bluuebunny/crossref_metadata_2025 huggingface dataset:
Small Modular ImmunoPharmaceutical (SMIPTM) Molecules Directed at the TCR Complex Block Acute Graft Versus Host Disease and Cause Minimal Cytokine Release In Vivo (145.30)
Authors: Beckett Travis , Wang Camilla , Kim Kei , Odegard Valerie , Mohler Ken , McMahan Catherine
Published: January 2023

The T cell receptor complex (TCR) is a powerful therapeutic target for modulating autoimmunity and transplantation biology. A number of studies have shown that TCR/CD3-directed molecules are beneficial in mouse models of disease (e.g, acute graft vs host disease, aGVHD). However, both 145-2C11 (anti-CD3ϵ) and H57-597 (anti-TCRβ) are T cell mitogens and stimulate the activation of T cells and cytokine release in vitro and in vivo . In contrast to conventional antibodies, SMIP molecules are single chain binding proteins based on a scFv-CH2-CH3 format. Compared to their corresponding parent antibodies, SMIP molecules utilizing the 2C11 and H57 binding domains demonstrate a more robust down modulation of the TCR complex, enhanced efficacy in in vitro assays of T cell function, and significantly reduced levels of pro-inflammatory cytokines. Administration of these compounds in vivo results in significantly lower levels of cytokine release with no measurable changes in animal weight loss or adverse clinical symptoms despite dosage of 60 fold molar excess compared to the original full-length antibody. In addition, our SMIPs significantly abrogate donor cell engraftment in spleens of host animals in a parent into F1 model of aGVHD. These observations indicate that features in the SMIP format could generate novel anti-human CD3-directed therapeutics with greatly reduced levels of cytokine release in vivo.
Dendritic cells and Regulatory T cells facilitate donor engraftment after hematopoietic stem cell transplantation (145.35)
Authors: Langfitt Deanna , Wallace Nikki , Gaines Sara , Dallas Mari
Published: January 2023

The role of thymic DCs in facilitating donor engraftment after hematopoietic stem cell transplantation (HSCT) has not been investigated. Here we show addition of ex-vivo generated DCs accelerates thymic engraftment as well as enhances T cell recovery after HSCT. We also show the ability of regulatory T cells (Tregs) to facilitate engraftment. Control group received 103 lin-sca-1+c-kit+ (LSK) HSC progenitors while DC and Treg groups received LSK cells along with ex-vivo generated DCs or ex vivo expanded Tregs. On day of HSCT, C57BL/6 recipients received lethal irradiation with 1000 cGy. Day 4 & 7 post HSCT, thymuses of the DC and T reg groups contained 1.5 and 2 fold, respectively, higher number of thymocytes compared to control group. Furthermore, at 4 and 7 days after HSCT, H&E slides of thymuses of showed increased cellularity with medullary lymphoid regeneration in the cohort receiving Tregs. We demonstrate that ex-vivo generated DCs efficiently migrate and home to the thymic medulla and hasten thymic recovery as demonstrated by the higher number of total thymoctyes. The addition of Tregs facilitates engraftment as demonstrated by total number of thymocytes, and lymphoid regeneration in the medullary region of the thymus. Lastly, recipients receiving Tregs were able to facilitate engraftment compared to control group as demonstrated by survival 50% and 0%, respectively (p=0.04).
A novel approach to deplete antigen-specific T cells (145.45)
Authors: Lee Sang Chul , Lee Sun Kyoung , Kim Eun Hwa , Kim Hye Jung , Cho Hong Rae , Kwon ByungSuk
Published: January 2023

CD137 is expressed on activated T cells and is believed to be a reliable surrogate marker for antigen-specific T cells. In this study, we examined the possibility for toxin-conjuated anti-CD137 mAb to kill CD137-expressing T cells. Saporin or doxorubicin was conjugated to anti-CD137 mAb. The efficacy of toxin-conjugated anti-CD137 mAb was examined in vitro and in vivo. First, we observed that fluorescence-conjugated anti-CD137 mAb was internalized into activated CD4+ T cells and CD8+ T cells within 4 hrs after its treatment as well as cell lines that constitutively express CD137. We next treated primary T cells with toxin-conjugated anti-CD137 mAb, following their activation with anti-CD3 mAb. Saporin- or doxorubicin-conjugated anti-CD137 mAb induced apoptosis in a majority of CD4+ T cells and CD8+ T cells within 48 hrs after its treatment, compare with control antibodies. We further confirmed the specificity of toxin-conjugated anti-CD137 mAb showing that only T cells that expressed CD137 underwent apoptosis by staining with toxin-FITC-anti-CD137 mAb conjugates. Finally, we found that toxin-conjugated anti-CD137 mAb was able to delete alloreactive T cells in vivo and prevent aGVHD in animal model. Our results indicate that CD137-targeted delivery of toxin is a good strategy to delete antigen-specific T cells. This approach may be useful for the prevention of rejection after transplantation and treatment of autoimmune diseases.
Donor-specific B cell quiescence following desensitization and transplantation (145.7)
Authors: Jackson Annette , Montgomery Robert
Published: January 2023

