bluuebunny/crossref_metadata_2025 huggingface dataset:
Cutting Edge: Memory B Cell Survival and Function in the Absence of Secreted Antibody and Immune Complexes on Follicular Dendritic Cells
Authors: Anderson Shannon M. , Hannum Lynn G. , Shlomchik Mark J.
Published: April 2014

Ag, in the form of immune complexes retained on follicular dendritic cells, has been implicated in the development and maintenance of B cell memory. We addressed this question using a H chain transgenic (Tg) mouse model that lacks secreted Ig (mIg), and thus does not deposit Ag-containing immune complexes. We compared the ability of the mIg strain and a control Tg strain, which secretes IgM, to develop and maintain long-lived memory cells. After immunization, there was an increase of Ag-specific B cells in both strains that was maintained for at least 20 wk. We labeled the long-lived Ag-specific cells with BrdU and found that this population was similarly maintained. In addition, both Tgs were able to maintain a functional memory response as measured by secondary germinal center reactions. Our studies indicate that localization of Ag on follicular dendritic cells is not necessary for development and maintenance of B cell memory.
Antigen Challenge Inhibits Thymic Emigration
Authors: Uldrich Adam P. , Berzins Stuart P. , Malin Mark A. , Bouillet Philippe , Strasser Andreas , Smyth Mark J. , Boyd Richard L. , Godfrey Dale I.
Published: April 2014

T cell development in the thymus involves a series of TCR-mediated control points including TCR-β selection and positive and negative selection. Approximately half of the thymic sojourn is spent in the medulla, where thymocytes undergo final maturation before emigrating to the periphery. Although it is acknowledged that thymic emigration is an active process, relatively little is known about how this is regulated, why it takes so long, and whether TCR-mediated signaling can influence this step. Using wild-type and TCR transgenic mice, we found that Ag injected i.v. or intrathymically led to a striking reduction in the number of recent thymic emigrants (RTE) in the periphery. This was caused by inhibition of T cell export rather than peripheral deletion, because a cohort of RTE that was already released before in vivo Ag challenge was not depleted, and similar results were observed in Bim-deficient mice, which have impaired T cell deletion. Within the thymus, the loss of RTE was associated with retention of medullary thymocytes rather than increased negative selection. In addition to Ag-specific inhibition of export, some TCR-independent suppression of emigration was also observed that appeared to be partly the result of the inflammatory cytokine TNF. Thus, in addition to its accepted role in intrathymic selection events, TCR signaling can also play an important role in the regulation of thymic emigration.
DNA Methylation by DNA Methyltransferase 1 Is Critical for Effector CD8 T Cell Expansion
Authors: Chappell Craig , Beard Caroline , Altman John , Jaenisch Rudolph , Jacob Joshy
Published: April 2014

Transcriptional silencing mediated by DNA methylation is a critical component of epigenetic regulation during early embryonic development in animals. However, the requirement for DNA methylation during activation and differentiation of mature CD8+ T cells into effector and memory cells is not clear. Using cre-mediated deletion of DNA methyltransferase 1 (Dnmt1) at the time of CD8+ T cell activation, we investigated the obligation for maintaining patterns of DNA methylation during the generation of Ag-specific effector and memory CD8+ T cells in response to acute viral infection of mice with lymphocytic choriomeningitis virus. Dnmt1−/− CD8+ T cells failed to undergo the massive CD8+ T cell expansion characteristic of lymphocytic choriomeningitis virus infection, leading to >80% reductions in Ag-specific effector CD8+ T cells at the height of the response. Despite this, Dnmt1−/− CD8+ T cells efficiently controlled the viral infection. Interestingly, the number of Ag-specific Dnmt1−/− memory CD8+ T cells was moderately reduced compared with the reductions seen at day 8 postinfection. Our data suggest that ablation of Dnmt1 and subsequent DNA methylation affect the finite proliferative potential of Ag-specific CD8+ T cells with moderate effects on their differentiation to effector and memory CD8+ T cells.
Intranasal Vaccination with Proinsulin DNA Induces Regulatory CD4+ T Cells That Prevent Experimental Autoimmune Diabetes
Authors: Every Alison L. , Kramer David R. , Mannering Stuart I. , Lew Andrew M. , Harrison Leonard C.
Published: April 2014

Insulin, an autoantigen in type 1 diabetes, when administered mucosally to diabetes-prone NOD mice induces regulatory T cells (Treg) that protect against diabetes. Compared with protein, Ag encoded as DNA has potential advantages as a therapeutic agent. We found that intranasal vaccination of NOD mice with plasmid DNA encoding mouse proinsulin II-induced CD4+ Treg that suppressed diabetes development, both after adoptive cotransfer with “diabetogenic” spleen cells and after transfer into NOD mice given cyclophosphamide to accelerate diabetes onset. In contrast to prototypic CD4+CD25+ Treg, CD4+ Treg induced by proinsulin DNA were both CD25+ and CD25− and not defined by markers such as glucocorticoid-induced TNFR-related protein (GITR), CD103, or Foxp3. Intriguingly, despite induction of Treg and reduced islet inflammation, diabetes incidence in proinsulin DNA-treated mice was unchanged. However, diabetes was prevented when DNA vaccination was performed under the cover of CD40 ligand blockade, known to prevent priming of CTL by mucosal Ag. Thus, intranasal vaccination with proinsulin DNA has therapeutic potential to prevent diabetes, as demonstrated by induction of protective Treg, but further modifications are required to improve its efficacy, which could be compromised by concomitant induction of pathogenic immunity.
Selective Requirement of p38α MAPK in Cytokine-Dependent, but Not Antigen Receptor-Dependent, Th1 Responses
Authors: Berenson Lisa S. , Yang Jianfei , Sleckman Barry P. , Murphy Theresa L. , Murphy Kenneth M.
Published: April 2014

