bluuebunny/crossref_metadata_2025 huggingface dataset:
Assembly, specific binding, and crystallization of a human TCR-alphabeta with an antigenic Tax peptide from human T lymphotropic virus type 1 and the class I MHC molecule HLA-A2.
Authors: Garboczi D N , Utz U , Ghosh P , Seth A , Kim J , VanTienhoven E A , Biddison W E , Wiley D C
Published: December 2022

T lymphocytes use TCR-alphabeta to bind and to recognize complexes of antigenic peptides bound to MHC proteins located at the surface of APCs. We have assembled and crystallized this intercellular complex of TCR/peptide/MHC from soluble human TCR-alphabeta and soluble peptide/HLA-A2 complexes. The soluble TCR-alphabeta binds specifically to its in vivo ligand, the complex of HLA-A2, and a peptide from the Tax protein of human T lymphotropic virus type 1. The soluble TCR also binds in vitro to an altered peptide ligand, which appears to be a partial agonist in T cell assays as determined by its ability to elicit different cytolytic and lymphokine secretion responses. Heterodimerization and the antigenic specificity of the TCR do not require its interchain disulfide bond, transmembrane segments, or glycosylations. Crystals of the TCR/peptide/HLA-A2 complex diffract x-rays, providing the means to study in atomic detail the mechanism of Ag-specific cell-cell recognition between T cells and target cells.
Cloning of the human platelet endothelial cell adhesion molecule-1 promoter and its tissue-specific expression. Structural and functional characterization.
Authors: Almendro N , Bellón T , Rius C , Lastres P , Langa C , Corbí A , Bernabéu C
Published: December 2022

Platelet endothelial cell adhesion molecule-1 (PECAM-1; CD31) is a cell adhesion molecule involved in transendothelial migration and expressed by hemopoietic and endothelial cells. To understand the mechanisms underlying its regulated expression, a genomic clone containing 1555 bp of the 5'-flanking region and the first exon of the human PECAM-1 gene has been isolated. The 5'-flanking region of the PECAM-1 gene lacks a consensus TATA box, but contains consensus motifs for Sp1, EGR1, ets, helix-loop-helix (HLH) box, GATA, AP-2, C/EBP, YY1, CCACC, LyF-1, imperfect octamer, heptamer, high mobility group proteins (HMG) box, and nuclear factor-kappaB, as well as shear stress-, retinoic acid-, glucocorticoid-, and acute phase-responsive elements, and an Alu sequence. Successive 5' to 3' or 3' to 5' deletions revealed tissue-specific promoter activity within the two contiguous 0.22-kb NheI/BglII and 0.44-kb BglII/PstI fragments. The transcriptional activity displayed by the 0.22-kb NheI/BglII fragment was specific for the myeloid lineage, whereas the promoter activity of the 0.44-kb BglII/PstI fragment was apparently restricted to endothelial cells. The transcriptional activity of the 0.22-kb NheI/BglII fragment was confirmed by 5' RACE (rapid amplification of 5' cDNA ends) and S1 nuclease protection experiments that revealed previously unidentified transcription start sites. The 0.22-kb NheI/BglII promoter exhibited PMA inducibility in myeloid cells and contained a PMA-responsive element recognized by Sp1 and EGR-1 transcription factors. Isolation and characterization of the human PECAM-1 promoter represent an initial step in elucidating the controlled expression of the PECAM-1 gene.
Localization to chromosome 22 of a gene encoding a human minor histocompatibility antigen.
Authors: Gubarev M I , Jenkin J C , Leppert M F , Buchanan G S , Otterud B E , Guilbert D A , Beatty P G
Published: December 2022

In human allogeneic bone marrow transplantation, graft-vs-host disease and graft rejection can occur even if the patient and donor are genotypically matched by inheritance for HLA. By definition, these allogeneic reactions are due to disparities in minor histocompatibility Ags (minor HAs). Minor HAs are presented to T lymphocytes as peptides bound to HLA molecules, and appear to be encoded by genes throughout the genome. We derived T lymphocyte clones from the PBL of a patient suffering from chronic graft-vs-host disease after bone marrow transplant from his HLA-identical sister. Clones reactive against minor HAs were selected on the basis of reactivity with pretransplant patient cells, and absence of reactivity with donor cells. One clone (MD2) was found to use HLA-B7 as a restricting element. A plasmid vector (pHEBo) containing cDNA encoding the HLA-B7 molecule was transfected into lymphoblastoid cell lines derived from two large families that previously had been saturation mapped for hundreds of polymorphic loci. When clone MD2 was tested against family K1362, it was found to be reactive with three of four grandparents, both parents, and eight of eleven offspring. The same clone was tested with family K1331, with two of three tested grandparents reactive, one of two parents, and nine of eleven offspring. Computer analysis showed that both family segregation patterns linked to an area on the long arm of chromosome 22, localizing the gene encoding this minor HA near the platelet-derived growth factor-beta and IL-2Rbeta genes.
Multiple regions of the transporter associated with antigen processing (TAP) contribute to its peptide binding site.
Authors: Nijenhuis M , Hämmerling G J
Published: December 2022

