bluuebunny/crossref_metadata_2025 huggingface dataset:
A beta polymorphic residues responsible for class II molecule recognition by alloreactive T cells.
Authors: Buerstedde J M , Nilson A E , Chase C G , Bell M P , Beck B N , Pease L R , McKean D J
Published: June 2004

In an effort to characterize the ligand that is bound by T helper lymphocyte antigen receptors, we have begun to identify class II polymorphic residues that comprise part of the allospecific TCR binding sites. Site-directed mutagenesis was used to construct mutant Ak beta (Ak beta*) genes that encode polypeptides into which single or multiple residues of the Ad beta polypeptide have been substituted in the beta 1 domain. A panel of cloned cell lines expressing the mutant Ak beta* Ak alpha or Ak beta* Ad alpha molecules was analyzed for the ability to stimulate Ak or Ad alloreactive T cell hybridomas. Substitution of d for k residues at specific positions in the beta 1 domain resulted not only in the loss of epitopes recognized by Ak-reactive T cells but, more importantly, in the gain of epitopes recognized by Ad-reactive T cells. Some of the polymorphic residues identified as contributing to the T cell epitopes are the same residues that contribute to the serologically immunodominant epitope. Other T cell epitopes map to positions predicted to be located either in an alpha-helix forming one side, or in a beta-pleated sheet forming the bottom of the putative antigen binding site. Thus, unlike serologic epitopes, TCR epitopes can be determined by A beta polymorphic residues in many different regions of the beta 1 domain and frequently depend upon contributions of A alpha polymorphic residues.
T cell-mediated inhibition of the transfer of autoimmune diabetes in NOD mice.
Authors: Boitard C , Yasunami R , Dardenne M , Bach J F
Published: June 2004

The nonobese diabetic (NOD) mouse has recently been introduced as a model for insulin-dependent diabetes mellitus. The role of regulatory T cells in the development of antipancreatic autoimmunity in this model remains unclear. To evaluate the presence of suppressive phenomena, we used disease transfer by spleen cells from diabetic NOD mice into preirradiated adult recipients as a model for accelerated disease. Suppressor phenomena were detected by testing the protection afforded by lymphoid cells from nondiabetic NOD mice against diabetes transfer in irradiated recipients. Transfer of diabetes was delayed by reconstituting recipients with spleen cells from nondiabetic NOD donors. The greatest protection against diabetes transfer was conferred by spleen cells from 8-wk-old nondiabetic female NOD mice. Depletion experiments showed that the protection was dependent on CD4+ cells. Protection was also detected within thymic cells from nondiabetic NOD mice and protection conferred by spleen cells was abrogated by thymectomy of nondiabetic female, but not male, NOD donors at 3 wk of age. These findings indicate that suppressive CD4+ T cells that are dependent on the presence of the thymus may delay the onset of diabetes in female diabetes-prone NOD mice.
Peptide-specific prevention of experimental allergic encephalomyelitis. Neonatal tolerance induced to the dominant T cell determinant of myelin basic protein.
Authors: Clayton J P , Gammon G M , Ando D G , Kono D H , Hood L , Sercarz E E
Published: June 2004

Experimental allergic encephalomyelitis (EAE) is a model of antigen-specific T cell-mediated autoimmune disease. The alpha-acetylated, NH2-terminal nine amino acids (1-9NAc) of myelin basic protein (MBP) represents the dominant T cell epitope for the induction of EAE in the B10.PL (H-2u) strain. We tolerized neonatal B10.PL mice to 1-9NAc and studied the proliferative responses to this peptide and to whole MBP. Mice exposed to 1-9NAc in the neonatal period were tolerant to subsequent challenge at the proliferative T cell level. Similarly, in the 1-9NAc-tolerant group, both the incidence and severity of 1-9NAc induced EAE were greatly reduced. The fact that we were able to tolerize mice normally responsive to MBP suggests that this self antigen is sequestered (within the central nervous system) and hence tolerance to it is not normally induced. No significant difference in disease incidence was seen in response to rat MBP between control animals and 1-9NAc-tolerized mice (50% in both groups), demonstrating the presence of at least one additional encephalitogenic determinant elsewhere on the molecule. We have successfully prevented disease induction by peptide-induced tolerization. Tolerance induction by peptides provides a new and specific strategy in the prevention of autoimmunity. However, it will be clearly necessary to fully define all epitopes potentially capable of inducing pathogenic T cells to ensure complete and effective therapy of T cell-mediated autoimmune disease.
Monoclonal antibodies reactive with the mouse interleukin 5 receptor.
Authors: Rolink A G , Melchers F , Palacios R
Published: June 2004

The rat mAbs R52.120 and R52.625 inhibit the action of IL-5 on both IL-5-sensitive cell lines and freshly isolated splenic B lymphocytes. Neither antibody inhibits the proliferative cell responses promoted by IL-2, IL-3, or IL-4. Purified R52.120+ lymphoid spleen cells contain 15-20-fold higher numbers of B lymphocytes responding to IL-5 in the form of maturation into antibody-producing cells. By immunofluorescence staining and flow fluorocytometry, the R52.120 and R52.625 antibodies bound to all 12 IL-5-sensitive cell lines tested. Both antibodies react with 2-4% cells in the spleen, 5% lymphoid cells, and 10-15% myeloid cells in the bone marrow, and 10-14% in the peritoneum of C57BL/6, DBA/2, and BALB/c adult mice. No positive cells for either antibody were detected in the thymus and lymph nodes of these mice. Both R52.120 and R52.625 antibodies specifically inhibit the binding of radiolabeled IL-5 to its receptor. Finally, R52.120 and R52.625 antibodies precipitate from 35S-methionine-labeled IL-5-R+ cell lysates three proteins with Mr 46,000, 130,000, and 140,000. Taken together from these results, we conclude that the R52.120 and R52.625 mAbs recognize epitopes on the IL-5-R complex very close or identical to the IL-5 binding sites.
Relative V beta transcript levels in thymus and peripheral lymphoid tissues from various mouse strains. Inverse correlation of I-E and Mls expression with relative abundance of several V beta transcripts in peripheral lymphoid tissues.
Authors: Okada C Y , Weissman I L
Published: June 2004

