bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes home preferentially to and induce selective regressions of autologous EBV-induced B cell lymphoproliferations in xenografted C.B-17 scid/scid mice.
Authors: Lacerda J F , Ladanyi M , Louie D C , Fernandez J M , Papadopoulos E B , O'Reilly R J
Published: June 2004

C.B-17 scid/scid (severe combined immunodeficiency [SCID]) mice inoculated with peripheral blood lymphocytes from Epstein-Barr virus (EBV)-seropositive donors, or with EBV-transformed lymphoblastoid B cell lines (EBV-LCL), develop lethal human EBV+ B cell lymphoproliferative disorders (EBV-LPD) with characteristics similar to those arising in immunodeficient patients. Using this model, we examined the capacity of human effector cells to control human EBV-LPD. SCID mice received rabbit anti-asialo GM1 antiserum to abrogate endogenous natural killer-cell function. Preliminary experiments showed that adoptive transfer of peripheral blood mononuclear cells (PBMC), purified T cells, interleukin (IL) 2-activated PBMC or anti-CD3-activated T cells derived from EBV-seropositive donors did not result in improved survival of treated mice (in vivo effector/target ratio 2:1 to 1:1). In contrast, EBV-specific cytotoxic T lymphocytes (CTL), derived from EBV-seropositive donors and expanded in vitro, exhibited strong EBV-specific and HLA-restricted activity both in vitro and in vivo. SCID mice inoculated intraperitoneally with autologous but not with HLA-mismatched EBV-LCL had significantly improved survival relative to untreated mice after inoculation of EBV-specific CTL either intraperitoneally (P<0.001) or intravenously (P<0.001) (in vivo effector/target ratio 1:1). SCID mice bearing large subcutaneous EBV+ tumors and treated intravenously with 10(7) EBV-specific CTL achieved complete tumor regression. Both CTL- and CTL-plus-IL-2-treated mice survived significantly longer than untreated animals or animals treated with IL-2 alone (P = 0.0004 and P<0.02, respectively). SCID mice bearing two subcutaneous EBV+ tumors, one autologous and the other HLA mismatched to the EBV-specific CTL donor, had regression of only the autologous tumor after intravenous infusion of 10(7) EBV-specific CTL. Moreover, we could demonstrate preferential homing of PKH26-labeled EBV-specific CTL to autologous but not to HLA-mismatched EBV+ tumors as early as 24 h after intravenous adoptive transfer. Immunophenotypic analyses also demonstrated preferential infiltration of T cells into the autologous EBV+ tumor in SCID mice bearing both the autologous and either fully HLA-mismatched or genotypically related haplotype-sharing EBV+ tumors. The human T cells infiltrating EBV+ tumors were CD3+ and, predominantly, CD8+CD4-. Our results indicate that EBV-specific CTL preferentially localize to and infiltrate EBV+ tumors bearing the appropriate HLA antigens and thereafter induce targeted regressions of disease.
Expression of variants of the major surface glycoprotein of Pneumocystis carinii.
Authors: Angus C W , Tu A , Vogel P , Qin M , Kovacs J A
Published: June 2004

Previously, we have shown that a multicopy family of related but unique genes encodes the major surface glycoprotein (MSG) of Pneumocystis carinii. To examine whether different members of this gene family are expressed by P. carinii, antisera were prepared against peptides whose sequences were determined from the deduced amino acid sequences of variants of rat-derived MSG. Immunohistochemical staining of serial sections of rat lungs of infected animals showed that at least three variants of MSG were expressed in an individual lobe, that there was a focal expression of these variants within the lung, and that the relative numbers of these foci were different. Indirect immunofluorescent staining of purified P. carinii organisms using these antisera revealed that at least three variants of MSG were present in organisms isolated from an individual rat and that both cysts and trophozoites reacted with each antiserum. A substantial difference in the fraction of organisms reacting with a specific antipeptide antiserum was seen when comparing organisms isolated from rats raised in a single colony over a period of two years as well as organisms isolated at one time point from rats raised in different colonies. This demonstration of antigenic variation in P. carinii supports the hypothesis that P. carinii utilizes such variation for evading host defense mechanisms.
Bone marrow-derived cells fail to induce positive selection in thymus reaggregation cultures.
Authors: Ernst B B , Surh C D , Sprent J
Published: June 2004

The requirements for inducing positive selection of T cells were examined in thymus reaggregation cultures, a system in which dispersed populations of immature CD4+8+ cells and purified thymic epithelial cells (TEC) are reaggregated in tissue culture. Studies with TEC from mice selectively lacking major histocompatibility complex (MHC) class I (I-II+), class II (I+II-), or both class I and II (I-II-) molecules showed that class II expression was essential for the differentiation of CD4+8+ cells into CD4+8- cells. Unexpectedly, the generation of TCRhi CD4-8+ cells from CD4+8+ cells was apparent with I-II+ TEC but not with I-II- TEC, perhaps reflecting cross-reactive specificity of CD4-8+ cells for class II molecules. Significantly, the failure of I-II- TEC to generate TCRhi CD4+8- or CD4-8+ cells could not be overcome by adding MHC+ bone marrow-derived cells. These findings, together with experiments on purified subsets of TEC, suggest that positive selection in thymus reaggregation cultures is an exclusive property of cortical TEC.
HLA-DQB1 codon 57 is critical for peptide binding and recognition.
Authors: Kwok W W , Domeier M E , Johnson M L , Nepom G T , Koelle D M
Published: June 2004

