bluuebunny/crossref_metadata_2025 huggingface dataset:
GATA-3 Regulates the Development and Function of Invariant NKT Cells
Authors: Kim Peter J. , Pai Sung-Yun , Brigl Manfred , Besra Gurdyal S. , Gumperz Jenny , Ho I-Cheng
Published: April 2014

Although invariant NKT (iNKT) cells participate in many aspects of immune responses, the molecular mechanisms regulating their development, maturation, and activation are still poorly understood. GATA-3 is a T cell-specific transcription factor that is also expressed in iNKT cells. The critical role of GATA-3 in conventional αβ T cells has been well documented, but whether GATA-3 also regulates the development and function of iNKT cells is unknown. In the present study, we report that deficiency of GATA-3 results in cell-intrinsic defects in the thymic development and peripheral maturation of murine iNKT cells. In addition, GATA-3 is also required for survival, activation, and effector functions of this unique population of T cells. Our data also reveal a previously unidentified peripheral maturation step that is GATA-3 dependent.
E2-2 Regulates the Expansion of Pro-B Cells and Follicular versus Marginal Zone Decisions
Authors: Wikström Ingela , Forssell Johan , Goncalves Mario , Colucci Francesco , Holmberg Dan
Published: April 2014

The E-proteins E2A, HeLa E-box binding protein, and E2-2 constitute a class of basic helix-loop-helix transcription factors that differentially affect B cell development. E2A is by far the most investigated and appears to operate at several levels during B cell ontogeny. Less is known concerning the role of the other E-proteins. To address the role of E2-2, we have performed transfers of fetal liver (FL) cells into irradiated Rag-deficient mice. Although the transfer of E2-2-deficient cells alone can reconstitute all B cell subpopulations, albeit with a moderate reduction in cellularity, E2-2-deficient cells have a disadvantage when transferred together with wild-type cells. Cultivation of E2-2−/− day 14.5 FL cells on stromal cells and IL-7 revealed a reduced frequency of responding B cell progenitors despite normal IL-7Rα surface expression. Real-time PCR analysis revealed that E2-2 mRNA expression is high at the pro-B cell stage and drops sharply at the pre-B cell stage, consistent with a role for E2-2 in pro-B cells. In contrast, E2A mRNA was most abundant in pre-B cells. Analysis of the peripheral repertoire revealed that mice reconstituted with E2-2−/− FL cells had an increased proportion of marginal zone (MZ) B cells. Interestingly, E2-2 mRNA was elevated ∼2-fold (p < 0.01) in follicular compared with MZ B cells. Although E2A mRNA showed a similar tendency, the difference was not significant. Collectively, our findings indicate that E2-2 is required for optimal expansion of pro-B cells, and also influences the follicular vs MZ decision.
Melan-A/MART-1-Specific CD4 T Cells in Melanoma Patients: Identification of New Epitopes and Ex Vivo Visualization of Specific T Cells by MHC Class II Tetramers
Authors: Bioley Gilles , Jandus Camilla , Tuyaerts Sandra , Rimoldi Donata , Kwok William W. , Speiser Daniel E. , Tiercy Jean-Marie , Thielemans Kris , Cerottini Jean-Charles , Romero Pedro
Published: April 2014

Over the past decade, many efforts have been made to identify MHC class II-restricted epitopes from different tumor-associated Ags. Melan-A/MART-126–35 parental or Melan-A/MART-126–35(A27L) analog epitopes have been widely used in melanoma immunotherapy to induce and boost CTL responses, but only one Th epitope is currently known (Melan-A51–73, DRB1*0401 restricted). In this study, we describe two novel Melan-A/MART-1-derived sequences recognized by CD4 T cells from melanoma patients. These epitopes can be mimicked by peptides Melan-A27–40 presented by HLA-DRB1*0101 and HLA-DRB1*0102 and Melan-A25–36 presented by HLA-DQB1*0602 and HLA-DRB1*0301. CD4 T cell clones specific for these epitopes recognize Melan-A/MART-1+ tumor cells and Melan-A/MART-1-transduced EBV-B cells and recognition is reduced by inhibitors of the MHC class II presentation pathway. This suggests that the epitopes are naturally processed and presented by EBV-B cells and melanoma cells. Moreover, Melan-A-specific Abs could be detected in the serum of patients with measurable CD4 T cell responses specific for Melan-A/MART-1. Interestingly, even the short Melan-A/MART-126–35(A27L) peptide was recognized by CD4 T cells from HLA-DQ6+ and HLA-DR3+ melanoma patients. Using Melan-A/MART-125–36/DQ6 tetramers, we could detect Ag-specific CD4 T cells directly ex vivo in circulating lymphocytes of a melanoma patient. Together, these results provide the basis for monitoring of naturally occurring and vaccine-induced Melan-A/MART-1-specific CD4 T cell responses, allowing precise and ex vivo characterization of responding T cells.
TCRα Genes Direct MHC Restriction in the Potent Human T Cell Response to a Class I-Bound Viral Epitope
Authors: Miles John J. , Borg Natalie A. , Brennan Rebekah M. , Tynan Fleur E. , Kjer-Nielsen Lars , Silins Sharon L. , Bell Melissa J. , Burrows Jacqueline M. , McCluskey James , Rossjohn Jamie , Burrows Scott R.
Published: April 2014

