bluuebunny/crossref_metadata_2025 huggingface dataset:
Human CD1d Functions as a Transplantation Antigen and a Restriction Element in Mice
Authors: Wang Bin , Chun Taehoon , Rulifson Ingrid C. , Exley Mark , Balk Steven P. , Wang Chyung-Ru
Published: April 2014

To study the potential functions of human CD1d (hCD1d), we developed transgenic (Tg) mice that ectopically express hCD1d under the control of H-2Kb promoter. High levels of hCD1d expression were detected in all Tg tissues tested. Skin grafts from the Kb/hCD1d Tg mice were rapidly rejected by MHC-matched non-Tg recipient mice, suggesting that hCD1d can act as transplantation Ags. Furthermore, we were able to elicit hCD1d-restricted CD8+ CTLs from mice immunized with Kb/hCD1d Tg splenocytes. These CTLs express TCR rearrangements that are distinct from invariant TCR of NK T cells, and secrete significant amounts of IFN-γ upon Ag stimulation. Analysis with various hCD1d-expressing targets and use of Ag presentation inhibitors indicated the recognition of hCD1d by CTLs did not involve species or tissue-specific ligands nor require the processing pathways of endosomes or proteasomes. Additionally, the reactivity of hCD1d-specific CTLs was not affected by acid stripping followed by brefeldin A treatment, suggesting that CTLs may recognize a ligand/hCD1d complex that is resistant to acid denaturation, or empty hCD1d molecules. Our results show that hCD1d can function as an alloantigen for CD8+ CTLs. The hCD1d Tg mice provide a versatile model for the study of hCD1d-restricted cytolytic responses to microbial Ags.
A Critical Role for p38 Mitogen-Activated Protein Kinase in the Maturation of Human Blood-Derived Dendritic Cells Induced by Lipopolysaccharide, TNF-α, and Contact Sensitizers
Authors: Arrighi Jean-François , Rebsamen Michela , Rousset Françoise , Kindler Vincent , Hauser Conrad
Published: April 2014

We investigated the involvement of mitogen-activated protein kinases (MAPKs) in the maturation of CD83− dendritic cells (DC) derived from human blood monocytes. Maturating agents such as LPS and TNF-α induced the phosphorylation of members of the three families of MAPK (extracellular signal-regulated kinase l/2, p46/54 c-Jun N-terminal kinase, and p38 MAPK). SB203580, an inhibitor of the p38 MAPK, but not the extracellular signal-regulated kinase l/2 pathway blocker PD98059, inhibited the up-regulation of CD1a, CD40, CD80, CD86, HLA-DR, and the DC maturation marker CD83 induced by LPS and TNF-α. In addition, SB203580 inhibited the enhancement of the allostimulatory capacity and partially prevented the down-regulation of FITC-dextran uptake induced by LPS and TNF-α. Likewise, SB203580 partially prevented the up-regulation of IL-1α, IL-1β, IL-lRa, and TNF-α mRNA upon stimulation with LPS and TNF-α, as well as the release of bioactive TNF-α induced by LPS. DC maturation induced by the contact sensitizers 2,4-dinitrofluorobenzene and NiSO4, as seen by the up-regulation of CD80, CD86, and CD83, was also coupled to the phosphorylation of p38 MAPK, and was inhibited by SB203580. The irritants SDS and benzalkonium chloride that do not induce DC maturation did not trigger p38 MAPK phosphorylation. Together, these data indicate that phosphorylation of p38 MAPK is critical for the maturation of immature DC. These results also suggest that p38 MAPK phosphorylation in DC may become useful for the identification of potential skin contact sensitizers.
Human Vascular Endothelial Cells Stimulate a Lower Frequency of Alloreactive CD8+ Pre-CTL and Induce Less Clonal Expansion than Matching B Lymphoblastoid Cells: Development of a Novel Limiting Dilution Analysis Method Based on CFSE Labeling of Lymphocytes
Authors: Dengler Thomas J. , Johnson David R. , Pober Jordan S.
Published: April 2014

We have previously shown that human endothelial cells (EC) are less efficient than professional APC, e.g., B lymphoblastoid cells (BLC), at stimulating allogeneic CD8+ T cells to develop into CTL. In this study we describe FACS-based limiting dilution analyses using the dilution of the intracellular dye CFSE as an indicator of CD8+ T cell alloactivation and expansion with significantly increased sensitivity compared with conventional, cytotoxicity-based assays. In addition, this assay permits the relative size of clonal CTL populations that are generated in individual CD8+ T cell cultures to be determined (clonal burst size). We have applied this method to quantitatively compare the generation of CTL at the clonal level following stimulation of allogeneic CD8+ T cells by either BLC or HUVEC derived from the same donor. CD8+ T cells expanded by allostimulation were identified as CD8+, CFSElow cells and were categorized as CTL by the expression of intracellular perforin and IFN-γ. Precursor frequencies for EC-stimulated CTL were 5- to 40-fold (mean, 7.5-fold) lower compared with BLC-stimulated CTL (p < 0.01). Concomitantly, the average clonal burst sizes in EC-stimulated CTL cultures were significantly smaller than those in conventional CTL cultures, primarily due to the occurrence of some very large clone sizes exclusively with BLC stimulation. Although EC-stimulated CTL were generated only from the memory subset of CD8+ T cells, BLC-stimulated very large burst sizes of CTL were observed from both naive and memory CD8+ T cell precursors. These data establish that both a lower frequency of reactive precursors and more limited clonal expansion, but not regulatory T cells, contribute to the reduced capacity of EC to promote alloreactive CTL differentiation compared with that of professional APC.
Inhibition of the MEK/ERK Signaling Pathway Blocks a Subset of B Cell Responses to Antigen
Authors: Richards James D. , Davé Shaival H. , Chou Chih-Hao G. , Mamchak Alusha A. , DeFranco Anthony L.
Published: April 2014