The transporter associated with Ag processing (TAP) translocates cytosolic peptides into the endoplasmic reticulum, where they can bind to MHC class I molecules. TAP does not translocate all peptides with equal efficiency, but selects peptides with regard to both their length and their sequence and, in this manner, affects the pool of peptides available for binding to MHC class I molecules. It has been demonstrated that peptide selection by TAP predominantly occurs during the first step in the translocation process, namely the association of the peptide with a binding site present on the TAP molecule. In this study, we identify four regions, two on the TAP1 and two on the TAP2 subunit, that make major contributions to this binding site. For both TAP1 and TAP2, the identified regions overlap with the cytosol-membrane boundaries of the two transmembrane segments closest to the ATP binding site. Our data are consistent with a model in which the transmembrane segments of TAP form a pore in the membrane, with the peptide binding site being formed by the cytosolic mouth of this pore.
Gene conversion contributes to Ig light chain diversity in cattle.
Authors: Parng C L , Hansal S , Goldsby R A , Osborne B A
Published: December 2022

In humans and mice, extensive gene rearrangement is the major mechanism of diversification of the primary Ig repertoire. This study shows that cattle depart from this pattern because rearrangement in the light chain locus is sharply limited. Furthermore, in cattle, gene conversion contributes to the diversification of the primary light chain repertoire. Sequencing of germ-line and expressed Vlambda genes revealed three important features. First, the germ line contained a number of Vlambda pseudogenes. In fact, 14 (70%) of the 20 germ-line genes identified and sequenced were pseudogenes, because they had one or more of the following defects: lack of recombination signal sequences at the 3' end, stop codons within the reading frame or truncations, and/or insertions or deletions that resulted in loss of reading frame. Second, Vlambda cDNA from ileal Peyer's patch B cells demonstrated that the light chain repertoire arises from only a small number of V(J) rearrangements. Even though two J genes were identified in the germ line, all of the expressed Vlambda genes examined contained the same J segment, indicating that only a single J gene participates in rearrangement at the lambda locus. Third, a significant number of departures from the germ-line sequences of rearranged Vlambda can be traced to donor sequences of one or more Vlambda pseudogenes. We conclude that a limited number of rearrangements and gene conversion play a role in contributing to the diversification of the primary lambda repertoire. Furthermore, while clear indications of a role for somatic mutation in lambda diversification was seen, V gene rearrangement was not a major factor.
HLA-DM is present in one-fifth the amount of HLA-DR in the class II peptide-loading compartment where it associates with leupeptin-induced peptide (LIP)-HLA-DR complexes.
Authors: Schafer P H , Green J M , Malapati S , Gu L , Pierce S K
Published: December 2022

HLA-DM has been shown in vitro to catalyze the release of invariant chain (Ii) derived peptides from the peptide-binding groove of class II molecules, thereby facilitating the binding of antigenic peptides. Previous studies showed that at steady state, the majority of DM resides in the class II peptide-loading compartment (IIPLC) where Ii dissociates from class II molecules and antigenic peptides are bound. Here we characterize the expression of DM in vivo in subcellular fractions containing the IIPLC. Using quantitative immunoblotting, we show that in the cell as a whole, class II molecules are expressed in 23-fold molar excess of DM. However, DM is concentrated in the IIPLC, where it is present in a considerably higher concentration relative to the class II molecules, in a molar ratio of 5DR:1 DM. This molar ratio of DM to DR in the IIPLC in vivo is consistent with the catalytic function proposed for DM from studies in vitro. We also provide both biochemical and genetic evidence that DM associates with complexes which contain Ii fragments and class II molecules in the IIPLC. Such complexes are only observed in leupeptin-treated cells in which Ii fails to be completely degraded and complexes containing the leupeptin-induced fragment of Ii (LIP) and class II molecules accumulate in the IIPLC. This observation is consistent with LIP-class II complexes being a substrate for DM in vivo and suggests that interactions of DM and LIP-class II are extremely transient under normal conditions.
Identification of a Borrelia burgdorferi OspA T cell epitope that promotes anti-OspA IgG in mice.
Authors: Bockenstedt L K , Fikrig E , Barthold S W , Flavell R A , Kantor F S
Published: December 2022

Lyme disease, due to infection with the tick-borne spirochete Borrelia burgdorferi, is a multisystem disorder that can lead to chronic disabling symptoms. Abs to the outer surface protein A (OspA) of B. burgdorferi provide protection against infection, and OspA is now the basis of a first generation recombinant vaccine undergoing phase III efficacy studies. Recent studies have suggested that T cells reactive with N-terminal epitopes in OspA could contribute to the development of treatment-resistant Lyme arthritis. In the present studies, we use the murine model of Lyme borreliosis to define an OspA T cell epitope located in the carboxyl terminus that accelerates anti-OspA IgG production after infection. In addition, we show that this T cell epitope is elicited by immunization with rOspA or with a truncated form of OspA that contains the B cell epitope targeted by protective OspA mAb. Polyclonal antisera to the truncated OspA fragment can protect mice from challenge infection. These results are the first demonstration of a B. burgdorferi-specific peptide that elicits a biologically important T cell response in vivo and have implications for the design of a second generation OspA-based subunit vaccine.
Induction of gammadelta T cells during acute murine infection with Toxoplasma gondii.
Authors: Kasper L H , Matsuura T , Fonseka S , Arruda J , Channon J Y , Khan I A
Published: December 2022

