bluuebunny/crossref_metadata_2025 huggingface dataset:
Colocalization of FcγRI-Targeted Antigen With Class I MHC: Implications for Antigen Processing
Authors: Guyre Cheryl A. , Barreda Marc E. , Swink Sharon L. , Fanger Michael W.
Published: April 2014

The high-affinity receptor for IgG (CD64 or FcγRI) is constitutively expressed exclusively on professional APCs (monocytes, macrophages, and dendritic cells). When Ag is targeted specifically to FcγRI, Ag presentation is markedly enhanced, although the mechanism of this enhancement is unknown. In an effort to elucidate the pathways involved in FcγRI targeting, we developed a model targeted Ag using enhanced green fluorescent protein (eGFP). This molecule, wH22xeGFP, consists of the entire humanized anti-FcγRI mAb H22 with eGFP genetically fused to the C-terminal end of each CH3 domain. wH22xeGFP binds within the ligand-binding region by its Fc end, as well as outside the ligand-binding region by its Fab ends, thereby cross-linking FcγRI. Confocal microscopy studies revealed that wH22xeGFP was rapidly internalized by the high-FcγRI-expressing cell line U937 10.6, but did not associate with intracellular proteins Rab4, Rab5a, or Lamp-1, suggesting that the targeted fusion protein was not localized in early endosomes, recycling vesicles, or lysosomes. Interestingly, wH22xeGFP was found colocalized with intracellular MHC class I, suggesting that FcγRI-targeted Ags may converge upon a class I processing pathway. These data are in agreement with studies in the mouse showing that FcγRI targeting can lead to Ag-specific activation of cytotoxic T cells. Data obtained from these studies should lead to a better understanding of how Ags targeted to FcγRI are processed and under what conditions they lead to presentation of antigenic peptides in MHC class I, as a foundation for the use of FcγRI-targeted Ags as vaccines.
Regulation of Inhibitory and Activating Killer-Cell Ig-Like Receptor Expression Occurs in T Cells After Termination of TCR Rearrangements
Authors: Vely Frédéric , Peyrat Marie-Alix , Couedel Christelle , Morcet Jean-François , Halary Franck , Davodeau François , Romagne François , Scotet Emmanuel , Saulquin Xavier , Houssaint Elisabeth , Schleinitz Nicolas , Moretta Alessandro , Vivier Eric , Bonneville Marc
Published: April 2014

A small fraction of T cells expresses killer-cell Ig-like receptors (KIR), a family of MHC class I-specific receptors that can modulate TCR-dependent activation of effector functions. Although KIR+ cells are enriched within Ag-experienced T cell subsets, the precise relationships between KIR+ and KIR− T cells and the stage of KIR induction on these lymphocytes remain unclear. In this study, we compared KIR− and KIR+ αβ T cell clones, sorted by means of the CD158b (KIR2DL2/KIR2DL3/KIR2DS2) specific mAb GL183. We isolated several pairs of CD158b+ and CD158b− αβ T cell clones sharing identical productive and nonproductive TCR transcripts. We showed that expression of functional KIR on T cells is regulated after termination of TCR rearrangements. Transcriptional regulation of KIR genes was documented in multiple T cell clones generated from the same donor, and the presence of KIR transcripts was also detected in KIR− T cells. These results document a complex regulation of KIR expression in T cells at both pre and posttranscriptional levels, under the control of yet undefined signals provided in vivo.
Comparison of a T Cell Clone and of T Cells from a TCR Transgenic Mouse: TCR Transgenic T Cells Specific for Self-Antigen Are Atypical
Authors: Dobbs Cathleen M. , Haskins Kathryn
Published: April 2014

It has been widely assumed that T cells from TCR-transgenic (Tg) mice better represent the behavior of T cells from normal mice than do in vitro cultures of T cell clones. We have found that autoreactive T cells arising in the presumably more physiological environment of the BDC-2.5 TCR-Tg mouse, despite being apparently “naive” in surface phenotype, are highly activated functionally and do not resemble CD4+ T cells from a spontaneously diabetic nonobese diabetic (NOD) mouse or the NOD-derived, diabetogenic CD4+ T cell clone of origin, BDC-2.5. Our results suggest that autoreactive T cells cloned from the spontaneously diabetic NOD mouse more closely resemble effector T cells arising during the natural disease process.
Selective Gene Expression and Activation-Dependent Regulation of Vasoactive Intestinal Peptide Receptor Type 1 and Type 2 in Human T Cells
Authors: Lara-Marquez Maria L. , O’Dorisio M. Sue , O’Dorisio Thomas M. , Shah Manisha H. , Karacay Bahri
Published: April 2014

Vasoactive intestinal peptide (VIP) has potent antiproliferative and anti-inflammatory functions in the immune system. Two structurally distinct G-protein-associated receptors, VIP receptor type 1 (VPAC1) and VIP receptor type 2 (VPAC2), mediate the biological effects of VIP. The regulation of VIP receptor gene expression and the distribution of these receptors in different compartments of the human immune systems are unknown. This study reports, for the first time, a quantitative analysis of VPAC1 and VPAC2 mRNA expression in resting and activated T cells as well as in resting monocytes. Purified human peripheral blood CD4+ T cells and CD8+ T cells were stimulated via the TCR/CD3 receptor complex. Using the novel fluorometric-based kinetic (real-time) RT-PCR, we determined that VPAC1 is constitutively expressed in resting T cells and monocytes; the levels of expression were significantly higher in monocytes and CD4+ T cells than in CD8+ T cells. VPAC1 mRNA expression is significantly higher relative to VPAC2 in resting CD4+ T cells and CD8+ T cells. VPAC2 is expressed at very low levels in resting T cells but is not detectable in resting monocytes. In vitro stimulation of Th cells with soluble anti-CD3 plus PMA induced a T cell activation-dependent down-regulation of VPAC1. VPAC1 is down-regulated under conditions of optimal T cell stimulation. Our results suggest that selective VIP effects on T cell function may be mediated via selective expression of VPAC1 and VPAC2 on T cells and monocytes. Furthermore, down-regulation of VPAC1 in CD4+ T cell subpopulations is highly correlated with T cell activation.
Low Dose Streptozotocin-Induced Diabetes in Rat Insulin Promoter-mCD80-Transgenic Mice Is T Cell Autoantigen-Specific and CD28 Dependent
Authors: Pechhold Klaus , Patterson Noelle B. , Blum Carmen , Fleischacker Christine L. , Boehm Bernhard O. , Harlan David M.
Published: April 2014

