bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of pro‐apoptotic signals via the CD28 in the thymus
Authors: Kishimoto Hidehiro , Ogawa Shuhei , Abe Ryo
Published: June 2021

Along with TCR signal, costimulatory signal given by CD28 plays a key roles in deciding the fate of T cells. Decision of death or survival of T cells by CD28 signal depends on the development stages of T cells. TCR+CD28 signals induce activation, proliferation and production of various cytokines in mature peripheral T cells whereas apoptosis is induced in CD4+8+ and CD24+CD4+8− thymocytes by TCR+CD28 signals. PI3K and Grb2/Gads have been shown to be recruited the YMNM motif in CD28 cytoplasmic portion, and LCK and ITK could associate with two PXXP motifs. To elucidate pro‐apoptotic signals induced by CD28, we established transgenic (Tg) mice expressing CD28 that have various mutations in CD28 cytoplasmic portion, and investigated susceptibility of CD4+8+ and CD24+CD4+8− thymocytes from those mutant CD28 mice to apoptosis induced by TCR+CD28 ligation. When immature and semi‐mature thymocytes form various CD28 mutant Tg mouse lines were stimulated with TCR+CD28 mAbs, thymocytes from the YF line and the ncPA line of CD28 mutant Tg mice showed complete abrogation of pro‐apoptotic function by CD28 signaling. The YF Tg line has no ability to recruit PI3K and Grb2/Gads, and the ncPA Tg line that is not designed to associate with neither LCK nor ITK. These results indicated that phosphorylation of the tyrosine residue in YMNM motif is indispensable for induction of pro‐apoptotic function in developing thymocytes by CD28.
Essential role for the beta3 regulatory subunit of L‐type calcium channel in the survival and functions of CD8 T cells
Authors: Jha Mithilesh Kumar , Badou Abdallah , Flockerzi Veit , Freichel Marc , Flavell Richard
Published: June 2021

Peripheral survival and homeostasis of naive T lymphocytes require sustained calcium influx mediated by interaction of naive T lymphocytes with dendritic cells in the absence of cognate antigen. The molecular mechanism which governs calcium influx in naive T lymphocytes is not well understood. Here we report a critical role of beta3‐Cav1.4 calcium channel complex in regulating the calcium influx in naive CD8 T lymphocytes. Beta3 and pore forming alpha subunit of Cav1.4 calcium channel are expressed in naive CD8 T cells. Beta3 KO naive CD8 T cells did not accumulate in vivo since these cells were susceptible to apoptosis due to increased Fas expression. TCR‐mediated global calcium entry and NFAT activation were reduced in beta3 KO naive CD8 T cells. IFN‐gamma, IL‐2 and TNF‐alpha production were significantly impaired in beta3KO CD8 cells challenged with BMDCs loaded with MHC class I‐specific ovalbumin peptide. Beta3 deficiency abrogated the pore forming Cav1.4 synthesis suggesting that it regulates the functional expression of Cav1.4 subunit in naive CD8 T cells. Our data demonstrate a critical role of Cav1.4‐beta3 channel complex in regulating the calcium entry required for the survival and functions of CD8 T cells.
Id3 controls the developmental window of γδ T cells
Authors: Hayakawa Ikuko , Zhuang Yuan
Published: June 2021

T cell progenitors permit active rearrangement at the TCRδ, TCRγ, and TCRβ loci prior to bifurcation of γδ and αβ T cell lineages. In postnatal thymus, majority of these bi‐potential progenitor cells develop into the αβ T cell lineage whereas only a small fraction chooses to adopt the γδ fate. Expression of a fully rearranged and functional TCRβ gene defines the checkpoint for αβ lineage commitment. A substantial fraction of β rearranging cells will not develop further due to out of frame rearrangements. We show here that loss of Id3, a transcription regulator, promotes γδ T cell development from progenitor T cells which have completed TCRβ rearrangement but without producing a functional TCRβ allele. Our data suggests that Id3 may play an important role in shutting off the window of γδ development after the completion of V‐DJβ rearrangement.
Probing the sensitivity of negative selection using two HEL TCR transgenic mice
Authors: Atibalentja Danielle Francoise , Unanue Emil R
Published: June 2021

Thymocytes that recognize self peptide‐MHC (pMHC) complexes with high avidity are eliminated by the process of negative selection. Using in vitro analysis, it has been shown that as few as three pMHC complexes can mediate the deletion of double positive thymocytes. Here, we examine negative selection in two TCR transgenic mice whose T‐cells recognize differentially presented epitopes of HEL (hen egg white lysozyme) on Ak. We show that 3A9 thymocytes which recognize the major HEL epitope, undergo efficient negative selection when mice are injected intravenously with soluble HEL. As little as 5 μg (350 pmol) intravenous HEL results in 3A9 thymocyte deletion. In contrast, LB11.3 thymocytes which recognize a minor epitope of HEL, require 50 μg (3.5 nmol) intravenous HEL to be deleted. Using 125I‐HEL, we show that only 0.25% of the injected HEL enters the thymus. This corresponds to 0.87 pmol or 5.2 x1011 molecules of HEL after a 5 μg intravenous injection. Together, with published data on HEL uptake, processing efficiency and Ak expression, we estimate 5.2 x106– 5.2 x107 pMHC complexes in the thymus are sufficient to delete 3A9 T‐cells. Assuming 106 APCs in the thymus, this suggest that negative selection can occur in response to single digit numbers of pMHC complexes, supporting previous in vitro findings and highlighting the remarkable sensitivity of this process. This work is supported by NIH 5T32AI007163.
Multiparameter flow cytometric analysis of GATA‐3 expression in T cell development and differentiation
Authors: Chronopoulou Efthalia , Agadir Anissa , Gu Jian , Nobida Naomi S. , Shih Jing Ping , Solsaa Guy M. , Paulsen Brad E. , Ernst David N.
Published: June 2021