The importance of the host gammadelta T cell response to microbial pathogens is not yet fully understood. We report that inbred mice infected with T. gondii developed a gammadelta T cell proliferative response to parasite Ag. Mice depleted of either the alphabeta or gammadelta TCR were found to be significantly more susceptible to infection than the parent mouse strain. Proliferation of gammadelta T cells was observed in mice deficient in the TCR-alphabeta in response to UV-irradiated parasites. These T cells lyse parasite-infected syngenic macrophages. Adoptive transfer of these gammadelta T cells into beta2 microglobulin-deficient mice that have been depleted of both CD4+ and NK cells prolongs survival against acute parasite challenge when compared with nontransferred controls. The gammadelta T cells isolated from infected alpha -/- mice express a 10-fold increase in mRNA and produce high titers of IFN-gamma. These data suggest that gammadelta T cells may play an important role in the early host response to infection with T. gondii.
TNF receptor p55 plays a major role in centrally mediated increases of serum IL-6 and corticosterone after intracerebroventricular injection of TNF.
Authors: Benigni F , Faggioni R , Sironi M , Fantuzzi G , Vandenabeele P , Takahashi N , Sacco S , Fiers W , Buurman W A , Ghezzi P
Published: December 2022

The aim of this work was to study the relative role of the two TNF receptors (p55 and p75) in the central actions of TNF, studying the elevation of serum corticosterone (CS) and IL-6 levels after injection of recombinant murine (rm)TNF (intracerebroventricularly (i.c.v.)) in normal or p55-deficient (p55 -/-) mice. rmTNF induced high serum IL-6 levels and doubled serum CS in normal mice, whereas no elevation of serum IL-6 or CS was induced in p55 -/- mice. However, a normal CS response was observed in p55 -/- mice after LPS (2.5 microg, i.c.v.). p55 -/- mice also responded, although to a lesser extent than p55 +/+, in terms of LPS-induced IL-6 production. We also injected two agonist Abs specific for the two receptors, alpha p55 and alpha p75. While alpha p55 injected i.c.v. induced a marked elevation in CS and IL-6, alpha p75 induced CS (although less than alpha p55) but no IL-6. rmTNF, which binds both receptors, was more potent in inducing IL-6 and CS than injection of rhTNF, which in mice binds only p55. Finally, we investigated the role of p55 and p75 in IL-6 induction by TNF in a murine brain endothelioma. The results resembled closely those obtained in vivo: rmTNF was more potent than rhTNF and only alpha p55, and not alpha p75, induced IL-6 production. These data indicate that p55 plays a major role in TNF activation of the hypothalamus-pituitary-adrenal axis and in the centrally mediated induction of peripheral IL-6 by TNF, but p75, despite having little IL-6 inductive properties by itself, seems to potentiate p55 induction of IL-6.
Differential regulation of the C3a and C5a receptors (CD88) by IFN-gamma and PMA in U937 cells and related myeloblastic cell lines.
Authors: Burg M , Martin U , Bock D , Rheinheimer C , Köhl J , Bautsch W , Klos A
Published: December 2022

We have analyzed the induction of the receptor for the anaphylatoxic peptide C3a (C3aR) by the immunomodulator IFN-gamma, the phorbol ester PMA, and dibutyryl cAMP (Bt2cAMP) in comparison with the C5a receptor (C5aR, CD88). For U937 cells, IFN-gamma and Bt2cAMP up-regulated the C3aR to the same extent, whereas Bt2cAMP was 20-fold more effective in C5aR induction. PMA increased the expression of the C5aR, and acted synergistically with IFN-gamma. In contrast, PMA did not increase specific 125I-hC3a binding, and actually antagonized C3aR induction by IFN-gamma. Two related human cell lines of the myeloblastic/monocytic lineage, HL-60 and Mono Mac 6, showed inducibility of the C3aR similar to U937 cells. The two receptors showed subtle differences in signal transduction. Despite comparable numbers of both receptors, IFN-gamma potentiated activation of the C5aR but not the C3aR, as measured by an increase in free cytosolic Ca2+ upon ligand activation. Interestingly, Bt2cAMP-treatment led to a functional response to C3a in U937 cells. Such differences in receptor regulation and signaling might underlie the partly differing physiologic effects of C3a and C5a on, for example, chemotaxis, induction of oxidative burst, or immunoregulatory functions.
CR3 (alphaM beta2; CD11b/CD18) restores IgG-dependent phagocytosis in transfectants expressing a phagocytosis-defective Fc gammaRIIA (CD32) tail-minus mutant.
Authors: Worth R G , Mayo-Bond L , van de Winkel J G , Todd R F , Petty H R
Published: December 2022

Previous studies have suggested that complement receptors cooperate with Fc receptors to mediate Ab-dependent effector functions. In the present study, we tested the capacity of complement receptor type 3 (CR3; alphaM beta2; CD11b/CD18) to participate in Fc gammaRIIA (CD32)-mediated phagocytosis. To test this hypothesis, we transfected a phagocytosis-defective tail-minus mutant of Fc gammaRIIA into fibroblasts that did or did not express CR3. As controls, cells exposed to the transfection protocol but not expressing either CR3 or Fc gammaRIIA constructs were also selected. Expression of cell surface receptors was confirmed by both flow cytometry and fluorescence microscopy. Cells were tested for their ability to bind and internalize IgG-opsonized erythrocytes and for surface proximity of CR3 and Fc gammaRIIA using resonance energy transfer techniques. Resonance energy transfer studies showed a physical proximity between CR3 and Fc gammaRIIA on cell surfaces. Cells expressing only the tail-minus mutant of Fc gammaRIIA were unable to internalize erythrocytes but showed significant binding ability. In contrast, cells expressing both mutant Fc gammaRIIA and CR3 internalized opsonized erythrocytes. These results show that CR3 can complement the phagocytic function of defective Fc gammaRIIA. These data suggest that CR3 is capable of transducing a phagocytic signal generated by Fc gammaRIIA recognition events that mediates internalization of IgG-opsonized targets.
The balance between persistent virus infection and immune cells determines demyelination.
Authors: Rodriguez M , Pavelko K D , Njenga M K , Logan W C , Wettstein P J
Published: December 2022