The underlying generic properties of αβ TCRs that control MHC restriction remain largely unresolved. To investigate MHC restriction, we have examined the CTL response to a viral epitope that binds promiscuously to two human leukocyte Ags (HLAs) that differ by a single amino acid at position 156. Individuals expressing either HLA-B*3501 (156Leucine) or HLA-B*3508 (156Arginine) showed a potent CTL response to the 407HPVGEADYFEY417 epitope from EBV. Interestingly, the response was characterized by highly restricted TCR β-chain usage in both HLA-B*3501+ and HLA-B*3508+ individuals; however, this conserved TRBV9+ β-chain was associated with distinct TCR α-chains depending upon the HLA-B*35 allele expressed by the virus-exposed host. Functional assays confirmed that TCR α-chain usage determined the HLA restriction of the CTLs. Structural studies revealed significant differences in the mobility of the peptide when bound to HLA-B*3501 or HLA-B*3508. In HLA-B*3501, the bulged section of the peptide was disordered, whereas in HLA-B*3508 the bulged epitope adopted an ordered conformation. Collectively, these data demonstrate not only that mobile MHC-bound peptides can be highly immunogenic but can also stimulate an extremely biased TCR repertoire. In addition, TCR α-chain usage is shown to play a critical role in controlling MHC restriction between closely related allomorphs.
CD8+ IL-17-Producing T Cells Are Important in Effector Functions for the Elicitation of Contact Hypersensitivity Responses
Authors: He Donggou , Wu Lizhi , Kim Hee Kyung , Li Hui , Elmets Craig A. , Xu Hui
Published: April 2014

Allergen-induced contact hypersensitivity (CHS) is a T cell-mediated delayed-type immune response which has been considered to be primarily mediated by CD8+ T cytotoxic type I (Tc1) cells. IFN-γ, the prototype Tc1 (Th1) cytokine, has been implicated as the primary inflammatory cytokine for CHS. In this study, we demonstrate that neutralization of IL-17 rather than IFN-γ suppresses the elicitation of CHS. The suppression does not result from inhibition of the proliferation of allergen-activated T cells. Allergen sensitization induces the development of distinct CD8+ T cell subpopulations that produce IFN-γ or IL-17. Although CD8+ IL-17-producing cells are stimulated by IL-23, they are inhibited by IL-12, a prototypical stimulator of IFN-γ-producing Tc1 cells. This indicates that CD8+ IL-17-producing cells are distinct from Tc1 cells and are important in effector functions at the elicitation of CHS. These studies provide insights into a novel mechanism for CHS.
The Study of High-Affinity TCRs Reveals Duality in T Cell Recognition of Antigen: Specificity and Degeneracy
Authors: Donermeyer David L. , Weber K. Scott , Kranz David M. , Allen Paul M.
Published: April 2014

TCRs exhibit a high degree of Ag specificity, even though their affinity for the peptide/MHC ligand is in the micromolar range. To explore how Ag specificity is achieved, we studied murine T cells expressing high-affinity TCRs engineered by in vitro evolution for binding to hemoglobin peptide/class II complex (Hb/I-Ek). These TCRs were shown previously to maintain Ag specificity, despite having up to 800-fold higher affinity. We compared the response of the high-affinity TCRs and the low-affinity 3.L2 TCR toward a comprehensive set of peptides containing single substitutions at each TCR contact residue. This specificity analysis revealed that the increase in affinity resulted in a dramatic increase in the number of stimulatory peptides. The apparent discrepancy between observed degeneracy in the recognition of single amino acid-substituted Hb peptides and overall Ag specificity of the high-affinity TCRs was examined by generating chimeric peptides between the stimulatory Hb and nonstimulatory moth cytochrome c peptides. These experiments showed that MHC anchor residues significantly affected TCR recognition of peptide. The high-affinity TCRs allowed us to estimate the affinity, in the millimolar range, of immunologically relevant interactions of the TCR with peptide/MHC ligands that were previously unmeasurable because of their weak nature. Thus, through the study of high-affinity TCRs, we demonstrated that a TCR is more tolerant of single TCR contact residue substitutions than other peptide changes, revealing that recognition of Ag by T cells can exhibit both specificity and degeneracy.
Reversing Tumor Immune Suppression with Intratumoral IL-12: Activation of Tumor-Associated T Effector/Memory Cells, Induction of T Suppressor Apoptosis, and Infiltration of CD8+ T Effectors
Authors: Kilinc Mehmet O. , Aulakh Karanvir S. , Nair Raji E. , Jones Stacy A. , Alard Pascale , Kosiewicz Michele M. , Egilmez Nejat K.
Published: April 2014

A single intratumoral injection of IL-12 and GM-CSF-loaded slow-release microspheres induces T cell-dependent eradication of established primary and metastatic tumors in a murine lung tumor model. To determine how the delivery of cytokines directly to the microenvironment of a tumor nodule induces local and systemic antitumor T cell activity, we characterized therapy-induced phenotypic and functional changes in tumor-infiltrating T cell populations. Analysis of pretherapy tumors demonstrated that advanced primary tumors were infiltrated by CD4+ and CD8+ T cells with an effector/memory phenotype and CD4+CD25+Foxp3+ T suppressor cells. Tumor-associated effector memory CD8+ T cells displayed impaired cytotoxic function, whereas CD4+CD25+Foxp3+ cells effectively inhibited T cell proliferation demonstrating functional integrity. IL-12/GM-CSF treatment promoted a rapid up-regulation of CD43 and CD69 on CD8+ effector/memory T cells, augmented their ability to produce IFN-γ, and restored granzyme B expression. Importantly, treatment also induced a concomitant and progressive loss of T suppressors from the tumor. Further analysis established that activation of pre-existing effector memory T cells was short-lived and that both the effector/memory and the suppressor T cells became apoptotic within 4 days of treatment. Apoptotic death of pre-existing effector/memory and suppressor T cells was followed by infiltration of the tumor with activated, nonapoptotic CD8+ effector T lymphocytes on day 7 posttherapy. Both CD8+ T cell activation and T suppressor cell purge were mediated primarily by IL-12 and required IFN-γ. This study provides important insight into how local IL-12 therapy alters the immunosuppressive tumor milieu to one that is immunologically active, ultimately resulting in tumor regression.
Antibodies to Complement Receptor 3 Treat Established Inflammation in Murine Models of Colitis and a Novel Model of Psoriasiform Dermatitis
Authors: Leon Francisco , Contractor Nikhat , Fuss Ivan , Marth Thomas , Lahey Edward , Iwaki Shoko , la Sala Andrea , Hoffmann Victoria , Strober Warren , Kelsall Brian L.
Published: April 2014