Signal transduction initiated by B cell Ag receptor (BCR) cross-linking plays an important role in the development and activation of B cells. Therefore, considerable effort has gone into determining the biochemical signaling events initiated by the BCR and delineating which events participate in specific biological responses to Ag. We used two inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) 1 and MEK2, PD98059, and U0126, to assess the role the Ras-mitogen-activated protein kinase pathway plays in several BCR-induced responses. PD98059 or U0126 treatment substantially inhibited the BCR-induced activation of the extracellular signal-regulated kinase (ERK) forms of mitogen-activated protein kinase in the immature B cell line WEHI-231, in immature splenic B cells, and in mature splenic B cells. However, MEK-ERK inhibition did not block BCR-induced growth arrest or apoptosis of WEHI-231 cells or apoptosis of immature splenic B cells, indicating that the MEK-ERK pathway is not required for these events. In contrast, PD98059 and U0126 treatment did inhibit the up-regulation of specific BCR-induced proteins, including the transcription factor Egr-1 in WEHI-231 and mature splenic B cells, and the CD44 adhesion molecule and CD69 activation marker in mature splenic B cells. Moreover, both inhibitors suppressed BCR-induced proliferation of mature splenic B cells, in the absence and in the presence of IL-4. Therefore, activation of the MEK-ERK pathway is necessary for a subset of B cell responses to Ag.
CD83 Is a Sialic Acid-Binding Ig-Like Lectin (Siglec) Adhesion Receptor that Binds Monocytes and a Subset of Activated CD8+ T Cells
Authors: Scholler Nathalie , Hayden-Ledbetter Martha , Hellström Karl-Erik , Hellström Ingegerd , Ledbetter Jeffrey A.
Published: April 2014

To help determine CD83 function, a cDNA encoding a soluble protein containing the CD83 extracellular domain was fused with a mutated human IgG1 constant region (CD83Ig) and expressed by stable transfection of Chinese hamster ovary cells. Purified CD83Ig bound to peripheral blood monocytes and a subset of activated CD3+CD8+ lymphocytes but did not bind to FcR. Monocytes that had adhered to plastic lost their ability to bind to CD83Ig after 90 min of in vitro incubation. CD83Ig bound to two of five T cell lines tested, HPB-ALL and Jurkat. The binding to HPB-ALL cells significantly increased when they were grown at a low pH (pH 6.5), whereas binding to Jurkat cells increased after apoptosis was induced with anti-Fas mAb. B cell and monocytic lines did not bind CD83Ig and neither did CD56+ NK cells or granulocytes. Full-length CD83 expressed by a transfected carcinoma line mediated CD83-dependent adhesion to HPB-ALL cells. CD83Ig immunoprecipitated and immunoblotted a 72-kDa protein from HPB-ALL cells. Binding of CD83Ig to HPB-ALL cells was eliminated by neuraminidase treatment of the cells. We conclude that CD83 is an adhesion receptor with a counterreceptor expressed on monocytes and a subset of activated or stressed T lymphocytes, and that interaction between CD83 and its counterreceptor is dependent upon the state of glycosylation of a 72-kDa counterreceptor by sialic acid residues. In view of the selectivity of the expression of CD83 and its ligand, we postulate that the interaction between the two plays an important role in the induction and regulation of immune responses.
Biphasic Regulation of NF-κB Activity Underlies the Pro- and Anti-Inflammatory Actions of Nitric Oxide
Authors: Connelly L. , Palacios-Callender M. , Ameixa C. , Moncada S. , Hobbs A. J.
Published: April 2014

Expression of inducible NO synthase (iNOS) by macrophages is a prerequisite for the production of high output NO, which mediates many bactericidal and tumoricidal actions of these immune cells. The expression of iNOS in mammalian cells is governed predominantly by the transcription factor, NF-κB, which regulates the expression of many host defense proteins. In the present study, we characterize a novel, biphasic effect of NO on NF-κB activity in murine macrophages. This mechanism depends on the local concentration of NO and enables it both to up- and down-regulate the expression of host defense proteins including iNOS, cyclooxygenase-2, and IL-6. This biphasic activity of NO appears to play a pivotal role in the time course of activation of these immune cells and, by inference, in facilitating the initiation of a defense response against pathogenic stimuli and in its termination to limit tissue damage. This mechanism may explain at least in part the reported ability of NO to act in both a pro- and anti-inflammatory manner.
The Fc Receptor for IgG Expressed in the Villus Endothelium of Human Placenta Is FcγRIIb2
Authors: Lyden Timothy W. , Robinson John M. , Tridandapani Susheela , Teillaud Jean-Luc , Garber Stacey A. , Osborne Jeanne M. , Frey Jürgen , Budde Petra , Anderson Clark L.
Published: April 2014

To evaluate the potential role of human placental endothelial cells in the transport of IgG from maternal to fetal circulation, we studied Fcγ receptor (FcγR) expression by immunohistology and immunoblotting. Several pan-FcγRII Abs that label the placental endothelium displayed a distribution pattern that correlated well with transport functions, being intense in the terminal villus and nil in the cord. In contrast, the MHC class 1-like IgG transporter, FcRn, and the classical FcγRIIa were not expressed in transport-related endothelium of the placenta. Our inference, that FcγRIIb was the likely receptor, we confirmed by analyzing purified placental villi, enriched in endothelium, by immunoblotting with a new Ab specific for the cytoplasmic tail of FcγRIIb. These experiments showed that the FcγRII expressed in villus endothelium was the b2 isoform whose cytoplasmic tail is known to include a phosphotyrosyl-based motif that inhibits a variety of immune responses. We suggest that this receptor is perfectly positioned to transport IgG although as well it may scavenge immune complexes.
IL-12 Inhibition of Endothelial Cell Functions and Angiogenesis Depends on Lymphocyte-Endothelial Cell Cross-Talk
Authors: Strasly Marina , Cavallo Federica , Geuna Massimo , Mitola Stefania , Colombo Mario P. , Forni Guido , Bussolino Federico
Published: April 2014

