bluuebunny/crossref_metadata_2025 huggingface dataset:
Deficiency of 5-Lipoxygenase Abolishes Sex-Related Survival Differences in MRL-lpr/lpr Mice
Authors: Goulet Jennifer L. , Griffiths Robert C. , Ruiz Phillip , Spurney Robert F. , Pisetsky David S. , Koller Beverly H. , Coffman Thomas M.
Published: January 2023

Leukotrienes, the 5-lipoxygenase (5LO) products of arachidonic acid metabolism, have many proinflammatory actions that have been implicated in the pathogenesis of a variety of inflammatory diseases. To investigate the role of LTs in autoimmune disease, we generated an MRL-lpr/lpr mouse line with a targeted disruption of the 5lo gene. MRL-lpr/lpr mice spontaneously develop autoimmune disease that has many features resembling human systemic lupus erythematosus, including sex-related survival differences; female MRL-lpr/lpr mice experience significant early mortality compared with males. Unexpectedly, we found that mortality was accelerated in male 5LO-deficient MRL-lpr/lpr mice compared with male wild-type MRL-lpr/lpr animals. In contrast, the 5lo mutation had no effect on survival in females. Mortality was also accelerated in male MRL-lpr/lpr mice that were treated chronically with a pharmacological inhibitor of LT synthesis. Furthermore, LT-dependent inflammatory responses are enhanced in male MRL-lpr/lpr mice compared with females, and the 5lo mutation has greater impact on these responses in males. Because immune complex-mediated glomerulonephritis is the major cause of death in MRL-lpr/lpr mice and has been related to arachidonic acid metabolites, we also assessed kidney function and histopathology. In male MRL-lpr/lpr mice, renal plasma flow was significantly reduced in the 5lo−/− compared with the 5lo+/+ group, although there were no differences in the severity of renal histopathology, lymphoid hyperplasia, or arthritis between the groups. These findings suggest that the presence of a functional 5lo gene confers a survival advantage on male MRL-lpr/lpr mice and that, when 5LO function is inhibited, either genetically or pharmacologically, this advantage is abolished.
A Complementary Peptide Vaccine That Induces T Cell Anergy and Prevents Experimental Allergic Neuritis in Lewis Rats
Authors: Araga Shigeru , Kishimoto Masahiro , Doi Satoko , Nakashima Kenji
Published: January 2023

We have developed and described a new method of altering T cell-mediated autoimmune diseases by immunization with the complementary peptide against T cell epitopes. The complementary peptide (denoted NAE 07-06) to the bovine P2 protein, residues 60–70 (denoted EAN 60–70), was tested in the Lewis rat model of experimental allergic neuritis (EAN). Immunization with NAE 07-06 induced polyclonal and monoclonal Abs that inhibited the proliferation of the P2-specific T cell line, stimulated with EAN 60–70, and recognized Vβ, but not Vα, of TCRs. Proliferation of T cells treated with anti-NAE 07-06 Abs could be partially restored by treatment with rIL-2, in accordance with an anergy model. A homologous sequence was found between NAE 07-06 and the VDJ junction of the TCR β-chain from an EAN 60–70-specific T cell line. Rats preimmunized with NAE 07-06 in vivo before EAN induction showed less disease severity clinically and histologically. These data suggest a new therapeutic approach for T cell-mediated autoimmune disorders through the induction of anti-TCR Abs with complementary peptide Ags.
Degradation of ZAP-70 Following Antigenic Stimulation in Human T Lymphocytes: Role of Calpain Proteolytic Pathway
Authors: Penna Doris , Müller Sabina , Martinon Fabio , Demotz Stephane , Iwashima Makio , Valitutti Salvatore
Published: January 2023

T cell activation by the specific Ag results in dramatic changes of the T cell phenotype that include a rapid and profound down-regulation and degradation of triggered TCRs. In this work, we investigated the fate of the TCR-associated ZAP-70 kinase in Ag-stimulated T cells. T cells stimulated by peptide-pulsed APCs undergo an Ag dose-dependent decrease of the total cellular content of ZAP-70, as detected by FACS analysis and confocal microscopy on fixed and permeabilized T cell-APC conjugates and by Western blot on total cell lysates. The time course of ZAP-70 consumption overlaps with that of ζ-chain degradation, indicating that ZAP-70 is degraded in parallel with TCR internalization and degradation. Pharmacological activation of protein kinase C (PKC) does not induce ZAP-70 degradation, which, on the contrary, requires activation of protein tyrosine kinases. Two lines of evidence indicate that the Ca2+-dependent cysteine protease calpain plays a major role in initiating ZAP-70 degradation: 1) treatment of T cells with cell-permeating inhibitors of calpain markedly reduces ZAP-70 degradation; 2) ZAP-70 is cleaved in vitro by calpain. Our results show that, in the course of T cell-APC cognate interaction, ZAP-70 is rapidly degraded via a calpain-dependent mechanism.
Endogenous CD8+ T Cell Expansion During Regression of Monoclonal EBV-Associated Posttransplant Lymphoproliferative Disorder
Authors: Khatri Vijay P. , Baiocchi Robert A. , Peng Ruoqi , Oberkircher Adam R. , Dolce Jean M. , Ward Pamela M. , Herzig Geoffrey P. , Caligiuri Michael A.
Published: January 2023