We have measured the relative levels of transcripts for 15 of the 22 known V beta gene segments. The level of transcripts for the highest and lowest expressed V beta gene segment differed by greater than 20-fold in the thymus and an even larger difference was observed in the periphery. The levels of expressions were unrelated to the order of the V beta genes on the chromosome. For most of the V beta gene segments, the relative transcript levels were the same in the thymus and periphery, suggesting that thymic selection in general does not act solely upon the V beta gene segment. One V beta gene segment in the BALB and B10 mice strains was an exception to this rule. V beta 5.2 expression in the periphery of BALB and B10 mice inversely correlated with the expression of the MHC class II molecule I-E. Five V beta gene segments had reduced transcript levels in the periphery of Mls-1a mice compared with their thymic levels or to the levels found in Mls-1b mice. The peripheral level of V beta 3 transcripts vary with MHC and Mls-2 haplotypes. The observation that certain V beta transcript levels are reduced in the periphery when compared with the thymus favors the hypothesis that self tolerance at the T cell level results in the elimination of self-reactive T cells, rather than paralysis by a block at some post-transcriptional step. Finally, the wide variability of V beta gene segment expression in the thymus suggests mechanisms exist to import an early bias to the repertoire. Whether this bias results from differential V beta segment rearrangement rates, differential V beta expression rates, or events occurring after TCR-alpha/beta expression on immature/nonmature thymocyte cell surfaces is yet to be determined.
A cell-killing monoclonal antibody (anti-Fas) to a cell surface antigen co-downregulated with the receptor of tumor necrosis factor.
Authors: Yonehara S , Ishii A , Yonehara M
Published: June 2004

We have prepared an mAb specific for a human cell surface component (termed anti-Fas mAb). Anti-Fas shows cell-killing activity that is indistinguishable from the cytolytic activity of TNF. Fas antigen was characterized by western blotting, indicating that Fas antigen is a cell surface protein with a molecular weight of 200,000, which is different from the molecular weight of TNF-R. Fas antigen, however, is co-downregulated with the TNF-R when cells sensitive to the cytolytic activity of TNF are incubated with either TNF or anti-Fas. In contrast, Fas antigen on cells insensitive to TNF is not co-downregulated with the TNF-R. We suggest that the cell-killing activity of TNF is mediated by Fas antigen associated with the TNF-R.
Molecular cloning of the human eosinophil peroxidase. Evidence for the existence of a peroxidase multigene family.
Authors: Ten R M , Pease L R , McKean D J , Bell M P , Gleich G J
Published: June 2004

Human eosinophil peroxidase (EPO) was purified from eosinophil granules derived from the peripheral blood of patients with eosinophilia. The molecular mass of the H and L subunits was determined by gel filtration to be 57,000 and 11,000 daltons, respectively. The partial amino acid sequences of both subunits were used to construct oligonucleotides for the screening of several cDNA libraries, including one derived from human-induced umbilical cord mononuclear cells. A cDNA clone was isolated corresponding to EPO. The nucleotide sequence revealed an open reading frame of 2,106 bp, corresponding to a prosequence, L chain, and H chain, in this order. Comparison of the EPO nucleotide sequence with other peroxidases, such as myeloperoxidase, suggests the existence of a multigene family.
Transient adhesion of neutrophils to endothelium.
Authors: Lo S K , Detmers P A , Levin S M , Wright S D
Published: June 2004

Fluorescently labeled polymorphonuclear leukocytes (PMN) were used to measure adhesion to human umbilical vein endothelial cells (EC) cultured in vitro. Stimulation of PMN with phorbol dibutyrate (PDB), TNF, or C5a caused an increase in adhesion followed by a return to prestimulation levels of adhesion of longer times of incubation. Maximal adhesion of PMN to EC occurred rapidly in response to C5a (5 min) and more slowly with TNF or PDB (15 min). PMN stimulated to adhere with C5a detached from EC by 15 min. PMN from CD11/CD18-deficient patients and PMN incubated with anti-CD18 mAbs failed to bind to EC despite maximal stimulation. Anti-CD11a/CD18 and anti-CD11b/CD18 each partially inhibited adhesion, and a combination of these two reagents completely blocked adhesion. The adhesion we measured was therefore completely dependent on CD11/CD18, and CD11a/CD18 and CD11b/CD18 each contributed to adhesion. Stimuli that enhanced adhesion of PMN to EC also enhanced expression of CD11b/CD18 on the cell surface, but the time course of expression correlated poorly with changes in adhesivity. To determine if changes in the expression of CD11b/CD18 are necessary for the changes in adhesivity, we used enucleate cytoplasts that did not increase expression of CD11b/CD18. Cytoplasts showed a normal rise and fall in adhesivity in response to PDB. We conclude that the transient adhesion of stimulated PMN to naive EC is regulated by changes in the nature of existing CD11/CD18 molecules on the PMN surface. Changes in expression of CD11b/CD18 may contribute to enhancement of adhesivity, but a definite role for this phenomenon has yet to be established.
Elimination of CD4+ suppressor T cells from susceptible BALB/c mice releases CD8+ T lymphocytes to mediate protective immunity against Leishmania.
Authors: Hill J O , Awwad M , North R J
Published: June 2004