The association of specific HLA-DQ alleles with autoimmunity is correlated with discrete polymorphisms in the HLA-DQ sequence that are localized within sites suitable for peptide recognition. The polymorphism at residue 57 of the DQB1 polypeptide is of particular interest since it may play a major structural role in the formation of a salt bridge structure at one end of the peptide-binding cleft of the DQ molecules. This polymorphism at residue 57 is a recurrent feature of HLA-DQ evolution, occurring in multiple distinct allelic families, which implies a functional selection for maintaining variation at this position in the class II molecule. We directly tested the amino acid polymorphism at this site as a determinant for peptide binding and for antigen-specific T cell stimulation. We found that a single Ala-->Asp amino acid 57 substitution in an HLA-DQ3.2 molecule regulated binding of an HSV-2 VP-16-derived peptide. A complementary single-residue substitution in the peptide abolished its binding to DQ3.2 and converted it to a peptide that can bind to DQ3.1 and DQ3.3 Asp-57-positive MHC molecules. These binding studies were paralleled by specific T cell recognition of the class II-peptide complex, in which the substituted peptide abolished T cell reactivity, which was directed to the DQ3.2-peptide complex, whereas the same T cell clone recognized the substituted peptide presented by DQ3.3, a class II restriction element differing from DQ3.2 only at residue 57. This structural and functional complementarity for residue 57 and a specific peptide residue identifies this interaction as a key controlling determinant of restricted recognition in HLA-DQ-specific immune response.
Reconstitution of human Fc gamma RIII cell type specificity in transgenic mice.
Authors: Li M , Wirthmueller U , Ravetch J V
Published: June 2004

The human low affinity receptors for the Fc domain of immunoglobulin G, Fc gamma RIII, are encoded by two genes (IIIA and IIIB) which share >95% sequence identity in both coding and flanking sequences. Despite this extraordinary sequence conservation, IIIA is expressed in natural killer (NK) cells and macrophages and is absent in neutrophils, whereas IIIB is expressed only in neutrophils. To determine the molecular basis for this differential expression, we have generated transgenic mice using the genomic sequences of IIIA and IIIB. IIIA and IIIB transgenic mice show faithful reconstitution of this human pattern of cell type specificity. To determine the cis acting sequence elements that confer this specificity, we constructed chimeric genes in which 5.8 kb of 5' sequences of the IIIB gene has been replaced with a homologous region from the IIIA gene, and conversely, IIIA 5' sequences have been substituted for the analogous region of the IIIB gene. Promoter swap transgenic mice that carry IIIA 5' flanking sequences express Fc gamma RIII in macrophages and NK cells. In contrast, promoter swap transgenic mice that contain IIIB 5' sequences express Fc gamma RIII in neutrophils only. These studies define the elements conferring the cell type-specific expression of the human Fc gamma RIII genes within the 5' flanking sequences and first intron of the human Fc gamma RIIIA and Fc gamma RIIIB genes.
A potential mechanism of "cross-talk" between the p55 tumor necrosis factor receptor and Fas/APO1: proteins binding to the death domains of the two receptors also bind to each other.
Authors: Varfolomeev E E , Boldin M P , Goncharov T M , Wallach D
Published: June 2004

The p55 tumor necrosis factor (TNF) receptor and Fas/APO1 induce cell death via distinct regions in their intracellular domains. Three cytoplasmic proteins that bind to these receptor regions have been identified recently. One, MORT1 (also called FADD), binds to Fas/APO1 but not to p55-R; another, TRADD, binds to the p55 TNF receptor but not to Fas/APO1; and the third, RIP, binds weakly to both receptors. The regions within these proteins that are involved in binding to the receptors and the receptor regions to which they bind share a common sequence motif, that of the "death domain." This study shows that the death domain motifs in MORT1, TRADD, and RIP bind effectively to each other, a mode of binding that may allow "cross-talk" between the functional expression of the p55 TNF receptor and Fas/APO1.
Stringent V beta requirement for the development of NK1.1+ T cell receptor-alpha/beta+ cells in mouse liver.
Authors: Ohteki T , MacDonald H R
Published: June 2004

The liver of C57BL/6 mice contains a major subset of CD4+8- and CD4-8- T cell receptor (TCR)-alpha/beta+ cells expressing the polymorphic natural killer NK1.1 surface marker. Liver NK1.1+TCR-alpha/beta+ (NK1+ T) cells require interaction with beta2-microglobulin-associated, major histocompatibility complex I-like molecules on hematopoietic cells for their development and have a TCR repertoire that is highly skewed to Vbeta8.2, Vbeta7, and Vbeta2. We show here that congenic C57BL/6.Vbeta(a) mice, which lack Vbeta8- expressing T cells owing to a genomic deletion at the Vbeta locus, maintain normal levels of liver NK1+ T cells owing to a dramatic increase in the proportion of cells expressing Vbeta7 and Vbeta2 (but not other Vbetas). Moreover, in C57BL/6 congenic TCR-V Vbeta3 and -Vbeta8.1 transgenic mice (which in theory should not express other Vbeta, owing to allelic exclusion at the TCR-beta locus), endogenous TCR-Vbeta8.2, Vbeta7, and Vbeta2 (but not other Vbetas) are frequently expressed on liver NK1+T cells but absent on lymph node T cells. Finally, when endogenous V beta expression is prevented in TCR-Vbeta3 and Vbeta8.1 transgenic mice (by introduction of a null allele at the C beta locus), the development of liver NK1+T cells is totally abrogated. Collectively, our data indicate that liver NK1+T cells have a stringent requirement for expression of TCR-Vbeta8.2, Vbeta7, or Vbeta2 for their development.
The cytoplasmic domain of CD4 promotes the development of CD4 lineage T cells.
Authors: Itano A , Salmon P , Kioussis D , Tolaini M , Corbella P , Robey E
Published: June 2004

