bluuebunny/crossref_metadata_2025 huggingface dataset:
Altered Behavior in the Novelty‐Induced Hypophagia Test in FoxG1+/− Mice
Authors: Flores Natasha Mahealani , Beech Robert
Published: June 2021

For this study we used mice with a heterozygous deletion of the FoxG1 gene to examine the relationship between adult neurogenesis and symptoms of depression as modeled by the Novelty‐Induced Hypophagia test. The mice were given a 0.2 mg/ml dosage of fluoxetine through drinking water for 3 weeks. Testing occurred in both the home cage and novel cage. We did not find an effect of antidepressant treatment on behavior in either the FoxG1+/− mice or wild type mice. Compared to wild type controls the FoxG1+/− mice displayed transgenic mice had decreased consumption of milk in the home cage as well as increased latency to feed. These are similar to two features of depression in humans, increased anxiety and decreased response to rewarding stimuli. Thus our results are consistent with the hypothesis that decreased neurogenesis is responsible for some symptoms of depression found in humans.

This study was funded by Yale University's Summer Undergraduate Research Fellowship, The Leadership Alliance and Chaminade University of Honolulu.
A career management program for basic science junior faculty
Authors: Sukalski Katherine A , Fox LaVonne , Moulton Patricia , Flower Ann M , Burns Elizabeth E , McHugo Jeanie , Larson Annette
Published: June 2021

A Career Management Program was implemented for new tenure track faculty (basic science and educator scholars) in a small community‐based medical school. The program was designed based on best practices in the literature to be a resource for faculty, complementing the mentoring provided by department chairs. Interactive panel presentations were led by senior faculty from basic science and allied health departments focusing on research, teaching, service, and strategic career planning. The basic science scholars also met as a cohort, with an experienced basic scientist to discuss specific issues raised in previous panel discussions. The program successfully completed year one with eleven junior faculty (five basic science) participating on a regular basis. The session evaluations resulted in 90% or higher for the value of the sessions and usefulness/transferability of the information to the participant's position and job responsibilities. This program demonstrates that a career management program can combine faculty from multiple tracks as long as they have opportunities to address career‐specific issues.

Sponsored by the North Dakota Women's Health CORE, A National Center of Excellence in Women's Health Region VIII Demonstration Project, Elizabeth Burns, PI.
Imaging thymocyte migration in real‐time during negative selection
Authors: Le Borgne Marie , Ladi Ena , Robey Ellen
Published: June 2021

Little is known about the the dynamic processes that contribute to thymocyte maturation. Using Two‐Photon Laser Scanning microscopy, we can determine the strategy employed by thymocytes to scan antigen presenting cells during negative selection.
Factors driving expansion of CD4+ CD25hi regulatory T cells in lymphopenic hosts
Authors: Lee Joon Youb , Ding Zhaoqing , Kobuley Dmytro E. , Yu Yimin , Cebra John J. , Surh Charles D.
Published: June 2021

The naturally occurring population of CD4+ CD25hi regulatory T cells (Tregs) is essential in suppressing potentially autoreactive T‐cells from inducing autoimmune diseases. Although Tregs are unresponsive to typical activation signals under in vitro conditions, these cells readily undergo proliferation upon adoptive transfer into syngeneic lymphopenic hosts. Recently, we have found that the source of antigens driving naïve T cell homeostatic expansion is determined by the recent immune status of the lymphopenic hosts. Consistent with this idea, the magnitude of Treg expansion differed considerably depending on the recent immune status of the host, i.e., in wild‐type hosts rendered acutely lymphopenic by irradiation, Tregs underwent moderate expansion, whereas a vastly stronger expansion was observed in congenic T cell‐deficient hosts, such as RAG– or SCID hosts. Experiments with various mutant mice raised under conventional and germ‐free conditions revealed that the moderate expansion of Tregs in acutely lymphopenic wild‐type hosts is largely driven by self‐MHC/peptide ligands with some contribution from IL‐2, but not from IL‐7 or IL‐15. In contrast, the bulk of the prodigious expansion of Tregs in immunodeficient mice is driven by MHC loaded with foreign peptides. These findings illustrate that Tregs resemble typical naïve T cells in their ability to respond to both self and foreign antigens.
Interleukin (IL)‐7 inactivates pro‐apoptotic protein Bim at the posttranslational level in association with peripheral T‐cell survival
Authors: Li Wenqing , Guszczynski T. , Xiao Z. , Veenstra T. , Durum S.
Published: June 2021

IL‐7 is critical for T‐cell development both in mouse and humans and is also important for peripheral T‐cell homeostasis. Survival of pro‐T cells and mature T cells depends on the trophic effects of IL‐7. The survival function of IL‐7 is partly through induction of the anti‐apoptotic protein Bcl‐2 and inhibition of pro‐apoptotic proteins Bax and Bad. In this study, we investigated the roles of Bim, a pro‐apoptotic protein member of the Bcl‐2 family, in IL‐7 signaling using an IL‐7‐dependent murine thymocyte line, D1. Overexpression of one of the three isoforms of Bim, BimS or BimL or BimEL, induced D1 cell apoptosis in the present of IL‐7. Knockdown of Bim by siRNA rescued D1 cells from death after 24 hours of IL‐7 withdrawal, but the protection could not extend to 48 hours of IL‐7 deprivation, suggesting that Bim is not a solo killer of T‐cell in the absence of IL‐7. Compared to WT CD4 or CD8 cells, Bim KO CD4 or CD8 cells showed much more resistance to IL‐7 deprivation when cultured in vitro. Peripheral T‐cell transfer experiments showed that Bim KO CD4 or CD8 cells survived better than WT cells in an IL‐7‐deficient host. T‐cell development in thymus was not observed in Rag2/IL‐7 double KO mice reconstituted with Bim KO bone marrow. IL‐7 deprivation did not increase Bim protein expression in pro‐T cells or mature T‐cell or D1 cells. Bim protein was constitutively localized in mitochondria and bound to Bcl‐2 in D1 cells. However, IL‐7 deprivation induced a BimEL molecule mobility shift by 2‐D electrophoresis in D1 cells. Our findings suggest that Bim is important for peripheral T‐cell apoptosis in the absence of IL‐7, but not critical for thymic development. IL‐7 inhibits Bim activity not at the transcriptional or translational level, but at the posttranslational level.
CD4 regulation of activation‐induced cell death
Authors: Tinsley Kevin William , Tatari‐Calderone Zoreh , Leitenberg David
Published: June 2021

