bluuebunny/crossref_metadata_2025 huggingface dataset:
Vasorin Expression During Mouse B cell Development (138.31)
Authors: Franco Daniel A. , Reardon Christopher L.
Published: January 2023

The stages of B cell development are delineated by stage-specific timing of gene and cell surface marker expression coinciding with exposure to cytokines and growth factors. Transforming growth factor beta (TGF-β) has been previously shown to affect lymphopoesis, as well as more mature processes, such as B-cell isotype switching. Receptors for TGF-β are diverse and widely distributed but display some tissue specificity. Here we describe expression of a Type-I TGF-β membrane receptor, vasorin, previously reported to be almost exclusively expressed by vascular tissue. Using RT-PCR analysis, the message for vasorin was identified in mouse pre-B cell lines prior to BCR gene rearrangement. Thus, vasorin expression may be closely linked to early lymphocyte development at the pro and pre-B stages. To verify that this finding was not an artifact of the cell lines, we looked for vasorin message in primary cell suspensions of spleen, thymus and liver from neonatal mice. Vasorin gene expression is readily detected in the primary repositories of B cell development, the spleen and liver, but not in primary thymocytes. These data suggest that receptor binding of TGF-β may be more vital to B-cell than to T-cell development. Vasorin expression by progenitor B cells may ensure measured proliferation via TGF-β receptor signaling.
VH Gene Usage and Restricted B cell Development (138.4)
Authors: Robbins Gregory R. , Knight Katherine L.
Published: January 2023

Signaling through the preBCR is an important checkpoint in B cell development in which the capacity for μ-chain to pair with surrogate light chain (SLC) is tested. Mutant ali/ali rabbits that lack the predominantly rearranged gene, VH1, use VH4 in most VDJ gene rearrangements. Surprisingly, the periphery of these rabbits is initially dominated by VHn-utilizing B cells, not VH4-utilizing B cells. We hypothesize that development of VH4-utilizing B cells is blocked in the bone marrow at the preB cell stage due to weak interaction of the μ-chain with SLC. The IgH CDR3 is important for formation of, and for signaling through the preBCR. Previous crystal structure analysis demonstrated that IgH CDR3 interacts with both components of the SLC, and raises the possibility that VH4-utilizing μ-chains require CDR3 of a particular size and/or amino acid sequence for SLC pairing. We compared IgH CDR3 sequences of VH4- and VHn-utilizing B cells and although we found no obvious differences in amino acid sequences, we did find that the CDR3 of VH4-utilizing B cells was significantly shorter than those found associated with VHn VDJ genes. We hypothesize that long IgH CDR3s encoded by VH4-utilizing VDJ genes prevent pairing with SLC. We are using in vitro experiments to test if pairing of VH4-derived μ-chains with SLC is restricted by CDR3 length. These studies will further identify the structural parameters required for preBCR formation.
IgE Signaling Selectively Suppresses FceRI Beta Chain Expression (139.2)
Authors: Ryan John J. , Barnstein Brian , Brenzovich Jennifer
Published: January 2023

Mast cells are critical players in allergic disease mediated by IgE and FcεRI interactions. In mice, FcεRI functions as a tetramer, composed of an IgE binding alpha subunit, two ITAM bearing gamma subunits, and a signal enhancing/amplifying beta subunit. Little is known about beta chain regulation. We activated mast cells with IgE + antigen and determined the resulting effects on beta chain mRNA, protein levels and surface expression. IgE crosslinkage inhibited beta chain mRNA expression, with a nadir at 5 hours post-stimulation. This was matched by reduced expression of the beta chain, with no change in the alpha or gamma subunits. A truncated form of beta, termed beta-t, may suppress IgE receptor expression. We found that IgE-mediated signaling also inhibited beta-t expression, and that beta-t was slower to return to baseline levels than conventional beta. We were able to classify the beta chain mRNA regulation pathway as primarily involving Fyn, Syk, PI3K, and NFκB. Monomeric IgE is known to greatly increase surface FceRI expression. Interestingly, we found that IgE crosslinkage returned membrane FceRI to basal levels in cells previously incubated with monomeric IgE. Furthermore, the presence of antigen during culture with IgE prevented FceRI upregulation. These data suggest that FceRI signaling not only activates mast cells but may also convey a homeostatic signal returning FceRI expression to a reduced basal level, perhaps to suppress continued signaling. Loss of this suppressive effect could be art of atopic disease etiology. Supported by NIH grants 1R01 AI59638 and U19A1077435.
The peptidyl-prolyl isomerase Pin1 suppresses Bax activation and mitochondrial targeting in human eosinophils (139.7)
Authors: Shen Zhong-Jian , Malter James S
Published: January 2023

Pulmonary eosinophilic inflammation is a defining feature of allergic asthma. Within a few days of allergen challenge, airway eosinophils (Eos) increase by ~20-fold in both human and animals. Genetic ablation of Eos markedly attenuates submucosal matrix deposition, airway smooth muscle hyperplasia as well as airway hyperresponsiveness, suggesting a critical role of these cells in asthmatic lung pathology. Normally short-lived (~3 days) peripheral blood Eos acquire prolonged survival (>7 days) in the lung by contacting with anti-apoptotic cytokines GM-CSF and IL-5, which increase after allergen exposure. How these cytokines suppress Eos apoptosis remains largely unknown. Here we show that Pin1 mediates GM-CSF/IL-5 signaling, inhibits activation of proapoptotic Bax and its mitochondrial disruption. Both cytokines considerably increased Pin1 isomerase activity while induced Erk1/2-mediated phosphorylation of Bax at Pin1 recognition sites, allowing Bax interaction with Pin1. Pin1 blockade disrupted this interaction, resulting in Bax N-terminal activation, mitochondrial translocation and downstream caspase 9 activation even in the presence of survival cytokines. Our study uncovers previously unknown mechanism by which Pin1 isomerization regulates Bax activation. Pin1 may be an attractive therapeutic target for the asthma and other eosinophilic diseases.
Negative Regulation of Human Th2 cells by Type I Interferon (140.15)
Authors: Huber Jonathan P , Chowdhury Fatema Z , Gill Michelle A , Gruchalla Rebecca S , Farrar J David
Published: January 2023

