bluuebunny/crossref_metadata_2025 huggingface dataset:
Impact of Growth Hormone (GH) Deficiency and GH Replacement upon Thymus Function in Adult Patients (138.26)
Authors: Morrhaye Gabriel , Kermani Hamid , Legros Jean-Jacques , Martens Henri , Geenen Vincent
Published: January 2023

Context: In rodents, growth hormone (GH), insulin-like growth factor-1 (IGF-1), and GH secretagogues (such as ghrelin) reverse age-related changes in thymus cytoarchitecture and increase thymopoiesis. GH administration also enhances thymic mass and function in HIV-infected patients. Until now, thymic function has not been investigated in adult GH deficiency (AGHD).

Objectives: The objective of this clinical study was to evaluate thymic function in AGHD, as well as the repercussion upon thymopoiesis of GH treatment for restoration of GH/IGF-1 physiological levels.

Design: Twenty-two patients with documented AGHD were enrolled in this study. The following parameters were measured: plasma IGF-1 concentrations, signal-joint T-cell receptor excision circle (sjTREC) frequency, and sj/ß TREC ratio. Analyses were performed at three time points: firstly on GH treatment at maintenance dose, secondly one month after GH withdrawal, and thirdly one month after GH resumption.

Results: After 1-month interruption of GH treatment, both plasma IGF-1 concentrations and sjTREC frequency were significantly decreased. Decreases in IGF-1 and sjTREC levels were significantly correlated. There was also a significant decrease in intrathymic T cell proliferation as indicated by the reduced sj/ß TREC ratio. One month after reintroduction of GH treatment, IGF-1 concentration and sjTREC frequency were restored at the level before arrest of GH treatment. The sj/ß TREC ratio also increased with GH resumption, but did not return to the level measured before GH withdrawal.

Conclusions: In patients with AGHD under GH treatment, GH withdrawal decreases thymic T cell output, as well as intrathymic T cell proliferation. These parameters of thymopoiesis are completely or partially restored one month after GH resumption.
Importance of MHCII expression on thymic epithelium versus dendritic cells for the positive and negative selection of CD4 T Cells (138.28)
Authors: Jenkinson S. Rhiannon , Zuklys Saulius , Hollander Georg A , Koni Pandelakis A , Reizis Boris , Hodes Richard J
Published: January 2023

Development of T cells from T cell precursors occurs in the thymus through interactions of T lineage cells with thymic stromal populations. These stromal populations are distributed in an ordered fashion that defines thymic architecture and provides the environment for thymic differentiation. The successful generation of a mature and self-tolerant CD4 T cell repertoire requires that developing T cells pass at least two checkpoints; positive and negative selection. Positive selection has been reported to be mediated by cortical epithelial cells, allowing T lineage cells that have rearranged a T cell receptor capable of recognizing self peptide-MHCII complexes at appropriately affinity, to survive and commit to the CD4 lineage. Positively selected cells can then move into the medulla where they must pass negative selection, which acts to remove highly self reactive cells. MHCII is expressed in the medulla on both medullary epithelial cells and dendritic cells and plays a critical role in negative selection. To further examine the cell populations in which MHCII expression is important in CD4 T cell development in unmanipulated mice, we are characterizing the effect of selective inactivation of MHCII, using a panel of mice expressing cre recombinase specifically in epithelial cells or in dendritic cells in combination with an MHCII floxed allele.
Hem-1 is essential for hematopoietic cell development and function by regulating the actin cytoskeleton (138.8)
Authors: Park Heon , Staehling-Hampton Karen , Appleby Mark W , Brunkow Mary E , Habib Tania , Ramsdell Fred , Liggit Denny , Freie Brian , Tsang Mark , Chan Maia , Carlson George , Friend Sherree , Iritani Brian
Published: January 2023

Hematopoietic protein-1 (Hem-1) is a hematopoietic cell-specific member of the Hem-family of cytoplasmic adaptor proteins. Orthologs of Hem-1 in flies and worms are essential for cytoskeletal reorganization, embryonic cell migration, and morphogenesis. However, the in vivo functions of mammalian Hem-1 are not known. Using a chemical mutagenesis strategy in mice to identify novel immune function genes, we positionally-cloned a nonsense mutation in the Hem1 gene. Loss of Hem-1 results in defective F-actin polymerization and actin capping in T lymphocytes and neutrophils, due to loss of the Rac GTPase-controlled actin-regulatory "WAVE" complex. T cell development and activation are impaired. Hem1-/- neutrophils are deficient in migration and phagocytic capacity. Conventional "B2" B cell development is inhibited at the pro-B and transitional T1 cell stages, and "B1" B cell development is also impaired. ELISA and ImageStream flow cytometric analyses reveal that NF-¿B-dependent transcription of proinflammatory cytokines, proceed normally in Hem1-/- mice, while TH17 differentiation is enhanced. These results demonstrate that Hem1 is essential for hematopoietic cell development and function by controlling cytoskeletal reorganization.
REGULATION OF SAPK/JNK PHOSPHORYLATION BY HISTAMINE IN SPLENOCYES (139.11)
Authors: Khan Manzoor M , Dandekar Radhika
Published: January 2023

Histamine is an inflammatory mediator in allergic disease and asthma. A number of histamine-mediated effects result from cytokine interactions and through their signal transduction mechanisms. SAPK/JNK are stress activated kinases which are stimulated by a variety of factors including cytokines and play a role in inflammation, cell proliferation and differentiation. This study was designed to study the effects of histamine on the phosphorylation of SAPK/JNK in splenocytes. Splenocytes were isolated from C57/BL 6 mice and after treatment with various agonists and antagonists phosphorylated SAPK/JNK levels were analyzed by Western Blot Analysis. While histamine at 10-4 inhibited the phosphorylation of SAPK/JNK , all histamine receptor subset agonists, H1, H2, H3, and H4 upregulated SAPK/JNK phosphorylation suggesting a complex interaction of histamine receptors in this process. Splenocytes from TNF-á knockout mice were employed to determine the role of TNF-á in histamine-mediated effects. Histamine further suppressed not only the effects of histamine but also that of histamine H1, H2, and H3 agonists but not that of H4 agonist. The effects of H4 agonists were further pronounced in TNF-á knockout splenocytes. Our studies demonstrate a role of TNF-á in histamine H1, H2 and H3 receptor- mediated regulation of SAPK/JNK phosphorylation. Other proinflammatory cytokines may be involved in mediating the effects of histamine on SAPK/JNK, via H4 receptors. In conclusion, the effects of histamine on SAPK/JNK phosphorylation involve complex interaction of its receptor subsets and cytokines which may have relevance in allergic disease.
SHIP is a repressor of mast cell hyperplasia, cytokine production, and allergic inflammation in vivo (139.12)
Authors: Haddon David James , Antignano Frann , Hughes Michael R , Blanchet Marie-Renée , Zbytnuik Lori , Krystal Gerald , McNagny Kelly Marshall
Published: January 2023