Although transgenic mice expressing murine B7-1 (mCD80) on their pancreatic β cells under the rat insulin-1 promoter (RIP-mCD80+ mice) rarely develop spontaneous β cell destruction and diabetes, we have previously reported the transgene-dependent induction of profound insulitis and lethal diabetes following multiple low dose injections of the β cell toxin streptozotocin (MLDS) in RIP-mCD80+ mice. Here, we have further characterized this MLDS-induced diabetes model using the RIP-mCD80+ mice and now demonstrate that disease is critically dependent on T cell signaling via CD28. Thus, although naive RIP-mCD80+ and nontransgenic littermates have comparable gross β cell mass, and immediately following MLDS induction the mice display similar degrees of insulitis and decrements in the β cell mass, only transgenic mice continued to destroy their β cells and develop insulin-dependent diabetes mellitus. Strikingly, MLDS-induced diabetes was completely prevented in CD28-deficient mice (RIP-mCD80+CD28−/−) due to abrogation of leukocytes infiltrating their pancreatic islets. We further characterized MLDS-induced diabetes in the RIP-mCD80+ mice by demonstrating that the MLDS-induced lymphocytic islet infiltrate contained a substantial frequency of autoantigen-specific, IFN-γ-secreting, CD8+ T cells. We conclude that MLDS-induced β cell destruction and subsequent insulin-dependent diabetes mellitus in RIP-mCD80+ mice is T cell-mediated as it involves both Ag-specific recognition of self-target molecules in the inflamed pancreatic islet (signal 1) and is CD28 costimulation dependent (signal 2).
Deletion of the DQ52 Element Within the Ig Heavy Chain Locus Leads to a Selective Reduction in VDJ Recombination and Altered D Gene Usage
Authors: Nitschke Lars , Kestler Jürgen , Tallone Tiziano , Pelkonen Sinikka , Pelkonen Jukka
Published: April 2014

The process of V(D)J recombination that leads to the assembly of Ig gene segments is tightly controlled during B cell differentiation. Two germline transcripts, one of which (μ0) originates from the promoter region of DQ52, may control the accessibility of the heavy chain locus. Here, we present the analysis of a mouse line in which the DQ52 gene together with its regulatory sequences is deleted by a Cre/loxP-based strategy. In F1 (DQ52+/−) mice, the use of the JH3 and JH4 elements in DJ or VDJ junctions of the DQ52− allele was strongly reduced in both the bone marrow pre-B and spleen cells, while the JH1 and JH2 elements were used with normal frequencies. In addition, IgM+ B cells of bone marrow and spleen used the DQ52− allele less frequently. On DJ joints of the DQ52− allele, there was 2 times less processing of JH3 ends, which resulted in clearly increased addition of P nucleotides. Although the use of D elements in DJ joints was quite similar, an altered D repertoire was found in VDJ joints of the DQ52− allele. In splenic B cells of the DQ52−/− mouse the amino acid distribution of the CDR3 was skewed, probably to compensate for the altered processing of JH3 ends. Thus, we have shown an interesting selective effect of the DQ52 region on controlling accessibility to 3′ JH elements on the Ig locus, which also seems to influence the processing of DJ joints. We propose a model in which the DQ52 promoter region enhances the induction of secondary DJ rearrangements.
Human T Cell Lymphotropic Virus Type I Tax Activates IL-15Rα Gene Expression Through an NF-κB Site
Authors: Mariner Jennifer M. , Lantz Valerie , Waldmann Thomas A. , Azimi Nazli
Published: April 2014

IL-15 mRNA levels are increased in diseases caused by human T cell lymphotropic virus type I (HTLV-I). In this study, we demonstrated that IL-15Rα, the IL-15-specific binding receptor, mRNA and protein levels were also elevated in HTLV-I-infected cells. We showed that transient HTLV-I Tax expression lead to increased IL-15Rα mRNA levels. In addition, by using a reporter construct that bears the human IL-15Rα promoter, we demonstrated that Tax expression increased promoter activity by at least 4-fold. Furthermore, using promoter deletion constructs and gel shift analysis, we defined a functional NF-κB-binding motif in the human IL-15Rα promoter, suggesting that Tax activation of IL-15Rα is due, in part, to the induction of NF-κB. These data indicate that IL-15Rα is transcriptionally regulated by the HTLV-I Tax protein through the action of NF-κB. These findings suggest a role for IL-15Rα in aberrant T cell proliferation observed in HTLV-I-associated diseases.
Fes Mediates the IL-4 Activation of Insulin Receptor Substrate-2 and Cellular Proliferation
Authors: Jiang Hong , Foltenyi Kristizna , Kashiwada Masaki , Donahue Liz , Vuong Bao , Hehn Boyd , Rothman Paul
Published: April 2014

