bluuebunny/crossref_metadata_2025 huggingface dataset:
DM Peptide-Editing Function Leads to Immunodominance in CD4 T Cell Responses In Vivo
Authors: Nanda Navreet K. , Bikoff Elizabeth K.
Published: April 2014

DM functions as a peptide editor for MHC class II-bound peptides. We examined the hypothesis that DM peptide editing plays a key role in focusing the in vivo CD4 T cell responses against complex pathogens and protein Ags to only one, or at most a few, immunodominant peptides. Most CD4 T cells elicited in the wild-type BALB/c (H-2d) mice infected with Leishmania major predominantly recognize a single epitope 158–173 within Leishmania homologue of activated receptor for c-kinase (LACK), as is the case when these mice are immunized with rLACK. Using DM-deficient (DM−/−) H-2d mice, we now show that in the absence of DM, the in vivo CD4 T cell responses to rLACK are skewed away from the immunodominant epitopes and are diversified to include two novel epitopes (LACK 33–48 and 261–276). DM−/− B10.BR (H-2k) mice showed similar results. These results constitute the first demonstration of the role of DM peptide editing in sculpting the specificity and immunodominance in in vivo CD4 T cell responses.
Rab5 and Rab7, but Not ARF6, Govern the Early Events of HIV-1 Infection in Polarized Human Placental Cells
Authors: Vidricaire Gaël , Tremblay Michel J.
Published: April 2014

Trophoblasts, the structural cells of the placenta, are thought to play a determinant role in in utero HIV type 1 (HIV-1) transmission. We have accumulated evidence suggesting that HIV-1 infection of these cells is associated with uptake by an unusual clathrin/caveolae-independent endocytic pathway and that endocytosis is followed by trafficking through multiple organelles. Furthermore, part of this trafficking involves the transit of HIV-1 from transferrin-negative to EEA1 and transferrin-positive endosomes, suggesting a merger from nonclassical to classical endocytic pathways in these cells. In the present article, the relationship between the presence of HIV-1 within specific endosomes and infection was studied. We demonstrate that viral infection is virtually lost when endosome inhibitors are added shortly after exposure to HIV-1. Thus, contrary to what is seen in CD4+ T lymphocytes, the initial presence of HIV-1 within the endosomes is mandatory for infection to take place. Importantly, this process is independent of the viral envelope proteins gp120 and gp41. The Rab family of small GTPases coordinates the vesicular transport between the different endocytic organelles. Experiments performed with various expression vectors indicated that HIV-1 infection in polarized trophoblasts relies on Rab5 and Rab7 without the contribution of Arf6 or Rab11. Furthermore, we conclude that Rab5 drives movements from raft-rich region to early endosomes, and this transit is required for subsequently reaching late endosomes via Rab7. This complex trafficking is mandatory for HIV-1 infection to proceed in human polarized trophoblasts.
A Single DH Gene Segment Creates Its Own Unique CDR-H3 Repertoire and Is Sufficient for B cell Development and Immune Function
Authors: Schelonka Robert L. , Ivanov Ivaylo I. , Jung David H. , Ippolito Gregory C. , Nitschke Lars , Zhuang Yingxin , Gartland G. Larry , Pelkonen Jukka , Alt Frederick W. , Rajewsky Klaus , Schroeder Harry W.
Published: April 2014

To test the contribution of individual D gene segments to B cell development and function, we used gene targeting to create mice that contain only DFL16.1 in the DH locus. We term this D-limited IgH allele ΔD-DFL. Although the absolute number of IgM+IgD− B cells in the bone marrow was decreased, homozygous ΔD-DFL BALB/c mice contained normal numbers of IgM+IgD+ B cells in bone marrow and spleen and normal numbers of B1a, B1b, and B2 cells in the peritoneal cavity. Bone marrow IgM+IgD+ B cells express a CDR-H3 repertoire similar in length and amino acid composition to the DFL16.1 subset of the wild-type BALB/c repertoire but divergent from sequences that do not contain DFL16.1. This similarity in content is the product of both germline bias and somatic selection, especially in the transition to the mature IgM+IgD+ stage of development. Serum Ig concentrations and the humoral immune response to a T-dependent Ag ([4-hydroxy-3-nitrophenyl]acetyl hapten) were nearly identical to wild-type littermate controls. A greater variance in the immune response to the T-independent Ag (α(1→3)-dextran) was observed in ΔD-DFL homozygotes, with half of the mice exhibiting levels below the range exhibited by controls. Although limited to a repertoire specific to DFL16.1, the presence of a single DH gene segment of normal sequence was sufficient for development of normal numbers of mature B cells and for robust humoral immune function.
Structural Requirements for the Interaction of Human IgA with the Human Polymeric Ig Receptor
Authors: Lewis Melanie J. , Pleass Richard J. , Batten Margaret R. , Atkin Julie D. , Woof Jenny M.
Published: April 2014

Transport of polymeric IgA onto mucosal surfaces to become secretory IgA is mediated by the polymeric Ig receptor (pIgR). To study the interaction of human dimeric IgA (dIgA) (the predominant form of IgA polymer) with the human pIgR (hpIgR), we generated recombinant wild-type dIgA1 and dIgA2m(1) and various mutant dIgA1 and analyzed their interaction with a recombinant human secretory component and membrane-expressed hpIgR. We found that wild-type dIgA1 and dIgA2m(1) bound to recombinant human secretory component with similar affinity and were transcytosed by the hpIgR to the same extent. Mutation of the IgA Cα2 domain residue Cys311 to Ser reduced binding to hpIgR, possibly through disruption of noncovalent interactions between the Cα2 domain and domain 5 of the receptor. Within the Cα3 domain of IgA1, we found that combined mutation of residues Phe411, Val413, and Thr414, which lie close to residues previously implicated in hpIgR binding, abolished interaction with the receptor. Mutation of residue Lys377, located very close to this same region, perturbed receptor interaction. In addition, 4 aa (Pro440-Phe443), which lie on a loop at the domain interface and form part of the binding site for human FcαRI, appear to contribute to hpIgR binding. Lastly, use of a monomeric IgA1 mutant lacking the tailpiece revealed that the tailpiece does not occlude hpIgR-binding residues in IgA1 monomers. This directed mutagenesis approach has thus identified motifs lying principally across the upper surface of the Cα3 domain (i.e., that closest to Cα2) critical for human pIgR binding and transcytosis.
Early Interaction of Yersinia pestis with APCs in the Lung
Authors: Bosio Catharine M. , Goodyear Andrew W. , Dow Steven W.
Published: April 2014