Defects in FAS‐mediated activation induced cell death (AICD) are associated with the development of autoimmunity in both mouse models and human disease. We have previously found that mature T cells with a mutant CD4 co‐receptor unable to associate with Lck are resistant to AICD. Since activated CD4 mutant T cells express similar levels of FasL, Fas, and FADD compared to wild‐type cells, we hypothesize that the CD4‐dependent signaling pathways provide a “competency” signal required by T cells to become susceptible to AICD. In support of this hypothesis, mixing experiments indicate that FasL expression by wild type CD4 T cell blasts could not rescue the defect in AICD seen in CD4 mutant T cells. Additionally, we find that the AICD resistant phenotype in CD4 mutant T cells is primarily observed following peptide restimulation, while wild‐type and mutant CD4 T cells are similarly sensitive to AICD upon re‐stimulation with plate bound anti‐CD3. These experiments suggest that death receptor signaling pathways are compromised in mutant CD4 T cells following physiologic stimulation with peptide that involves CD4 signaling. Since CD4 association with Lck alters the compartmentalization of cellular Lck pools, these data suggest that that distinct pools of Lck may be differentially involved in modulating sensitivity to Fas signaling.

Supported in part by a grant from the American Cancer Society.
The interdependent role of YMNM and PxxP motifs in CD28 costimulation
Authors: Ogawa Shuhei , Kishimoto Hidehiro , Abe Ryo
Published: June 2021

CD28‐mediated costimualtion is known to be critical required for T cell activation. There are Y189MNM motif and two PxxP motifs in CD28 cytoplasmic region that are thought to play a key role for the costimulatory function. It has been shown that the YMNM motif functions as a binding site for PI3K and Grb2/Gads, and the N‐terminal and C‐terminal PxxP motif play a critical role for binding to Itk and Lck. We generated transgenic mice expressing the Y‐>F mutant CD28 which lack both PI3K and Grb2/Gads binding and P‐>A mutant CD28 which lack binding of Lck and/or Itk in the CD28 deficient background. Previously, we reported that a point mutation of Y189 diminished proliferation and IL‐2 production. In this study, we found that T cells from ncPA mutant mice which have mutations in both N‐ and C‐terminal PxxP motifs showed striking defect in proliferation and IL‐2 production similar to those from Y‐>F mutant. T cells from mice having individual mutation in N‐ or C‐terminal PxxP motif demonstrated a defect of proliferative response to suboptimal dose of stimulation. These results suggested that both N‐ and C‐terminal PxxP motifs are required for CD28 mediated costimulatory functions of T cell activation.
Non‐apoptotic caspase‐8 activation balances T lymphocyte autophagy
Authors: Bell Bryan Daniel , Leverrier Sabrina , Weist Brian , Morrissette Naomi , Walsh Craig
Published: June 2021

FADD and caspase‐8 (casp8) transduce an apoptotic signal through tumor necrosis factor family members. Paradoxically, lymphocytes lacking FADD or casp8 function fail to undergo normal clonal expansion and instead succumb to caspase‐independent cell death. However, the exact nature of the survival defects of these T cells has remained elusive. We show that T cells lacking FADD or casp8 are subject to hyperactive autophagic signaling. FADD‐deficient embryonic fibroblasts and Jurkats also possessed elevated levels of autophagy compared to wild type cells. T cell autophagy induced casp8 activation through an interaction with FADD:Atg5‐Atg12 complexes. Inhibition of autophagic signaling with 3‐methyladenine, or by expression of dominant‐negative Vps34, rescued T cells expressing a dominant‐negative FADD protein. Since the necroptosis inhibitor Nec‐1 also rescued T cells lacking FADD or casp8 function, our findings demonstrate that while autophagy is necessary for rapid T cell proliferation, FADD and casp8 form a feedback loop to limit autophagy and prevent this salvage pathway from inducing type‐II cell death. These results suggest that, by linking autophagy mediators to cell death executioners, hyperautophagic conditions promote apoptotic cell death, thus avoiding an inflammatory response induced by type‐II cell death in vivo. Support from NIH (T32CA09054, BDB; R01AI50506, CMW).
Targeting PD‐1 or ICOS pathways does not rescue decreased CD3‐induced proliferation of aged T cells
Authors: Lages Celine Silva , Lewkowich Ian P , Sproles Alyssa A , Wills‐Karp Marsha , Chougnet Claire
Published: June 2021