Type I interferon (IFN-α/β) is a potent anti-viral cytokine that regulates multiple aspects of innate and adaptive responses to viral infections. As some respiratory viral infections have been linked to the etiology and exacerbation of asthma, we wished to determine the effects of IFN-α/β on driving the development of human Th2 cells. In these studies, we found that IFNα/β inhibited the ability of IL-4 to promote Th2 differentiation and significantly de-stablized cytokine expression from fully polarized Th2 cells. Further, this effect was specific to IFNα/β as neither IL-12 nor IFNγ inhibited IL-4-regulated Th2 development or stability. IFNα/β inhibited Th2 development by blocking the induction of GATA-3, and this effect was found to bypass the induction of GATA3 via IL-4-mediated STAT6 activation. Finally, IFNα/β also potently inhibited Th2 cytokine secretion from fully committed CRTH2+ cells isolated directly from peripheral blood. Thus, we have found a novel function of IFNα/β in the inhibition of Th2 responses that may be useful as a therapeutic approach for the treatment of asthma.
Humanization of an anti-CεmX monoclonal antibody 4B12 for potential clinical use in targeting mIgE+B cells (140.19)
Authors: Wu Pheidias Che-Hao , Chen Jiun-Bo , Hung Alfur Fu-Hsin , Hsueh Ying-Chao , Chang Tse Wen
Published: January 2023

The CεmX domain of 52 a.a. residues, located between the CH4 domain and membrane-anchor segment of human membrane-bound ? chain on mIgE+ B cells, is a unique antigenic site for the immunological targeting of those B cells. We have demonstrated that a chimeric form of anti-CεmX monoclonal antibody (mAb), 4B12, can induce apoptosis and ADCC against a human mIgE.Fc+ B cell transfectoma via effector cells from PBMC in vitro (presented by another abstract in this meeting). To enhance the clinical applicability of 4B12, we have humanized it by first grafting its six complementary-determining regions (CDRs) into human Ig VH subgroup IV and V? subgroup II frameworks (FWs). Those residues in the FWs of VH and VL of the parental murine 4B12 that are essential for Fv dimer interaction and CDR conformation were identified by analyzing the binding affinity of a number of designed Ab variants with site-directed mutations, using competitive ELISA and surface plasmon resonance analysis. A humanized 4B12 retaining the affinity of its parent was obtained by adopting all FWs of the selected human V? template without any change and all FWs of the selected VH with only one residue change. The Val at position 71 of the human VH was replaced by Arg at the corresponding position of the murine VH. Based on molecular modeling analysis, it is likely that such a change is probably crucial for maintaining the proper structural packing of Arg71 with Ile29 in the CDRH1 and Gly55 in the CDRH2. The KD of the chimeric and humanized 4B12 are 5.37×10-9 M-1 and 5.76×10-9 M-1, respectively. Affinity enhancement of humanized 4B12 by selective CDR mutations is in progress.
Anti-CmεX monoclonal antibodies potentially applicable for targeting mIgE-expressing B cells (140.20)
Authors: Chen Jiun-Bo , Wu Pheidias C. , Hung Alfur Fu-Hsin , Yu Hui-Ming , Chang Tse Wen
Published: January 2023

On B lymphocytes, membrane-bound IgE (mIgE) is part of BCR and essential for generating isotype-specific IgE response. On mIgE+ B cells in man, the membrane-bound ε chain (mε) exists predominantly in the long form (mεL), containing 52-a.a. CεmX domain between CH4 and the C-terminal membrane-anchoring segment; the conventional form of mε (mεS) exists in minor proportions. CεmX thus provides an attractive site for immunological targeting of mIgE+ cells. Here we have shown that 9 newly prepared CεmX-specific monoclonal antibodies (mAbs), as well as previously reported a20, can bind to mIgE.FcL-expressing CHO cells, while only 4B12 and 26H2 can bind to mIgE.FcL-expressing B cell line Ramos cells. In epitope mapping studies using synthetic peptides representing different parts of CεmX, 4B12 was shown to bind to the N-terminal part, 26H2 the middle part, and all others the C-terminal part of CεmX. These results suggest that the C-terminal part of CεmX is sterically hindered for mAb binding by associated components in the BCR. The chimerized mAbs c4B12 and c26H2, when cross-linked by secondary antibodies, could lyse mIgE.FcL-expressing Ramos cells by apoptosis through a BCR-dependent caspase pathway. Using PBMCs as the source of effector cells, c4B12 and c26H2 could cause ADCC against mIgE.FcL-expressing Ramos cells in a does-dependent fashion. These results indicate that anti-CεmX mAbs, 4B12 and 26H2, can bind to native mIgE on B cells and are potentially useful for targeting mIgE+ B cells for controlling IgE production.
Antigen –specific immunotherapy inhibited Th2 via GATA-3 and Th17 cells in the absence of T-bet in an allergic asthma model (140.6)
Authors: Zhivkova Nataliya , Karwot Roman , Maxeiner Joachim , Soó-Becker Kinga , Lehr Hans-Anton , Susetta Finotto
Published: January 2023

Asthma is caused by T-helper 2 (Th2)-driven immune-responses in which upregulation of GATA-3 has been shown to play a crucial role in both in humans and in murine models of this disease.