Transplantation improves the health and quality of life for patients suffering from renal failure, but for many, antibodies specific for HLA antigens create a substantial barrier. Our center has developed a desensitization protocol to remove HLA-specific antibodies and successfully transplant these sensitized patients. This protocol involves alternate day plasmapheresis, intravenous immunoglobulin (IvIg), and immunosuppressants. We use a Luminex assay to detect HLA-specific antibody and developed a flow cytometric technique using HLA tetramer molecules to quantitate HLA-specific B cells. Following desensitization and transplantation, we observe a sustained loss of donor-specific HLA antibodies, while antibodies specific for 3rd party HLA return to pre-treatment levels. Interestingly, B cells specific for donor-specific HLA persist. We hypothesize that transplantation in the absence of inflammation, due to continued desensitization treatment, allows for the induction of B cell anergy toward donor-HLA. We have isolated HLA-specific B cells post-transplant from desensitized patients using HLA tetramers and performed in vitro cultures for 10-12 days using media containing CD40L, IL2, IL21, IL10, and CpG. B cells specific for donor HLA did not differentiate into antibody secreting cells, while B cells specific for 3rd party HLA did. Further research is underway to understand the mechanisms involved in keeping donor-specific B cells quiescent in these patients post-transplant.
IL-17 promotes the conversion of normal neutrophils to PMN-I, an immunostimulating subset of neutrophils (34.14)
Authors: Inatsu Akihito , Yoshida Shohei , Asai Akira , Kobayashi Makiko , Herndon David , Suzuki Fujio
Published: January 2023

Neutrophils (PMN) influenced by Th1 cytokines or Th17 pathway are essential effector cells in host’s anti-Candida resistance. However, the properties of PMN influenced by IL-17 remain unclear. IL-17 is a cytokine produced by Th17 cells. In this paper, PMN-I with the abilities to kill Candida albicans were shown to be generated from normal neutrophils (PMN-N) after stimulation with IL-17. PMN were considered to be PMN-I when resident Mϕ or naive T cells transwell-cultured with PMN samples were converted to M1Mϕ (CCL3+ IL-10- Mϕ) or Th1 cells (IFN-γ+ IL-4- T cells). PMN (1 × 105 cells/ml), isolated from peripheral blood of healthy donors, were cultured with rIL-17 (0.1 to10 ng/ml) for 9 h. Thereafter, these PMN were transwell-cultured for 9 h with resident Mϕ or T cells syngeneic to PMN donors (1 × 106 cells/ml, lower chamber). Cells in the lower chamber were re-cultured for 24 to 48 h, and culture fluids obtained were assayed for M1Mϕ markers (CCL3 and IL-10) or Th1 cell markers (IFN-γ and IL-4) by ELISA. In the results, M1Mϕ were generated from resident Mϕ transwell-cultured with IL-17-stimulated PMN. Also, Th1 cells were generated from naive T cells transwell-cultured with IL-17-stimulated PMN. These results indicate that IL-17 stimulates the neutrophil conversion from PMN-N to PMN-I.
Toll-like receptor 4 (TLR4) and interferon-gamma (IFN-r) are required for HMGB1-induced TNF release from macrophages. (34.18)
Authors: Yang Huan , Rosas Mauricio , Ochani Mahendar , Li Jianhua , Lu Ben , Olofsson Peder , Chavan Sangeeta , Tracey Kevin
Published: January 2023

High mobility group box 1 (HMGB1) has been implicated as a cytokine mediator in the pathogenesis of inflammatory diseases (Yang et al. BBA, 2009). To study mechanisms of HMGB1 action, we produced mammalian HMGB1, devoid of bacterial products, and examined receptor signaling for HMGB1 mediated macrophage cytokine release. HMGB1 induced significant TNF release in macrophages from TLR2 KO, RAGE KO and wild type (WT) mice (n = 4-5 experiments). HMGB1 induced TNF release was completely abolished in TLR4 KO macrophages as compared to WT. Interestingly, we observed that HMGB1 induced TNF mRNA expression, but not TNF protein release, from TLR4 KO mouse macrophages. Pre-treatment of TLR4 KO macrophages with IFN-r restored HMGB1-induced TNF protein release. Thus, our results indicate that TLR4 is required for HMGB1-mediated cytokine release from macrophages, and that IFN-r can restore HMGB1-induced TNF release in TLR4 KO macrophages. (supported in part by grant from NIH, NIGMS, to KJT).
Characterization of the role of indirect TRAF6 association in CD40 signaling (34.2)
Authors: Decker Dima , Bishop Gail
Published: January 2023

CD40 is a member of the TNFR superfamily expressed on the surface of B cells. The activation of CD40 by its ligand CD154 results in signaling pathways that are of major importance for the activation of B lymphocytes. CD40 lacks direct kinase activity; therefore TNFR-associated factors (TRAFs) are adapter proteins which initiate CD40-mediated signaling by associating with its cytoplasmic (CY) domain. One of these proteins, TRAF6, promotes CD40-mediated NF-κB, JNK and ERK activation. Interestingly, previous work has shown that although TRAF6 binds the CD40 CY domain, and this binding is required for a subset of CD40-mediated functions, TRAF6 can also contribute to CD40 signaling without direct receptor association. To understand the potential regulatory functions mediated by CY TRAF6, we utilized a TRAF6 mutant that cannot bind CD40 (TRAF6ΔC). Expression of this mutant in TRAF6-/- B cells showed that TRAF6 binding was not needed to induce CD40-mediated NF-κB activation. Our results indicated that cytoplasmic signaling of TRAF6 without direct association to CD40 was also sufficient for AKT, p38 and TAK1 activation. Ongoing studies probe additional signaling pathways, and seek to determine the detailed mechanism used by TRAF6 to indirectly mediate CD40 effects.
Anti-inflammatory property of the cannabinoid receptor-2-selective agonist in spinal cord injury (35.11)
Authors: Adhikary Sabina , Hongbo Li , Skarica Mario , Tuma Ronald , Ganea Doina
Published: January 2023

