bluuebunny/crossref_metadata_2025 huggingface dataset:
Re-evaluating the Generation of a “Proteasome-Independent” MHC Class I-Restricted CD8 T Cell Epitope
Authors: Wherry E. John , Golovina Tatiana N. , Morrison Susan E. , Sinnathamby Gomathinayagam , McElhaugh Michael J. , Shockey David C. , Eisenlohr Laurence C.
Published: April 2014

The proteasome is primarily responsible for the generation of MHC class I-restricted CTL epitopes. However, some epitopes, such as NP147–155 of the influenza nucleoprotein (NP), are presented efficiently in the presence of proteasome inhibitors. The pathways used to generate such apparently “proteasome-independent” epitopes remain poorly defined. We have examined the generation of NP147–155 and a second proteasome-dependent NP epitope, NP50–57, using cells adapted to growth in the presence of proteasome inhibitors and also through protease overexpression. We observed that: 1) Ag processing and presentation proceeds in proteasome-inhibitor adapted cells but may become more dependent, at least in part, on nonproteasomal protease(s), 2) tripeptidyl peptidase II does not substitute for the proteasome in the generation of NP147–155, 3) overexpression of leucine aminopeptidase, thymet oligopeptidase, puromycin-sensitive aminopeptidase, and bleomycin hydrolase, has little impact on the processing and presentation of NP50–57 or NP147–155, and 4) proteasome-inhibitor treatment altered the specificity of substrate cleavage by the proteasome using cell-free digests favoring NP147–155 epitope preservation. Based on these results, we propose a central role for the proteasome in epitope generation even in the presence of proteasome inhibitors, although such inhibitors will likely alter cleavage patterns and may increase the dependence of the processing pathway on postproteasomal enzymes.
Temporal and Dose-Dependent Relationships between In Vivo B Cell Receptor-Targeted Proliferation and Deletion-Induced by a Microbial B Cell Toxin
Authors: Goodyear Carl S. , Sugiyama Fujimi , Silverman Gregg J.
Published: April 2014

The effective functioning of the adaptive immune system requires careful clonal regulation within the B cell compartment. Some microbial pathogens produce virulence factors, like staphylococcal protein A, which interact at high frequencies with B lymphocyte through unconventional binding sites in BCR variable region frameworks conserved during evolution. We have characterized the in vivo effect of staphylococcal protein A treatment on peripheral B cells bearing susceptible BCR, and found a dose-dependent direct relationship over the range of 2 mg to <0.2 μg in the magnitude of induced BCR-targeted supraclonal cell death. Significantly, some level of targeted B cell proliferation was always detectable, with greatest interim supraclonal expansion demonstrated at 2 days after 20-μg treatment. Subsequently, this transient expansion always collapsed. In direct comparisons, i.p. treatment was more efficacious than i.v. treatment, although at higher doses this finding was less marked. These studies elucidate a general paradigm in which in vivo encounters with a B cell superantigen are uniformly associated with proliferative expansion followed by deletion that is more rapid and complete with higher doses, whereas lower doses lead to greater transient in vivo expansion with delayed deletion to levels at later times that are still quantitatively proportional to the dose. Our results document the potent in vivo B cell-targeted properties of a microbial B cell superantigen, even at submicrogram doses associated with great molar excess of circulating Ig, and clearly illustrate the intertwined relationships between targeted proliferative cycling and apoptotic death that is induced by a microbial B cell superantigen.
Antigen Exposure during Enhanced CTLA-4 Expression Promotes Allograft Tolerance In Vivo
Authors: Salvalaggio Paolo R. O. , Camirand Geoffrey , Ariyan Charlotte E. , Deng Songyan , Rogozinski Linda , Basadonna Giacomo P. , Rothstein David M.
Published: April 2014

The role of CTLA-4 in tolerance is primarily inferred from knockout and blocking studies. Anti-CD45RB mediates allograft tolerance in mice by inducing CTLA-4 expression on CD4 cells, providing a novel opportunity to determine how therapeutic enhancement of CTLA-4 promotes tolerance. We now show that induced CTLA-4 expression normally resolves by day 17. Although thymectomy prolongs enhanced CTLA-4 expression, long-term engraftment is unaffected. To address the temporal relationship between increased CTLA-4 expression and engraftment, transplantation was delayed for various times after anti-CD45RB treatment. Delaying transplantation for 7 days (when CTLA-4 expression had peaked but treatment mAb was no longer detectable), resulted in long-term engraftment comparable to transplantation with no delay (day 0). Delaying transplantation from 10 to 18 days led to a progressively poorer outcome as CTLA-4 expression returned to baseline. This suggested that Ag exposure while CTLA-4 expression is enhanced is sufficient to induce long-term engraftment. To substantiate this, on day 0, anti-CD45RB-treated mice received BALB/c vs unrelated alloantigen, followed by transplantation of BALB/c islets 10 days later. Whereas recipients exposed to unrelated Ag experienced acute rejection, recipients exposed to donor Ag achieved long-term engraftment. Anti-CD45RB-treated mice exposed to alloantigen exhibited anergic CD4+CD25− effector cells and regulatory CD4+CD25+ cells. Moreover, CD25 depletion in the peritransplant period prevented anti-CD45RB-mediated engraftment. Thus, exposure of CD4 cells expressing CTLA-4 to donor Ag is necessary and sufficient to induce long-term engraftment which appears to be mediated by both regulation and anergy.
CD4-Dependent Potentiation of a High Molecular Weight-Melanoma-Associated Antigen-Specific CTL Response Elicited in HLA-A2/Kb Transgenic Mice
Authors: Peng Liaomin , Ko Eric , Luo Wei , Wang Xinhui , Shrikant Protul A. , Ferrone Soldano
Published: April 2014