Thymocytes must bind major histocompatibility complex (MHC) proteins on thymic epithelial cells in order to mature into either CD8+ cytotoxic T cells or CD4+ helper T cells. Thymic precursors express both CD8 and CD4, and it has been suggested that the intracellular signals generated by CD8 or CD4 binding to class I or II MHC, respectively, might influence the fate of uncommitted cells. Here we test the notion that intracellular signaling by CD4 directs the development of thymocytes to a CD4 lineage. A hybrid protein consisting of the CD8 extracellular and transmembrane domains and the cytoplasmic domain of CD4 (CD884) should bind class I MHC but deliver a CD4 intracellular signal. We find that expression of a hybrid CD884 protein in thymocytes of transgenic mice leads to the development of large numbers of class I MHC-specific, CD4 lineage T cells. We discuss these results in terms of current models for CD4 and CD8 lineage commitment.
Importance of low affinity Elf-1 sites in the regulation of lymphoid-specific inducible gene expression.
Authors: John S , Marais R , Child R , Light Y , Leonard W J
Published: June 2004

Elf-1 is an Ets family transcription factor that regulates a number of inducible lymphoid-specific genes, including those encoding interleukin 3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF), and the IL-2 receptor (IL-2R) alpha chain. A minimal oligonucleotide spanning the IL-2R alpha Elf-1 site (-97/-84) bound Elf-1 poorly, but binding activity markedly increased when this oligonucleotide was multimerized or flanking sequences were added. This result is consistent with the requirement of accessory proteins for efficient Elf-1 binding, as has been demonstrated for the GM-CSF and IL-3 promoters. A binding site selection analysis revealed the optimal Elf-1 consensus motif to be A(A/t)(C/a)CCGGAAGT(A/S), which is similar to the consensus motif for the related Drosophila E74 protein. This minimal high affinity site could bind Elf-1 and functioned as a stronger transcription element than the -97/-84 IL-2R alpha oligonucleotide when cloned upstream of a heterologous promoter. In contrast, in the context of the IL-2R alpha promoter, conversion of the naturally occurring low affinity Elf-1 site to an optimal site decreased inducible activation of a reporter construct in Jurkat cells. This finding may be explained by the observation that another Ets family protein, ER GB/Fli-1, can efficiently bind only to the optimal site, and in this context, interferes with Elf-1 binding. Therefore, high affinity Elf-1 sites may lack sufficient binding specificity, whereas naturally occurring low affinity sites presumably favor the association of Elf-1 in the context of accessory proteins. These findings offer an explanation for the lack of optimal sites in any of the known Elf-1-regulated genes.
Does B7-1 expression confer antigen-presenting cell capacity to tumors in vivo?
Authors: Huang A Y , Bruce A T , Pardoll D M , Levitsky H I
Published: June 2004

Tumors engineered to express the costimulatory molecule B7-1 can elicit CD8+ cytotoxic T lymphocyte (CTL)-dependent antitumor responses in immunocompetent mice. It has been postulated that this result reflects direct priming of CTL by the modified tumor in vivo. Previous studies of the immune response to a B7-1- murine colon carcinoma expressing influenza nucleoprotein (NP) as a model tumor antigen have demonstrated the crucial role of bone marrow-derived antigen-presenting cells (APCs) in the priming of NP-specific CTL in vivo. In this system, no evidence of direct CTL priming by tumor was detected. We have performed a similar analysis to determine if B7-1 transfectant of this tumor results in the direct priming of CTL, and to compare this response to that primed by host APCs. When H-2b-->H-2bxd bone marrow chimeras were immunized with a single injection of CT26/NP/B7-1 (H-2d), NP-specific CTL were detected that were restricted to the bone marrow haplotype (H-2b), but not to the tumor haplotype. In contrast, CTL recognizing the NP antigenic epitope in the context of the tumor's major histocompatibility complex were detectable only after multiple immunizations. These results suggest that whereas B7-1+ tumor vaccines result in some degree of direct presentation to CD8+ T cells, the dominant mechanism of CTL priming is through the uptake and presentation of tumor antigens by bone marrow-deprived APCs. However, repeated immunization with B7-1+ tumor cells can efficiently expand the directly primed CD8+ CTL population.
A cell type-specific enhancer in the human B7.1 gene regulated by NF-kappaB.
Authors: Zhao J , Freeman G J , Gray G S , Nadler L M , Glimcher L H
Published: June 2004

The costimulatory molecule B7.1 provides a second signal critical for T cell activation. The distribution of this integral membrane protein is restricted to certain tissues where its level of expression is modulated by multiple exogenous stimuli. To identify the molecular basis for specificity and inducibility, the chromatin configuration of the human B7.1 gene was examined in intact nuclei from various cell types. The identification of a tissue-specific deoxyribonuclease I hypersensitive site approximately 3kb upstream of the transcription start site led to the characterization of a cell type-specific enhancer region. This 183-bp region was both cell type specific and responsive to two distinct stimuli, lipopolysaccharide and dibutyryl cAMP, known to regulate B7.1 expression. Deletional and site-directed mutagenesis revealed the presence of multiple functionally critical cis elements within this region, one of which was a nuclear factor (NF)-kappaB consensus sequence. In B7.1-positive B cells, this element bound several members of the NF-kappaB family, transcription factors already implicated in signal transduction pathways relevant to B7.1 expression. This is the first description, to our knowledge, of regulatory elements that control expression of a gene encoding a B7 costimulatory molecule.
B7-CD28 costimulation unveils the hierarchy of tumor epitopes recognized by major histocompatibility complex class I-restricted CD8+ cytolytic T lymphocytes.
Authors: Johnston J V , Malacko A R , Mizuno M T , McGowan P , Hellström I , Hellström K E , Marquardt H , Chen L
Published: June 2004