Recently cannabinoids are being studied in treatment of various animal models of CNS disorders such as MS, AD, PD and stroke. They are also being used in treating neuropathic pain in patients with spinal cord injury (SCI). Two cannabinoid receptors have been cloned; CB1, mainly expressed in the CNS and CB2, predominantly expressed in the periphery including immune cells. Previous studies from our laboratory showed that treatment with the CB2 specific agonist O-1966 improved recovery of motor and bladder function in mouse model of acute spinal cord injury. In SCI, increased infiltration and activation of inflammatory immune cells that follow the initial injury exacerbate neuronal damage. In this study we analyzed expression of inflammatory molecules that serve as markers for activation and infiltration of immune cells and their modulation by the CB2 agonist treatment. A murine thoracic spinal cord contusion model of SCI was used and spinal cords were analyzed for expression of various cytokines and chemokines. We found suppression of a number of molecules in spinal cords that were subjected to the CB2 agonist treatment. There was decreased expression of the CXCL family chemokine members CXCL1, 2, 3, 9 and 11, the CC family chemokine members CCL 8, 11, 20, T cell activation cytokines: IFN-γ, IL-23, IL-22 and some of the Toll-like receptors such as TLR1, 4, 6, and 7. Thus, it appears that CB2 agonist treatment may aid in recovery in acute SCI via its immunomodulating effects.
An essential function of FADD in hematopoietic stem cells and progenitors (36.10)
Authors: Zhang Jianke , Rosenberg Stephen
Published: January 2023

Signal transduction mediated by FADD represents a paradigm of co-regulation of both apoptosis and proliferation. Death receptors signal through FADD to activate the downstream caspase 8, and are required for homeostasis in the immune system, yet dispensable during embryonic development. In contrast, a FADD deficiency results in embryonic lethality. In previous studies, a lack of FADD in embryonic stem cells led to severe defects in lymphocyte development. However, conditional deletion of FADD following lymphoid lineage commitment still allows for the generation of T and B cell compartments. Whereas Fas-mediated apoptosis is blocked, a lymphoproliferation disease is lacking in lymphocyte-specific FADD-deficient mice due in part to defective lymphocyte activation. Recent studies indicated that a loss of Fas in dendritic cells is sufficient to cause lymphoproliferation diseases, while the impact of a FADD deficiency in non-lymphiod cells has not been determined. The current study analyzed the effect of simultaneous deletion of FADD in multiple cell types in adult mice by using the IFN-inducible MX1-Cre transgene. Unlike Fas deficient mice, the resulting FADD mutant mice do not develop lymphoproliferation diseases. However, the data demonstrated that FADD plays a critical role in the differentiation and maintenance of hematopoietic stem cells and progenitors.
B cell lineage progression and identity are impeded synergistically in EBF1 and Runx1 haploinsuficient mice (36.13)
Authors: Lukin Kara , Fields Scott , Guerrettaz Lisa , Lopez Desiree , Mansson Robert , Cherrier Marie , Cambier John , Feeney Ann , Sigvardsson Mikael , Hagman James
Published: January 2023

The transcription factors Early B cell factor 1 (EBF1) and Runx1 are essential for B cell development. B lymphopoiesis is arrested at an early state and hematopoiesis is ablated in the absence of EBF1 and Runx1, respectively. Previously, we demonstrated synergistic activity of EBF1 and Runx1 in the activation of the B cell-specific gene mb-1(Cd79a). To identify cooperative functions of these factors in vivo, we generated Ebf(Ehet)Runx(Rhet)-double haploinsufficient(ERhet) mice. Significant compound effects were observed in ERhet mice. B cell numbers in the bone marrow and spleen were reduced greatly. Generation of pre-B and immature B cells in ERhet mice is impeded by: 1) reduced frequencies of Ig light chain gene rearrangements and 2) significant alterations in B cell-specific gene expression. Notably, repression of c-kit and activation of Aiolos were delayed. Interestingly, a high percentage of pro- and pre-B cells from ERhet mice expressed the NK cell markers NK1.1, CD160 and 2B4. Co-expressions of these NK cell markers with genes indicative of B cell commitment was demonstrated using multiplex, single cell PCR. Retroviral complimentation of Ebf1 and Runx1 deficiencies eliminated the promiscuous expression of NK cell genes in ERhet pro-B cells. Thus, appropriate dosage of Ebf1 and Runx1 is necessary for efficient activation of the B cell-specific program and suppression of lineage inappropriate genes. Our data solidify EBF1’s role in controlling B cell lineage commitment.
Identifying chemokine receptor signals required for thymic settling (36.26)
Authors: Zlotoff Daniel , Sambandam Arivazhagan , Logan T. , Bell J. , Schwarz Benjamin , Bhandoola Avinash
Published: January 2023

T cell generation requires the recruitment of bone marrow derived hematopoietic precursors to the thymus via the bloodstream. The chemokine receptor CCR9 and the selectin ligand PSGL-1 each independently support progenitor recruitment to the unirradiated thymus, but progenitors lacking each are not restricted from thymic entry. Hence, additional molecules are likely at work. We now show that the chemokine receptor CCR7 is also involved in this process, as CCR7-/- progenitors are impaired in thymic settling. Furthermore, mice lacking both CCR7 and CCR9 have severe reductions in the number of early intrathymic progenitors, and in competitive assays CCR7-/-CCR9-/- progenitors are almost completely restricted from thymic settling. The compensatory proliferation of rare intrathymic progenitors can allow CCR7-/-CCR9-/- thymi to achieve near-normal cellularity despite severely impaired settling by circulating progenitors. These studies, however, focused on the signals used in unirradiated mice. The mechanisms involved in reconstituting a lethally-irradiated thymus remain poorly defined. We found that during a brief period following irradiation, CCR7-/-CCR9-/- cells are now capable of thymic setttling. Together, these observations illustrate the critical role of chemokine receptor signaling in thymic settling and help to clarify the cellular identity of the physiologic thymic settling progenitors.
FoxN1 in K14 promoter-driven epithelium is required for generation and maintenance of 3D-thymus medulla and preventing nude phenotype in the skin (36.33)
Authors: Guo Jianfei , Rahman Moshiur , Zhang Shuangmin , Zhang Zhijie , Tvinnereim Amy , Su Dong-Ming
Published: January 2023