The human high m.w.-melanoma-associated Ag (HMW-MAA) is an attractive target for the immunotherapy of melanoma, due to its relatively high expression in a high percentage of melanoma lesions and its restricted distribution in normal tissues. Active immunization with HMW-MAA mimics has been previously shown to induce a HMW-MAA-specific, T cell-dependent Ab response associated with an apparent clinically beneficial effect in advanced melanoma patients. Although T cells play an important role in controlling tumor growth, only limited information is available to date about the induction of HMW-MAA-specific CTL. In this report, we show that immunization of HLA-A2/Kb transgenic mice with HMW-MAA cDNA-transfected syngeneic dendritic cells elicited a CD8+ CTL response specific for HMW-MAA peptides with HLA-A2 Ag-binding motifs. The elicited CTL lysed HLA-A2+HMW-MAA+ melanoma cells in vitro, and mouse HLA-A2/Kb cells pulsed with HMW-MAA-derived peptides in vitro and in vivo. Although this CTL response could be generated in the absence of CD4+ T cell help, harnessing CD4+ T cell help in a noncognate Ag-specific manner with the polyclonal activator staphylococcal enterotoxin A augmented the CTL response. These results imply that dendritic cell-based immunization, in combination with CD4+ T cell help, represents an effective strategy to implement T cell-based immunotherapy targeting HMW-MAA in patients with HMW-MAA-bearing tumors.
Effector and Memory CD8+ T Cells Can Be Generated in Response to Alloantigen Independently of CD4+ T Cell Help
Authors: Jones Nick D. , Carvalho-Gaspar Manuela , Luo Shiqiao , Brook Matthew O. , Martin Laurent , Wood Kathryn J.
Published: April 2014

There is now considerable evidence suggesting that CD8+ T cells are able to generate effector but not functional memory T cells following pathogenic infections in the absence of CD4+ T cells. We show that following transplantation of allogeneic skin, in the absence of CD4+ T cells, CD8+ T cells become activated, proliferate, and expand exclusively in the draining lymph nodes and are able to infiltrate and reject skin allografts. CD44+CD8+ T cells isolated 100 days after transplantation rapidly produce IFN-γ following restimulation with alloantigen in vitro. In vivo CD44+CD8+ T cells rejected donor-type skin allografts more rapidly than naive CD8+ T cells demonstrating the ability of these putative memory T cells to mount an effective recall response in vivo. These data form the first direct demonstration that CD8+ T cells are able to generate memory as well as effector cells in response to alloantigen during rejection in the complete absence of CD4+ T cells. These data have important implications for the design of therapies to combat rejection and serve to reinforce the view that CD8+ T cell responses to allografts require manipulation in addition to CD4+ T cell responses to completely prevent the rejection of foreign organ transplants.
Chemokine Receptor CXCR3 Desensitization by IL-16/CD4 Signaling Is Dependent on CCR5 and Intact Membrane Cholesterol
Authors: Rahangdale Shilpa , Morgan Roger , Heijens Claudia , Ryan Thomas C. , Yamasaki Hisato , Bentley Elizabeth , Sullivan Elizabeth , Center David M. , Cruikshank William W.
Published: April 2014

Previous work has shown that IL-16/CD4 induces desensitization of both CCR5- and CXCR4-induced migration, with no apparent effect on CCR2b or CCR3. To investigate the functional relationship between CD4 and other chemokine receptors, we determined the effects of IL-16 interaction with CD4 on CXCR3-induced migration. In this study we demonstrate that IL-16/CD4 induced receptor desensitization of CXCR3 on primary human T cells. IL-16/CD4 stimulation does not result in surface modulation of CXCR3 or changes in CXCL10 binding affinity. This effect does require p56lck enzymatic activity and the presence of CCR5, because desensitization is not transmitted in the absence of CCR5. Treatment of human T cells with methyl-β-cyclodextrin, a cholesterol chelator, prevented the desensitization of CXCR3 via IL-16/CD4, which was restored after reloading of cholesterol, indicating a requirement for intact cholesterol. These studies demonstrate an intimate functional relationship among CD4, CCR5, and CXCR3, in which CCR5 can act as an adaptor molecule for CD4 signaling. This process of regulating Th1 cell chemoattraction may represent a mechanism for orchestrating cell recruitment in Th1-mediated diseases.
Langerhans-Type Dendritic Cells Genetically Modified to Express Full-Length Antigen Optimally Stimulate CTLs in a CD4-Dependent Manner
Authors: Yuan Jianda , Latouche Jean-Baptiste , Hodges Joanna , Houghton Alan N. , Heller Glenn , Sadelain Michel , Riviere Isabelle , Young James W.
Published: April 2014

Oncoretroviral vectors encoding either full-length Ag or a corresponding immunodominant peptide were expressed in Langerhans-type dendritic cells (LCs) differentiated from CD34+ progenitors. We used human CMV as a model Ag restricted by HLA-A*0201 to define parameters for eventual expression of cancer Ags by LCs for active immunization against tumors. Stimulation by CMVpp65495–503-pulsed LCs, CMVpp65495–503-transduced LCs, and full-length CMVpp65-transduced LCs respectively increased tetramer-reactive T cells with an effector memory phenotype by 10 ± 11, 34 ± 21, and 51 ± 24-fold (p < 0.05) from CMV-seropositive donors. CMV-specific CD8+ CTLs achieved respective frequencies of 231 ± 102, 583 ± 219, and 714 ± 281 spot-forming cells per 105 input cells (p < 0.01) in ELISPOT assays for IFN-γ secretion. LCs expressing full-length Ag stimulated greater lytic activity than either peptide-transduced or peptide-pulsed LCs (p < 0.05), all in the absence of exogenous cytokines. pp65-transduced LCs presenting class I and II MHC-restricted epitopes expanded IFN-γ-secreting CD4+ T cells, whereas pp65495–503-transduced LCs did not. CD4+ T cell numbers even declined after stimulation by pp65495–503 peptide-pulsed LCs. CD4+ T cell depletion confirmed their contribution to the more robust CTL responses. LCs, transduced with a retroviral vector encoding full-length Ag, stimulate potent CTLs directed against multiple epitopes in a CD4+ Th cell-dependent manner.
Involvement of Protein Kinase Cε in the Negative Regulation of Akt Activation Stimulated by Granulocyte Colony-Stimulating Factor
Authors: Liu Hong , Qiu Yaling , Xiao Lei , Dong Fan
Published: April 2014