Src homology 2-containing inositol 5'-phosphatase (SHIP) inhibits immune receptor signaling through hydrolysis of the phosphatidylinositol-3 kinase (PI3K) product PI-3,4,5-P3, forming PI-3,4-P2. SHIP represses FcεRI- and cytokine-mediated mast cell activation in vitro, but little is known regarding the function of SHIP in mast cells in vivo, or the susceptibility of Ship-/- mice to mast cell-associated diseases. In this study, we found that Ship-/- mice have systemic mast cell hyperplasia, increased serum levels of IL-6, TNF, and IL-5, and heightened anaphylactic response. Further, by reconstituting mast cell-deficient mice with Ship+/+ or Ship-/- mast cells, we found that the above defects were due to loss of SHIP in mast cells. Additionally, we found that loss of SHIP in mast cells alone resulted in increased allergic asthma pathology. In summary, our data show that SHIP represses allergic inflammation and mast cell hyperplasia in vivo, and that SHIP exerts these effects specifically in mast cells.
PGI2, and its analog iloprost, regulate CD4+ Th2 lung inflammation and B cell production of IgE (140.11)
Authors: Ferrini Maria , Girtsman Teri , Jaffar Zeina , Roberts Kevan
Published: January 2023

Our previous findings have shown that PGI2 plays an important role in regulating Th2-mediated lung inflammation. Here, we have examined the mechanism by which PGI2, and its analog iloprost, limit allergic inflammation and the IgE response. Effector DO11.10 CD4+ Th2 cells, generated in the presence of the iloprost, produced normal levels of IL-4 and IL-5, but expressed higher levels of ICOS and CD62L than untreated Th2 cells. Adoptive transfer of iloprost-treated Th2 cells into BALB/c mice caused a markedly reduced airway inflammation following OVA inhalation, compared to untreated Th2 cells. CFSE labeling of CD4+ Th2 cells prior to injection revealed that the iloprost-treated cells proliferated less effectively in vivo in response to inhaled OVA. Moreover, intranasal instillation of FITC-OVA showed that iloprost-treated Th2 cells were less effective at promoting dendritic cell migration to the draining lymph nodes and subsequent expansion OVA-specific T cells. These effects were confirmed using mice lacking the IP receptor for PGI2 (IPKO). Following OVA aerosol challenge, primed IPKO mice developed increased eosinophilic inflammation, with elevated levels of airway CD4+ T cells and serum levels of OVA-specific IgE, compared to WT mice. Collectively, these data demonstrate that generation of PGI2, during allergic lung inflammation serves to limit both Th2 cell expansion and IgE production by B cells. A critical role for IL-4 in promoting expression of PGI2 receptor, IP, on Th2 cells and B cells that produce IgE is proposed.
Streptococcus pneumoniae vaccine, Prevenar, utilises Tregs to suppress Asthma (140.2)
Authors: Hansbro Philip M , Thorburn Alison N , Foster Paul S , Gibson Peter G
Published: January 2023

Our recent finding that exposure to Streptococcus pneumoniae may suppress pro-asthmatic responses led us to investigate whether the current human S. pneumoniae vaccines may be used to suppress asthma using mouse models of ovalbumin-(OVA)-induced allergic airways disease (AAD).

AAD was induced by intraperitoneal sensitisation and intranasal challenge with OVA. At the time of OVA sensitisation, Prevenar or Pneumovax were delivered intranasally either with or without CpG.

Prevenar, but not Pneumovax, suppressed hallmark features of AAD, including eosinophil influx, OVA-specific T helper (Th) 2 cytokine release, mucus hypersecretion, total serum IgE and airways hyperresponsiveness. We then investigated whether regulatory T cells (Tregs) were important in the suppression of AAD. Prevenar increased the number of CD4+CD25+FoxP3+ expressing Tregs in the lungs, lymph nodes and spleens. The release of immuno-suppressive cytokines IL-10 and TGF-â from lymph nodes was reduced. However, Prevenar increased expression of CD103 and CTLA-4 on CD4+CD25+FoxP3+ cells. Proliferation assays demonstrated that Tregs suppressed allergic effector T cell proliferation regardless of Prevenar treatment.

In conclusion, a currently available human vaccine increases the infiltration of Tregs and suppresses AAD, and may be useful as a novel therapy for asthma.
Reciprocal differentiation and tissue-specific pathogenesis of Th1, Th2, and Th17 cells in graft versus host disease (141.13)
Authors: Yi Tangsheng , Chen Ying , Wang Lin , Du Gong , Huang Daniel , zhao Dongchang , young James , Todorov Ivan , Lieping Chen , Iwakura Yoichiro , Stephen Forman , Zeng Defu
Published: January 2023