Although Jak kinases are essential for initiating cytokine signaling, the role of other nonreceptor tyrosine kinases in this process remains unclear. We have examined the role of Fes in IL-4 signaling. Examination of Jak1-deficient cell lines demonstrates that Jak1 is required for the activation of Fes by IL-4. Experiments studying signaling molecules activated by IL-4 receptor suggest that IL-4 signaling can be subdivided into Fes-dependent and Fes-independent pathways. Overexpression of kinase-inactive Fes blocks the IL-4 activation of insulin receptor substrate-2, but not STAT6. Fes appears to be a downstream kinase from Jak1/Jak3 in this process. Further examination of downstream signaling demonstrates that kinase-inactive Fes inhibits the recruitment of phosphoinositide 3-kinase to the activated IL-4 receptor complex and decreases the activation of p70S6k kinase in response to IL-4. This inhibition correlates with a decrease in IL-4-induced proliferation. In contrast, mutant Fes does not inhibit the activation of Akt by IL-4. These data demonstrate that signaling pathways activated by IL-4 require different tyrosine kinases. This differential requirement predicts that specific kinase inhibitors may permit the disruption of specific IL-4-induced functions.
The WI-1 Adhesin Blocks Phagocyte TNF-α Production, Imparting Pathogenicity on Blastomyces dermatitidis
Authors: Finkel-Jimenez Bea , Wüthrich Marcel , Brandhorst Tristan , Klein Bruce S.
Published: April 2014

The WI-1 adhesin is indispensable for pathogenicity of Blastomyces dermatitidis and is thought to promote pulmonary infection by fixing yeast to lung tissue and cells. Recent findings suggest that WI-1 confers pathogenicity by mechanisms in addition to adherence. Here, we investigated whether WI-1 modulates host immunity by altering production of pro-inflammatory cytokines. Production of TNF-α in lung alveolar fluids of mice infected with B. dermatitidis was severalfold higher for WI-1 knockout yeast compared with wild-type yeast, and in vitro coculture of unseparated lung cells with these isogenic yeast disclosed similar differences. Upon coculture with purified macrophages and neutrophils, wild-type yeast blocked TNF-α production, yet WI-1 knockout yeast stimulated production. Coating knockout yeast with purified WI-1 converted them from stimulating TNF-α production to inhibiting production. Addition of purified WI-1 into stimulated phagocyte cultures led to concentration-dependent inhibition of TNF-α production. Neutralization of TNF-α in vivo exacerbated experimental pulmonary infection, particularly for the nonpathogenic WI-1 knockout yeast. Inducing increased TNF-α levels in the lung by adenovirus-vectored gene therapy controlled infection with wild-type yeast. Thus, the WI-1 adhesin on yeast modulates host immunity through blocking TNF-α production by phagocytes, which fosters progression of pulmonary infection.
CXC Chemokine Receptor 4 Expression and Function in Human Astroglioma Cells
Authors: Oh Jae-Wook , Drabik Kathryn , Kutsch Olaf , Choi Chulhee , Tousson Albert , Benveniste Etty N.
Published: April 2014

Chemokines constitute a superfamily of proteins that function as chemoattractants and activators of leukocytes. Astrocytes, the major glial cell type in the CNS, are a source of chemokines within the diseased brain. Specifically, we have shown that primary human astrocytes and human astroglioma cell lines produce the CXC chemokines IFN-γ-inducible protein-10 and IL-8 and the CC chemokines monocyte chemoattractant protein-1 and RANTES in response to stimuli such as TNF-α, IL-1β, and IFN-γ. In this study, we investigated chemokine receptor expression and function on human astroglioma cells. Enhancement of CXC chemokine receptor 4 (CXCR4) mRNA expression was observed upon treatment with the cytokines TNF-α and IL-1β. The peak of CXCR4 expression in response to TNF-α and IL-1β was 8 and 4 h, respectively. CXCR4 protein expression was also enhanced upon treatment with TNF-α and IL-1β (2- to 3-fold). To study the functional relevance of CXCR4 expression, stable astroglioma transfectants expressing high levels of CXCR4 were generated. Stimulation of cells with the ligand for CXCR4, stromal cell-derived factor-1α (SDF-1α), resulted in an elevation in intracellular Ca2+ concentration and activation of the mitogen-activated protein kinase cascade, specifically, extracellular signal-regulated kinase 2 (ERK2) mitogen-activated protein kinase. Of most interest, SDF-1α treatment induced expression of the chemokines monocyte chemoattractant protein-1, IL-8, and IFN-γ-inducible protein-10. SDF-1α-induced chemokine expression was abrogated upon inclusion of U0126, a pharmacological inhibitor of ERK1/2, indicating that the ERK signaling cascade is involved in this response. Collectively, these data suggest that CXCR4-mediated signaling pathways in astroglioma cells may be another mechanism for these cells to express chemokines involved in angiogenesis and inflammation.
Brain Dendritic Cells and Macrophages/Microglia in Central Nervous System Inflammation
Authors: Fischer Hans-Georg , Reichmann Gaby
Published: April 2014

Microglia subpopulations were studied in mouse experimental autoimmune encephalomyelitis and toxoplasmic encephalitis. CNS inflammation was associated with the proliferation of CD11b+ brain cells that exhibited the dendritic cell (DC) marker CD11c. These cells constituted up to 30% of the total CD11b+ brain cell population. In both diseases CD11c+ brain cells displayed the surface phenotype of myeloid DC and resided at perivascular and intraparenchymatic inflammatory sites. By lacking prominent phagocytic organelles, CD11c+ cells from inflamed brain proved distinct from other microglia, but strikingly resembled bone marrow-derived DC and thus were identified as DC. This brain DC population comprised cells strongly secreting IL-12p70, whereas coisolated CD11c− microglia/brain macrophages predominantly produced TNF-α, GM-CSF, and NO. In comparison, the DC were more potent stimulators of naive or allogeneic T cell proliferation. Both DC and CD11c− microglia/macrophages from inflamed brain primed naive T cells from DO11.10 TCR transgenic mice for production of Th1 cytokines IFN-γ and IL-2. Resting microglia that had been purified from normal adult brain generated immature DC upon exposure to GM-CSF, while CD40 ligation triggered terminal maturation. Consistently, a functional maturation of brain DC was observed to occur following the onset of encephalitis. In conclusion, these findings indicate that in addition to inflammatory macrophage-like brain cells, intraparenchymatical DC exist in autoimmune and infectious encephalitis. These DC functionally mature upon disease onset and can differentiate from resident microglia. Their emergence, maturation, and prolonged activity within the brain might contribute to the chronicity of intracerebral Th1 responses.
Selective Inhibition of Inducible Nitric Oxide Synthase Exacerbates Erosive Joint Disease
Authors: McCartney-Francis Nancy L. , Song Xiao-yu , Mizel Diane E. , Wahl Sharon M.
Published: April 2014