T-cell tolerance, induced by respiratory exposure to allergen prior to allergen sensitization, can inhibit the development of airway hyperractivity (AHR), a cardinal feature of asthma. However, the subset of T cells that mediates this protective function in allergic asthma has not been clearly identified yet. We recently set up a tolerogenic model of allergic asthma. In this model, peripheral T cell tolerance is induced by respiratory exposure to allergen and is characterized by decreased Th2 (IL-4,IL-5 and IL-13)-based responses via downregulation of the main Th2 transcription factor GATA-3 in the lung which results in decreased allergen induced AHR and lung eosinophilia.

In addition, we found that our tolerogenic model is characterized also by decreased differentiation of Th17 cells. Moreover respiratory exposure to allergen prior to sensitization protected also airway hyperreactivity induced in the absence of T-bet. Thus this model suppressed effector Th2 and Th17 mediated immune responses also in the absence of IFN-gamma and could therefore provide a new avenue also for genetically determined allergic diseases.

This work is supported by a SFB 548-B8 project in Mainz, Germany
Dendritic cell (DC)- derived exosomes spread donor allo-antigen between recipient's DC following cardiac transplantation (141.11)
Authors: Montecalvo Angela , Divito Sherrie J , Shufesky William J , Beer-Stolz Donna , Larregina Adriana T , Morelli Adrian E
Published: January 2023

Exosomes are nanovesicles generated in multivesicular endosomes that are released to the extracellular space by different cell types. Since DC-derived exosomes (dexosomes) express MHC-peptide and costimulatory molecules, we investigated the role of dexosomes in elicitation of the anti-donor response in transplantation. Dexosomes were generated from bone marrow-derived DC and labeled with PKH67 for traffic studies. CD4+ TCRtg T cells (Thy1.1+) specific for the IEα52-68 (BALB/c)-IAb (B10) complex were CFSE-labeled and transferred i.v. to host Thy1.2+ B6 mice. Graft infiltrating DC were isolated from BALB/c (CD45.2+) cardiac grafts 3 days after transplantation in B6 (CD45.2+) recipients and genetically-engineered ex vivo to release exosomes expressing the reporter marker eGFP, and then transferred i.v. into host CD45.1+ B6 mice. We demonstrated that although graft-infiltrating leukocytes release exosomes ex vivo, they do not secrete enough exosomes in circulation to stimulate donor-reactive T-cells in lymphoid organs. Instead, migrating DCs (generated in vitro or isolated from allografts), once they home in the spleen, they transfer exosomes expressing eGFP to spleen-resident DCs. Thus, exchange of exosomes between DCs in lymphoid organs constitutes a mechanism by which passenger leukocytes transfer alloAg to recipient's APC and amplify generation of donor-reactive T-cells.
Bioimaging to monitor the in vivo Distribution of administered Mesenchymal stem cells (MSCs) for treating Graft versus Host Disease (GVHD) (141.12)
Authors: Ju Sun-Young , Cho Kyung-Ah , Jung Yun-Jae , Park Seong-Yeol , Choi Yong-Bock , Hong Kyeong-Man , Kim Han-Seong , Seoh Ju-Young , Woo So-Youn , Ryu Kyung-Ha
Published: January 2023

Acute Graft-versus-Host Disease (GVHD) remains a major complication after allogeneic hematopoietic cell transplantation (HCT). Several publications show a potential benefit of human MSCs for the treatment of refractory GVHD; however, their cellular fate and distribution remain unclear. We set out to develop an animal model that can be used to study the effect of MSCs on GVHD. GVHD was induced by transplantating C3H/he or C57BL/6 donor bone marrow cells (5 × 106) and CD3+ spleen cells (1 × 106) into lethally irradiated BALB/c recipient mice. C3H/10T1/2 cell lines were transfected with a pcDNA3.1-luciferase construct (luc-MSCs). Bioluminescence of these cells was measured (IVIS-Xenogen system) after treatment with luciferin, showing a linear increase of photon emission with rising cell numbers. To track these cells in vivo, groups of mice were injected with various dose luc-MSCs per animal and imaged with bioluminescence imaging at various time points. After transplantation, mice were monitored daily for weight and survival. Differences in symptom severity were compared using a clinical GVHD scoring system. We conclude that this mouse model can be used to study the effects of MSCs on acute GVHD and the described bioluminescence technology provides a sensitive and safe tool for the repeated in vivo tracking of infused luc-MSCs in GVHD target organs.
Selective deletion of HY-reactive CD8+ T cells by saporin-coupled MHC class I tetramers (141.15)
Authors: Hess Paul R , Murray Sabrina L , Buntzman Adam S , Young Ellen F , Steele Shaun P , Collins Edward J , Frelinger Jeffrey A
Published: January 2023

CD8+ T cells are the principal effectors mediating rejection of allografts performed across minor histocompatibility (H) antigen (Ag) barriers. Previously we have shown that cytotoxic class I tetramers are capable of selectively deleting cognate T cells in vivo; in this study, we hypothesized that tetramers targeting minor H Ag-reactive T cells would permit long-term tolerance of mismatched grafts. To investigate this prediction, we used the male HY Ag in C57BL/6 mice as a model of a minor H barrier. A single immunization of female mice with male bone marrow cells reliably primed T cells against the two major H2-Db-restricted HY epitopes, Uty and Smcy, with peak expansion at 14 days. We prepared Db-Uty and Db-Smcy tetramers coupled to the ribosome-inactivating toxin, saporin. When administered prior to immunization, cognate CD8+ T cell responses were no longer evident in the blood at day 14, and cytotoxic T lymphocyte (CTL) responses measured against peptide-pulsed and native targets in vivo were significantly diminished. Treatment with saporin-coupled HY tetramers allowed prolonged survival of male bone marrow allografts in female mice. This work demonstrates that cytotoxic tetramers can exert meaningful biologic effects in vivo, and illustrates a potentially useful means of obtaining Ag-specific transplantation tolerance. This project is supported by grants from the NIH (1K08DK082264-01A1) and NCSU-CVM.
CD40L-specific mAb mediates its tolerogenic effects through engagement of FcgRIIB, not via depletion of activated T cells (141.17)
Authors: Lucas Carrie L. , Fehr Thomas , Haspot Fabienne , Sykes Megan
Published: January 2023

Objective: To determine the mechanism by which a widely used anti-mouse CD40L mAb (clone MR1) given with allogeneic bone marrow cells (BMCs) tolerizes peripheral CD8 T cells.