In vivo IL-12-dependent tumor inhibition rests on the ability of IL-12 to activate a CD8-mediated cytotoxicity, inhibit angiogenesis, and cause vascular injury. Although in vivo studies have shown that such inhibition stems from complex interactions of immune cells and the production of IFN-γ and other downstream angiostatic chemokines, the mechanisms involved are still poorly defined. Here we show that IL-12 activates an anti-angiogenic program in Con A-activated mouse spleen cells (activated spc) or human PBMC (activated PBMC). The soluble factors they release in its presence arrest the cycle of endothelial cells (EC), inhibit in vitro angiogenesis, negatively modulate the production of matrix metalloproteinase-9, and the ability of EC to adhere to vitronectin and up-regulate ICAM-1 and VCAM-1 expression. These effects do not require direct cell-cell contact, yet result from continuous interaction between activated lymphoid cells and EC. We used neutralizing Abs to show that the IFN-inducible protein-10 and monokine-induced by IFN-γ chemokines are pivotal in inducing these effects. Experiments with nu/nu mice, nonobese diabetic-SCID mice, or activated spc enriched in specific cell subpopulations demonstrated that CD4+, CD8+, and NK cells are all needed to mediate the full anti-angiogenetic effect of IL-12.
Absence of CTLA-4 Lowers the Activation Threshold of Primed CD8+ TCR-Transgenic T Cells: Lack of Correlation with Src Homology Domain 2-Containing Protein Tyrosine Phosphatase
Authors: Gajewski Thomas F. , Fallarino Francesca , Fields Patrick E. , Rivas Fabiola , Alegre Maria-Luisa
Published: April 2014

To examine the role of CTLA-4 in controlling Ag-specific CD8+ T cell activation, TCR-transgenic/CTLA-4 wild-type or -deficient mice were generated in a recombination-activating gene 2-deficient background. Naive T cells from these mice responded comparably whether or not CTLA-4 was expressed. In contrast, primed T cells responded more vigorously if they lacked CTLA-4 expression. We took advantage of the difference between naive and primed T cell responses to approach the mechanism of CTLA-4 function. Single-cell analyses demonstrated that a greater fraction of CTLA-4-deficient cells responded to a fixed dose of Ag compared with CTLA-4-expressing cells, whereas the magnitude of response per cell was comparable. A shift in the dose-response curve to APCs was also observed such that fewer APCs were required to activate CTLA-4-deficient T cells to produce intracellular IFN-γ and to proliferate. These results suggest that CTLA-4 controls the threshold of productive TCR signaling. Biochemical analysis comparing stimulated naive and primed TCR-transgenic cells revealed no obvious differences in expression of total CTLA-4, tyrosine-phosphorylated CTLA-4, and associated Src homology domain 2-containing protein tyrosine phosphatase. Thus, the biochemical mechanism explaining the differential inhibitory effect of CTLA-4 on naive and primed CD8+ T cells remains unclear.
CTLA-4 Blockade Enhances the CTL Responses to the p53 Self-Tumor Antigen
Authors: Hernández Javier , Ko Alice , Sherman Linda A.
Published: April 2014

p53 is an attractive target for cancer immunotherapy because it is overexpressed in a high proportion of many different types of tumors. However, it is also expressed in normal tissues and acts as a toleragen in vivo. Previously, detailed examination of the repertoire specific for the murine p53261–269 epitope in conventional and p53-deficient mice demonstrated that because of expression of p53, the CD8+ T cells that respond to this epitope express low-affinity TCRs. It has been reported that tolerance to tumor Ags can be broken by in vivo administration of anti-CTLA-4 mAb. With the goal of overriding tolerance and achieving optimal activation of p53-specific CTL, the current study has assessed the effect of anti-CTLA-4 mAb on the p53-specific repertoire. It was found that blockade of CTLA-4 engagement at the time of antigenic stimulation induced a vigorous amplification of the CTL responses to p53 as well as proportionate expansion of the memory T cell pool. This effect was dependent on the presence of CD4+ T cell help and correlated with an enhancement of helper function. However, anti-CTLA-4 treatment did not enhance the avidity of the resultant p53-specific CTL populations and, therefore, could not reverse this important consequence of tolerance.
Novel Single Nucleotide Polymorphisms in the Distal IL-10 Promoter Affect IL-10 Production and Enhance the Risk of Systemic Lupus Erythematosus
Authors: Gibson Andrew W. , Edberg Jeffrey C. , Wu Jianming , Westendorp Rudi G. J. , Huizinga Tom W. J. , Kimberly Robert P.
Published: April 2014

Family studies of first-degree relatives and analysis of twins indicate that as much as 75% of the differences in quantitative IL-10 production in man derive from heritable genetic factors. Studies of single nucleotide polymorphisms (SNP) in the proximal 1.0 kb of the IL-10 promoter have yielded inconsistent association with IL-10 production and variable results in promoter-reporter studies. However, in normal donors, an association of quantitative production with certain alleles of the IL-10.R short tandem repeat polymorphism at −4.0 kb suggested that SNPs in the more distal promoter might be informative. We have identified seven novel SNP sites in the genomic sequence of the first 4 kb of the IL-10 promoter region 5′ to the ATG start site from Caucasian individuals with either a high or a low IL-10 production phenotype. We have also identified eight SNP haplotypes in the distal promoter that segregate with significant differences in quantitative IL-10 production in normal donors. These SNPs are significantly associated with systemic lupus erythematosus in African-Americans and may define one component of the genetic susceptibility to systemic lupus erythematosus in this group.
The Repertoire of Killer Cell Ig-Like Receptor and CD94:NKG2A Receptors in T Cells: Clones Sharing Identical αβ TCR Rearrangement Express Highly Diverse Killer Cell Ig-Like Receptor Patterns
Authors: Uhrberg Markus , Valiante Nicholas M. , Young Neil T. , Lanier Lewis L. , Phillips Joseph H. , Parham Peter
Published: April 2014