Stimulation of cells with G-CSF activates multiple signaling cascades, including the serine/threonine kinase Akt pathway. We show in this study that G-CSF-induced activation of Akt in myeloid 32D was specifically inhibited by treatment with PMA, a protein kinase C (PKC) activator. PMA treatment also rapidly attenuated sustained Akt activation mediated by a carboxy truncated G-CSF receptor, expressed in patients with acute myeloid leukemia evolving from severe congenital neutropenia. The inhibitory effect of PMA was abolished by pretreatment of cells with specific PKC inhibitor GF109203X, suggesting that the PKC pathway negatively regulates Akt activation. Ro31-8820, a PKCε inhibitor, also abrogated PMA-mediated inhibition of Akt activation, whereas rottlerin and Go6976, inhibitors of PKCδ and PKCαβI, respectively, exhibited no significant effects. Furthermore, overexpression of the wild-type and a constitutively active, but not a kinase-dead, forms of PKCε markedly attenuated Akt activation, and inhibited the proliferation and survival of cells in response to G-CSF. The expression of PKCε was down-regulated with G-CSF-induced terminal granulocytic differentiation. Together, these results implicate PKCε as a negative regulator of Akt activation stimulated by G-CSF and indicate that PKCε plays a negative role in cell proliferation and survival in response to G-CSF.
Scaffolding Adapter Grb2-Associated Binder 2 Requires Syk to Transmit Signals from FcεRI
Authors: Yu Min , Lowell Cliff A. , Neel Benjamin G. , Gu Haihua
Published: April 2014

Scaffolding adapter Grb2-associated binder 2 (Gab2) is a key component of FcεRI signaling in mast cells, required for the activation of PI3K. To understand how Gab2 is activated in FcεRI signaling, we asked which protein tyrosine kinase is required for Gab2 phosphorylation. We found that Gab2 tyrosyl phosphorylation requires Lyn and Syk. In agreement with published results, we found that Fyn also contributes to Gab2 tyrosyl phosphorylation. However, Syk activation is defective in Fyn−/− mast cells, suggesting that Syk is the proximal kinase responsible for Gab2 tyrosyl phosphorylation. Then, we asked which domains in Gab2 are required for Gab2 tyrosyl phosphorylation. We found that the Grb2-Src homology 3 (SH3) binding sites are required for, whereas the pleckstrin homology (PH) domain contributes to, Gab2 tyrosyl phosphorylation. Using a protein/lipid overlay assay, we determined that the Gab2 PH domain preferentially binds the PI3K lipid products, PI3, 4,5P3 and PI3, 4P2. Furthermore, the Grb2-SH3 binding sites and PH domain binding to PI3K lipid products are required for Gab2 function in FcεRI-evoked degranulation and Akt activation. Our data strongly suggest a model for Gab2 action in FcεRI signaling. The Grb2 SH3 binding sites play a critical role in bringing Gab2 to FcεRI, whereupon Gab2 becomes tyrosyl-phosphorylated in a Syk-dependent fashion. Phosphorylated Gab2 results in recruitment and activation of PI3K, whose lipid products bind the PH domain of Gab2 and acts in positive feedback loop for sustained PI3K recruitment and phosphatidylinositol-3,4,5-trisphosphate production, required for FcεRI-evoked degranulation of mast cells.
The Molecular Basis for Galα(1,3)Gal Expression in Animals with a Deletion of the α1,3Galactosyltransferase Gene
Authors: Milland Julie , Christiansen Dale , Lazarus Brooke D. , Taylor Simon G. , Xing Pei Xiang , Sandrin Mauro S.
Published: April 2014

The production of homozygous pigs with a disruption in the GGTA1 gene, which encodes α1,3galactosyltransferase (α1,3GT), represented a critical step toward the clinical reality of xenotransplantation. Unexpectedly, the predicted complete elimination of the immunogenic Galα(1,3)Gal carbohydrate epitope was not observed as Galα(1,3)Gal staining was still present in tissues from GGTA1−/− animals. This shows that, contrary to previous dogma, α1,3GT is not the only enzyme able to synthesize Galα(1,3)Gal. As iGb3 synthase (iGb3S) is a candidate glycosyltransferase, we cloned iGb3S cDNA from GGTA1−/− mouse thymus and confirmed mRNA expression in both mouse and pig tissues. The mouse iGb3S gene exhibits alternative splicing of exons that results in a markedly different cytoplasmic tail compared with the rat gene. Transfection of iGb3S cDNA resulted in high levels of cell surface Galα(1,3)Gal synthesized via the isoglobo series pathway, thus demonstrating that mouse iGb3S is an additional enzyme capable of synthesizing the xenoreactive Galα(1,3)Gal epitope. Galα(1,3)Gal synthesized by iGb3S, in contrast to α1,3GT, was resistant to down-regulation by competition with α1,2fucosyltransferase. Moreover, Galα(1,3)Gal synthesized by iGb3S was immunogenic and elicited Abs in GGTA1 −/− mice. Galα(1,3)Gal synthesized by iGb3S may affect survival of pig transplants in humans, and deletion of this gene, or modification of its product, warrants consideration.
Cytolytic CD8+ T Cells Directed against a Cryptic Epitope Derived from a Retroviral Alternative Reading Frame Confer Disease Protection
Authors: Ho On , Green William R.
Published: April 2014