Despite the importance of pneumonic plague, little is known of the early pulmonary immune responses that occur following inhalation of Yersinia pestis. Therefore, we conducted studies to identify the early target cells for uptake of Y. pestis in the lungs following intratracheal or i.v. inoculation. Following intratracheal inoculation, Y. pestis was rapidly internalized primarily by a distinctive population of CD11c+DEC-205+CD11b− cells in the airways, whereas i.v. inoculation resulted in uptake primarily by CD11b+CD11c− macrophages and granulocytes in lung tissues. The airway cells internalized and were infected by Y. pestis, but did not support active replication of the organism. Intratracheal inoculation of Y. pestis resulted in rapid activation of airway CD11c+ cells, followed within 24 h by the selective disappearance of these cells from the airways and lungs and the accumulation of apoptotic CD11c+ cells in draining lymph nodes. When CD11c+ cells in the airways were depleted using liposomal clodronate before infection, this resulted in a significantly increased replication of Y. pestis in the lungs and dissemination to the spleen and draining lymph nodes. These findings suggest that CD11c+ cells in the airways play an important role in suppressing the initial replication and dissemination of inhaled Y. pestis, although these results will also require confirmation using fully virulent strains of Y. pestis. Depletion of these airway cells by Y. pestis may therefore be one strategy the organism uses to overcome pulmonary defenses following inhalation of the organism.
Differentiation to the CCR2+ Inflammatory Phenotype In Vivo Is a Constitutive, Time-Limited Property of Blood Monocytes and Is Independent of Local Inflammatory Mediators
Authors: Xu Heping , Manivannan Ayyakkannu , Dawson Rosemary , Crane Isabel J. , Mack Matthias , Sharp Peter , Liversidge Janet
Published: April 2014

It is proposed that CCR2+ monocytes are specifically recruited to inflammatory sites, whereas CCR2− monocytes are recruited to normal tissue to become resident macrophages. Whether these subsets represent separate lineages, how differential trafficking is regulated and whether monocytes undergo further differentiation is uncertain. Using a mouse model of autoimmune uveoretinitis we examined monocyte trafficking to the inflamed retina in vivo. We show that bone marrow-derived CD11b+ F4/80− monocytes require 24 to 48 h within the circulation and lymphoid system before acquiring the CCR2+ phenotype and trafficking to the inflamed retina is enabled. This phenotype, and the capacity to traffic were lost by 72 h. Monocyte CCR2 expression followed a similar time course in normal mice indicating that differentiation to an inflammatory phenotype is a constitutive, time-limited property, independent of local inflammatory mediators. Phenotypic analysis of adoptively transferred cells indicated that circulating inflammatory monocytes also differentiate into CD11c+ and B220+ dendritic cells and F4/80+ tissue macrophages in vivo. Our data supports the hypothesis of continuous extravasation and progressive differentiation over time of inflammatory monocytes in the circulation rather than replication within the actively inflamed tissue, and supports the concept of myeloid dendritic cell differentiation from trafficking monocytes under physiological conditions in vivo.
CXCR2/CXCR2 Ligand Biology during Lung Transplant Ischemia-Reperfusion Injury
Authors: Belperio John A. , Keane Michael P. , Burdick Marie D. , Gomperts Brigitte N. , Xue Ying Ying , Hong Kurt , Mestas Javier , Zisman David , Ardehali Abbas , Saggar Rajan , Lynch Joseph P. , Ross David J. , Strieter Robert M.
Published: April 2014

Lung transplantation is a therapeutic option for a number of end-stage pulmonary disorders. Early lung allograft dysfunction (ischemia-reperfusion injury) continues to be the most common cause of early mortality after lung transplantation and a significant risk factor for the development of bronchiolitis obliterans syndrome. Ischemia-reperfusion injury is characterized histopathologically by lung edema and a neutrophil predominate leukocyte extravasation. The specific mechanism(s) that recruit leukocytes to the lung during post-lung transplantation ischemia-reperfusion injury have not been fully elucidated. Because the ELR+ CXC chemokines are potent neutrophil chemoattractants, we investigated their role during post-lung transplantation ischemic-reperfusion injury. We found elevated levels of multiple ELR+ CXC chemokines in human bronchoalveolar lavage fluid from patients with ischemia-reperfusion injury. Proof of concept studies using a rat orthotopic lung transplantation model of “cold” ischemic-reperfusion injury demonstrated an increase in lung graft neutrophil sequestration and injury. In addition, lung expression of CXCL1, CXCL2/3, and their shared receptor CXCR2 paralleled lung neutrophil infiltration and injury. Importantly, inhibition of CXCR2/CXCR2 ligand interactions in vivo led to a marked reduction in lung neutrophil sequestration and graft injury. Taken together these experiments support the notion that increased expression of ELR+ CXC chemokines and their interaction with CXCR2 plays an important role in the pathogenesis of post-lung transplantation cold ischemia-reperfusion injury.
HIV-1-Specific CD8+ T Cell Responses and Viral Evolution in Women and Infants
Authors: Sanchez-Merino Victor , Nie Siwei , Luzuriaga Katherine
Published: April 2014