The role of the p38 MAPK pathway in Th1 development has been controversial, because indirect manipulations of either upstream p38 activators or modifiers of p38 activity have had variable effects on IFN-γ production in CD4+ T cells. Uncertainties regarding the specificity of pharmacologic inhibition or p38 dominant negative mutants diminish the strength of conclusions about the role of the p38α isoform in Th1 development. Also, the effects of some upstream p38 activators, such as MAPK kinase 3, on Th1 development are not as strong as the effects of other manipulations, such as the expression of a dominant negative p38 mutant. Finally, embryonic lethality has prevented a direct examination of p38α-deficient T cells. To test the requirement for p38α in Th1 differentiation, we generated Ag-specific p38α+/− and p38α−/− CD4+ T cells using RAG2−/− blastocyst complementation and retroviral expression of the DO11.10 TCR. IFN-γ production in response to TCR signaling is normal in p38α−/− T cells cultured in Th1 conditions, implying normal Th1 development. However, p38α−/− Th1 cells have a much greater defect in IFN-γ secretion stimulated by IL-12/IL-18 compared with TCR-induced IFN-γ secretion. These results suggest that the activity of p38α in Th1 cells is relatively restricted to acting in one of two alternative pathways (i.e., cytokine induced) that can induce the production of IFN-γ in differentiated Th1 cells, but that p38α is not required for the process of Th1 commitment and development itself.
Accelerated Memory Cell Homeostasis during T Cell Depletion and Approaches to Overcome It
Authors: Neujahr David C. , Chen Chuangqi , Huang Xiaolun , Markmann James F. , Cobbold Stephen , Waldmann Herman , Sayegh Mohamed H. , Hancock Wayne W. , Turka Laurence A.
Published: April 2014

Partial T cell depletion is used in solid organ transplantation as a valuable strategy of peritransplant induction immunosuppression. Using a murine cardiac allograft model, we recently demonstrated that this led to lymphopenia-induced (homeostatic) proliferation among the residual nondepleted lymphocytes. Rather than promoting tolerance, peritransplant T cell-depleting Abs actually resulted in resistance to tolerance induction by costimulatory blockade. In this study we show that memory T cells predominate shortly after subtotal lymphodepletion due to two distinct mechanisms: relative resistance to depletion and enhanced homeostatic proliferation. In contrast, regulatory cells (CD4+CD25+Foxp3+) are depleted as efficiently as nonregulatory cells and exhibit reduced homeostatic expansion compared with memory cells. The resistance to tolerance induction seen with subtotal T cell depletion can be overcome in two different ways: first, by the adoptive transfer of additional unprimed regulatory cells at the time of transplant, and second, by the adjunctive use of nondepleting anti-CD4 and anti-CD8 mAbs, which effectively block homeostatic expansion. We conclude that the resistance to tolerance induction seen after subtotal lymphocyte depletion can be attributed to alterations in the balance of naive, memory, and regulatory T cells. These data have clinically relevant implications related to the development of novel strategies to overcome resistance to tolerance.
CD48 Stimulation by 2B4 (CD244)-Expressing Targets Activates Human NK Cells
Authors: Messmer Birgitta , Eissmann Philipp , Stark Sebastian , Watzl Carsten
Published: April 2014

Human NK cells can be activated by a variety of different cell surface receptors. Members of the SLAM-related receptors (SRR) are important modulators of NK cell activity. One interesting feature of the SRR is their homophilic interaction, combining receptor and ligand in the same molecule. Therefore, SRR cannot only function as activating NK cell receptors, but also as activating NK cell ligands. 2B4 (CD244) is the only SRR that does not show homophilic interaction. Instead, 2B4 is activated by binding to CD48, a GPI-anchored surface molecule that is widely expressed in the hemopoietic system. In this study, we show that 2B4 also can function as an activating NK cell ligand. 2B4-expressing target cells can efficiently stimulate NK cell cytotoxicity and IFN-γ production. Using soluble receptor fusion proteins and SRR-transfected cells, we show that 2B4 does not bind to any other SRR expressed on NK cells, but only interacts with CD48. Lysis of 2B4-expressing target cells can be blocked by anti-CD48 Abs and triggering of CD48 in a redirected lysis assay can stimulate NK cell cytotoxicity. This demonstrates that 2B4 can stimulate NK cell cytotoxicity and cytokine production by interacting with NK cell expressed CD48 and adds CD48 to the growing number of activating NK cell receptors.
Dendritic Cell Differentiation and Immune Tolerance to Insulin-Related Peptides in Igf2-Deficient Mice
Authors: Hansenne Isabelle , Renard-Charlet Chantal , Greimers Roland , Geenen Vincent
Published: April 2014

There is some evidence that insulin-like growth factor 2 (IGF-2) may intervene in the control of T cell differentiation. To further study the immunoregulatory function of this growth factor, we analyzed the immune system of Igf2−/− mice. Phenotypically, some immunological parameters such as lymphoid organ morphology and cellularity were unaltered in Igf2−/− mice, but an increase of CD8+ cells and a decrease of B220+ cells were observed in spleen. In vitro, the development of bone marrow-derived dendritic cells was affected by the absence of Igf2 expression. After maturation, a higher percentage of immature dendritic cells was observed in Igf2−/− population, together with a secondary decrease in allogenic T cell proliferation. Activation of T cells was also affected by the lack of expression of this growth factor. The profile of B cell response in mutant mice immunized with IGF-2 evidenced a T-dependent profile of anti-IGF-2 Abs that was absent in Igf2+/+ mice. The influence of IGF-2 upon tolerance to insulin was also assessed in this model, and this showed that IGF-2 also intervenes in tolerance to insulin. The presence of a T-dependent response in Igf2-deficient mice should allow cloning of specific “forbidden” T CD4+ lymphocytes directed against IGF-2, as well as further investigation of their possible pathogenic properties against insulin family.
Conditioning of Langerhans Cells Induced by a Primary CD8 T Cell Response to Self-Antigen In Vivo
Authors: Mayerova Dita , Wang Liangchun , Bursch Laura S. , Hogquist Kristin A.
Published: April 2014