The co‐stimulatory molecules ICOS and PD‐1, and their ligands (ICOSL and PD‐L1/PD‐L2 respectively), regulate T cell activation and tolerance in young animals. However, their role in the immune deficiency associated with aging is still unknown. We therefore compared the expression of these molecules in the spleen, lymph nodes and lungs from young and aged mice. In aged mice the expression of PD‐L1 was higher on plasmacytoid dendritic cells (DC), whereas the expression of PD‐L2 was higher on all DC subsets examined. While constitutive levels of ICOS and PD‐1 were more elevated on CD4+ T cells from aged mice, in vitro anti‐CD3‐mediated activation increased both PD‐1 and ICOS expression on young, but not old, CD4+ T cells. Interestingly, the lower proliferation of old T cells was not rescued by ICOS stimulation or blockade of the PD‐1 pathway. PD‐1 or PD‐L1 blockade enhanced IFNγ production by old T cells, but had limited impact on IL‐13 and IL‐17 contrary to what we observed in young T cells. Our data suggest that, despite higher basal ICOS and PD‐1 expression, these pathways may be less efficient at regulating T cell activation in aged animals. This finding could be due to intrinsic defects in the signaling pathways and/or to extrinsic mechanisms of dysregulation, such as the increased number of functional regulatory T cells in aged mice. Supported by AG025149, HL67736, ALA grant to IPL.
CD40 ligand expression abrogates infectious tolerance induced by apoptotic cells
Authors: Gurung Prajwal , Ferguson Thomas A. , Griffith Thomas S.
Published: June 2021

The decision to generate a productive immune response or tolerance after pathogenic insult often depends on the context in which T cells first encounter Ag. Ag encountered in the presence of apoptotic cells leads to immune tolerance, while the presence of necrotic cells promotes immunity. Using a classical system of tolerance induction, we re‐examined the tolerance induced by i.v. injection of apoptotic cells. In this system, cross‐presentation of Ag associated with apoptotic cells fails to prime CD4+ T cell‐mediated help and induces CD8+ suppressor T cells that mediate unresponsiveness. While apoptotic spleen cells induce immune tolerance when injected i.v., activation by PMA/ionomycin or anti‐CD3/CD28 mAb prior to apoptosis induction abrogated the ability to induce tolerance. Moreover, the activated apoptotic cells directly primed CD4+ T cells leading to immunity rather than tolerance. Further studies revealed a role for the costimulatory molecule CD40 ligand (CD40L), as tolerance resulted from i.v. injection of either naïve or activated apoptotic CD40L−/− cells and co‐injection of an agonistic anti‐CD40 mAb with naïve apoptotic cells induced robust immunity. In sum, these results show the key role of CD40L in determining the immune response to Ag in the presence of apoptotic cells, and suggest naïve apoptotic cells induce tolerance because CD8+ T cells fail to receive “help” via CD40‐CD40L binding.
SLE progression in human and murine SLE is accompanied by cell and pathway specific negative regulation of cytokine signal transduction
Authors: Hale Matthew , Nolan Garry P
Published: June 2021

Systemic lupus erythematosus (SLE) is a cyclic, persistent disease of unknown etiology, with multiple lines of evidence supporting a model in which aberrant regulation of cytokine signaling pathways promote and sustain SLE. Postulating that such mechanisms may be disturbed in SLE patients, we used phospho‐specific flow cytometry to perform an extensive profile of cytokine signaling in a murine model of SLE and 47 human patients, revealing disease course‐dependent, cell type, and pathway specific negative regulation of the JAK‐STAT signal transduction pathways induced by the interferons. Specific down‐regulation of Stat1, Stat3, Stat5 responses in different immune cell subtypes was correlated with upregulated SOCS1 protein expression. SLE pathology involving the prominent IFNα model is here expanded to include additional signaling nodes, coupling these systems to negative feedback regulation. Pathogenic coupling of positive and negative feedback regulation may explain the cyclic nature of human SLE.

This work was supported by NHLBI Proteomics Center contract N01‐HV‐28183 and NIH grant 8‐PA1036535C
IL‐4δ2, a natural splice variant of IL‐4, is a mediator of T cell recruitment
Authors: Luzina Irina G. , Lockatell Virginia , Highsmith Kendrick , Nacu Natalia , Choi Jung , Papadimitriou John C. , Atamas Sergei P.
Published: June 2021

We previously described a natural splice variant of IL‐4 that lacks exon 2, and reported that recombinant IL‐4δ2 antagonizes functional effects of IL‐4 on cultured T and B lymphocytes and monocytes. Changes in the absolute and relative expression levels of IL‐4 and IL‐4δ2 have been reported in the lungs of patients with asthma, tuberculosis, and HIV infection. Functional effects of IL‐4δ2 in vivo have not been characterized. Adenoviral gene delivery of IL‐4 or IL‐4δ2 to mouse lungs in vivo in comparison with AdV‐NULL infections was utilized. Overexpression of IL‐4 or IL‐4δ2 was confirmed by ELISA and LC/MS. Delivery of either IL‐4 or IL‐4δ2 caused profound peribronchial and perivascular infiltration of mostly T cell, equally CD4+ and CD8+, with some granulocytes, macrophages, and plasma cells. In the bronchoalveolar lavage cells, the relative contribution of lymphocytes changed from normal 1.7±1.2% to 33±8% in IL‐4‐expressing and 53±6% in IL‐4δ2‐expressing mice. Less than 1% of the infiltrating CD8+ cells but 17±3% of CD4+ cells and were also CD25+. These changes peaked at 7–14 days following the infections, and declined by 21–28 days. The implication based on experiments in cell culture that IL‐4δ2 is simply a passive inhibitor of IL‐4 effects is likely incorrect. The splice variant IL‐4δ2 is an active cytokine mediating pulmonary infiltration of mostly T cells in vivo. Support: R01HL074067, VA Merit (SPA).
Treatment of Inflammatory Autoimmune Disease Using a Small Molecule Cytokine Inhibitor
Authors: Kithcart Aaron P , Sielecki T , Williams J , Gienapp I , Shawler T , Smith K , Song F , Whitacre C C
Published: June 2021