NO is an essential cytotoxic agent in host defense, yet can be autotoxic if overproduced, as evidenced in inflammatory lesions and tissue destruction in experimental arthritis models. Treatment of streptococcal cell wal1-induced arthritis in rats with NG-monomethyl-l-arginine (l-NMMA), a competitive nonspecific inhibitor of both constitutive and inducible isoforms of NO synthase (NOS), prevents intraarticular accumulation of leukocytes, joint swelling, and bone erosion. Because increased inducible NOS (iNOS) expression and NO generation are associated with pathogenesis of chronic inflammation, we investigated whether a selective inhibitor of iNOS, N-iminoethyl-l-lysine (l-NIL), would have more directed anti-arthritic properties. Whereas both l-NMMA and l-NIL inhibited nitrite production by streptococcal cell wall-stimulated rat mononuclear cells in vitro and systemic treatment of arthritic rats with l-NMMA ablated synovitis, surprisingly l-NIL did not mediate resolution of inflammatory joint lesions. On the contrary, daily administration of l-NIL failed to reduce the acute response and exacerbated the chronic inflammatory response, as reflected by profound tissue destruction and loss of bone and cartilage. Although the number of iNOS-positive cells within the synovium decreased after treatment with l-NIL, immunohistochemical analyses revealed a distinct pattern of endothelial and neuronal NOS expression in the arthritic synovium that was unaffected by the isoform-specific l-NIL treatment. These studies uncover a contribution of the constitutive isoforms of NOS to the evolution of acute and chronic inflammation pathology which may be important in the design of therapeutic agents.
IL-12-Dependent Vascular Cell Adhesion Molecule-1 Expression Contributes to Airway Eosinophilic Inflammation in a Mouse Model of Asthma-Like Reaction
Authors: Wang Shuhe , Fan Yijun , Han Xiaobing , Yang Jie , Bilenki Laura , Yang Xi
Published: April 2014

Bronchial-alveolar eosinophilic inflammation is among the characteristic pathological changes in asthma, which has been shown to be correlated with type 2 cytokine and chemokine production. Exogenous IL-12 has been found to be inhibitory for pulmonary eosinophilia in reported studies. Using a murine asthma-like model induced by OVA, we found in the present study that IL-12 gene knockout (KO) mice showed substantially reduced airway recruitment of eosinophils compared with wild-type control mice following OVA sensitization/challenge, although the levels of circulating eosinophils were comparable in these two groups of mice. Cytokine analysis showed Ag-driven Th1 (IFN-γ) and Th2 (IL-4, IL-5, IL-10, and IL-13) cytokine production by CD4 T cells from local draining lymph nodes and spleen. Similarly, local eotaxin production was comparable in wild-type and IL-12 KO mice. In contrast, immunohistochemical analysis showed that the expression of VCAM-1 on the lung endothelium of IL-12 KO mice was dramatically less than that in wild-type mice. Furthermore, administration of rIL-12 at the stage of sensitization and challenge with OVA restored airway eosinophilia and VCAM-1 expression in IL-12 KO mice. The results suggest that endogenous IL-12 contributes to the recruitment of eosinophils into airways observed in asthma, possibly via enhancement of the expression of VCAM-1 on local vascular endothelial cells.
IL-4 Promotes Airway Eosinophilia by Suppressing IFN-γ Production: Defining a Novel Role for IFN-γ in the Regulation of Allergic Airway Inflammation
Authors: Cohn Lauren , Herrick Christina , Niu Naiqian , Homer Robert J. , Bottomly Kim
Published: April 2014

Airway eosinophilia in asthma is dependent on cytokines secreted by Th2 cells, including IL-5 and IL-4. In these studies we investigated why the absence of IL-4 led to a reduction in airway, but not lung tissue, eosinophils. Using adoptively transferred, in vitro-generated TCR-transgenic Th2 cells deficient in IL-4, we show that this effect is independent of IL-5 and Th2 cell generation. Airway eosinophilia was no longer inhibited when IL-4−/− Th2 cells were transferred into IFN-γR−/− mice, indicating that IFN-γ was responsible for reducing airway eosinophils in the absence of IL-4. Intranasal administration of IFN-γ to mice after IL-4+/+ Th2 cell transfer also caused a reduction in airway, but not lung parenchymal, eosinophils. These studies show that IL-4 indirectly promotes airway eosinophilia by suppressing the production of IFN-γ. IFN-γ reduces airway eosinophils by engaging its receptor on hemopoietic cells, possibly the eosinophil itself. These studies capitalize on the complex counterregulatory effects of Th1 and Th2 cytokines in vivo and clarify how IL-4 influences lung eosinophilia. We define a new regulatory role for IFN-γ, demonstrating that eosinophilic inflammation is differentially regulated at distinct sites within the respiratory tract.
IL-4 and IL-13, But Not IL-10, Protect Human Synoviocytes from Apoptosis
Authors: Relić Biserka , Guicheux Jérome , Mezin Françoise , Lubberts Erik , Togninalli Danilo , Garcia Irène , van den Berg Wim B. , Guerne Pierre-André
Published: April 2014