This study examined the capacity of BALB/c mice that had been depleted of T cell subpopulations to generate a protective immune response to Leishmania major. Thymectomized mice were depleted of either L3T4+ (CD4+) T lymphocytes, Ly2+ (CD8+) T lymphocytes, or both, by treatment with appropriate mAbs. It was found that susceptible mice were rendered resistant to Leishmania by an intravenous infusion of anti-L3T4 mAb. These mice generated an immune response that destroyed the parasite in the primary lesion and in visceral metastatic foci. CD4+ cell-depleted mice also acquired a capacity to mount a sustained delayed-type hypersensitivity (DTH) response to parasite antigens, indicating that DTH, per se, is not a disease-promoting mechanism in the susceptible murine host as has been suggested. Depleting BALB/c mice of CD8+, as well as CD4+ T cells, left them highly susceptible to Leishmania infection, thereby indicating that CD8+ lymphocytes are key protective cells. Our results can be interpreted as showing that the susceptibility of BALB/c mice is due to the generation of CD4+ cells that suppress either the generation or expression of CD8+ T cell-mediated antiLeishmania immunity.
Plasmodium falciparum-infected erythrocytes form spontaneous erythrocyte rosettes.
Authors: Udomsangpetch R , Wåhlin B , Carlson J , Berzins K , Torii M , Aikawa M , Perlmann P , Wahlgren M
Published: June 2004

Erythrocytes infected with trophozoites or schizonts of Plasmodium falciparum bind uninfected erythrocytes, leading to rosette formation. Both established laboratory strains and fresh isolates from patients form such rosettes, but at widely different frequencies. IgG preparations from the serum of some P. falciparum-immune donors and heparin inhibited rosette formation. The results indicate that cytoadherence of infected erythrocytes to endothelial cells and rosetting represent distinct genetic traits.
Expression of the third member of the serum amyloid A gene family in mouse adipocytes.
Authors: Benditt E P , Meek R L
Published: June 2004

Three homologous genes that code for three related proteins comprise the serum amyloid A (SAA) family in the mouse. Endotoxin induces equally vigorous expression of mRNAs for the three SAA genes in liver. In extrahepatic tissues SAA1 and/or SAA2 mRNAs have been found only in kidney and intestine, however, SAA3 is expressed in all extrahepatic tissues thus far examined. This observation raised the question: is SAA3 mRNA expressed by a single cell system dispersed throughout all tissues, or by differentiated cells of each tissue? This question was explored in various tissues by in situ hybridization with a single-stranded cRNA probe specific for SAA3 mRNA. We found expression in the liver of SAA3 mRNA by other cells as well as by hepatocytes. A common feature among extrahepatic tissues was SAA3 mRNA expression in adipocytes. SAA3 mRNA was also found in two nonadipose cells, Leydig cells of the testis, and some of the cells located in parafollicular zones of the spleen.
Human TCR-gamma+/delta+, CD8+ T lymphocytes recognize tetanus toxoid in an MHC-restricted fashion.
Authors: Kozbor D , Trinchieri G , Monos D S , Isobe M , Russo G , Haney J A , Zmijewski C , Croce C M
Published: June 2004

We have analyzed the ability of human gamma+/delta+ T cells to recognize a nominal antigen in association with MHC molecules. A TT-specific T cell line with approximately 40% gamma+/delta+ T cells was established from a hyperimmunized donor, D.F., by stimulation with antigen and autologous APC. Three DF-derived gamma+/delta+ clones were CD8+ as determined by immunofluorescence staining, and by Southern and Northern blotting with probes detecting delta chain rearrangement and delta and gamma chain transcripts, respectively. The gamma+/delta+ clones responded to stimulation with TT, but not TNP-BSA, and autologous APC by proliferation and IFN-gamma production. No proliferation or IFN-gamma production was detected when TT-specific T cell clones were stimulated with either TT or autologous APC only. The response to TT was enhanced by addition of exogenous IL-2. The use of allogeneic APC from 19 donors sharing one HLA-determinant with the autologous donor D.F., showed that the gamma+/delta+ T cells responded to TT with HLA-DR4-related restriction as measured by proliferation and IFN-gamma production. These results demonstrate that gamma/delta receptors can recognize non-MHC-encoded foreign antigen in a self-MHC-restricted fashion.
Murine V kappa gene expression does not follow the VH paradigm.
Authors: Kaushik A , Schulze D H , Bona C , Kelsoe G
Published: June 2004

V kappa gene family expression among LPS-reactive murine B lymphocytes, unlike that of VH gene families, is not proportional to genomic complexity, i.e., nonstoichiometric. Furthermore, no positional bias for the overexpression of J-proximal V kappa genes (V kappa 21) is observed among neonatal B lymphocytes. Yet, the V kappa 1 and V kappa 9 families located in the center of V kappa locus are preferentially used by neonatal B splenocytes. Thus, the mechanisms of V kappa gene rearrangement and expression appear to differ significantly from those controlling the VH locus.
Restricted immunoglobulin variable region gene usage by normal Ly-1 (CD5+) B cells that recognize phosphatidyl choline.
Authors: Mercolino T J , Locke A L , Afshari A , Sasser D , Travis W W , Arnold L W , Haughton G
Published: June 2004