An organized 3D thymic epithelial cell (TEC) meshwork is crucial for T-lymphocyte development. Expansion of thymic epithelial cystic cavities represents disruption of this 3D-structure. Abnormal culture, lacking lympho-stromal interactions, thymic aging, and gene mutation induce 2D-thymus cysts. Since germline FoxN1 gene mutation develops a 2D-cystic thymic rudiment, while abnormal culture caused 2D-thymus cyst-lining was reported to be non-FoxN1-dependent, it is unclear whether loss of FoxN1 promotes generation of thymic cysts, whether FoxN1 regulates maintenance of the 3D-thymus structure. Using loxP-floxed FoxN1 mouse model, we demonstrated that deletion of FoxN1 in K-14 promoter-driven epithelium induced loss of 3D-thymus medullary structure by producing a large number of alveolus-like 2D-epithelial cysts. The cyst-lining epithelium showed immature TEC phenotype by expressing Cld3,4 and K8, but low levels of MHC-II and K5. These 2D-thymus cysts expanded with ages, associated with reduced UEA-1+ and Aire+ TECs, and FoxN1 mutant Cld3,4+ TECs cannot develop mature medullary TECs. Meanwhile, K14-specific FoxN1 deletion sufficiently induced hair follicle defect and caused FoxN1-null nude phenotype in the skin, but did not cause athymia. These findings provide new insight into gene regulation on 3D-thymus epithelial structure and separate roles of FoxN1 in the thymus and skin. Supported by the NIH grants (R01AI081995, R21AG031880, and R21AI079747) to D-M S.
Cbl Inhibits Vav-1-Independent T Cell Differentiation (36.56)
Authors: Chiang Jeffrey , Hodes Richard
Published: January 2023

Cbl, an E3 ubiquitin ligase, negatively regulates signal transduction events involved in thymic development. Most recently, we reported that inactivation of Cbl rescues the defect of T cell development in mice expressing a mutant SLP-76 that lacks the ability to bind Vav. The defect in T cell development in these SLP-76 mutant mice is similar to that of Vav1 deficient mice, marked by a block at the DN3 stage. It was therefore possible that Cbl inactivation facilitates T cell development through a Vav signaling pathway independent of SLP-76. To assess this possibility, we generated Cbl-/-Vav1-/- double knockout mouse and hypothesized that Cbl inactivation would not be able to reverse the defects of T cell development in Vav1-/- mice. Unexpectedly, the inactivation of Cbl rescued T cell development of Vav1-/- mice. Rescued development in Cbl-/-Vav1-/- DP thymocytes is associated with enhanced tyrosine-phosphorylation of signaling molecules, including Vav2, PLC-gama1 and ERKs in response to TCR stimulation. Therefore, Cbl normally inhibits a T cell differentiation pathway which is independent of Vav-1. The role of Vav2 and other components of this pathway remain to be elucidated.
Expression of CD38 during ontogeny of murine B cells. (36.59)
Authors: Romero-Ramirez Hector , Pelayo-Camacho Rosana , Lopez-Santiago Ruben , Santos-Argumedo Leopoldo
Published: January 2023

CD38 is a 45 KD transmembrane protein, expressed by immature and mature lymphocytes. Agonistic antibodies directed to this molecule induce several biological responses: cellular proliferation, protein phosphorylation, intracellular Ca2+ mobilization, rescue from and induction of apoptosis. CD38 ligation with a mAb anti-CD38 (NIM-R5) induces apoptosis of the murine pro-B cell line (Ba/F3); this phenomenon depends on tyrosine kinases activation and the MAP kinase ERK. The expression and function of CD38 during B-cell ontogeny has been poorly analyzed in mice. We analyzed the expression of CD38 during the ontogeny of B cells in murine bone marrow. Bone marrow cells from C57BL/6J mice were stained with antibodies against: CD19, CD45R, IgM, CD43 and CD38. The samples were captured in a Beckman coulter’s CyAn flow cytometer and each subpopulation was analyzed with FlowJo v.7.5 software. CD38 is expressed from the preproB stage. ProB, preB and immature B cells from murine bone marrow were all positive for the expression of CD38, but immature B cells had the highest fluorescence intensity. The results suggest that CD38 may play a role during the ontogeny of murine B cells. Then, the next step is to analyze whether CD38 ligation on these subpopulations, induces proliferation, maturation or apoptosis.
Id3 is a critical mediator of lineage commitment and functional maturation of γδ-T cells (36.63)
Authors: Lee Sang-Yun , Zuniga-Pflucker Juan-Carlos , Wiest David
Published: January 2023

αβ and γδ T cells are thought to arise from a common precursor during development in the thymus. However, the basis for separation of these lineages is poorly understood. Previously, we showed that strong TCR signals promote γδ lineage choice, while weaker signals favor adoption of the αβ lineage. Here we show that the HLH factor Id3 plays a defining role in this process. We found that Id3 is required for strong TCR signals to both promote adoption of the γδ-fate and oppose the αβ-fate outcome. Importantly, the effects of Id3-deficiency on γδ lineage cells appear to differ depending on the γδ subset. In Id3-/- mice, development of the Vγ2 subset and Vγ3 dendritic epidermal T cell (DETC) subset are both reduced, while innate type NK γδ T cells bearing a Vγ1.1/Vδ6.3 TCR are dramatically expanded. Our analysis suggests that the γδ subsets whose development is impaired by Id3-deficiency are dependent on TCR signals of intermediate intensity. In contrast, those which are expanded (Vγ1.1Vδ6.3) in Id3-deficient mice may normally be deleted, perhaps by high-affinity ligand. In support this view, KN6 γδ TCR transgenic thymocytes normally deleted by high affinity ligand (T-10/22b) are rescued by Id3-defciency. Likewise, the junctional sequences of the TCR subunits of Vγ1.1Vδ6.3 T cells in Id3-/- exhibited almost no variation, suggesting strong selective pressure. These results indicate that Id3 not only in regulates fate selection, but also in shapes the γδ TCR repertoire.
The expression of semaphorins 4A, 4D and 7A in early hematopoietic T progenitors in the bone marrow of C57BL/6 mice. (36.65)
Authors: Chatterjea Devavani , Añó Leonor , Linder Grace , Obodo Udochukwu , Matthes David
Published: January 2023