CD8+ T lymphocyte responses play an important role in controlling HIV-1 replication but escape from CD8+ T cell surveillance may limit the effectiveness of these responses. Mother-to-child transmission of CD8+ T cell escape variants may particularly affect CD8+ T cell recognition of infant HIV-1 epitopes. In this study, amino acid sequence variation in HIV-1 gag and nef was examined in five untreated mother-infant pairs to evaluate the potential role of CD8+ T cell responses in the evolution of the viral quasispecies. Several CD8+ T cell escape variants were detected in maternal plasma. Evaluation of infant plasma viruses at 1–3 mo documented heterogeneity of gag and nef gene sequences and mother-to-child transmission of CD8+ T cell escape variants. Infant HLA haplotype and viral fitness appeared to determine the stability of the escape mutants in the infant over time. Changes in CD8+ T cell epitope sequences were detected in infants’ sequential plasma specimens, suggesting that infants are capable of generating virus-specific CD8+ T cell responses that exert selective pressures in vivo. Altogether, these studies document that HIV-1-specific CD8+ T cell responses contribute to the evolution of the viral quasispecies in HIV-1-infected women and their infants and may have important implications for vaccine design.
Modulation of Ovalbumin-Induced Airway Inflammation and Hyperreactivity by Tolerogenic APC
Authors: Zhang-Hoover Jie , Finn Patricia , Stein-Streilein Joan
Published: April 2014

Allergic asthma is mediated in part by unregulated Th2 inflammation in response to an allergen. Induction of peripheral tolerance by inoculation of Ags into the anterior chamber of the eye (ocular tolerance) before sensitization blocks Th2 responses. Thus, we proposed that induction of ocular tolerance to the allergen might modulate an ongoing allergen-induced Th2 pathogenesis in the lung. We initiated ocular tolerance in previously immunized mice in a classic mouse model of OVA-induced pulmonary allergic inflammation. In the model of ocular tolerance, the need for inoculation of Ag into the anterior chamber can be bypassed by i.v. inoculation of in vitro-generated tolerogenic (TGF-β2-treated, Ag-pulsed) APC (tol-APC). We observed that with i.v. inoculation, such tolerogenic APC, but not control APC, reduced eosinophil and lymphocyte pulmonary infiltration in experimental mice. Similarly, production of Th2 cytokines (IL-4, -5, and -13), but not IFN-γ, was reduced. Importantly, airway hyperresponsiveness and mucus production were significantly reduced after treatment with the tol-APC. We also show that in vitro suppression of IL-13 production from OVA-sensitized effector T cells was mediated by CD8+, not CD4+, T regulatory cells. Thus, i.v. inoculation of the tol-APC induced peripheral tolerance that suppressed Th2-mediated pathogenesis in the lungs of presensitized mice. The ability of the tol-APC to induce peripheral tolerance and suppress existing Th2 immune inflammation may lead to novel therapies for pulmonary allergic inflammation and its related pathology.
RasGRP1 and RasGRP3 Regulate B Cell Proliferation by Facilitating B Cell Receptor-Ras Signaling
Authors: Coughlin Jason J. , Stang Stacey L. , Dower Nancy A. , Stone James C.
Published: April 2014

The RasGRPs are a family of Ras activators that possess diacylglycerol-binding C1 domains. In T cells, RasGRP1 links TCR signaling to Ras. B cells coexpress RasGRP1 and RasGRP3. Using Rasgrp1 and Rasgrp3 single and double null mutant mice, we analyzed the role of these proteins in signaling to Ras and Erk in B cells. RasGRP1 and RasGRP3 both contribute to BCR-induced Ras activation, although RasGRP3 alone is responsible for maintaining basal Ras-GTP levels in unstimulated cells. Surprisingly, RasGRP-mediated Ras activation is not essential for B cell development because this process occurs normally in double-mutant mice. However, RasGRP-deficient mice do exhibit humoral defects. Loss of RasGRP3 led to isotype-specific deficiencies in Ab induction in immunized young mice. As reported previously, older Rasgrp1−/− mice develop splenomegaly and antinuclear Abs as a result of a T cell defect. We find that such mice have elevated serum Ig levels of several isotypes. In contrast, Rasgrp3−/− mice exhibit hypogammaglobulinemia and show no signs of splenomegaly or autoimmunity. Double-mutant mice exhibit intermediate serum Ab titers, albeit higher than wild-type mice. Remarkably, double-mutant mice exhibit no signs of autoimmunity or splenomegaly. B cell proliferation induced by BCR ligation with or without IL-4 was found to be RasGRP1- and RasGRP3-dependent. However, the RasGRPs are not required for B cell proliferation per se, because LPS-induced proliferation is unaffected in double-mutant mice.
Membranous Glomerulonephritis Development with Th2-Type Immune Deviations in MRL/lpr Mice Deficient for IL-27 Receptor (WSX-1)
Authors: Shimizu Sakiko , Sugiyama Naonobu , Masutani Kohsuke , Sadanaga Atsushi , Miyazaki Yoshiyuki , Inoue Yasushi , Akahoshi Mitsuteru , Katafuchi Ritsuko , Hirakata Hideki , Harada Mine , Hamano Shinjiro , Nakashima Hitoshi , Yoshida Hiroki
Published: April 2014

MRL/lpr mice develop spontaneous glomerulonephritis that is essentially identical with diffuse proliferative glomerulonephritis (World Health Organization class IV) in human lupus nephritis. Lupus nephritis is one of the most serious complications of systemic lupus erythematosus. Diffuse proliferative glomerulonephritis is associated with autoimmune responses dominated by Th1 cells producing high levels of IFN-γ. The initial mounting of Th1 responses depends on the function of the WSX-1 gene, which encodes a subunit of the IL-27R with homology to IL-12R. In mice deficient for the WSX-1 gene, proper Th1 differentiation was impaired and abnormal Th2 skewing was observed during infection with some intracellular pathogens. Disruption of the WSX-1 gene dramatically changed the pathophysiology of glomerulonephritis developing in MRL/lpr mice. WSX-1−/− MRL/lpr mice developed disease resembling human membranous glomerulonephritis (World Health Organization class V) with a predominance of IgG1 in glomerular deposits, accompanied by increased IgG1 and IgE in the sera. T cells in WSX-1−/− MRL/lpr mice displayed significantly reduced IFN-γ production along with elevated IL-4 expression. Loss of WSX-1 thus favors Th2-type autoimmune responses, suggesting that the Th1/Th2 balance may be a pivotal determinant of human lupus nephritis development.
Analysis of Anthrax and Plague Biowarfare Vaccine Interactions with Human Monocyte-Derived Dendritic Cells
Authors: Skowera Anna , de Jong Esther C. , Schuitemaker Joost H. N. , Allen Jennifer S. , Wessely Simon C. , Griffiths Gareth , Kapsenberg Martien , Peakman Mark
Published: April 2014

