsis was developed in normal mice inoculated with B-F4/80 Mϕ. N-F4/80 Mϕ produced IL-12 (but not IL-10) and were characterized as M1Mϕ, whereas B-F4/80 Mϕ produced IL-10 (but not IL-12) and were characterized as M2Mϕ. These results indicate that the abscess formation is a major host defense against MRSA i.d. infection. M1Mϕ at tissues surrounding the infection site play a pivotal role in the abscess formation. An abscess is not formed in burn mice predominated with M2Mϕ, which are inhibitory on the M1Mϕ generation. This study was supported by SHC NA #8840. A.A was supported by The James W. McLaughlin Fellowship Fund.
Mannose-binding lectin deficiency increases susceptibility to influenza A virus infection. (37.20)
Authors: Takahashi Kazue , Chang Wei-Chuan , White Mitchell , Moyo Patience , McClear Sheree , Thiel Steffen , Hartshorn Kevan
Published: January 2023

Influenza A virus (IAV) infection can cause life-threatening condition even in individuals who are otherwise healthy. IAV infects the airway and the lung, where lung-surfactant protein-A (SP-A), SP-D and alveolar macrophages are thought to be primary innate immune protection. SP-A and SP-D belong to the collectin family that also includes a serum protein, mannose-binding lectin (MBL), that shares structural and functional similarities to lung collectins. MBL is a broad-spectrum pattern recognition molecule that plays a key role in first line host defense as an activator of the lectin complement pathway. In this study, we have found that MBL null mice have increased susceptibility to IAV infection and that recombinant MBL improves the infection. The anti-viral mechanisms of MBL include initiation of the lectin complement pathway as well as a thrombin-like activity that requires the cooperation of other serum factors. These results also demonstrate the ability of MBL to modulate leukocyte recruitment and inflammation in infected lungs. These findings reveal that MBL plays a key role in clearing IAV and maintaining lung homeostasis. In addition, our results suggest that MBL deficiency may be a risk factor in IAV infection and that MBL may be useful as a therapy against IAV infection.
Antibody can mediate protective immunity at early tissue-dwelling stages of intestinal helminth infection (37.27)
Authors: Bowdridge Scott , Liu Qian , Kreider Timothy , Liu Zhugong , Song Youngmia , Gaydo Andrew , Urban Joseph , Gause William
Published: January 2023

B cells may be critical in mediating protective immunity by aiding Th2 cell development and/or antibody production. To examine the role of B cells during helminth infection B cell-deficient mice were inoculated with one of two intestinal nematode parasites: Nippostrongylus brasiliensis (Nb) and Heligmosomoides polygyrus (Hp). A similar pattern in elevation of Th2 cytokine gene expression was seen in B cell-deficient and WT mice after inoculation with either species. No difference in worm expulsion was found between B cell-deficient and WT mice after Nb infection, but differences in worm expulsion occurred after Hp inoculation. At day 4 and day 7 after secondary Hp challenge impaired larval migration and development occurred in WT but not B cell-deficient mice suggesting that both early invasion and in situ larval development were inhibited through B cell-dependent mechanisms. Administration of immune serum to B cell-deficient mice restored protective immunity and when given to naïve mice mediated protective effects, suggesting a primary role for antibody in protective immunity during Hp infection. Thus, these data indicate that B cells contribute to protective immunity during Th2-types responses primarily through their production of antibodies and that their importance varies among different species of intestinal helminthes.
Distinct signaling pathways during innate and adaptive immune responses to Aspergillus fumigatus induce alternatively activated macrophages expressing Th2 cytokines (37.37)
Authors: Bhatia Shikha , Fei Mingjian , Oriss Timothy , Yarlagadda Manohar , Rooijen Nico , Ray Anuradha , Ray Prabir
Published: January 2023