GATA‐3 is a key zinc‐finger transcription factor essential for T cell development and differentiation. In this study, a monoclonal antibody (mAb) was raised against mouse and human GATA‐3. This mAb was used to analyze GATA‐3 expression in intrathymic T cell progenitors and polarized Th1 and Th2 cells by Western blotting, and multicolor immunofluorescent staining, and flow cytometric analysis. Our results confirm that GATA‐3 is expressed at every stage of intrathymic mouse T‐cell development and that its expression levels vary among the T cell progenitor subsets. The highest levels of GATA‐3 are expressed by a subset of double negative CD4−CD8− cells and by the single positive CD4+CD8− cell population. In addition, we found that mature T cells express relatively low levels of GATA‐3 upon in vitro CD3 and CD28 costimulation. Moreover, their levels of GATA‐3 remain unchanged or rise significantly if these cells are skewed towards the Th1 or Th2 phenotypes in the presence of exogenous IL‐12 or IL‐4, respectively. In summary, we show that this antibody is compatible with both saponin and methanol permeabilization methods. It can be used in combination with fluorescent antibodies specific for staining various cell surface markers, as well as other intracellular transcription factors or cytokines to elucidate the T cell signaling pathways utilized during development and differentiation.
Thymocyte‐dendritic cell interactions near sources of CCR7 ligands in the thymic cortex
Authors: Ladi Ena , Schwickert Tanya , Chtanova Tanya , Aaron Holly , Killeen Nigel , Robey Ellen
Published: June 2021

Thymocyte maturation requires the migration through distinct regions in the thymus and interation with diverse cell types. In characterizing structures of the thymus, we describe Dendritic Cells (DCs) that are tightly associated with a cortical capillary network. We find that thymocytes expressing a transgenic, positive selecting TCR (P14) interact more extensively with DCs compared to thymocytes bearing polyclonal TCRs. In addition, overexpression of the chemokine receptor CCR7 in thymocytes results in a significant increase in DC‐thymocyte interactions in the cortex. Intriguingly, the cortical source of CCR7 ligand, CCL21, is not DCs but rather the blood vessels of the cortex which are in close contact with the cortical DCs. These observations suggest that CCR7 ligands produced by vasculature in the thymic cortex attract thymocytes that are undergoing positive selection, thus increasing the potential for cell‐cell interactions involved in T cell repertoire selection.
Cell cycle‐dependent caspase‐8 enzymatic activity is necessary for rapid lymphocyte proliferation
Authors: Walsh Craig Michael , Leverrier Sabrina
Published: June 2021

Caspase‐8 is a cysteine protease recruited to ligated death receptors and is an essential intermediary in the generation of apoptotic signals. Caspase‐8 has also been found to be necessary for the clonal expansion of lymphocytes under certain mitogenic conditions. Consistent with this, caspase‐8 enzymatic activity was strongly induced in non‐apoptotic T cells, as assessed using a fluorogenic probe specific for caspase‐8. This enzymatic activation was found to be essential for efficient T cell proliferation, since caspase‐8 deficient T cell mitogenesis was rescued by enzymatically active, but not inactive caspase‐8 expressing retroviruses. The induction of caspase‐8 activity was found to be highest in proliferating CD8+ T cells or in B cells activated by lipopolysaccharide. While caspase‐8 activity did not depend on death receptors, it was dependent upon cell cycle progression, since inhibitors of various stages of the cell cycle afforded a graded blockade in caspase‐8 enzymatic activity. Although Fas ligation led to caspase‐8 autoproteolytic processing, proliferation‐induced caspase‐8 activation did not induce such processing. These results suggest that in rapidly dividing lymphocytes, caspase‐8 enzymatic activity is induced in a manner distinct from its activation by ligated death receptors and is required for rapid lymphocyte proliferation. This work was funded by an award from NIAID (R01AI50506).
Differing Roles for ERK1 and ERK2 in CD8 T cell Activation and Survival
Authors: D'souza Warren N. , Chang Chiung‐Fang , Fischer April M. , Hedrick Stephen M.
Published: June 2021

The ERK1 and ERK2 MAP kinases regulate proliferation, survival and differentiation in a variety of cell types. Herein, we investigated the requirement for these molecules during CD8 T cell activation. We observed that ERK2, but not ERK1 was critical for cell cycle initiation in the absence of co‐stimulation in vitro. Surprisingly, CD8 T cell proliferation following optimal stimulation in vitro or following two different pathogenic infections in vivo proceeded uninhibited in the absence of either ERK1 or ERK2. However, ERK2 signaling remained indispensable for the survival and accumulation of the effector CD8 T cells. This function was mediated by the Bcl‐2 family members Bcl‐2, Bcl‐XL and Bim. As a result, ERK2 deficient CD8 T cell viability was restored by the additional ablation of Bim. These results ascribe multifaceted functions specific to ERK2 in CD8 T cell activation and survival.
4‐1BBL is a critical component in the costimulation dependent rescue of functionally impaired HIV‐specific CTL
Authors: Wang Chao , Wen Tao , Routy Jean‐Pierre , Bernard Nicole , Sekaly Rafick , Watts Tania
Published: June 2021

During chronic infection, HIV‐specific CD8 T cells exhibit progressive signs of functional impairment, attributed to persistent antigenic stimulation, up‐regulation of the inhibitory receptor PD‐1, and declining T cell help. Strategies that directly improve CD8 T cell function offer the potential of restoring immune control of HIV. Although PD‐1 expression has been identified as a cause of functional impairment in HIV, in this study, PD‐1 expression was observed on only a subfraction of HIV‐specific CTL in a subfraction of donors, whereas HIV‐specific CTL from all donors exhibited a limited repertoire of effector functions. 4‐1BBL is emerging as an important stimulator of antiviral CD8 T cell responses. Regardless of the PD‐1 status of the donors, here we show that 4‐1BBL, when combined with CD80 or CD70, expands a population of Ag‐specific CD8 T cells expressing multiple markers of effector function, from the functionally impaired starting population. In contrast, CD70 in combination with CD80 was insufficient for these effects and the related TNF family ligand, LIGHT, had negligible activity. The unique contribution of 4‐1BBL correlated with down‐regulation of the proapoptotic molecule Bim in activated CD8 T cells. Decreasing the level of TRAF1 in T cells using siRNA resulted in increased levels of Bim in the 4‐1BBL‐stimulated CD8s. Thus, costimulation via 4‐1BBL leads to TRAF1‐dependent Bim down‐modulation in T cells, resulting in increased T cell expansion. These studies identify 4‐1BBL as a critical component in therapeutic strategies aimed at improving CD8 T cell function.
Modulation effect of in vivo induced type I immune responses on airway inflammation in an asthma model
Authors: Tang Yafang
Published: June 2021