Methods: B6 mice received 3Gy total body irradiation one day prior to ip injection of 2mg a-CD40L & iv infusion of 25x106 MHC-mismatched B10.A BMCs. This regimen induces long-term mixed chimerism, as measured by durable presence of donor leukocytes, without depletion of recipient CD4 or CD8 T cells.

Results: Although genetic deficiency of CD40L alone allowed graft acceptance in recipients depleted of CD8 cells, it did not prevent rejection in mice replete with CD8 T cells. Thus, Fc-dependent functions of a-CD40L were studied. A role for complement was excluded, as 4/5 C3 KO recipients became chimeric. CD8 T cell-intrinsic CD40L was dispensable (3/5 chimeras each in mice with WT or KO CD8 cells), indicating that a-CD40L need not act directly on CD8 cells. However, FcgRIIB KO recipients promptly rejected donor BM unless CD8 T cells were depleted (5/7, 0/5, 7/8, & 5/7 chimeras for WT, KO, WT + a-CD8, & KO + a-CD8 groups, respectively). Recipient B cells are the likely source of FcgRIIB, as DC-derived FcgRIIB was not essential (4/6 chimeras each in mice with WT or KO DC).

Conclusions: A-CD40L with allogeneic BMCs generates immune complexes that ligate FcgRIIB, inducing as yet undefined downstream effects essential for CD8 T cell tolerance.

Supported by NIH Grant RO1 49915 & NDSEG Fellowship.
Analysis of CD4 Gene Expression in Hematopoietic Cell Transplant (HCT) Tolerant Patients (141.20)
Authors: Tabellini Laura , Fan Wenhong , Bumgarner Tara E , Flowers Mary E , Grogan Bryan M , Martin Paul J , Zhao Lue Ping , Hansen John A
Published: January 2023

Although graft-vs-host disease (GVHD) remains a significant problem following allo HCT, tolerance eventually occurs in many patients. To better understand the mechanism(s), RNA from purified CD4 T cells from 22 tolerant patients (T) and 20 normal controls (NC) was hybridized on Illumina Ref-8 expression arrays. Transcriptional profiles of tolerant patients were very different from NC. A cluster analysis of genes showing differential expression revealed 2 distinct subgroups based on the time of study post-HCT: "early" (mean 1.0, 1.0-2.6) years (TE, n=10), and "late" (mean 4.0, 2.5-7.4) years (TL, n=12).

1073 genes were differentially expressed between TE and NC, 810 genes were differentially expressed between TL and NC, and there were 203 genes overlapping TE and TL. Analysis of TE revealed increased gene expression of the IFNG, TCR and IL2 signaling pathways, with increased expression of CTLA4. Analysis of TL revealed down regulation of genes involved in glycolysis, nucleotides biosynthesis and cell cycle, and down regulation of the IL2 and TCR signaling pathways compared to NC and TE.

These data suggest that 1) tolerance is an active and dynamic process, and 2) that this adaptive response evolves over time. The changes observed suggest elements of both immune regulation and immune exhaustion. Functional studies will be necessary to further interpret these transcriptional changes and define mechanisms.
Quiescent recipient splenic dendritic cells (DC) re-process therapeutic DC into alloantigen for presentation to and down-regulation of anti-donor T cells to prolong allograft survival (141.31)
Authors: Divito Sherrie J. , Montecalvo Angela , Wang Zhiliang , Shufesky William J. , Erdos Geza , Larregina Adriana T. , Morelli Adrian E.
Published: January 2023

Current dogma in transplantation presumes that therapeutic maturation-resistant dendritic cells (MR-DC) prevent allograft rejection by directly interacting with donor-reactive T-cells in vivo. However, this assumption remains untested. Our study therefore aimed to elucidate the mechanism by which donor MR-DC down-regulate the anti-donor response to prolong allograft survival in mice. We employed as prototypic donor MR-DC, bone marrow-derived DC treated with the active form of vitamin D3, 1α,25(OH)2D3. We demonstrate that donor-derived MR-DC prolonged cardiac allograft survival similarly as donor apoptotic or viable splenocytes, suggesting that different donor-derived cellular therapies share a common mechanism of action. We determined that once injected i.v., MR-DC were short-lived and failed to directly tolerize donor-reactive T-cells. Rather, apoptotic MR-DC were internalized and re-processed by quiescent recipient splenic CD11chiCD8α+ and CD11chiCD8- DC, which in turn induced deletion of effector T-cells and outgrowth of regulatory T-cells via the indirect pathway. Importantly, activated recipient DC induced robust T-cell activation via indirect presentation. Our data suggest that therapeutic DC, like other more practical cellular therapies, serve as a source of alloantigen for recipient quiescent DC that in turn down-regulate the anti-donor response to prolong allograft survival.
Leukaemia Inhibitory Factor (LIF) is a Treg cytokine (141.35)
Authors: Metcalfe Su M , Gao Wenda , Muthukumarana Poorni , Zhou Qiang , Fahmy Tarek , Strom Terry B
Published: January 2023