Killer cell Ig-like receptor (KIR) and CD94:NKG2A molecules were first defined as human NK cell receptors (NKR), but now are known to be expressed and to function on subpopulations of T cells. Here the repertoires of KIR and CD94:NKG2A expression by T cells from two donors were examined and compared with their previously defined NK cell repertoires. T cell clones generated from peripheral blood of both donors expressed multiple NKR in different combinations and used the range of receptors expressed by NK cells. In both donors αβ T cells less frequently expressed the inhibitory receptors CD94:NKG2A and KIR2DL1 than either γδ T cells or NK cells. In contrast to NK cells, not all NKR+ T cells expressed an inhibitory receptor for autologous HLA class I. This lack of specific inhibitory NKR was especially apparent on αβ T cells of one donor. Overall, αβ T cells exhibited a distinct pattern of NKR expression different from that of γδ T and NK cells, which expressed highly similar NKR repertoires. In one donor, analysis of TCR rearrangement revealed a dominant subset of NKR+ T cells sharing identical TCR α- and β-chains. Remarkably, among 55 T cell clones sharing the same TCRαβ rearrangement 18 different KIR phenotypes were seen, suggesting that KIR expression was initiated subsequently to TCR rearrangement.
Differential Expression of Leukocyte Receptor Complex-Encoded Ig-Like Receptors Correlates with the Transition from Effector to Memory CTL
Authors: Young Neil T. , Uhrberg Markus , Phillips Joseph H. , Lanier Lewis L. , Parham Peter
Published: April 2014

The human leukocyte receptor complex (LRC) on chromosome 19q13.4 encodes Ig superfamily receptors expressed on hemopoietic cells. Killer Ig-like receptors (KIR) are expressed in cytotoxic lymphocytes but other LRC molecules (Ig-like transcript(ILT)/leukocyte Ig-like receptor (LIR)) are more ubiquitous. We investigated expression of the ILT2/LIR1 inhibitory receptor compared with the related KIR. Both ILT2/LIR1 and KIR were expressed by peripheral CD8+ T cells with a memory/effector phenotype. ILT2/LIR1+ T cells demonstrated diverse TCRBV repertoires in contrast to KIR+ T cells, while numbers of peripheral ILT2/LIR1+ T cells were greater than KIR+ T cells and the majority of ILT2/LIR1+ T cells did not coexpress KIR. Analysis of CD8+ T cells with specific HLA class I tetramers confirmed this pattern of expression, indicating differential regulation of LRC gene expression in T lymphocytes. Only a minor proportion of ILT2/LIR1+ KIR− clones survived in vitro cloning, were more susceptible to anti-CD3 or cognate peptide induced cell death than KIR+ T cells and exhibited lower levels of the Bcl-2 survival molecule. Our results indicate a sequential program of LRC-encoded receptor expression with initial ILT2/LIR1 expression in effector T cells and KIR gene transcription in the minor proportion of expanded clones which survives activation-induced cell death to become long term memory T cells.
Organization and Functional Analysis of the Mouse Transporter Associated with Antigen Processing 2 Promoter
Authors: Arons Evgeny , Kunin Victor , Schechter Chana , Ehrlich Rachel
Published: April 2014

In accordance with the key role of MHC class I molecules in the adaptive immune response against viruses, they are expressed by most cells, and their expression can be enhanced by cytokines. The assembly and cell surface expression of class I complexes depend on a continuous peptide supply. The peptides are generated mainly by the proteasome and are transported to the endoplasmic reticulum by a peptide transport pump consisting of two subunits, TAP1 and TAP2. The proteasome low molecular weight polypeptide (2 and 7), as well as TAP (1 and 2) genes, are coordinately regulated and are induced by IFNs. Despite this coordinate regulation, examination of tumors shows that these genes can be discordantly down-regulated. In pursuing a molecular explanation for these observations, we have characterized the mouse TAP2 promoter region and 5′-flanking sequence. We show that the 5′ untranslated regions of TAP2 genes have a characteristic genomic organization that is conserved in both the mouse and the human. The mouse TAP2 promoter belongs to a class of promoters that lack TATA boxes but contain a MED1 (multiple start site element downstream) sequence. Accordingly, transcription is initiated from multiple sites within a 100-nucleotide window. An IFN regulatory factor 1 (IRF1)/IRF2 binding site is located in this region and is involved in both basal and IRF1-induced TAP2 promoter activity. The implication of the extensive differences found among the promoters of class I heavy chain, low molecular weight polypeptide, and TAP genes, all encoding proteins involved in Ag presentation, is discussed.
Anti-Peptide Antibody Blocks Peptide Binding to MHC Class I Molecules in the Endoplasmic Reticulum
Authors: Hilton Craig J. , Dahl Astrid M. , Rock Kenneth L.
Published: April 2014