Immunization of mice with tumors genetically engineered to express the B7 costimulatory molecules amplifies the antitumor immune response mediated by CD8+ cytolytic T lymphocytes (CTL). In this report, we examined the effect of B7-CD28 costimulation on the hierarchy of tumor epitopes. Using a combination of affinity chromatography/reversed-phase high performance liquid chromatography and CTL cloning, we show that major histocompatibility complex (MHC) class I molecules from EL4 lymphoma cells can present at least six distinct CTL epitopes presented by MHC class I molecules. Nevertheless, mice immunized with wild-type B7-negative EL4 cells develop CTL only to one immunodominant epitope. In contrast, immunization with B7-transduced EL4 cells led to not only the amplification of the CTL response to this immunodominant epitope, but also to the recognition of five otherwise silent subdominant epitopes. The adoptive transfer of a CTL clone against such a subdominant epitope cured mice bearing EL4 lymphoma growing as an ascites tumor. The fact that CTL response can be spread to normally silent epitopes as a result of B7-CD28 costimulation suggests a novel approach to manipulate the hierarchy of CTL epitopes and offers an opportunity to explore novel targets for T cell-mediated cancer therapy.
The relative contribution of the CD28 and gp39 costimulatory pathways in the clonal expansion and pathogenic acquisition of self-reactive T cells.
Authors: Griggs N D , Agersborg S S , Noelle R J , Ledbetter J A , Linsley P S , Tung K S
Published: June 2004

The zona pellucida (ZP), an ovarian extracellular structure, contains three major glycoproteins: ZP1, ZP2, and ZP3. A ZP3 peptide contains both an autoimmune oophoritis-inducing T cell epitope and a B cell epitope that induces autoantibody to ZP. This study investigates two major T cell costimulation pathways in this disease model. Herein we show that blockage of glycoprotein (gp)39 and CD40 interaction with gp39 monoclonal antibody (mAb) results in the failure to induce both autoimmune oophoritis and autoantibody production. Inhibition of ligand binding to the CD28 receptor with the fusion protein, murine CTLA4-immunoglobulin (Ig), also results in failure to generate antibody to ZP and significantly reduces disease severity and prevalence. Surprisingly, the frequencies of antigen-specific T cells in anti-gp39 mAb-treated mice, CTLA4-Ig treated mice, and in mice given control hamster IgG or control fusion protein L6, were equivalent as determined by limiting dilution analysis (approximately equals 1:5,000). These T cells, which produced comparable amounts of interleukin 4 and interferon gamma in vitro, were able to transfer oophoritis to normal recipients. When anti-gp39 mAb and CTLA4-Ig were given together, the effect was additive, leading to inhibition of T cell activation as determined by in vitro proliferation and limiting dilution analysis (approximately equals 1:190,000); disease and antibody responses were absent in these mice. By studying these two costimulatory pathways in parallel, we have shown that autoimmune disease and autoantibody production are inhibitable by blocking either the gp39 or the CD28 pathway, whereas inhibition of clonal expansion of the effector T cell population occurs only when both pathways are blocked.
Tyrosyl phosphorylation and DNA binding activity of signal transducers and activators of transcription (STAT) proteins in hematopoietic cell lines transformed by Bcr/Abl.
Authors: Carlesso N , Frank D A , Griffin J D
Published: June 2004

Bcr/Abl is a chimeric oncogene that can cause both acute and chronic human leukemias. Bcr/Abl-encoded proteins exhibit elevated kinase activity compared to c-Abl, but the mechanisms of transformation are largely unknown. Some of the biological effects of Bcr/Abl overlap with those of hematopoietic cytokines, particularly interleukin 3 (IL-3). Such effects include mitogenesis, enhanced survival, and enhanced basophilic differentiation. Therefore, it has been suggested that p210Bcr/Abl and the IL-3 receptor may activate some common signal transduction pathways. An important pathway for IL-3 signaling involves activation of the Janus family kinases (JAKs) and subsequent tyrosyl phosphorylation of STAT proteins (signal transducers and activators of transcription). This pathway directly links growth factor receptors to gene transcription. We analyzed JAK activation, STAT protein phosphorylation, and the formation of specific DNA-binding complexes containing STAT proteins, in a series of leukemia cell lines transformed by Bcr/Abl or other oncogenes. We also examined these events in cell lines transformed by a temperature sensitive (ts) mutant of Bcr/Abl, where the kinase activity of Abl could be regulated. STAT1 and STAT5 were found to be constitutively phosphorylated in 32D, Ba/F3, and TF-1 cells transformed by Bcr/Abl, but not in the untransformed parental cell lines in the absence of IL-3. Phosphorylation of STAT1 and STAT5 was also observed in the human leukemia cell lines K562 and BV173, which express the Bcr/Abl oncogene, but not in several Bcr/Abl-negative leukemia cell lines. Phosphorylation of STAT1 and STAT5 was directly due to the tyrosine kinase activity of Bcr/Abl since it could be activated or deactivated by temperature shifting of cells expressing the Bcr/Abl ts mutant. DNA-STAT complexes were detected in all Bcr/Abl-transformed cell lines and they were supershifted by antibodies against STAT1 and STAT5. DNA-STAT complexes in 32Dp210Bcr/Abl cells were similar, but not identical, to those formed after IL-3 stimulation. It is interesting to note that JAK kinases (JAK1, JAK2, JAK3, and Tyk2) were not consistently activated in Bcr/Abl-positive cells. These data suggest that STATs can be activated directly by Bcr/Abl, possibly bypassing JAK family kinase activation. Overall, our results suggest a novel mechanism that could contribute to some of the major biological effects of Bcr/Abl transformation.
Arginine at positions 13 or 70-71 in pocket 4 of HLA-DRB1 alleles is associated with susceptibility to tuberculoid leprosy.
Authors: Zerva L , Cizman B , Mehra N K , Alahari S K , Murali R , Zmijewski C M , Kamoun M , Monos D S
Published: June 2004