We addressed the contributions of persistent virus infection and immune cells to the pathogenesis of Theiler's virus-induced demyelination, a model for human multiple sclerosis. We developed a model involving the transfer of spleen cells into immunodeficient C.B-17-scid (SCID) mice, which normally die of overwhelming virus encephalitis without demyelination when infected with Theiler's virus. Adoptive transfer of nonimmune spleen cells from BALB/c mice into SCID mice resulted in the survival of all mice. However, these mice developed extensive demyelination and virus Ag/RNA persistence in the spinal cord white matter. The most demyelination was observed when mice received an intermediate number of spleen cells (1.8-7.5 x 10(6)), whereas too few cells (0.5 x 10(6)) did not ameliorate the SCID phenotype, and too many cells (30 x 10(6)) resulted in almost complete viral clearance with minimal demyelination. Adoptive transfer of spleen cells depleted of either CD4+ or CD8+ T cells produced vacuolar demyelination associated with virus persistence. In contrast, reconstitution with both CD4+ and CD8+ T cells produced less severe demyelination and partial clearance of virus. These experiments support the hypothesis that demyelination is the result of a balance between persistent virus infection and immune injury mediated by either CD4+ or CD8+ T cells.
Sophisticated strategies for information encounter in the lymph node: the reticular network as a conduit of soluble information and a highway for cell traffic.
Authors: Gretz J E , Kaldjian E P , Anderson A O , Shaw S
Published: December 2022

The lymph node is the crossroad in which soluble signals and cells carried by lymph meet lymphocytes emigrating from blood. Efficient interactions among these elements depend on the reticular network, which comprises reticular fibers, related extracellular matrix components, and associated fibroblastic reticular cells. This network provides a three-dimensional scaffold for attachment of APCs and pathways for the migration of T cells to these APCs. In addition, the network constitutes a miniature conduit system for bulk flow delivery of soluble molecules to distinct sites in the paracortex, particularly the high endothelial venule. The delivered mediators, such as chemokines, regulate the phenotype of the high endothelial venule, the recruitment of lymphocytes, and the behavior of the recruited lymphocytes. Thus, the reticular network is a multifunctional infrastructure that facilitates encounters of cells with other cells and factors necessary for effective and efficient immune surveillance.
The weak CD8+ CTL response to an influenza hemagglutinin epitope reflects limited T cell availability.
Authors: Cao W , Myers-Powell B A , Braciale T J
Published: December 2022

One of the two class I MHC (H-2Kd)-restricted immunogenic regions identified on the influenza virus strain A/Japan/57 (H2N2) hemagglutinin (HA) encompasses two distinct, partially overlapping epitopes. These epitopes map to residues 204 to 212 (JHA 204-212) and 210 to 219 (JHA 210-219), respectively. When we investigated the magnitude of the CTL responses of H-2d BALB/c mice to these two epitopes, we found that the JHA 204-212 nonamer epitope is immunodominant, eliciting vigorous CTL responses in BALB/c mice immunized with A/Japan/57 virus. In contrast, the CTL response to the JHA 210-219 decamer epitope was weak and variable. The subdominance of the JHA 210-219 was not due to low affinity binding of JHA 210-219 to H-2Kd or to inefficient processing of this epitope in vivo. Rather, an analysis of CTL precursor (pCTL) frequency by limiting dilution showed that the frequency of pCTL to the JHA 210-219 epitope was at least 10-fold lower than the frequency of pCTL to the JHA 204-212 epitope, implying that the low and variable response to the JHA 210-219 epitope was due to a limited number of CD8+ T cell precursors directed to JHA 210-219. This hypothesis was further supported by the finding of limited heterogeneity in reactivity pattern displayed by short term bulk cultures of the JHA 210-219-specific CTLs for cross-reactive epitopes. Implications of these findings for vaccine design and for T lymphocyte function and repertoire development are discussed.
IFN-alpha induces autoimmune T cells through the induction of intracellular adhesion molecule-1 and B7.2.
Authors: Chakrabarti D , Hultgren B , Stewart T A
Published: December 2022

Insulin-dependent diabetes is an autoimmune disease characterized by the loss of the insulin-producing beta cells and the appearance of autoreactive (anti-islet) T cells. The mechanism by which these autoimmune T cells become activated has not been resolved. We demonstrate that the expression of IFN-alpha by the pancreatic beta cells leads to the development of CD4+ T cells that proliferate in the presence of islet Ags. These autoreactive T cells have a Th1 phenotype and are able to lyse islet cells, possibly through an indirect, cytokine-mediated mechanism. We also demonstrate that the pancreatic infiltrating leukocytes in the transgenic mice express increased levels of 87.2 and ICAM-1 and that IFN-alpha can directly induce these two costimulatory molecules on nontransgenic splenic APCs. Treatment of the transgenic mice with Abs against these costimulatory molecules demonstrated that B7.2 is essential for the induction of autoreactive T cells, whereas ICAM-1 contributes to but is not essential for the formation of these autoreactive cells. As B7.2 and ICAM-1 are able to synergize to provide costimulatory signals to naive T cells under conditions of limiting Ag, we propose that IFN-alpha expression normally contributes to the development of an anti-viral cellular immune response, but that uncontrolled expression of this cytokine will induce autoimmunity.
Delayed-type hypersensitivity in mast cell-deficient mice: dependence on platelets for expression of contact sensitivity.
Authors: Geba G P , Ptak W , Anderson G M , Paliwal V , Ratzlaff R E , Levin J , Askenase P W
Published: December 2022