Priming of the T lymphocyte results in either regulatory (Treg), or effector (Teff), lineage development. During priming the immediate micro-environment contributes to outcome and LIF, a pluripotent cytokine of the IL6 family, appears to be linked to Treg: in contrast IL6 is associated with Teff. Here we ask, is LIF a Treg cytokine? We found in vitro (i) exogenous LIF (10ng/ml) increased transcription of both Foxp3 - the Treg lineage transcription factor - and LIF: and (ii) when naive CD4+ Foxp3- T cells were activated by anti-CD3/anti-CD28, the presence of exogenous LIF enhanced numbers of Foxp3+ cells. We therefore asked, can LIF be used as a tolerogenic therapy? To avoid LIF's rapid degradation by serum proteases we developed a delivery system wherein LIF was encapsulated within nanoparticles and targeted to CD4+ T cells by surface-attached anti-CD4. In vitro LIF-nano proved efficacious, increasing Foxp3+CD4+ T cell numbers. In vivo, donor-specific Foxp3 Treg expansion was promoted by LIF-nano therapy in recipients of an allogeneic spleen cell transfusion. Confirmation of LIF's involvement in Treg differentiation in vivo was sought by removal of LIF: anti-LIF therapy significantly inhibited antigen-specific nTreg expansion. We conclude that LIF is a Treg cytokine, able to support the Treg lineage and Foxp3 expression during antigen-specific T cell responses. Funding Support: BHF, NIHR, NIH, JDRF, NSFCA.
TH17 ANTAGONIZE TREG TO REJECT ALLOGRAFTS (141.38)
Authors: Yang Yu , Xu Jiangnan , Ochando Jordi C , Bromberg Jonathan S , Ding Yaozhong
Published: January 2023

Treg are central in controlling immune responses and transplant tolerance, and TGFβ plays an important role in inducing and maintaining Treg. However, when TGFβ is combined with IL-6, CD4 T cells develop along the Th17 pathway, and Treg have been suggested to be one of the main sources for TGFβ in Th17 differentiation. We determined how Treg affected Th17 induction, function, and allograft rejection. Vascularized cardiac allografts were performed in SCID mice, and Treg and/or Th17 were adoptively transferred 4 days later and graft survival monitored. We found that in the presence of Treg, proliferation of CD4 T cells was inhibited, and IL-6 partially abolished proliferative suppression by Treg. However, Treg did not inhibit but actually increased IL-6 driven IL-17 expression, suggesting that Treg did not suppress Th17 differentiation. Allograft survival was significantly prolonged when effector T cells were co-transferred with Treg, with mean survival increasing from about 14 to > 50 days. Treg were not as effective when co-transferred with Th17, allograft survival increasing from 10 to only 15 days. These findings have important implications for understanding the signals necessary for preventing Th17 development and function in order to prolong graft survival and induce tolerance.
Preformed Alloantibodies Prevent the Induction of Allograft Tolerance by Enhancing Alloreactive T cell Priming (141.4)
Authors: Troutman Audrea M , Chong Anita
Published: January 2023

The presence of alloantibodies (alloAbs) in transplant recipients is often associated with increased Ab-mediated as well as cellular rejection. We hypothesize that alloAbs can facilitate cellular rejection by enhancing T cell activation and have tested this hypothesis by investigating the effect of alloAbs in a BALB/c to C57BL/6 skin transplant model. Long-term acceptance was induced with anti-CD154 (MR1) plus DST. The impact of alloAbs was investigated using anti-Kd (HB159; mouse IgG2a) mAbs, administered at the time of transplantation. Anti-Kd mAbs prevented anti-CD154-mediated graft acceptance (MST=12d). We hypothesize that alloAbs form immune complexes that facilitate allo-antigen uptake and presentation by APCs. We used CFSE-labeled T cells from three different TCR-Tg mice (TCR75-CD4+, OT-II -CD4+, and OT-I -CD8+ TCR Tg T cells to measure T cell responses in BL/6 recipients of OVA-BALB/c skin ± anti-CD154/DST ± anti-Kd mAbs. Anti-Kd mAbs enhanced proliferation of all three TCR-Tg T cells in anti-CD154/DST-treated recipients compared to anti-CD154/DST-treated recipients. In conclusion, alloAbs promote the CD40-CD154-independent activation of allo-T cells, thus preventing allograft tolerance.
Insights into the Mechanism Responsible for Spontaneous Acceptance of Liver Allografts (141.41)
Authors: Zhuo Ming , Ito Takashi , Wang Mouer , Esquivel Carlos O , Martinez Olivia M. , Krams Sheri M.
Published: January 2023

Transplantation of fully histoincompatable livers between strains of rodents often results in spontaneous acceptance of the graft. Lewis rats reject DA livers in 14 days while DA recipients of Lewis livers have long-term graft survival. We sought to determine the mechanism of this spontaneous graft survival.

NK cells and subsets of T cells express NKG2D. On NK cells, NKG2D functions as a stimulatory receptor that induces effector functions. We examined NKG2D receptor and ligand expression post-transplant. Groups of Lewis rats received livers from DA (rejection combination) or Lewis donors (syngeneic) and DA rats received livers from Lewis (non-rejection combination) or DA donors (syngeneic). NKG2D expression was low in syngeneic grafts but increased 7-fold in Lewis→DA grafts and 17-fold in DA→Lewis grafts by day 7 post-transplant.