There are experimental data which suggest that the primary immune effector cell responsible for maintaining immune surveillance against the outgrowth of EBV-transformed B cells in humans is the CTL, but in vivo proof of this is lacking. In this study we perform a series of cellular and molecular assays to characterize an autologous, endogenous immune response against a transplantation-associated, monoclonal, EBV+ posttransplant lymphoproliferative disorder (PTLD). Following allogeneic bone marrow transplantation, a patient developed a monoclonal PTLD of donor B cell origin. With a decrease in immune suppression, we document the emergence of endogenous, donor-derived CD3+CD8+ CTLs, followed by regression of the PTLD. The TCR Vβ repertoire went from a polyclonal pattern prior to the development of PTLD to a restricted TCR Vβ pattern during the outgrowth and regression of PTLD. Donor-derived CD3+CD8+ T lymphocytes displayed MHC class I-restricted cytolytic activity against the autologous EBV+ B cells ex vivo without additional in vitro sensitization. The striking temporal relationship between the endogenous expansion of a TCR Vβ-restricted, CD3+CD8+ population of MHC class I-restricted CTL, and the regression of an autologous monoclonal PTLD, provides direct evidence in humans that endogenous CD3+CD8+ CTLs can be responsible for effective immune surveillance against malignant transformation of EBV+ B cells.
Early Autoantibody Responses in Prediabetes Are IgG1 Dominated and Suggest Antigen-Specific Regulation
Authors: Bonifacio Ezio , Scirpoli Miriam , Kredel Katharina , Füchtenbusch Martin , Ziegler Anette-G.
Published: January 2023

The islet autoimmunity of preclinical type 1 diabetes remains poorly characterized in humans. In this paper, the IgG subclass response to the islet autoantigens insulin, glutamic acid decarboxylase, and IA-2 was studied sequentially from birth to diabetes onset or current follow-up in 26 autoantibody positive offspring of parents with diabetes. Islet autoantibody appearance was characterized by an early IgG1 peak response to one or more Ags, most commonly to insulin, at a median age of 2.2 yr (interquartile range, 2–2.9 yr). In five offspring, an acute fulminant β-cell destruction and diabetes onset occurred during this initial Ab response. In the remainder, early Ab levels declined markedly, and Ab peaks against other β cell Ags arose sequentially over several years suggesting regulation and spreading of autoimmunity. Second peak Ab responses to the same Ag were observed in only two offspring, both developing diabetes at this time. Two others developed diabetes with declining Ab levels. Abs of IgG1 subclass dominated against each Ag, and other subclasses, were usually only detected during peak IgG1 responses. The IgG4 response to insulin was exceptional, being dominant over IgG1 in four offspring and in five others appeared and/or persisted after IgG1 levels declined. These Th2-associated IgG4 responses were not correlated with protection from diabetes. The presence of IgG1-restricted responses to DA2 were associated with diabetes development. These findings suggest that type 1 diabetes has an early acute destructive phase of β cell autoimmunity, which may be regulated and which spreads chronically until diabetes onset.
Expression of a Variant of CD28 on a Subpopulation of Human NK Cells: Implications for B7-Mediated Stimulation of NK Cells
Authors: Galea-Lauri Joanna , Darling David , Gan Shu-Uin , Krivochtchapov Leonid , Kuiper Marcel , Gäken Joop , Souberbielle Bernard , Farzaneh Farzin
Published: January 2023

The ability of NK cells to kill tumor cells is controlled by a balance between activating and inhibitory signals transduced by distinct receptors. In murine tumor models, the costimulatory molecule B7.1 not only acts as a positive trigger for NK-mediated cytotoxicity but can also overcome negative signaling transduced by MHC class I molecules. In this study, we have evaluated the potential of human B7.1-CD28 interaction as an activating trigger for human blood NK cells. Using multiparameter flow cytometric analysis and a panel of different CD28 mAbs, we show that human peripheral blood NK cells (defined by CD56+, CD16+, and CD3− surface expression) express the CD28 costimulatory receptor, with its detection totally dependent on the mAb used. In addition, the level of CD28 varies among individuals and on different NK cell lines, irrespective of CD28 steady-state mRNA levels. By performing Ab binding studies on T cells, our data strongly suggest that binding of two of the anti-CD28 Abs (clones 9.3 and CD28.2) is to a different epitope to that recognized by clones L293 and YTH913.12, which is perhaps modified in the CD28 molecule expressed by the NK cells. We also show that B7.1 enhances the NK-mediated lysis of NK-sensitive but not of NK-resistant tumor cells and that this increased lysis is dependent on CD28-B7 interactions as shown by the ability of Abs to block this lysis. Coculture of the B7.1-positive NK-sensitive cells also led to the activation of the NK cells, as determined by the expression of CD69, CD25, and HLA class II.
The Regulatory Role of CD45 on Rat NK Cells in Target Cell Lysis
Authors: Giezeman-Smits Katinka M. , Gorter Arko , van Vlierberghe Ronald L. P. , v. Eendenburg Jaap D. H. , Eggermont Alexander M. M. , Fleuren Gert Jan , Kuppen Peter J. K.
Published: January 2023

To investigate the role of CD45 in rat NK cell function, we developed new mAbs directed against rat CD45. mAb ANK12 binds to a high molecular isoform of CD45 and mAb ANK74 binds to the common part on all known CD45 isoforms, as has been described for the anti-rat CD45 mAb OX1. The ability of these mAbs to affect NK cell-mediated lysis was tested using the Fc receptor-positive target cell line P815. mAb ANK12 was found to significantly enhance the lysis of P815, whereas ANK74 and the anti-CD45 mAb OX1 did not. In addition, cross-linking of the CD45 isoform by ANK12 induced tyrosine phosphorylation of specific proteins in NK cells. Subsequently, the involvement of CD45 in the negative signaling after “self” MHC class I recognition by rat NK cells was investigated. The anti-CD45 mAbs were found to affect NK cell-mediated lysis of syngeneic tumor cell lines, depending upon the expression level of MHC class I on target cells. mAbs ANK74 and OX1 only inhibited lysis of the syngeneic tumor cell lines that expressed low levels of MHC class I. Furthermore, both mAbs caused an inhibition of NK cell-mediated lysis of these tumor cell lines when MHC class I molecules on the tumor cell lines were masked by an Ab. These results suggest that CD45 regulates the inhibitory signal pathway after self MHC class I recognition, supposedly by dephosphorylation of proteins.
Cutting Edge: IFN-γ Regulated Germline Transcripts Are Expressed from γ2a Transgenes Independently of the Heavy Chain 3′ Enhancers
Authors: Collins John T. , Dunnick Wesley A.
Published: January 2023