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). Macrophage migration inhibitory factor (MIF), a ubiquitous inflammatory cytokine, is elevated in the CSF of MS patients during relapse. We have shown that MIF−/− (KO) mice show markedly suppressed clinical signs of experimental autoimmune encephalomyelitis (EAE) relative to wild type (WT) controls. The purpose of this study was to identify the timing in which MIF is important for progression of disease. Using adoptive transfer, we showed that activated lymphocytes from KO mice could transfer EAE to WT recipients, but that WT cells were unable to transfer EAE to KO recipients, suggesting that the CNS environment was protective in the absence of MIF. We used a small molecule inhibitor of MIF (INH) at two different times to selectively test the induction of EAE in the absence of MIF. Mice receiving INH at Day 0 were not protected, but mice treated after disease onset had less severe EAE. Despite the reduced severity of disease, lymphocytes from INH‐treated mice secreted substantial IFN‐γ and IL‐17. INH‐treated mice had a larger population of CD4+25+foxP3 lymphocytes. These findings suggest that MIF is important for disease progression but is not critical for T cell activation; an inhibitor could be a novel therapeutic approach for treating ongoing MS and other autoimmune diseases. (Supported by NIH grant AI 064320 and NMSS grant RG3272)
Priming of the immune response to Respiratory Syncytial Virus takes place in the lung in CCR7−/− mice
Authors: Kallal Lara E , Lukacs Nicholas W
Published: June 2021

Chemokine receptor CCR7 is expressed on naïve T cells and is responsible for their trafficking into secondary lymphoid tissue to sample antigen. Conversely, CCR7 is upregulated on dendritic cells (DCs) upon antigen uptake and activation, permitting maturation and migration of DCs to the lymph nodes to prime T cell responses. We sought to investigate the role of DCs and T cells deficient in CCR7 during a primary pulmonary viral infection caused by Respiratory Syncytial Virus, a negative strand RNA virus that can cause severe complications in infants and immunocompromised individuals. We found that CCR7−/− mice had significantly elevated numbers of CD69+ CD4+ and CD8+ T cells in the lung. Consistent with this, expression of the Th2 cytokines IL‐4 and IL‐13, as well as the Th1 cytokine, IFNg, was upregulated in CCR7−/− mice. When we assayed for DC recruitment, we found significantly higher numbers of CD11b+CD11c+ cells in the lungs of CCR7−/− mice. Upon histological evaluation, the airways of CCR7−/− mice exhibited considerable mucus accumulation in the airways compared to WT. In accord with this, RT‐PCR for the mucus inducing genes, muc and gob5a, were highly upregulated in CCR7−/− mice. In the lymph node, however, total cellularity was decreased, suggesting that the immune response to RSV may be mediated locally in the lung by DCs and T cells that are unable to migrate properly in the absence of CCR7.
Expression of osteopontin in human decidua: its role in decidual recruitment of natural killer cells
Authors: Qu Xun , Yang Meixiang , Kong Beihua , Liang Lu , Zhang Yan , Liu Jia , Gao Wenjuan , Yang Qifeng , Gong Feili
Published: June 2021

Accumulation of decidual natural killer cells (dNK) during early gestation is evident in successful pregnancy, and some chemokines have been recognized to be involved in this event. Osteopontin, as a chemokine, is proposed to involve in keeping successful pregnancy in mice and ruminants. To date, there is little information on osteopontin expression in human pregnant process and RPL patients. In this study, we detected osteopontin expression in decidua of early pregnant women and RPL patients by RT‐PCR analysis and immunohistochemical staining. Osteopontin was expressed both in human decidual stromal cells and dNK cells, and upregulated in the decidual tissue from later gestational phase than that from early gestational phase. The strong osteopontin expression in perivascular decidual stromal cells corresponded with a large amount of dNK cells, whereas osteopontin expression and accumulation of dNK cells were reduced significantly in RPL patients. Moreover, we found that progesterone significantly induced osteopontin secretion from cultured decidual stromal cells. These results indicate that osteopontin may play an important role in dNK recruit through autocrine and/or prairie pathways during human early pregnancy. Our findings provide further understanding of the role of osteopontin in dNK recruitments and the complex mechanism of progesterone on decidual stromal cells in the control of human pregnant maintenance.
The role of nuclear factor erythroid 2‐related factor 2 (Nrf2) in cytokine production by CD4+ T cells
Authors: Rockwell Cheryl Elizabeth , Fields Patrick E , Klaassen Curtis D
Published: June 2021

Nrf2 is a transcription factor that is activated by oxidative stress and electrophilic stimuli that has been best characterized for its role in cytoprotective mechanisms. Because it has been demonstrated that Nrf2‐null female mice develop a systemic lupus erythematosus (SLE)‐like pathology that is characterized by multi‐organ inflammation and immunoglobulin complex formation and deposition, the purpose of the present studies was to determine the role of Nrf2 in T cells. The initial studies of the current project showed that enriched CD4+ Nrf2‐null T cells produced nearly 100‐fold more IFNγ than wild‐type T cells 24 hr after anti‐CD3/anti‐CD28 activation and IFNγ levels from Nrf2‐null T cells remained 20‐fold higher compared to wild‐type T cells 72 hr after activation. In addition, IFNγ production by Th1‐polarized Nrf2‐null T cells was markedly greater (20‐fold) than that produced by wild‐type Th1‐polarized cells. The present studies also demonstrate that IL‐17 production was 60‐fold greater in supernatants from Nrf2‐null CD4+ T cells compared to wild‐type CD4+ T cells 24 hr after primary activation and 10‐fold greater 72 hr after activation. Collectively, these data suggest that Nrf2 plays a role in the regulation of IFNγ and IL‐17 production in CD4+ T cells. In addition, these studies suggest that Nrf2 also regulates IFNγ in Th1‐polarized cells. (Supported by NIH grants ES09649, ES09716, ES07079, and RR021940.)
TRAIL‐TRAIL Receptor interactions between donor T cells and host B cells are required for induction of autoimmunity in chronic graft‐versus‐host disease
Authors: Nguyen Vinh , Shorter Shayla , Saltis Mark , El‐Deiry Wafik S. , Davidson Wendy , Rus Violeta
Published: June 2021