Using a previously described model of autoimmune skin disease, we addressed the question of how CD8 T cell responsiveness to self-Ag is regulated during chronic inflammation. In this model, CD8 T cells expand and induce tissue pathology directed at an epidermal self-Ag. However, we show here that this primary CD8 T cell response prevented subsequent expansion of a second CD8 T cell population with the same specificity. This lack of T cell accumulation was not due to Ag elimination, nor was it due to competition between the two T cell populations. However, skin-specific dendritic cells that present Ag in this model–Langerhans cells–underwent significant phenotypic changes associated with a compromised ability to stimulate naive T cells. Our study suggests that conditioning of dendritic cells may play a role in maintaining unresponsiveness to self-Ag during chronic inflammation.
NF-κB-Inducing Kinase Is Involved in the Activation of the CD28 Responsive Element through Phosphorylation of c-Rel and Regulation of Its Transactivating Activity
Authors: Sánchez-Valdepeñas Carmen , Martín Angel G. , Ramakrishnan Parameswaran , Wallach David , Fresno Manuel
Published: April 2014

Previous evidence suggested that NF-κB-inducing kinase (NIK) might regulate IL-2 synthesis. However, the molecular mechanism is not understood. In this study, we show that NIK is involved in CD3 plus CD28 activation of IL-2 transcription. Splenic T cells from aly/aly mice (that have a defective NIK protein) have a severe impairment in IL-2 and GM-CSF but not TNF secretion in response to CD3/CD28. This effect takes place at the transcriptional level as overexpression of alyNIK inhibits IL-2 promoter transcription. NIK activates the CD28 responsive element (CD28RE) of the IL-2 promoter and strongly synergizes with c-Rel in this activity. We found that NIK interacts with the N-terminal domain of c-Rel, mapping this interaction to aa 771–947 of NIK. Moreover, NIK phosphorylates the c-Rel C-terminal transactivation domain (TAD) and induces Gal4-c-Rel-transactivating activity. Anti-CD28 activated Gal4-c-Rel transactivation activity, and this effect was inhibited by a NIK-defective mutant. Deletion studies mapped the region of c-Rel responsive to NIK in aa 456–540. Mutation of several serines, including Ser471, in the TAD of c-Rel abrogated the NIK-enhancing activity of its transactivating activity. Interestingly, a Jurkat mutant cell line that expresses one of the mutations of c-Rel (Ser471Asn) has a severe defect in IL-2 and CD28RE-dependent transcription in response to CD3/CD28 or to NIK. Our results support that NIK may be controlling CD28RE-dependent transcription and T cell activation by modulating c-Rel phosphorylation of the TAD. This leads to more efficient transactivation of genes which are dependent on CD28RE sites where c-Rel binds such as the IL-2 promoter.
Nitric Oxide Protects Macrophages from Hydrogen Peroxide-Induced Apoptosis by Inducing the Formation of Catalase
Authors: Yoshioka Yasuhiro , Kitao Tatsuya , Kishino Takashi , Yamamuro Akiko , Maeda Sadaaki
Published: April 2014

We investigated the cytoprotective effect of NO on H2O2-induced cell death in mouse macrophage-like cell line RAW264. H2O2-treated cells showed apoptotic features, such as activation of caspase-9 and caspase-3, nuclear fragmentation, and DNA fragmentation. These apoptotic features were significantly inhibited by pretreatment for 24 h with NO donors, sodium nitroprusside and 1-hydroxy-2-oxo-3,3-bis-(2-aminoethyl)-1-triazene, at a low nontoxic concentration. The cytoprotective effect of NO was abrogated by the catalase inhibitor 3-amino-1,2,4-triazole but was not affected by a glutathione synthesis inhibitor, l-buthionine-(S,R)-sulfoximine. NO donors increased the level of catalase and its activity in a concentration-dependent manner. Cycloheximide, a protein synthesis inhibitor, inhibited both the NO-induced increase in the catalase level and the cytoprotective effect of NO. These results indicate that NO at a low concentration protects macrophages from H2O2-induced apoptosis by inducing the production of catalase.
Motif Inference Reveals Optimal CTL Epitopes Presented by HLA Class I Alleles Highly Prevalent in Southern Africa
Authors: Honeyborne Isobella , Rathod Almas , Buchli Rico , Ramduth Dhanwanthie , Moodley Eshia , Rathnavalu Prinisha , Chetty Senica , Day Cheryl , Brander Christian , Hildebrand William , Walker Bruce D. , Kiepiela Photini , Goulder Philip J. R.
Published: April 2014