The finding that MHC class I molecules are physically associated with the TAP transporter has suggested that peptides may be directly transported into the binding groove of the class I molecules rather than into the lumen of the endoplasmic reticulum (ER) where they subsequently would encounter class I molecules by diffusion. Such a mechanism would protect peptides from peptidases in the ER and/or escaping back into the cytoplasm. However, we find that an anti-peptide Ab that is cotranslationally transported into the ER prevents TAP-transported peptides from being presented on class I molecules. The Ab only blocks the binding of its cognate peptide (SIINFEKL) but not other peptides (KVVRFKDL, ASNENMETM, and FAPGNYPAL). Therefore, most TAP-transported peptides must diffuse through the lumen of the ER before binding stably to MHC class I molecules.
Physical Map and Expression Profile of Genes of the Telomeric Class I Gene Region of the Rat MHC
Authors: Ioannidu Sofia , Walter Lutz , Dressel Ralf , Günther Eberhard
Published: April 2014

The rat is an important model for studying organ graft rejection and susceptibility to certain complex diseases. The MHC, the RT1 complex, plays a decisive role in controlling these traits. We have cloned the telomeric class I region of the RT1 complex, RT1-C/E/M, of the BN inbred rat strain in a contig of overlapping P1-derived artificial chromosome clones encompassing ∼2 Mb, and present a physical map of this MHC region. Forty-five class I exon 4-hybridizing BamHI fragments were detected, including the previously known rat class I genes RT1-E, RT-BM1, RT1-N, RT1-M2, RT1-M3, and RT1-M4. Twenty-six non-class I genes known to map to the corresponding part of the human and mouse MHC were tested and could be fine mapped in the RT1-C/E/M region at orthologous position. Four previously known microsatellite markers were fine mapped in the RT1-C/E/M region and found to occur in multiple copies. In addition, a new, single-copy polymorphic microsatellite has been defined. The expression profiles of several class I genes and the 26 non-class I genes were determined in 13 different tissues and exhibited restricted patterns in most cases. The data provide further molecular information on the MHC for analyzing disease susceptibility and underline the usefulness of the rat model.
A 3′-Transcribed Region of the HLA-A2 Gene Mediates Posttranscriptional Stimulation by IFN-γ
Authors: Snyder Steven R. , Waring Jeffrey F. , Zhu Sheng Zu , Kaplan Sarah , Schultz Julie , Ginder Gordon D.
Published: April 2014

The expression of several MHC class I genes is up-regulated at the transcriptional level by IFN-γ. Posttranscriptional mechanisms also have been implicated, but not well characterized. To investigate the mechanism of IFN-γ stimulation of the human MHC class I gene HLA-A2, several human tumor cell lines were transfected with reporter gene constructs driven by the HLA-A2 promoter. We have previously shown that the extended 525-bp HLA-A2 promoter alone, which includes a 5′ IFN-stimulated response element consensus sequence, is not sufficient for IFN-γ response in either K562 or Jurkat cells. In the current study, stable transfection of a genomic HLA-A2 gene construct, containing both 5′- and 3′-flanking sequences, resulted in stimulation of the gene by IFN-γ. Nuclear run-on assays revealed that, unlike other class I genes, IFN-γ stimulation of HLA-A mRNA accumulation occurs almost entirely through posttranscriptional mechanisms. RNA stability assays showed that the effect is not mediated by alteration of the half-life of the HLA-A2 mRNA. Formation of the 3′ end was unaffected by IFN-γ treatment. Sequences that mediate the majority of IFN-γ induction of HLA-A2 mRNA reside in a 127-bp 3′-transcribed region of the gene. This region contains the terminal splice site, the usage of which is not affected by IFN-γ treatment. These results demonstrate a novel posttranscriptional mechanism of regulation of MHC class I genes by IFN-γ.
Targeting Src Homology 2 Domain-Containing Tyrosine Phosphatase (SHP-1) into Lipid Rafts Inhibits CD3-Induced T Cell Activation
Authors: Su Michael Wei-Chih , Yu Chao-Lan , Burakoff Steven J. , Jin Yong-Jiu
Published: April 2014

To study the mechanism by which protein tyrosine phosphatases (PTPs) regulate CD3-induced tyrosine phosphorylation, we investigated the distribution of PTPs in subdomains of plasma membrane. We report here that the bulk PTP activity associated with T cell membrane is present outside the lipid rafts, as determined by sucrose density gradient sedimentation. In Jurkat T cells, ∼5–10% of Src homology 2 domain-containing tyrosine phosphatase (SHP-1) is constitutively associated with plasma membrane, and nearly 50% of SHP-2 is translocated to plasma membrane after vanadate treatment. Similar to transmembrane PTP, CD45, the membrane-associated populations of SHP-1 and SHP-2 are essentially excluded from lipid rafts, where other signaling molecules such as Lck, linker for activation of T cells, and CD3ζ are enriched. We further demonstrated that CD3-induced tyrosine phosphorylation of these substrates is largely restricted to lipid rafts, unless PTPs are inhibited. It suggests that a restricted partition of PTPs among membrane subdomains may regulate protein tyrosine phosphorylation in T cell membrane. To test this hypothesis, we targeted SHP-1 into lipid rafts by using the N-terminal region of Lck (residues 1–14). The results indicate that the expression of Lck/SHP-1 chimera inside lipid rafts profoundly inhibits CD3-induced tyrosine phosphorylation of CD3ζ/ε, IL-2 generation, and nuclear mobilization of NF-AT. Collectively, these results suggest that the exclusion of PTPs from lipid rafts may be a mechanism that potentiates TCR/CD3 activation.
Class I MHC Expression in the Yellow Baboon
Authors: Sidebottom David A. , Kennedy Ronald , Hildebrand William H.
Published: April 2014