Prior studies indicated the ability of Abs to complement receptor 3 (CR3, CD11b/CD18) to suppress the production of IL-12 from immune cells. Therefore, we tested the ability of an anti-CR3 Ab (clone M1/70) to treat established IL-12-dependent Th1-mediated inflammation in murine models. Systemic administration of anti-CR3 significantly ameliorated established intestinal inflammation following the intrarectal administration of trinitrobenzene sulfonic acid (TNBS-colitis), as well as colitis and skin inflammation in C57BL/10 RAG-2−/− mice reconstituted with CD4+CD45RBhigh T cells. The hyperproliferative skin inflammation in this novel murine model demonstrated many characteristics of human psoriasis, and was prevented by the adoptive transfer of CD45RBlow T cells. In vitro and in vivo studies suggest that anti-CR3 treatment may act, at least in part, by directly inhibiting IL-12 production by APCs. Administration of anti-CR3 may be a useful therapeutic approach to consider for the treatment of inflammatory bowel disease and psoriasis in humans.
CD4+CXCR4highCD69+ T Cells Accumulate in Lung Adenocarcinoma
Authors: Wald Ori , Izhar Uzi , Amir Gail , Avniel Shani , Bar-Shavit Yochai , Wald Hanna , Weiss Ido D. , Galun Eithan , Peled Amnon
Published: April 2014

The chemokine receptor CXCR4 is involved in the growth and metastasis of tumor cells. However, the expression of its ligand, the chemokine CXCL12, in tumors and its role in regulating the accumulation of immune cells within the tumors is not clear. Using ELISA and immunohistochemistry we found that CXCL12 is expressed in the majority of nonsmall cell lung cancer tissue sections obtained from stage IA to IIB nonsmall cell lung cancer patients undergoing operation. Histopathologic examination of these sections indicated that high CXCL12 expression correlated with increased tumor inflammation. In addition, disease recurrence rates in a subgroup of adenocarcinoma patients showed a tendency to correlate with high CXCL12 expression in the tumor. Isolation of adenocarcinoma-infiltrating immune cells demonstrated an increase in the percentage of CD4+CD69+CXCR4+ T cells as compared with normal lung tissue. About 30% of these cells expressed the regulatory T cell markers CD25high and FoxP3. The percentage of CD8 T cells within the tumor did not change, however; the percentage of NK and NK T cells was significantly reduced. In correlation with CXCR4 expression, CD4 T cells showed increased migration in response to CXCL12 compared with CD8 T cells and NK cells. Overall, these observations suggest that CXCL12 expression may influence tumor progression by shaping the immune cell population infiltrating lung adenocarcinoma tumors.
Genes within the Idd5 and Idd9/11 Diabetes Susceptibility Loci Affect the Pathogenic Activity of B Cells in Nonobese Diabetic Mice
Authors: Silveira Pablo A. , Chapman Harold D. , Stolp Jessica , Johnson Ellis , Cox S. Lewis , Hunter Kara , Wicker Linda S. , Serreze David V.
Published: April 2014

Autoreactive T cells clearly mediate the pancreatic β cell destruction causing type 1 diabetes (T1D). However, studies in NOD mice indicate that B cells also contribute to pathogenesis because their ablation by introduction of an Igμnull mutation elicits T1D resistance. T1D susceptibility is restored in NOD.Igμnull mice that are irradiated and reconstituted with syngeneic bone marrow plus NOD B cells, but not syngeneic bone marrow alone. Thus, we hypothesized some non-MHC T1D susceptibility (Idd) genes contribute to disease by allowing development of pathogenic B cells. Supporting this hypothesis was the finding that unlike those from NOD donors, engraftment with B cells from H2g7 MHC-matched, but T1D-resistant, nonobese-resistant (NOR) mice failed to restore full disease susceptibility in NOD.Igμnull recipients. T1D resistance in NOR mice is mainly encoded within the Idd13, Idd5.2, and Idd9/11 loci. B cells from NOD congenic stocks containing Idd9/11 or Idd5.1/5.2-resistance loci, respectively, derived from the NOR or C57BL/10 strains were characterized by suppressed diabetogenic activity. Immature autoreactive B cells in NOD mice have an impaired ability to be rendered anergic upon Ag engagement. Interestingly, both Idd5.1/5.2 and Idd9/11-resistance loci were found to normalize this B cell tolerogenic process, which may represent a mechanism contributing to the inhibition of T1D.
IL-10 Deficiency Promotes Increased Borrelia burgdorferi Clearance Predominantly through Enhanced Innate Immune Responses
Authors: Lazarus John J. , Meadows Michelle J. , Lintner Robert E. , Wooten R. Mark
Published: April 2014

Borrelia burgdorferi is capable of persistently infecting a variety of hosts despite eliciting potent innate and adaptive immune responses. Preliminary studies indicated that IL-10-deficient (IL-10−/−) mice exhibit up to 10-fold greater clearance of B. burgdorferi from target tissues compared with wild-type mice, establishing IL-10 as the only cytokine currently known to have such a significant effect on spirochetal clearance. To further delineate these IL-10-mediated immune effects, kinetic studies indicated that spirochete dissemination to target tissues is similar in both wild-type and IL-10−/− mouse strains, and that enhanced clearance of B. burgdorferi in IL-10−/− mice is correlated with increased B. burgdorferi-specific Ab as early as 2 wk postinfection. Immunoblot analysis indicated that Abs produced by infected IL-10−/− and wild-type mice recognize similar ranges of spirochetal Ags. Immune sera from IL-10−/− and wild-type mice also exhibited similar bactericidal activity in vitro, and passive transfer of these immune sera into B. burgdorferi-infected SCID mice caused similar reductions of bacterial numbers in target tissues. Infectious dose studies indicated that 8-fold more B. burgdorferi were needed to efficiently infect naive IL-10−/− mice, suggesting these animals possess higher innate barriers to infection. Moreover, macrophages derived from IL-10−/− mice exhibit enhanced proinflammatory responses to B. burgdorferi stimulation compared with wild-type controls, and these responses are not significantly affected by the presence of immune serum. These findings confirm that B. burgdorferi clearance by innate immune responses is more efficient in the absence of IL-10, and these activities are not directly related to increased levels of B. burgdorferi-specific Ab.
Bruton’s Tyrosine Kinase Is Required for TLR-Induced IL-10 Production
Authors: Schmidt Nathan W. , Thieu Vivian T. , Mann Brandon A. , Ahyi Ayele-Nati N. , Kaplan Mark H.
Published: April 2014