Evaluation of human histocompatibility leukocyte antigen (HLA) class II genes in 54 cases of tuberculoid leprosy (TL) and 44 controls has shown a positive association with HLA-DRB1 alleles that contain Arg13 or Arg70-Arg71. Among TL patients, 87% carry specific alleles of DRB1 Arg13 or Arg70-Arg71 as compared to 43% among controls (p = 5 x 10(-6)) conferring a relative risk of 8.8. Thus, susceptibility to TL involves three critical amino acid positions of the beta chain, the side chains of which, when modeled on the DR1 crystal structure, line a pocket (pocket 4) accommodating the side chain of a bound peptide. This study suggests that disease susceptibility may be determined by the independent contribution of polymorphic residues participating in the formation of a functional arrangement (i.e., pocket) within the binding cleft of an HLA molecule.
Erythropoietin-independent erythrocyte production: signals through gp130 and c-kit dramatically promote erythropoiesis from human CD34+ cells.
Authors: Sui X , Tsuji K , Tajima S , Tanaka R , Muraoka K , Ebihara Y , Ikebuchi K , Yasukawa K , Taga T , Kishimoto T , Nakahata T
Published: June 2004

Erythropoietin (EPO) is the primary humoral regulator of erythropoiesis and no other factor has previously been reported to support proliferation and terminal maturation of erythroid cells from hemopoietic stem cells. Here we show that stimulation of glycoprotein (gp130) by a combination of recombinant human soluble interleukin 6 receptor (sIL-6R) and IL-6 but not sIL-6R or IL-6 alone can support proliferation, differentiation, and terminal maturation of erythroid cells in the absence of EPO from purified human CD34+ cells in suspension culture containing stem cell factor (SCF). A number of erythroid bursts and mixed erythroid colonies also developed in methylcellulose culture under the same combination. The addition of anti-gp130 monoclonal antibodies but not anti-EPO antibody to the same culture completely abrogated the generation of erythroid cells. These results clearly demonstrate that mature erythroid cells can be emerged from hemopoietic progenitors without EPO in vitro. Together with the previous reports that human sera contain detectable levels of sIL-6R, IL-6, and SCF, current data suggest that gp130 signaling in association with c-kit activation may play a role in human erythropoiesis in vivo.
Cytokine imbalance and autoantibody production in T cell receptor-alpha mutant mice with inflammatory bowel disease.
Authors: Mizoguchi A , Mizoguchi E , Chiba C , Spiekermann G M , Tonegawa S , Nagler-Anderson C , Bhan A K
Published: June 2004

Spontaneous inflammatory bowel disease (IBD) resembling human ulcerative colitis develops in mice mutant for the T cell receptor alpha gene (TCR-alpha-/-). TCR-alpha-/- mice lack TCR-alpha/beta+ cells but contain TCR-gamma/delta+ cells and a small population of a unique CD4+, TCR-alpha-/beta+(low) cells. Since all the immunoglobulin (Ig) classes are present in these mice, help to B cells must be provided by cells other than TCR-alpha/beta+ cells. In the present study, we found serum levels of IgG1 and IgG2 to be markedly increased in TCR-alpha-/- mice with IBD as compared to TCR-alpha-/- mice without IBD or TCR-alpha+/- controls. An increase in IgG1-, IgG2a- and IgA- but not IgM-secreting mesenteric lymph node (MLN) B cells was detected in TCR-alpha-/- mutant mice. There was also a marked increase in MLN B cells secreting autoantibody (IgG) to tropomyosin, a cytoskeletal protein. Examination of the hyperplastic MLN showed a marked increase in the number of B, TCR-delta+, and CD4+ TCR-alpha-/beta+ cells, similar to the cell population observed at the site of colonic inflammation. Analysis of spontaneous cytokine production by MLN cells using an enzyme-linked immunospot assay, immunohistochemistry, and reverse transcription/polymerase chain reaction showed a decrease of interleukin 2 (IL-2) but a marked increase of IL-4 and interferon gamma (IFN-gamma) production in TCR-alpha-/- mice with IBD as compared to TCR-alpha-/- mice without IBD and TCR alpha+/- control mice. Both TCR-alpha-/beta+ and TCR-delta+ cells were found to be capable of producing IL-4; IFN-gamma was produced mostly by non-T cells, many of which were shown to be CD3- NK 1.1+ cells. We propose that the cytokine imbalance present in these mice results in expansion of B cells, production and switching of autoantibodies to IgG2 subclass, and development of IBD. It is possible that the unusual CD4+ TCR-alpha-/beta+ population and expanded TCR-gamma/delta+ population present in TCR-alpha-/- mice plays a central role in this abnormal immune response.
An insulin peptide that binds an alternative site in class II major histocompatibility complex.
Authors: Tompkins S M , Moore J C , Jensen P E
Published: June 2004