Cytolytic CD8+ T cells (CTL) are key to the immune response that controls virus infections and mediates disease protection. The ability of CTL to induce apoptosis of infected cells and/or limit viral replication is determined by recognition of processed viral peptide epitopes on the surface of the target cell. An understudied source of MHC class I-presented peptides is the aptly named “cryptic epitopes,” defined by their nontraditional methods of generation, including derivation from alternative reading frames (ARFs). Although ARF-encoded epitopes have now been documented in a few systems, their potential functional relevance in vivo has been debated. In this study, we demonstrate the physiological significance of an ARF-derived CTL epitope in a retrovirus-induced disease model. We show that disease-susceptible CD8-deficient mice reconstituted with CTL specific for the retroviral ARF-derived SYNTGRFPPL epitope controlled an infection by the LP-BM5 retrovirus isolate, evidently at the level of viral clearance, resulting in protection of these mice from disease. These data indicate that ARF-derived epitopes are indeed relevant inducers of the immune system and demonstrate the importance of atypically generated peptides as functional Ag with a physiologic role in disease protection.
Neutrophils Regulate Airway Responses in a Model of Fungal Allergic Airways Disease
Authors: Park Stacy J. , Wiekowski Maria T. , Lira Sergio A. , Mehrad Borna
Published: April 2014

Neutrophils infiltrate airway walls in patients with allergic airway diseases and in animal models of these illnesses, but their contribution to the pathogenesis of airway allergy is not established. We hypothesized that, in a mouse model of airway allergy to the ubiquitous environmental mold, Aspergillus fumigatus, airway neutrophils contribute to disease severity. Ab-mediated neutrophil depletion resulted in reduced airway hyperresponsiveness and remodeling, whereas conditional transgenic overexpression of the neutrophil chemotactic molecule, CXCL1, in airway walls resulted in worsened allergic responses. This worsened phenotype was associated with a marked increase in the number of airway neutrophils but not other lung leukocytes, including eosinophils and lymphocyte subsets, and depletion of neutrophils in sensitized mice with transgenic overexpression of CXCL1 resulted in attenuated airway responses. The number of lung neutrophils correlated with lung matrix metalloproteinase 9 (MMP-9) activity both in the context of neutrophil depletion and with augmented neutrophil recruitment to the airways. Although wild-type and MMP-9-deficient neutrophils homed to the inflamed airways to a similar extent, transfer of wild-type, but not MMP-9-deficient, neutrophils to MMP-9-deficient animals resulted in augmented allergic airway responses. Taken together, these data implicate neutrophils in the pathogenesis of fungal allergic airway disease.
Involvement of Group VIA Calcium-Independent Phospholipase A2 in Macrophage Engulfment of Hydrogen Peroxide-Treated U937 Cells
Authors: Pérez Rebeca , Balboa María A. , Balsinde Jesús
Published: April 2014

Hydrogen peroxide-induced apoptosis of U937 cells results in substantial hydrolysis of membrane phospholipids by calcium-independent group VIA phospholipase A2 (iPLA2-VIA). However, abrogation of cellular iPLA2-VIA neither delays nor decreases apoptosis, suggesting that, beyond a mere destructive role, iPLA2-VIA may serve other specific roles. In this study, we report that phagocytosis of apoptosing U937 cells by macrophages is blunted if the cells are depleted of iPLA2-VIA by treatment with an inhibitor or an antisense oligonucleotide, and it is augmented by overexpression of iPLA2-VIA in the dying cells. Thus, the magnitude of macrophage phagocytosis correlates with the level of iPLA2-VIA activity of the dying cells. Eliminating by antisense oligonucleotide technology of cytosolic group IVA phospholipase A2 does not attenuate phagocytosis of U937 dying cells by macrophages. Incubation of U937 cells with different fatty acids has no effect on either the extent of hydrogen peroxide-induced apoptosis or the degree of phagocytosis of the dying cells by macrophages. However, preincubation of the macrophages with lysophosphatidylcholine before exposing them to the dying cells blocks phagocytosis of the latter. These results indicate that formation of lysophosphatidylcholine by iPLA2-VIA in hydrogen peroxide-treated U937 cells to induce apoptosis directly contributes to their efficient clearance by macrophages.
Deletion of CCR1 Attenuates Pathophysiologic Responses during Respiratory Syncytial Virus Infection
Authors: Miller Allison L. , Gerard Craig , Schaller Matthew , Gruber Achim D. , Humbles Allison A. , Lukacs Nicholas W.
Published: April 2014

The role of chemokines in chronic inflammatory responses are central to the recruitment of particular subsets of leukocytes. In the present studies, we have examined the role of CCR1 in the developing pathogenesis of respiratory syncytial virus (RSV) in the lungs of infected BALB/c mice. Although we did not observe significant differences in clearance of RSV, we were able to identify decreased pathophysiologic responses in CCR1−/− mice. CCR1−/− mice displayed a significant reduction in both airway hyperresponsiveness and mucus production that corresponded to significant increases in IFN-γ and CXCL10. The goblet cell hyper/metaplasia and the expression of mucus-associated gene, gob5, were correspondingly reduced in the CCR1−/− mice. In addition, the Western blot analysis of gob5 protein indicated that CCR1−/− mice have virtually no up-regulation of the protein at day 6 of infection compared with wild-type-infected mice. Results from bone marrow chimeric mice indicated that partial reconstitution of the response could be achieved in the CCR1−/− mice with wild-type bone marrow cells, suggesting that these cells have a role in the response. However, transplanting of CCR1−/− bone marrow into wild-type mice did demonstrate an incomplete deficit in RSV-induced responses, indicating that CCR1+ parenchymal cells may also play a significant role in the process. Thus, the presence of CCR1 appears to have a significant role in the development of detrimental airway physiologic responses during RSV infection. These data suggest that CCR1 may be a potential target during detrimental pulmonary responses during infection.
Inhalation Tolerance Is Induced Selectively in Thoracic Lymph Nodes but Executed Pervasively at Distant Mucosal and Nonmucosal Tissues
Authors: Alvarez David , Swirski Filip K. , Yang Teng-Chih , Fattouh Ramzi , Croitoru Ken , Bramson Jonathan L. , Stämpfli Martin R. , Jordana Manel
Published: April 2014