Several results indicate that transcriptional enhancers lying 3′ of the Cα gene regulate RNA expression and switch recombination of heavy chain genes. To investigate this regulation we prepared transgenic mice with a 10.5-kb transgene that included the germline form of the murine γ2α gene, including promoter, I, S, and C regions. RNA was expressed from these γ2a transgenes with correct IFN-γ regulation, in spite of the fact that they lacked the 3′ enhancers. This RNA expression was independent of insertion site and dependent on copy number, indicating that the γ2a gene includes locus control region-like elements. Addition of either a cassette containing 3′ enhancer DNase I hypersensitive sites 1, 2, 3B, and 4 or the intronic μ enhancer increased transcription from the γ2a transgene by ∼75-fold in B cells. However, this increased transcription was not responsive to IFN-γ treatment of the transgenic B cells.
Crosslinking of the Human Fc Receptor for IgA (FcαRI/CD89) Triggers FcR γ-Chain-Dependent Shedding of Soluble CD89
Authors: van Zandbergen Ger , Westerhuis Ralf , Mohamad Ngaisah Klar , van de Winkel Jan G. J. , Daha Mohamed R. , van Kooten Cees
Published: January 2023

CD89/FcαRI is a 55- to 75-kDa type I receptor glycoprotein, expressed on myeloid cells, with important immune effector functions. At present, no information is available on the existence of soluble forms of this receptor. We developed an ELISA for the detection of soluble CD89 (sCD89) forms and investigated the regulation of sCD89 production. PMA/ionomycin stimulation of monocytic cell lines (U937, THP-1, and MM6), but not of neutrophils, resulted in release of sCD89. Crosslinking of CD89 either via its ligand IgA or with anti-CD89 mAbs similarly resulted in sCD89 release. Using CD89-transfected cells, we showed ligand-induced shedding to be dependent on coexpression of the FcR γ-chain subunit. Shedding of sCD89 was dependent on signaling via the γ-chain and prevented by addition of inhibitors of protein kinase C (staurosporine) or protein tyrosine kinases (genistein). Western blotting revealed sCD89 to have an apparent molecular mass of 30 kDa and to bind IgA in a dose-dependent fashion. In conclusion, the present data document a ligand-binding soluble form of CD89 that is released upon activation of CD89-expressing cells. Shedding of CD89 may play a role in fine-tuning CD89 immune effector functions.
A Role for a Melanosome Transport Signal in Accessing the MHC Class II Presentation Pathway and in Eliciting CD4+ T Cell Responses
Authors: Wang Siqun , Bartido Shirley , Yang George , Qin Jie , Moroi Yoichi , Panageas Katherine S. , Lewis Jonathan J. , Houghton Alan N.
Published: January 2023

Melanosomal membrane proteins are frequently recognized by the immune system of patients with melanoma and vitiligo. Melanosomal glycoproteins are transported to melanosomes by a dileucine-based melanosomal transport signal (MTS). To investigate whether this sorting signal could be involved in presentation of melanosome membrane proteins to the immune system, we devised a fusion construct containing the MTS from the mouse brown locus product gp75/tyrosinase-related protein-1 and full-length OVA as a reporter Ag. The fusion protein was expressed as an intracellular membrane protein, sorted to the endocytic pathway, processed, and presented by class II MHC molecules. DNA immunization with this construct elicited CD4+ T cell proliferative responses in vivo. Ag presentation and T cell responses in vitro and in vivo required a functional MTS. Mutations of either the upstream leucine in MTS or elimination of the entire MTS negated in vitro Ag presentation and in vivo T cell responses. In a mouse melanoma model, DNA immunization with MTS constructs protected mice from tumor challenge in a CD4+ T cell-dependent manner, but complete deletion of MTS decreased tumor rejection. Therefore, MTS can target epitopes to the endocytic pathway leading to presentation by class II MHC molecules to helper T cells.
IL-15 Promotes Survival But Not Effector Function Differentiation of CD8+ TCRαβ+ Intestinal Intraepithelial Lymphocytes
Authors: Lai Yein-Gei , Gelfanov Vasily , Gelfanova Valentina , Kulik Liudmila , Chu Ching-Liang , Jeng Sheau-Wen , Liao Nan-Shih
Published: January 2023

CD8 single-positive cells, including CD8αα+ and CD8αβ+ subsets, constitute the majority of TCRαβ+ intestinal intraepithelial lymphocytes (αβ iIEL) in mice. CD8+ αβ iIEL show significantly weaker responses to TCR stimulation in the presence of exogenous IL-2 than do CD8+ T cells of the central immune system. IL-15 is a T cell growth factor likely expressed in the intestine mucosa. To understand the role of IL-15 in CD8+ αβ iIEL biology, we compared the effects of exogenous IL-15 and IL-2 on the survival and primary responses of the two CD8+ αβ iIEL subsets in vitro. In contrast to the death of ∼60% of both CD8αα+ and CD8αβ+ iIEL cultured in IL-2 with or without TCR stimulation, IL-15 promoted survival of the CD8αα+ subset in the presence of TCR stimulation and promoted survival of both subsets in the absence of TCR stimulation. The higher proliferation level of TCR stimulated CD8αα+ αβ iIEL cultured in IL-15 compared with those cultured in IL-2 is likely due to IL-15’s prosurvival effects. In addition, unlike exogenous IL-2, exogenous IL-15 did not support the effector functions of either iIEL subsets, including IFN-γ production, IL-4-induced Th2 cytokine production, and anti-TCR mAb-redirected cytotoxicity. These findings demonstrate that IL-15 and IL-2 are functionally distinct and suggest that IL-15 plays a unique role in the maintenance of the CD8+ αβ iIEL pool in the absence of Ag stimulation and in the survival and expansion of CD8αα+ αβ iIEL upon Ag stimulation.
Distinct Proteolytic Processes Generate the C and N Termini of MHC Class I-Binding Peptides
Authors: Mo X. Y. , Cascio Paolo , Lemerise Kristen , Goldberg Alfred L. , Rock Kenneth
Published: January 2023