Bruton’s tyrosine kinase (Btk) is a critical signaling mediator downstream of the B cell Ag receptor. X-linked agammaglobulinemia is caused by mutations in Btk resulting in multiple defects in B cell development and function, and recurrent bacterial infections. Recent evidence has also supported a role for Btk in TLR signaling. We demonstrate that Btk is activated by TLR4 in primary macrophages and is required for normal TLR-induced IL-10 production in multiple macrophage populations. Btk-deficient bone marrow-derived macrophages secrete decreased levels of IL-10 in response to multiple TLR ligands, compared with wild-type (WT) cells. Similarly, Btk-deficient peritoneal and splenic macrophages secrete decreased IL-10 levels compared with WT cultures. This phenotype correlates with Btk-dependent induction of NF-κB and AP-1 DNA binding activity, and altered commensal bacteria populations. Decreased IL-10 production may be responsible for increased IL-6 because blocking IL-10 in WT cultures increased IL-6 production, and supplementation of IL-10 to Btk-deficient cultures decreased IL-6 production. Similarly, injection of IL-10 in vivo with LPS decreases the elevated IL-6 serum levels during endotoxemia in Btk-deficient mice. These data further support a role for Btk in regulating TLR-induced cytokine production from APCs and provide downstream targets for analysis of Btk function.
Evidence of a Functional Role for Mast Cells in the Development of Type 1 Diabetes Mellitus in the BioBreeding Rat
Authors: Geoffrey Rhonda , Jia Shuang , Kwitek Anne E. , Woodliff Jeffrey , Ghosh Soumitra , Lernmark Åke , Wang Xujing , Hessner Martin J.
Published: April 2014

Human type 1 diabetes mellitus (T1DM) arises through autoimmune destruction of pancreatic β cells and is modeled in many respects by the lymphopenic and spontaneously diabetic BioBreeding (BB) DRlyp/lyp rat. Previously, preonset expression profiling of whole DRlyp/lyp pancreatic lymph nodes (PLN) revealed innate immune activity, specifically that of mast cells and eosinophils. Furthermore, we observed that pancreatic islets of DRlyp/lyp rats as well as those of diabetes-inducible BB DR+/+ rats potentially recruit innate cells through eotaxin expression. Here we determine that lifelong eotaxin expression begins before 40 days of life and is localized specifically to β cells. In this report, we find that PLN mast cells are more abundant in DRlyp/lyp compared with related BB DR+/+ rats (2.1 ± 0.9% vs 0.9 ± 0.4% of total cells, p < 0.0001). DRlyp/lyp PLN mast cell gene expression profiling revealed an activated population and included significant overrepresentation of transcripts for mast cell protease 1, cationic trypsinogen, carboxypeptidase A, IL-5, and phospholipase Cγ. In the DR+/+ rat, which develops T1DM upon depletion of T regulator cells, mast cells displayed gene expression consistent with the negative regulation of degranulation, including significant overrepresentation of transcripts encoding tyrosine phosphatase SHP-1, lipid phosphatase SHIP, and E3 ubiquitin ligase c-Cbl. To recapitulate the negative mast cell regulation observed in the DR+/+ rats, we treated DRlyp/lyp rats with the mast cell “stabilizer” cromolyn, which significantly (p < 0.05) delayed T1DM onset. These findings are consistent with a growing body of evidence in human and animal models, where a role for mast cells in the initiation and progression of autoimmune disease is emerging.
Expression and Functions of the Vascular Endothelial Growth Factors and Their Receptors in Human Basophils
Authors: de Paulis Amato , Prevete Nella , Fiorentino Isabella , Rossi Francesca Wanda , Staibano Stefania , Montuori Nunzia , Ragno Pia , Longobardi Amelia , Liccardo Bianca , Genovese Arturo , Ribatti Domenico , Walls Andrew F. , Marone Gianni
Published: April 2014

Angiogenesis is a multistep complex phenomenon critical for several inflammatory and neoplastic disorders. Basophils, normally confined to peripheral blood, can infiltrate the sites of chronic inflammation. In an attempt to obtain insights into the mechanism(s) underlying human basophil chemotaxis and its role in inflammation, we have characterized the expression and function of vascular endothelial growth factors (VEGFs) and their receptors in these cells. Basophils express mRNA for three isoforms of VEGF-A (121, 165, and 189) and two isoforms of VEGF-B (167 and 186). Peripheral blood and basophils in nasal polyps contain VEGF-A localized in secretory granules. The concentration of VEGF-A in basophils was 144.4 ± 10.8 pg/106 cells. Immunologic activation of basophils induced the release of VEGF-A. VEGF-A (10–500 ng/ml) induced basophil chemotaxis. Supernatants of activated basophils induced an angiogenic response in the chick embryo chorioallantoic membrane that was inhibited by an anti-VEGF-A Ab. The tyrosine kinase VEGFR-2 (VEGFR-2/KDR) mRNA was expressed in basophils. These cells also expressed mRNA for the soluble form of VEGFR-1 and neuropilin (NRP)1 and NRP2. Flow cytometric analysis indicated that basophils express epitopes recognized by mAbs against the extracellular domains of VEGFR-2, NRP1, and NRP2. Our data suggest that basophils could play a role in angiogenesis and inflammation through the expression of several forms of VEGF and their receptors.
Epitope Specificity of Autoreactive T and B Cells Associated with Experimental Autoimmune Encephalomyelitis and Optic Neuritis Induced by Oligodendrocyte-Specific Protein in SJL/J Mice
Authors: Kaushansky Nathali , Zhong Ming-Chao , Kerlero de Rosbo Nicole , Hoeftberger Romana , Lassmann Hans , Ben-Nun Avraham
Published: April 2014