We report that a peptide from the B chain of insulin, B(10-30), binds with high affinity to multiple class II proteins, including IAb,d,k, IEd,k, and DR1. The ability of B(10-30) to inhibit the binding of other peptide antigens to class II does not correlate with its affinity for class II. B(10-30) only weakly inhibits the binding of antigenic peptides. Conversely, peptides with high affinity for the peptide-binding groove of various class II proteins do not inhibit B(10-30) binding. The rate of association of B(10-30) with class II is unusually rapid, approaching saturation in 1-2 h compared with 1-2 d for classical peptide antigens in the same conditions. The dissociation rate is also relatively rapid. The B(10-30) peptide inhibits the binding of the super-antigen staphylococcal enterotoxin B (SEB) to IAk. It also inhibits SEB-mediated T cell activation. These observations support the conclusion that B(10-30) binds to a site outside the peptide-binding groove. Our findings indicate that short-lived peptide-class II complexes can be formed through interactions involving the SEB-binding site and raise the possibility that alternative complexes may serve as T cell receptor ligands.
Signaling through the lymphotoxin beta receptor induces the death of some adenocarcinoma tumor lines.
Authors: Browning J L , Miatkowski K , Sizing I , Griffiths D , Zafari M , Benjamin C D , Meier W , Mackay F
Published: June 2004

Surface lymphotoxin (LT) is a heteromeric complex of LT-alpha and LT-beta chains that binds to the LT-beta receptor (LT-beta-R), a member of the tumor necrosis factor (TNF) family of receptors. The biological function of this receptor-ligand system is poorly characterized. Since signaling through other members of this receptor family can induce cell death, e.g., the TNF and Fas receptors, it is important to determine if similar signaling events can be communicated via the LT-beta-R. A soluble form of the surface complex was produced by coexpression of LT-alpha and a converted form of LT-beta wherein the normally type II LT-beta membrane protein was changed to a type I secreted form. Recombinant LT-alpha 1/beta 2 was cytotoxic to the human adenocarcinoma cell lines HT-29, WiDr, MDA-MB-468, and HT-3 when added with the synergizing agent interferon (IFN) gamma. When immobilized on a plastic surface, anti-LT-beta-R monoclonal antibodies (mAbs) induced the death of these cells, demonstrating direct signaling via the LT-beta-R. Anti-LT-beta-R mAbs were also identified that inhibited ligand-induced cell death, whereas others were found to potentiate the activity of the ligand when added in solution. The human WiDr adenocarcinoma line forms solid tumors in immunocompromised mice, and treatment with an anti-LT-beta-R antibody combined with human IFN-gamma arrested tumor growth. The delineation of a biological signaling event mediated by the LT-beta-R opens a window for further studies on its immunological role, and furthermore, activation of the LT-beta-R may have an application in tumor therapy.
Inactivation of human immunodeficiency virus (HIV)-1 envelope-specific CD8+ cytotoxic T lymphocytes by free antigenic peptide: a self-veto mechanism?
Authors: Takahashi H , Nakagawa Y , Leggatt G R , Ishida Y , Saito T , Yokomuro K , Berzofsky J A
Published: June 2004

Free peptide has been found to inhibit cytotoxic T lymphocyte (CTL) activity, and veto cells bearing peptide-major histocompatibility complex (MHC) complexes have been found to inactivate CTL, but the two phenomena have not been connected. Here we show that a common mechanism may apply to both. CD8+ CTL lines or clones specific for a determinant of the human immunodeficiency virus (HIV) 1 IIIB envelope protein gp160, P18IIIB, are inhibited by as little as 10 min exposure to the minimal 10-mer peptide, I-10, within P18IIIB, free in solution, in contrast to peptide already bound to antigen-presenting cells (APC), which does not inhibit. Several lines of evidence suggest that the peptide must be processed and presented by H-2Dd on the CTL itself to the specific T cell receptor (TCR) to be inhibitory. The inhibition was not killing, in that CTL did not kill 51Cr-labeled sister CTL in the presence of free peptide, and in mixing experiments with CTL lines of different specificities restricted by the same MHC molecule, Dd, the presence of free peptide recognized by one CTL line did not inhibit the activity of the other CTL line that could present the peptide. Also, partial recovery of activity could be elicited by restimulation with cell-bound peptide, supporting the conclusion that neither fratricide nor suicide (apoptosis) was involved. The classic veto phenomenon was ruled out by failure of peptide-bearing CTL to inactivate others. Using pairs of CTL lines of differing specificity but similar MHC restriction, each pulsed with the peptide for which the other is specific, we showed that the minimal requirement is simultaneous engagement of the TCR and class I MHC molecules of the same cell. This could occur in single cells or pairs of cells presenting peptide to each other. Thus, mechanistically, the inhibition is analogous to veto, and might be called self-veto. As a clue to a possible mechanism, we found that free I-10 peptide induced apparent downregulation of expression of specific TCR as well as interleukin 2 receptor, CD69, lymphocyte function-associated antigen 1, and CD8. This self-veto effect also has implications for in vivo immunization and mechanisms of viral escape from CTL immunity.
The life span of major histocompatibility complex-peptide complexes influences the efficiency of presentation and immunogenicity of two class I-restricted cytotoxic T lymphocyte epitopes in the Epstein-Barr virus nuclear antigen 4.
Authors: Levitsky V , Zhang Q J , Levitskaya J , Masucci M G
Published: June 2004