Most of the MHC class I peptides presented to the immune system are generated during the course of protein breakdown by the proteasome. However, the precise role of the proteasome, e.g., whether this particle or some other protease generates the carboxyl (C) and amino (N) termini of the presented 8- to 10-residue peptides, is not clear. Here, we show that presentation on Db of ASNENMETM, a peptide from influenza nucleoprotein, and on Kb of FAPGNYPAL, a peptide from Sendai virus nucleoprotein, was blocked by the proteasome inhibitor, lactacystin. Using plasmid minigene constructs encoding oligopeptides of various lengths, we found that presentation of ASNENMETM from C-terminally extended peptides that contain this antigenic peptide plus three or five additional amino acids and presentation of FAPGNYPAL from a peptide containing FAPGNYPAL plus one additional C-terminal residue required the proteasome. In contrast, the proteasome inhibitor did not reduce presentation of cytosolically expressed ASNENMETM or FAPGNYPAL or N-terminally extended versions of these peptides, suggesting involvement of aminopeptidase(s) in trimming these N-extended variants. Accordingly, when the N termini of these 3N-extended peptides were blocked by acetylation, they were resistant to hydrolysis by cellular aminopeptidases and pure leucine aminopeptidase. Moreover, if introduced into the cytosol, Ag presentation of these peptides occurred to a much lesser extent than from their nonacetylated counterparts. Thus, the proteasome is essential for the generation of ASNENMETM and FAPGNYPAL peptides from the full-length nucleoproteins. Although it generates the C termini of these presented peptides, distinct aminopeptidase(s) can trim the N termini of these presented peptides to their proper size.
A Distinct IL-18-Induced Pathway to Fully Activate NK T Lymphocytes Independently from TCR Engagement
Authors: Leite-de-Moraes Maria C. , Hameg Agathe , Arnould Anne , Machavoine François , Koezuka Yasuhiko , Schneider Elke , Herbelin André , Dy Michel
Published: January 2023

NK T lymphocytes are characterized by their ability to promptly generate IL-4 and IFN-γ upon TCR engagement. Here, we demonstrate that these cells can also be fully activated in the absence of TCR cross-linking in response to the proinflammatory cytokine IL-18 associated with IL-12. NK T cells stimulated with IL-18 plus IL-12 proliferated, killed Fas+ target cells, and produced high levels of IFN-γ without IL-4. In these conditions, IFN-γ production was at least 10-fold higher than that upon TCR cross-linking. Interestingly, a 2-h pretreatment with IL-12 plus IL-18 sufficed to maintain the high IFN-γ-producing potential during subsequent stimulation with anti-TCR mAbs or with the specific Ag α-galactosylceramide. Similar effects were observed in vivo, because splenic CD4+ NK T cells from MHC class II-deficient mice secreted IFN-γ without further stimulation when removed 2 h after a single injection of IL-12 plus IL-18. In conclusion, our evidence for activation of NK T lymphocytes in response to IL-18 plus IL-12 in the absence of TCR engagement together with the maintenance of preferential IFN-γ vs IL-4 production upon subsequent exposure to specific Ags is consistent with the active participation of this cell population in innate as well as acquired cellular immune responses.
Altered Donor and Recipient Ly49+ NK Cell Subsets in Allogeneic H-2d → H-2b and H-2b → H-2d Bone Marrow Chimeras
Authors: Korten Simone , Wilk Esther , Gessner J. Engelbert , Meyer Dirk , Schmidt Reinhold E.
Published: January 2023

NK cells reject non-self hematopoietic bone marrow (BM) grafts via Ly49 receptor-mediated MHC class I-specific recognition and calibration of receptor expression levels. In this paper we investigated how Ly49+ subset frequencies were regulated dependent on MHC class I expression. The development of donor and host Ly49A+ (recognizes H-2Dd and H-2Dk ligands) and Ly49C/I+ (Ly49CBALB/c recognizes H-2Kb, H-2Kd, and H-2Dd, and Ly49CB6 recognizes only H-2Kb) NK cell frequencies were monitored for 120 days in murine-mixed allogeneic BM chimeras. C57BL/6 (H-2b) BM was transplanted into BALB/c (H-2d) mice and vice versa. Peripheral NK cell populations were examined every 5 days. Chimerism was found to be stable with 80–90% donor NK cells. In contrast to syngeneic controls reexpressing pretransplant patterns, donor and host NK cells revealed new and mainly reduced subset frequencies 55 days after allogeneic transplantation. Recipient NK cells acquired these later than donor NK cells. In H-2d → H-2b chimeras Ly49A+, Ly49C/I+, and Ly49A+/Ly49C/I+ proportions were mainly diminished upon interaction with cognate ligands. Also in H-2b → H-2d chimeras, Ly49A+ and Ly49A+/Ly49C/I+ subsets were reduced, but there was a transient normalization of Ly49C/I+ proportions in the noncognate host. After 120 days all subsets were reduced. Therefore, down-regulation of developing Ly49A+ and Ly49C/I+ chimeric NK cell frequencies by cognate ligands within 7–8 wk after BM transplantation may be important for successful engraftment.
Antigen-Experienced T Cells Undergo a Transient Phase of Unresponsiveness Following Optimal Stimulation
Authors: De Mattia Fabrizio , Chomez Susanna , Van Laethem François , Moulin Véronique , Urbain Jacques , Moser Muriel , Leo Oberdan , Andris Fabienne
Published: January 2023