HIV-specific CTL play a central role in immune control of HIV. The basis for understanding the success or failure of this immune response requires identification of the specific epitopes targeted by CTL. However, in populations most severely affected by the global epidemic, this fundamental knowledge is hindered by the lack of characterization of many of the HLA class I alleles highly prevalent in such populations. Overall, the peptide-binding motif has been determined for a small minority (9%) of HLA class I alleles, with a strong bias toward those alleles prevalent in Caucasoid populations. These studies therefore set out to define, in a South African Zulu/Xhosa population at the epicenter of the epidemic, the epitopes presented by alleles highly prevalent, but for which the peptide-binding motif had not been characterized. Using a method of motif inference, epitopes presented by four such alleles prevalent in the Zulu/Xhosa population of Durban, South Africa, namely, B*3910, B*4201, B*8101, and Cw*1801, are described. Importantly, this approach may additionally facilitate optimization of epitopes in certain instances where conflicting reports in the literature exist regarding the peptide-binding motif, such as for HLA-A*2902, also highly prevalent in southern African populations. These data indicate that the previously anomalous position of HLA-A*2902 among HLA-A alleles, outside any recognized HLA-A supertype, is artifactual, and the true position of the A*2902 motif overlaps those of the A1 and A24 supertypes.
Immunosensitization of Tumor Cells to Dendritic Cell-Activated Immune Responses with the Proteasome Inhibitor Bortezomib (PS-341, Velcade)
Authors: Schumacher Lana Y. , Vo Dan D. , Garban Hermes J. , Comin-Anduix Begoña , Owens Sharla K. , Dissette Vivian B. , Glaspy John A. , McBride William H. , Bonavida Benjamin , Economou James S. , Ribas Antoni
Published: April 2014

Proteasome inhibition results in proapoptotic changes in cancer cells, which may make them more sensitive to immune effector cells. We established a murine model to test whether the proteasome inhibitor bortezomib could sensitize established B16 melanoma tumors to dendritic cell (DC)-activated immune effector cells. Day 3-established s.c. B16 tumors had significantly decreased tumor outgrowth when treated with a combination of bortezomib and DC, regardless of whether the DC were loaded or not with a tumor Ag. In vivo Ab-depletion studies demonstrated that the effector cells were NK and CD8+ cells, but not CD4+ cells. NF-κB nuclear transcription factor assay and gene-expression profiling of B16 treated with bortezomib was consistent with inhibition of NF-κB target genes leading to a proapoptotic phenotype. In vitro lytic assays demonstrated that TNF-α, but not perforin, Fas-ligand, or TRAIL, was responsible for bortezomib-sensitized B16 cytotoxicity. In conclusion, the proteasome inhibitor bortezomib can pharmacologically sensitize tumor cells to the lytic effects of DC-activated immune effector cells.
Engagement of CD28 Outside of the Immunological Synapse Results in Up-Regulation of IL-2 mRNA Stability but Not IL-2 Transcription
Authors: Sanchez-Lockhart Mariano , Miller Jim
Published: April 2014

During T cell activation by APC, CD28 is colocalized with TCR in the central supramolecular activation cluster (cSMAC) region of the immunological synapse. CD28 signaling through PI3K results in the recruitment of protein kinase C (PKC)θ to the cSMAC, activation of NF-κB, and induction of IL-2 transcription. These results suggest that localized engagement of CD28 within the cSMAC may be required for CD28 activation and/or signal integration with TCR signals. To test this model we have examined the mechanism of CD28-mediated induction of IL-2 secretion when CD28 is engaged outside of the immunological synapse. CD4 T cells were stimulated with Ag presented by B7-negative APC and CD28 costimulation was provided in trans by anti-CD28-coated beads or by class II-negative, B7-positive cells. We show that induction of IL-2 secretion under these conditions did not require expression of PKCθ and did not induce NF-κB activation or IL-2 transcription. In contrast, CD28 costimulation in trans did induce IL-2 mRNA stability, accounting for the up-regulation of IL-2 secretion. These data indicate that the ability of CD28 to up-regulate IL-2 transcription requires colocalization of TCR and CD28 at the plasma membrane, possibly within the cSMAC of the immunological synapse. In contrast, the ability of CD28 to promote IL-2 mRNA stability can be transduced from a distal site from the TCR, suggesting that signal integration occurs downstream from the plasma membrane. These data support the potential role of trans costimulation in tumor and allograft rejection, but limit the potential functional impact that trans costimulation may have on T cell activation.
Selective Regulation of IL-10 Signaling and Function by Zymosan
Authors: Du Zhimei , Kelly Erin , Mecklenbräuker Ingrid , Agle Lucila , Herrero Carmen , Paik Paul , Ivashkiv Lionel B.
Published: April 2014

Balanced activity of pro- and anti-inflammatory cytokines during innate immune responses is required to allow effective host defense while avoiding tissue damage and autoimmunity. Induction of cytokine production after recognition of pathogen-associated molecular patterns (PAMPs) by innate immune cells has been well demonstrated, but modulation of cytokine function by PAMPs is not well understood. In this study we show that stimulation of macrophages with zymosan, which contains PAMPs derived from yeast, rapidly extinguished macrophage responses to IL-10, a suppressive cytokine that limits inflammatory tissue damage but also compromises host defense. The mechanism of inhibition involved protein kinase Cβ and internalization of IL-10R, and was independent of TLR2 and phagocytosis. Inhibition of IL-10 signaling and function required direct contact with zymosan, and cells in an inflammatory environment that had not contacted zymosan remained responsive to the paracrine activity of zymosan-induced IL-10. These results reveal a mechanism that regulates IL-10 function such that antimicrobial functions of infected macrophages are not suppressed, but the activation of surrounding noninfected cells and subsequent tissue damage are limited. The fate of individual cells in an inflammatory microenvironment is thus specified by dynamic interactions among host cells, microbes, and cytokines that determine the balance between protection and pathology.
Coordination of NF-κB and NFAT Antagonism by the Forkhead Transcription Factor Foxd1
Authors: Lin Ling , Peng Stanford L.
Published: April 2014