In acute graft versus host disease (GVHD), donor CD4+ T cells recognize alloantigens on host antigen-presenting cells and differentiate into T helper (Th) subsets (i.e. Th1, Th2, and Th17), but the role of Th subsets in GVHD pathogenesis is still elusive. We found in an MHC-mismatched model of C57BL/6 donor to BALB/c recipient, wild-type donor CD4+ T cells predominantly differentiate into Th1 and preferentially mediate GVHD tissue damage in gut and liver. However, lack of IFN-γ in donor CD4+ T cells results in augmented Th2 and Th17 differentiation and exacerbated tissue damage in lung and skin; lack of both IL-4 and IFN-γ results in augmented Th17 differentiation and exacerbated tissue damage in skin; and lack of both IFN-γ and IL-17 leads to further augmentation of Th2 differentiation, expansion of TNF-α -producing proinflammatory Th2, and idiopathic pneumonia. The organ-specific GVHD mediated by differential Th subsets is associated with differential expression of chemokine receptors by different Th subsets and different chemokines released by various organ tissues. Furthermore, lack of tissue expression of IFN-γ -inducible co-inhibitory molecule B7-H1 play a critical role in augmenting the Th2-mediated idiopathic pneumonia. These results indicate that donor CD4+ T cells reciprocally differentiate into Th1, Th2, and Th17 in acute GVHD recipients; different Th subsets mediate organ-specific tissue damage.
The PD-L1 Signal is Important to Liver Dendritic Cells in Induction of Foxp3+CD4+CD25+ Treg and Liver Transplant Tolerance (141.33)
Authors: Li Wei , Bakthavatsalam Ramasamy , Meng zihui , Perkins James D , Latchman Yvette , Reyes Jorge D
Published: January 2023

Our previous studies have demonstrated that Foxp3+CD25+CD4+ regulatory T cells (Treg) play an important role in the induction of murine liver spontaneous transplant tolerance. How Treg are induced and what their mechanisms are in the regulation of liver transplant tolerance remain undefined. Here, we investigated the role of liver dendritic cells (DCs) on the induction of Treg and liver graft tolerance in vivo and in vitro. Fms-like tyrosine kinase 3 ligand (Flt3L) mutation mice, which have severe reduction in all types of DCs, and PD-L1 deficient mice were used to examine the role of liver DCs and the PD-L1 signal in Treg induction and liver transplant tolerance. Our results showed that liver DCs, which expressed a high number of PD-L1 molecules, induced more Foxp3+CD25+CD4+ Treg in vitro compared to spleen DCs. However, DCs from PD-L1 deficient mice failed to expand Foxp3+CD25+CD4+ Treg in vitro. Adoptive transfer of Foxp3+CD25+CD4+ Treg expanded from liver DCs prolonged heart allograft survival significantly more than in the CD4 treated controls. The liver grafts from Flt3L-/- and PD-L1-/- mice were rejected acutely in the C3H recipients. Foxp3+ cells were reduced, but IL-2, IL-10, and IFN-γ producing cells were significantly increased in the liver graft and recipient spleen sections of Flt3L-/- and PD-L1-/- donors. Thus, liver DCs play a critical role in the induction of Treg, which underpin spontaneous acceptance of liver allografts. The function of DCs on Treg induction appears to depend on the PD-L1 signal.
Epstein-Barr Virus Latent Membrane Protein 1 Modulates Host MicroRNAs in B Cell Lymphomas. (141.37)
Authors: Harris Aleishia , Lambert Stacie , Krams Sheri , Martinez Olivia M
Published: January 2023

Epstein-Barr Virus (EBV) infection is usually benign but immunosuppression can promote post-transplant lymphoproliferative disease (PTLD) and B cell lymphomas in transplant recipients. Latent membrane protein 1 (LMP1) is an important EBV-encoded oncogene that usurps cellular pathways to drive B cell transformation. MicroRNA (miRNA) are tiny, non-coding RNA that regulate cellular gene expression. Since aberrant expression of miRNA has been observed in a variety of malignancies, we hypothesized that modulation of cellular miRNA expression by EBV may play a role in the development of EBV+ B cell lymphomas. Infection of B cell lymphoma lines with EBV significantly modulated expression of miRNA-7, -15b, -221, and -222. Importantly, a similar pattern of microRNA expression was observed in an EBV+ B cell line derived from a patient with PTLD. Activation of a chimeric LMP1 signaling system in EBV negative B lymphoma cell lines also led to induction of miRNA-221 and -222 expression. These mRNA target the cell cycle regulators p27 and p57. Thus, LMP1 can promote cell cycle progression in B cells through miRNA expression. Modulation of miRNA may be a novel mechanism by which EBV drives lymphomagenesis.
Interleukin-10 delivery via mesenchymal stem cells (MSC) to prevent ischemia/reperfusion injury in lung transplantation (141.46)
Authors: Salgar Shashikumar K , Manning E , Li S , Vazquez-Padron R , Mathew J , Ruiz P , Pham S
Published: January 2023

Ischemia-reperfusion injury (IR) is an important cause for lung graft loss (~30%). In this study, MSC & viral interleukin-10 (vIL-10) engineered MSC were tested for their ability to prevent lung IR injury. Bone marrow derived MSC from Lewis rat were transduced with rvIL-10-retrovirus & selected on neomycin. Following 120 min of left lung ischemia induction, Group A, rats received vIL-10-MSC (~15 x 106; i.v.); Group B, empty vector engineered MSC; Group C, MSC; Group D, saline; and Group E, no ischemia or MSC. Mean blood oxygenation (PaO2/FiO2 ratio, mmHg) was reduced (P<0.05) at 24h post-IR injury in Group B (138±86; n=9) & Group D (87±39; n=10), compared to MSC-vIL10 (353±105; Group A; n=10) group. By days 3 & 7 with MSC-vIL10 oxygenation was normal (475±55 & 435±33; n>9); by 4h it was 319±94 (n=7). MSC (passage ≤6) increased PaO2/FiO2 (454 ± 59; n=5) by 24h post-IR. Bronchoalveolar lavage at 24h post-MSC-vIL10 therapy reduced (P<0.05) granulocytes, CD4 & CD8 T cells. Lung injury score (histopathology) was higher (P<0.05) with no treatment (3.5 ± 1.3; n=5) compared to MSC-vIL10 (1.21± 0.6; n=7) & MSC (1.6±0.9; n=6) treated groups. Lung microvascular permeability & wet:dry ratio were lower (P<0.05) in MSC-vIL10 group. IL-1α, MCP-1α, MIP-1α, & IL1-β were increased in IR injured lung. ISOL (in situ staining for DNA fragmentation) & CASPACE-3 demonstrated reduced (p<0.05) number of apoptotic cells in MSC-vIL10 treated lungs. Ex vivo, expanded MSC were CD34-, CD31+ & CD45+ (5-10%), CD29+, CD90+ & CD44+ (65-95%), CD80 (0%), CD 86 (8%), MHC Class I+ (23-57%), & MHC Class II-. MSC & IL-10 delivery via MSC to prevent lung transplant IR injury seems promising.
Islet Allograft Protection by Low Levels of Circulating Transgenic Lung-Specific Human Alpha-1-Antitrypsin (141.49)
Authors: Shahaf Galit , DeFelice Nathaniel , Ficek Chad , Friedman Frida , Lewis Eli C
Published: January 2023