TNF‐related apoptosis‐inducing ligand (TRAIL) mediates the apoptosis of transformed and immune cells and also exerts immunoregulatory functions. Our studies in the parent‐into‐F1 model of chronic graft‐vs‐host disease (cGVHD) demonstrated that TRAIL expression on T cells exacerbates lupus by positively regulating CD4+ T helper cell numbers and sustaining T cell help for B cells. To determine whether direct interactions between TRAIL on donor T cells and TRAIL‐R on autoreactive host B cells are required for induction of autoimmunity we used TRAIL‐R KO as recipients in the bm12‐>B6 model of cGVHD. When cGVHD was induced by injecting Bm12 donor cells into TRAIL R1/DR5‐KO B6 mice, parameters of cGVHD were attenuated compared to cGVHD induced in WT B6 mice. Specifically, the expression of activation markers on host B cells were significantly lower in TRAIL R1/DR5‐KO B6 compared to WT B6 hosts (80±20 vs 362±51 for MHC class II MCF, 19± 6.7 vs 54.1±9.2 for Fas and 38.9±8 vs 65.3±6.4 for B7‐2; p=0.001 for all three comparisons). In addition anti ss‐DNA autoantibody levels were significantly lower in TRAIL R1/DR5‐KO B6 hosts compared to WT B6 hosts. In conclusion, these results suggest that the direct interactions between TRAIL on donor CD4+ T cells and TRAIL‐R on host B cells may be essential for the activation of autoreactive B cells and their differentiation into autoantibody producing cells.
Transgenic overexpression of anti‐apoptotic FLIP on lymphocytes inhibits resolution of granulomatous experimental autoimmune thyroiditis (G‐EAT)
Authors: Fang Yujiang , Braley‐Mullen Helen
Published: June 2021

G‐EAT isinduced by transfer of mouse thyroglobulin (MTg)‐primed in vitro activated splenocytes. We recently showed that transgenic FLIP expressed on thyroid epithelial cells (TEC) promotes earlier resolution of G‐EAT by protecting TEC from Fas‐mediated apoptosis. We hypothesized that if transgenic FLIP were expressed on lymphocytes, CD4+ effectors would be protected from Fas‐mediated apoptosis, and resolution would be inhibited resulting in chronic inflammation. Transgenic (Tg) mice overexpressing FLIP under the CD2 promoter were generated. Transgenic FLIP was expressed on all CD4+ and CD8+ T cells and B cells. Tg+ mice and Tg‐ littermates were immunized with MTg and adjuvant, and donor CD8+ T cells were depleted. Splenocytes were activated in vitro with MTg and IL‐12, and transferred to syngeneic recipients. Recipients of both Tg+ and Tg‐ donor cells developed severe G‐EAT (4–5+) 20 days later. Thyroid lesions in recipients of Tg‐ donor cells were resolving or had begun to resolve at day 60, but lesions in recipients of Tg+ cells did not resolve. Studies are in progress to determine if transgenic FLIP protects T cells from apoptosis and if expression of FLIP on T cells alters cytokine profiles in thyroids. The results indicate that a single anti‐apoptotic molecule can determine if an autoimmune inflammatory response resolves or becomes chronic depending on where it is expressed (TEC or lymphocytes) (NIH DK35527).
BAFF Overexpression Promotes Loss of Tolerance by Rescuing Anergic B Cells
Authors: Thorn Mitchell , Mumbey Alice , Spatz Linda Ann
Published: June 2021

Overexpression of BAFF has been shown to promote B cell survival and lead to the production of autoantibodies. The mechanism by which BAFF drives B cell autoimmunity remains unknown. By overexpressing BAFF in a transgenic (Tg) anti‐dsDNA mouse model, we induced the breakdown of tolerance and secretion of Tg autoantibody in a dose dependent manner. We observed that BAFF overexpression leads to the expansion of mature Tg B cells, without significantly attenuating central deletion. It also leads to an enrichment of Tg anti‐dsDNA B cells in the marginal zone, that respond to mitogen stimulation by secreting autoantibody. We also observed that mice overexpressing BAFF contain a population of Tg anti‐dsDNA B cells with an anergic phenotype. These B cells express low levels of surface immunoglobulin, low levels of BAFF receptor and cannot be activated by BCR cross‐linking concomitant with CD40 signaling. In addition these B cells display the phenotype of T3 transitional B cells as they are AA4hi IgMlo and CD23+. We have demonstrated that adoptive transfer of these Tg T3 B cells into BAFF mice, leads to their maturation into B cells capable of secreting autoantibody. This suggests that BAFF may aid in the loss of tolerance by rescuing anergic B cells from the T3 population.
iNKT cells regulate B cell development and homeostasis in the periphery
Authors: Wen Xiangshu , Yang Junqi , Singh Ram Raj
Published: June 2021

Invariant natural killer T (iNKT) cells trans‐activate B cells. We found that this activation caused different fates of different B cell subsets. In the presence of activated iNKT cells, splenic follicular B cells proliferated and expanded whereas the proportion of marginal zone B (MZB) cells was selectively reduced. The reduction in MZB cells was via their activation induced cell death and suppression of proliferation in the presence of iNKT cells. To further understand the role of iNKT cells in peripheral B cell development, we analyzed immature B cells in iNKT cell‐KO and Tg mice as well as in Rag1−/− mice reconstituted with T and B cells. We found that the proportion of AA4.1+ IgM+ transitional immature B cells was increased by 2‐3‐fold in Jα18−/− and CD1d−/− mice as compared to the WT and Vα14 Tg mice. In resonance with this, the proportion of immature B cells particularly the T2 fraction was reduced in WT and Vα14 Tg mice, but not in Jα18−/− and CD1d−/− mice, in the presence of activated iNKT cells both in vivo and in vitro. Activation of iNKT cells also reduced the proportion of immature B cells in reconstituted Rag1−/− mice in vivo. Finally, activated iNKT cells reduced the proportion of MZB cell precursors (T2‐MZP) and up‐regulated CXCR5 expression on MZB cells. In summary, we propose that iNKT cells play an important role in the development and maturation of MZB cells, thus regulating their homeostasis in the periphery. Supported in part by NIH and Arthritis Foundation Southern California Chapter
pConsensus peptide induces tolerogenic CD8+T cells in lupus‐prone (NZB x NZW) F1 mice by differentially regulating Foxp3 and PD1 molecules
Authors: Singh Ram P , Tamayo Alejandro M , La Cava Antonio , Hahn Bevra
Published: June 2021