Asthma is a Th2‐mediated inflammatory disease of the airways characterized by reversible obstruction of respiratory airway flow caused by airway thickening, mucus hyper‐secretion and bronchoconstriction. Production of antigen‐specific IgE, type II cytokines such as IL‐4, IL‐5 and IL‐13 as well as eosinophil infiltration into the lung are important markers of asthma. It is widely accepted that Th1 responses induced by viral or bacterial infections can antagonize allergic diseases. However, there is also evidence showing that virus or bacteria‐induced Th1 immune responses can promote the development or exacerbation of allergic diseases. Genetic background was recognized as a possible explanation on this contradictory issue. We show here that ovalbumin (OVA) induced stronger Th2 immune in Th2‐biased BALB/c mice while Th1 responses induced by influenza A virus were similar in both BALB/c and Th1‐biased C57BL/6 mice. When the asthmatic mice were infected with Influenza A, despite for the severer netrophilia induced in BALB/c mice than in C57BL/6 mice, OVA‐induced eosinophilia, IgE production and Th2 cytokine secretion were not affected by viral infection in either strain of mouse. Apart from Th1 cells, cytotoxic CD8 T cells (CTLs) also play an important role in immune defence against viral infection. To study the role of CTLs in asthma development, OVA‐specific CTLs isolated from OT‐I mice were activated in vitro and then intravenously injected into OVA sensitized C57BL/6 mice before intranasal challenge with OVA. We found that transferred CTLs led to an increase in IFN‐γ level in the bronchoalveolar larvage (BAL) fluid without apparent inhibition effect on IgE production.

This work is funded by Immunology Programme (Grant no. C182‐002‐002‐001). Tang YF is supported by NUS Graduate School for Integrative Science and Engineering Scholarship.
Breaking of CD8+ T cell tolerance by in vivo ligation of CD40 results in inhibition of chronic graft‐vs‐host disease (GVHD) and complete donor cell engraftment without inducing acute GVHD
Authors: Kim Juyang , Kim Hye Jeong
Published: June 2021

In the DBA/2□ unirradiated (C57BL/6 × DBA/2)F1 model of systemic lupus erythematosus‐like chronic graft‐vs‐host disease (cGVHD), donor CD4+ T cells play a critical role in breaking host B cell tolerance, while donor CD8+ T cells are removed after transfer into the host and the remaining cells fall into anergy. Previously we have demonstrated that in vivo ligation of GITR, a costimulatory receptor, can activate donor CD8+ T cells, which subsequently result in converting the disease pattern from cGVHD to an acute form (aGVHD). In this study, we investigated the effect of an agonistic mAb against CD40 on cGVHD. Treatment of anti‐CD40 mAb inhibited the production of anti‐DNA IgG1 autoantibody and the development of glomerulonephritis, typical symptoms of cGVHD. The inhibition of cGVHD occurred because anti‐CD40 mAb prevented donor CD8+ T cell anergy such that subsequently activated donor CD8+ T cells deleted host cells including host CD4+ T cells and host B cells involved in autoantibody production. In addition, functionally active donor CD8+ T cells induced full engraftment of donor cells and exhibited an increased graft‐vs‐leukemia (GVL) effect. However, induction of aGVHD by donor CD8+ T cells seemed to be not so apparent. Further CTL analysis indicated that there were lower levels of donor CTL activity against host cells in mice that received anti‐CD40 mAb, compared with mice that received anti‐ GITR mAb. Taken together, our results suggest that a different intensity of donor CTL activity is required for removal of host hematopoietic cells including leukemia vs induction of aGVHD. Therefore, it is possible to segregate a GVL effect from GVHD by modulate the cytotoxic activity of donor CD8+ T cells
CD5, CD28 and CD40 as interconnected co‐stimulatory/immune‐modulators of T cells responses in the NOD, NOR and BALB mouse strains
Authors: Pennock Nathan David , Craig Dan B , Vaitaitis Gisela M , Waid Dan M , Wagner David H
Published: June 2021

Previously we have illustrated a significant expansion of the CD4+CD40+ T cell subset in both mouse and human type 1‐diabetic subjects as compared to non‐autoimmune controls. To mechanistically associate disease onset with the observed expansions in this cell population we have suggested that CD40 engagement promotes alteration of peripheral TCR (revision) as well as induces the anti‐apoptotic protein BcL‐xL. Recently it has been documented that CD40 can serve as co‐stimulatory molecule further enhancing the classical signal 1 & 2 activation stimulus of CD3 & CD28 in the C57BL/6 model of CIA illustrating that CD40 signaling has a direct effect on effector T cell function. Here we elaborate on the notion of CD40 as a co‐stimulatory molecule illustrating that in NOD, NOR and BALB, CD40 dampens the IL‐2 response instigated by CD3&CD28 while elevating IL‐2 response induced by the unconventional stimulus of CD3&CD5, revealing CD40 to have a role as an immunomodulator whose effects are dependent upon activation conditions and intimately connected with signals propagated through CD3, CD28 and CD5. Importantly we illustrate that CD5 a molecule long associated with augmentation of TCR signaling, reverses the inhibitory effects of CD40. Additionally we observe that CD5 activation in combination with signals from CD3, CD28 and CD40 induces surface expression of CD40 on the surface of previously CD40 negative T cells.
Adjuvants enhance the cognate function of aged CD4 T cells via a mechanism involving IL‐17 production
Authors: Eaton Sheri , Lanthier Paula A , Maue Alexander C , Haynes Laura
Published: June 2021