Under immunogenic conditions, both the site of initial Ag exposure and consequent T cell priming in specific draining lymph nodes (LNs) imprint the ensuing immune response with lasting tissue-selective tropism. With respect to immune tolerance, whether the site of tolerance induction leads to compartmentalized or, alternatively, pervasive tolerance has not been formally investigated. Using a murine model of inhalation tolerance, we investigated whether the induction of respiratory mucosal tolerance precludes the development of de novo Th2 sensitization upon subsequent exposure to the same Ag at distant mucosal (gut) and nonmucosal (cutaneous) sites. By tracking the proliferation of CFSE-labeled OVA-TCR transgenic CD4+ T cells upon OVA inhalation in vivo, we defined the site of tolerance induction to be restricted to the thoracic LNs. Expectedly, inhalation tolerance prevented de novo Th2 sensitization upon subsequent exposure to the same Ag at the same site. Importantly, although gut- and skin-draining LNs were not used during tolerance induction, de novo Ag-specific proliferation and Th2 differentiation in these LNs, as well as memory/effector Th2 responses in the gut (allergic diarrhea) and skin (late-phase cutaneous responses) were inhibited upon immunogenic challenge to the same Ag. Interestingly, this pervasive tolerogenic phenotype was not associated with the presence of suppressive activity throughout the lymphatics; indeed, potent suppressive activity was detected solely in the spleen. These data indicate that while inhalation tolerance is selectively induced in local thoracic LNs, its tolerogenic activity resides systemically and leads to pervasive immune tolerance in distant mucosal and nonmucosal sites.
Epithelial CXCR3-B Regulates Chemokines Bioavailability in Normal, but Not in Sjögren’s Syndrome, Salivary Glands
Authors: Sfriso Paolo , Oliviero Francesca , Calabrese Fiorella , Miorin Marta , Facco Monica , Contri Antonella , Cabrelle Anna , Baesso Ilenia , Cozzi Franco , Andretta Marilisa , Cassatella Marco Antonio , Fiocco Ugo , Todesco Silvano , Konttinen Yrjö T. , Punzi Leonardo , Agostini Carlo
Published: April 2014

Expression of CXCR3-targeting chemokines have been demonstrated in several diseases, suggesting a critical role for CXCR3 in recruiting activated T cells to sites of immune-mediated inflammation. Sjögren’s syndrome (SS) is an autoimmune disease characterized by a mononuclear cell infiltrate of activated T cells around the duct in the salivary gland. Analysis of minor salivary gland biopsy specimens from 20 healthy subjects and 18 patients with primary SS demonstrated that CXCR3, in particular, the B form of this receptor, is constitutively expressed by human salivary gland epithelial cells. Salivary gland epithelial cell cultures demonstrated that CXCR3 participate in removing relevant amount of agonists from the supernatant of exposed cells without mediating calcium flux or chemotaxis while retaining the ability to undergo internalization. Although in normal salivary gland epithelial cells, CXCR3 behaves as a chemokine-scavenging receptor, its role in SS cells is functionally impaired. The impairment of this scavenging function might favor chemotaxis, leading to heightened immigration of CXCR3-positive T lymphocytes. These findings suggest that epithelial CXCR3 may be involved in postsecretion regulation of chemokine bioavailability. They also support a critical role for CXCR3 in the pathogenesis of SS and identify its agonists as potential therapeutic targets.
The Shaving Reaction: Rituximab/CD20 Complexes Are Removed from Mantle Cell Lymphoma and Chronic Lymphocytic Leukemia Cells by THP-1 Monocytes
Authors: Beum Paul V. , Kennedy Adam D. , Williams Michael E. , Lindorfer Margaret A. , Taylor Ronald P.
Published: April 2014

Clinical investigations have revealed that infusion of immunotherapeutic mAbs directed to normal or tumor cells can lead to loss of targeted epitopes, a phenomenon called antigenic modulation. Recently, we reported that rituximab treatment of chronic lymphocytic leukemia patients induced substantial loss of CD20 on B cells found in the circulation after rituximab infusion, when rituximab plasma concentrations were high. Such antigenic modulation can severely compromise therapeutic efficacy, and we postulated that B cells had been stripped (shaved) of the rituximab/CD20 complex by monocytes or macrophages in a reaction mediated by FcγR. We developed an in vitro model to replicate this in vivo shaving process, based on reacting rituximab-opsonized CD20+ cells with acceptor THP-1 monocytes. After 45 min at 37°C, rituximab and CD20 are removed from opsonized cells, and both are demonstrable on acceptor THP-1 cells. The reaction occurs equally well in the presence and absence of normal human serum, and monocytes isolated from peripheral blood also promote shaving of CD20 from rituximab-opsonized cells. Tests with inhibitors and use of F(ab′)2 of rituximab indicate transfer of rituximab/CD20 complexes to THP-1 cells is mediated by FcγR. Antigenic modulation described in previous reports may have been mediated by such shaving, and our findings may have profound implications for the use of mAbs in the immunotherapy of cancer.
Human NK Cells Positively Regulate γδ T Cells in Response to Mycobacterium tuberculosis
Authors: Zhang Ruijun , Zheng Xiaodong , Li Baiqing , Wei Haiming , Tian Zhigang
Published: April 2014