5-15% of lymphocytes in the peritoneums of normal adult B10.H-2aH-4bp/Wts (2a4b) mice are CD5+ (Ly-1) B cells that recognize phosphatidyl choline (PtC), a phospholipid component of all mammalian cells. We produced a set of IgM-secreting hybridomas from the peritoneal cells of normal, adult 2a4b mice. We found that this set of hybridomas shows a similarly high frequency of antibodies specific for PtC (21 of 86) that also react with bromelain-treated mouse erythrocytes. Restriction fragment analysis of Ig gene rearrangements and analysis of expressed Ig idiotypes reveal that these cells use a restricted set of variable region genes to generate the PtC-specific antibodies. The Ig genes used by the PtC-specific hybridomas appear to be the same as those found in the PtC-specific Ly-1 B cell lymphomas, CH27 and CH34.
Histones have high affinity for the glomerular basement membrane. Relevance for immune complex formation in lupus nephritis.
Authors: Schmiedeke T M , Stöckl F W , Weber R , Sugisaki Y , Batsford S R , Vogt A
Published: June 2004

An effort has been made to integrate insights on charge-based interactions in immune complex glomerulonephritis with nuclear antigen involvement in lupus nephritis. Attention was focussed on the histones, a group of highly cationic nuclear constituents, which could be expected to bind to fixed anionic sites present in the glomerular basement membrane (GBM). We demonstrated that all histone subfractions, prepared according to Johns (4), have a high affinity for GBM and the basement membrane of peritubular capillaries. Tissue uptake of 125I-labeled histones was measured by injecting 200 micrograms of each fraction into the left kidney via the aorta and measuring organ uptake after 15 min. In glomeruli isolated from the left kidneys, the following quantities of histones were found: f1, 13 micrograms; f2a (f2al + f2a2), 17 micrograms; f2b, 17 micrograms; and f3, 32 micrograms. Kinetic studies of glomerular binding showed that f1 disappeared much more rapidly than f2a. The high affinity of histones (pI between 10.5 and 11.0; mol wt 10,000-22,000) for the GBM correlates well with their ability to form aggregates (mol wt greater than 100,000) for comparison lysozyme (pI 11, mol wt 14,000), which does not aggregate spontaneously bound poorly (0.4 micrograms in isolated glomeruli). The quantity of histones and lysozyme found in the isolated glomeruli paralleled their in vitro affinity for a Heparin-Sepharose column (gradient elution studies). This gel matrix contains the sulfated, highly anionic polysaccharide heparin, which is similar to the negatively charged heparan sulfate present in the GBM. Lysozyme eluted with 0.15 M NaCl, f1 with 1 M NaCl, and f2a, f2b, and f3 could not be fully desorbed even with 2 M NaCl; 6 M guanidine-HCl was necessary. Two further findings of great relevance for the concept of induction of immune complex glomerulonephritis by histones were: (a) glomerular-bound histone was accessible for specific antibody given intravenously; and (b) prior binding of histones promoted glomerular deposition of anionic antigens, as could be shown with ssDNA fragments. These data justify the proposal that glomerular deposition of histones can induce immune complex formation, start an inflammatory process, and produce tissue damage.
RIII S/J (H-2r). An inbred mouse strain with a massive deletion of T cell receptor V beta genes.
Authors: Haqqi T M , Banerjee S , Anderson G D , David C S
Published: June 2004

We have identified an inbred strain of mouse, RIII S/J (H-2r), that has the largest known deletion of the TCR V beta genes by screening with mAb and TCR V beta specific probes. Upon screening of PBL with mAb F23.1, which is specific for V beta 8 TCR, RIII S/J was found to be negative. On further screening with mAb KJ 23a, which is specific for V beta 17a TCR, RIII S/J was completely negative. We next tested RIII S/J with mAb 44-22-1, which is specific for V beta 6 TCR, and found it also to be negative. The (B10 X RIII)F1 mice showed a 50% expression of V beta 6 gene, indicating a genomic rather than a clonal deletion. mAb KJ25, detecting V beta 3, was positive in RIII S/J, denoting the downstream boundary for the deletion. Southern blot analysis of liver DNA using TCR V beta-specific probes confirmed the deletion of V beta 8 gene subfamily and V beta 5 gene subfamily, along with V beta 9, V beta 11, V beta 12, and V beta 13 genes similar to the known TCR V beta deletion mutants (SWR, SJL, C57L, and C57Br). In addition, RIII S/J is missing V beta 6, V beta 15, and V beta 17 genes. Our mapping of the deletion indicates that RIII S/J has lost approximately 130 kb of V beta chromosome and with it 13 V beta genes out of the known 21 V beta genes of the TCR. The deletion is marked by the presence of V beta 10 gene upstream and V beta 3 gene downstream.
Identification of an immunodominant and highly immunopathogenic determinant in the retinal interphotoreceptor retinoid-binding protein (IRBP).
Authors: Sanui H , Redmond T M , Kotake S , Wiggert B , Hu L H , Margalit H , Berzofsky J A , Chader G J , Gery I
Published: June 2004