The humoral response in aged humans and mice is significantly reduced compared to young and is a contributing factor in the reduced efficacy of vaccines seen in aged populations. Using an adoptive transfer model comparing young and aged T cell receptor transgenic (TCR Tg) CD4 T cells in young hosts, we have shown that age‐related declines in CD4 T cell function contributed greatly to the reduced humoral responses observed in the aged. This decline leads to reduced B cell expansion and differentiation as well as reduced IgG production. The helper function of aged CD4 T was enhanced using a combination of inflammatory cytokines (TNF alpha, IL‐1 and IL‐6) or adjuvants that induce these cytokines. In addition, the helper function of aged CD4 T cells was also enhanced when the inflammatory cytokines were added during in vitro CD4 effector generation. In both of these cases, the helper activity of aged CD4 T cells was significantly enhanced, leading to better expansion and differentiation of antigen‐specific B cells. Using real time PCR, we found that these cytokines induced significant production of IL‐17 by the aged CD4 T cells in vitro. We have also gone on to show that Th17 effectors generated in vitro from naive aged CD4 T cells exhibit potent helper activity in our adoptive transfer model. We propose that one of the major mechanisms involved in the ability of adjuvants to enhance T cell responses involves induction of IL‐17 production by the responding CD4 T cells.

This work was funded by NIH grant AG02160 to LH.
IL‐13Rα2 and IL‐10 coordinately suppress Th2‐dependent Inflammation and Immunopathology.
Authors: Wilson Mark Stephen , Elnekave Eldad , Mentink‐Kane Margaret , Pesce John , Ramalingam Thirumalai , Thompson Robert , Flavell Richard , Cheever Allen , Wynn Thomas
Published: June 2021

Immunopathology; such as airway hyper responsiveness (AHR) in asthmatics and fibrotic lesions following infection with Schistosoma sp. often result from excessive inflammation. However, the transition from inflammation to immunopathology is poorly understood. This study aimed to dissect critical mediators during Th2‐driven inflammation, following infection or airway allergen provocation, which mediate down‐stream pathology and to subsequently identify the regulatory mechanisms that control them. To separate inflammation from immunopathology, we used two in‐vivo models ‐ a model of asthma and Schistosomiasis, both of which present distinctive inflammation and downstream pathological changes. Following allergen challenge or infection, we observed an accumulation of IL‐10+ and Foxp3+ regulatory T cells (Treg) in the lungs or liver respectively. In the absence of IL‐10, Th2 inflammation was exacerbated, as well as the emergence of Th1 responses. Paradoxically, despite increased inflammation in IL‐10–/– mice pathology was curtailed. In both the lung and the liver, IL‐13R α 2, an endogenous neutralizer of IL‐13, was elevated. To test the role of IL‐13R α 2 in regulating Th2 pathology we generated mice deficient in both IL‐13R α 2 and IL‐10 (dKO). The absence of IL‐13R α 2, on an IL‐10–/– background, increased lung and liver pathology, compared to IL‐10–/– mice, indicating that IL‐13R α 2 is a key regulator of pathology, even during uncontrolled inflammation. This study presents a novel two‐pronged model of immunoregulation by IL‐10‐secreting Treg's and IL‐13R α 2, which could be exploited to control a variety of important Th2‐dominant diseases.
Bim and Fas Function Concurrently to Control Autoimmunity and CD8+ T Cell Contraction
Authors: Weant Ashley , Michalek Ryan D , Khan Islam U , Holbrook Beth C , Willingham Mark C , Grayson Jason M
Published: June 2021

Throughout most of adult life lymphocyte number remains constant due to a balance between proliferation and death. To determine whether loss of genes in both the extrinsic and intrinsic cell death pathways would exacerbate defects in immune homeostasis we created Bim−/−Faslpr/lpr mice. Mice were born at the expected Mendelian ratios and appeared to develop normally. However, by 14 weeks of age they developed massive lymphadenopathy compared to either single mutant or wildtype animals. Loss of both Bim and Fas also resulted in dramatic increases in autoantibodies and deposition of immunoglobulin in the kidneys. When antiviral immune responses were quantitated following acute LCMV infection, double mutants contained 100‐fold more antigen‐specific memory CD8+ T cells in their lymph nodes compared to wildtype mice. This increase in cell numbers was not the result of increased proliferation, therefore it must be due to decreased apoptosis. Although contraction was blocked in the lymph nodes of double mutants, it was similar to the Bim parental strain in the spleen and liver. Following chronic LCMV infection, the number of antigen‐specific CD8+ T cells was similar between Bim−/−Faslpr/lpr and the Bim−/− parental genotype. Our results demonstrate that multiple death pathways function concurrently to prevent autoimmunity and downsize T cell responses.

Supported by ACS‐ #RSG‐04‐066‐01‐MBC to Jason Grayson.
Non‐antigenic signals alter memory CD8 T cell development during chronic infection
Authors: Shin Haina , Beiting Daniel , Roos David , Wherry E. John
Published: June 2021

Memory CD8 T cells are a critical component of protective immunity. After an acute infection or vaccination, a small pool of antigen‐specific CD8 T cells differentiate from effector to memory CD8 T cells. They acquire several properties, including the ability to rapidly produce anti‐viral cytokines and a high proliferative capacity, that allow them to confer protection. During chronic infection, however, CD8 T cells undergo altered memory differentiation and do not acquire the properties that are attributed to memory CD8 T cells. While antigen load has been shown to correlate with the severity of CD8 T cell dysfunction, it is not clear if persistent antigen signaling alone is responsible for all the defects that occur during chronic infection. We have found that the non‐antigenic signals present during chronic infection influence the survival, phenotype and proliferative capacity of antigen‐specific CD8 T cells differentiating from the effector to memory stage, but did not have a profound effect on cytokine production. Furthermore, the chronic inflammatory environment may be sufficient to delay the differentiation of effector memory CD8 T cells to central memory. These findings indicate that TCR and non‐TCR signals may alter different aspects of memory differentiation during chronic infection.
Ustekinumab neutralizes human IL‐12 and IL‐23 mediated Th1 and Th17 cell function and provides clinical benefit to patients with plaque psoriasis
Authors: Benson Jacqueline , Orlovsky Yevgeniya , Luo Jinquan , Wu Sheng‐Jiun , Lacy Eilyn , Li Jian
Published: June 2021