Interaction of the Ag-specific receptor of T lymphocytes with its Ag/MHC ligand can lead either to cell activation or to a state of unresponsiveness often referred to as anergy. It has been generally assumed that anergy develops as a consequence of inadequate stimulation, such as in response to altered peptide ligands or to agonists presented by costimulatory-deficient accessory cells. The present study uncovers an alternative way of inducing an unresponsive state in T cells. Indeed, we demonstrate herein that Ag-stimulation of murine CD4+ Th clones induces cellular activation, characterized by cytokine production and cell proliferation, followed by a state of transient (lasting up to 6 days) unresponsiveness to further antigenic stimulation. This state of activation-induced unresponsiveness 1) is not a consequence of inadequate costimulation, as it occurs when cells are stimulated in the presence of dendritic cells or anti-CD28 Abs; 2) develops after an optimal response to Ag; 3) is not due to cell death/apoptosis or CTLA-4 engagement; 4) down-regulates the proliferation and cytokine production of both Th1- and Th2-like clones; and 5) does not affect the early steps of signal transduction. Finally, naive T cells are not sensitive to this novel form of unresponsiveness, but become gradually susceptible to activation-induced unresponsiveness upon Ag stimulation. Collectively, these data suggest that activation-induced T cell unresponsiveness may represent a regulatory mechanism limiting the clonal expansion and effector cell function of Ag-experienced T cells, thus contributing to the homeostasis of an immune response.
Antigen-Driven Selection of TCR In Vivo: Related TCR α-Chains Pair with Diverse TCR β-Chains
Authors: Mikszta John A. , McHeyzer-Williams Louise J. , McHeyzer-Williams Michael G.
Published: January 2023

Ag-driven selection mediates effective T cell help and the development of Th cell memory in vivo. To analyze the dynamics of interclonal competition during the selection process in vivo, we use the I-Ek-restricted murine response to pigeon cytochrome c (PCC). The dominant PCC-specific clonotype expresses Vα11Vβ3 V regions with preferred sequence features in the third hypervariable regions (CDR3). In the current study we define and quantitatively monitor four subdominant PCC-specific clonotypes that express Vα11 paired with non-Vβ3 TCR β-chains (Vβ6, Vβ8.1/8.2, Vβ8.3, and Vβ14). The subdominant clonotypes emerge with similar dynamics to the dominant clonotype and together amount to similar numbers as the dominant clonotype in vivo. These subdominant clonotypes do not efficiently enter germinal centers, although they enter the memory compartment and rapidly re-emerge upon secondary challenge. Analysis of CDR3 diversity in the TCR α-chains identifies many preferred sequence features expressed by the dominant clonotype. These studies quantitatively demonstrate selection for diverse Th cells in vivo and highlight TCR α-chain dominance in Ag-driven selection for best fit.
A New Monoclonal Antibody Detects a Developmentally Regulated Mouse Ecto-ADP-Ribosyltransferase on T Cells: Subset Distribution, Inbred Strain Variation, and Modulation Upon T Cell Activation
Authors: Koch-Nolte Friedrich , Duffy Ted , Nissen Marion , Kahl Sarah , Killeen Nigel , Ablamunits Vitaly , Haag Friedrich , Leiter Edward H.
Published: January 2023

ADP-ribosylation of membrane proteins on mouse T cells by ecto-ADP-ribosyltransferase(s) (ARTs) can down-regulate proliferation and function. The lack of mAbs against mouse ARTs has heretofore prevented analysis of ART expression on T cell subsets. Using gene gun technology, we immunized a Wistar rat with an Art2b expression vector and produced a novel mAb, Nika102, specific for ART2.2, the Art2b gene product. We show that ART2.2 is expressed as a GPI-anchored protein on the surface of mature T cells. Inbred strain-dependent differences in ART2.2 expression levels were observed. C57BL/6J and C57BLKS/J express the Ag at high level, with up to 70% of CD4+ and up to 95% of CD8+ peripheral T cells expressing ART2.2. CBA/J and DBA/2J represent strains with lowest expression levels. T cell-deficient mice and NZW/LacJ mice with a defective structural gene for this enzyme were ART2.2 negative. In the thymus, ART2.2 expression is restricted to subpopulations of mature cells. During postnatal ontogeny, increasing percentages of T cells express ART2.2, reaching a peak at 6–8 wk of age. Interestingly, ART2.2 and CD25 are reciprocally expressed: activation-induced up-regulation of CD25 is accompanied by loss of ART2.2 from the cell surface. Nika102 thus defines a new differentiation/activation marker of thymic and postthymic T cells in the mouse and should be useful for further elucidating the function of the ART2.2 cell surface enzyme.
TCR α-Chain Repertoire in pTα-Deficient Mice Is Diverse and Developmentally Regulated: Implications for Pre-TCR Functions and TCRA Gene Rearrangement
Authors: Mancini Stéphane , Candéias Serge M. , Fehling Hans Jorg , von Boehmer Harald , Jouvin-Marche Evelyne , Marche Patrice N.
Published: January 2023

Pre-TCR expression on developing thymocytes allows cells with productive TCRB gene rearrangements to further differentiate. In wild-type mice, most TCRA gene rearrangements are initiated after pre-TCR expression. However, in pTα-deficient mice, a substantial number of αβ+ thymocytes are still produced, in part because early TCR α-chain expression can rescue immature thymocytes from cell death. In this study, the nature of these TCR α-chains, produced and expressed in the absence of pre-TCR expression, have been analyzed. We show, by FACS analysis and sequencing of rearranged transcripts, that the TCRA repertoire is diverse in pTα−/− mice and that the developmental regulation of AJ segment use is maintained, yet slightly delayed around birth when compared with wild-type mice. We also found that T cell differentiation is more affected by pTα inactivation during late gestation than later in life. These data suggest that the pre-TCR is not functionally required for the initiation and regulation of TCRA gene rearrangement and that fetal thymocytes are more dependent than adult cells on pTα-derived signals for their differentiation.
Effect of Anti-B7-1 and Anti-B7-2 mAb on Theiler’s Murine Encephalomyelitis Virus-Induced Demyelinating Disease
Authors: Inoue Atsushi , Koh Chang-Sung , Yamazaki Masashi , Yagita Hideo
Published: January 2023