Ligands of NKG2D were not expressed in normal liver but were up regulated in all liver grafts by day 1 post-transplant, suggesting that ischemia/reperfusion injury and trauma induce expression of these ligands. RAE1L and RRLT were significantly up regulated specifically in DA→Lewis liver grafts by day 7 post-transplant. In marked contrast, the expression of RAE1L and RRLT in Lewis→ DA liver grafts was similar to the levels detected in syngeneic liver grafts suggesting that signals that lead to an increase in NKG2D ligand expression are absent in this strain combination. In conclusion, the absence of NKG2D ligand expression in the liver graft may play a role in preventing rejection, and decreasing the expression of these proteins may be a useful strategy to promote graft non-responsiveness. Supported by AI044095
Combined mTOR inhibition and post-transplant infusion of alloantigen-specific Treg promotes long-term graft survival in otherwise unmanipulated hosts (141.42)
Authors: Raimondi Giorgio , Sumpter Tina L , Matta Benjamin , Corbitt Natasha , Tokita Daisuke , Wang Zhiliang , Thomson Angus W
Published: January 2023

Protocols that achieve donor-specific tolerance of MHC-mismatched organ grafts remain an unmet clinical goal. We report a strategy that combines post-transplant administration of alloAg-specific Treg (AAsTreg) with inhibition of the mammalian target of rapamycin (Rapa) (mTOR) to promote long-term, donor-specific heart graft survival.

AAsTreg selection was achieved by co-culture of CD4+CD25+ Treg with immature dendritic cells (DC) in the presence of conditioned media. This rendered cells that potently inhibited T cell proliferation in an Ag-specific manner. However, AAsTreg remained unable to control T cell activation when stimulated by mature DC, - a phenomenon dependent on IL-6 release. Exploiting the in vivo inhibitory effects of Rapa on inflammatory cytokine (IL-6) production, T cell proliferation and graft infiltration, AAsTreg were administered post-transplant (d7) in combination with short-term Rapa. AAsTreg exerted a pro-tolerogenic effect: >80% long-term graft survivors [LTS] (MST>150d). Polyclonal Treg exerted an inferior protective effect: 40% LTS (MST=45d). Moreover, the pro-tolerogenic effect was Ag-specific as AAsTreg selected against third party DC failed to prolong graft survival. Significantly, LTS exhibited alloreactive T cell anergy. Thus, combination of mTOR inhibition with AAsTreg is an effective, clinically-applicable approach to promote long-term graft survival.
Inverse relationship between liver γδT cells and Foxp3+CD25+CD4+ Tregs contributes to liver allograft tolerance and rejection (141.44)
Authors: Malone Frances R , Wang Weigang , Meng Zihui , Reyes Jorge D , Li Wei
Published: January 2023

Our previous studies have demonstrated that Foxp3+CD25+CD4+ regulatory T (Treg) cells play an important role in inducing liver transplant tolerance. It is unknown whether γdT cells are involved in liver tolerance and how they interact with other types of cells. In this study, we examined the involvement of γdT cells in liver transplant tolerance by using an orthotopic liver transplantation model in which liver allografts are accepted spontaneously across the MHC barrier. Controls received either anti-CTLA4 mAb or anti-CD25 mAb pre- or peri-operatively, which induced acute liver allograft rejection. The majority of γdT cells were located in the liver and were CD4 and CD8 negative, did not express TCRαβ and NK receptors. In contrast to Foxp3+CD4+CD25+ Treg, γdT cells were decreased in the spontaneously tolerant liver allografts throughout the time courses from day 1 to day 100 posttransplantation and increased in the rejected liver grafts at day 5 posttransplantation. The CD4 and CD8 marker expression on γdT cells were increased in the tolerated livers post transplantation (10-20%). We speculate that the decreased number of γdT cells in the tolerated liver allografts may be due to the suppressive effect of Foxp3+CD4+CD25+ Treg. Therefore, γδ T cells, expressed in opposite frequency to Foxp3+CD4+CD25+ Treg, are involved in the processes of liver transplant tolerance.
NKG2D-mediated activation of NK cells by allogeneic CD34+ blood cells. (141.9)
Authors: Arpinati Mario , Nicolini Benedetta , Steinle Alexander , Rondelli Damiano
Published: January 2023

Background: We have previously demonstrated that human CD34+ cells and allogeneic T lymphocytes cross-interact in-vitro. In this study, we addressed the interaction bewteen CD34+ cells and allogeneic NK cells.

Methods: Purified CD3-CD56+ NK cells were cultured with allogeneic CD34+ or CD14+ blood cells or with 1000 U/ml IL-2. NK cells were then used to lyse NK-sensitive K562 (NK activity) or NK-resistant Daudi cells (LAK activity) in a 51Cr release assay.

Results: CD34+ cells induced greater NK activity than monocytes (p=0.02). While LAK activity was negligible before culture, it increased upon culture with CD34+ cells, but not monocytes (p=0.01). Activation of NK cells by CD34+ cells was comparable to high dose IL-2. NK cell activation was also documented by CD69 and CD25 in NK cells and by production of IFN? and TNF?. CD34+-induced NK activation was inhibited by anti-NKG2D antibody. However, anti-NKG2D antibody did not prevent NK activation by IL-2. Moreover, surface expression of NKG2D on NK cells was downregulated by CD34+ cells but not IL-2. Expression of NKG2D ligands (MICA-B and ULBP1-3) is currently being tested in CD34+ cells. Finally, NK cells affected the phenotype of CD34+ cells, that upregulated their expression of CD40 and CD86.