The anti-biowarfare anthrax and plague vaccines require repeated dosing to achieve adequate protection. To test the hypothesis that this limited immunogenicity results from the nature of vaccine interactions with the host innate immune system, we investigated molecular and cellular interactions between vaccines, dendritic cells (DCs), and T cells and explored the potential for adjuvants (pertussis) to boost induction of host immunity. Human monocyte-derived DCs were matured in the presence of vaccines and analyzed for their ability to induce Th1/Th2 development from naive T cells, expression of cell surface maturation/costimulation molecules, and cytokine production. The vaccines showed different behavior patterns. Although the plague vaccine is equivalent to control maturation factors in maturation and stimulation of DCs and induces strong MLR and Th outgrowth, the anthrax vaccine is a poor inducer of DC maturation, as indicated by low levels of HLA-DR, CD86, and CD83 induction and minimal proinflammatory cytokine production. Interestingly, however, anthrax vaccine-treated DCs stimulate Th1 and Th2 outgrowth and a limited MLR response. There was no sustained negative modulatory effects of the anthrax vaccine on DCs, and its limited stimulatory effects could be overridden by coculture with pertussis. These results were supported by analysis of anthrax vaccine recall responses in subjects vaccinated using pertussis as an adjuvant, who demonstrate anthrax-specific effector T cell responses. These data show that the anthrax vaccine is a suboptimal DC stimulus that may in part explain the observation that it requires repeated administration in vivo and offer a rational basis for the use of complementary DC-maturing adjuvants in combined immunotherapy.
Programmed Death-1 (PD-1):PD-Ligand 1 Interactions Inhibit TCR-Mediated Positive Selection of Thymocytes
Authors: Keir Mary E. , Latchman Yvette E. , Freeman Gordon J. , Sharpe Arlene H.
Published: April 2014

Positive selection during thymocyte development is driven by the affinity and avidity of the TCR for MHC-peptide complexes expressed in the thymus. In this study, we show that programmed death-1 (PD-1), a member of the B7/CD28 family of costimulatory receptors, inhibits TCR-mediated positive selection through PD-1 ligand 1 (PD-L1):PD-1 interactions. Transgenic mice that constitutively overexpress PD-1 on CD4+CD8+ thymocytes display defects in positive selection in vivo. Using an in vitro model system, we find that PD-1 is up-regulated following TCR engagement on CD4+CD8+ murine thymocytes. Coligation of TCR and PD-1 on CD4+CD8+ thymocytes with a novel PD-1 agonistic mAb inhibits the activation of ERK and up-regulation of bcl-2, both of which are downstream mediators essential for positive selection. Inhibitory signals through PD-1 can overcome the ability of positive costimulators, such as CD2 and CD28, to facilitate positive selection. Finally, defects in positive selection that result from PD-1 overexpression in thymocytes resolve upon elimination of PD-L1, but not PD-1 ligand 2, expression. PD-L1-deficient mice have increased numbers of CD4+CD8+ and CD4+ thymocytes, indicating that PD-L1 is involved in normal thymic selection. These data demonstrate that PD-1:PD-L1 interactions are critical to positive selection and play a role in shaping the T cell repertoire.
CD8 T Cell Sensory Adaptation Dependent on TCR Avidity for Self-Antigens
Authors: Marquez Maria-Elena , Ellmeier Wilfried , Sanchez-Guajardo Vanesa , Freitas Antonio A. , Acuto Oreste , Di Bartolo Vincenzo
Published: April 2014

Adaptation of the T cell activation threshold may be one mechanism to control autoreactivity. To investigate its occurrence in vivo, we engineered a transgenic mouse model with increased TCR-dependent excitability by expressing a Zap70 gain-of-function mutant (ZAP-YEEI) in postselection CD8 thymocytes and T cells. Increased basal phosphorylation of the Zap70 substrate linker for activation of T cells was detected in ZAP-YEEI-bearing CD8 T cells. However, these cells were not activated, but had reduced levels of TCR and CD5. Moreover, they produced lower cytokine amounts and showed faster dephosphorylation of linker for activation of T cells and ERK upon activation. Normal TCR levels and cytokine production were restored by culturing cells in the absence of TCR/spMHC interaction, demonstrating dynamic tuning of peripheral T cell responses. The effect of avidity for self-ligand(s) on this sensory adaptation was studied by expressing ZAP-YEEI in P14 or HY TCR transgenic backgrounds. Unexpectedly, double-transgenic animals expressed ZAP-YEEI prematurely in double-positive thymocytes, but no overt alteration of selection processes was observed. Instead, modifications of TCR and CD5 expression due to ZAP-YEEI suggested that signal tuning occurred during thymic maturation. Importantly, although P14 × ZAP-YEEI peripheral CD8 T cells were reduced in number and showed lower Ag-induced cytokine production and limited lymphopenia-driven proliferation, the peripheral survival/expansion and Ag responsiveness of HY × ZAP-YEEI cells were enhanced. Our data provide support for central and peripheral sensory T cell adaptation induced as a function of TCR avidity for self-ligands and signaling level. This may contribute to buffer excessive autoreactivity while optimizing TCR repertoire usage.
The Exopolysaccharide Alginate Protects Pseudomonas aeruginosa Biofilm Bacteria from IFN-γ-Mediated Macrophage Killing
Authors: Leid Jeff G. , Willson Carey J. , Shirtliff Mark E. , Hassett Daniel J. , Parsek Matthew R. , Jeffers Alyssa K.
Published: April 2014