Interleukin-4, which has been contemplated for the treatment of rheumatoid arthritis and/or osteoarthritis because of its anticatabolic properties, has also been shown to modulate apoptosis. Because inadequate apoptosis is thought to contribute to synovial hyperplasia, we have investigated the ability of IL-4 and other Th2 cytokines to protect human synovial cells from apoptosis. Human synoviocytes or synovial explants were pretreated with IL-4, IL-10, and IL-13 before exposure to NO donor sodium-nitro-prusside (SNP). Apoptosis was evaluated by microscopy, annexin V-FITC, 3-(4,5-dimethylthiazol-2-gl)-5-(3-carboxymethoxylphenyl)-2-(4-sulphophenyl-2H tetrazolium inner salt (MTS) test, pulse field gel electrophoresis, and a method proposed in this study based on 32P Klenow end labeling of high m.w. DNA. Pretreatment by IL-4 or IL-13, but not IL-10, protected human synoviocytes from apoptosis induced by SNP. Even at doses as high as 2 mM SNP, up to 86% and 56% protection was achieved, after IL-4 and IL-13 treatment, respectively. Cell survival was dependent on IL concentration. IL-4 and IL-13 also had antiapoptotic effects on SNP-treated human synovial explants. Effects of IL-4 and IL-13 varied in the presence of phosphatidylinositol-3 kinase and protein kinase C inhibitors, implying the involvement of these pathways in antiapoptotic signaling. Antiapoptotic effects were dramatically inhibited by LY294002, and partially by the protein kinase C inhibitor Gö 6976, while insulin-like growth factor increased synoviocyte survival. The possibility that IL-4 and IL-13 may enhance synovial expansion in vivo by their antiapoptotic effects is discussed.
Type I Diabetes and Multiple Sclerosis Patients Target Islet Plus Central Nervous System Autoantigens; Nonimmunized Nonobese Diabetic Mice Can Develop Autoimmune Encephalitis
Authors: Winer Shawn , Astsaturov Igor , Cheung Roy K. , Gunaratnam Lakshman , Kubiak Violetta , Cortez Miguel A. , Moscarello Mario , O’Connor Paul W. , McKerlie Colin , Becker Dorothy J. , Dosch H.-Michael
Published: April 2014

Type I diabetes and multiple sclerosis (MS) are distinct autoimmune diseases where T cells target either islet or CNS self-proteins. Unexpectedly, we found that autoreactive T cells in diabetic patients, relatives with high diabetes risk, nonobese diabetic (NOD) mice, and MS patients routinely target classical islet as well as CNS autoantigens. The pathogenic potential of CNS autoreactivity was testable in NOD mice. Pertussis holotoxin, without additional Ags or adjuvants, allowed development of an NOD mouse-specific, autoimmune encephalitis with variable primary-progressive, monophasic, and relapsing-remitting courses. T cells from diabetic donors transferred CNS disease to pertussis toxin-pretreated NOD.scid mice, with accumulation of CD3/IFN-γ transcripts in the brain. Diabetes and MS appear more closely related than previously perceived. NOD mouse-specific, autoimmune encephalitis provides a new MS model to identify factors that determine alternative disease outcomes in hosts with similar autoreactive T cell repertoires.
Expression of Chemokine Receptors by Lung T Cells from Normal and Asthmatic Subjects
Authors: Campbell James J. , Brightling Christopher E. , Symon Fiona A. , Qin Shi , Murphy Kristine E. , Hodge Mmarty , Andrew David P. , Wu Lijun , Butcher Eugene C. , Wardlaw Andrew J.
Published: April 2014

The lung is an important tertiary lymphoid organ with constant trafficking of T cells through the lung in both health and disease. T cell migration is controlled by a combination of adhesion receptors and chemokines expressed on vascular endothelium and in the tissue, often in an organ-specific manner. This leads to selective accumulation of different T cell subsets, a process called lymphocyte homing. There is evidence for a distinct lung-homing pathway, but no specific lung-homing receptors have been described. We analyzed the chemokine receptor profile of lung T cells to determine the extent to which lung T cells shared homing pathways with other organs such as the gut and skin. In addition, we compared expression of receptors in normal and asthmatic individuals to determine whether different pathways were used in health and disease. We observed that lung T cells expressed a profile of chemokine and adhesion receptors distinct from that of gut- and skin-homing T cells although no chemokine receptor specific for the lung was found. In particular, lung T cells expressed CCR5 and CXCR3, but not CCR9 or cutaneous lymphocyte Ag, and only low levels of CCR4 and α4β7. No differences were observed between lung T cells from normal vs asthmatic subjects. This study provides added support for the concept of a lung-homing pathway separate from other mucosal organs such as the gut and suggests that the chemokine pathways that control T cell migration in normal homeostasis and Th2-type inflammatory responses are similar.
Anti-Tumor Immunity Provided by a Synthetic Multiple Antigenic Glycopeptide Displaying a Tri-Tn Glycotope
Authors: Lo-Man Richard , Vichier-Guerre Sophie , Bay Sylvie , Dériaud Edith , Cantacuzène Danièle , Leclerc Claude
Published: April 2014