Ustekinumab (aka CNTO 1275) is a fully human IgG1k monoclonal antibody that specifically binds to human and non‐human primate IL‐12/23p40, the 40 kDa protein subunit of the heterodimeric cytokines IL 12 and IL 23. The precise molecular interactions between ustekinumab and IL‐12/23p40 are well characterized through crystal structure, protein mutagenesis, and stoichiometry analysis. Ustekinumab demonstrates equivalent neutralization of IL‐12 and IL‐23 bioactivity by binding to IL‐12/23p40 and preventing interaction with the cell surface IL‐12Rb1 receptor protein. Ustekinumab does not bind to receptor bound IL‐12 or IL‐23 and does not stimulate complement dependent cytotoxicity. In addition, equivalent neutralization is achieved with intact IgG or Fab fragments of ustekinumab. Thus, there is no evidence that the Fc component of ustekinumab contributes to the mechanism of action. Ustekinumab effectively neutralizes all human IL‐12 (Th1) and IL‐23 (Th17) mediated cellular responses tested using in vitro bioassays. Th1 and Th17 cascades are associated with a variety of human immune‐mediated diseases. Thus, ustekinumab may serve as a first‐in‐class therapy with broad applications in areas of unmet medical need. Indeed, ustekinumab provides a significant level of efficacy in the majority of subjects in clinical studies of plaque psoriasis. (All funding was provided by Centocor R&D)
G‐CSF induces leukocyte recruitment in vivo via the leukocyte G‐CSF receptor
Authors: Hickey Michael , Norman M. Ursula , Croker Ben , Roberts Andrew , Wicks Ian , Eyles Jo
Published: June 2021

Granulocyte colony stimulating factor (G‐CSF) is in routine clinical use but has been shown to activate neutrophil adhesion in vitro. The aim of this study was to determine if G‐CSF is capable of inducing leukocyte‐endothelial cell interactions in vivo. Mice were treated with G‐CSF either by acute, local administration, or systemically over 4 hrs, and leukocyte‐endothelial cell interactions assessed via intravital microscopy of the cremasteric microcirculation. Local application of G‐CSF caused a reduction in leukocyte rolling but induced a significant increase in leukocyte adhesion within 15 mins, a process dependent on Mac‐1. The adherent cells were exclusively Gr‐1+ neutrophils. Adherent leukocytes also underwent transmigration within 45 mins. To assess the effect of G‐CSF under conditions mimicking its clinical use, G‐CSF was also administered systemically. Systemic G‐CSF induced increased leukocyte rolling, adhesion and transmigration after 4 hrs. Using mice rendered chimeric for the G‐CSF receptor (G‐CSFR) via bone marrow transfer between wild‐type and G‐CSFR−/− mice, this response was found to be dependent on leukocyte‐expressed G‐CSFR. These findings indicate a pro‐inflammatory role for G‐CSF and provide a potential mechanism for the association of G‐CSF administration with exacerbation of diseases such as rheumatoid arthritis.

Supported by the NHMRC (Australia)
Identification of formyl peptides from S. aureus and L. monocytogenes as highly potent chemoattractants for mouse neutrophils
Authors: Southgate Erica Lynn , He Rong , Ye Richard D
Published: June 2021

The prototypic formyl peptide fMet‐Leu‐Phe (fMLF) is an E. coli‐derived chemoattractant for neutrophils. fMLF is a potent agonist of human formyl peptide receptor 1 (FPR1) and induces bactericidal functions of human neutrophils at nanomolar concentrations. However, mouse neutrophil activation requires fMLF concentrations in the micromolar range. To determine whether the mouse FPR is a physiologically relevant receptor for the detection of invading bacteria, formyl peptides derived from S. aureus and L. monocytogenes were examined. It was found that a tetrapeptide (fMIFL) from S. aureus and a pentapeptide (fMIVIL) from L. monocytogenes are potent agonists for the activation of mouse neutrophils. Both fMIFL and fMIVIL induced chemotaxis at concentrations of 1–10 nM and superoxide production at concentrations of 10–100 nM. Additionally, fMIFL and fMIVIL induced greater calcium mobilization than fMLF in mouse neutrophils. Mouse FPR1‐transfected rat basophilic leukemia cells (RBL) also responded to these peptides with greater induction of calcium mobilization and phosphorylation of ERK1/2 compared to fMLF. In conclusion, the S. aureus and L. monocytogenes derived peptides are approximately 100‐fold more potent than fMLF for the mouse FPR1. Differential recognition of formyl peptides from different bacterial strains by mouse neutrophils may be important for the innate immunity to these microorganisms.
Pro‐inflammatory immune complexes are sufficient to induce a lupus‐like phenotype in R4AxFcRIIb−/− BALB/c mice
Authors: Jeganathan Venkatesh , Kawabata Daisuke , Xu Xiaonan , Kim Sunjung , Diamond Betty , Grimaldi Christine
Published: June 2021