We examined the role of B7-1 and B7-2, costimulatory molecules critical to full activation of T cells, in the development of Theiler’s murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD). Treatment with mAbs to B7-1 resulted in significant suppression of the development of this disease both clinically and histologically. In mice treated with these mAbs, the production of TNF-α and IFN-γ in the spleen cells was decreased. The delayed-type hypersensitivity and T cell proliferative response specific for TMEV were decreased by this treatment. In contrast, treatment with Abs to B7-2, resulted in no effect on TMEV-IDD. These data suggest that B7-1 is critically involved in the pathogenesis of TMEV-IDD and that Abs to B7-1 could be a novel therapeutic approach in the clinical treatment of demyelinating diseases such as human multiple sclerosis.
BALB/c Mice Produce Blister-Causing Antibodies Upon Immunization with a Recombinant Human Desmoglein 3
Authors: Fan Ji-Lao , Memar Omeed , McCormick Daniel J. , Prabhakar Bellur S.
Published: January 2023

Pemphigus vulgaris (PV) is an Ab-mediated autoimmune blistering disease of mucotaneous surfaces. Over 95% of the patients with PV express DR4 or DRw6, and the disease is characterized by the presence of autoantibodies directed against desmoglein 3 (Dsg 3), a protein expressed on keratinocytes. An appropriate animal model is required to understand immunoregulation and to address the role of immunogenetic components in the production of pathogenic Abs that are characteristic of PV. Therefore, we turned to the development of a mouse model. Four strains of female mice (BALB/c, DBA/1, SJL/J, and HRS/J) were screened for their ability to produce pathogenic anti-Dsg 3 Abs. We demonstrated that only BALB/c mice immunized with a full-length Dsg 3 can produce pathogenic Abs capable of causing acantholysis of human foreskin in culture and blistering in neonatal mice. This observation suggested that either H-2d or the BALB background contains the immunogenetic makeup necessary for the production of pathogenic anti-Dsg 3 Abs. No correlation was noted between a given isotype and the pathogenic potential of autoantibodies from different strains of mice. Similarly, the pattern of reactivity of Abs with a panel of 46 synthetic peptides that span the entire Dsg 3 failed to reveal any association between binding specificity and the pathogenic potential, and suggested that pathogenic Abs might recognize conformational epitopes. Moreover, our studies showed that the epitopes recognized by pathogenic Abs are contained within the extracellular Dsg 3.
WASP Levels in Platelets and Lymphocytes of Wiskott-Aldrich Syndrome Patients Correlate with Cell Dysfunction
Authors: Shcherbina Anna , Rosen Fred S. , Remold-O’Donnell Eileen
Published: January 2023

Wiskott-Aldrich syndrome, an inherited blood cell disorder due to mutations of the X-chromosome gene WASP (Wiskott-Aldrich syndrome protein), was characterized originally by thrombocytopenia, immunodeficiency, and eczema. Whereas platelet dysfunction is severe and consistent, immune defects are clinically variable, ranging from negligible to life threatening. To understand this heterogeneity, we quantified WASP in PBMC and platelets, and also in neutrophils, of patients with diverse mutations. A surprisingly complex pattern of WASP expression found for lymphoid cells formed the basis for dividing the patient mutations into four groups. Group A have low WASP levels in PBMC and higher levels in EBV cell lines, as well as near normal WASP RNA levels (7 patients, most with mild disease), suggesting that group A WASP molecules are hypersusceptible to proteolysis. Group B have low WASP levels in PBMC and EBV cells and similar low RNA levels (2 patients, moderate disease). Group C have discordant expression: WASP-positive peripheral T cells and WASP-negative peripheral B cells and EBV cell lines (9 patients, variable disease severity). Noteworthy among group C kindred are several instances of B cell lymphomas. In group D, PBMC and EBV cell lines are WASP negative (7 patients, severe disease). In contrast to the complex lymphoid cell expression patterns, all patient platelets examined were WASP negative (18 diverse patients). WASP absence in platelets provides an apparent molecular explanation for the universally severe platelet dysfunction in this disease, and the cumulative lymphoid cell findings suggest that WASP levels play a substantial role in determining immune outcome.
A Rapid Quantitative Precipitin Technic
Authors: Boyden Alan , Baier Joseph G.
Published: January 2023

The measurement of volume of precipitate in the precipitin reaction has been found to be fairly simple, rapid, and reliable when Van Allen thrombocytocrits are used. The technique described is recommended for the serological study of biological relationships where more measurements and fewer family trees are needed.
The Specificity of Mammalian Spermatozoa, with Especial Reference to Electrophoresis as a Means of Serological Differentiation
Authors: Mudd Stuart , Mudd Emily B. H.
Published: January 2023

The literature on spermatotoxins repeatedly raises the question of the species specificity of mammalian spermatozoa (1–8); however, some authors conclude or imply that spermatozoa are, and some that they are not species specific. The reason for this discrepancy lies in part perhaps in the difficulty of obtaining quantitative estimates of the potencies of antisperm sera by the methods ordinarily used; in part perhaps the trouble has been that too few sperm species have been used, in experiments only incidental to some other purpose.1

Landsteiner and van der Scheer (10) have recently reported the tissue specificity of bull spermatozoa, which they could distinguish serologically from all somatic tissues studied. Do mammalian spermatozoa, then, like lens protein, possess tissue but not species specificity, or do they, as Dunbar has shown for plant pollens and fish spermatozoa (11) possess both tissue and species specificity? The first object of the present report is to answer the question of species specificity.
The Significance of Brucella Abortus Agglutinins in Human Serum
Authors: Carpenter C. M. , Boak Ruth , Chapman O. D.
Published: January 2023