Forkhead transcription factors play critical roles in the maintenance of immune homeostasis. In this study, we demonstrate that this regulation most likely involves intricate interactions between the forkhead family members and inflammatory transcription factors: the forkhead member Foxd1 coordinates the regulation of the activity of two key inflammatory transcription factors, NF-AT and NF-κB, with Foxd1 deficiency resulting in multiorgan, systemic inflammation, exaggerated Th cell-derived cytokine production, and T cell proliferation in autologous MLRs. Foxd1-deficient T cells possess increased activity of both NF-AT and NF-κB: the former correlates with the ability of Foxd1 to regulate casein kinase 1, an NF-AT inhibitory kinase; the latter with the ability of Foxd1 to regulate Foxj1, which regulates the NF-κB inhibitory subunit IκBβ. Thus, Foxd1 modulates inflammatory reactions and prevents autoimmunity by directly regulating anti-inflammatory regulators of the NF-AT pathway, and by coordinating the suppression of the NF-κB pathway via Foxj1. These findings indicate the presence of a general network of forkhead proteins that enforce T cell quiescence.
IFN-γ Enhances Production of Nitric Oxide from Macrophages via a Mechanism That Depends on Nucleotide Oligomerization Domain-2
Authors: Tötemeyer Sabine , Sheppard Mark , Lloyd Adrian , Roper David , Dowson Christopher , Underhill David , Murray Peter , Maskell Duncan , Bryant Clare
Published: April 2014

Pattern recognition receptors are central to the responsiveness of various eukaryotic cell types when they encounter pathogen-associated molecular patterns. IFN-γ is a cytokine that is elevated in humans and other animals with bacterial infection and enhances the LPS-induced production of antibacterial mediators by macrophages. Mice lacking the pattern recognition receptor, TLR4, respond very poorly to stimulation by LPS, but administration of IFN-γ has been described as restoring apparent sensitivity to this stimulatory ligand. In this study, we show that IFN-γ primes murine macrophages stimulated by crude LPS preparations to produce the antibacterial mediator NO, a proportion of which is independent of TLRs 2 and 4. This response is lost in tlr4−/− IFN-γ-primed murine macrophages when the LPS preparation is highly purified. NO is also induced if chemically synthesized muramyl dipeptide, an intermediate in the biosynthesis of peptidoglycan, is used to stimulate macrophages primed with IFN-γ. This is absolutely dependent on the presence of a functional nucleotide oligomerization domain-2 (NOD-2) protein. IFN-γ increases NOD-2 expression and dissociates this protein from the actin cytoskeleton within the cell. IFN-γ priming of macrophages therefore reveals a key proinflammatory role for NOD-2. This study also shows that the effect of IFN-γ in restoring inflammatory responses to Gram-negative bacteria or bacterial products in mice with defective TLR4 signaling is likely to be due to a response to peptidoglycan, not LPS.
Differential T Cell-Mediated Regulation of CD23 (FcεRII) in B Cells and Follicular Dendritic Cells
Authors: Sukumar Selvakumar , Conrad Daniel H. , Szakal Andras K. , Tew John G.
Published: April 2014

Differences in murine follicular dendritic cells (FDC)-CD23 expression under Th1 vs Th2 conditions prompted the hypothesis that T cells help regulate the phenotype of FDCs. FDCs express CD40, suggesting that T cell-CD40L and lymphokines may be involved in regulating FDC-CD23. To test this, highly enriched FDCs were incubated with CD40L trimer or anti-CD40 to mimic T cell signaling in the presence of IFN-γ or IL-4. Surface expression of CD23 was determined by flow cytometry, whereas mRNA levels of CD23 and its isoforms CD23a and CD23b were independently measured by quantitative PCR. When FDCs were incubated with either CD40L trimer or agonistic anti-CD40 Ab, the expression of FDC-CD23 was increased both at the mRNA and protein levels. Moreover, engagement of FDC-CD40 enhanced mRNA levels for both CD23a and CD23b isoforms. In addition, IFN-γ substantially enhanced CD23a and CD23b mRNA levels in CD40-stimulated FDCs. Curiously, IL-4 could also up-regulate FDC-CD23a but not -CD23b. Anti-IFN-γ dramatically inhibited FDC-CD23 in mice immunized with CFA, whereas anti-IL-4 had only a modest inhibitory effect. In contrast with FDCs, IFN-γ inhibited surface expression of murine B cell-CD23 as well as mRNA for B cell CD23a and -CD23b, whereas IL-4 dramatically enhanced message for both isoforms as well as protein expression. In short, CD23 was regulated very differently in FDCs and B cells. Previous studies suggest that high levels of FDC-CD23 inhibit IgE production, and this IFN-γ and CD40L-mediated up-regulation of FDC-CD23 may explain, at least in part, why Th1 responses are associated with low IgE responses in vivo.
Wip1 Phosphatase-Deficient Mice Exhibit Defective T Cell Maturation Due To Sustained p53 Activation
Authors: Schito Marco L. , Demidov Oleg N. , Saito Shin’ichi , Ashwell Jonathan D. , Appella Ettore
Published: April 2014

The PP2C phosphatase Wip1 dephosphorylates p38 and blocks UV-induced p53 activation in cultured human cells. Although the level of TCR-induced p38 MAPK activity is initially comparable between Wip1−/− and wild-type thymocytes, phosphatase-deficient cells failed to down-regulate p38 MAPK activity after 6 h. Analysis of young Wip1-deficient mice showed that they had fewer splenic T cells. Their thymi were smaller, contained significantly fewer cells, and failed to undergo age-dependent involution compared with wild-type animals. Analysis of thymocyte subset numbers by flow cytometry suggested that cell numbers starting at the double-negative (DN)4 stage are significantly reduced in Wip1-deficient mice, and p53 activity is elevated in cell-sorted DN4 and double-positive subpopulations. Although apoptosis and proliferation was normal in Wip1−/− DN4 cells, they appeared to be in cell cycle arrest. In contrast, a significantly higher percentage of apoptotic cells were found in the double-positive population, and down-regulation of thymocyte p38 MAPK activation by anti-CD3 was delayed. To examine the role of p38 MAPK in early thymic subpopulations, fetal thymic organ cultures cultured in the presence/absence of a p38 MAPK inhibitor did not correct the thymic phenotype. In contrast, the abnormal thymic phenotype of Wip1-deficient mice was reversed in the absence of p53. These data suggest that Wip1 down-regulates p53 activation in the thymus and is required for normal αβ T cell development.
Nuclear Magnetic Resonance Structure-Based Epitope Mapping and Modulation of Dust Mite Group 13 Allergen as a Hypoallergen
Authors: Chan Siew Leong , Ong Seow Theng , Ong Su Yin , Chew Fook Tim , Mok Yu Keung
Published: April 2014