FcRIIb, a low‐affinity inhibitory receptor for the Fc portion of IgG, is expressed on B cells and is required for the elimination of long‐lived plasma cells. FcRIIb−/− C57BL/6 mice spontaneously develop high titers of pathogenic IgG anti‐DNA antibodies and die prematurely of immune complex (IC)‐mediated renal failure, whereas BALB/c mice deficient in FcRIIb show no evidence of autoimmune disease. We have developed a mouse that is transgenic (Tg) for the heavy (H) chain of a nephritogenic anti‐DNA antibody (R4A‐γ2b) that maintains B cell tolerance when expressed on a non‐autoimmune background. Since the R4A H chain can generate pro‐inflammatory anti‐DNA antibodies, we hypothesized that the R4A Tg may be sufficient to break tolerance in FcRIIb−/− BALB/c mice. Young R4A Tg BALB/c FcRIIb−/− mice displayed negligible anti‐DNA titers; however by 6 months of age they developed elevated anti‐DNA antibodies, proteinuria and IC deposition in the kidney. An increase in the number of Tg‐expressing plasma cells was observed. In addition IFN‐α inducible genes, IFI202b and OASLE were upregulated in spleen consistent with the presence of pro‐inflammatory DNA‐containing ICs. Interestingly, splenic BAFF mRNA and serum BAFF protein levels were significantly increased. We propose that FcRIIb deficiency induces a feedback loop wherein the failure to eliminate plasma cells facilitate the generation of DNA‐containing pro‐inflammatory ICs, which induce BAFF, and enhance the maturation of high affinity anti‐DNA B cells.
Gender specific disease propensities of bone marrow cells from New Zealand hybrid mice
Authors: Jorgensen Trine N. , MacLeod Megan K.L. , David Alexandria , Kappler John W. , Marrack Philippa
Published: June 2021

The (NZBxNZW)F1 (BWF1) hybrid mouse is the best analyzed mouse model of SLE. Most female, but only ∼25% of male, BWF1 mice develop SLE‐related symptoms by 1 year of age.

Sex hormones may play a role in this disease. To test this, we transferred BM cells from 9 wk old BWF1 mice into lethally irradiated, age‐matched recipients of the same or opposite gender, thus obtaining four groups of chimeras: F→F, M→F, F→M and M→M. Eighteen weeks later, F→F and F→M chimeras had higher levels of serum IgG (3.7 ± 1.8 and 3.4 ± 2.6 mg/ml respectively) than M→F or M→M chimeras (1.9 ± 1.2 or 1.9 ± 1.1 mg/ml respectively). The same was true for the development of renal disease, as it developed statistically earlier in F→F and F→M mice (27 and 30 wk post transfer, respectively) than in M→M and M→F chimeras (>33 wk, p < 0.02). Thus, BM cells from 9 week old female mice have the capacity to drive autoantibody production and renal disease regardless of their subsequent hormonal environment.

This study was funded by The Denver Autoimmune Center of Excellence and HHMI.
The role of BAFF in Autoimmunity is T cell‐independent but requires MyD88 signalling.
Authors: Mackay Fabienne , Groom Joanna R , Fletcher Carrie A , Mackay Charles R , Sweet Mathew J , Smyth Mark J
Published: June 2021

The TNF‐like ligand BAFF is a fundamental survival factor for B cells, essential during their maturation. However, overproduction of BAFF in transgenic (Tg) mice triggers autoimmune disorders similar to Systemic Lupus Erythematosus (SLE) and Sjögren's syndrome (SS), possibly as the result of abnormal self‐reactive B cell survival. Intriguingly, we showed that excess BAFF only mildly affected B cell immune tolerance, could not prevent deletion of high affinity self‐reactive B cells but could rescue some low affinity self‐reactive B cells, in particular marginal zone (MZ) B cells. As germinal center formation and antibody affinity maturation were not essential for disease in these mice, we questioned the real implication of low affinity autoreactive B cells in driving the disease. Indirect and direct effects of BAFF on T cell activation and expansion of the effector T cell compartment in BAFF Tg mice were then thought to perhaps contribute to nephritis in these animals. Surprisingly, BAFF Tg mice lacking T cells develop the same autoimmune disease as the original BAFF Tg mice. Interestingly, MyD88−/− B cells had impaired autoantibody production in BAFF Tg mice. Thus, this work revealed that autoimmunity in BAFF Tg mice is the result of an abnormal innate B cell response involving the combined effects of excess BAFF and TLR signalling.
CD4+ T cells use IFN‐γ, not IL‐17, to drive neutrophil‐mediated liver damage in murine autoimmune hepatitis
Authors: Wang Jing , Cripps James G , Robinson Richard T , Gorham James D
Published: June 2021

Autoimmune hepatitis (AIH) is a poorly understood disease caused by an immune‐mediated attack on hepatocytes. BALB/c‐TGF‐β1−/− mice spontaneously develop fulminant AIH that recapitulates important features of the human disease. Liver disease is dependent upon both CD4+ T cells and IFN‐γ. Intracellular cytokine analysis as well as cell subset depletion showed that CD4+ T cells, but not CD8+ T cells or NK cells, are the relevant source of IFN‐γ in this model system. Histological examination of TGF‐β1−/− livers revealed an abundance of neutrophils, supported by microarray analysis showing increased expression of neutrophil‐associated gene products. Liver neutrophilia was dependent upon the CD4+ T cell subset as well as on the Th1 cytokine IFN‐γ. Interestingly, TGF‐β1−/− liver CD4+ T cells produced abundant IFN‐γ, but no detectable IL‐17. The relevance of the neutrophil subset for pathology was confirmed directly, as depletion of neutrophils using a subsetspecific monoclonal antibody protected TGF‐β1−/− mice from liver disease. Thus AIH in TGF‐β1−/− mice is driven by a CD4+ Th1 – IFN‐γ – neutrophil pathway, with no role for IL‐17.
Toll‐like receptor control of IL‐6 production by dendritic cells and B cells
Authors: Sisk Bryan , Krogstad Aric , Kannan Krishnaswamy , Ortmann Robert
Published: June 2021

Collagen‐induced arthritis can be elicited in resistant C57BL/6 mice by increasing the amount of adjuvant (dessicated Mycobacterium tuberculosis) during immunization. This adjuvant contains ligands for toll‐like receptors (TLRs), especially TLR2 and TLR9, which are known to be critical for innate immune responses and are expressed on Antigen Presenting Cells (APCs) such as dendritic cells and B‐cells. Our current studies are aimed at elucidating mechanisms by which TLR signaling may promote arthritis. We investigate how cross‐linking TLR2 and TLR9 receptors might promote pathogenic Th‐17 cells by regulating the production of IL‐6 by these APCs.