MHC class I molecules play a crucial role in the immune response to pathogens and vaccines and in self/non-self recognition. Therefore, characterization of MHC class I gene expression of Papio subspecies is a prerequisite for studies of immunology and transplantation in the baboon (Papio hamadryas). To elucidate MHC class I expression and variation within Papio subspecies and to further investigate the evolution of A and B loci in Old World primates, we have characterized the expressed class I repertoire of the yellow baboon (Papio hamadryas cynocephalus) by cDNA library screening. A total of nine distinct MHC class I cDNAs were isolated from a spleen cDNA library. The four A alleles and four B alleles obtained represent four distinct loci indicating that a duplication of the A and B loci has taken place in the lineage leading to these Old World primates. No HLA-C homologue/orthologue was found. In addition a single, nonclassical homologue of HLA-E was characterized. Examination of nucleotide and extrapolated protein sequences indicates that alleles at the two B loci are much more diversified than the alleles at the A loci. One of the A loci in particular appears to display very limited polymorphism in both Papio hamadryas cynocephalus and Papio hamadryas anubis subspecies. The failure to detect a homologue of HLA–C in the baboon provides additional evidence for the more recent origin of this locus in the Pongidae and Hominidae. Further comparative analysis with MHC sequences among the primate species reveals specific patterns of divergence and conservation within class I molecules of the yellow baboon.
A Structural Difference Limited to One Residue of the Antigenic Peptide Can Profoundly Alter the Biological Outcome of the TCR-Peptide/MHC Class I Interaction
Authors: Thomson Cole T. , Kalergis Alexis M. , Sacchettini James C. , Nathenson Stanley G.
Published: April 2014

The vesicular stomatitis virus (VSV) octapeptide RGYVYQGL binds to H-2Kb and triggers a cytotoxic T cell response in mice. A variant peptide, RGYVYEGL (E6) with a glutamic acid for glutamine replacement at position 6 of the VSV peptide, elicits a T cell response with features that are quite different from those elicited by the wild-type VSV peptide. The differences found in the nature of the T cells responding to the E6 peptide include changes in both the Vβ elements and the sequences of the complementarity-determining region 3 loops of their TCRs. Further experiments found that the E6 peptide can act as an antagonist for VSV-specific T cell hybridomas. To determine whether these differences in Vβ usage, complementarity-determining region 3 sequences, and the switch from agonism to antagonism are caused by a conformational change on the MHC, the peptide, or both, we determined the crystal structure of the variant E6 peptide bound to H-2Kb. This structure shows that the only significant structural difference between H-2Kb/E6 and the previously determined H-2Kb/VSV is limited to the side chain of position 6 of the peptide, with no differences in the MHC molecule. Thus, a minor conformational change in the peptide can profoundly alter the biological outcome of the TCR-peptide/MHC interaction.
Topology and Structure of the C1q-Binding Site on C-Reactive Protein
Authors: Agrawal Alok , Shrive Annette K. , Greenhough Trevor J. , Volanakis John E.
Published: April 2014

The host defense functions of human C-reactive protein (CRP) depend to a great extent on its ability to activate the classical complement pathway. The aim of this study was to define the topology and structure of the CRP site that binds C1q, the recognition protein of the classical pathway. We have previously reported that residue Asp112 of CRP plays a major role in the formation of the C1q-binding site, while the neighboring Lys114 hinders C1q binding. The three-dimensional structure of CRP shows the presence of a deep, extended cleft in each protomer on the face of the pentamer opposite that containing the phosphocholine-binding sites. Asp112 is part of this marked cleft that is deep at its origin but becomes wider and shallower close to the inner edge of the protomer and the central pore of the pentamer. The shallow end of the pocket is bounded by the 112–114 loop, residues 86–92 (the inner loop), the C terminus of the protomer, and the C terminus of the pentraxin α-helix 169–176, particularly Tyr175. Mutational analysis of residues participating in the formation of this pocket demonstrates that Asp112 and Tyr175 are important contact residues for C1q binding, that Glu88 influences the conformational change in C1q necessary for complement activation, and that Asn158 and His38 probably contribute to the correct geometry of the binding site. Thus, it appears that the pocket at the open end of the cleft is the C1q-binding site of CRP.
Two MHC Surface Amino Acid Differences Distinguish Foreign Peptide Recognition from Autoantigen Specificity
Authors: Basu Devraj , Horvath Stephen , O’Mara Leigh , Donermeyer David , Allen Paul M.
Published: April 2014

KRN T cells can recognize two self MHC alleles with differing biological consequences. They respond to the foreign peptide RN(42–56) bound to I-Ak or alternatively initiate autoimmune arthritis by interacting with a self Ag, GPI(282–294), on I-Ag7. Five surface amino acid differences between the two MHC molecules collectively alter which peptide side chains are recognized by the KRN TCR. In this study, it is shown that mutation of only two of these residues, α65 and β78, in I-Ak to their I-Ag7 counterparts is sufficient to allow recognition of the TCR contacts from GPI(282–294). To provide a detailed mechanism for the specificity change, the distinct contributions of each of these two mutations to the global effect on peptide specificity were analyzed. The α65 mutation is shown to broaden the spectrum of amino acids permissible at P8 of the peptide. In contrast, the β78 mutation alone blocks KRN TCR interaction with I-Ak and requires the simultaneous presence of the α65 mutation to preserve recognition. In the presence of the α65 mutation, the β78 residue broadens peptide recognition at P3 and prevents recognition of the P8 L in RN(42–56), thus producing the observed specificity shift. These results localize the functionally relevant differences between the surfaces of two self-restricted MHC molecules to two residues that have counterbalanced positive and negative contributions to interaction with a single TCR. They highlight how subtle structural distinctions attributable to single amino acids can stand at the interface between foreign Ag responsiveness and pathogenic autoreactivity.
Rapidly Fatal Leishmaniasis in Resistant C57BL/6 Mice Lacking TNF
Authors: Wilhelm Patricia , Ritter Uwe , Labbow Stefanie , Donhauser Norbert , Röllinghoff Martin , Bogdan Christian , Körner Heinrich
Published: April 2014