Alpha-1-antitrypsin (AAT) exerts anti-inflammatory activities during islet allograft transplantation in several diabetes mouse models. We sought to examine inflammatory pathways in the previously described lung-specific human AAT (hAAT) transgenic mice, and establish whether transgenic circulating hAAT is sufficient to protect islet allografts from acute rejection. Homozygote mice exhibited circulating hAAT levels that were unaffected by age and by inflammatory stimuli (50-200 ng/ml). Transgene expression was limited to lung epithelia and was absent from macrophages and islets. Homozygote mice exhibited marked anti-inflammatory serum and lung tissue cytokine profiles (high IL-1 receptor antagonist and low IL-6 levels). In vivo, 48 hours after streptozotocin challenge (STZ, 225 mg/kg), blood glucose levels were 50% lower then those elicited in wild-type mice (n = 3). In islet allograft transplantation model, grafts were accepted in homozygote mice and rejected in wild-type mice (n = 4). We conclude that as low as 50-200 ng/ml transgenic circulating hAAT is sufficient to generate an anti-inflammatory cytokine milieu, improve islet viability and induce islet allograft immune protection. These low levels of the transgenic product are below its physiological anti-protease inhibitory threshold, suggesting that AAT may contain anti-inflammatory activities independent of protease inhibition.
Memory B cells and Alloantibodies Synergistically Prevent Anti-CD154-Mediated Cardiac Allograft Acceptance (141.5)
Authors: Troutman Audrea M , Ma Lianli , Xu Jing , Li Yijin , Chong Anita S.
Published: January 2023

The impact of memory B cells and preformed antibody in tolerance induction has yet to be elucidated. We have developed a heart transplant model that specifically generates allospecific memory B-cells and not memory T cells. 3.83 BCR (BALB/c) knock-in mice harboring 3.83 B-cells with dual-specificity for H-2Kk and H-2Kb were used as recipients of C3H hearts. To test whether memory 3.83 B-cells could prevent tolerance induction, C3H-primed-3.83 recipients (60d post-Htx) received a 2nd heart and α-CD154(MR1). Second B/6 hearts were acutely rejected (MST 12d). To confirm the importance of memory B cells in facilitating α-CD154-resistant rejection, we transferred memory B-cells from C3H-presensitized 3.83 mice into naïve BALB/c treated with α-CD154 and rejection occurred in only half of recipients. Surprisingly, transfer of low doses of serum from C3H-presensitized 3-83 recipients could not overcome α-CD154 therapy; however, transfer of low dose serum alloAbs with memory 3-83 B cells elicited rejection in one 100% of BALB/c recipients treated with α-CD154. In conclusion, memory B cells and alloAbs synergize to induce CD40-CD40L-independent activation of allo-T cells, which prevent graft tolerance.
Human memory FOXP3+ Treg secrete IL-17 ex-vivo and constitutively express the TH17 lineage specific transcription factor RORγt (33.14)
Authors: Ayyoub Maha , Deknuydt Florence , Raimbaud Isabelle , Dousset Christelle , Leveque Lucie , Bioley Gilles , Valmori Danila
Published: January 2023

Recent studies have suggested a close relationship between CD4+FOXP3+ regulatory T cells (Treg) and pro-inflammatory IL-17-producing T helper cells (TH17) expressing the lineage-specific transcription factor RORγt. We report here the unexpected finding that human memory Treg secrete IL-17 ex-vivo and constitutively express RORγt. IL-17 secreting Treg share some phenotypic and functional features with conventional TH17 cells, expressing high levels of CCR4 and CCR6 and low levels of CXCR3. However, at variance with conventional TH17 cells, they express low levels of CD161 and mostly fail to co-secrete IL-22 and TNF-α ex-vivo. Ex-vivo secretion of IL-17 and constitutive expression of RORγt by human memory Treg suggest that, beside their well known suppressive functions, these cells likely play additional yet un-described pro-inflammatory functions.
Dynamic equilibrium of B7-1 dimers and monomers is important for regulation of TCR/CD28 – mediated T cell activation (33.28)
Authors: Bhatia Sumeena , Almo Steven C , Nathenson Stanley G , Hodes Richard J
Published: January 2023

Under steady state conditions, B7-1 is present as a mixed population of non-covalent dimers and monomers on the cell surface. Here we have examined the physiological significance of this unique dimer-monomer equilibrium state of B7-1. We demonstrate that altering B7-1 to uniformly covalent dimeric state results in increased frequency of CD28 mediated T cell-APC conjugates. This augmented T cell-APC conjugate formation correlates with persistent concentration of signaling molecules, PKC-θ and lck, at the immunological synapse (IS) and with a higher calcium flux in T cells. In contrast, B7-1 acquisition by T cells, an event that occurs as a consequence of CD28 engagement with B7-1/B7-2, is highly reduced when B7-1 is present in covalently dimeric state. The ability of covalently dimeric and wild type B7-1 to costimulate antigen-specific T cell proliferation was also assessed. In contrast to the enhanced ability of dimeric B7-1 to support proximal T cell activation events, sensitivity to competitive inhibition by soluble CTLA-4:Ig indicated that the covalent dimeric form of B7-1 is in fact less efficient in costimulating T cell proliferation. These findings suggest a novel model in which optimal T cell costimulatory function of B7-1 requires CD28 engagement by B7-1 in a non-covalent dimeric state, followed by dissociation of these B7-1 dimers, facilitating downregulation of CD28 and internalization of B7-1.
The lifespan of dendritic cells (DC) affects DC-mediated regulation of B cells (34.16)
Authors: Ma Daphne Y , Draves Kevin E , Clark Ed A
Published: January 2023