We isolated bone‐marrow derived dendritic cells (BM‐DCs) and splenic B‐cells from wild type, TLR2 and TLR9 deficient B6 mice. Wild type BM‐DCs produce IL‐6 in response to TLR2, TLR4, and TLR9 cross‐linking. Immune (but not naïve) B‐cells make IL‐6 in response to TLR2, TLR4 and TLR9 ligands. As compared to B6 wt, however, TLR2‐KO derived DCs produced several fold more IL‐6 when stimulated with TLR4 or TLR9 ligands and TLR9‐KO derived DCs produced several fold more IL‐6 when stimulated with TLR4 or TLR2 ligands. These findings suggest that DCs and B‐cells are potent cellular sources of IL‐6. IL‐6 production in response to adjuvants appears to be regulated by the coordinated input of TLR signaling and may be important in the generation of pathogenic Th‐17 cells.
Pristane‐induced alveolar hemorrhage in C57BL/6 mice is not mediated by immune complex, T or B‐cells, or Fas
Authors: Barker Tolga , Lee Pui Y. , Weinstein Jason S. , Nacionales Dina C. , Kelly‐Scumpia Kindra M. , Sobel Eric S. , Reeves Westley H. , Satoh Minoru
Published: June 2021

Intraperitoneal (ip) injection of pristane in normal mice induces antinuclear antibodies and immune‐complex (IC) glomerulonephritis similar to human systemic lupus erythematosus (SLE). In addition, C57BL/6 (B6) mice frequently develop alveolar hemorrhage (AH), leading to 10–50% early mortality. AH is an uncommon but often fatal complication of human SLE and is thought to be mediated by IC. The role of IC, apoptosis and T‐ and B‐cells in pristane‐induced AH was investigated. B6, B6 RAG1−/−, B6 FcR γ1 −/−, and B6/lpr, received ip pristane and the lungs were examined for pathology, IC deposition, apoptosis and cytokines and chemokines. In some experiments, bone marrow, spleen, or peritoneal cells from pristane‐treated B6 mice were transferred to RAG1−/− mice to examine their ability to induce AH. Two‐weeks after pristane ip, nearly all of B6, B6 RAG1−/− or B6 FcRγ1 −/− mice developed AH, indicating IC, autoantibody‐binding to lung tissue, or T‐ or B‐cells is not necessary for AH. B6/lpr are resistant to pristane‐induced lupus yet developed AH, indicating that Fas‐mediated apoptosis is not essential and that AH and other pathology induced by pristane are regulated differently. Short term transfer of pristane treated B6 cells into RAG1−/− mice did not induce AH while some long term transfers did induce AH. We conclude that the induction of AH by pristane does not require IC or antibodies, B‐ or T cells, or Fas.

Supported by NIH.
Autoimmune responses to the Goodpasture antigen are driven primarily by IL‐23 and are IL‐12 independent
Authors: Ooi Joshua Daniel , Holdsworth Stephen R. , Kitching A. Richard
Published: June 2021

Goodpasture's disease is an autoimmune renal disease with a known target autoantigen, the non‐collagenase domain of the alpha 3 chain of Type IV collagen [α3(IV)NC1]; the Goodpasture antigen. Cellular and humoral mediated autoimmune responses towards the Goodpasture antigen play an important role in disease. To investigate the role of IL‐12 and IL‐23 in the autoimmune responses towards this autoantigen, we sensitized C57 wildtype mice, IL‐12p40 KO mice (IL‐12 and IL‐23 deficient), IL‐12p35 KO mice and IL‐23p19 KO mice with mouse α3(IV)NC1. C57 wildtype mice and IL‐12p35 KO mice developed comparable levels of autoantigen specific cellular mediated responses, as measured by footpad DTH responses, lymphocyte proliferative responses, and cytokine secretion (IFNγ, IL‐17 and IL‐4), as wells as humoral mediated responses, measured by α3(IV)NC1 specific serum IgG, IgG1, IgG2b, IgG3 ELISAs and intraglomerular IgG staining. These responses were markedly reduced in both IL‐12p40 KO and IL‐23p19 KO mice. These results suggest that cell mediated and humoral autoimmune responses towards the Goodpasture antigen are driven primarily by IL‐23 and are independent of IL‐12. This work supported by the Australian NHMRC.
Characterization of the Specificity of Anti‐Neuronal Antibodies in Sera of Multiple Sclerosis Patients
Authors: Takalo Elisa , Fox Autumn , DeVries George H. , Kennett Roger H.
Published: June 2021

Screening of 30 sera from multiple sclerosis (MS) patients against enriched axolemma fractions (AEF) indicated that 11 could be classified as having high titers, 14 as intermediate and 5 as low or negative. (J. of Neurochem. 96 (Suppl. 1), 134) Two of these sera were pooled, diluted, bound to microtiter plates and used to screen a phage display library expressing random peptide epitopes 5–7 amino acids in length. Peptides bound corresponded to sequences in several proteins including ankyrin (3 epitopes), spectrin, K+ and Ca++ ion channels and the extra‐cellular matrix protein AGC1 (2 epitopes). We have continued these experiments by binding antibodies in MS sera to human neuroblastoma cells and using the eluted antibodies to screen a phage display library expressing cDNA clones from human brain. This new procedure detects antibody epitopes on cell surface proteins that may represent targets for antibodies found in MS sera. Screening of the phage display targets with additional MS sera should provide an indication of whether or not they may be involved in the pathogenesis of MS.