The ability of Pseudomonas aeruginosa to form biofilms and cause chronic infections in the lungs of cystic fibrosis patients is well documented. Numerous studies have revealed that P. aeruginosa biofilms are highly refractory to antibiotics. However, dramatically fewer studies have addressed P. aeruginosa biofilm resistance to the host’s immune system. In planktonic, unattached (nonbiofilm) P. aeruginosa, the exopolysaccharide alginate provides protection against a variety of host factors yet the role of alginate in protection of biofilm bacteria is unclear. To address this issue, we tested wild-type strains PAO1, PA14, the mucoid cystic fibrosis isolate, FRD1 (mucA22+), and the respective isogenic mutants which lacked the ability to produce alginate, for their susceptibility to human leukocytes in the presence and absence of IFN-γ. Human leukocytes, in the presence of recombinant human IFN-γ, killed biofilm bacteria lacking alginate after a 4-h challenge at 37°C. Bacterial killing was dependent on the presence of IFN-γ. Killing of the alginate-negative biofilm bacteria was mediated through mononuclear cell phagocytosis since treatment with cytochalasin B, which prevents actin polymerization, inhibited leukocyte-specific bacterial killing. By direct microscopic observation, phagocytosis of alginate-negative biofilm bacteria was significantly increased in the presence of IFN-γ vs all other treatments. Addition of exogenous, purified alginate to the alginate-negative biofilms restored resistance to human leukocyte killing. Our results suggest that although alginate may not play a significant role in bacterial attachment, biofilm development, and formation, it may play an important role in protecting mucoid P. aeruginosa biofilm bacteria from the human immune system.
A Predominant Role for Antibody in Acquired Immunity to Chlamydial Genital Tract Reinfection
Authors: Morrison Sandra G. , Morrison Richard P.
Published: April 2014

Acquired immunity to murine Chlamydia trachomatis genital tract reinfection has long been assumed to be solely dependent on cell-mediated immunity. However, in this study, we identify a previously unrecognized protective role for Ab. Immunity develops in Ab-deficient mice following the resolution of primary chlamydial genital infection. Subsequent depletion of CD4+ T cells, but not CD8+ T cells, in those immune Ab-deficient mice before secondary infectious challenge, resulted in an infection that did not resolve. Passive immunization with immune (convalescent) serum conferred a marked level of protective immunity to reinfection, which was characterized by a striking decrease in bacterial shedding, from >100,000 inclusion forming units to fewer than 10 inclusion forming units, and a shortened duration of infection. Furthermore, mAbs to the chlamydial major outer membrane protein and LPS conferred significant levels of immunity to reinfection and reduced chlamydial shedding by >100-fold. Anti-heat shock protein 60 mAb had no protective effect. In contrast to the marked protective efficacy of immune serum on reinfection, the course of primary infection was essentially unaltered by the passive transfer of immune serum. Our results convincingly demonstrate that Abs contribute importantly to immunity to chlamydial genital tract reinfection, and that Ab-mediated protection is highly dependent on CD4+ T cell-mediated adaptive changes that occur in the local genital tract tissues during primary infection. These results impact our understanding of immunity to chlamydial genital infection and may provide important insight into vaccine development.
Potentiation of Caspase-1 Activation by the P2X7 Receptor Is Dependent on TLR Signals and Requires NF-κB-Driven Protein Synthesis
Authors: Kahlenberg J. Michelle , Lundberg Kathleen C. , Kertesy Sylvia B. , Qu Yan , Dubyak George R.
Published: April 2014

The proinflammatory cytokines IL-1β and IL-18 are inactive until cleaved by the enzyme caspase-1. Stimulation of the P2X7 receptor (P2X7R), an ATP-gated ion channel, triggers rapid activation of caspase-1. In this study we demonstrate that pretreatment of primary and Bac1 murine macrophages with TLR agonists is required for caspase-1 activation by P2X7R but it is not required for activation of the receptor itself. Caspase-1 activation by nigericin, a K+/H+ ionophore, similarly requires LPS priming. This priming by LPS is dependent on protein synthesis, given that cyclohexamide blocks the ability of LPS to prime macrophages for activation of caspase-1 by the P2X7R. This protein synthesis is likely mediated by NF-κB, as pretreatment of cells with the proteasome inhibitor MG132, or the IκB kinase inhibitor Bay 11-7085 before LPS stimulation blocks the ability of LPS to potentiate the activation of caspase-1 by the P2X7R. Thus, caspase-1 regulation in macrophages requires inflammatory stimuli that signal through the TLRs to up-regulate gene products required for activation of the caspase-1 processing machinery in response to K+-releasing stimuli such as ATP.
A Role for the Neuronal Protein Collapsin Response Mediator Protein 2 in T Lymphocyte Polarization and Migration
Authors: Vincent Peggy , Collette Yves , Marignier Romain , Vuaillat Carine , Rogemond Véronique , Davoust Nathalie , Malcus Christophe , Cavagna Sylvie , Gessain Antoine , Machuca-Gayet Irma , Belin Marie-Françoise , Quach Tam , Giraudon Pascale
Published: April 2014