DCs can directly regulate B cells without T cell help. We investigated the physiological properties of DCs that dictate their ability to prime and direct B cell responses. We used DCs lacking the pro-apoptotic molecule Bim (Bim-/-). Bim-/- DCs survive longer than DCs from wildtype (WT) control mice. Also, CD8α+DEC205+ DCs are elevated in Bim-/- mice. This suggested that Bim-/- DCs may be functionally distinct from WT DCs in regulating B cells. We thus examined effects of WT and Bim-/- DCs on α-IgM-induced proliferation of WT splenic B cells. LPS-matured DCs from Bim-/- mice are less effective than WT DCs in their ability to promote α-IgM-induced B cell proliferation. As with α-IgM, DCs from Bim-/- mice pulsed with chicken gamma globulin conjugated to (4-hydroxy-3-nitrophenyl)acetyl hapten (NP-CGG) were less capable of promoting proliferation in B cells with an NP-specific-Ig heavy chain "knock-in" (B1-8hi) B cell receptor. Finally, we transferred congenically marked CFSE-labeled B1-8hi B cells into C57BL/6 recipients by i.v. injection. 24 hours later, we transferred NP-CGG pulsed DCs from WT or Bim-/- mice into B1-8hi B cell recipients. 7 days later, B1-8hi B cells had proliferated less in recipients that had received antigen-pulsed Bim-/- DCs versus WT DCs. Together this illustrates a functional difference between WT and Bim-/- DCs in regulation of B cell activation and proliferation. We are currently investigating whether antibody responses are also altered by the effects of Bim-/- DCs.

(This work is supported by NIH grants 5 T32 CA009537 and DE16381.)
CD30 / CD30L interactions promote class-switched antibody responses to T-dependent antigens (34.3)
Authors: Willis Cynthia R , Hu Yi-Ling , Leith Anh , Rottman James B
Published: January 2023

The cell-surface glycoproteins CD30 and CD30L are members of the TNFR and TNF superfamilies, respectively. CD30 is expressed on subpopulations of activated T and B cells. CD30L is expressed on activated T cells, macrophages, and dendritic cells, as well as germinal center B cells. CD30 / CD30L interactions provide activation-induced costimulatory signals that sustain T-cell responses, mediate B-cell activity, and generate long-lived memory T cells for chronic B-cell help. Although other activation-induced costimulatory molecules induce antibody isotype class switching, it is less clear what the function is of CD30 signaling on class switching and antibody production. Therefore, we tested the effect of in vivo blockade of the CD30 / CD30L pathway on antibody class switching in response to T-dependent antigens using two systems that require activated-T cells to stimulate B-cell responses. First, BALB/c mice immunized with a T-dependent antigen and treated with anti-CD30L antibodies produced less antigen-specific antibodies of IgG1 and IgE isotypes as compared to control-treated mice, whereas antigen-specific antibodies of the IgM isotype were similar in all groups. Likewise, NZB/W F1 lupus-prone mice treated with anti-CD30L antibodies produced less anti-dsDNA antibodies of the IgG1 and IgG2a isotypes as compared to control-treated mice, whereas anti-dsDNA antibodies of the IgM isotype were similar in all groups. In both systems, specific subpopulations of lymphocytes necessary for responses to T-dependent antigens were reduced by blockade of the CD30 / CD30L pathway. Our results demonstrate that CD30 / CD30L interactions positively regulate T-cell dependent B-cell responses necessary for class-switched antibody responses.
Migration and retention of CD8 T cells within the intestinal epithelium (39.19)
Authors: Casey Kerry Anne , Vezys Vaiva , Masopust David
Published: January 2023

Memory CD8 T cells persist in the small intestinal epithelium in a highly activated state. To test whether an antigen reservoir or other enduring byproduct of infection was required for the maintenance of this phenotype, we transferred TCR transgenic CD8 T cells of fixed specificity into Rag deficient hosts. Firstly, introduction to a lymphopenic environment was sufficient to induce dissemination of donor T cells to the intestinal mucosa. Once there, they adopted the quintessential properties of small intestine lymphocytes including expression of CD69 and Granzyme B, suggesting that the maintenance of an activated phenotype within intestinal epithelium was independent of residual byproducts of infection. Notably, memory CD8 T cells within small intestinal epithelium express CD103 integrin, a ligand for epithelium expressed E-cadherin. Using virus specific TCR transgenic cells deficient for CD103, we assessed the role of this molecule in migration and maintenance. While cells deficient for CD103 migrated to intestinal epithelium without defect shortly after infection, they exhibited a profoundly reduced capacity to persist in that compartment after clearance of the infection. Thus, the establishment of a resident memory CD8 T population with an activated phenotype within the intestinal epithelium occurs in the absence of residual infectious byproducts and is largely dependent upon expression of CD103.
Effect of B7.1 costimulation on T-cell based immunity against TAP-negative cancer can be facilitated by TAP1 expression (40.11)
Authors: Zhang Qian-Jin , Li Xiao-Lin , Liu Yong-Yu , Knight David , Odaka Yoshi , Mathis J. Michael , Shi Runhua , Glass Jonathan
Published: January 2023