Systemic lupus erythematosus (SLE) is an autoimmune disease caused primarily by autoantibodies (including IgG anti‐DNA) and immune complexes. Tolerization with an artificial peptide (pCONSENSUS, pCons) based on murine anti‐DNA IgG sequences containing MHC class I and class II T cell determinants, lupus‐prone NZB/NZW F1 female (BWF1) mice develop regulatory CD4+CD25+T cells and inhibitory CD8+T cells, both of which suppress anti‐DNA Ig production and immune glomerulonephritis. In the present work, we show that splenocytes from BWF1 mice treated with pCons had significant expansion of primarily CD8+T cells. CD4+ T cells and B cells were each directly suppressed by CD8+T cells from tolerized mice in a contact‐independent manner. Both pCons‐ induced CD8+CD28+ and CD8+CD28− T cells suppressed production of anti‐DNA in vitro. Silencing with siRNA of Foxp3 abrogated the suppression mediated by both CD8+ T cell subsets. In addition, CD8+ T cells from tolerized mice were weakly cytotoxic against syngeneic B cells from old anti‐DNA‐producing mice, but not from young mice. Importantly, CD8+ suppressor T cells from tolerized mice, increases the intracellular expression of Foxp3 while decreases the surface expression of PD1 molecules. Blocking PD1/PDL1 interactions in the CD8+ T cells from tolerized mice reduced their expression of Foxp3 and their ability to suppress CD4+CD25− proliferation.
Functional Silencing of Immature B cells by Insulin
Authors: Henry Rachel A. , Acevedo‐Suárez Carlos A. , Thomas James W.
Published: June 2021

The majority of nascent B cells possess autoreactive specificities, thus constant maintenance of B cell tolerance is critical. While receptor editing and deletion effectively remove many autoimmune specificities from the repertoire, clonal anergy functions to censor autoreactive B cells without sacrificing repertoire diversity. Mature spleen B cells that harbor an anti‐insulin BCR transgene (125Tg) are rendered anergic in the periphery, as evidenced by impaired calcium mobilization and proliferation to various stimuli. To understand the ontogeny of this anergic state, an IL‐7‐driven bone marrow culture system was employed to generate antigen‐naïve or antigen‐experienced 125Tg immature B cells in vitro. When immature B cells encounter antigen during their genesis, they acquire many key hallmarks of anergy, including surface IgM downregulation and impaired intracellular calcium mobilization. Mimicking chronic antigen exposure in vitro also leads to impaired proliferative responses; thus immature B cell function is compromised by exposure to a moderate affinity, non‐crosslinking antigen. These data suggest that the small protein hormone, insulin, induces anergy in developing B cells in the bone marrow, and identify a therapeutic window for preventing insulin autoimmunity.
Generation of IgE B cells in‐vitro and pathway analysis by microarray reveals specific lack of NF‐kB survival pathway
Authors: Tsai Angela , Zhang Tingting , Zhang Zhaomei , Wang Jianmei , Allard John , Fuentes Maria , Dahl Martin E.
Published: June 2021

IgE B cells that differentiate into plasma cells capable to secreting IgE antibodies play a central role in allergic disease. The development of a therapeutic to neutralize IgE is well documented and modulates allergic asthma. However, effective therapies that target IgE B cells specifically have not been identified. Immature murine B cells induced to undergo class switching via stimulation with IL‐4 and anti‐CD40 antibodies become either IgG1 or IgE expressing B cells, although the survival of IgE B cells is significantly reduced in‐vitro. We sought to elucidate potential mechanisms that would account for the significant cell death of IgE B cells during early development. After inducing IgG1 or IgE class switching of naïve B cells, we enriched for each B cell population and compared their gene expression profile by microarray to identify unique pathways. A total of 397 genes were differentially expressed. Among those genes, we identified the lack of a survival pathway in IgE B cells characterized by reduced mRNA expression of IKKβ, NF‐kB and cIAP, as well as BCL‐XL and BCL‐2. Furthermore, a large number of genes associated with the downstream effects of NF‐kB were specifically down regulated in IgE B cells compared to IgG1 B cells. The differential expression of this signaling pathway reflects a fundamental difference in the development of two distinct B cell subsets after identical stimulation. Source of support: Roche
The IL‐3/IL‐5/GM‐CSF Common β Receptor Plays a Pivotal Role in Regulating Th2 Immunity and Allergic Airway Inflammation
Authors: Asquith Kelly Lyn , Ramshaw Hayley , Lopez Angel , Foster Paul Stephen
Published: June 2021