Previous studies of cutaneous T cell-mediated responses in mice have obtained pharmacologic, morphologic, and immunologic evidence pointing to a critical role for local mast cells in release of the vasoactive amine serotonin (5-HT) to mediate early, initiating events that are required for elicitation of these responses. However, the role of mast cells in initiating these T cell-mediated cutaneous responses has been questioned due to the presence of relatively intact delayed-type hypersensitivity responses, such as contact sensitivity (CS), in mast cell-deficient mice whose skin contains only 1 % normal mast cell numbers. The contribution of other potential local sources of 5-HT, such as circulating platelets, at the site of a delayed-type hypersensitivity or CS response in these mast cell-deficient strains, has not been investigated. Therefore, we studied the effect of systemic platelet depletion, produced with an anti-platelet Ab, on blood and tissue levels of 5-HT, and on in vivo T cell-mediated cutaneous sensitivity responses, in W/Wv and Sl/Sld mast cell-deficient mice. The results showed that: 1) platelet depletion severely reduced whole blood 5-HT; 2) tissue levels of 5-HT, in mast cell-deficient mice, depended in large part on the presence of circulating platelets, and 3) specific depletion of platelets markedly suppressed CS responses in both W/Wv and Sl/Sld mast cell-deficient mice, and only moderately reduced CS in normal +/+ congenic mast cell-sufficient controls, but did not decrease CS in beige mice, with platelet granules that are defective in storage of 5-HT. We concluded that platelets may provide 5-HT crucial for the initiation of cutaneous T cell-mediated immune responses, such as CS.
Th1 development of naive CD4+ T cells is inhibited by co-activation with anti-CD4 monoclonal antibodies.
Authors: Goedert S , Germann T , Hoehn P , Koelsch S , Palm N , Rüde E , Schmitt E
Published: December 2022

Co-activation of CD4+ T cells by anti-CD4 mAb strongly enhances the proliferation of these T cells. We have analyzed the influence of CD4-mediated co-activation on Th cell differentiation. Our data demonstrate that activation of high density (HD)-CD4+ T cells by immobilized anti-CD3 mAb in combination with immobilized anti-CD4 mAb led to a strong inhibition of Th1 differentiation and to a variable but significant induction of Th2 development. Priming of highly enriched Mel-14highCD4+ T cells in the presence of anti-CD4 mAb also resulted in a pronounced suppression of secondary IFN-gamma production, indicating that the Th1 development of naive CD4+ T cells is inhibited by co-activation via CD4. In contrast to HD-CD4+ T cells, CD4-induced costimulation of MEL-14highCD4+ T cells did not result in a primary and secondary IL-4 production. Hence, these results suggest that a MEL-14low population within the HD-CD4+ T cell fraction was the source of the endogenous IL-4 and imply, in addition, that co-activation via CD4 inhibits the development of Th1 cells from naive CD4+ T cells independently from endogenous IL-4. This assumption is further corroborated by the fact that neither the application of neutralizing anti-IL-4 mAb nor the use of T cells from IL-4 knockout mice could prevent CD4-mediated inhibition of Th1 development. The Th1-inhibiting effect of anti-CD4 mAb could not be reversed by the application of the Th1 inducer IL-12. On the contrary, the secondary IL-4 production of HD-CD4+ T cells as an indicator of Th2 differentiation, which was promoted by anti-CD4 mAb, was enhanced even in the presence of IL-12. Therefore, our results suggest that co-activation of naive CD4+ T cells by anti-CD4 mAb directly and selectively inhibits Th1 differentiation of naive dense CD4+ T cells.
Bcmd governs recruitment of new B cells into the stable peripheral B cell pool in the A/WySnJ mouse.
Authors: Lentz V M , Cancro M P , Nashold F E , Hayes C E
Published: December 2022

The A/WySnJ mouse provides a genetic model for studying new B cell selection into the stable peripheral B cell pool. Unlike the related A/J strain, the A/WySnJ has a single, autosomal codominant gene defect, Bcmd, resulting in a profound peripheral B cell deficiency. Here, continuous in vivo bromodeoxyuridine labeling and immunofluorescence analysis showed normal bone marrow B cell genesis but excessive B cell loss from the marrow and each peripheral pool in A/WySnJ. The A/WySnJ immature B220low/HSAhigh splenic B cell pool was 79% smaller, had a 69% slower renewal rate, and its cells had a 29% shorter average half-life than A/J. The A/WySnJ mature B220high/HSAlow splenic B cell pool was 92% smaller, had an 83% slower renewal rate, and its cells had a 56% shorter average half-life. In reciprocal chimeras, the A/WySnJ marrow failed to repopulate the peripheral B cell pool in A/J mice, whereas the A/J marrow fully reconstituted the A/WySnJ mice. Histochemistry revealed disordered splenic architecture in A/WySnJ, with few primary lymphoid follicles and a second abnormal phenotype, mastocytosis. There was no common genetic basis for B cell deficiency and mastocytosis in the F2 progeny of an (A/WySnJ x CAST/Ei)F1 intercross. We conclude that Bcmd is expressed in bone marrow cells, most likely B cells, where it hinders short-lived B cell maturation to a long-lived phenotype with the potential to form memory B cells.
Peripheral blood dendritic cells express Fc epsilon RI as a complex composed of Fc epsilon RI alpha- and Fc epsilon RI gamma-chains and can use this receptor for IgE-mediated allergen presentation.
Authors: Maurer D , Fiebiger S , Ebner C , Reininger B , Fischer G F , Wichlas S , Jouvin M H , Schmitt-Egenolf M , Kraft D , Kinet J P , Stingl G
Published: December 2022