The resolution of infections with the protozoan parasite Leishmania major in mice requires a Th1 response that is closely associated with the expression of IL-12, IFN-γ, and inducible NO synthase. Previous Ab neutralization studies or the use of mice deficient for both TNF receptors suggested that TNF plays only a limited role in the control of parasite replication in vivo. In this study we demonstrate that resistant C57BL/6 (B6.WT) mice locally infected with L. major rapidly succumb to progressive visceral leishmaniasis after deletion of the TNF gene by homologous recombination. A reduction of the parasite inoculum to 3000 promastigotes did not prevent the fatal outcome of the disease. An influence of the altered morphology of secondary lymphoid organs in C57BL/6-TNF−/− (B6.TNF−/−) mice on the course of disease could be excluded by the generation of reciprocal bone marrow chimeras. Although infected B6.TNF−/− mice mounted an L. major-specific IFN-γ response and expressed IL-12, the onset of the immune reaction was delayed. After in vitro stimulation, B6.TNF−/− inflammatory macrophages released 10-fold less NO in response to IFN-γ than B6.WT cells. However, in the presence of a costimulus, e.g., L. major infection or LPS, the production of NO by B6.WT and B6.TNF−/− macrophages was comparable. In vivo, inducible NO synthase protein was readily detectable in skin lesions and draining lymph nodes of B6.TNF−/− mice, but its expression was more disperse and less focal in the absence of TNF. These are the first data to demonstrate that TNF is essential for the in vivo control of L. major.
Successful Therapy of Lethal Murine Visceral Leishmaniasis with Cystatin Involves Up-Regulation of Nitric Oxide and a Favorable T Cell Response
Authors: Das Lopamudra , Datta Neeta , Bandyopadhyay Santu , Das Pijush K.
Published: April 2014

The virulence of Leishmania donovani in mammals depends at least in part on cysteine proteases because they play a key role in CD4+ T cell differentiation. A 6-fold increase in NO production was observed with 0.5 μM chicken cystatin, a natural cysteine protease inhibitor, in IFN-γ-activated macrophages. In a 45-day BALB/c mouse model of visceral leishmaniasis, complete elimination of spleen parasite burden was achieved by cystatin in synergistic activation with a suboptimal dose of IFN-γ. In contrast to the case with promastigotes, cystatin and IFN-γ inhibited the growth of amastigotes in macrophages. Although in vitro cystatin treatment of macrophages did not induce any NO generation, significantly enhanced amounts of NO were generated by macrophages of cystatin-treated animals. Their splenocytes secreted soluble factors required for the induction of NO biosynthesis, and the increased NO production was paralleled by a concomitant increase in antileishmanial activity. Moreover, splenocyte supernatants treated with anti-IFN-γ or anti-TNF-α Abs suppressed inducible NO generation, whereas i.v. administration of these anticytokine Abs along with combined therapy reversed protection against infection. mRNA expression and flow cytometric analysis of infected spleen cells suggested that cystatin and IFN-γ treatment, in addition to greatly reducing parasite numbers, resulted in reduced levels of IL-4 but increased levels of IL-12 and inducible NO synthase. Not only was this treatment curative when administered 15 days postinfection, but it also imparted resistance to reinfection. These studies provide a promising alternative for protection against leishmaniasis with a switch of CD4+ differentiation from Th2 to Th1, indicative of long-term resistance.
Bactericidal Properties of Group IIA and Group V Phospholipases A2
Authors: Grönroos Juha O. , Laine Veli J. O. , Janssen Marcel J. W. , Egmond Maarten R. , Nevalainen Timo J.
Published: April 2014

Group V phospholipase A2 (PLA2) is a recently characterized 14-kDa secretory PLA2 of mammalian heart and macrophage-derived cells. Group IIA PLA2, which is structurally close to group V PLA2, has been shown to kill Gram-positive bacteria in vitro and to prevent symptoms of Gram-positive infection in vivo. We studied the antibacterial properties of fully active recombinant rat group IIA and V PLA2s. Both group IIA and V PLA2s were highly bactericidal against Gram-positive bacteria, including methicillin-resistant staphylococci and vancomycin-resistant enterococci. Only high concentrations of group IIA PLA2 showed some bactericidal effect against the Gram-negative bacterium Escherichia coli. Our results confirm that group IIA PLA2 is a potent antibacterial enzyme against Gram-positive bacteria. Moreover, we show here that group V PLA2 is a novel antibacterial mammalian protein, but is less potent than group IIA PLA2. Both enzymes may be considered as future therapeutic agents against bacterial infections.
CXC Chemokine Receptor 2 But Not C-C Chemokine Receptor 1 Expression Is Essential for Neutrophil Recruitment to the Cornea in Helminth-Mediated Keratitis (River Blindness)
Authors: Hall Laurie R. , Diaconu Eugenia , Patel Roopa , Pearlman Eric
Published: April 2014

Infiltration of neutrophils and eosinophils into the mammalian cornea can result in loss of corneal clarity and severe visual impairment. To identify mediators of granulocyte recruitment to the corneal stroma, we determined the relative contribution of chemokine receptors CXC chemokine receptor (CXCR)-2 (IL-8R homologue) and CCR1 using a murine model of ocular onchocerciasis (river blindness) in which neutrophils and eosinophils migrate from peripheral vessels to the central cornea. CXCR2−/− and CCR1−/− mice were immunized s.c. and injected into the corneal stroma with Ags from the parasitic helminth Onchocerca volvulus. We found that production of macrophage-inflammatory protein (MIP)-2, KC, and MIP-1α was localized to the corneal stroma, rather than to the epithelium, which was consistent with the location of neutrophils in the cornea. CCR1 deficiency did not inhibit neutrophil or eosinophil infiltration to the cornea or development of corneal opacification. In marked contrast, neutrophil recruitment to the corneas of CXCR2−/− mice was significantly impaired (p < 0.0001 compared with control, BALB/c mice) with only occasional neutrophils detected in the central cornea. Furthermore, CXCR2−/− mice developed only mild corneal opacification compared with BALB/c mice. These differences were not due to impaired KC and MIP-2 production in the corneal stroma of CXCR2−/− mice, which was similar to BALB/c mice. Furthermore, although MIP-1α production was lower in CXCR2−/− mice than BALB/c mice, eosinophil recruitment to the cornea was not impaired. These observations demonstrate the critical role for CXCR2 expression in neutrophil infiltration to the cornea and may indicate a target for immune intervention in neutrophil-mediated corneal inflammation.
Early Response Cytokines and Innate Immunity: Essential Roles for TNF Receptor 1 and Type I IL-1 Receptor During Escherichia coli Pneumonia in Mice
Authors: Mizgerd Joseph P. , Spieker Matt R. , Doerschuk Claire M.
Published: April 2014