We have investigated the reactivity to two human histocompatibility leukocyte antigen (HLA) A11-restricted cytotoxic T lymphocyte (CTL) epitopes derived from amino acids 416-424 (IVTDFSVIK, designated IVT) and 399-408 (AVFDRKSVAK, designated AVF) of the Epstein-Barr virus (EBV) nuclear antigen (EBNA) 4. A strong predominance of CTL clones specific for the IVT epitope was demonstrated in polyclonal cultures generated by stimulation of lymphocytes from the EBV-seropositive donor BK with the autologous B95.8 virus-transformed lymphoblastoid cell line (LCL). This was not due to intrinsic differences of CTL efficiency since clones specific for the two epitopes lysed equally well A11-positive phytohemagglutinin blasts and LCLs pulsed with the relevant synthetic peptide. Irrespective of the endogenous levels of EBNA4 expression, untreated LCLs were lysed more efficiently by the IVT-specific effectors, suggesting that a higher density of A11-IVT complexes is presented at the cell surface. In accordance, 10-50-fold higher amounts of IVT peptides were found in high-performance liquid chromatography fractions of acid extracts corresponding to an abundance of about 350-12,800 IVT and 8-760 AVF molecules per cell. Peptide-mediated competition of CTL sensitization, transport assays in streptolysin-O permeabilized cells, and induction of A11 expression in the transporter associated with antigen presentation-deficient T2/A11 transfectant demonstrated that the IVT and AVF peptides bind with similar affinities to A11, are translocated with equal efficiency to the endoplasmic reticulum, and form complexes of comparable stability over a wide range of temperature and pH conditions. A rapid surface turnover of A11 molecules containing the AVF peptide was demonstrated in metabolically active T2/A11 cells corresponding to a half-life of approximately 3.5 as compared to approximately 2 h for molecules induced at 26 degrees C in the absence of exogenous peptides and >12 h for IVT-containing complexes. This difference in persistence is likely to determine the representation of individual class I-restricted CTL epitopes within the cell surface pool of molecules, and may be an important factor contributing to their immunogenicity.
Suppression of proinflammatory cytokines in monocytes by a tetravalent guanylhydrazone.
Authors: Bianchi M , Bloom O , Raabe T , Cohen P S , Chesney J , Sherry B , Schmidtmayerova H , Calandra T , Zhang X , Bukrinsky M , Ulrich P , Cerami A , Tracey K J
Published: June 2004

An overproduction of proinflammatory cytokines by activated macrophages/monocytes mediates the injurious sequelae of inflammation, septic shock, tissue injury, and cachexia. We recently synthesized a tetravalent guanylhydrazone compound (CNI-1493) that inhibits cytokine-inducible arginine transport and nitric oxide (NO) production in macrophages, and protects mice against lethal endotoxemia and carrageenan-induced inflammation. During these investigations we noticed that CNI-1493 effectively prevented lipopolysaccharide (LPS)-induced NO production, even when added in concentrations 10-fold less than required to competitively inhibit L-arginine uptake, suggesting that the suppressive effects of this guanylhydrazone compound might extend to other LPS-induced responses. Here, we report that CNI-1493 suppressed the LPS-stimulated production of proinflammatory cytokines (tumor necrosis factor [TNF], interleukins 1beta and 6, macrophage inflammatory proteins 1alpha and 1beta) from human peripheral blood mononuclear cells. Cytokine suppression was specific, in that CNI-1493 did not inhibit either the constitutive synthesis of transforming growth factor beta or the upregulation of major histocompatibility complex class II by interferon gamma (IFN-gamma). In contrast to the macrophage suppressive actions of dexamethasone, which are overridden in the presence of IFN-gamma, CNI-1493 retained its suppressive effects even in the presence of IFN-gamma. The mechanism of cytokine-suppressive action by CNI-1493 was independent of extracellular L-arginine content and NO production and is not restricted to induction by LPS. As a selective inhibitor of macrophage activation that prevents TNF production, this tetravalent guanylhydrazone could be useful in the development of cytokine-suppressive agents for the treatment of diseases mediated by overproduction of cytokines.
Relative contribution of T and B cells to hypermutation and selection of the antibody repertoire in germinal centers of aged mice.
Authors: Yang X , Stedra J , Cerny J
Published: June 2004

The immune system of aged individuals often produces antibodies that have lower affinity and are less protective than antibodies from young individuals. Recent studies in mice suggested that antibodies produced by old individuals may be encoded by distinct immunoglobulin (Ig) genes and that the somatic hypermutation process in these individuals is compromised. The present study employed Ighb scid mice reconstituted with normal lymphocytes from young (2-3-mo-old) and aged (20-25-mo-old) donors and immunized with a protein conjugate of the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) to determine whether the molecular changes in antibody repertoire reflect senescence in the B cells or whether they are mediated by the aging helper T lymphocytes. The NP-reactive B cells from splenic germinal centers (GC) were recovered by microdissection of frozen tissue sections and their rearranged Ig heavy chain variable region (VH) genes of the V186.2/V3 families were sequenced. It was found that the VH gene repertoire of the GC B cells was strongly influenced by the source of the CD4+ T cells. When T cells were donated by young mice, the anti-NP response in GC was dominated by the canonical V186.2 gene, even if the responder B cells came from aged donors. However, when the mice were reconstituted with T cells from aged donors, the expression of the V186.2 gene by young B cells was diminished and the response was dominated by the C1H4 gene, another member of the V186.2/V3 family. In contrast, the somatic hypermutation process in the GC B cells followed a different pattern. The mutation frequencies in the animals that were reconstituted with both B and T cells from young donors (1/50 to 1/150 bp) were comparable to the frequencies previously reported for NP-immunized intact young/adult mice. However, when either lymphocyte subset was donated by the aged mice, the mutation frequencies declined. Thus, mice reconstituted with T cells from the aged and B cells from the young had severely compromised mutational mechanism. Likewise, the recipients of aged B and young T cells had diminished mutations even though the repertoire of their anti-NP response was dominated by the canonical V186.2 gene. It appears that the change in germine-encoded repertoire and the decrease of somatic hypermutation represent distinct mechanisms of immunosenescence and that the aging of helper T cells plays a pivotal role in both of these processes.
Human germinal center B cells express the apoptosis-inducing genes Fas, c-myc, P53, and Bax but not the survival gene bcl-2.
Authors: Martinez-Valdez H , Guret C , de Bouteiller O , Fugier I , Banchereau J , Liu Y J
Published: June 2004