The eosinophil is a central effector cell in allergic asthma. Differentiation and function of eosinophils is regulated by interleukin (IL)‐3, IL‐5 and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), which all signal through a common β receptor subunit (βc). Recent therapeutic approaches targeting IL‐5 alone have had limited effects on disease progression. By employing mice lacking the βc and βIL‐3 receptor subunits we were able to interrupt signalling through all three eosinophilopoietic cytokines and prevent allergen‐induced expansion and accumulation of eosinophils in the lung in a mouse model of allergic airways inflammation. Moreover, βc deficiency resulted in inhibition of hallmark features of asthma including airways hypersensitivity, mucus hypersecretion and antigen‐specific IgE. Surprisingly, we also identified a critical role for the βc in regulating type 2 immunity. Th2 cells in the lung of allergen‐challenged βc−/− mice were limited in their ability to proliferate, produce cytokines and migrate to effector sites, which was attributed to reduced numbers of myeloid dendritic cells in the lung compartment. Thus the βc subunit plays a critical role in allergen‐induced eosinophil responses and is pivotal in regulating molecules that promote both early and late phases of allergic inflammation, representing a novel target for therapy. (Supported by the NIH and NHMRC)
Understanding the dynamics of an endogenous ova specific CD8+ T‐cell response in a murine model of allergic airway disease
Authors: McNamara Jeffery Thomas , Singh Anurag , Secor Eric , Guernsey Linda , Thrall Roger , Lefrancois Leo
Published: June 2021

Elegant studies examining the endogenous antigen specific CD8+ T‐cell responses to bacterial infections have recently been published utilizing confocal imaging and tetramer technology. The current study was designed to elucidate the role of antigen specific CD8+ T‐cells in a well characterized murine model of allergic airway disease (AAD). In this AAD model acute aerosol exposure (3–7days) with 1% ovalbumin results in a clonal expansion of T and B‐cells in the local lung compartments (BAL, Lung tissue, HLN). Chronic aerosol exposure (42days) results in results in resolution of AAD. We have labeled this as local inhalational tolerance (LIT), which is characterized by the maintenance of CD8+ T cells. We hypothesis that antigen specific CD8+ T‐cells expand during the acute phase of the model and contribute to the development of AAD and are diminished at LIT. This makes them an important indicator of disease but suggests they have a limited role in the resolution of disease. Utilizing flow cytometry we have found an increase in tetramer positive CD8+ T cells in local lung compartments during AAD and a decrease during LIT. We are currently correlating this data using confocal microscopy.
A Role for Indoleamine 2,3‐Dioxygenase in Lung Dendritic Cell Activation in Response to Allergens Impacting Allergic Airways Disease
Authors: Xu Hui , Oriss Timothy B , Henry Adam C , Melgert Barbro N , Chen Li , Mellor Andrew L , Munn David H , Irvin Charles G , Ray Prabir , Ray Anuradha
Published: June 2021

Indoleamine 2,3 dioxgenase (IDO) has been shown to inhibit Th1‐mediated immune responses. However, the role of IDO in Th2 cell‐mediated diseases has not been adequately addressed. We have investigated the role of IDO in allergic airways disease. Interestingly, IDO protein expression was easily detected in lung myeloid DCs from naïve wild‐type (WT) mice which was not true for spleen DCs. Upon antigen sensitization via the lung, fewer mature DCs and fewer adoptively transferred CD4+ T cells from DO11.10 mice were recovered from lung‐draining lymph nodes (LNs) of IDO−/− mice compared to that from LNs of WT mice. In line with the lower net accumulation of adoptively transferred CD4+ T cells in the lung‐draining LNs of IDO−/− mice, Th2 cytokine levels in the lung tissue of these mice was also lower. In a chronic model of experimental asthma, all features of allergic airways disease were significantly attenuated in the IDO−/− mice. Collectively, these data show an important role for IDO in lung myeloid DC activation in response to allergens and suggest that the reduced Th2 type immune response in IDO−/− mice is due, at least in part, to less effective co‐stimulation between DCs and T cells. Supported by NIH grants HL 77430 (to A.R) and HL60207 (to P.R.)
IL‐1β is required for TLR‐mediated antimicrobial activity
Authors: Liu Philip T. , Schenk Mirjam , Kanchanapoomi Melissa , Krutzik Stephan , Modlin Robert L.
Published: June 2021

Toll‐like receptor 2/1 activation on human monocytes induces vitamin D receptor (VDR) dependent expression of the antimicrobial peptide (AMP) cathelicidin, which is the only known TLR‐regulated AMP expressed in monocytes. We hypothesize that DEFB4, a VDR‐dependent AMP, will also be regulated by TLR2/1. Here we demonstrate that TLR2/1 activated monocytes in vitamin D sufficient human serum (HS) but not fetal calf serum (FCS) expresses DEFB4. Knockdown of either DEFB4 or cathelicidin ablates the TLR‐mediated antimicrobial activity against M. tb, indicating that both peptides are required. Supplementation of FCS with the vitamin D prohormone (25D3) rescues TLR‐induction of cathelicidin but not DEFB4. Induction of DEFB4 in HS is dependent on the VDR, but the active vitamin D (1,25D3) alone was not sufficient for DEFB4 expression, suggesting another factor is required, such as IL‐1β. TLR2/1 activated monocytes cultured in HS secrete significantly higher levels of IL‐1β than cells in FCS. TLR‐induced DEFB4 but not cathelicidin expression was synergistically increased with addition of IL‐1β or ablated when IL‐1β activity was inhibited. These data suggest that autocrine production of IL‐1β is required for the TLR‐induced antimicrobial activity.
Toll‐like receptor signaling increases production of 1,25‐dihydroxyvitamin D3 in bovine macrophages
Authors: Nelson Corwin D. , Beitz Donald C. , Reinhardt Tim A. , Lippolis John D.
Published: June 2021