IgE-mediated allergic response involves cross-linking of IgE bound on mast cells by specific surface epitopes of allergens. Structural studies on IgE epitopes of allergens are essential in understanding the characteristics of an allergen and for development of specific allergen immunotherapy. We have determined the structure of a group 13 dust mite allergen from Dermatophagoides farinae, Der f 13, using nuclear magnetic resonance. Sequence comparison of Der f 13 with homologous human fatty acid-binding proteins revealed unique surface charged residues on Der f 13 that may be involved in IgE binding and allergenicity. Site-directed mutagenesis and IgE binding assays have confirmed four surface charged residues on opposite sides of the protein that are involved in IgE binding. A triple mutant of Der f 13 (E41A_K63A_K91A) has been generated and found to have significantly reduced IgE binding and histamine release in skin prick tests on patients allergenic to group 13 dust mite allergens. The triple mutant is also able to induce PBMC proliferation in allergic patients with indices similar to those of wild-type Der f 13 and shift the secretion of cytokines from a Th2 to a Th1 pattern. Mouse IgG serum raised using the triple mutant is capable to block the binding of IgE from allergic patients to wild-type Der f 13, indicating potential for the triple mutant as a hypoallergen for specific immunotherapy. Findings in this study imply the importance of surface charged residues on IgE binding and allergenicity of an allergen, as was also demonstrated in other major allergens studied.
Expression of Endoplasmic Reticulum Aminopeptidases in EBV-B Cell Lines from Healthy Donors and in Leukemia/Lymphoma, Carcinoma, and Melanoma Cell Lines
Authors: Fruci Doriana , Ferracuti Silvia , Limongi Maria Zaira , Cunsolo Veronica , Giorda Ezio , Fraioli Rocco , Sibilio Leonardo , Carroll Oliver , Hattori Akira , van Endert Peter M. , Giacomini Patrizio
Published: April 2014

Peptide trimming in the endoplasmic reticulum (ER), the final step required for the generation of most HLA class I-binding peptides, implicates the concerted action of two aminopeptidases, ERAP1 and ERAP2. Because defects in the expression of these peptidases could lead to aberrant surface HLA class I expression in tumor cells, we quantitatively assayed 14 EBV-B cell lines and 35 human tumor cell lines of various lineages for: 1) expression and enzymatic activities of ERAP1 and ERAP2; 2) ER peptide-trimming activity in microsomes; 3) expression of HLA class I H chains and TAP1; and 4) surface HLA class I expression. ERAP1 and ERAP2 expression was detectable in all of the EBV-B and tumor cell lines, but in the latter it was extremely variable, sometimes barely detectable, and not coordinated. The expression of the two aminopeptidases corresponded well to the respective enzymatic activities in most cell lines. A peptide-trimming assay in microsomes revealed additional enzymatic activities, presumably contributed by other unidentified aminopeptidases sharing substrate specificity with ERAP2. Interestingly, surface HLA class I expression showed significant correlation with ERAP1 activity, but not with the activity of either ERAP2 or other unidentified aminopeptidases. Transfection with ERAP1 or ERAP2 of two tumor cell lines selected for simultaneous low expression of the two aminopeptidases resulted in the expected, moderate increases of class I surface expression. Thus, low and/or imbalanced expression of ERAP1 and probably ERAP2 may cause improper Ag processing and favor tumor escape from the immune surveillance.
Transcriptional Regulation of CD4 Gene Expression by T Cell Factor-1/β-Catenin Pathway
Authors: Huang Zhaofeng , Xie Huimin , Ioannidis Vassilio , Held Werner , Clevers Hans , Sadim Maureen S. , Sun Zuoming
Published: April 2014

By interacting with MHC class II molecules, CD4 facilitates lineage development as well as activation of Th cells. Expression of physiological levels of CD4 requires a proximal CD4 enhancer to stimulate basic CD4 promoter activity. T cell factor (TCF)-1/β-catenin pathway has previously been shown to regulate thymocyte survival via up-regulating antiapoptotic molecule Bcl-xL. By both loss and gain of function studies, in this study we show additional function of TCF-1/β-catenin pathway in the regulation of CD4 expression in vivo. Mice deficient in TCF-1 displayed significantly reduced protein and mRNA levels of CD4 in CD4+CD8+ double-positive (DP) thymocytes. A transgene encoding Bcl-2 restored survival but not CD4 levels of TCF-1−/− DP cells. Thus, TCF-1-regulated survival and CD4 expression are two separate events. In contrast, CD4 levels were restored on DP TCF-1−/− cells by transgenic expression of a wild-type TCF-1, but not a truncated TCF-1 that lacks a domain required for interacting with β-catenin. Furthermore, forced expression of a stabilized β-catenin, a coactivator of TCF-1, resulted in up-regulation of CD4. TCF-1 or stabilized β-catenin greatly stimulated activity of a CD4 reporter gene driven by a basic CD4 promoter and the CD4 enhancer. However, mutation of a potential TCF binding site located within the enhancer abrogated TCF-1 and β-catenin-mediated activation of CD4 reporter. Finally, recruitment of TCF-1 to CD4 enhancer was detected in wild-type but not TCF-1 null mice by chromatin-immunoprecipitation analysis. Thus, our results demonstrated that TCF/β-catenin pathway enhances CD4 expression in vivo by recruiting TCF-1 to stimulate CD4 enhancer activity.
TLR3 Can Directly Trigger Apoptosis in Human Cancer Cells
Authors: Salaun Bruno , Coste Isabelle , Rissoan Marie-Clotilde , Lebecque Serge J. , Renno Toufic
Published: April 2014