Conclusions: Our study suggest a cross talk between NK cells and allogeneic CD34+ cells through a NKG2D-related mechanism.
Real-time imaging of TCR dynamics in vivo reveals rapid TCR clustering and internalization in response to antigen (33.13)
Authors: Friedman Rachel S. , Beemiller Peter J. , Sorensen Caitlin M. , Krummel Matthew F.
Published: January 2023

Real-time in vivo imaging has given insight into the cellular dynamics of T cell activation, but less is known about receptor and signaling dynamics in vivo. Live imaging of subcellular signaling complexes has been challenging in living tissues due to low physiological molecular densities, inherent limitations of fluorophores, and strong autofluorescence of tissues. Our goal was to characterize T cell receptor (TCR) dynamics in naïve T cells in the lymph node during their first encounter with dendritic cells (DCs) bearing cognate antigen. To do so we developed a mouse expressing a GFP tagged OT-I TCR to track surface and intracellular TCR, and used image processing facilitated by co-labeling of the T cells to isolate specific fluorescence. In the absence of antigen, the TCR was mainly confined to the cell surface without consistent polarity; however, a small percentage of T cells transiently clustered their TCR. Within 30 min of antigen recognition, many T cells stopped crawling and clustered their TCR. Surprisingly, detectable antigen-dependent TCR clustering duration was short (7.1±5.1 min) and as TCR clustering declined, TCR internalization increased. Thus, naïve T cells in the lymph node can signal through the TCR in response to their first interaction with DCs bearing antigen and these contacts lead to rapid TCR clustering and internalization.

This work was funded by the Larry L. Hillblom Foundation and NIH R21.
Identification through ENU genome-wide mutagenesis of a novel mutant mouse that expresses a non-functional induced T cell kinase (Itk) with 8-amino acid insertion (33.30)
Authors: Kung John T
Published: January 2023

With the human genome sequenced, the race is on to understand the functions of the 30,000 or so genes. Ethyl-nitrosourea (ENU) was used to treat C57BL/6 (B6) male mice, followed by a 3-generation breeding scheme to generate G3 mice that were screened for immunological phenotypes under recessive genetic control. The primary phenotypes of one mutant, P-135, were increased CD44hi representation among both CD4 and CD8 T cells and highly elevated serum IgE. Recessive mode of inheritance was confirmed by mating an affected G3 male to wildtype B6 females to generate F2 offspring. Affected G3 mice were outcrossed to generated F2 offspring and the affected F2 mice were subjected to linkage analysis by haplotype interval mapping (Neuhaus and Beier, 1998. Mammalian Genome 9, 150-154). The mutant gene was found to be linked to chromosome 11. Through DNA sequencing of genes located within the linked region, a point mutation (T to C) in the 12th intron was identified. This point mutation resulted in the creation of a new splice donor site which in turn resulted in a 24-bp (8 amino acids, RVRAGMCR) in-frame insertion. P-135 mutant mice thus carries a non-functional Itk protein with 8 amino acid insertion and may be useful in defining structural-functional relationships of Itk.
Pregnancy primes an antigen specific maternal regulatory T cell response to fetal male antigen (33.6)
Authors: Kahn Daniel A , Baltimore David
Published: January 2023

Maternal CD4+ T cells awareness of fetal antigens is not well studied. T regulatory (Treg) cells, a powerful subset of CD4+ T cells, are capable of limiting robust immune responses. Tregs are implicated in maternal tolerance of the fetus.

Methods

Virgin female 6 week old C57BL/6 mice bred with male littermates or immunized with KLH/CFA. Splenocytes from virgin, parous, and pregnant mice (day post coitus 6.5, 12.5, and 18.5) and from KLH/CFA immunized mice on day 7. Proliferative response to CD4+ T cell peptide epitope of the H-Y antigen (DBY), control peptide (LSO), or KLH determined in co-cultures. Treg isolation by magnetic bead separation of CD25+ cells.

Results

Pregnant and parous splenocytes proliferate in response to DBY. Virgin splenocytes do not respond to DBY. Depletion of Tregs augments response to DBY in the context of pregnancy; not in virgin mice. Earlier in pregnancy, more male fetuses are required for a detectable response than later in pregnancy. Tregs isolated from pregnant mouse splenocytes co-cultured with Treg depleted splenocytes from KLH/CFA immunized mice are able to suppress proliferative response to KLH only in the presence of DBY.

ConclusionsPregnancy leads to maternal CD4+ T cell awareness of the fetus in proportion to the antigenic dose and is associated with a regulatory T cell response. Tregs are functional (suppressive) only in the presence of the antigen to which they were developed.
Critical role of CIN85 in Cbl-mediated regulation of B cell function after antigen receptor stimulation (34.11)
Authors: Niiro Hiroaki , Jabbarzadeh Tabrizi Siamak , Aizawa Kumiko , Inoue Yasushi , Arinobu Yojiro , Akashi Koichi
Published: January 2023

Upon B-cell receptor (BCR) ligation by autoantigens, B cells normally undergo deletion (apoptosis), inactivation (anergy), or receptor editing. These processes are finely tuned by positive and negative signals downstream of B cell receptor (BCR). Aberrant regulation of BCR signals may thus lead to activation and expansion of autoreactive B cells.

B cell adaptors function as molecular conduits to effectively link upstream signals to specific downstream branches. Cbl is an E3 ubiquitin ligase that is highly expressed in self-tolerant lymphocytes. Notably, B cell-specific ablation of Cbl in mice manifests SLE-like autoimmune disease, suggesting a pivotal role of Cbl in BCR-mediated tolerance induction. A novel Cbl-interacting adaptor termed CIN85 (c-Cbl interacting protein of 85 kDa) was recently identified, however little is known about a role of CIN85 in B cell function.