Activation of macrophages can occur through Toll‐like receptor (TLR) recognition of pathogen associated molecular patterns (PAMP). Recently, it has been discovered that TLR signaling can increase 1α‐hydroxylase (Cyp27B1) expression in human and mouse macrophages. The enzymatic activity of 1α‐hydroxylase produces the steroid hormone 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3) from its precursor 25‐hydroxyvitamin D3 (25(OH)D3. Thus, we hypothesized that TLR recognition of PAMP can increase Cyp27B1 expression and conversion of 25(OH)D3 to 1,25(OH)2D3 in bovine macrophages. We supplemented bovine monocyte derived macrophages (BMDM) with 25(OH)D3 and stimulated them with ligands for TLR2 and TLR4. Quantitative real‐time PCR was used to measure changes in Cyp27B1 and 24‐hydroxylase (Cyp24A1) mRNA expression. Stimulation of BMDM with TLR2 and TLR4 ligands in the presence of 25(OH)D3 resulted in increased Cyp27B1 and Cyp24A1 mRNA expression. Conversion of 25(OH)D3 to 1,25(OH)2D3 in BMDM was demonstrated by increased expression of Cyp24A1, which is dependent on binding of the vitamin D receptor/1,25(OH)2D3 complex to the Cyp24A1 promoter. In conclusion, our results show that an autocrine vitamin D signaling pathway is present in bovine macrophages. Future studies will focus on the impact of vitamin D on the bovine immune system at the cellular and molecular level.

Funded by USDA‐ARS CRIS 3625‐32000‐094‐00D
Airway epithelial versus immune cell STAT‐1 function for innate defense against respiratory viral infection
Authors: Shornick Laurie P , Wells Audrey G , Zhang Yong , Patel Anand C , Huang Guangming , Kazutaka Takami , Sosa Moises , Shukla Nikhil A , Agapov Eugene A , Holtzman Michael J
Published: June 2021

The epithelial surface is often proposed to actively participate in host defense, but the evidence remains circumstantial. Here we use a mouse paramyxovirus (Sendai virus; SeV) to show that a prominent early event in respiratory infection is activation of Stat1 in airway epithelial cells. Stat1−/– mice developed illness that resembled severe paramyxoviral respiratory infection in humans and was characterized by increased viral replication and neutrophilic inflammation in concert with overproduction of TNF‐α and MIP‐2. Poor control of viral replication as well as TNF‐α and MIP‐2 overproduction were mimicked by infection of Stat1−/– airway epithelial cells in vitro. TNF‐α drives the MIP‐2 response, since it can be reversed by TNF‐α blockade both in vitro and in vivo. These findings pointed to an epithelial defect in Stat1−/– mice. Indeed, Stat1−/– mice reconstituted with wild‐type bone marrow were still susceptible to infection with SeV whereas wild‐type mice that received Stat1−/– bone marrow retained resistance to infection. The susceptible epithelial Stat1−/– chimeric mice also exhibited increased viral replication as well as excessive neutrophils, MIP‐2, and TNF‐α in the airspace. These findings provide some of the most definitive evidence for the role of barrier epithelial cells in innate immunity to common pathogens, particularly in controlling viral replication. Funding: NIH & Parker B. Francis Foundation
CpG oligodeoxynucleotides induce cyclooxygenase‐2 and prostaglandin synthesis in human B lymphocytes: Implications for antibody production and innate immunity.
Authors: Phipps Richard P. , Bernard Matthew
Published: June 2021

Many chronic inflammatory diseases are characterized by infiltrates of B cells and plasma cells coupled with high prostaglandin (PG) levels. B cells were not previously thought to express cyclooxygenase‐2 (Cox‐2) and PGs. Interest in CpG oligodeoxynucleotides (ODN) is fueled by clinical trials that show CpG ODN enhance antibody production and act as adjuvants. Since antibodies play a key role in fighting infections, it is important to understand how CpG ODN affect human B cells and influence antibody production. We recently demonstrated that activated human B cells highly express Cox‐2 and that Cox‐2 activity is crucial for optimal antibody production (J. Immunology, 177:7811, 2006). Herein, we tested the hypothesis that CpG ODNs induce human B cell Cox‐2 and that Cox‐2 activity is important for optimal antibody production. Exposure of human B lymphocytes to synthetic CpG ODN sequences increased steady‐state Cox‐2 mRNA levels and induced Cox‐2 protein. Importantly, CpG‐induced IgM and IgG production was attenuated following inhibition of Cox‐2 activity by selective drug inhibitors. CpG motifs therefore enhance antibody production by directly stimulating B cells and by inducing Cox‐2. These findings support the concept that drugs that attenuate Cox‐2 activity can reduce optimal antibody response to adjuvants/vaccination.
Novel function of complement C3d as an autologous helper T cell target
Authors: Knopf Paul , Hai Si‐Han , Rivera Daniel S. , Martin William , McMurray Julie , De Groot Anne S.
Published: June 2021

The C3d fragment of complement component C3 has been shown to enhance immune responses to antigens that lack T‐cell epitopes such as bacterial polysaccharides by binding to the B cell complement receptor (CD21). Paradoxically, C3d has also been shown to enhance B cell and T cell responses in the CD21 KO mouse model, suggesting that that an auxiliary CR2‐independent pathway of immune activation may exist. We hypothesized that in addition to its molecular adjuvant property that enhances signal 1 during B cell activation (co‐signal 1), C3d also contains T cell epitopes that are able to stimulate autoreactive C3d peptide‐specific helper T cells which we term “co‐signal 2”.

Using the EpiMatrix T‐cell epitope‐mapping algorithm, we identified 11 putative T‐cell epitopes in C3d. Stimulation of human PBMC with C3d‐derived peptides leads to dramatic increases IFN‐γ secretion. These IFN‐γ secreting cells were exclusively found to reside in the CD4+ T helper subset by FACS analysis.

We believe that the discovery of these autologous T cells autoreactive for C3d provides evidence supporting the “co‐signal 2” hypothesis and may offer a novel explanation of the CD21 KO paradox. This data suggests a novel mechanism for the adjuvant effect of C3d and raises questions regarding self‐tolerance and suggests the possibility of AIRE‐independent differentiation into non‐pathogenic, self‐reactive T cells.

All work funded by EpiVax, Inc.