Semaphorins are a group of transmembrane, secreted, and GPI-linked proteins that have been thoroughly studied in the nervous system where they function as ‘traffic’ enzymes in guiding axonal growth. Although most exhaustively characterized in this realm, they are also expressed in several other physiological systems. We have characterized the expression and localization of T cell semaphorins 4A, 4D and 7A in developing thymocytes. We show here that hematopoietic T cell progenitor cells (CD90+) enriched from the Bone Marrow have high levels of sema4A and 4D expression, but no sema7A. PlexinD1 and C1 (the receptors for sema4A and 7A) were also expressed in this subset, but there was no PlexinB1 or CD72 (the receptors for sema4A and 4D respectively). This provides further evidence of the dynamic expression and regulation of sema4A, 4D and 7A in early T cell development.
GABPalpha is required for normal T cell development (36.67)
Authors: Yu Shuyang , Zhao Dongmei , Jothi Raja , Xue Hai-hui
Published: January 2023

GA binding protein (GABP) consists of GABPalpha and GABPbeta subunits. GABPalpha is a member of Ets family transcription factors and binds DNA via its conserved Ets domain, whereas GABPbeta does not bind DNA but possesses transactivation activity. In T cells, GABP has been demonstrated to regulate the gene expression of interleukin-7 receptor alpha chain (IL-7Ralpha) and postulated to be critical in T cell development. To directly investigate its function in early thymocyte development, we used GABPalpha conditional knockout mice where the exons encoding the Ets DNA-binding domain are flanked with LoxP sites. Ablation of GABPalpha with the Lck-Cre transgene greatly diminished thymic cellularity, blocked thymocyte development at the double negative (DN)-3 stage, and resulted in reduced expression of T cell receptor (TCR) beta chain in DN4 thymocytes. By chromatin immunoprecipitation, we demonstrated in DN thymocytes that GABPalpha is associated with transcription initiation sites of genes encoding key molecules in TCR rearrangements. Among these GABP-associated genes, knockdown of GABPalpha expression by RNA interference diminished expression of DNA ligase IV, Artemis, and Ku80 components in DNA-dependent protein kinase complex. Interestingly, forced expression of pre-arranged TCR but not IL-7Ralpha can alleviate the DN3 block in GABPalpha-targeted mice. Our observations collectively GABP is critically required for TCR rearrangements and hence normal T cell development.
ThPOK induction by gdTCR signaling promotes gd maturation (36.68)
Authors: Park Kyewon , He Xi , Lee Hyung-Ok , Hua Xiang , Wiest David , Kappes Dietmar
Published: January 2023

Current models suggest that the transcription factor ThPOK, the “master regulator” of CD4 commitment, is selectively induced in developing abTCR+ thymocytes by strong/persistent TCR signals, although direct evidence for this conjecture is lacking. We show that strong TCR signals mediated by antibody treatment or high affinity endogenous ligands cause ThPOK induction in thymocytes of the distinct gd T cell lineage. A key ThPOK cis-acting element, the distal regulatory element (DRE), is sufficient to mediate TCR dependent gene activation in gd thymocytes. Additionally, we demonstrate that ThPOK controls maturation and repertoire selection of developing gd thymocytes. Thus, lack of functional ThPOK causes a severe reduction in mature gdTCR+ thymocytes and a shift in Vg usage, while constitutive ThPOK expression leads to a striking increase in mature gd thymocytes. These results demonstrate that ThPOK expression in gd thymocytes is regulated by the strength of TCR signaling, define an important new role for ThPOK in gd T cell development/maturation, and lend strong support to the view that development of most gd T cells depends on TCR engagement/signaling.
Complement activation on Chlamydia pneumoniae is accelerated by direct binding of native properdin to its surface (37.10)
Authors: Cortes Claudio , Ferreira Viviana , Pangburn Michael
Published: January 2023

Activation of complement represents one means of natural resistance to infection from a wide variety of potential pathogens. Recently properdin, a positive regulator of the alternative pathway of complement, has been shown to bind to surfaces and activate complement de novo. Here we studied the molecular mechanisms of properdin-mediated complement activation on Chlamydia pneumoniae, an obligate intracellular gram-negative bacterium that causes upper respiratory tract infections. We analyzed the ability of properdin to initiate complement activation by binding to C. pneumoniae directly, providing a platform for de novo convertase assembly. We determined for the first time that the physiological P2-P4 forms of human properdin bind to the surface of C. pneumoniae. The binding of these physiological forms initiates and accelerates complement activation on the C. pneumoniae surface, as measured by C3b and C9 deposition. Finally, properdin-depleted serum could not control C. pneumoniae infection of HEp-2 cells when compared with normal human serum. However, addition of native properdin to properdin depleted serum recovered the ability of serum to control the infection. Altogether, our data suggest that properdin is a pattern recognition molecule that plays a role in resistance to Chlamydia infection.
IL-18 is critical to cytotoxic NK and CD8+T cell activation and susceptibility to Ehrlichia-induced septic shock-like syndrome (37.19)
Authors: Ismail Nahed , Fang Rong , Ghose Purnima , Pulliam Stephanie
Published: January 2023