The decrease in NK cell activity and the loss of γδ T cells in active pulmonary tuberculosis patients have been reported. In this study, we observed that the proliferating response of γδ T cells to the heat-treated Ags of Mycobacterium tuberculosis from different individuals was noted to be dependent on the content or function of NK cells in PBMC in a population study. We also found that NK cells were directly rapidly activated by the heat-treated Ags from M. tuberculosis (H37Ra) in vitro; in turn, the activated NK cells improved γδ T cell proliferation both by CD54-mediated cell-cell contact through the forming immune synapse and by soluble factors TNF-α, GM-CSF, and IL-12, but not IFN-γ. Our results demonstrated that an interaction between NK cells and γδ T cells existed in antituberculosis immunity. Up-regulating the function of NK cells might be beneficial to the prevention and control of pulmonary tuberculosis.
Tolerance Induction by Transcutaneous Immunization through Ultraviolet-Irradiated Skin Is Transferable through CD4+CD25+ T Regulatory Cells and Is Dependent on Host-Derived IL-10
Authors: Ghoreishi Mehran , Dutz Jan P.
Published: April 2014

UV radiation of the skin impairs immune responses to haptens and to tumor Ags. Transcutaneous immunization (TCI) is an effective method of inducing immune responses to protein and peptide Ag. We explore the effect of UV irradiation on TCI. The generation of Ag-specific CTL to OVA protein, but not class I MHC-restricted OVA peptide, is inhibited by TCI through UV-irradiated skin. Consequently, the induction of protein contact hypersensitivity and in vivo Ag-specific CTL activity following OVA protein immunization is prevented. Application of haptens to UV-exposed skin induces hapten-specific tolerance. We demonstrate that application of protein or class II MHC-restricted OVA peptide to UV-irradiated skin induces transferable Ag-specific tolerance. This tolerance is mediated by CD4+CD25+ T regulatory (Treg) cells. These Ag-specific Treg cells inhibit the priming of CTL following protein immunization in the presence of CpG adjuvant. IL-10 deficiency is known to prevent hapten-specific tolerance induction. In this study, we demonstrate, using IL-10-deficient mice and adoptive T cell transfer, that IL-10 is required for the direct inhibition of CTL priming following immunization through UV-irradiated skin. However, IL-10 is not required for the induction of Treg cells through UV-irradiated skin as IL-10-deficient Treg cells are able to mediate tolerance. Rather, host-derived IL-10 is required for the function of UV-generated Treg cells. These experiments indicate that protein and peptide TCI through UV-irradiated skin may be used to induce robust Ag-specific tolerance to neo-Ags and that UV-induced Treg cells mediate their effects in part through the modulation of IL-10.
Longitudinal Studies of Clonally Expanded CD8 T Cells Reveal a Repertoire Shrinkage Predicting Mortality and an Increased Number of Dysfunctional Cytomegalovirus-Specific T Cells in the Very Elderly
Authors: Hadrup Sine Reker , Strindhall Jan , Køllgaard Tania , Seremet Tina , Johansson Boo , Pawelec Graham , thor Straten Per , Wikby Anders
Published: April 2014

The age-associated decrease in functionality of the human immune system is thought to have a negative impact on the capacity to provide protection against infection, in turn leading to increased incidence of mortality. In a previous longitudinal study of octogenarians, we identified an immune risk phenotype (IRP) in the very elderly defined by an inverted CD4/CD8 ratio, which was associated with increased mortality and persistent CMV infection. In this study, we analyzed the CD8 clonal composition of nonagenarians and middle-aged individuals. An increased number of CD8 T cell clones was observed in the nonagenarians, and was associated with CMV-seropositivity. Surprisingly, CMV-seropositive nonagenarians with the IRP had a significantly lower number of clones compared with non-IRP individuals. The decrease in clone numbers in IRP individuals was associated with shorter survival time. MHC/peptide multimer staining indicated that the frequency of CMV-specific T cells was higher in nonagenarians than in the middle-aged, but the ratio of functionally intact cells was significantly lower. The lowest ratio of functional CMV-specific T cells was found in an IRP individual. A thorough longitudinal analysis of the CMV-specific T cells in nonagenarians showed a stable pattern with respect to frequency, phenotype, and clonal composition. We hypothesize that the number of different CD8 T cell clonal expansions increases as the individual ages, possibly, as a compensatory mechanism to control latent infections, e.g., CMV, but eventually a point is reached where clonal exhaustion leads to shrinkage of the CD8 clonal repertoire, associated with decreased survival.
HIV-Specific Cytotoxic Cell Frequencies Measured Directly Ex Vivo by the Lysispot Assay Can Be Higher or Lower Than the Frequencies of IFN-γ-Secreting Cells: Anti-HIV Cytotoxicity Is Not Generally Impaired Relative to Other Chronic Virus Responses
Authors: Snyder-Cappione Jennifer E. , Divekar Anagha A. , Maupin Genny M. , Jin Xia , Demeter Lisa M. , Mosmann Tim R.
Published: April 2014

CD8+ T cells in HIV-infected patients are believed to contribute to the containment of the virus and the delay of disease progression. However, the frequencies of HIV-specific CD8+ T cells, as measured by IFN-γ secretion and tetramer binding, often do not correlate with a delay in disease progression during chronic infection. Using the Lysispot and ELISPOT assays, we measured the frequencies of cytotoxic and IFN-γ-secreting T cells responding to overlapping peptides from Gag, Nef, Env, and Pol consensus HIV-1 clade B sequences. PBMC from the majority of HIV-infected subjects have significant frequencies of HIV-specific cells that killed targets within 5 h directly ex vivo. The relative frequencies of IFN-γ-secreting and cytotoxic cells varied markedly between different HIV peptide pools within the same patient, and some T cells lysed targets without secreting IFN-γ. These results indicate that measurement of IFN-γ production alone may be insufficient to evaluate the breadth of the HIV-specific T cell response. Also, neither the CTL to IFN-γ ratios nor the ex vivo CTL frequencies specific for different HIV proteins were consistently lower than responses specific for two other chronic viral infections, human CMV and EBV, within the same subjects. Thus ex vivo cytotoxic T cell frequencies do not provide evidence for a model of “preterminal differentiation” of HIV-specific CD8+ T cells during chronic HIV infection. Analysis of the frequency of directly cytotoxic HIV-specific T cells may be of considerable value in the assessment of disease progression and the potential efficacy of HIV vaccines.
Cutting Edge: Diminished T Cell TLR Expression and Function Modulates the Immune Response in Human Filarial Infection
Authors: Babu Subash , Blauvelt Carla P. , Kumaraswami V. , Nutman Thomas B.
Published: April 2014