The encephalitogenic potential of oligodendrocyte-specific protein (OSP) in mice, its specific localization in the intralamellar tight junctions in CNS myelin, and the detection of autoreactivity against OSP in multiple sclerosis (MS) strongly suggest the relevance of autoreactivity against OSP in the pathogenesis of MS. In this study, we have characterized the autoimmune T and B cells that are associated with clinicopathological manifestations of OSP-induced MS-like disease in mice by using recombinant soluble mouse OSP (smOSP) and synthetic overlapping peptides spanning smOSP. SJL/J mice immunized with smOSP developed chronic relapsing clinical experimental autoimmune encephalomyelitis accompanied with intense perivascular and parenchymal inflammatory infiltrates, widespread demyelination, axonal loss, and remarkable optic neuritis. The smOSP-primed lymph node cells reacted predominantly against OSP55–80 and to a lesser extent also to OSP22–46 and OSP179–207. Unexpectedly, in vitro selection with smOSP resulted in pathogenic smOSP-specific CD4+ T cells that reacted equally well against OSP55–80, OSP22–46, OSP45–66, and OSP179–207. Fine analysis of the anti-OSP autoimmunity revealed that the disease is primarily associated with CD4+ T cells directed against the major (OSP55–80) and the minor (OSP179–207) encephalitogenic regions that were further delineated, both in vitro and in vivo, to OSP55–66 and OSP194–207, respectively. In contrast, the OSP-induced Abs were predominantly directed against OSP22–46; these Abs were mostly of IgG1 isotype, but high levels of IgG2a and IgG2b and significant levels of IgE were also observed. The reactivity of pathogenic T cells to two encephalitogenic regions, OSP55–80 and OSP179–207, and their diverse TCRVβ gene repertoire may impose difficulties for epitope-directed or TCR-targeting approaches to immune-specific modulation of OSP-related pathogenesis.
CD4+CD25high T Cells Are Enriched in the Tumor and Peripheral Blood of Prostate Cancer Patients
Authors: Miller Ashley M. , Lundberg Kajsa , Özenci Volkan , Banham Alison H. , Hellström Magnus , Egevad Lars , Pisa Pavel
Published: April 2014

In this study, we investigated whether CD4+CD25high regulatory T cells (Treg) are increased in the tumor tissue and peripheral blood of early-stage prostate cancer patients undergoing prostatectomy. We show that the prevalence of CD4+CD25high T cells inside the prostate was significantly higher in the tumor compared with benign tissue from the same prostate. Furthermore, the frequency of CD4+CD25high T cells in peripheral blood was significantly higher in prostate cancer patients compared with normal donors. A proportion of the CD4+CD25high T cells was also shown to be glucocorticoid-induced TNF receptor, ICOS, and FOXP3 positive. Moreover, CD4+CD25+ T cells from blood and supernatants from cultured prostate tumor tissue samples exhibited immunosuppressive function in vitro. Furthermore, supernatants from cultured prostate tissue samples and prostate cancer ascites fluid induced migration of CD4+CD25+ T cells and were shown to contain the regulatory T cell chemokine CCL22 by ELISA. Our findings indicate that Tregs are an important cellular component of early-stage prostate tumors, and thus new therapeutic strategies aimed at inhibition or depletion of Tregs may improve prostate cancer immunotherapy.
Role of MHC-Linked Genes in Autoantigen Selection and Renal Disease in a Murine Model of Systemic Lupus Erythematosus
Authors: Sekine Hideharu , Graham Kareem L. , Zhao Shenru , Elliott Margaret K. , Ruiz Philip , Utz Paul J. , Gilkeson Gary S.
Published: April 2014

We previously described a renal protective effect of factor B deficiency in MRL/lpr mice. Factor B is in the MHC cluster; thus, the deficient mice were H2b, the haplotype on which the knockout was derived, whereas the wild-type littermates were H2k, the H2 of MRL/lpr mice. To determine which protective effects were due to H2 vs factor B deficiency, we derived H2b congenic MRL/lpr mice from the 129/Sv (H2b) strain. Autoantibody profiling using autoantigen microarrays revealed that serum anti-Smith and anti-small nuclear ribonucleoprotein complex autoantibodies, while present in the majority of H2k/k MRL/lpr mice, were absent in the H2b/b MRL/lpr mice. Surprisingly, 70% of MRL/lpr H2b/b mice were found to be serum IgG3 deficient (with few to no IgG3-producing B cells). In addition, H2b/b IgG3-deficient MRL/lpr mice had significantly less proteinuria, decreased glomerular immune complex deposition, and absence of glomerular subepithelial deposits compared with MRL/lpr mice of any H2 type with detectable serum IgG3. Despite these differences, total histopathologic renal scores and survival were similar among the groups. These results indicate that genes encoded within or closely linked to the MHC region regulate autoantigen selection and isotype switching to IgG3 but have minimal effect on end-organ damage or survival in MRL/lpr mice.
Defective Th1 Cytokine Gene Transcription in CD4+ and CD8+ T Cells from Wiskott-Aldrich Syndrome Patients
Authors: Trifari Sara , Sitia Giovanni , Aiuti Alessandro , Scaramuzza Samantha , Marangoni Francesco , Guidotti Luca G. , Martino Silvana , Saracco Paola , Notarangelo Luigi D. , Roncarolo Maria-Grazia , Dupré Loïc
Published: April 2014