In many cancer cells the alteration of glycosylation processes leads to the expression of cryptic carbohydrate moieties, which make them good targets for immune intervention. Identification of cancer-associated glycotopes as well as progress in chemical synthesis have opened up the way for the development of fully synthetic immunogens that can induce anti-saccharide immune responses. Here, we synthesized a dendrimeric multiple antigenic glycopeptide (MAG) containing the Tn Ag O-linked to a CD4+ T cell epitope. This MAG is based on three consecutive Tn moieties (tri-Tn) corresponding to the glycotope recognized by an mAb (MLS 128) produced against the LS180 colon carcinoma cell line. The Abs induced by this MAG recognized murine and human tumor cell lines expressing the Tn Ag. Prophylactic vaccination using MAG provided protection of mice against tumor challenge. When used in active specific immunotherapy, the MAG carrying the tri-Tn glycotope was much more efficient than the mono-Tn analogue in promoting the survival of tumor-bearing mice. Furthermore, in active specific immunotherapy, a linear glycopeptide carrying two copies of the tri-Tn glycotope was shown to be poorly efficient compared with the dendrimeric MAG. Therefore, both the clustering of carbohydrate Ags and the way they are displayed seem to be important parameters for stimulating efficient anti-saccharide immune responses.
Expression of a Tolerizing Tumor Antigen in Peripheral Tissue Does Not Preclude Recovery of High-Affinity CD8+ T Cells or CTL Immunotherapy of Tumors Expressing the Antigen
Authors: Öhlén Claes , Kalos Michael , Hong Doley J. , Shur Aaron C. , Greenberg Philip D.
Published: April 2014

Transgenic (TG) mice were generated selectively expressing the gag protein of Friend murine leukemia virus (FMuLV) in the liver. FMuLVgag is also expressed by the FBL leukemia, and is the immunodominant tumor Ag of the CD8+ T cell response in C57BL/6 mice. gag-TG mice expressing FMuLVgag in the liver were tolerant to the protein and failed to generate a CTL response to either FBL or FMuLVgag. This tolerance reflected anergy rather than deletion, as CTL responsiveness could be recovered after four cycles of in vitro stimulation. Adoptively transferred gag-specific T cells were not anergized in gag-TG recipients, as revealed by antitumor activity in vivo. Also, such T cells did not induce detectable autoimmune injury in gag-TG liver cells. These results suggest that the requirements for a tissue Ag to provide a tolerizing stimulus are distinct from those for being the target of a T cell-mediated autoimmune response and that the requirements for induction and maintenance of peripheral tolerance are distinct for naive and primed T cells. That anergic T cells reactive with tumor-associated Ags can be recovered by repetitive in vitro stimulation and can mediate tumor therapy suggests strategies that use such Ags to generate CTL for adoptive immunotherapy should be further developed.
Isolation of a New Melanoma Antigen, MART-2, Containing a Mutated Epitope Recognized by Autologous Tumor-Infiltrating T Lymphocytes
Authors: Kawakami Yutaka , Wang Xiang , Shofuda Tomoko , Sumimoto Hidetoshi , Tupesis Janis P. , Fitzgerald Ellen , Rosenberg Steven A.
Published: April 2014

Using cDNA expression cloning, a cDNA encoding a novel human melanoma Ag, MART-2 (melanoma Ag recognized by T cells-2), recognized by HLA-A1-restricted CD8+ T cells from tumor-infiltrating lymphocytes (TIL1362) was isolated from an autologous melanoma cell line, 1362 mel. Homologous sequences to the cDNA had been registered in the EST database. This gene encoded an uncharacterized protein expressed ubiquitously in most normal and cancer cells. A mutation (A to G transition) was found in the cDNA obtained from the1362 mel melanoma cell line in the sequences encoding the phosphate binding loop (P-loop) that resulted in loss of the ability to bind GTP. Transfection of NIH-3T3 with the mutated MART-2 did not result in the development of significant foci. By screening 36 various cancer cell lines using single-strand conformation polymorphism, a possible mutation in the P-loop of MART-2 was found in one squamous cell lung cancer cell line, EBC1. The T cell epitope for TIL1362, FLEGNEVGKTY, was identified to be encoded by the mutated sequence of the MART-2 Ag. The mutation substituted glycine in the normal peptide with glutamic acid at the third amino acid of the epitope, which is an important primary anchor amino acid for HLA-A1 peptide binding. The normal peptide, FLGGNEVGKTY, was not recognized by TIL1362, suggesting that this T cell response was specific for the autologous tumor. Although transforming activity was not detected in the NIH-3T3 assay, MART-2 with the mutation in the P-loop may be involved in the generation of melanoma through a loss of GTP binding activity.
Psoriatic Arthritis Joint Fluids Are Characterized by CD8 and CD4 T Cell Clonal Expansions that Appear Antigen Driven
Authors: Costello Patrick J. , Winchester Robert J. , Curran Shane A. , Peterson Karin S. , Kane David J. , Bresnihan Barry , FitzGerald Oliver M.
Published: April 2014

The CD8 αβT cell receptor repertoire in joint fluid of individuals with active psoriatic arthritis contained an average of 32 major oligoclonal expansions in many variable genes of the TCR β chain (BV) families, as shown by β-chain CDR3 length analysis. Interestingly, a small number of oligoclonal expansions were shared between simultaneous samples of joint fluid and blood; however, most expansions found in joint fluid were not identifiable in blood emphasizing the immunologic specificity of the clonal events for the inflamed joint at a given point of time. The CD4 T cell joint fluid repertoire contained fewer and smaller oligoclonal expansions also largely restricted to the joint, suggesting that CD4 T cells participate perhaps by interacting cognitively to generate the CD8 clones. The inferred amino acid sequence of a single CD8 oligoclonal expansion revealed that they usually are composed of one or a few structurally related clones at the amino acid sequence level with β-chains that encode identical or highly homologous CDR3 motifs. These were not shared among patients. Moreover, several clones that encoded the same amino acid sequence were found to be structurally distinct at the nucleotide level, strongly implying clonal selection and expansion is operating at the level of specific TCR-peptide interactions. The findings support a model of psoriatic arthritis inflammation involving extensive and selective Ag, likely autoantigen, driven intra-articular CD4, and CD8 T cell clonal expansions.
Cutting Edge: Reversal of Murine Lupus Nephritis with CTLA4Ig and Cyclophosphamide
Authors: Daikh David I. , Wofsy David
Published: April 2014