Tumors deficient in TAP expression cannot efficiently induce T cell immunity, and are resistant to T cell lysis. Here we show that introduction of B7.1 molecule into TAP-negative (TAP-) and TAP1-transfected (TAP1+) CMT.64 tumor cells dramatically augments T cell immunity against TAP- tumors. Differences in the immune responses were observed between TAP- and TAP1+ CMT.64 cells depending on the dose of inoculation. While mice immunized with B7.1 expressing TAP1+ cells rejected TAP- tumors with high and low dose inoculations, only high dose inoculations with B7.1 expressing TAP- cells resulted in tumor rejection. The induced protective immunities were T cell dependent as indicated by depletion of CD8 or CD4 cells in immune competent mice. We also found that B7.1 and TAP1 coexpression decreased tumorigenicity. In addition, when CMT.64 cells infected with vaccinia viruses carrying B7.1 and/or TAP1 were used for immunization, we found that B7.1 and TAP1 coexpression, but not B7.1 expression alone, played a critical role in induction of protective immunity after a high dose inoculation, suggesting that viral antigens significantly affected TAP negative and B7.1 expressing cells for tumor antigen presentation and T cell priming. Our results indicate that B7.1-provoked antitumor immunity against TAP- cancer is facilitated by TAP1 expression and thus, both genes should be considered for cancer therapy in the future.
IL-18-elicited regulatory NK cells with immunoablative functions (40.22)
Authors: Ullrich Evelyn , Terme Magali , Aymeric Laetitia , Chaput Nathalie , Schultze Joachim , Mackensen Andreas , Zitvogel Laurence
Published: January 2023

Cancer-induced tolerance involves multiple immunosuppressive pathways, which subvert adaptive immune responses. Extensive phenotypic characterization of NK cells from tumor-bearing mice led us to the observation of a consistent expansion of c-kit(CD117)-expressing NK cells (Kit+NK) compromising the NK cell arm of tumor immunosurveillance.

Purified Kit+NK cells failed to produce IFNγ in response to IL-2 and could not promote DC maturation. Adoptive transfer of Kit+NK cells into tumor-bearing mice promoted tumor progression. Micro array comparison of CD3-NK1.1+Kit+ and CD3-NK1.1+Kit- cells revealed that genes involved in tolerance (B7-H1, CTLA4, Lag3, Hmox1) or bone marrow homeostasis (Cxcl2, CD81, CD63, Csf2) were markedly up-regulated in Kit+NK. B7-H1 appeared particularly intriguing as we found that Kit+NK killed Kit-NK cells in a B7-H1/PD-1-dependent manner.

Moreover, IL-18 produced by B16F10 tumors converts Kit- into Kit+NK cells endowed with immunoablative functions in lymph nodes. Upon tumor inoculation, Kit+NK, which upregulate B7-H1, accumulate in lymphoid organs in an IL-18R/MyD88-dependent manner and directly kill conventional NK cells, thereby promoting the progression of NK-controlled cancers.

In summary, these data suggest that IL-18 promotes the expansion of Kit+NK cells endowed with immunosuppressive functions that could act as negative regulator of general NK responses.
Attenuation effects of CD4+CD25+ regulatory T cells on cisplatin induced nephrotoxicity in mice (40.24)
Authors: Lee Hyojung , Keum Dongjune , Lee Youngseop , Hong Moo-Chang , Shin Min-Kyu , Kim Yangseok , Bae Hyunsu
Published: January 2023

Cisplatin is a chemotherapeutic agent, which is widely used for a variety of malignancies, such as ovarian, head and neck cancers. However, its nephrotoxicity limits the use of this drug. This research examines whether CD4+CD25+ Regulatory T cells (Treg) have protective effects on cisplatin induced acute renal failure in mice. For the experiment, 1x 106 CD4+CD25+ T cells, CD4+CD25- T cell, were reconstituted into each group of T cell-deficient (nu/nu) mice. After 10 days, all groups of mice received a single dose of cisplatin at 40mg/kg intraperitoneally. Treg adoptive transferred nu/nu mice had a marked attenuation in serum creatine, BUN, and survival. At 48h time point, the Treg group had a significantly reduced increase in TNF-a, IL-1b cytokines when compared with the control group and CD4+CD25- group. In addition, the renal tubular injury worsened less in Treg reconstituted mice. These results suggest that CD4+CD25+ regulatory T cell can attenuate cisplatin induced acute kidney injury.
Bi-functional fusion proteins: Induction of effective apoptosis of malignant cell lines by dual signaling (41.14)
Authors: Orbach Ariel , Rachmilewitz Jacob , Tykocinski Mark L , Dranitzki Elhalel Michal
Published: January 2023

One mechanism involved in evolution of death resistant malignant cells is upregulation of anti-apoptotic signals. CTLA-4FasL, that integrates the capacities to bind to B7 and to Fas receptor (CD95), induces effective apoptosis of activated T cells by activating the caspases in parallel to decreasing the expression of the anti-apoptoic protein cFLIP. Aims: Explore the ability of CTLA-4FasL and CD40FasL (binds to CD40L and CD95) to induce apoptosis of malignant B and T cell lines, and test whether they decrease cFLIP in conjunction with caspases activation. Results: CTLA-4FasL induced effective apoptosis of malignant T and B cell lines that express CD95 (tested by annexin/PI). Activation of caspase 3, 8 and 9 was verified by western blots. Only B7 expressing B cells responded to CTLA-4FasL presence with rapid abrogation of cFLIP expression. CD40FasL also induced apoptosis of T and B cell lines, and was most toxic to T cell line that expressed high levels of CD40L in addition to CD95. These CD40L expressing cells decrease cFLIP in response to CD40FasL. CTLA-4FasL and CD404FasL were more potent than either of their components, alone or in combination. Conclusion: Both fusion proteins activate Fas receptor, but maximal potency is seen when target malignant cells express a binding site for the second domain of the fusion proteins (B7 or CD40L). This potency is probably related to inhibition of anti-apoptotic signals.
Antitumor efficacy of palmitylated IL-2 in adoptive and active immunotherapies (41.30)
Authors: Chou Sharon , Xu Lipeng , Chen Aoshuang , Zheng Guoxing
Published: January 2023