Interphotoreceptor retinoid-binding protein (IRBP), a glycoprotein specific for the retina and pineal gland, induces inflammatory changes in these two organs in immunized animals. We report here on the identification of an immunodominant determinant of bovine IRBP that is highly immunogenic and immunopathogenic in the Lewis rat. The peptide, which comprises the sequence 1169-1191 of bovine IRBP, was shown to be immunodominant by its capacity to stimulate lymphocytes sensitized against whole IRBP. A comparison was made between peptide 1169-1191 and another peptide, 1158-1180, which is nondominant but is immunogenic and immunopathogenic in the Lewis rat. Peptide 1169-1191 was found to be superior in its immunological capacities; the minimal dose of 1169-1191 needed to induce cellular immune response or disease in Lewis rats (0.02-0.1 nmol/rat) is congruent to 1,000 times smaller than that of 1158-1180. In addition, unlike the ocular disease induced by 1158-1180, the disease produced by 1169-1191 resembled that induced by whole IRBP in its kinetics and histopathological features. The immunological activity of 1169-1191 in the Lewis rat was localized to the 10 residues at the COOH terminus; no such activity was exhibited by the truncated peptide 1169-1188, which comprises the 20 residues at the NH2 terminus of the full peptide. The usefulness of this unique experimental system in analyzing the role of immunodominance in peptide immunogenicity and immunopathogenicity is underscored.
T cell memory. Long-term persistence of virus-specific cytotoxic T cells.
Authors: Jamieson B D , Ahmed R
Published: June 2004

This study documents that virus-specific CTL can persist indefinitely in vivo. This was accomplished by transferring Thy-1.1 T cells into Thy-1.2 recipient mice to specifically identify the donor T cell population and to characterize its antigenic specificity and function by using a virus-specific CTL assay. Thy-1.1+ T cells from mice previously immunized with lymphocytic choriomeningitis virus (LCMV) were transferred into Thy-1.2 mice persistently infected with LCMV. The transferred LCMV-specific CTL (Thy-1.1+ CD8+) eliminate virus from the chronically infected carriers and persist in the recipient mice in small numbers, comprising only a minor fraction of the total T cells. Upon re-exposure to virus, these long-lived "resting" CD8+ T cells proliferate in vivo to become the predominant cell population. These donor CD8+ T cells can be recovered up to a year post-transfer and still retain antigenic specificity and biological function. They kill LCMV infected H-2-matched cells in vitro and can eliminate virus upon transfer into a second infected host. In addition, these long-lived CD8+ T cells appear not to be dependent on help from CD4+ T cells, since depletion of CD4+ T cells has minimal or no effect on their biological properties (proliferation, CTL response, viral clearance). These donor CTL also exhibit an immunodominance over the host-derived LCMV-specific CTL response. When both host and donor T cells are present, the donor CTL response is dominant over the potential CTL response of the cured carrier host. Taken together, these results suggest that virus-specific CTL can persist for the life span of the host as memory cells.
Early onset of somatic mutation in immunoglobulin VH genes during the primary immune response.
Authors: Levy N S , Malipiero U V , Lebecque S G , Gearhart P J
Published: June 2004

The dynamics of somatic mutation in Ig variable genes was investigated in order to define a population of B cells undergoing mutation. BALB/cJ mice were injected with PC-KLH, and splenic RNA was prepared 5, 7, and 13 d later. The mRNA was annealed to gamma constant region primers to make cDNA transcripts encoding VH genes. 103 cDNA clones corresponding to 18 different genes from the VH7183, VH3660, and VHS107 subfamilies were sequenced to identify mutation. VH genes had a low level of mutation on day 5 after immunization and accumulated more mutation by day 7 at a rate of 10(-3) mutations per nucleotide per generation. However, by day 13, the number of mutations per gene did not increase, and most of the substitutions encoded replacement amino acid changes that were clustered in the hypervariable regions, indicating that the mutational process was less active during the second week and that antigen selection had occurred. The data are consistent with a developmentally regulated mechanism in which mutation is activated during the first week of the primary immune response for a limited time period, after which selection acts to preserve the beneficial mutants.
Isolated human follicular dendritic cells display a unique antigenic phenotype.
Authors: Schriever F , Freedman A S , Freeman G , Messner E , Lee G , Daley J , Nadler L M
Published: June 2004

In the present study, follicular dendritic cells (FDCs) were purified to homogeneity in order to define the lineage and function of these cells. FDCs were identified by their characteristic morphology and by their expression of receptors for the third complement component, the myeloid-restricted antigen CD14, and the FDC antigen DRC-1. Unclustered FDCs displayed a unique antigenic phenotype since they expressed several B- and myeloid lineage-restricted antigens, but lacked T and NK cell antigens as well as the leukocyte common antigen. FDCs expressed adhesion molecules, including most of the VLA proteins, intercellular adhesion molecule 1 (ICAM-1), and CD11b. FDCs could be isolated to homogeneity by their intense staining with anti-CD14 using flow cytometric cell sorting. These highly purified FDCs expressed CD14 and CD21 but lacked CD20. This antigen pattern and characteristic morphology confirmed that these cells were, in fact, homogeneous FDC preparations. Analysis of polymerase chain reaction-amplified cDNA from highly purified FDCs showed no transcripts for IL-6. The isolation of homogeneous FDC populations will be important for the analysis of the functional role of FDCs within the lymphoid follicle.
T cell receptor beta gene has two downstream DNase I hypersensitive regions. Possible mechanisms of tissue- and stage-specific gene regulation.
Authors: Hashimoto Y
Published: June 2004

Two DNase I-hypersensitive regions were identified downstream of the TCR gene constant region. One of these regions is located at the site of a putative enhancer element and was observed only in T cell lines and not in cell lines derived from other tissues. The other DNase-hypersensitive region was also detected only in T cell lines but only in those expressing TCR-beta RNA. Thus, the first region is probably tissue specific, while the second region is probably tissue and stage specific. The DNA sequence of the second DNase I-hypersensitive region revealed several stretches of nucleotides that are characteristic of consensus sequences for regulatory elements. These results, together with the observations in transgenic mice that indicate a requirement for two distinct regions for optimal TCR gene expression, suggest the presence of at least two regulatory regions downstream of the C-beta-2 region; one is an enhancer region and the other is a transcriptionally related regulatory region. The tissue/stage specificity of these DNase I-hypersensitive regions supports the notion that changes in chromatin structure control tissue-specific gene expression.
Interleukins and IgA synthesis. Human and murine interleukin 6 induce high rate IgA secretion in IgA-committed B cells.
Authors: Beagley K W , Eldridge J H , Lee F , Kiyono H , Everson M P , Koopman W J , Hirano T , Kishimoto T , McGhee J R
Published: June 2004