Macrophages adopt an ‘alternative phenotype’ in a Th2-mediated inflammatory environment. Although identified in a wide variety of disease conditions, alternatively activated macrophages (AAMs) have been functionally best studied in helminth infections. The heterogeneity of functions performed by AAMs is attributed to the diverse molecules they express. Since infection with the fungus, Aspergillus fumigatus induces a chronic allergic response in patients with cystic fibrosis and asthma, we investigated the participation of AAMs in the process. We have found that Aspergillus infection promotes early recruitment of AAMs to the lung and the cells possess features of both AAMs and Th2 cells. The AAMs produce Th2 cytokines, which is independent of Dectin-1 signaling. Interestingly, unlike in helminth infections, both AAM recruitment and Th2 cytokine production were found to be independent of IL-4Rα and STAT6. Besides being part of the innate immune response to A.fumigatus, AAMs were also induced during adaptive immune response to the fungus. However, at this later time, AAM recruitment and their typical features were abolished in the absence of IL-4Rα and STAT6. Collectively, our studies show that different signaling pathways regulate the development of AAMs in the innate and adaptive arms of the immune response to A. fumigatus. Being early producers of Th2 cytokines, the presence of AAMs would be deleterious to the host unless regulated efficiently.
RSV-induced polarization of lung DCs towards plasmacytoid DCs with an associated Th2 response is dependent on NKT cells in the neonatal lungs (37.46)
Authors: Habeeb Keith , Durre Kainath , Sun Jonathan , Qureshi Mahboob
Published: January 2023

RSV bronchiolitis and pneumonia during early infancy is associated with increased susceptibility to asthma; and is characterized by a hyper-responsiveness airway and a predominant Th2 response. Using a BALB/C neonatal mouse model we have observed a skewed polarization of DCs towards plasmacytoid DCs (pDCs: CD11blo CD11clo CD45R+) over myeloid DC (mDCs: CD11blo CD11chi/med CD45R-), as determined by flow cytometry (FACS). This was associated with increased and decreased production of Th2 (IL-10) and Th1 (IFN-g) cytokines, respectively, in the alveolar spaces, as measured by ELISA. A temporal observation of enhanced NKT (CD3-CD19-CD1d+) cell response was also determined by FACS. In addition, FACS revealed that RSV infection of NKT cell knock out (CD1d-/-) neonatal mouse had lower proportions of pDCs and a higher proportions of mDCs compared to the wild type (WT) mouse of same age. Amount of IL-10 in bronchoalveolar lavage fluid was lower in the CD1d-/- mouse than that in the WT mouse. This data suggests that NKT cells are instrumental in skewed polarization of DC in response to RSV infection, which in turn may influence the T cell responses and hence, development of susceptibility to asthma in later life.
Distinct migratory behavior of myelomonocytic cells and antigen specific CD8 T cells during infections with Listeria monocytogenes (37.48)
Authors: Williams Cassandra , Velázquez Peter , Dustin Michael
Published: January 2023

Listeria Monocytogenes is a facultative, gram-positive, intra-cytosolic pathogen that causes severe infections in immunocompromised individuals. Listeria infections are characterized by the formation of leukocyte rich infectious foci in the liver and spleen. In order to establish the spatiotemporal interactions that must occur between innate and adaptive immune cells during the formation of infectious foci in the liver, intravital microscopy was employed. The quantitative analysis of acute changes in LysM+ neutrophil migration in response to Listeria challenge showed an increase in directed migration and direct contacts made with Listeria positive Kupffer cells. Additionally, Listeria foci filled with LysM+ granulocytes exclude blood, indicating that both a physical barrier and hypoxia may facilitate bacterial resistance. Upon adoptive transfer of Listeria specific CD8+ T cells it was found that T cell receptor specificity did not affect patrolling of effector CD8+ T cells near foci. This suggests that non-specific adhesion mechanisms are dominant players in lymphocyte behavior near foci. Together, these data provide insight into the spatiotemporal interactions that direct leukocyte behavior and the physical mechanisms underlying protection from Listeriosis.
RELM-beta is required for optimal development of theTh2 cytokine response to the intestinal parasite Heligmosomoides polygyrus (37.53)
Authors: Patel Nirav , Mishra Pankaj , Gause William
Published: January 2023

Previous studies have suggested that resistin-like molecule beta (RELM-beta) contributes to protective immunity against intestinal nematode parasites by directly interfering with adult worm feeding in the lumen. Other studies have suggested that RELM-beta may also function in the mucosal milieu by regulating Th1/Th2 cytokine production. In our studies, we examined the development of the memory Th2-type response to the murine intestinal nematode parasite, Heligmosomoides polygyrus (Hp). Mice were inoculated with Hp, drug-cleared at day 14, and several weeks later given an Hp secondary inoculation. Consistent with previous studies, adult worm burden was increased in Hp inoculated RELM-beta KO compared to inoculated WT controls. Analysis of cytokine gene (RT-PCR) and protein (ELISPOT) expression in draining mesenteric lymph nodes (MLNs) showed reductions in Th2 cytokine levels in RELM-beta KO compared to WT mice at day 7 after Hp secondary inoculation. At this time point spleen weight and total numbers of MLN CD4+ T cells and B cells were also reduced in RELM-beta KO compared to WT mice. Analysis of sorted CD4+ T cells showed reduced IL-4 expression in RELM-beta KO mice compared to WT mice at day 7 after secondary inoculation. These studies suggest that RELM-beta may affect protective immunity through multiple mechanisms, including enhancement of theTh2 cytokine response.
A strategy to identify human monoclonal antibodies that neutralize a broad spectrum of influenza A subtypes (38.12)
Authors: Olsen Ole , Frey Steve , Boozer Christy , Grandea Glen , Cicarelli Mike , Lily Rebecca , Ward Courtney , Fitch Alison , Feng I-wei , Jellyman David , Grondin Roxanne , Gibbs Aaron , Chan-hui Po-ying , Whitman Darbie , Fox Ray , Mosley Bruce , Hammond Phil , Branum Mark , Cieplak Witold , Moyle Matthew
Published: January 2023