Ehrlichia chaffeensis is a Gram negative bacteria that cause septic shock where disease pathogenesis is mediated by IFN-γ and TNF-α producing, cytotoxic NK and CD8+ T cells, and late apoptosis of CD4+Th1 cells. We tested here the hypothesis that IL-18 signaling through IL-18Rα is involved in the pathogenesis of Ehrlichia-induced shock. Mice deficient of IL-18 or treated with anti-IL-18Rα blocking antibody were infected with virulent Ehrlichia that cause fatal disease. IL-18-/-mice have enhanced resistance to Ehrlichia infection as indicated by lower bacterial burden, which was accompanied by increased number of CD11b+ macrophages, decreased maturation of dendritic cells and B cells, and decreased number of granulocytes on day 3 post-infection (p.i.) compared to infected wild type controls. On day 7 p.i, absence of IL-18 or signaling via IL-18Rα resulted in decreased number of Fas-expressing, TNF-α producing-CD8+T cells and NK cells, increased number of IFN-γ producing NKT and CD4+Th1 cells, reduced expression of Fas on CD4+T and non T cells. Interestingly, infected mice treated with anti-IL-18Rα exhibited an increased number of TNF-α by NK and CD3+ Tcells, suggesting that a TNF-α inhibitory -endogenous ligand engages IL-18Rα following lethal ehrlichial infection. These data suggest that IL-18 play a pathogenic role in Ehrlichia-induced shock via activation of pathogenic NK and CD8 T cell responses and down regulation of protective IFN-γ producing NKT and CD4+ Th1 responses.
The opportunistic intracellular parasite Toxoplasma gondii infects and functionally inactivates human plasmacytoid dendritic cells. (37.30)
Authors: Pierog Piotr , Zhao Yanlin , Singh Sukwinder , Yap George , Fitzgerald-Bocarsly Patricia
Published: January 2023

Plasmacytoid dendritic cells (pDC) are a rare population of peripheral blood mononuclear cells with important functions during viral infection. In response to viral nucleic acids, pDC produce IFN-α which induces an antiviral state in neighboring cells, controls viral replication, and serves as an important link between innate and adaptive immunity. This immune response is attenuated in AIDS patients as previously reported by our lab, and can lead to susceptibility of these immunocompromised individuals to opportunistic infections like Toxoplasma gondii. Here, we investigated the effects of T.gondii infection on pDC function. We found that T.gondii infects pDC and establishes a parasitophorous vacuole membrane (PVM) without affecting pDC viability. pDC did not produce IFN-α or TNF-α in response to T.gondii; however, T.gondii-infected pDC were negatively affected in their ability to respond to viral stimulation. T.gondii-infected pDC exposed to HIV-MN or HSV-I secreted significantly lower quantities of IFN-α and TNF-α as compared to virus alone. To investigate the mechanism of IFN-alpha inhibition we used ImageStream, an imaging flow cytometer, and found that HSV-induced nuclear translocation of IRF-7 was inhibited in T.gondii-infected pDC. These results indicate that T.gondii evades innate immune responses by infecting and functionally inactivating plasmacytoid dendritic cells.
Intravital imaging of lymphocyte dynamics and signaling during immune Response to Listeria infection in the spleen (37.31)
Authors: Waite Janelle , Leiner Ingrid , Lauer Peter , Zheng Huan , Rae Chris , Portnoy Daniel , Pamer Eric , Dustin Michael
Published: January 2023

T cells play a major role in the adaptive immune response. T cells are primed by professional antigen presenting cells (APC), frequently dendritic cells (DC). T-DC interactions occur in lymphoid tissue where T cells are highly motile. Activation of the T cell receptor by cognate antigen triggers arrest in motility to allow stable and specialized contact with APC, termed the immunological synapse. Signaling that initiates and maintains stable T-DC contact has yet to be fully characterized. We employed intravital microscopy to image lymphocyte dynamics in the spleen, a major secondary lymphoid organ important for clearance of blood borne pathogens. We directly image a DC network in the subcapsular red pulp (scDC) where effector T cells migrate and arrest acutely in response to antigen. scDC internalized intravascular Listeria. Myelomonocytic cells were recruited and interdigitated with non-motile infected scDC as dynamic swarms and tight foci to contain Lm. Effector CD8+ T cells displayed a two-tiered reduction in motility with antigen independent and dependent components, including stable interactions with scDC. In addition, we monitored antigen induced intracellular calcium ([Ca2+]i) elevation in vivo. While [Ca2+]i elevation is sufficient to arrest crawling T cells, blocking [Ca2+]i elevation did not inhibit arrest. These data suggest there is an alternative antigen induced stopping pathway that allows stable T-APC contacts in the absence of [Ca2+]i elevation.
Yersinia pestis LOS stimulation of U937 cells shows different miRNA expression compared to Escherichia coli LPS stimulation (37.32)
Authors: Gibbons Jennifer , Pelletier Mark , Ernst Robert
Published: January 2023