The early response cytokines, TNF and IL-1, have overlapping biologic effects that may function to propagate, amplify, and coordinate host responses to microbial challenges. To determine whether signaling from these early response cytokines is essential to orchestrating innate immune responses to intrapulmonary bacteria, the early inflammatory events induced by instillation of Escherichia coli into the lungs were compared in wild-type (WT) mice and mice deficient in both TNF receptor 1 (TNFR1) and the type I IL-1 receptor (IL1R1). Neutrophil emigration and edema accumulation induced by E. coli were significantly compromised by TNFR1/IL1R1 deficiency. Neutrophil numbers in the circulation and within alveolar septae did not differ between WT and TNFR1/IL1R1 mice, suggesting that decreased neutrophil emigration did not result from decreased sequestration or delivery of intravascular neutrophils. The nuclear translocation of NF-κB and the expression of the chemokine macrophage inflammatory protein-2 did not differ between WT and TNFR1/IL1R1 lungs. However, the concentration of the chemokine KC was significantly decreased in the bronchoalveolar lavage fluids of TNFR1/IL1R1 mice compared with that in WT mice. Thus, while many of the molecular and cellular responses to E. coli in the lungs did not require signaling by either TNFR1 or IL1R1, early response cytokine signaling was critical to KC expression in the pulmonary air spaces and neutrophil emigration from the alveolar septae.
CD8 CTL from Genital Herpes Simplex Lesions: Recognition of Viral Tegument and Immediate Early Proteins and Lysis of Infected Cutaneous Cells
Authors: Koelle David M. , Chen Hongbo B. , Gavin Marc A. , Wald Anna , Kwok William W. , Corey Lawrence
Published: April 2014

HSV-2 causes chronic infections. CD8 CTL may play several protective roles, and stimulation of a CD8 response is a rational element of vaccine design for this pathogen. The viral Ags recognized by CD8 T cells are largely unknown. It has been hypothesized that HSV inhibition of TAP may favor recognition of virion input proteins or viral immediate early proteins. We tested this prediction using HSV-specific CD8 CTL clones obtained from genital HSV-2 lesions. Drug and replication block experiments were consistent with specificity for the above-named classes of viral proteins. Fine specificity was determined by expression cloning using molecular libraries of viral DNA, and peptide epitopes recognized at nanomolar concentrations were identified. Three of four clones recognized the viral tegument proteins encoded by genes UL47 and UL49. These proteins are transferred into the cytoplasm on virus entry. Processing of the tegument Ag-derived epitopes was TAP dependent. The tegument-specific CTL were able to lyse HLA class I-appropriate fibroblasts after short times of infection. Lysis of keratinocytes required longer infection and pretreatment with IFN-γ. Another clone recognized an immediate early protein, ICP0. Lymphocytes specific for these lesion-defined epitopes could be reactivated from the PBMC of additional subjects. These data are consistent with an influence of HSV immune evasion genes upon the selection of proteins recognized by CD8 CTL in lesions. Tegument proteins, identified for the first time as Ags recognized by HSV-specific CD8 CTL, are rational candidate vaccine compounds.
Mice Lacking the Multidrug Resistance Protein 1 Are Resistant to Streptococcus pneumoniae-Induced Pneumonia
Authors: Schultz Marc J. , Wijnholds Jan , Peppelenbosch Maikel P. , Vervoordeldonk Margriet J. B. M. , Speelman Peter , van Deventer Sander J. H. , Borst Piet , van der Poll Tom
Published: April 2014

Leukotrienes (LTs) are considered important for antibacterial defense in the lung. Multidrug resistance protein 1 (mrp1) is a transmembrane protein responsible for the cellular extrusion of LTC4. To determine the role of mrp1 in host defense against pneumonia, mrp1−/− and wild-type mice were intranasally inoculated with Streptococcus pneumoniae. mrp1−/− mice displayed a diminished outgrowth of pneumococci in lungs and a strongly reduced mortality. These findings were related to an effect of mrp1 on LT metabolism, because survival was similar in mrp1−/− and wild-type mice treated with the 5-lipoxygenase-activating protein inhibitor MK-886. Although LTC4 levels remained low in the bronchoalveolar lavage fluid of mrp1−/− mice, LTB4 concentrations were higher than in wild-type mice. These elevated LTB4 concentrations were important for the relative protection of mrp1−/− mice, because the LTB4 antagonist LTB4-dimethyl amide abolished their survival advantage. In vitro experiments suggested that the intracellullar accumulation of LTC4 in mrp1−/− mice results in product inhibition of LTC4-synthase, diminishing substrate competition between LTA4-hydrolase (which yields LTB4) and LTC4-synthase for the available LTA4. We conclude that mrp1−/− mice are resistant against pneumococcal pneumonia by a mechanism that involves increased release of LTB4. These results identify mrp1 as a novel target for adjunctive therapy in pneumonia.