During T cell-dependent antibody responses, B cells within germinal centers (GC) alter the affinity of their antigen receptor by introducing somatic mutations into variable region of immunoglobulin (IgV) genes. During this process, GC B cells are destined to die unless positively selected by antigens and CD40-ligand. To understand survival/death control of germinal center B cell, the expression of four apoptosis-inducing genes, Fas, c-myc, Bax, and P53, together with the survival gene bcl-2, has been analyzed herein among purified tonsillar naive, GC, and memory B cells. IgD+CD38- naive B cells were separated into CD23- (mature B cell [Bm]1) subset and CD23+ (Bm2), IgD-CD38+ GC B cells were separated into subsets of CD77+ centroblasts (Bm3) and CD77- centrocytes (Bm4), whereas IgD-CD38- cells represented the Bm5 memory B cell subset. Sequence analysis of IgV region genes indicated that somatic hypermutation was triggered in the Bm3 centroblast subset. Here we show that bcl-2 is only detectable with naive (Bm1 and 2) and memory B cell (Bm5) subsets, whereas all four apoptosis-inducing genes were most significantly expressed within GC B cells. Fas was equally expressed in Bm3 centroblasts and Bm4 centrocytes, whereas Bax was most significantly expressed in Bm4 centrocytes. c-myc, a positive regulator of cell cycle, was most significantly expressed in proliferating Bm3 centroblasts, whereas P53, a negative regulator of cell cycle, was most signficantly expressed in nonproliferating Bm4 centrocytes. The present results indicate that the survival/death of GC B cells are regulated by the up- and downregulation of multiple genes, among which the expression of c-myc and P53 in the absence of bcl-2 may prime the proliferating Bm3 centroblasts and nonproliferating Bm4 centrocytes to apoptosis.
Interaction of Ipa proteins of Shigella flexneri with alpha5beta1 integrin promotes entry of the bacteria into mammalian cells.
Authors: Watarai M , Funato S , Sasakawa C
Published: June 2004

Shigella is a genus of highly adapted bacterial pathogens that cause bacillary dysentery in humans. Bacteria reaching the colon invade intestinal epithelial cells by a process of bacterial-directed endocytosis mediated by the Ipa proteins: IpaB, IpaC, and IpaD of Shigella. The invasion of epithelial cells is thought to be a receptor-mediated phenomenon, although the cellular components of the host that interact with the Ipa proteins have not yet been identified. We report here that in a Shigella flexneri invasive system and Chinese hamster ovary (CHO) cell monolayers, the Ipa proteins were capable of interacting directly with alpha5beta1 integrin. The invasive capacity of S. flexneri for CHO cells increased as levels of alpha5beta1 integrin were elevated. When CHO cells were infected with S. flexneri, the tyrosine phosphorylation both of pp 125FAK, an integrin-regulated 125 K focal adhesion kinase, and of paxillin was stimulated. In contrast, an isogenic strain of S. flexneri that was defective in invasion owing to a mutation in its spa32 gene failed to induce such phosphorylation. Under in vitro and in vivo conditions, the released IpaB, IpaC, and IpaD proteins bound to alpha 5 beta 1 integrin in a manner different from that of soluble fibronectin but similar to that of the tissue form of fibronectin. At the site of attachment of S. flexneri to CHO cells, alpha5beta1 integrin converged with polymerization of actin. These data thus suggest that the capacity of Ipa proteins to interact with alpha5beta1 integrin may be an important Shigella factor in triggering the reorganization of actin cytoskeletons.
Endogenous altered peptide ligands can affect peripheral T cell responses.
Authors: Vidal K , Hsu B L , Williams C B , Allen P M
Published: June 2004

T cells potentially encounter a large number of endogenous self-peptide/MHC ligands in the thymus and the periphery. These endogenous ligands are critical to both positive and negative selection in the thymus; however, their effect on peripheral T cells has not been directly ascertained. Using the murine allelic Hbd (64-76)/I-Ek self-antigen model, we have previously identified altered peptide ligands (APLs) which are able to stimulate some but not all TCR-mediated effector functions. To determine directly the effect of endogenously synthesized APL/MHC complexes on peripheral T cells, we used a TCR transgenic mouse which had reversed our normal antigen system, with Ser69 peptide now being the agonist and Hbd(64-76) being the APL. In this report, we show that the constitutive level of endogenous Hbd(64-76)/I-Ek complexes presented by APCs in vivo is too low to affect the response of Ser69 reactive T cells. However, by increasing the number of Hbd(64-76)/I-Ek complexes expressed by the APCs, TCR antagonism is observed for both primary T cells and T cell hybridomas. In addition, the level of the CD4 coreceptor expressed on T cells and T cell hybridomas. In addition, the level of the CD4 coreceptor expressed on T cells changes the response pattern to endogenously presented Hbd(64-76)/I-Ek ligand. These findings demonstrate that T cells are selected to ignore the constitutive levels of endogenous complexes they encounter in the periphery. T cell responses can be affected by endogenous APLs in the periphery under limited but attainable circumstances which change the efficacy of the TCR/ligand interaction. Thus, endogenous APLs play a role in both the selection of T cells in the thymus and the responses of peripheral T cells.