Lipopolysaccharide (LPS)-stimulated activation of Toll-like Receptor 4 (TLR4) initiates downstream effects central to the inflammatory response that are important for host-pathogen interaction. In eukaryotes, microRNAs or miRNAs are small regulatory RNAs with multiple mRNA targets. There are ~1000 miRNAs encoded in the human genome, with the potential to regulate ~30% of the transcripts. The lipid A component of E. coli LPS is hexa-acylated, resulting in a highly proinflamatory response in human cells. Yersinia pestis synthesizes different lipid A structures depending on growth temperature, correlating to host conditions. Purified tetra-acylated lipooligosaccharide (LOS) of Y. pestis grown at 37○C, mimicking human infection, does not stimulate the innate immune system, whereas purified hexa-acylated LOS of Y. pestis grown at 25○C, mimicking flea infection, stimulates the innate immune system via TLR4, similar to E. coli LPS. Our hypothesis was that miRNA expression would be altered when human cells are incubated with purified LOS from Y. pestis grown at 25○C vs. 37○C. Initial studies in U937 cells using SABiosciences’ miRNA whole genome PCR arrays showed fewer miRNA expression changes in both LOS treatments than reported for LPS-stimulation. However, one modulation of interest is the 10-fold upregulation of miR-601 with 37○C LOS, compared to a 2-fold upregulation with 25○C. These data hint at a novel LOS-related mechanism, given recent reports of NFκB repression via miR-601.
Aberrant innate immune response to Leishmania major in the absence of secretory leukocyte protease inhibitor (SLPI) (37.38)
Authors: McCartney-Francis Nancy , Jin Wenwen , McGrady George , Wahl Sharon
Published: January 2023

Resistance to intracellular pathogens such as leishmania relies upon both innate and adaptive immune responses. Overexuberant host responses can lead to uncontrolled inflammation, highlighting a critical need for suppressive mechanisms to limit tissue damage. SLPI is an epithelial-derived protease inhibitor with anti-microbial and anti-inflammatory functions and is inducible in rodent phagocytes. Moreover, SLPI is induced by L. major and increased SLPI expression is evident within lesions following local infection with L. major. While resistance in C57Bl/6 mice is attributed to IFNγ-dominant Th1 responses, SLPI-deficient C57Bl/6 mice also develop strong Th1 responses, yet infection with L. major rapidly rages out of control with progression to systemic disease. To determine the mechanism of this dysregulated response, we infected SLPI-deficient and wildtype phagocytes with L. major and detected increased levels of inducible NO synthase (iNOS) and myeloperoxidase (MPO) coincident with altered arginase expression in the absence of SLPI. Intradermal infection with L. major revealed a rapid influx of phagocytes and modulation of iNOS, MPO and arginase. However, despite persistence of activated phagocytic cells, SLPI-deficient mice fail to resolve the inflammation or clear the infection. Our results define a novel contribution of SLPI to innate host defense against intracellular parasites and its potential use to hasten resolution of infection and inflammatory pathogenesis.
IL-4Rα signaling may enhance resolution of parasite-induced lung damage by inhibiting inflammation and mediating acute wound healing (37.8)
Authors: Chen Fei , Liu Zhugong , Liu Qian , Wu Wenhui , Bowdridge Scott , Millman Ariel , Rozo Cristina , Gause William
Published: January 2023

The parasitic nematode Nippostrongylus brasiliensis (Nb) third-stage larvae (L3) migrate to the host lung at about 24 to 48 hrs after infection, causing lung damage, inflammation and a potent Th2-type response. Yet, little is known about the mechanism causing lung damage and whether components of the Th2-type immune response contribute to acute lung wound repair. In this study we report that migrating Nb larvae (L3) induce lung inflammation that results in increased hemorrhaging in addition to mechanical damage in the lung. Neutrophil depletion reduced lung hemorrhage and both hemorrhaging and neutrophil infiltration were decreased in Nb-infected IL-17R KO mice. Resolution of acute hemorrhage and inflammation was dependent on either IL-4 or IL-13 as IL-4Rα KO mice, but not IL-13Rα or IL-4 deficient mice, induced significantly higher hemorrhaging, neutrophil influx, and IL-17 expression. Elevations in Arg1, IGF-1, and IL-10 were also inhibited in IL-4RαKO mice. In IL-10KO mice, resolution of inflammation was also blocked, control of hemorrhaging was partly suppressed, and Arg1 and IGF-1 elevations were not affected. These data suggest that the Th2-type immune response mediates acute wound healing and control of IL-17-dependent inflammation during helminth infection.
Activation of adenosine 2a receptor (A2aR) blocks IL-23/IL-17 mediated neutrophil migration in kidney ischemia-reperfusion injury (37.9)
Authors: Li Li , Huang Liping , Okusa Mark
Published: January 2023

Polymorphoneutrophils (PMNs) infiltration is the characteristic of kidney ischemia-reperfusion injury (IRI). Endogenous adenosine accumulating in kidney following IRI has a crucial role in terminating an overactive immune response through A2aRs. Our studies have demonstrated that the protective effect of the A2aR agonist, ATL146e, was mediated through bone marrow derived leukocytes. However, the mechanism by which A2aR agonists block neutrophil migration is unknown. Both kidney pedicles were clamped in C57BL/6 mice for 28 min and then released for 24 hrs. Kidneys from mice deficient of IL-23 (p19/40KO), IL-17A or IL-17R were protected from IRI with less neutrophil infiltration,CXCL1 and CXCL2 production and lower plasma creatinine levels (P<0.01, compared to WT IRI). Blocking of p19 or 17A by using neutralization mAb reduced plasma creatinine following IRI compared to isotype control-treated mice (P<0.001). Greater neutrophil accumulation was found in A2aRKO mouse kidneys subjected to IRI (P<0.001) than in WT mice, suggesting an important role of A2aR on neutrophil migration. ATL313 significantly decreased Gr-1+ neutrophil infiltration 24h following kidney IRI (P<0.001). Co-administration of IL-23+ATL313 or IL-17A+ATL313 significantly suppressed CD11b+GR-1+ peritoneal neutrophil migration (P<0.01). A2AR activation is a unique and potent strategy in attenuating IRI by blocking IL-23/IL-17 mediated neutrophil migration.
Detection of Crimean-Congo hemorrhagic fever virus glycoprotein antigens (38.19)
Authors: Bagriacik Emin , Albayrak Nurhan , Uslu Kadriye , Ertek Mustafa
Published: January 2023