Cyclophosphamide (CTX) prevents progression of nephritis and prolongs survival in (NZB × NZW)F1 (B/W) mice and is used to treat humans with lupus nephritis. To compare the efficacy of CTLA4Ig with CTX and determine whether there is an incremental benefit to combining CTLA4Ig with CTX, we treated B/W mice with CTX, CTLA4Ig, or both agents. In mice with mild renal disease, treatment delayed the onset of proteinuria and prolonged survival in all groups. In mice with advanced renal disease, treatment with both agents reduced proteinuria in 71% of mice, whereas mice treated with either agent alone had no such improvement. Survival was also markedly improved among mice treated with both agents. Thus, combination treatment with CTX and CTLA4Ig is more effective than either agent alone in reducing renal disease and prolonging survival of B/W mice with advanced nephritis. This striking reversal of proteinuria is unprecedented in animal models of SLE.
Gastrointestinal Cells of IL-7 Receptor Null Mice Exhibit Increased Sensitivity to Irradiation
Authors: Welniak Lisbeth A. , Khaled Annette R. , Anver Miriam R. , Komschlies Kristin L. , Wiltrout Robert H. , Durum Scott , Ruscetti Francis R. , Blazar Bruce R. , Murphy William J.
Published: April 2014

IL-7 is a critical cytokine in the development of T and B cells but little is known about its activity on nonhematopoietic cells. An unexpected finding was noted in allogeneic bone marrow transplant studies using IL-7 receptor null (IL-7Rα−/−) mice as recipients. These mice exhibited a significantly greater weight loss after total body irradiation compared with wild type, IL-7Rα+/+, mice. Pathological assessment indicated greater intestinal crypt damage in IL-7Rα−/− recipients, suggesting these mice may be predisposed to gut destruction. Therefore, we determined the effect of the conditioning itself on the intestinal tract of these mice. IL-7Rα−/− mice and IL-7Rα+/+ mice were irradiated and examined for lesions and apoptosis within the small intestine. In moribund animals, IL-7Rα−/− mice had extensive damage in the small intestine, including marked ablation of the crypts and extreme shortening of villi following 1500 cGy total body irradiation. In contrast, by 8 days after irradiation, the small intestines of IL-7Rα+/+ mice had regenerated as distinguished by normal villus length and hyperplastic crypts. Following 750 cGy irradiation, IL-7Rα−/− mice had a higher proportion of apoptotic cells in the crypts and an accompanying increase in the pro-apoptotic protein Bak was expressed in intestinal epithelial cells. These results demonstrate the increased radiosensitivity of intestinal stem cells within the crypts in IL-7Rα−/− mice and a role for IL-7 in the protection of radiation-induced apoptosis in these same cells. This study describes a novel role of IL-7 in nonhematopoietic tissues.
The Balance Between CD45RChigh and CD45RClow CD4 T Cells in Rats Is Intrinsic to Bone Marrow-Derived Cells and Is Genetically Controlled
Authors: Subra Jean-Francois , Cautain Bastien , Xystrakis Emmanuel , Mas Magali , Lagrange Dominique , van der Heijden Harry , van de Gaar Marie-Jose , Druet Philippe , Fournié Gilbert J. , Saoudi Abdelhadi , Damoiseaux Jan
Published: April 2014

The level of CD45RC expression differentiates rat CD4 T cells in two subpopulations, CD45RChigh and CD45RClow, that have different cytokine profiles and functions. Interestingly, Lewis (LEW) and Brown Norway (BN) rats, two strains that differ in their ability to mount type 1 and type 2 immune responses and in their susceptibility to autoimmune diseases, exhibit distinct CD45RChigh/CD45RClow CD4 T cell ratios. The CD45RChigh subpopulation predominates in LEW rats, and the CD45RClow subpopulation in BN rats. In this study, we found that the antiinflammatory cytokines, IL-4, IL-10, and IL-13, are exclusively produced by the CD45RClow CD4 T cells. Using bone marrow chimeras, we showed that the difference in the CD45RChigh/CD45RClow CD4 T cell ratio between naive LEW and BN rats is intrinsic to hemopoietic cells. Furthermore, a genome-wide search for loci controlling the balance between T cell subpopulations was conducted in a (LEW × BN) F2 intercross. Genome scanning identified one quantitative trait locus on chromosome 9 (∼17 centiMorgan (cM); log of the odds ratio (LOD) score 3.9). In addition, two regions on chromosomes 10 (∼28 cM; LOD score 3.1) and 20 (∼40 cM; LOD ratio score 3) that contain, respectively, a cytokine gene cluster and the MHC region were suggestive for linkage. Interestingly, overlapping regions on these chromosomes have been implicated in the susceptibility to various immune-mediated disorders. The identification and functional characterization of genes in these regions controlling the CD45RChigh/CD45RClow Th cell subpopulations may shed light on key regulatory mechanisms of pathogenic immune responses.
Differential Regulation of Human Blood Dendritic Cell Subsets by IFNs
Authors: Ito Tomoki , Amakawa Ryuichi , Inaba Muneo , Ikehara Susumu , Inaba Kayo , Fukuhara Shirou
Published: April 2014