Wiskott-Aldrich syndrome (WAS) protein (WASP) plays a key role in TCR-mediated activation and immunological synapse formation. However, the effects of WASP deficiency on effector functions of human CD4+ and CD8+ T cells remain to be determined. In this study, we report that TCR/CD28-driven proliferation and secretion of IL-2, IFN-γ, and TNF-α are strongly reduced in CD8+ T cells from WAS patients, compared with healthy donor CD8+ T cells. Furthermore, WAS CD4+ T cells secrete low levels of IL-2 and fail to produce IFN-γ and TNF-α, while the production of IL-4, IL-5, and IL-10 is only minimally affected. Defective IL-2 and IFN-γ production persists after culture of naive WAS CD4+ T cells in Th1-polarizing conditions. The defect in Th1 cytokine production by WAS CD4+ and CD8+ T cells is also present at the transcriptional level, as shown by reduced IL-2 and IFN-γ mRNA transcripts after TCR/CD28 triggering. The reduced transcription of Th1 cytokine genes in WAS CD4+ T cells is associated with a defective induction of T-bet mRNA and a reduction in the early nuclear recruitment of NFAT-1, while the defective activation of WAS CD8+ T cells correlates with reduced nuclear recruitment of both NFAT-1 and NFAT-2. Together, our data indicate that WASP regulates the transcriptional activation of T cells and is required specifically for Th1 cytokine production.
Cutting Edge: Pivotal Function of Ubc13 in Thymocyte TCR Signaling
Authors: Yamamoto Masahiro , Sato Shintaro , Saitoh Tatsuya , Sakurai Hiroaki , Uematsu Satoshi , Kawai Taro , Ishii Ken J. , Takeuchi Osamu , Akira Shizuo
Published: April 2014

The Ubc13 E2 ubiquitin-conjugating enzyme is essential for BCR-, TLR-, and IL-1 receptor (IL-1R)-mediated immune responses. Although Ubc13-deficient mice show defects in BCR-, TLR/IL-1R-, or CD40-mediated activation of mitogen-activated protein kinases, the function of Ubc13 in TCR-mediated signaling and responses remains uncertain. To address this, we here generated T cell-specific conditional Ubc13-deficient mice. The frequency of T lymphocytes was severely reduced in spleens from Ubc13-deficient mice. Moreover, Ubc13-deficient thymocytes displayed defective proliferation in response to anti-CD3/CD28 or PMA/ionophore stimulation. Regarding the signal transduction, although NF-κB activation was modestly affected, PMA/ionophore-induced activation of Jnk and p38 was profoundly impaired in Ubc13-deficient thymocytes. In addition, PMA/ionophore-mediated ubiquitination of NF-κB essential modulator (NEMO)/IκB kinase γ (IKKγ) and phosphorylation of TGF-β-activated kinase 1 (TAK1) were nearly abolished in Ubc13-deficient thymocytes. Thus, Ubc13 plays an important role in thymocyte TCR-mediated signaling and immune responses.
Regulation of Immunity by a Novel Population of Qa-1-Restricted CD8αα+TCRαβ+ T Cells
Authors: Tang Xiaolei , Maricic Igor , Purohit Nikunj , Bakamjian Berge , Reed-Loisel Lisa M. , Beeston Tara , Jensen Peter , Kumar Vipin
Published: April 2014

Regulatory mechanisms involving CD8+ T cells (CD8 regulatory T cells (Tregs)) are important in the maintenance of immune homeostasis. However, the inability to generate functional CD8 Treg clones with defined Ag specificity has precluded a direct demonstration of CD8 Treg-mediated regulation. In the present study, we describe the isolation of functional lines and clones representing a novel population of TCRαβ+ Tregs that control activated Vβ8.2+ CD4 T cells mediating experimental autoimmune encephalomyelitis. They express exclusively the CD8αα homodimer and recognize a peptide from a conserved region of the TCR Vβ8.2 chain in the context of the Qa-1a (CD8αα Tregs). They secrete type 1 cytokines but not IL-2. CD8αα Tregs kill activated Vβ8.2+ but not Vβ8.2− or naive T cells. The CD8αα Tregs prevent autoimmunity upon adoptive transfer or following in vivo activation. These findings reveal an important negative feedback regulatory mechanism targeting activated T cells and have implications in the development of therapeutic strategies for autoimmune diseases and transplantation.
Oncostatin M Enhances CCL21 Expression by Microvascular Endothelial Cells and Increases the Efficiency of Dendritic Cell Trafficking to Lymph Nodes
Authors: Sugaya Makoto , Fang Lei , Cardones Adela R. , Kakinuma Takashi , Jaber Samer H. , Blauvelt Andrew , Hwang Sam T.
Published: April 2014

CCL21, a lymphatic endothelial cell (LEC)-derived chemokine, and its receptor CCR7 regulate dendritic cell (DC) trafficking to lymph nodes (LN), but it is unclear how CCL21 expression is regulated. Oncostatin M (OSM) is an IL-6-like cytokine synthesized by activated DC and other leukocytes. In vitro, OSM (but not TNF-α) stimulated CCL21 mRNA and protein expression by human dermal microvascular EC (DMEC) in an ERK1/2-dependent fashion. Conditioned medium from OSM-treated DMEC stimulated CCL21-dependent chemotaxis of mouse bone marrow-derived DC (BMDC). Cultured BMDC expressed OSM, which was increased with the addition of LPS. Topical application of the contact-sensitizing hapten, trinitrochlorobenzene, resulted in enhanced OSM expression in the skin, whereas cutaneous injection of TNF-α did not. Injection of OSM into the footpad increased CCL21 mRNA expression in the draining LN by ∼10-fold and in mouse skin by ∼4-fold without increasing CCR7 mRNA. In vitro, OSM increased the permeability of DMEC and lung microvascular EC monolayers to FITC-dextran beads, and, in vivo, it enhanced accumulation of Evans blue dye in draining LN by ∼3-fold (p = 0.0291). Of note, OSM increased trafficking of BMDC injected in footpads to draining LN by 2-fold (p = 0.016). In summary, OSM up-regulates CCL21 expression in skin and draining regional LN. We propose that OSM is a regulator of CCL21 expression and endothelial permeability in skin, contributing to efficient migration of DC to regional LN.
Control of Memory CD4 T Cell Recall by the CD28/B7 Costimulatory Pathway
Authors: Ndejembi Modesta P. , Teijaro John R. , Patke Deepa S. , Bingaman Adam W. , Chandok Meena R. , Azimzadeh Agnes , Nadler Steven G. , Farber Donna L.
Published: April 2014