Crimean-Congo hemorrhagic fever (CCHF) is a viral infection caused by Crimean-Congo hemorrhagic fever virus (CCHFV), a tick-borne virus (Nairovirus) of Bunyaviridae. Viral antigens of CCHFV can be detected by using various immunologic methodologies. We developed two methodologies to detect viral antigens of CCHFV using polyclonal antibodies isolated either from human plasma or from hyperimmunized horse serum. The first methodology is a western blot using human polyclonal antibodies. The second one is an antigen ELISA using horse antibodies. CCHFV isolates from vero-cell cultures were used as a source of the antigens. Additionally, CCHFV isolates obtained from Centers for Disease Control and Prevention (CDC) of USA were used as positive controls. The human polyclonal antibody pool detected several glycoproteins of various sizes ranging from approximately 35 KDa to 80 KDa or higher indicating that humans can develop antibody responses to different epitopes of the viral glycoproteins. Also, the ELISA can be used successfully to detect CCHFV with quite reproducible results. Our results indicated that both antibody sources could be convenient for rapid detection of CCHFV glycoprotein antigens.
CD73 and extracellular adenosine confer susceptibility to Toxoplasma gondii infection (38.7)
Authors: Mahamed Deeqa , Wang Yongmei , Bynoe Margaret
Published: January 2023

After tissue injury or infection, high levels of ATP inside cells is liberated and can serve as a danger signal that activates immune cells and promotes inflammation. The subsequent breakdown of extracellular ATP to adenosine by the cell-surface enzyme CD73 plays an important role in the resolution of the inflammatory response. Interestingly, the protozoan pathogen Toxoplasma gondii requires host adenosine for its purine needs as it cannot synthesize purines de novo. The role of extracellular adenosine in the immune response to T. gondii infection has not been previously elucidated. To investigate the role of CD73-generated adenosine in the immune response to infection with Toxoplasma gondii, wild-type C57BL/6 and CD73-/- mice were inoculated with T. gondii cysts by intragastric gavage. CD73-/- mice were less susceptible to T. gondii infection, with reduced intestinal inflammation and tissue damage during the acute stages of infection, and reduced parasite burden in the brain during chronic infection. In addition, cysts isolated from infected CD73-knockout mice were attenuated in their ability to cause pathology and mortality when inoculated into naïve wildtype or CD73-/- mice. In vitro, peritoneal macrophages and bone-marrow derived dendritic cells lacking CD73 were less permissive to the intracellular replication of T. gondii tachyzoites. These findings are the first to demonstrate that lack of CD73-generated adenosine impairs T. gondii pathogenesis.
Infection with Cytomegalovirus but not Herpes Simplex Virus induces the accumulation of late differentiated “senescent” T-cells in humans (39.13)
Authors: Pawelec Graham , Maier Andrea , Jahn Gerhard , Beck Robert , Haehnel Karin , Westendorp Rudi , Derhovanessian Evelyna
Published: January 2023

Infection with the persistent reactivating human cytomegalovirus (CMV) is associated with an age-associated increasing accumulation of late-differentiated CD8+ T-cells even in asymptomatic healthy carriers. It has been postulated that chronic exposure to any repetitively stimulating antigen (”chronic antigenic stress”) will have similar effects. Here, we have analyzed CMV and HSV infection status in the general population. Detailed analysis of T-cell subsets was performed using 10-colour flow cytometry in 100 individuals aged 40-80 yr. T-cells were characterized according to surface expression of CD45RA, CCR7, CD27 and CD28. Our results demonstrate that T-cell subset distribution was similar in CMV-matched, HSV-seropositive and HSV-seronegative individuals, whereas, in line with our and others previous data, individuals harboring CMV had lower frequencies of naïve T-cells (CD45RA+CCR7+CD27+CD28+) ad a reciprocal accumulation of late-differentiated effector memory (CD45RA-CCR7-CD27-CD28-) and TEMRA (CD45RA+CCR7-CD27-CD28-) subsets. These findings emphasize the apparently unique effect of CMV infection on the human immune system, providing evidence that infection with a different persisting herpesvirus does not show any effects similar to the observed large impact of CMV on the proportions of different T-cell subsets. This work was supported by EU 7FP LifeSpan (LSHG-CT-2007-036894), DFG-SFB685-B4 and DFG-PA 361-11/1
Unravelling CD4 T cell Dysfunction During Chronic Infection (39.17)
Authors: Crawford Alison , Wherry E John
Published: January 2023

Chronic infections are a major cause of mortality and morbidity worldwide and are often associated with CD8 T cell exhaustion. T cell dysfunction in these settings is likely a major reason for failed control of infection. In many chronic infections, CD4 T cells are also of major importance (e.g., HIV, HCV and LCMV). Compared to CD8 T cells, however, there is relatively little information on dysfunction of virus-specific CD4 T cell responses. This prompted us to ask whether CD4 T cells also become dysfunctional and how the differentiation of virus-specific CD4 T cells during chronic viral infection compares to CD8 T cells. We have defined the pattern of differentiation, hierarchy of dysfunction and diversity of costimulatory and inhibitory pathways involved in CD4 T cells exhaustion by comparing responses during acute and chronic LCMV infection. The results show that while CD4 T cells do become exhausted, they do not follow the exhaustive profile that has been established for CD8 T cells. While CD4 T cells do upregulate multiple inhibitory receptors during chronic infection, the inhibitory receptors they upregulate and their expression profile differs from CD8 T cells. These studies highlight that the well-characterized phenotype of CD8 T cell exhaustion cannot be broadly applied to CD4 T cells and suggests that CD4 and CD8 T cells may require different therapeutic treatments to rescue exhaustion.