Although soluble IL-2 has shown antitumor activity in preclinical studies, clinical use of IL-2 (usually applied systemically) has been limited by its severe toxicity. This necessitates efforts on minimizing toxicity of IL-2-based therapies. Previously, we showed that recombinant immunoregulatory proteins (such as costimulators), after being chemically palmitylated, can be incorporated into cell membranes via the lipid anchorage. Hence, we hypothesized that palmitylation of IL-2 may permit controlled targeting of IL-2, to either antitumor T cells or cells in the tumor microenvironment, thereby bypassing the need to apply IL-2 systemically. In this study, we demonstrated that palmitylated human IL-2 (pal-IL-2) can be incorporated into the surfaces of antitumor T cells, and the pal-IL-2-modified T cells show increases in proliferation and survival in vitro. Subsequently, we showed that pal-IL-2 modification of CD8+ TCR-transgenic OT-1 T cells enhances therapeutic efficacy of the T cells, upon adoptive transfer, against established E.G7-OVA tumors. Also, we showed that intratumoral injection of pal-IL-2 into B16F10 tumor-bearing mice inhibits the growth of tumors and prolongs the survival of treated animals, whereas injection of soluble IL-2 (as control) has minimal efficacy. Collectively, our study suggests that palmitylation of cytokines may provide a novel approach to target cytokines to antitumor T cells or the tumor microenvironment, to minimize the systemic toxicity and, thus, improve the therapeutic efficacy of the cytokines.
Loss of pre-existing tumor-specific T-cells and increase in regulatory T-cells after peptide-vaccination in combination with PADRE, CpG 7909, and Montanide ISA51 (41.42)
Authors: Kuball Jurgen , deBoer Karin , Wagner Eva , Wattad Mohammed , Antunes Edite , Lotz Carina , vanDorp Suzanne , Hol Samantha , Huber Christoph , Greenberg Philip D , Heit Wolfgang , Theobald Matthias
Published: January 2023

T-cells with specificity for antigens derived from Wilms-Tumor-gene (WT1), Proteinase3(Pr3), and mucin1(MUC1) have been demonstrated to lyse AML blasts and MM cells and strategies to enhance or induce such tumor-specific T-cells by vaccination are currently explored in multiple clinical trials. To test the safety and immunogenicity of a vaccine composed of WT1-, Pr3-, and MUC1-derived Class I-restricted peptides in combination with CpG 7909 and Montanide ISA51 adjuvants and the PADRE helper epitope, 9 patients with AML or MM were repetitively vaccinated. No clinical responses were observed. Comprehensive immunologic analyses revealed that 5 out of 9 patients harboured non-functional and 2 out of 9 functional MUC1- or WT1-specific CD8+ T-cells prior vaccination. However, neither the pre-existing nor naive de novo WT1-/Pr3-/MUC1-specific CD8+ cells were expanded in vivo by vaccination. Moreover, the pre-existing WT1-/MUC1-specific T-cells became no longer detected following vaccination, whereas the frequency and function of CMV-specific CD8+ T-cells was unchanged. A more than 2-fold increase in PADRE-specific CD4+ T-helper-cells was observed but these did not produce IL2, and CD4+ T-cells with a regulatory phenotype increased more than 2-fold. These results are in stark contrast to previous vaccination studies, and should be carefully taken into consideration in the context of multiple ongoing as well as future clinical vaccination trials.
Denileukin diftitox (DT) and interferon (IFN)-alpha boost numbers and maturation of a novel stimulatory CD11b+Gr-1+ cell in ovarian cancer (41.62)
Authors: Thibodeaux Suzanne R , Shin Tahiro , Daniel Benjamin , Lin Pei Yi , Livi Carolina , Wall Shawna , Brumlik Michael , Kious Mark , Jeansonne Duane , Ludwig Sara , Curiel Tyler
Published: January 2023

CD11b+Gr-1+ cells increase in spleen, ascites and mesenteric lymph nodes in BL6 mice challenged with intraperitoneal OVA-expressing ID8 ovarian carcinoma cells, and boost rather than suppress tumor (OVA)-specific immunity. We studied regulation of CD11b+Gr-1+ cells. ID8-challenged mice treated with DT to deplete regulatory T cells, or DT plus IFN-α had improved survival. Surprisingly, DT or IFN-α each increased percentage and numbers of CD11b+Gr-1+ cells in spleen and ascites. IFN-α, but not DT, increased CD11b+Gr-1+ cell CD86 and DR expression, suggesting maturation and differential DT versus IFN-α effects. Effects are not tumor-specific as DT or IFN-α each also increased CD11b+Gr-1+ cells in BL6 mice without tumor. ID8 tumor challenge in B7-H1-/- BL6 mice resulted in ~50% fewer CD11b+Gr-1+ cells compared to after ID8 challenge in wild type mice, suggesting that B7-H1 signals regulate CD11b+Gr-1+ cell generation. Intradermal EG7 tumor in BL6 mice boosts suppressive CD11b+Gr-1+ cells. Strikingly, DT or IFN-α each reduced CD11b+Gr-1+ cells in EG7-bearing mice ~60%. Increasing stimulatory CD11b+Gr-1+ cells or reducing inhibitory CD11b+Gr-1+ cells could represent novel mechanisms of treatment effects of Treg depletion or IFN-α.

Supported by the NCI, the Rippel Foundation, and Voelcker and Greehey Funds.
Modulation of immune response by varying ligand density on the surface of nanoparticles (42.22)
Authors: Bandyopadhyay Arunima , Bockenstedt Linda , fahmy Tarek
Published: January 2023

Targeting antigen presenting cells with biodegradable nanoparticles provides an excellent opportunity for the development of vaccines. Polymeric nanoparticles with entrapped antigens serve as an excellent platform for controlled release of antigens. Immune response can be optimized and enhanced by selectively targeting the antigen loaded nanoparticles to dendritic cells. Here we report our findings using the PLGA nanoparticulate system to target DCs by targeting their surface receptor DEC205. These nanoparticles encapsulate the model antigen ovalbumin and are surface modified with anti DEC205 at different ligand densities. Our studies demonstrate that targeting bone marrow derived DCs using different modifications of nanoparticles but targeting the same receptor lead to altered T cell responses. These studies have implications in designing a novel nanoparticle based vaccine delivery system.
Isolation of high affinity human scFv antibodies against mouse and human tumor endothelial marker 1 (TEM1) using yeast-display scFv library derived from an autoimmune patient. (42.4)
Authors: Zhao Aizhi , Li Chunsheng , Coukos George , Siegel Don L , Scholler Nathalie
Published: January 2023

TEM1 is predominantly expressed on neovasculature, thus ideally suited for targeted imaging and therapy for cancer. Yeast has emerged as an alternative technology for display and selection of recombinant antibody fragments (scFv). Because autoimmune disorders may produce self-reactive specificities, an autoimmune repertoire derived from a patient with thrombotic thrombocytopenic purpura (TTP) was screened to isolate anti-TEM1 scFv.