Influenza A continues to be a serious public health threat as it continuously evolves to evade and escape immune surveillance through genetic drift, shift, and re-assortment. Hemagglutinin (HA) is the main glycoprotein target of the humoral immune response and elicits protective, neutralizing, antibodies during infection. However, these antibodies are usually specific for the influenza subtype causing infection and exhibit a narrow spectrum of protectiveness. To date, many of the influenza-neutralizing monoclonal antibodies described are cross-specific for subtypes H1 and H5 but do not neutralize subtypes H3 or H7. Therefore, we have designed an approach to survey the human B cell repertoire to identify monoclonal antibodies that neutralize influenza viruses of both the H1 and H3 subtypes. Our strategy is to identify humans that have broadly reactive serum antibodies, culture IgG+ memory B cells from these individuals at near clonal density, and then assay the culture supernatants for the presence of antibodies that neutralize both H1 and H3 influenza subtypes. By combining functional and binding assays we will determine if there exist in nature highly conserved neutralizing epitopes that are represented on most or all of the influenza HA subtypes tested.
Viral induced T cell independent B cell activation (38.13)
Authors: Blutt Sarah , Conner Margaret
Published: January 2023

Many acute viral infections induce early T cell independent polyclonal B cell activation and production of antigen-specific antibody prior to the formation of classical germinal centers. Rotavirus, a well characterized intestinal virus, induces Peyer’s patch (PP) T cell independent B cell activation and viral-specific IgA production in mice within 48 hours after viral exposure. We have utilized an in vitro culture system to identify important factors required for viral-induced B cell activation. We found that CD11c+ dendritic cells were essential for viral-induced B cell activation and that conditioned medium from virus-treated dendritic cells was sufficient to induce B cell activation indicating that direct contract between the dendritic cell and B cell is not required. PP dendritic cells contained rotavirus antigen and were activated during viral infection in vivo, concurrent with B cell activation, suggesting that dendritic cells are important modulators of the B cell response to rotavirus in vivo as well as in vitro. Our findings suggest that dendritic cells modulate the production of rapid T cell independent pathogen-specific antibody in the PP. Defining how pathogens induce rapid but specific antibody has tremendous potential to define the role of early antibody in limiting viral replication and dissemination and providing protection.
Regaining of the functions of antibodies in HIV-1-infected individuals (38.2)
Authors: Yu Qigui , Hu Weiguo , Hu Ningjie , Byrd Daniel , Amet Tohti , Qin Xuebin
Published: January 2023

Patients infected with HIV-1 are well known to mount a vigorous and sustained antibody (Ab) response to the virus. This response is also well known to have a limited effect on controlling virus proliferation or on protecting the patients from developing AIDS. This puzzling and frustrating phenomenon has been explained as the consequence of the incorporation and hijacking of regulators of complement activation (RCA) on HIV-1 virions and infected cells, which blocks Ab-dependent complement-mediated lysis (ADCML) of HIV-1 virions and infected cells by inhibiting the formation of the membrane attack complex (MAC) at the terminal stage of the complement activation cascades. Here, we report a potent and specific inhibitor of human CD59 (hCD59), one major member of human RCA. This inhibitor (designated rILYd4) was developed as a recombinant protein (114 aa) from the fourth domain of the bacterial toxin intermedilysin. We showed that rILYd4 in conjunction with polyclonal Abs to HIV-1 envelope, or with sera from HIV-1-infected patients at different stages of viral infection, efficiently abrogated hCD59 function and rendered complement-resistant HIV-1 virions including lab strains and primary isolates sensitive to ADCML. Toxicity assay using erythrocytes suggested rILYd4 has no or minimal non-specific cytotoxic effect. These results indicate that rILYd4 may represent a novel therapeutic agent for regaining the functions of Abs in HIV-1-infected patients to combat HIV-1 infection.
The kinetics of B-cell trafficking and long-term maintenance in the CNS induced by Sindbis virus infection (39.14)
Authors: Metcalf Talibah , Griffin Diane
Published: January 2023