The work of several investigators has shown that agglutinins specific for Brucella abortus may be demonstrated in human serum. In many cases, such antibodies are present in the serum from an individual who is showing no visible symptoms of undulant fever. Because of such findings, the diagnostic value of the agglutination test is questioned, particularly by those who have not made extensive examinations and who are not familiar with the procedure. The interpretation of the agglutination by human serum of an antigen prepared from cultures of Br. abortus demands careful scrutiny. We, therefore, considered it very important to obtain definite information regarding the following questions. Are the agglutinins specific for Br. abortus, Br. melitensis or some other closely related specie?Is the presence of abortus agglutinins in human serum caused by an infection with Br. abortus from which the individual has recovered?
The adaptor molecule MyD88 directly promotes CD8 T cell responses to vaccinia virus (128.12)
Authors: Zhao Yuan , Trez Carl De , Flynn Rachel , Ware Carl , Croft Michael , Salek-Ardakani Shahram
Published: January 2023

Vaccinia Virus (VACV) elicits a robust CD8 T cell response that plays an important role in protection. To date, there is little information on the molecules that are essential to generate large pools of VACV-specific CD8 T cells. Here we show that the adaptor molecule myeloid differentiation primary-response gene 88 (MyD88) is critical for the magnitude of primary CD8 T cell responses to both dominant and subdominant VACV epitopes, with MyD88-/- mice exhibiting profoundly reduced CD8 T cell expansion and anti-viral cytokine production. Interestingly, the defect was not due to impaired antigen-presenting cell function. MyD88-/- DC matured normally and were able to promote strong CD8 T cell priming following VACV infection. Rather, adoptive transfer experiments demonstrated that MyD88-dependent pathways in CD8 T cells were critical. MyD88-deficient CD8 T cells were defective in surviving in wild-type hosts, and poor expansion of MyD88-deficient VACV-specific CD8 T cells resulted after virus infection. In contrast, no defect was evident in the absence of TRIF, TLR2, TLR9, and IL-1R. Together, our results highlight an important role for MyD88 in initial anti-viral CD8 T cell responses and suggest that targeting this pathway may be useful in promoting and sustaining anti-VACV immunity. This work was supported by NIH grants AI77079 to S.S.A and AI67341 to M.C.
The VSVΔG/ZEBOV GP vaccine protects against an Ebola infection and induces humoral and cellular immunity (128.16)
Authors: Alimonti Judie B. , Qiu Xiangguo , Fernando Lisa , Melito Leno , Feldmann Friedericke , Stroher Ute , Feldmann Heinz , Jones Steven
Published: January 2023

Ebola virus (EBOV) is a highly infectious filovirus causing a severe hemorrhagic fever in humans with fatality rates approaching 90%. We used the live attenuated recombinant vaccine vector vesicular stomatitis virus (VSV) to create a vaccine for EBOV. Ten cynomolgus macaques were immunized either intramuscularly (IM), orally (OR), or intranasally (IN) with VSVΔG/ZEBOVGP 28 days before receiving a lethal challenge of Zaire ebolavirus (ZEBOV). There were no signs of illness in the vaccinated animals, and no loss of lymphocytes typically seen during infection. ZEBOVGP-specific humoral and cell mediated immune responses (CMIR) were generated by all immunization routes. Surprisingly, in general, the IN route generated more potent humoral responses, while the IM route generated early IL-2 and IFN-γ CMIR. We demonstrate that a single dose of the VSVΔG/ZEBOVGP vaccine delivered by any of the three routes induces cellular and humoral immune responses and protects non-human primates against a lethal challenge of ZEBOV.
Tegument- and glycoprotein-reactive HSV-1-specific CD4 and CD8 T-cells localize to human trigeminal ganglia (128.24)
Authors: Jing Lichen , Verjans G.M.G.M , Haas Juergan , Bailer Susanne , McClurkan Christopher L , Chong Tiana M , Koelle David M
Published: January 2023

Chronic HSV infections of the natural human host are characterized by lifelong latency in dorsal root ganglia. It is not clear which viral proteins can access antigen presentation pathways in the human ganglia. HSV-1-infected ganglia showed increased inflammation compared to virus-negative ganglia. The leukocyte infiltrate in human HSV-infected TG was shown to include HSV-specific CD4 and CD8 T-cells using interferon-gamma (IFN-g) readouts. By cloning HSV-1 ORFeome in a large set of eGFP fusion plasmids, We have interrogated the fine specificity of TG-derived T-cell lines. Antigen presenting cells for CD8 assays were HLA class I heavy chain /HSV-1 ORF co-transfectants, while HLA class II-matched PBMC were used as APC to stimulate CD4 responses. CD8 recognition of discrete HLA class I-restricted epitopes was documented for two distinct HSV-1 glycoproteins, gL and gK, expressed with late kinetics, as well as for the major transactivator tegument protein, VP16. Tetramer and IFN-g staining showed that up to 20% of TG CD8 cells recognized single HSV-1 epitopes. Glycoprotein-specific CD8 T-cells displayed nanomolar avidity and cross-recognized the homologous HSV-2 nonamer peptides. Interestingly, VP16 was also recognized by TG-infiltrating CD4 T-cells. HSV-1 lytic cycle proteins can access ganglionic HLA class I and II antigen presentation pathways during chronic TG infection, implying that local APC somehow sample viral proteins from neuronal reactivation events. Local antigen-specific T-cells may contribute to the life-long equilibrium between latency and clinical reactivation, and influence disease phenotype. Supported by NIH grant AI50132.
The contributions of glycosylation and GPI-linkage of the murine cytomegalovirus immune evasin, m157, to functional interactions with its lectin-like Ly49 receptors (128.25)
Authors: Carlin-Brown Lindsey E. , Guseva Natalya V. , Heusel Jonathan W.
Published: January 2023