The semaphorin-signaling transducer collapsin response mediator protein 2 (CRMP2) has been identified in the nervous system where it mediates Sema3A-induced growth cone navigation. In the present study, we provide first evidence that CRMP2 is present in the immune system and plays a critical role in T lymphocyte function. CRMP2 redistribution at the uropod in polarized T cells, a structural support of lymphocyte motility, suggests that it may regulate T cell migration. This was evidenced in primary T cells by small-interfering RNA-mediated CRMP2 gene silencing and blocking Ab, as well as CRMP2 overexpression in Jurkat T cells tested in a chemokine- and semaphorin-mediated transmigration assay. Expression analysis in PBMC from healthy donors showed that CRMP2 is enhanced in cell subsets bearing the activation markers CD69+ and HLA-DR+. Heightened expression in T lymphocytes of patients suffering from neuroinflammatory disease with enhanced T cell-transmigrating activity points to a role for CRMP2 in pathogenesis. The elucidation of the signals and mechanisms that control this pathway will lead to a better understanding of T cell trafficking in physiological and pathological situations.
Evidence for Multiple Shared Antigenic Determinants within Ro60 and Other Lupus-Related Ribonucleoprotein Autoantigens in Human Autoimmune Responses
Authors: Pal Rahul , Deshmukh Umesh S. , Ohyama Yukiko , Fang Qiang , Kannapell Carol C. , Gaskin Felicia , Fu Shu Man
Published: April 2014

Ab responses directed against several ribonucleoprotein (RNP) Ags are a characteristic feature of systemic lupus erythematosus (SLE). Previous work in our laboratory using mouse model systems had revealed that both epitope spreading and inherent cross-reactivity between ribonucleoproteins contributes to the observed multiple specificities in autoimmune sera. We have now extended these studies to human autoimmune responses. Using purified polyclonal and mAbs derived from SLE patients, cross-reactivity between Ro60 and SmD was demonstrated. The cross-reactive epitope was mapped to nonhomologous regions on Ro60481–505 and SmD88–102. Five mAbs specifically recognized apoptotic cells, demonstrated variable levels of cross-reactivity toward other nonhomologous ribonucleoprotein targets and bound multiple, nonoverlapping and nonhomologous epitopes on Ro60. Our study demonstrates that cross-reactivity between frequently targeted autoantigens is an important aspect of human systemic autoimmune responses. The presence of multiple cross-reactive epitopes on Ro60 might be important for the generation of anti-Ro60 Ab in SLE patients and in normal individuals displaying no evidence of clinical disease.
Cell- Rather Than Antibody-Mediated Immunity Leads to the Development of Profound Thrombocytopenia during Experimental Plasmodium berghei Malaria
Authors: Gramaglia Irene , Sahlin Herman , Nolan John P. , Frangos John A. , Intaglietta Marcos , van der Heyde Henri C.
Published: April 2014

Experimental malarial thrombocytopenia can reach life-threatening levels and is believed to be due to Abs targeting platelets for destruction by the reticuloendothelial system. However, we report that Abs account for at most 15% of platelet destruction as Plasmodium berghei-infected B cell-deficient mice exhibited profound thrombocytopenia (83%) as did C57BL/6 controls (98%). Further, no significant increase in Abs bound to intact platelets was observed during infection. P. berghei infection can enhance the activity of anti-platelet Abs as indicated by a significantly (p < 0.005) increased thrombocytopenia on day 4 of infection in mice that were administered a low dose anti-CD41 mAb compared with rat IgG1-injected controls. RAG1−/− and CD4- plus CD8-deficient mice were markedly protected from thrombocytopenia (p < 0.005) and malarial pathogenesis. CD8- or TCRγδ-deficient mice were not protected from thrombocytopenia and CD4-deficient mice were modestly protected. RAG1−/− mice exhibited significantly (p < 0.05) lower levels of plasma TNF, IFN-γ, and IL-12 during infection. IFNγ−/− and IL-12−/− mice exhibited increased survival but similar thrombocytopenia to C57BL/6 controls. Collectively, these data indicate that thrombocytopenia is necessary but not sufficient for malarial pathogenesis and Abs are not the major contributors to malarial thrombocytopenia. Rather, we propose that both CD4+ and CD8+ T cell populations play key roles in malarial thrombocytopenia; a complex bidirectional interaction between cell-mediated immunity and platelets exists during experimental severe malaria that regulates both responses.
Tolerogenic Treatment of Lupus Mice with Consensus Peptide Induces Foxp3-Expressing, Apoptosis-Resistant, TGFβ-Secreting CD8+ T Cell Suppressors
Authors: Hahn Bevra H. , Singh Ram Pyare , La Cava Antonio , Ebling Fanny M.
Published: April 2014

Lupus-prone (NZB × NZW)F1 mice spontaneously develop elevated titers of anti-DNA Abs that contain T cell determinants in their VH regions. We have previously shown that tolerization with an artificial peptide based on these T cell determinants (pConsensus (pCons)) can block production of anti-DNA Abs and prolong survival of the mice. In this study, we show that this protection depends in part on the generation of peripheral TGFβ- and Foxp3-expressing inhibitory CD8+ (Ti) cells. These CD8+ Ti cells suppress anti-DNA IgG production both in vitro and in vivo and require up-regulated expression of both Foxp3 and TGFβ to exert their suppressive function, as indicated by microarray analyses, small interfering RNA inhibition studies, and blocking experiments. Additionally, CD8+ Ti cells from pCons-tolerized mice were longer-lived suppressors that up-regulated expression of Bcl-2 and were more resistant to apoptosis than similar cells from naive mice. These data indicate that clinical suppression of autoimmunity after administration of pCons depends in part on the generation of CD8+ Ti cells that suppress secretion of anti-DNA Ig using mechanisms that include Foxp3, TGFβ, and resistance to apoptosis.
Analysis of the Cellular Mechanism of Antitumor Responses and Autoimmunity in Patients Treated with CTLA-4 Blockade
Authors: Maker Ajay V. , Attia Peter , Rosenberg Steven A.
Published: April 2014