Freshly isolated murine PP B cells were cultured with 10 different cytokines, including IL-1 alpha, IL-2, IL-4, IL-5, IL-6, IL-7, IFN-gamma, TNF-alpha, and TGF-beta, to investigate a possible role for these cytokines in induction of Ig synthesis. Of interest was the finding that only IL-5 and both mouse recombinant (mr) and human recombinant (hr) IL-6 enhanced IgA synthesis. The effect was greater with either mrIL-6 or hrIL-6 than with mrIL-5. IL-6 induced cycling mIgA+ PP B cells to secrete high levels of IgA (approximately 7-fold increase over control). Of importance was the finding that mrIL-6 had little effect on secretion of IgM or IgG by PP B cell cultures. hrIL-6 also increased IgA secretion by PP B cells and this enhancement was abolished by a goat anti-hrIL-6 antiserum. mrIL-6 did not cause B cell proliferation but induced a sharp increase in numbers of B cells secreting IgA. Isotype-switching was not a mechanism for this marked increase in IgA synthesis since mIgA- PP B cells were not induced to secrete IgA by mrIL-6. From these studies we conclude that IL-6 plays an important role in promoting the terminal differentiation of PP B cells to IgA-secreting plasma cells.
In vivo expression of perforin by CD8+ lymphocytes during an acute viral infection.
Authors: Young L H , Klavinskis L S , Oldstone M B , Young J D
Published: June 2004

CTL and NK cells cultured in vitro have been shown to contain a cytolytic pore-forming protein (PFP/perforin/cytolysin). To date, it has not been determined whether perforin is expressed by CTL that have been primed in vivo. Here, we have infected mice with two strains of lymphocytic choriomeningitis virus (LCMV), one of which mainly produces choriomeningitis and, the other, hepatitis. Brain and liver cryostat sections obtained from LCMV-infected mice were stained for various lymphocyte markers, including perforin. We were able to detect a large accumulation of perforin antigen in CD8+/Thy-1+/asialo GM1+/CD4- lymphocytes, which in fact represent the main infiltrating cell type found in brain and liver sections obtained during the late acute stage of LCMV infection. Perforin was also detected in a smaller population of CD8-/asialo GM1+/NK 1.1+/F480- cells, presumably corresponding to NK cells. Perforin-positive cells were found to have the morphology of blasts or large granular lymphocytes (LGL). These observations, together with in vitro studies performed in the past, indicate that perforin may be associated exclusively with LGL-like CTL blasts and NK cells. Our results demonstrate for the first time the presence of perforin in CTL that have been primed in vivo and suggest that perforin-positive CTL may be directly involved in producing the immunopathology associated with the LCMV infection.
Murine T cells express a cell surface receptor for multiple extracellular matrix proteins. Identification and characterization with monoclonal antibodies.
Authors: Maxfield S R , Moulder K , Koning F , Elbe A , Stingl G , Coligan J E , Shevach E M , Yokoyama W M
Published: June 2004

Cell-cell and cell-extracellular (ECM) protein interactions are mediated through heterodimers termed integrins. We have demonstrated that dendritic epidermal T cell (DETC) lines adhere to the ECM proteins, fibronectin, fibrinogen, and vitronectin but not to collagen, laminin, or control proteins. This adhesion was blocked by the tetrapeptide RGDS, but not the control peptide, RGES. We have derived a hamster mAb H9.2B8, and a rat mAb, 8.18E12, from immunizations with DETC lines. The mAbs in combination, but not individually, specifically inhibited the adhesion of DETC lines to fibronectin, fibrinogen, and vitronectin. Immunoprecipitation analysis revealed that both mAbs reacted with a heterodimer composed of noncovalently linked 140- and 95-kD subunits. The 140-kD subunit can be reduced to 120- and 23-kD fragments. Although the two mAbs did not cross-compete for binding to DETC, sequential immunoprecipitation studies indicated that they react with the same 120-kD fragment. While all DETC cell lines and several T cell clones were reactive with the mAbs, the mAbs were not reactive with normal spleen, lymph node, thymus, or skin. Stimulation of splenic T cells with Con A or allogeneic cells induced mAb reactivity after 1 wk in vitro. These data demonstrate that a single lymphocyte receptor, with biochemical features characteristic of integrins, mediates RGD-dependent binding to the ECM proteins, fibronectin, fibrinogen, and vitronectin. Furthermore, since this integrin is expressed by long-term activated T cells, this receptor may play a physiological role in T cell function.
Putative amino acid sequence of HLA-DRB chain contributing to rheumatoid arthritis susceptibility.
Authors: Watanabe Y , Tokunaga K , Matsuki K , Takeuchi F , Matsuta K , Maeda H , Omoto K , Juji T
Published: June 2004