Originally limited to basophils and mast cells, the spectrum of high affinity IgE receptor (Fc epsilon RI-bearing cells has expanded recently to include Langerhans cells, dermal dendritic cells (DC), monocytes, and eosinophils. As a result of studies on the distribution, structure, and function of Fc epsilon RI on APCs, we discovered a minor nonbasophil, nonmonocyte PBMC population that can bind IgE via Fc epsilon RI. This receptor occurs on the surface of these cells as a multimeric structure containing Fc epsilon RI alpha- and Fc epsilon RI gamma-chains but, unlike its counterpart on basophils, lacking Fc epsilon RI beta. Further experiments revealed that these Fc epsilon RI alpha gamma-expressing cells closely resemble peripheral blood DC by immunophenotype (HLA-DRhigh, HLA-DQhhigh; CD4+, CD11a+, CD32+, CD33+, B7/2 (CD86)+; CD11blow, CD14low, CD40low, CD54low, CD64low) and cell morphology. These features allowed us to isolate Fc epsilon RI-expressing DC from the peripheral blood and to investigate their immunostimulatory properties. We found Fc epsilon RI-positive DC to be efficient stimulators of both primary (allogeneic MLR) and Fc epsilon RI/IgE-dependent, secondary T cell responses at low cell numbers. Thus, Fc epsilon RI-expressing DC may not only amplify established type I allergic immune reactions but, unlike Fc epsilon RI-positive semiprofessional APCs, may be able to prime naive T cells to common and/or cryptic epitopes of IgE-reactive Ags.
IFN-gamma-induced MHC class II gene expression is suppressed in endothelial cells by dextran sulfate.
Authors: Lian R H , Kotwal G J , Wellhausen S R , Hunt L A , Justus D E
Published: December 2022

IFN-gamma-activated endothelial cells actively participate in initiating immune responses by interacting with immunocompetent cells via class II MHC proteins. In this study, dextran sulfate, a synthetic heparin analogue, was shown to selectively inhibit IFN-gamma-induced surface expression of HLA-DR molecules by human umbilical cord vascular endothelial cells, but not other cytokine-induced molecules such as ELAM-1 or ICAM-1. Inhibition occurred regardless of whether dextran sulfate was added 24 h before, at the same time as, or 24 h after IFN-gamma stimulation of cells. In addition, both high (500 kDa) and low (5 kDa) molecular mass dextran sulfate molecules were able to block class II expression, whereas treating cells with naturally occurring polysulfated glycosaminoglycans such as heparin, heparan, and chondroitin sulfate did not produce any suppressive effects. Radiolabeling of cells with [35S]methionine followed by radioimmunoprecipitation using anti-HLA-DR alpha mAb demonstrated that biosynthesis of class II proteins was specifically blocked. RT-PCR and Southern blotting were utilized to examine transcription of the HLA-DR alpha gene and demonstrated an absence of HLA-DR alpha mRNA from dextran sulfate-treated and IFN-gamma-induced cells. Dextran sulfate also prevented transcription of the gene encoding CIITA, a transactivator protein required for IFN-gamma-inducible expression of class II genes. Thus, dextran sulfate apparently inhibited this step or an earlier one in the intracellular signaling pathway for IFN-gamma in human endothelial cells, subsequent to IFN-gamma binding to its cell surface receptor.
Accelerated amyloid deposition in mice treated with the aspartic protease inhibitor, pepstatin.
Authors: Yamada T , Liepnieks J , Benson M D , Kluve-Beckerman B
Published: December 2022

The development of amyloidotic diseases is believed to be determined in large part by the structure and metabolism of the amyloid subunit protein. The amino-terminal region of serum amyloid A (SAA), the subunit precursor protein in reactive amyloidosis, appears to confer fibrillogenic potential. Here we present data consistent with the hypothesis that amyloid A fibrillogenesis is favored when proteolysis of the amino-terminal region of SAA is impaired. Murine tissue extracts were found to contain pepstatin-inhibitable protease activity that cleaved mouse SAA2 between Glu8 and Ala9. Tissues obtained from mice that had been treated with pepstatin for 3 days lacked this activity. To investigate a possible relationship between inhibition of aspartic proteases and amyloidogenesis, mice were treated with pepstatin while concurrently undergoing a standard amyloid induction protocol (repeated casein injections). Pepstatin-treated mice showed amyloid deposition significantly sooner than the control group, which had received only casein. During the preamyloidotic phase, pepstatin-treated mice had higher concentrations of SAA in serum and spleen than control mice. In addition, clearance of injected 125I-labeled SAA from plasma was significantly delayed. Based on these findings, it is reasonable to postulate that inhibition of aspartic protease activity can lead to an accumulation of amino-terminally intact SAA molecules and thereby accelerate amyloid fibril formation.
Analysis of the fine B cell specificity during the chronic/relapsing course of a multiple sclerosis-like disease in Lewis rats injected with the encephalitogenic myelin oligodendrocyte glycoprotein peptide 35-55.
Authors: Ichikawa M , Johns T G , Liu J , Bernard C C
Published: December 2022