We have demonstrated previously that the administration of CTLA-4 blockade has mediated objective cancer regression and autoimmunity in patients with metastatic melanoma. To explore the mechanism of these in vivo effects, we have studied the changes in lymphocyte phenotype and function in patients receiving anti-CTLA-4 Ab (MDX-010). Patients with stage IV melanoma or renal cell cancer were treated every 3 wk with an anti-CTLA-4 Ab with or without peptide immunization. Pheresis samples were analyzed using flow cytometry to determine lymphocyte cell surface markers. Gene expression analyses and proliferation assays were conducted on purified T cell subsets. Anti-CTLA-4 Ab did not inhibit the suppressive activity of CD4+CD25+ cells in vitro or in vivo. In addition, there was no decrease in the expression of CD4+CD25+ cells in whole PBMC, nor a decrease in Foxp3 gene expression in the CD4+ or CD4+CD25+ purified cell populations posttreatment. The percentage of CD4+, CD8+, CD4+CD25+, and CD4+CD25− T cells in PBMC expressing the activation marker HLA-DR increased following anti-CTLA-4 Ab administration. Therefore, our results suggest that the antitumor effects of CTLA-4 blockade are due to increased T cell activation rather than inhibition or depletion of T regulatory cells.
Cutting Edge: Lectin-Like Transcript-1 Is a Ligand for the Inhibitory Human NKR-P1A Receptor
Authors: Rosen David B. , Bettadapura Jayaram , Alsharifi Mohammed , Mathew Porunelloor A. , Warren Hilary S. , Lanier Lewis L.
Published: April 2014

Increasingly, roles are emerging for C-type lectin receptors in immune regulation. One receptor whose function has remained largely enigmatic is human NKR-P1A (CD161), present on NK cells and subsets of T cells. In this study, we demonstrate that the lectin-like transcript-1 (LLT1) is a physiologic ligand for NKR-P1A. LLT1-containing liposomes bind to NKR-P1A+ cells, and binding is inhibited by anti-NKR-P1A mAb. Additionally, LLT1 activates NFAT-GFP reporter cells expressing a CD3ζ-NKR-P1A chimeric receptor; reciprocally, reporter cells with a CD3ζ-LLT1 chimeric receptor are stimulated by NKR-P1A. Moreover, LLT1 on target cells can inhibit NK cytotoxicity via interactions with NKR-P1A.
CD18 Is Required for Optimal Development and Function of CD4+CD25+ T Regulatory Cells
Authors: Marski Marissa , Kandula Sravanthi , Turner Jerrold R. , Abraham Clara
Published: April 2014

CD4+CD25+ T regulatory (Treg) cells inhibit immunopathology and autoimmune disease in vivo. CD4+CD25+ Treg cells’ capacity to inhibit conventional T cells in vitro is dependent upon cell-cell contact; however, the cell surface molecules mediating this cell:cell contact have not yet been identified. LFA-1 (CD11a/CD18) is an adhesion molecule that plays an established role in T cell-mediated cell contact and in T cell activation. Although expressed at high levels on murine CD4+CD25+ Treg cells, the role of LFA-1 in these cells has not been defined previously. We hypothesized that LFA-1 may play a role in murine CD4+CD25+ Treg function. To evaluate this, we analyzed LFA-1-deficient (CD18−/−) CD4+CD25+ T cells. We show that CD18−/− mice demonstrate a propensity to autoimmunity. Absence of CD18 led to diminished CD4+CD25+ T cell numbers and affected both thymic and peripheral development of these cells. LFA-1-deficient CD4+ CD25+ T cells were deficient in mediating suppression in vitro and in mediating protection from colitis induced by the transfer of CD4+CD25− T cells into lymphopenic hosts. Therefore, we define a crucial role for CD18 in optimal CD4+CD25+ Treg development and function.
Identification of Specific Proteins and Peptides in Mycobacterium leprae Suitable for the Selective Diagnosis of Leprosy
Authors: Spencer John S. , Dockrell Hazel M. , Kim Hee Jin , Marques Maria A. M. , Williams Diana L. , Martins Marcia V. S. B. , Martins Marcio L. F. , Lima Monica C. B. S. , Sarno Euzenir N. , Pereira Geraldo M. B. , Matos Haroldo , Fonseca Leila S. , Sampaio Elisabeth P. , Ottenhoff Thomas H. M. , Geluk Annemieke , Cho Sang-Nae , Stoker Neil G. , Cole Stewart T. , Brennan Patrick J. , Pessolani Maria C. V.
Published: April 2014

Diagnosis of leprosy is a major obstacle to disease control and has been compromised in the past due to the lack of specific reagents. We have used comparative genome analysis to identify genes that are specific to Mycobacterium leprae and tested both recombinant proteins and synthetic peptides from a subset of these for immunological reactivity. Four unique recombinant proteins (ML0008, ML0126, ML1057, and ML2567) and a panel of 58 peptides (15 and 9 mer) were tested for IFN-γ responses in PBMC from leprosy patients and contacts, tuberculosis patients, and endemic and nonendemic controls. The responses to the four recombinant proteins gave higher levels of IFN-γ production, but less specificity, than the peptides. Thirty-five peptides showed IFN-γ responses only in the paucibacillary leprosy and household contact groups, with no responses in the tuberculosis or endemic control groups. High frequencies of IFN-γ-producing CD4+ and CD8+ T cells specific for the 15- and 9-mer peptides were observed in the blood of a paucibacillary leprosy patient. 9-mer peptides preferentially activated CD8+ T cells, while the 15-mer peptides were efficient in inducing responses in both the CD4+ and CD8+ T cell subsets. Four of the six 9-mer peptides tested showed promising specificity, indicating that CD8+ T cell epitopes may also have diagnostic potential. Those peptides that provide specific responses in leprosy patients from an endemic setting could potentially be developed into a rapid diagnostic test for the early detection of M. leprae infection and epidemiological surveys of the incidence of leprosy, of which little is known.
Long-Term MHC Class II Presentation of the EBV Lytic Protein BHRF1 by EBV Latently Infected B Cells following Capture of BHRF1 Antigen
Authors: Landais Elise , Saulquin Xavier , Bonneville Marc , Houssaint Elisabeth
Published: April 2014