Anti-viral antibodies are essential for both the clearance of Sindbis virus (SINV) and long-term suppression of reactivation in the CNS. To characterize the changing functional characteristics of B-cells and identify important determinants of B-cell trafficking and long-term maintenance, CNS tissue from SINV-infected mice was assessed at various times post-infection (pi) using flow cytometry and qRT-PCR. Plasmablasts (PBs) and memory B-cells (MBs) (CD19+CD38+CD138-IgM-IgD-) were 70% of the B-cell population for at least 6-mo pi, while plasma cells (CD19+/-CD38-CD138-) were less than 5% of the population. Staining for intracellular and surface IgG at day 26 pi detected 50% PBs and 60% MBs. A small population of CD19+CD38-CD138- cells was detected indicating possible germinal center (GC) B-cells in the brain. Further staining for GC marker GL-7 detected GL-7+ CD38+/- cells in the brain for at least 1-mo pi. mRNA levels of CXCL9/CXCL10 (trafficking), CCL19/CXCL13 (follicle formation), and BAFF (B-cell survival) peaked on days 5-7 pi. Levels gradually decreased and returned close to baseline by 6-mo pi except for BAFF that was maintained at a low level. The expression of CXCR3, CCR7, and BAFF receptor were detected on B-cells for at least 1-mo pi. These results suggest that PBs and MBs play a role in the long-term suppression of SINV replication and that the brain may provide a microenvironment for differentiation and survival of B-cells.
T-cell receptor repertoire diversity during persistent and fatal ehrlichial infections (40.17)
Authors: Thirumalapura Nagaraja , Thomas Sunil , Soong Lynn , Walker David
Published: January 2023

Human monocytotropic ehrlichiosis (HME), an emerging tick-transmitted bacterial disease, is caused by Ehrlichia chaffeensis. HME is modeled in C57BL/6 mice using Ehrlichia muris, which causes persistent infection, and Ixodes ovatus Ehrlichia (IOE), which is either acutely lethal or sub-lethal depending on the dose and route of inoculation. To better define the roles of T cells in protection and pathogenesis, we analyzed TCR Vβ usage in activated T cells from mice infected with E. muris or IOE by TCR-CDR3 spectratyping. Our data indicated that E. muris, but not IOE, induced oligoclonal expansion of CD4+ T cells within the several Vβ families (Vβ1, Vβ2, Vβ8.1, Vβ8.2, Vβ12, Vβ16 and Vβ19) early during the effector phase of T cell response. Further, oligoclonal expansion of CD8+ T cells within the Vβ8.2, Vβ4 and Vβ19 families was observed during E. muris infection compared to oligoclonal expansion of Vβ16 and Vβ19 CD8+ T cells in mice infected with IOE. Functional analysis of T cell subsets by flow cytometry indicated that E. muris induced a strong Th1 response involving several Vβ families compared to a weak Th1 response induced by IOE. The data suggested that unlike IOE, E. muris induces a strong Th1 response which tends to focus on certain Vβ families early during the effector phase of T cell response. Future studies will focus on functional characterization of memory T cell subsets induced following infection with E. muris.
Using team-based learning (TBL) to teach immunology to biomedical science graduate students (41.2)
Authors: McCormack Wayne , Garvan Cynthia
Published: January 2023

Purpose: The focus of team-based learning (TBL) on problem-solving and integration of information has tremendous potential but has not been explored for improving biomedical science graduate education. We assessed the TBL experience of PhD students in a graduate level immunology course. Methods: TBL sessions replaced 9 of 42 lectures in “Principles of Immunology”, a 3-credit graduate level course. Course evaluation results and student performance were compared before and after TBL implementation. Results: After TBL implementation, students gave higher ratings for the course overall, general format (teaching methods used), fairness of test grading, appropriateness of test content, and overall rating of lectures. Most students preferred TBL over other small group teaching methods, spent an hour or more preparing for class, and agreed that they were better prepared and their classmates seemed better prepared for class, and that TBL helped them prepare for exams. Most agreed that TBL resulted in more interaction and that they could learn better in TBL compared to other small group settings. Students showed modest gains in performance on exams. Conclusion: Based on student feedback, TBL appears to improve course evaluations and to promote active learning in graduate-level courses. Implementation of TBL in graduate education may improve application and problem-solving skills, generate enthusiasm in the classroom, and promote collaboration and teamwork among graduate students.
Connecting laboratories and classrooms - Easy and inexpensive educational outreach (41.4)
Authors: Lukin Kara
Published: January 2023