Patent lymphatic filariasis is characterized by profound Ag-specific T cell hyporesponsiveness with impaired IFN-γ and IL-2 production. Because T cells have been shown to express a number of TLR and to respond to TLR ligands, we hypothesized that diminished T cell TLR function could partially account for the T cell hyporesponsiveness in filariasis. T cells expressed TLR1, TLR2, TLR4, and TLR9, and the baseline expression of TLR1, TLR2, and TLR4, but not TLR9 was significantly lower in T cells of the filarial-infected individuals compared with the uninfected individuals (both endemic and nonendemic). TLR function was significantly diminished in the T cells of filarial-infected individuals based on decreased T cell activation/cytokine production in response to TLR ligands. Thus, diminished expression and function of T cell TLR is a novel mechanism underlying T cell immune tolerance in lymphatic filariasis.
Cutting Edge: Enhanced Pulmonary Clearance of Pseudomonas aeruginosa by Muc1 Knockout Mice
Authors: Lu Wenju , Hisatsune Akinori , Koga Takeshi , Kato Kosuke , Kuwahara Ippei , Lillehoj Erik P. , Chen Wilbur , Cross Alan S. , Gendler Sandra J. , Gewirtz Andrew T. , Kim K. Chul
Published: April 2014

MUC1 (MUC1 in human and Muc1 in nonhumans) is a membrane-tethered mucin that interacts with Pseudomonas aeruginosa (PA) through flagellin. In this study, we compared PA pulmonary clearance and proinflammatory responses by Muc1−/− mice with Muc1+/+ littermates following intranasal instillation of PA or flagellin. Compared with Muc1+/+ mice, Muc1−/− mice showed increased PA clearance, greater airway recruitment of neutrophils, higher levels of TNF-α and KC in bronchoalveolar lavage fluid, higher levels of TNF-α in media of flagellin-stimulated alveolar macrophages, and higher levels of KC in media of tracheal epithelial cells. Knockdown of MUC1 enhanced flagellin-induced IL-8 production by primary human bronchial epithelial cells. Expression of MUC1 in HEK293T cells attenuated TLR5-dependent IL-8 release in response to flagellin, which was completely ablated when its cytoplasmic tail was deleted. We conclude that MUC1/Muc1 suppresses pulmonary innate immunity and speculate its anti-inflammatory activity may play an important modulatory role during microbial infection.
Cutting Edge: Elimination of an Endogenous Adjuvant Reduces the Activation of CD8 T Lymphocytes to Transplanted Cells and in an Autoimmune Diabetes Model
Authors: Shi Yan , Galusha Shelly A. , Rock Kenneth L.
Published: April 2014

The generation of adaptive immune responses is thought to require the presence of adjuvants. Although microbial adjuvants are well characterized, little is known about what provides the adjuvant effect in responses to transplanted cells or in autoimmune diseases. It had been postulated that, in these situations, injured cells instead released “endogenous adjuvants.” We previously identified uric acid as an endogenous adjuvant for coinjected Ags. We now report that elimination of uric acid reduced the generation of CTL to an Ag in transplanted syngeneic cells and the proliferation of autoreactive T cells in a transgenic diabetes model. In contrast, uric acid depletion did not reduce the stimulation of T cells to mature APCs or when endogenous APCs were activated with anti-CD40 Ab. These findings support the concept that danger signals contribute to the T cell responses to cell-associated Ags by activating APCs and identify uric acid as one of these signals.
Cutting Edge: B and T Lymphocyte Attenuator and Programmed Death Receptor-1 Inhibitory Receptors Are Required for Termination of Acute Allergic Airway Inflammation
Authors: Deppong Christine , Juehne Twyla I. , Hurchla Michelle , Friend Lindzy D. , Shah Dulari D. , Rose Christine M. , Bricker Traci L. , Shornick Laurie P. , Crouch Erika C. , Murphy Theresa L. , Holtzman Michael J. , Murphy Kenneth M. , Green Jonathan M.
Published: April 2014

T cell activation is regulated by coordinate interaction of the T cell Ag receptor and costimulatory signals. Although there is considerable insight into processes that regulate the initiation of inflammation, less is known about the signals that terminate immune responses. We have examined the role of the inhibitory receptors programmed death receptor-1 and B and T lymphocyte attenuator in the regulation of allergic airway inflammation. Our results demonstrate that there is a temporally regulated expression of both the receptors and their ligands during the course of allergic airway inflammation. Following a single inhaled challenge, sensitized wild-type mice exhibit peak inflammation on day 3, which resolves by day 10. In contrast, mice deficient in the expression of programmed death receptor-1 or B and T lymphocyte attenuator have persistent inflammation out to 15 days following challenge. Thus, these receptors are critical determinants of the duration of allergic airway inflammation.
Phenotypic and Functional Similarity of Gut Intraepithelial and Systemic T Cells in a Teleost Fish
Authors: Bernard David , Six Adrien , Rigottier-Gois Lionel , Messiaen Sébastien , Chilmonczyk Stefan , Quillet Edwige , Boudinot Pierre , Benmansour Abdenour
Published: April 2014