Although T lymphocytes are considered essential for the control of EBV infection, it remains uncertain how this control occurs. We previously reported unexpected killing of EBV-transformed B-lymphoblastoid cells (LCLs) that did not express BHRF1 by CD4+ T cells specific for BHRF1, an EBV lytic cycle protein. Using LCLs transformed with an EBV mutant, in which the BHRF1 gene was deleted, we showed that killing of latently infected cells through the recognition of a protein produced during the lytic cycle is due to transfer of BHRF1 from lytically infected to latently infected cells, which occurs in culture. Accordingly, LCLs efficiently presented exogenous BHRF1 protein. Furthermore, we present evidence for persistence of captured BHRF1 Ag for several days. Due to this long-term persistence, repeated loading of suboptimal amounts of BHRF1 led to accumulation of BHRF1 Ags in LCLs and, ultimately, to their optimal recognition by BHRF1-specific CD4+ T cells. These results unveil an MHC class II-dependent pathway that could be important for the control of EBV latent infection through recognition of lytic cycle Ags.
Protein Tyrosine Phosphatase α Regulates Fyn Activity and Cbp/PAG Phosphorylation in Thymocyte Lipid Rafts
Authors: Maksumova Lola , Le Hoa T. , Muratkhodjaev Farkhad , Davidson Dominique , Veillette André , Pallen Catherine J.
Published: April 2014

A role for the receptor protein tyrosine phosphatase α (PTPα) in immune cell function and regulation of Src family kinases was investigated using thymocytes from PTPα-deficient mice. PTPα-null thymocytes develop normally, but unstimulated PTPα−/− cells exhibit increased tyrosine phosphorylation of specific proteins, increased Fyn activity, and hyperphosphorylation of Cbp/PAG that promotes its association with C-terminal Src kinase. Elevated Fyn activity in the absence of PTPα is due to enhanced phosphorylation of Fyn tyrosines 528 and 417. Some PTPα is localized in lipid rafts of thymocytes, and raft-associated Fyn is specifically activated in PTPα−/− cells. PTPα is not a Cbp/PAG phosphatase, because it is not required for Cbp/PAG dephosphorylation in unstimulated or anti-CD3-stimulated thymocytes. Together, our results indicate that PTPα, likely located in lipid rafts, regulates the activity of raft Fyn. In the absence of PTPα this population of Fyn is activated and phosphorylates Cbp/PAG to enhance association with C-terminal Src kinase. Although TCR-mediated tyrosine phosphorylation was apparently unaffected by the absence of PTPα, the long-term proliferative response of PTPα−/− thymocytes was reduced. These findings indicate that PTPα is a component of the complex Src family tyrosine kinase regulatory network in thymocytes and is required to suppress Fyn activity in unstimulated cells in a manner that is not compensated for by the major T cell PTP and SFK regulator, CD45.
The Sphingosine 1-Phosphate Receptor Agonist FTY720 Differentially Affects the Sequestration of CD4+/CD25+ T-Regulatory Cells and Enhances Their Functional Activity
Authors: Sawicka Elzbieta , Dubois Gerald , Jarai Gabor , Edwards Matthew , Thomas Matthew , Nicholls Andy , Albert Rainer , Newson Catherine , Brinkmann Volker , Walker Christoph
Published: April 2014

The sphingosine 1-phosphate (S1P) receptor agonist FTY720 is well known for its immunomodulatory activity, sequestering lymphocytes from blood and spleen into secondary lymphoid organs and thereby preventing their migration to sites of inflammation. Because inflammation is critically dependent on a balance between Ag-specific Th/effector cells and T-regulatory cells, we investigated the effect of FTY720 on T-regulatory cell trafficking and functional activity. An increased number of CD4+/CD25+ T cells was found in blood and spleens of FTY720-treated mice, and transfer of these cells resulted in a significantly more pronounced accumulation in spleens but not lymph nodes after treatment, suggesting that this compound differentially affects the homing properties of T-regulatory cells compared with other T cell subsets. Indeed, CD4+/CD25+ T cells express lower levels of S1P1 and S1P4 receptors and demonstrate a reduced chemotactic response to S1P. Moreover, analysis of the functional response of FTY720-treated CD4+/CD25+ T cells revealed an increased suppressive activity in an in vitro Ag-specific proliferation assay. This correlated with enhanced function in vivo, with T-regulatory cells obtained from FTY720-treated mice being able to suppress OVA-induced airway inflammation. Thus, FTY720 differentially affects the sequestration of T-regulatory cells and importantly, increases the functional activity of T-regulatory cells, suggesting that it may have disease-modifying potential in inflammatory disorders.
IL-2 Regulates Perforin and Granzyme Gene Expression in CD8+ T Cells Independently of Its Effects on Survival and Proliferation
Authors: Janas Michelle L. , Groves Penny , Kienzle Norbert , Kelso Anne
Published: April 2014

Perforin and the serine protease granzymes are key effectors of CD8+ T cell granule-mediated cytotoxicity, but the requirements for their expression remain largely undefined. We show in this study that IL-2 increased the expression of perforin and granzyme A, B, and C mRNA; intracellular granzyme B protein levels; and cytolytic function in a dose-dependent manner during primary activation of murine CD8+ T cells in vitro. Two approaches showed that these responses were not a consequence of the effects of IL-2 on cell survival and proliferation. First, IL-2 enhancement of perforin and granzyme expression was equivalent in CD8+ T cells from wild-type and bcl-2 transgenic mice, although only the latter cells survived in low concentrations or the absence of added IL-2. This property of bcl-2 transgenic T cells also allowed the demonstration that induction of granzyme A, B, and C mRNA and granzyme B protein required exogenous IL-2, whereas induction of perforin and IFN-γ expression did not. Second, analysis of perforin and granzyme mRNA levels in cells separated according to division number using the dye CFSE showed that the effects of IL-2 were unrelated to division number. Together, these findings indicate that IL-2 can directly regulate perforin and granzyme gene expression in CD8+ T cells independently of its effects on cell survival and proliferation.