The association between HLA-DR4 and rheumatoid arthritis (RA) has been established in many ethnic groups. To clarify the determinant of susceptibility to RA, a polymorphic segment of the HLA-DRB gene was amplified in vitro by polymerase chain reaction and analyzed with oligonucleotide probes specific for the HLA-DR4 DNA sequences. A particular sequence encoding amino acids Gln70-Arg71-Arg72-Ala73-Ala74 showed a strong association with RA (p less than 0.005, relative risk 6.0). This amino acid sequence occurs in the DRB molecules with three RA-associated specificities, DR4/Dw14, DR4/Dw15, and DR1. DR4/Dw4, which is common in Caucasian RA patients, has a strikingly similar amino acid sequence Gln70-Lys71-Arg72-Ala73-Ala74 in terms of polarity and charge profiles. Other RA nonassociated sequences differ from this sequence by at least one amino acid substitution that causes the change of the net charge. The composition of amino acid residues at the positions 70-74 may play a crucial role in the pathogenesis of RA.
Functional immunoglobulin light chain genes are replaced by ongoing rearrangements of germline V kappa genes to downstream J kappa segment in a murine B cell line.
Authors: Levy S , Campbell M J , Levy R
Published: June 2004

A murine B cell lymphoma (38C13) was subjected to immunoselection with mAbs directed against the idiotypic determinants of its cell surface Ig. Variants emerged with altered Ig receptors containing identical heavy chains but different light chains. The functional light chain genes in these variants were composed of V kappa segments drawn from the V kappa Ox-1 family, which had replaced the V kappa gene expressed by the parental tumor by rearranging to downstream J kappa segments. Rearrangement at the kappa locus continued to occur spontaneously, giving rise to secondary and tertiary variants at a rate of 1.9 x 10(-4) per cell per generation. Variants were isolated that had ceased production of surface Ig but went on to rearrange again and to become surface Ig+. The Ig- state may be an intermediate step providing a stimulus for continued rearrangement. This process provides an additional mechanism for generating diversity within B cell clones and expands the use of the available repertoire of Ig genes.
Regulation of T lymphocyte proliferation. Interleukin 2-mediated induction of c-myb gene expression is dependent on T lymphocyte activation state.
Authors: Churilla A M , Braciale T J , Braciale V L
Published: June 2004

We previously reported that with time, after antigenic stimulation of antigen-regulated murine T lymphocyte clones, total IL-2-R expression decayed 10-50-fold, commensurate with a decline in the ability of the cells to proliferate to IL-2. However, late after antigenic stimulation, when the cells were refractory to the IL-2-proliferative stimulus, high levels of high affinity IL-2-R remained. In this report we further explore the basis of unresponsiveness to IL-2 in the quiescent clones. We show that the proto-oncogene c-myc is induced in the late cell population by IL-2 to comparable levels observed early after antigen stimulation. IL-2-dependent c-myb induction, however, is seen only early after activation but not in the late-activated population. Analysis of the IL-2-dependent expression of c-myb mRNA with time after antigenic stimulation showed that steadystate c-myb expression declines dramatically with kinetics closely paralleling a decay in IL-2-dependent proliferative ability. In contrast, steadystate c-myc expression remains high throughout this period. Expression of c-myb is critical for proliferation of these cells since antisense oligodeoxy-nucleotide to c-myb can inhibit their IL-2-dependent proliferation. We present evidence for a pathway of c-myb induction via the TCR that is independent of the IL-2/IL-2-R interaction. In addition, the inhibition of IL-2-R-induced c-myb expression by 2-aminopurine and enhanced induction of c-myb via the TCR demonstrate that TCR activation and IL-2-R activation lead to induction of c-myb by different mechanisms.
Isolation and chromosomal localization of cDNAs encoding a novel human lymphocyte cell surface molecule, LAM-1. Homology with the mouse lymphocyte homing receptor and other human adhesion proteins.
Authors: Tedder T F , Isaacs C M , Ernst T J , Demetri G D , Adler D A , Disteche C M
Published: June 2004

A cDNA encoding a new human lymphocyte cell surface molecule has been isolated and shown to identify a fourth member of a recently discovered family of adhesion proteins. This lymphocyte-associated molecule (LAM-1) is uniquely composed of multiple distinct domains, one domain homologous with animal lectins, one homologous with epidermal growth factor, and two short consensus repeat units similar to those found in C3/C4 binding proteins. This cDNA clone hybridized with RNAs found in B cell lines and T lymphocytes, but not with RNA from other cell types. The amino acid sequence of LAM-1 is 77% homologous with the sequence of the mouse lymphocyte homing receptor, suggesting that LAM-1 may function in human lymphocyte adhesion. The LAM-1 gene is located on chromosome 1q23-25, as is another member of this adhesion family, suggesting that this new family of proteins may be encoded by a cluster of "adhesion protein" loci.
Positive selection determines T cell receptor V beta 14 gene usage by CD8+ T cells.
Authors: Liao N S , Maltzman J , Raulet D H
Published: June 2004

We report here a mAb, 14-2, reactive with TCRs that include V beta 14. The frequency of V beta 14+ T cells varies with CD4 and CD8 subset and is controlled by the H-2 genes. Thus CD8+ T cells from H-2b mice include approximately 2.3% V beta 14+ T cells while CD8+ T cells from mice expressing K kappa include greater than 8% V beta 14+ T cells. In all strains examined, 7-8% of CD4+ T cells express V beta 14. The frequent usage of V beta 14 in CD8+ T cells of K kappa-expressing mice is a result of preferential positive selection of V beta 14+ CD8+ T cells as demonstrated by analysis of radiation chimeras. These studies demonstrate that H-2-dependent positive selection occurs in unmanipulated mice. Furthermore, the results imply that positive selection, and possibly H-2 restriction, can be strongly influenced by a V beta domain, with some independence from the beta-junctional sequence and alpha chain.
Identification of pre-T cells in human peripheral blood. Extrathymic differentiation of CD7+CD3- cells into CD3+ gamma/delta+ or alpha/beta+ T cells.
Authors: Preffer F I , Kim C W , Fischer K H , Sabga E M , Kradin R L , Colvin R B
Published: June 2004