Based on the relative expression of CD11c and CD1a, we previously identified subsets of dendritic cells (DCs) or DC precursors in human peripheral blood. A CD1a+/CD11c+ population (CD11c+ DCs), also called myeloid DCs, is an immediate precursor of Langerhans cells, whereas a CD1a−/CD11c− population (CD11c− DCs), sometimes called lymphoid DCs but better known as plasmacytoid DCs, is composed of type I IFN (IFN-αβ)-producing cells. Here, we investigate the effects of IFN-αβ and IFN-γ as well as other cytokines on CD11c+ and CD11c− DC subsets, directly isolated from the peripheral blood, instead of in vitro-generated DCs. IFN-γ and IFN-α, rather than GM-CSF, were the most potent cytokines for enhancing the maturation of CD11c+ DCs. Incubation of CD11c+ DCs with IFN-γ also resulted in increased IL-12 production, and this IL-12 allowed DCs to increase Th1 responses by alloreactive T cells. In contrast, IFN-α did not induce IL-12 but, rather, augmented IL-10 production. IFN-α-primed matured CD11c+ DCs induced IL-10-producing regulatory T cells; however, this process was independent of the DC-derived IL-10. On the other hand, IFN-α by itself neither matured CD11c− DCs nor altered the polarization of responding T cells, although this cytokine was a potent survival factor for CD11c− DCs. Unlike IFN-α, IL-3 was a potent survival factor and induced the maturation of CD11c− DCs. The IL-3-primed CD11c− DCs activated T cells to produce IL-10, IFN-γ, and IL-4. Thus, CD11c+ and CD11c− DC subsets play distinct roles in the cytokine network, especially their responses to IFNs.
CD4 T Cell-Mediated Alloresistance to Fully MHC-Mismatched Allogeneic Bone Marrow Engraftment Is Dependent on CD40-CD40 Ligand Interactions, and Lasting T Cell Tolerance Is Induced by Bone Marrow Transplantation with Initial Blockade of this Pathway
Authors: Ito Hiroshi , Kurtz Josef , Shaffer Juanita , Sykes Megan
Published: April 2014

Costimulatory blockade can be used to promote allogeneic marrow engraftment and tolerance induction, but on its own is not 100% reliable. We sought to determine whether one or the other of the CD4 or CD8 T cell subsets of the recipient was primarily responsible for resistance to allogeneic marrow engraftment in mice receiving costimulatory blockade, and to use this information to develop a more reliable, minimal conditioning regimen for induction of mixed chimerism and transplantation tolerance. We demonstrate that a single anti-CD40 ligand mAb treatment is sufficient to completely overcome CD4 cell-mediated resistance to allogeneic marrow engraftment and rapidly induce CD4 cell tolerance, but does not reliably overcome CD8 CTL-mediated alloresistance. The data suggest that costimulation, which activates alloreactive CTL, is insufficient to activate alloreactive CD4 cells when the CD40 pathway is blocked. The addition of host CD8 T cell depletion to anti-CD40 ligand treatment reliably allows the induction of mixed chimerism and donor-specific skin graft tolerance in 3 Gy-irradiated mice receiving fully MHC-mismatched bone marrow grafts. Thus, despite the existence of multiple costimulatory pathways and pathways of APC activation, our studies demonstrate an absolute dependence on CD40-mediated events for CD4 cell-mediated rejection of allogeneic marrow. Exposure to donor bone marrow allows rapid tolerization of alloreactive CD4 cells when the CD40 pathway is blocked, leading to permanent marrow engraftment and intrathymic tolerization of T cells that develop subsequently.
Regulatory and Effector CD4 T Cells in Nonobese Diabetic Mice Recognize Overlapping Determinants on Glutamic Acid Decarboxylase and Use DistinctVβGenes
Authors: Quinn Anthony , McInerney Brigid , Reich Eva P. , Kim Olivia , Jensen Kent P. , Sercarz Eli E.
Published: April 2014

The 524–543 region of glutamic acid decarboxylase (GAD65), GAD65(524–543), is one of the first fragments of this islet Ag to induce proliferative T cell responses in the nonobese diabetic (NOD) mouse model of spontaneous autoimmune diabetes. Furthermore, NOD mice given tolerogenic doses of GAD65(524–543) are protected from spontaneous and cyclophosphamide-induced diabetes. In this study, we report that there are at least two I-Ag7-restricted determinants present in the GAD65(524–543) sequence, each capable of recruiting unique T cell repertoires characterized by distinct TCR Vβ gene use. CD4+ T cells arise spontaneously in young NOD mice to an apparently dominant determinant found within the GAD65 peptide 530–543 (p530); however, T cells to the overlapping determinant 524–538 (p524) dominate the response only after immunization with GAD65(524–543). All p530-responsive T cells used the Vβ4 gene, whereas the Vβ12 gene is preferentially used to encode the TCR of p524-responsive T cell populations. T cell clones and hybridomas from both of these T cell groups were responsive to APC pulsed with GAD65(524–543) or whole rGAD65. p524-reactive cells appeared to be regulatory upon adoptive transfer into young NOD mice and could inhibit insulin-dependent diabetes mellitus development, although they were unable to produce IL-4, IL-10, or TGFβ upon antigenic challenge. Furthermore, we found that i.p. injection with p524/IFA was very effective in providing protection from cyclophosphamide-induced insulin-dependent diabetes mellitus. These data demonstrate that the regulatory T cells elicited by immunizing with GAD65(524–543) are unique and distinct from those that arise from spontaneous endogenous priming, and that T cells to this limited region of GAD65 may be either regulatory or pathogenic.
Cumulative Inhibition of NK Cells and T Cells Resulting from Engagement of Multiple Inhibitory Ly49 Receptors
Authors: Hanke Thomas , Raulet David H.
Published: April 2014

Inhibitory receptors specific for MHC class I molecules are expressed on partially overlapping subpopulations of NK cells and memory T cells. A central question pertinent to NK cell development and function is how the combinatorial expression of different receptors with distinct class I specificities affects functional recognition. We therefore studied the quantitative effects resulting from class I engagement of multiple inhibitory Ly49 receptors. We used a transgenic mouse model in which all NK cells and T cells express two different Ly49 receptors with shared class I specificity. Comparisons of cells from these mice with cells from single transgenic mice and wild-type mice revealed that Ly49 receptors cumulatively inhibit lymphocyte effector functions. Multiple Ly49 interactions also had a cumulative impact on NK cell development. The findings suggest that the interactions of inhibitory receptors with class I are interpreted quantitatively rather than as on/off switches. They have intriguing implications concerning NK cell tolerance and reactivity toward cells with extinguished expression of a limited number of class I molecules.