We thank D. L. Feinstein and C. Kaiser, Department of Anesthesiology, Univ. of IL for the MS sera samples used in the study. The work was supported by Wheaton College Ruth Kraft Strohschein research funds and a VA Merit Grant†.
Selective Deregulation of EBNA1‐Specific T Cells in Multiple Sclerosis
Authors: Lunemann Jan , Jelcic Ilijas , Roberts Susanne , Lutterotti Andreas , Tackenberg Bjorn , Martin Roland , Munz Christian
Published: June 2021

Symptomatic primary EBV infection and elevated humoral immune responses to EBV are associated with an increased risk to develop Multiple Sclerosis (MS). We explored mechanisms leading to this change in EBV‐specific immunity in patients with MS and healthy virus carriers matched for expression of MS‐associated HLA alleles. MS patients showed a selective increase of T cell responses to the EBV nuclear antigen‐1 (EBNA1), but not to other EBV‐encoded proteins. Influenza and human cytomegalovirus specific immune control was unchanged in MS. The enhanced response to EBNA1 was mediated by an expanded reservoir of EBNA1‐specific central memory CD4+ Th1 precursors and Th1 (but not Th17)‐polarized effector memory cells which displayed a phenotype indicative of frequent antigen recognition. In addition, EBNA1‐specific T cells recognized myelin antigens more frequently than other autoantigens. Myelin cross‐reactive T cells produced IFN‐g, but differed from EBNA1‐monospecific ones in their capability to produce IL‐2 compatible with a polyfunctional T cell phenotype as found in controlled chronic viral infections. Our data support the concept that clonally expanded EBNA1‐specific T cells potentially contribute to the development of MS by cross‐recognition of myelin antigens.
CD4+ T cell independent LPS induced experimental autoimmune myasthenia gravis (EAMG).
Authors: Allman Windy Rose , Qi Huibin , Saini Shamsher S. , Christadoss Premkumar
Published: June 2021

Myasthenia gravis (MG) is an autoimmune disease defined by muscle weakness due to autoantibody mediated depletion of acetylcholine receptors (AChR) at the neuromuscular junction (NMJ). Isotype switching (IS) to complement binding antibodies (IgG2b and IdG2c) is thought to be mediated by activated CD4+ T cells. However, it is not understood how B and T cells break tolerance during antibody‐mediated immune responses. To determine whether CD4+ cells are required for antibody IS, a new mouse model was developed. The T independent mitogen, LPS (5 μg), was used in place of desiccated Mycobacterium as an adjuvant for immunization with AChR (20 μg) in incomplete Freund's adjuvant. Mice developed MG‐like disease after boosting at day 28. Muscle weakness (measured in a grip strength meter) was associated with production of sera anti‐AChR IgG2b and IgG2c, IgG and C3 deposits at the NMJ, and greater than 30% loss of AChR. CD4 −/− mice immunized with LPS and AChR had no significant differences in onset and severity of disease and produce significant amounts of anti‐AChR IgG1, IgG2b and IgG2c. The absence of CD4+ cells in the blood, spleen, and lymph nodes was confirmed by flow cytometry. The results indicate that IS in vivo is not dependent on CD4+ interaction with B cells. Consequently, in this model, breaks in CD4+ cell tolerance may not be necessary for the initial triggering of autoreactive B cells.* MGFA Viets Fellow and AFM Fellow.
Autoreactive B and T cell response leading to arthritis
Authors: Mandik‐Nayak Laura , DuHadaway James B. , Muller Alexander J. , Scott Grant N.
Published: June 2021

Rheumatoid arthritis (RA) is a chronic inflammatory disease that affects approximately 1% of the population. Although both T and B cell‐mediated autoimmune components have been implicated, the mechanism leading to the initiation of disease is not known. K/BxN mice spontaneously develop a joint inflammatory response that mimics many of the features of human RA. Importantly, in this murine model, disease induction is synchronous and predictable, allowing for the study of the factors that contribute to disease initiation and pathogenesis. In K/BxN mice, arthritis is induced by autoantibodies against the glycolytic enzyme glucose‐6‐phosphate‐isomerase (GPI). How autoreactive B and T cells escape tolerance induction to this ubiquitously expressed self‐Ag and initiate a joint‐specific autoimmune response remains unclear. Previously, our laboratory generated low affinity anti‐GPI Ig tg mice to investigate how GPI‐reactive B cells are normally tolerized by the immune system. Surprisingly, although anti‐GPI B cells show evidence of autoantigen encounter beginning in the BM, they are not tolerant to GPI. The anti‐GPI B cells remain functionally competent and can be induced to secrete autoantibodies in response to cognate T cell help both in vitro and in vivo. This suggests that they have the potential to trigger the arthritogenic autoimmune response.
Unique Processing and Presentation of a Region of Glutamic Acid Decarboxylase (GAD) 65 Is Required for Activation of a Diabetic T Cell Clone
Authors: Dai Yang David , Marrero Idania , Li Yangmei , Rottero Anthony E. , Schroder Kim , Wilson Darcy B. , Sercarz Eli E.
Published: June 2021

A diabetic T cell clone, BDC2.5, reacts to a GAD65‐mimicking peptide in the picomolar range, but remains unresponsive to all GAD65 peptides tested. However, APC from GAD65‐primed non‐obese diabetic (NOD) mice were found effective in presenting a native GAD65 peptide, p524–543, to stimulate this clone (Dai et al. JI 175:3621). Mouse beta cell lines express different levels of GAD65 protein, and vary in stimulating BDC2.5 T cells. Surprisingly, overexpression of GAD65 in one beta cell line increased its antigenicity over 100‐fold in stimulating the T cells, whereas control plasmids had no effect; mutagenesis within the GAD65 plasmids confirmed that the same region, p524–543, is essential for the enhanced stimulation of BDC2.5 cells, and a mutation outside this region was ineffective. Synthetic peptides covering different sequences within p524–543 were non‐stimulatory for the T cells; however, a single replacement, K534W, within this peptide was sufficient to convert the non‐stimulatory native peptide to a strong agonist that activates BDC2.5 cells at 1.0 μg/ml. These data suggest that unique intracellular events occur to GAD65 molecule after its transcription or translation, which result in altered processing and presentation of otherwise tolerogenic T cell determinants and thus activating diabetogenic T cells, such as BDC2.5. (Supported by grants from NIH, JDRF and DNRG to EES)