TLRs function as molecular sensors to detect pathogen-derived products and trigger protective responses ranging from secretion of cytokines that increase the resistance of infected cells and chemokines that recruit immune cells to cell death that limits microbe spreading. Viral dsRNA participate in virus-infected cell apoptosis, but the signaling pathway involved remains unclear. In this study we show that synthetic dsRNA induces apoptosis of human breast cancer cells in a TLR3-dependent manner, which involves the molecular adaptor Toll/IL-1R domain-containing adapter inducing IFN-β and type I IFN autocrine signaling, but occurs independently of the dsRNA-activated kinase. Moreover, detailed molecular analysis of dsRNA-induced cell death established the proapoptotic role of IL-1R-associated kinase-4 and NF-κB downstream of TLR3 as well as the activation of the extrinsic caspases. The direct proapoptotic activity of endogenous human TLR3 expressed by cancerous cells reveals a novel aspect of the multiple-faced TLR biology, which may open new clinical prospects for using TLR3 agonists as cytotoxic agents in selected cancers.
The LFA-1 Adhesion Molecule Is Required for Protective Immunity during PulmonaryMycobacterium tuberculosisInfection
Authors: Ghosh Shamik , Chackerian Alissa A. , Parker Christina M. , Ballantyne Christie M. , Behar Samuel M.
Published: April 2014

Host immunity to Mycobacterium tuberculosis is mediated by T cells that recognize and activate infected macrophages to control intracellular bacterial replication. The early appearance of T cells in the lungs of infected mice correlates with greater resistance to infection. However, it is unknown whether the trafficking of T cells to the lung following infection is dependent upon the expression of certain adhesion molecules. To address this question, we infected knockout (KO) mice that have defective expression of CD11a, CD11b, CD18, CD62, CD103, or β7. We found that the integrins CD11a and CD18 are absolutely required for host resistance following infection with aerosolized M. tuberculosis. Although Ag-specific T cells are generated following infection of CD11a KO mice, T cell priming is delayed, T cell trafficking to the lung is impaired, and fewer ESAT6-specific CD4+ T cells are found in the lungs of CD11a KO mice compared with control mice. Thus, LFA-1 (CD11a/CD18) plays an essential role in immunity to M. tuberculosis infection.
Targeted Immunomodulation of the NF-κB Pathway in Airway Epithelium Impacts Host Defense against Pseudomonas aeruginosa
Authors: Sadikot Ruxana T. , Zeng Heng , Joo Myungsoo , Everhart M. Brett , Sherrill Taylor P. , Li Bo , Cheng Dong-sheng , Yull Fiona E. , Christman John W. , Blackwell Timothy S.
Published: April 2014

We investigated the impact of inflammatory signaling in airway epithelial cells on host defense against Pseudomonas aeruginosa, a major cause of nosocomial pneumonia. In mice, airway instillation of P. aeruginosa resulted in NF-κB activation in the lungs that was primarily localized to the bronchial epithelium at 4 h, but was present in a variety of cell types by 24 h. We modulated NF-κB activity in airway epithelium by intratracheal delivery of adenoviral vectors expressing RelA (AdRelA) or a dominant inhibitor of NF-κB before P. aeruginosa infection. Bacterial clearance was enhanced by up-regulation of NF-κB activity following AdRelA administration and was impaired by treatment with a dominant inhibitor of NF-κB. The TNF-α concentration in lung lavage was increased by AdRelA treatment and beneficial effects of NF-κB up-regulation were abrogated in TNF-α-deficient mice. In contrast, NF-κB inhibition reduced MIP-2 expression and neutrophil influx following P. aeruginosa infection. Therefore, inflammatory signaling through the NF-κB pathway in airway epithelial cells critically regulates the innate immune response to P. aeruginosa.
Inducible Activation of TLR4 Confers Resistance to Hyperoxia-Induced Pulmonary Apoptosis
Authors: Qureshi Salman T. , Zhang Xuchen , Aberg Erika , Bousette Nicolas , Giaid Adel , Shan Peiying , Medzhitov Ruslan M. , Lee Patty J.
Published: April 2014

TLRs are essential mediators of host defense against infection via recognition of unique microbial structures. Recent observations indicate that TLR4, the principal receptor for bacterial LPS, may also be activated by noninfectious stimuli including host-derived molecules and environmental oxidant stress. In mice, susceptibility to ozone-induced lung permeability has been linked to the wild-type allele of TLR4, whereas deficiency of TLR4 predisposes to lethal lung injury in hyperoxia. To precisely characterize the role of lung epithelial TLR4 expression in the host response to oxidant stress, we have created an inducible transgenic mouse model that targets the human TLR4 signaling domain to the airways. Exposure of induced transgenic mice to hyperoxia revealed a significant reduction in pulmonary apoptosis compared with controls. This phenotype was associated with sustained up-regulation of antiapoptotic molecules such as heme oxygenase-1 and Bcl-2, yet only transient activation of the transcription factor NF-κB. Specific in vivo knockdown of pulmonary heme oxygenase-1 or Bcl-2 expression by intranasal administration of short interfering RNA blocked the effect of TLR4 signaling on hyperoxia-induced lung apoptosis. These results define a novel role for lung epithelial TLR4 as a modulator of cellular apoptosis in response to oxidant stress.
STAT1 in Peripheral Tissue Differentially Regulates Homing of Antigen-Specific Th1 and Th2 Cells
Authors: Mikhak Zamaneh , Fleming Carolyn M. , Medoff Benjamin D. , Thomas Seddon Y. , Tager Andrew M. , Campanella Gabriele S. , Luster Andrew D.
Published: April 2014