Providing high school and middle school students with first-hand scientific experiences is a powerful way in which to stimulate their interests in the biomedical sciences. Many GK-12 teachers lack the resources, personnel and/or expertise to integrate experimental procedures into their curricula. By contributing just a few hours of their time, academics at institutions of higher education can introduce simple demonstrations that will transform the manner in which students view biology and increase their academic interests and achievement. I have developed a simple, inquiry-based, lesson-plan that can be adapted readily to comply with district and/or state science standards. Investigators can adopt the exercise easily for their efforts in educational outreach. Educators seeking to incorporate novel experiences in their classrooms can accommodate the lesson to a wide range of biology courses and a diverse student population. Additionally, educators may share the lesson when soliciting scientists and professors with which to partner.
Immune modulation enhances the macrophage-mediated clearance of Pneumocystis without the severe inflammatory consequences of Pneumocystis pneumonia. (42.11)
Authors: Wang Jing , Gigliotti Frank , Bhagwat Samir , Wright Terry
Published: January 2023

Pneumocystis (Pc) causes high mortality Pneumocystis pneumonia (PcP) in immunocompromised patients. The Pc-driven immune response is essential for host defense against Pc. However, this response is also a key pathologic component of Immune Reconstitution Inflammatory Syndrome (IRIS), an acute form of PcP. NF-κB signaling is critical to many aspects of immune function and inflammatory responses. Its role in regulating immune-mediated inflammatory lung injury, however, is unknown. We hypothesized that specifically interrupting this pathway would diminish the Pc-driven immune response, resulting in reduced inflammatory disease at the expense of impaired immunity. As expected, when Pc-infected immunodeficient mice were immunologically reconstituted they exhibited the marked pulmonary inflammation and rapid respiratory decompensation characteristic of IRIS. In contrast, mice treated with sulfasalazine (SSZ), a potent inhibitor of IκB kinase, showed attenuated pulmonary inflammation and dramatic physiologic improvement. Unexpectedly, SSZ did not abrogate but actually enhanced the clearance of Pc from the host, while promoting an accelerated resolution of inflammatory lung disease. The enhanced Pc clearance is associated with the increased AM phagocytosis. These results suggest that a divergence exists between the mechanisms of lung injury and host defense. NF-κB and AM are potentially important therapeutic targets for the treatment of PcP.
Severe burn injury leads to spontaneous generation of Th17 CD4+ T cells in wound draining lymph nodes (42.18)
Authors: Neely Crystal , Maile Rob , Wang Ming-jin , Wolfgang Matthew , Cairns Bruce
Published: January 2023

Severe burn injury leads to early and prolonged immune dysfunction resulting in a reduced resistance to bacterial infection. In a model of burn injury, we have previously shown that burn mice challenged with Pseudomonas aeruginosa develop systemic infection and have high mortality rates whereas sham mice do not. Wound tissue TGF-β levels and serum IL-6 levels are elevated in the infected burn (IL-6: 32.68 +/- 9.53 pg/mL, n=4) compared to infected sham (2.87 +/- 1.43, n=3) mice. IFN-γ levels are similar between the groups. Since TGF-β and IL-6 are key for differentiation of naïve CD4+ T cells into the Th17 phenotype, we hypothesized that Th17 T cells are generated following burn injury. Female C57BL/6 mice were anesthetized and subjected to 20% full thickness contact burn or sham procedure. After 3, 7 or 14 days, CD4+ T cells selected from wound-draining lymph nodes were in vitro stimulated with mitogen. Flow cytometry was used to identify intracellular IL-17 in CD3+ CD4+ T cells. At all time points there was a statistically significant burn-dependent increase in both percentage of CD4+ T cells that were Th17 cells and absolute Th17 cell number (e.g. Day 3, 7384 +/- 1370 cells, n=3) compared to sham (1604 +/- 499, n=5). While burn injury spontaneously induces Th17 cells, known to recruit neutrophils, this does not protect against subsequent P. aeruginosa challenge. This may provide a novel mechanism for post-burn immunosuppression.