The CD28/B7 costimulatory pathway is generally considered dispensable for memory T cell responses, largely based on in vitro studies demonstrating memory T cell activation in the absence of CD28 engagement by B7 ligands. However, the susceptibility of memory CD4 T cells, including central (CD62Lhigh) and effector memory (TEM; CD62Llow) subsets, to inhibition of CD28-derived costimulation has not been closely examined. In this study, we demonstrate that inhibition of CD28/B7 costimulation with the B7-binding fusion molecule CTLA4Ig has profound and specific effects on secondary responses mediated by memory CD4 T cells generated by priming with Ag or infection with influenza virus. In vitro, CTLA4Ig substantially inhibits IL-2, but not IFN-γ production from heterogeneous memory CD4 T cells specific for influenza hemagglutinin or OVA in response to peptide challenge. Moreover, IL-2 production from polyclonal influenza-specific memory CD4 T cells in response to virus challenge was completely abrogated by CTLA4Ig with IFN-γ production partially inhibited. When administered in vivo, CTLA4Ig significantly blocks Ag-driven memory CD4 T cell proliferation and expansion, without affecting early recall and activation. Importantly, CTLA4Ig treatment in vivo induced a striking shift in the phenotype of the responding population from predominantly TEM in control-treated mice to predominantly central memory T cells in CTLA4Ig-treated mice, suggesting biased effects of CTLA4Ig on TEM responses. Our results identify a novel role for CD28/B7 as a regulator of memory T cell responses, and have important clinical implications for using CTLA4Ig to abrogate the pathologic consequences of TEM cells in autoimmunity and chronic disease.
Expression of Integrin β3 Is Correlated to the Properties of Quiescent Hemopoietic Stem Cells Possessing the Side Population Phenotype
Authors: Umemoto Terumasa , Yamato Masayuki , Shiratsuchi Yoshiko , Terasawa Masao , Yang Joseph , Nishida Kohji , Kobayashi Yoshiro , Okano Teruo
Published: April 2014

With significant attention paid to the field of tissue-specific stem cells, the identification of stem cell-specific markers is of considerable importance. Previously, the side population (SP) phenotype, with the capacity to efflux the DNA-binding dye Hoechst 33342, has been recognized as a common feature of adult tissue-specific stem cells. In this study, we show that high expression of integrin β3 (CD61) is an attribute of SP cells isolated from mouse bone marrow. Additionally, we confirmed that the expression of integrin β3 is correlated with properties of quiescent hemopoietic stem cells (HSCs) including the strength of the SP phenotype, cell cycle arrest, expression of HSC markers, and long-term hemopoiesis. Importantly, Lineage− (Lin−)/integrin β3high (β3high) SP cells have as strong a capacity for long-term hemopoiesis as c-Kit+/Sca-1+/Lin− SP cells, which are regarded as one of the most highly enriched HSC populations. Finally, the integrin β3 subunit that is present in SP cells having the properties of HSCs, is associated with integrin αv (CD51). Therefore, our results demonstrate that high expression of integrin β3 is correlated to the properties of quiescent HSCs and suggest that the integrin β3 subunit is available as a common surface marker of tissue-specific stem cells.
TLR2/TLR4-Independent Neutrophil Activation and Recruitment upon Endocytosis of Nucleosomes Reveals a New Pathway of Innate Immunity in Systemic Lupus Erythematosus
Authors: Rönnefarth Viktoria M. , Erbacher Annika I. M. , Lamkemeyer Tobias , Madlung Johannes , Nordheim Alfred , Rammensee Hans-Georg , Decker Patrice
Published: April 2014

The nucleosome is a major autoantigen in systemic lupus erythematosus (SLE); it can be detected as a circulating complex in the serum, and nucleosomes have been suggested to play a key role in disease development. In the present study, we show for the first time that physiological concentrations of purified nucleosomes trigger innate immunity. The nucleosomes are endocytosed and induce the direct activation of human neutrophils (polymorphonuclear leukocytes (PMN)) as revealed by CD11b/CD66b up-regulation, IL-8 secretion, and increased phagocytic activity. IL-8 is a neutrophil chemoattractant detected in high concentrations in the sera of patients, and IL-8 secretion might thus result in enhanced inflammation, as observed in lupus patients, via an amplification loop. Nucleosomes act as free complexes requiring no immune complex formation and independently of the presence of unmethylated CpG DNA motifs. Both normal and lupus neutrophils are sensitive to nucleosome-induced activation, and activation is not due to endotoxin or high-mobility group box 1 contamination. In mice, i.p. injection of purified nucleosomes induces neutrophil activation and recruitment in a TLR2/TLR4-independent manner. Importantly, neutrophils have been suggested to link innate and adaptive immunity. Thus, nucleosomes trigger a previously unknown pathway of innate immunity, which may partially explain why peripheral tolerance is broken in SLE patients.
Generation and Growth of CD28nullCD8+ Memory T Cells Mediated by IL-15 and Its Induced Cytokines
Authors: Chiu Wai Kan , Fann Monchou , Weng Nan-ping
Published: April 2014