We have recently shown that a single injection of myelin oligodendrocyte glycoprotein (MOG), or the MOG35-55 peptide, produces a relapsing-remitting neurologic disease with extensive plaque-like demyelination. Given the features that this new autoimmune demyelinating model has in common with the clinicopathologic manifestations of multiple sclerosis, we have examined the Ab reactivity to native MOG and MOG35-55 peptide during the course of the disease in Lewis rats. Following immunization with MOG35-55, varied clinical symptoms were observed; these included hind and foreleg paralysis and various degrees of balance impairment. Disease progression also varied: 3 out of 21 animals had a single mild disease episode; 4 out of 21 had a mild relapsing-remitting disease; and 14 out of 21 had severe relapsing-remitting disease. Ab reactivity to MOG35-55 and native MOG was first detected in all rats 4 wk postimmunization and persisted throughout the 12 wk of observation. The Ab response was highly restricted with no reactivity to other peptides encompassing different extracellular segments of MOG. Fine epitope mapping showed that Ab from serum and cerebrospinal fluid of injected rats reacted strongly to MOG37-46 and to a lesser extent to MOG43-50. Although significant levels of anti-MOG Abs appeared necessary for the development of demyelinating lesions, their presence in blood and cerebrospinal fluid alone was not sufficient to produce severe clinical symptoms. These results demonstrate that the MOG35-55 peptide is highly encephalitogenic and can induce strong T and B cell responses. It is probably the complex interaction between these T and B cells that determines the severity of disease in individual rats.
Cloning and analysis of IgG kappa and IgG lambda anti-thyroglobulin autoantibodies from a patient with Hashimoto's thyroiditis: evidence for in vivo antigen-driven repertoire selection.
Authors: McIntosh R S , Asghar M S , Watson P F , Kemp E H , Weetman A P
Published: December 2022

Antibodies to thyroglobulin (Tg) are commonly found in patients with the autoimmune thyroid diseases Graves' disease and Hashimoto's thyroiditis as well as in individuals with apparently normal thyroid function. Although it is not clear how Tg Abs are involved in the pathology of the diseases, the study and analysis of these Abs may nevertheless be instructive in allowing the development of an Ab response to an autoimmune disease-associated self Ag to be followed. We have prepared IgG kappa and lambda phage display combinatorial libraries from the cervical lymph node of a single Hashimoto's thyroiditis patient with a high anti-Tg titer. These were selected with purified human Tg, and 10 IgG kappa and 9 IgG lambda clones were analyzed further. Sequence analysis of the clones showed a very highly restricted heavy chain usage and a less restricted light chain usage. There was a variable degree of divergence from germ-line sequence in the light chain sequences, with a clear relationship between relatively higher affinity of the Fab for human Tg and an increased degree of somatic hypermutation. The Tg-selected Fab did not bind to Tg from other species, to reduced denatured Tg, or to thyroid peroxidase. The Fab inhibited patient serum binding to human Tg by between 39 and 79%. In summary, we have isolated 19 high affinity, human Tg-specific Fab and shown that the relative affinity of the Fab is directly related to the pattern of somatic hypermutation.
Different CD40-mediated signaling events require distinct CD40 structural features.
Authors: Hostager B S , Hsing Y , Harms D E , Bishop G A
Published: December 2022

Signals delivered to B cells through CD40 are critical to the development of humoral immune responses. In this study we characterize regions of the 62-amino acid cytoplasmic domain of human CD40 (hCD40) that are essential for signal transduction and examine the functional consequences of mutations in these regions. A panel of mutant hCD40 molecules was stably expressed in mouse B cell lines and tested for its ability to stimulate Ab secretion, homotypic adhesion, and increased surface expression of B7, Fas, CD23, LFA-1, and intracellular adhesion molecule-1. Our results indicate that CD40 contains at least two major signaling determinants in the cytoplasmic domain: one disrupted by a truncation of 22 amino acids, and a second disrupted by the removal of 10 additional amino acids. The second determinant includes threonine 234, a residue previously shown to be important in CD40 signal transduction. Our functional analysis of alanine and serine substitutions at position 234 indicates that phosphorylation of this residue may be important for full CD40 signaling activity.
Cyclic adenosine 3',5'-monophosphate increases the open probability of potassium channels in activated human T cells.
Authors: Oleson D R , DeFelice L J , Quinn M F , Donahoe R M
Published: December 2022