Gut-associated lymphocytes were described in fish, but their involvement in immune responses is still unknown. In rainbow trout, intraepithelial lymphocytes (IELs) are scattered between gut epithelial cells, but neither Peyer’s patches nor mesenteric lymph nodes were identified. Rainbow trout IELs contain mainly T cells, because they expressed transcripts of T cell marker homologs of CD8, CD4, CD28, CD3ε, TCRζ, TCRγ, and TCRβ and lacked IgM. However, trout IELs did not show specific homing to the gut mucosa, which in mammals defines IELs as a distinctive mucosal population. A detailed analysis of the TCRβ repertoire of rainbow trout IELs was performed in both naive and virus-infected animals. TCRβ transcripts of rainbow trout IELs were highly diverse and polyclonal in adult naive individuals, in sharp contrast with the restricted diversity of IEL oligoclonal repertoires described in birds and mammals. Significant modifications of the trout IEL TCRβ repertoire were observed after a systemic infection with a fish rhabdovirus and were especially marked for Vβ4-bearing receptors as previously reported for spleen cells. Thus, we could not find any specific properties of the trout IEL TCRβ repertoire compared with the spleen and pronephros TCRβ repertoire, which questions the reality of a distinct IEL compartment in teleosts. Our findings suggest that a highly diversified αβ ΤCR repertoire is maintained in fish IELs in the absence of Peyer’s patches and mesenteric lymph nodes, whereas the restricted diversity of mouse αβ IELs is attributed to multiple cycles of activation and recirculation, allowing a progressive narrowing of the repertoire.
Apoptotic Death Concurrent with CD3 Stimulation in Primary Human CD8+ T Lymphocytes: A Role for Endogenous Granzyme B
Authors: Laforge Mireille , Bidère Nicolas , Carmona Sylvie , Devocelle Aurore , Charpentier Bernard , Senik Anna
Published: April 2014

We exposed primary CD8+ T cells to soluble CD3 mAb plus IL-2 and limited numbers of monocytes (3%). These cells were activated but concurrently subjected to ongoing apoptosis (∼25% were apoptotic from day 2 of culture). However, their costimulated CD4+ counterparts were much less prone to apoptosis. The apoptotic signaling pathway bypassed Fas and TNFRs, and required the activity of cathepsin C, a protease which performs the proteolytic maturation of granzyme (Gr) A and GrB proenzymes within the cytolytic granules. Silencing the GrB gene by RNA interference in activated CD8+ T cells prevented the activation of procaspase-3 and Bid, and indicated that GrB was the upstream death mediator. A GrB-specific mAb immunoprecipitated a ∼70-kDa molecular complex from cytolytic extracts of activated CD8+ (but not resting) T cells, that was specifically recognized by a nucleocytoplasmic protease inhibitor 9 (PI-9) specific mAb. This complex was also detected after reciprocal immunoprecipitation of PI-9. It coexisted in the cytosol with the 32-kDa form of GrB. As neither were detected in the cytosol of CD4+ bystander T cells (which poorly synthesized GrB), and as silencing the perforin (Pf) gene had no effect in our system, endogenous GrB was likely implicated. Immunoprecipitation experiments failed to reveal Pf in the cytosol of CD8+ T cells, and only a tiny efflux of granular GrA was detected by ELISA. We propose that some GrB is released from cytolytic granules to the cytosol of CD8+ T lymphocytes upon CD3/TCR stimulation and escapes PI-9, thereby mediating apoptotic cell death.
IL-7 Is a Critical Factor in Modulating Lesion Development in Skn-Directed Autoimmunity
Authors: Staton Pamela J. , Carpenter A. Betts , Jackman Susan H.
Published: April 2014

In a murine model of autoimmunity targeted against the epidermal cell Ags, Skn, adoptive transfer of Skn-immune T cells to immunosuppressed recipients elicits skin lesions in areas of mild epidermal trauma. In this study, we examined peripheral regulation of Skn-induced autoreactivity disrupted by rendering the mice immunoincompetent. We found that regulation of Skn-directed autoimmunity was restored by cotransfer of normal syngeneic spleen cells at twice the concentration of Skn-immune cells and was evidenced by significantly reduced lesion severity by days 5–7 post-cotransfer compared with animals given injections of Skn-immune cells alone. Enrichment and depletion of normal CD4+ or CD8+ spleen cells and RT-PCR analysis of selected cytokines identified CD4+ cells as the regulatory cells in the cotransfer inoculum; however, significant reduction in lesion severity was observed only when there was a concomitant increase in levels of IL-7. The role of IL-7 was further supported in that mice cotransferred with Skn-immune cells plus normal spleen cells, but also treated with anti-IL-7 Ab, no longer exhibited reduced lesion severity. To determine whether IL-7 expression without normal spleen cell cotransfer could modulate lesion development, an IL-7-encoding plasmid (pCMV-Tag1-IL-7) was topically delivered to sites flanking the stressed skin site in Skn-induced autoimmune mice. Daily application of 15 μg of pCMV-Tag1-IL-7 significantly suppressed lesion severity. Our results support a mechanism for CD4+ T cells and IL-7 in contributing to the control of autoreactivity.
Protein Kinase Bα Is Required for Vesicle Trafficking and Class II Presentation of IgA Fc Receptor (CD89)-Targeted Antigen
Authors: Lang Gillian A. , Lang Mark L.
Published: April 2014

Ag presentation stimulates Ag-specific adaptive immune responses. FcαR (CD89)-mediated capture of IgA-bound exogenous Ag leads to efficient MHC class II Ag presentation by APCs. CD89 signaling is required for trafficking of internalized Ag to specialized multivesicular bodies known as MHC class II compartments (MIIC) and subsequent class II presentation. In the present study, we tested the hypothesis that the vesicle trafficking regulator protein kinase Bα (PKBα) is required for CD89-mediated trafficking to MIIC and Ag presentation. We observed by two independent methods (chemical inhibitors and specific RNA interference) that PKBα was required for CD89 trafficking to MIIC and class II Ag presentation. Expression of constitutively active PKBα in APCs expressing a mutant CD89 accessory signaling molecule (deficient in CD89/Ag trafficking, processing, and presentation) induced trafficking of CD89 to lamp1-containing late endocytic vesicles, but not class II-containing vesicles (MIIC), or class II Ag presentation. These studies show for the first time that PKBα is required for receptor-mediated Ag presentation and suggest the mechanism of action includes regulation of vesicle trafficking.