Accumulation of CD28nullCD8+ T cells and the defects of these cells in response to antigenic stimulation are the hallmarks of age-associated decline of T cell function. However, the mechanism of these age-associated changes is not fully understood. In this study, we report an analysis of the growth of human CD28null and CD28+CD8+ memory T cells in response to homeostatic cytokine IL-15 in vitro. We showed that 1) there was no proliferative defect of CD28nullCD8+ memory T cells in response to IL-15 compared with their CD28+ counterparts; 2) stable loss of CD28 expression occurred in those actively dividing CD28+CD8+ memory T cells responding to IL-15; 3) the loss of CD28 was in part mediated by TNF-α that was induced by IL-15; and 4) CCL4 (MIP-1β), also induced by IL-15, had a significant inhibitory effect on the growth of CD28null cells, which in turn down-regulated their expression of CCL4 receptor CCR5. Together, these findings demonstrate that CD28nullCD8+ memory T cells proliferate normally in response to IL-15 and that IL-15 and its induced cytokines regulate the generation and growth of CD28nullCD8+ T cells, suggesting a possible role of IL-15 in the increase in CD28nullCD8+ T cells that occurs with aging.
Heat Shock Protein 70 Is Secreted from Tumor Cells by a Nonclassical Pathway Involving Lysosomal Endosomes
Authors: Mambula Salamatu S. , Calderwood Stuart K.
Published: April 2014

Heat shock protein (HSP)70 can be released from tumor cells and stimulate a potent antitumor immune response. However, HSP70 does not contain a consensus secretory signal and thus cannot traverse the plasma membrane by conventional mechanisms. We have observed HSP70 release from intact human prostate carcinoma cell lines (PC-3 and LNCaP) by a mechanism independent of de novo HSP70 synthesis or cell death. This pathway is similar to one used by the leaderless protein IL-1β. Our studies show that HSP70 release involves transit though an endolysosomal compartment and is inhibited by lysosomotropic compounds. In addition, the rate of HSP70 secretion correlates well with the appearance of the lysosomal marker LAMP1 on the cell surface, further suggesting the role for endolysosomes. The entry of HSP70 into this secretory compartment appears to involve the ABC family transporter proteins and ABC transporter inhibitor glibenclamide antagonizes secretion. Although the cell signals involved in triggering stress induced HSP70 release though this lysosomal pathway are largely unknown, our experiments suggest a regulatory role for extracellular ATP. These mechanisms appear to be shared by IL-1β secretion. Following release, we observed the binding of extracellular HSP70 to the cell surface of the prostate carcinoma cells. These findings suggest that secreted HSP70 can take part in paracrine or autocrine interactions with adjacent cell surfaces. Our experiments therefore suggest a mechanism for HSP70 secretion and binding to the surface of other cells that may be involved in recognition of the tumor cells by the immune system.
Microarrays Reveal Distinct Gene Signatures in the Thymus of Seropositive and Seronegative Myasthenia Gravis Patients and the Role of CC Chemokine Ligand 21 in Thymic Hyperplasia
Authors: Le Panse Rozen , Cizeron-Clairac Géraldine , Bismuth Jacky , Berrih-Aknin Sonia
Published: April 2014

Myasthenia gravis (MG) is an autoimmune disease mainly caused by antiacetylcholine receptor autoantibodies (seropositive (SP) disease) or by Abs against unknown autoantigenic target(s) (seronegative (SN) disease). Thymectomy is usually beneficial although thymic hyperplasia with ectopic germinal centers is mainly observed in SP MG. To understand the role of thymus in the disease process, we compared the thymic transcriptome of non-MG adults to those of SP patients with a low or high degree of hyperplasia or SN patients. Surprisingly, an overexpression of MHC class II, Ig, and B cell marker genes is observed in SP but also SN MG patients. Moreover, we demonstrate an overexpression of CXCL13 in all MG thymuses leading probably to the generalized B cell infiltration. However, we find different chemotactic properties for MG subgroups and, especially, a specific overexpression of CCL21 in hyperplastic thymuses triggering most likely ectopic germinal center development. Besides, SN patients present a peculiar signature with an abnormal expression of genes involved in muscle development and synaptic transmission, but also genes implicated in host response, suggesting that viral infection might be related to SN MG. Altogether, these results underline differential pathogenic mechanisms in the thymus of SP and SN MG and propose new research areas.
Analysis of the Individual Contributions of Igα (CD79a)- and Igβ (CD79b)-Mediated Tonic Signaling for Bone Marrow B Cell Development and Peripheral B Cell Maturation
Authors: Fuentes-Pananá Ezequiel M. , Bannish Gregory , Karnell Fredrick G. , Treml John F. , Monroe John G.
Published: April 2014

The individual contribution of Igα and Igβ for BCR-triggered fates is unclear. Prior evidence supports conflicting ideas concerning unique as well as redundant functions for these proteins in the context of BCR/pre-BCR signaling. Part of this ambiguity may reflect the recent appreciation that Igα and Igβ participate in both Ag-independent (tonic) and Ag-dependent signaling. The present study undertook defining the individual requirement for Igα and Igβ under conditions where only ligand-independent tonic signaling was operative. In this regard, we have constructed chimeric proteins containing one or two copies of the cytoplasmic domains of either Igα or Igβ and Igα/Igβ heterodimers with targeted Tyr→Phe modifications. The ability of these proteins to act as surrogate receptors and trigger early bone marrow and peripheral B cell maturation was tested in RAG2−/− primary pro-B cell lines and in gene transfer experiments in the μMT mouse model. We considered that the threshold for a functional activity mediated by the pre-BCR/BCR might only be reached when two functional copies of the Igα/Igβ ITAM domain are expressed together, and therefore the specificity conferred by these proteins can only be observed in these conditions. We found that the ligand-independent tonic signal is sufficient to drive development into mature follicular B cells and both Igα and Igβ chains supported formation of this population. In contrast, neither marginal zone nor B1 mature B cell subsets develop from bone marrow precursors under conditions where only tonic signals are generated.