Th1 and Th2 effector CD4+ T cells orchestrate distinct counterregulatory biological responses. To deliver effective tissue Th1- and Th2-type responses, Th1 and Th2 cell recruitment into tissue must be differentially regulated. We show that tissue-derived STAT1 controls the trafficking of adoptively transferred, Ag-specific, wild-type Th1 cells into the lung. Trafficking of Th1 and Th2 cells is differentially regulated as STAT6, which regulates Th2 cell trafficking, had no effect on the trafficking of Th1 cells and STAT1 deficiency did not alter Th2 cell trafficking. We demonstrate that STAT1 control of Th1 cell trafficking is not mediated through T-bet. STAT1 controls the recruitment of Th1cells through the induction of CXCL9, CXCL10, CXCL11, and CXCL16, whose expression levels in the lung were markedly decreased in STAT1−/− mice. CXCL10 replacement partially restored Th1 cell trafficking in STAT1-deficient mice in vivo, and deficiency in CXCR3, the receptor for CXCL9, CXCL10, and CXCL11, impaired the trafficking of adoptively transferred Th1 cells in wild-type mice. Our work identifies that STAT1 in peripheral tissue regulates the homing of Ag-specific Th1 cells through the induction of a distinct subset of chemokines and establishes that Th1 and Th2 cell trafficking is differentially controlled in vivo by STAT1 and STAT6, respectively.
Role of CCR5 in the Pathogenesis of IL-13-Induced Inflammation and Remodeling
Authors: Ma Bing , Liu Wei , Homer Robert J. , Lee Patty J. , Coyle Anthony J. , Lora Jose M. , Lee Chun Geun , Elias Jack A.
Published: April 2014

IL-13 is a major effector at sites of Th2 inflammation and tissue remodeling. In these locations, it frequently coexists with the CCR5 chemokine receptor and its ligands MIP-1α/CCL3 and MIP-1β/CCL4. We hypothesized that CCR5 induction and activation play important roles in the pathogenesis of IL-13-induced tissue responses. To test this hypothesis, we evaluated the effects of IL-13 on the expression of CCR5 in the murine lung. We also compared the effects of lung-targeted transgenic IL-13 in mice treated with anti-CCR5 or an Ab control and mice with wild-type or null CCR5 loci. These studies demonstrate that IL-13 is a potent stimulator of epithelial cell CCR5 expression. They also demonstrate that CCR5 neutralization or a deficiency of CCR5 significantly decreases IL-13-induced inflammation, alveolar remodeling, structural and inflammatory cell apoptosis, and respiratory failure and death. Lastly, these studies provide mechanistic insights by demonstrating that CCR5 is required for optimal IL-13 stimulation of select chemokines (MIP-1α/CCL3, MIP-1β/CCL4, MCP-1/CCL-2), matrix metalloproteinase-9 and cell death regulators (Fas, TNF, TNFR1, TNFR2, Bid), optimal IL-13 inhibition of α1-antitrypsin, and IL-13-induction of and activation of caspases-3, -8, and-9. Collectively, these studies demonstrate that CCR5 plays a critical role in the pathogenesis of IL-13-induced inflammation and tissue remodeling.
ASC Directs NF-κB Activation by Regulating Receptor Interacting Protein-2 (RIP2) Caspase-1 Interactions
Authors: Sarkar Anasuya , Duncan Michelle , Hart Judy , Hertlein Erin , Guttridge Denis C. , Wewers Mark D.
Published: April 2014

Receptor interacting protein-2 (RIP2) is a caspase recruitment domain (CARD)-containing kinase that interacts with caspase-1 and plays an important role in NF-κB activation. Apoptosis-associated speck-like protein containing a CARD (ASC) is a PYRIN and CARD-containing molecule, important in the induction of apoptosis and caspase-1 activation. Although RIP2 has also been linked to caspase-1 activation, RIP2 knockout animals fail to show a defect in caspase-1-mediated processing of proIL-1β to its active form. Therefore, RIP2 function in binding to caspase-1 remains poorly understood. We hypothesized that caspase-1 may serve as a scaffolding molecule that promotes RIP2 interaction with IκB kinase-γ thus inducing NF-κB activation. We further hypothesized that ASC, which also interacts with caspase-1 via its CARD, may interfere with the caspase-1 RIP2 interaction. In HEK293 cells, ASC induced prominent activation of caspase-1 and proIL-1β processing. RIP2 transient transfection induced transcription of an NF-κB reporter gene. This RIP2-induced NF-κB activity and caspase-1 binding was inhibited in a dose-dependent fashion by ASC. Consistent with a role for caspase-1 as a scaffold for RIP2, caspase-1 knockout macrophages were suppressed in their ability to activate NF-κB, and septic caspase-1 knockout animals produced less IL-6, a functional marker of NF-κB activity. Lastly, THP-1 cells treated with small interfering RNA for ASC decreased their caspase-1 activity while enhancing their NF-κB signal. These data suggest that ASC may direct caspase-1 away from RIP2-mediated NF-κB activation, toward caspase-1-mediated processing of proIL-1β by interfering with the RIP2 caspase-1 interaction.