CD7+CD3- cells purified (greater than 99.99%) by FACS from the peripheral blood of healthy adults include precursors for mature T cells that have the capacity to differentiate into TCR-1+ or TCR-2+ CD3+ cells. Extrathymic differentiation was demonstrable from all eight healthy donors in the presence of a high concentration of IL-2, mitogenic levels of PHA, and irradiated blood mononuclear feeder cells, after a lag of approximately 40 d in vitro. The extrathymic T (ET) cells were predominantly TCR-1+, although TCR-2+ cells were also derived. ET TCR-1+ cells were CD4-CD8-, CD4-CD8DIM+, and CD4+CD8-, and were distinguished from natural T TCR-2+ cells by a variety of cell surface markers. The ET cells had phenotypes generally displayed by normal mature T cells, although the CD5DIM+ on ET cells was more typical of thymocytes. Acquisition of CD3 on purified CD7+CD3- cells was not due to antigenic modulation or growth of contaminants, and ET cells could be demonstrated at the clonal level. Studies in athymic mice and bone marrow recipients support the view that extrathymic maturation does occur in vivo. Whether the CD7+CD3- cell population was unexposed to the thymus, or exposed but not processed, is unknown. In any case, unusual or "forbidden" autoreactive specificities are predicted since ET cells differentiate without thymic selection of the TCR.
Identification of a subset of murine natural killer cells that mediates rejection of Hh-1d but not Hh-1b bone marrow grafts.
Authors: Sentman C L , Hackett J , Kumar V , Bennett M
Published: June 2004

NK cells demonstrate many immune functions both in vitro and in vivo, including the lysis of tumor or virus-infected cells and the rejection of bone marrow allografts. However it remains unclear whether or not all NK cells can mediate these various functions or if NK cells exist in functionally distinct subsets. We have developed a new NK-specific mAb, SW5E6, which binds to approximately 50% of murine NK cells. The 5E6 antigen identifies a distinct and stable subset of NK cells and is expressed on about one-half of fresh or rIL-2-activated murine NK cells. Both 5E6+ and 5E6- NK cells are capable of lysing YAC-1 tumor cells in vitro and in vivo. By treating animals with SW5E6, we demonstrate that the 5E6+ subset is necessary for the rejection of H-2d/Hh-1d but not H-2b/Hh-1b bone marrow cells. Thus NK cells exist as functionally separable subsets in vivo.
Reconstruction of the immunogenic peptide RNase(43-56) by identification and transfer of the critical residues into an unrelated peptide backbone.
Authors: Lorenz R G , Tyler A N , Allen P M
Published: June 2004

The involvement of each of the amino acid residues of the I-Ak-restricted T cell determinant RNase(43-56) was examined in detail using a series of peptides containing single amino acid substitutions. Four positions were identified as being essential for the formation of the determinant, Phe-46, Val-47, His-48, and Leu-51. When these four residues were substituted into the backbone of the unrelated peptide HA(130-144), a nonstimulatory peptide was obtained. The inclusion of an additional residue, Val-54, resulted in a chimeric peptide, RN/HA2, which was nearly as active as the native molecule. The peptide RN/HA2 was able to prime in vivo for RNase reactivity, confirming that these five residues contained all of the specificity of the RNase(43-56) determinant. The role of three of these critical residues was examined using both a functional competition assay and an in vivo priming assay. It was ascertained that the Phe-46 was directly involved in contacting the TCR, while the His-48 and Leu-51 were either involved in binding to the I-Ak molecule or in determining the conformation of the peptide. Thus, by critically evaluating the contribution of each of the amino acid residues in a T cell determinant, we were able to generate a chimeric peptide only containing 5 of 15 residues from the RNase(43-56) sequence that was functionally identical to the native RNase(43-56) molecule both in vitro and in vivo.
Tumor antigens defined by cloned immunological probes are highly polymorphic and are not detected on autologous normal cells.
Authors: Ward P L , Koeppen H , Hurteau T , Schreiber H
Published: June 2004

We have isolated UV light-induced and spontaneous tumors along with nonmalignant cells and tissues from each host. CD8+ CTL clones generated to a number of highly immunogenic UV-induced tumors did not react with autologous normal fibroblasts nor with autologous second tumors. Using up to 25 independently induced tumors as targets, these CTL clones were found to be uniquely specific for the particular tumor used for immunization even when multiple tumors isolated from the same animals were used as targets. In addition to this extensive antigenic diversity of independently induced tumors, we found that a single cancer cell can express multiple independent antigens that were uniquely expressed on the tumor but were not detectable on autologous nonmalignant fibroblasts. A poorly immunogenic spontaneous tumor was also found to express an antigen that was uniquely specific for the immunizing tumor in that it was absent from any of 25 other tumors tested. This antigen was recognized by a mAb and not detected on autologous nonmalignant fibroblasts or on an autologous second spontaneous tumor. These findings demonstrate that syngeneic CTL clones or mAbs can define unique antigens on UV-induced or spontaneous tumors. The use of autologous nonmalignant fibroblast targets made it unlikely that these antigens were widely expressed on normal cells. The availability of cloned immunological probes to antigens on tumors isolated with autologous normal cells will allow a reliable identification of the genetic origins of unique antigens on experimentally induced and spontaneous tumors and permit a decisive answer to whether these unique antigens are encoded by normal genes or by genes that have undergone somatic mutations; i.e., whether these antigens are truly tumor specific.