In this study we demonstrate that upon BCR stimulation, CIN85 strongly associated with Cbl as well as BLNK. This association required the SH3 domain of CIN85. The experiments using CIN85-overexpressing and CIN85 knockdown B cells showed that CIN85 is critical for Cbl phosphorylation, which in turn regulates BCR-induced calcium flux and activation of Syk and PLCγ2. CIN85 knockdown in human primary B cells resulted in their enhanced survival and proliferation by upregulating expression of bcl-2 family and cyclin genes following BCR stimulation. Together, these findings suggest a critical role of CIN85 in Cbl-mediated regulation of B cell function after BCR stimulation.
Tissue-Specific Cues Direct Fate Decisions of Mucosal Memory T Cells (39.21)
Authors: HUANG Yujun , Wang-Zhu Yiran , Schoenberger Stephen , Kronenberg Mitchell , Cheroutre Hilde
Published: January 2023

Mucosal memory CD8 T cells are characterized by their location at peripheral interfaces and the ability to immediately exhibit effector functions. By contrast, lymphoid memory T cells reside within lymphoid tissues and require further differentiation to become effectors. Both memory subtypes contribute to protective immunity, but the accumulation of mucosal effector memory T cells at pathogen portals forms a central component of protective immunity. The process that drives the compartmentalization and functional differentiation of this memory subset is poorly understood. Here we show that the thymus leukemia (TL) antigen, a ligand for the TCR co-repressor, CD8αα, serves a key role in the molecular mechanism that determines the fate of mucosal CD8αβ+ effector T cells. TL specifically controls the survival of CD8αα+CD8αβ+ effectors, but eliminates CD8αα negative CD8αβ effectors. This TL-CD8αα-mediated process allows the intestinal environment to selectively preserve the "fittest" effector cells to become mucosal memory T cells that form the first line of immune protection.
Alcohol consumption induces regional immune dysfunction (39.22)
Authors: Brand Rhonda M , Donahue Cara , Miller Megan , Plowey Jeff , Falo Louis D
Published: January 2023

Substantial clinical evidence suggests that alcohol abuse suppresses both innate and adaptive immune responses leading to more infections, greater susceptibility to cancer, and delayed recovery from trauma. The mechanisms underlying alcohol induced immune dysfunction are largely unknown and potentially include effects on dendritic cell function. Though published studies have focused on alcohol effects on systemic immune responses, it is well established that alcohol ingestion results in regional differences in ethanol concentration. This suggests the hypothesis that regional differences in ethanol induced DC dysfunction may lead to differences in regional immunity. Using the well defined Meadows-Cook model of alcohol ingestion, we immunized ethanol and control fed mice either cutaneously or hepatically with plasmid DNA encoding the model antigen ovalbumin. CTL and DTH responses were significantly reduced in ethanol fed mice compared to controls after liver targeted immunization. In contrast, minimal differences were seen between ethanol fed mice and controls after skin targeted immunization. These data suggest that the immunosuppressive effects of alcohol ingestion may be limited to regions of high ethanol exposure, and that intact immune systems in ethanol spared regions such as the skin could be used to induce effective immunity in individuals with significant alcohol ingestion. Funded by NIH.
IL-17 is required for protective immunity to nasal Brucella infections in an IFN-γ-dependent fashion (39.23)
Authors: Skyberg Jerod A. , Thornburg Theresa , Pascual David W.
Published: January 2023

Th1 and Th17 cells are the principal mediators of the inflammatory pathways. While the role of Th1 cells have been extensively studied for Brucella infections, there is little information on the function of IL-17 in response to Brucella infections. To evaluate such role, mice functionally deficient in IL-17 or IL-17 receptor (IL-17R-/-) mice were more susceptible to nasal Brucella infection than immunocompetent mice as evidenced by increased splenic weights and splenic CFUs. Interestingly, inhibition of IL-17 failed to enhance the IFN-γ response, but instead RT-PCR analysis revealed greater lung IL-13 mRNA transcripts than lungs from IL-17-sufficient mice. Splenic mononuclear cells from Brucella-infected mice depleted of IL-17 produced more IL-10, but less IFN-γ and IL-17 upon subsequent restimulation with heat-killed brucellae than the same cells from IL-17-sufficient mice. Interestingly, splenic mononuclear cells from infected IFN-γ-/- mice produced less IL-17, indicating that mice blocked in either IL-17 or IFN-γ pathways are impaired in their ability to produce either cytokine when infected with Brucella. These results implicate IL-17 is important for protection against nasal Brucella infections, and that IL-17 in combination with IFN-γ modulates the host immune responses to Brucella. This work is supported by USDA 2007-0612 and 2008-03776.
Expansion of CD4loCD14+CD40+ LKLF Hi CD4+ T cells correlates with acute pulmonary inflammation and provides a mechanism for impaired pathogen clearance in Cystic Fibrosis (39.24)
Authors: Pennock Nathan D , Vaitaitis Gisela M , Waid Dan M , Saavedra Milene T , Wagner, Jr David H
Published: January 2023

Cystic Fibrosis (CF) is characterized by chronic infection of the lung by bacterial pathogens with a subsequent robust but ineffectual neutrophilic immune response. As a result, patients' airways sustain irreversible injury, with respiratory failure accounting for >80% of deaths from the disease. Examination of lymphocyte/monocyte subsets in the peripheral blood of CF patients revealed a greatly expanded CD4+ T cell subset characterized by a very high expression level of the cell survival/quiescence transcription factor Lung Kruppel Like Factor, LKLF. Furthermore, cells exhibited co-expression of CD40 as well as the pattern recognition coreceptor CD14. Importantly, this observation was mirrored in airway submucosa and pulmonary lymph nodes isolated from CF lung transplant recipients. In a majority of patients, this expanded T cell subset from the peripheral blood was found to produce high levels of IL-4, which coincides with previous observations of Th-2 bias in CF patients infected with Pseudomonas aeruginosa. In vitro assays illustrated that simultaneous cross-linking of CD40 and CD3 on this T cell subset results in the elevation of LKLF. Paired with previous observations of elevated circulating soluble CD40 ligand in CF patients that correlate with worse pulmonary function, this data suggests a mechanism for the persistence of the deleterious Th-2 cytokine producing T cells that hamper the effectual clearance of lung pathogen. Importantly alterations in cell number within this T cell subtype from the peripheral blood correlate with observed severity of pulmonary inflammation and provide a means of evaluating efficacy of treatment. Research support;NIH:DDK (Wagner),Cystic Fibrosis Foundation(Saavedra)