The MCMV-encoded glycoprotein, m157, is recognized by the activating Ly49HC57Bl/6 and inhibitory Ly49I129S lectin-like NK cell receptors. We and others have shown that Ly49H is tolerant to multiple amino acid changes in m157 with the exception of K161, which is predicted to anchor a unique N-terminal α0-helix to the main m157 chain. We undertook site-directed mutagenesis to address the contributions of N-linked glycosylation and the GPI anchor of m157 to Ly49H and Ly49I recognition. Variants of m157 containing single or multiple N-to-Q substitutions at positions N178, N187, N213, and N267, or fusions between the m157 extracellular domain and the transmembrane + cytoplasmic domains of DAP12 or CD4, were expressed in myeloid or fibroblast cell lines. We find that 1) only fully deglycosylated m157 has reduced capacity to activate Ly49H or chimeric Ly49HI reporter cells; 2) m157-CD4 transmembrane versions of m157 fully activate Ly49H and chimeric Ly49HI reporter lines; and 3) Ly49H+ NK cells are activated by all N-glycosylation and transmembrane variants of m157. Thus, neither N-glycosylation nor GPI anchoring of m157 is required for functional trans interactions with lectin-like Ly49 receptors.
Lethal poxvirus infection can be treated at later stages of infection using Abs to the non-structural type I IFN binding protein (128.30)
Authors: Xu Renhuan , Sigal Luis
Published: January 2023

Ectromelia virus (ECTV) is an Orthopoxvirus (OPV) that causes mousepox, the murine equivalent of human smallpox caused by the OPV Variola virus (VARV). Both ECTV and VARV encode a Type I IFN binding protein (EVM166 in ECTV) that inactivates IFNƒÑ. We have previously shown that ECTV deficient in EVM166 is avirulent and that immunization of mice with recombinant EVM166 prevented mousepox in BALB/c mice challenged with WT ECTV. Here, we tested whether EVM166 protein can also be used as a target for post-exposure therapy. Strikingly, we found that rabbit polyclonal or mouse mAb to EVM166 given in a single dose 1, 3 or even 5 days post infection (PI) were highly protective. Moreover, treatment with anti-EVM 166 significantly reduced virus loads and liver pathology. Furthermore, we found that EVM166 protein is present in the sera of mice from day 5 PI and that this was significantly reduced following anti-EVM 166 treatment. Analysis of gene expression demonstrated that mice treated with anti-EVM166 Ab produced ~5-fold less Type I IFN in the liver but had ~5-fold higher expression of the IFN inducible genes such as Mx1. This suggests that the amount of type I IFN production in the liver is dependent on virus loads and that anti-EVM 166 Ab treatment decreases virus loads in the liver by permitting IFN signaling and an anti-viral state of the organ. Our study provides a new promising strategy for post-exposure treatment of poxvirus diseases and yields important basic information about the mechanisms whereby Type I IFNs protect in vivo and the host-pathogen interaction.

This work was supported by NIH grant R01AI065544 to LJS
Inhibition of HIV-1 infectivity across subtypes by the green tea catechin, epigallocatechin gallate, without altered immune function (128.6)
Authors: Nance Christina L , Siwak Edward B , D'Souza Melinda , Songa Sonia , McMullen Ashley M , Shearer William T
Published: January 2023

Previously, we presented evidence that the green tea catechin, epigallocatechin gallate (EGCG), binds to the CD4 molecule at the gp120 attachment site on T cells. CD4+ T cells, B cells and macrophages were isolated from HIV-1 uninfected donors. HIV-1 infectivity by HIV-1 p24 ELISA on M-tropic (R5) subtypes B, C and G; T-tropic (X4) subtypes B and D; and dual tropic (R5/X4) subtype B. Immune function studies: cytotoxicity by ViCell, apoptosis by flow cytometry, immunoglobulin (Ig) production by ELISA and cytokine profiling by multiplex platform. Statistical significance by Student's t test. EGCG significantly inhibited HIV-1 infectivity on human immune cells in a dose-dependent manner (6-100μM) (p<0.01-0.001). At physiologic concentration of 6μM, EGCG significantly inhibited HIV-1 p24 antigen production across HIV-1 subtypes [B (p<0.001), C, D, and G (p<0.01)]. The specificity of the EGCG-induced inhibition was substantiated by the failure of EGCG derivatives lacking galloyl and/or pyrogallol side groups to alter HIV-1 p24 levels. EGCG-induced inhibition of HIV-1 infectivity was not due to cytotoxcity, cell growth inhibition, nor apoptosis. Neither Ig nor cytokine production were significantly altered. EGCG inhibits HIV-1 infectivity across a broad spectrum of HIV-1 subtypes without significant effects on immune function, thereby supporting EGCG as a candidate for HIV-1 therapy.

(NIH AT003084)
Identification of a Mamu Class I-restricted listeriolysin T-cell epitope in rhesus macaques infected with Listeria monocytogenes (LM) (129.13)
Authors: Williams Kristina M. , Bigley Elmer C. , Babu Uma S.
Published: January 2023

LM is a gram positive foodborne pathogen that causes a flu-like illness in immunocompetent patients, but immunocompromised patients can develop septicemia and meningitis resulting in a high mortality rate. The primate model of LM infection is being used to investigate both innate and adaptive components of immunity in an animal model that is more relevant to human disease. Cytotoxic T lymphocytes were isolated from rhesus monkeys infected with LM by nasogastric intubation and used to identify Class I-restricted LM epitopes. Peptides were eluted from the surface of syngeneic B lymphocytes that were incubated overnight with heat-killed LM, and then peptides were separated by reversed-phase HPLC. An 11-mer peptide was identified that induced significant target cell lysis. The amino acid sequence of this peptide was determined by Edman degradation and confirmed by mass spectrometry. The sequence of this peptide encompassed amino acids 220-230 of the listeriolysin protein. Synthetic peptides were synthesized according to this sequence. The CTL responses induced by these peptides were comparable to the eluted native peptide.