A yeast-display scFv library was constructed by homologous recombination of the TTP patient repertoire originally expressed on M13 bacteriophage. The library (10^9 members) was flow sorted with decreasing antigen concentration until a scFv pool detecting 2 nM of rhTEM1/GST was obtained. Secreted scFv were expressed and 470 scFv were screened by ELISA for binding to rhTEM1/GST. Nearly 50% of the scFv gave colorimetric signals higher than 3 standard deviations above background. No cross-reactivity with GST control protein was detected. Sequencing of 29 scFv identified 8 unique antibodies. Two of these clones exhibited Kd's in the 100 pM range and bound to both mouse and human TEM1. Such cross-reactive anti-mouse/human TEM will facilitate proof of concept for the development of novel anti-angiogenic targeted therapies.

Our data suggest that the use of large scFv libraries derived from autoimmune patient repertoires may provide a rich source of high affinity antibody reagents to therapeutically relevant molecules.

This work was supported by the Claneil Foundation
Mimogen restores the HBV specific cytotoxic T lymphocytes responses in chronic HBV carriers (42.9)
Authors: Wu Yuzhang
Published: January 2023

Despite effective prophylactic vaccines against hepatitis B virus existing for over 20 years, there is no effective therapy in the treatment of chronic hepatitis B (CHB). Viral persistence is thought to be related to poor HBV-specific T-cell responses. Whereas we recently showed that the frequency of HBV-specific T-cells could be detectable but not effective to clear virus which reflecting an early intermediate stage of differentiation. A phase II clinical trial was performed in CHB carriers to investigate whether the artificial antigen (mimogen) could restore T-cell responsiveness and change the stage of differentiation. 24 patients with chronic hepatitis B nonresponder to approved treatments for HBV infection were enrolled in the trial. . HBV-specific T cell responses were assessed by proliferation, tetramer staining, intracellula cytokine staining (ICCS) and cytometric bead array (CBA). The study provides evidence that mimogen is safe and immunologically effective. Despite increasing the frequency of HBV-specific CTLs, mimogen could restore the function and convert the premature state of HBV-specific CTLs. Thus mimogen may be a new immunotherapeutic for the cure of CHB.
Exercise reduces inflammatory responses to influenza virus infectoin in aged mice (43.12)
Authors: Yu Shan , Sim Young-Je , Singh Navrozedeep , Kohut Marian
Published: January 2023

In order to examine the extent to which exercise alters the inflammatory and clinical responses to respiratory infection in old mice, 19-month old male BALB/cJ mice were assigned to exercise (EX) and non-exercise (non-EX) groups. After 3 months of exercise or control treatment, mice were intranasally infected with influenza virus. Bronchoalveolar lavage (BAL) fluid was collected at 4 and 7 days post-infection (DPI) to assess viral titer and cytokines/chemokine profiles. Sera were collected for antibody measurement at 7 and 10 DPI. Lungs were collected for lesion scores at 10DPI. Weight loss and food intake were recorded daily. The results showed that the virus titer in EX mice at 4 DPI was significantly lower than non-EX mice. At 7DPI, 14% of the mice in EX had virus in lungs whereas 35% of non-EX had detectable virus. IL-12p70 & IL-1α in BAL were significantly lower in EX than in non-EX, with trends for reduced IP-10, MIP-1α and RANTES at 4 DPI. At 7 DPI, EX mice had less IL-12p70, IL-1β, and IFNγ in BAL as compared to non-EX mice, whereas IL-10 tended to be higher in EX mice. Anti-influenza IgG titer was significantly lower in EX compared to non-EX at 7 and 10 DPI. Lung lesion scores were significantly lower in EX compared to that in non-EX at 10 DPI. Non-EX lost more weight and ate less food than EX over time. In summary, exercise improved influenza viral clearance, improved clinical scores, and reduced lung inflammation in aged mice.
Investigating the immunomodulatory effects of Chinese herbs on mouse (43.16)
Authors: Li Ching , Chu I-Ching , Liao Hui-Fen
Published: January 2023

Chinese herbs Radix Isatidis and Viola yedoensis Makino have been implicated to possess antiviral activity. This study is to test whether these herbs are capable of modulating the immune function in Balb/cByJ mouse that supports such activity. We first confirmed that the extract from Viola yedoensis Makino, but not Radix Isatidis or a homeopathic medicinal drink Método Canova, could inhibit the replication of HSV-1 and enterovirus 71 in human neuroblastoma SK-N-SH cell line. Subsequently, the herbal extracts and Método Canova were subjected to the immunomodulatory tests with 120 6-8 weeks old mice. The animals were randomly divided into to 8 groups, of which 6 groups were fed with the half human-to-mouse-body-weight-adjusted doses of the herbal medicine and Método Canova for 9 weeks. At 8th week, these 6 groups and an unfed group of mice were injected peritoneally with one dose of the immune suppressant 5'-fluorouracil, and all 8 groups of animals were sacrificed a week later for immune function evaluations, which included the measurements of the WBC counts and the IgG levels, the numbers for B-, T-, and I-A/I-E-splenocytes, the proliferative rates for B- and T-splenocytes upon stimulation, and the serum cytokine and chemokine levels with protein arrays. Among the tests, we detected that serum BLC and MIP-2 levels were dramatically increased in mice fed with Radix Isatidis. This result indicates that taking the herb extract can improve the responsiveness of immune cells to inflammation, as these two chemokines possess the capability of inducing neutrophils, macrophages, and B-cells chemotaxis. However, no mouse in any group showed a clear improvement in cellular immunity that can suppress viral infections.