In the present report we describe a cAMP-responsive K channel in activated human T cells. Single channel events were recorded using the patch-clamp technique in cell-attached-patch configuration. The channel was K selective, as determined by reversal potentials under different K gradients, and displayed voltage-independent gating. When the applied potential (Vp) was equal to zero, the conductance of the channel was 21.8 +/- 0.9 pS with 150 mM K in the electrode. Typical patches contained between two and seven channels that were relatively quiet, or silent, before agonist stimulation. Adenosine (20-30 microM) increased the average open time probability from 0.017 +/- 0.008 to 0.108 +/- 0.054 over a period of 108 s. Subsequent addition of the phosphodiesterase inhibitor Ro 20-1724 (0.5 mM) increased the probability of being in the open state to 1.155 +/- 0.407 over a period of 180 s. Channel kinetics were well described by assuming two open and two closed states. Exponential time constants for the open states were 0.51 +/- 0.06 and 4.34 +/- 0.31 ms, and closed state time constants were 0.58 +/- 0.05 and 10.1 3 +/- 2.32 ms. In addition, extracellular ATP (0.3-1.0 mM) decreased channel activity. Moreover, Rp-cAMP (0.5-1.0 mM), an antagonist that specifically blocks the ability of cAMP to bind and activate protein kinase A, failed to inhibit adenosine- and Ro 20-1724-induced increases in channel activity, implying a direct action of cAMP on channel gating.
Identification of a novel regulatory region critical for expression of the RANTES chemokine in activated T lymphocytes.
Authors: Nelson P J , Ortiz B D , Pattison J M , Krensky A M
Published: December 2022

The RANTES chemokine is a T cell-expressed, proinflammatory cytokine recently implicated as a suppressive agent of HIV replication. We have identified tandem kappaB-like sequences within the promoter for RANTES that are critical for RANTES promoter-reporter gene activity in both the T cell tumor line Hut78 and in PHA-activated PBL. This region binds not only Rel family members (including p50-p65 heterodimers and p50-p50 homodimers) but also non-Rel factors up-regulated in PBL 3 to 5 days following activation. The expression of these "late" expressed nuclear factors correlates with an up-regulation of RANTES message found at this point in T cell activation. These factors are also constitutively expressed in functionally mature CD8+ T cells. We hypothesize that these apparently novel proteins are responsible in part for the temporal regulation of RANTES seen in peripheral blood T cells and represent a component of transcriptional regulatory machinery newly expressed at this "late" stage of peripheral T cell development.
Differential regulation of c-Rel translocation in activated B and T cells.
Authors: Venkataraman L , Wang W , Sen R
Published: December 2022

c-Rel induction in activated lymphocytes is suppressed by the immunosuppressive drug, FK506. Here we show that FK506-suppressible, delayed c-Rel induction is similar in B and T cells and is regulated by mRNA production. In contrast, rapid nuclear translocation of pre-existing cytoplasmic c-Rel occurs only in B cells, but not in T cells. Analysis of I-kappaBalpha and -beta in these cells showed that both I-kappaBalpha and I-kappaBbeta were rapidly degraded in response to stimulation in B cells, but only I-kappaBalpha was affected in T cells. These observations suggest that 1) different Rel proteins in the same cell may be sequestered in the cytoplasm differently, 2) the sequestration mechanism is cell-type specific, and 3) differential sensitivities of I-kappaBalpha and beta in B and T cells may regulate, in part, the rapid response of family members. We propose that subunit-specific and cell-specific regulation of nuclear translocation may help determine the varied cellular responses to different stimuli.
Genetic regulation of protective immune response in congenic strains of mice vaccinated with a subunit malaria vaccine.
Authors: Tian J H , Miller L H , Kaslow D C , Ahlers J , Good M F , Alling D W , Berzofsky J A , Kumar S
Published: December 2022

The C-terminal 19-kDa, epidermal growth factor-like region of the merozoite surface protein 1 (MSP1) has been used as a vaccine to induce protective immunity to Plasmodium yoelii in mice and to Plasmodium falciparum in monkeys. To analyze the mechanisms and genetic regulation of this MSP1 vaccine-induced protection, we studied the immunologic correlates of protection in H-2 recombinant and congenic mouse strains on the B10 background. Multiple H-2-linked loci were found to contribute, each with a different mechanism. One locus mapped to the I-A region based on the strong protection in C57BL/10 mice compared with intermediate protection in B10.A(4R) mice and the lack of a difference between B10.AKM and B10.MBR mice. Differences in efficacy of passively transferred antisera from vaccinated C57BL/10 vs B10.A(4R) mice indicated that the protection regulated by the I-A locus was at least in part Ab dependent. Two loci mapped to the right of I-A (FE, H-2S, or H-2D) based on a correlation with the number of H-2k loci to the right of I-A in mice that were I-Ak. One effect was Ab independent and may correspond to a possible negative effect of the I-Ek locus. T cells from protected and nonprotected strains differed in their production of IFN-gamma and TNF-alpha following immunization with MSP1(19), but it was unclear how the differential patterns of cytokine expression related to the level of protection. Thus, MSP1(19) vaccine-induced protection is regulated by H-2-linked loci corresponding to two different immune mechanisms. These findings may indicate the need for more than one Ag in a vaccine to protect an HLA-diverse population.
TNF-alpha bidirectionally modulates the viability of primitive murine hematopoietic progenitor cells in vitro.
Authors: Jacobsen F W , Veiby O P , Stokke T , Jacobsen S E
Published: December 2022

It is well established that TNF-alpha can induce apoptosis in many normal and transformed cell types. The effects of TNF-alpha on cytokine-induced proliferation and differentiation of normal hematopoietic progenitors have been characterized extensively, whereas little is known about how TNF-alpha can affect their viability. The present studies suggest, based on experiments using delayed addition of growth-promoting cytokines as well direct viability assays, that TNF-alpha bidirectionally affects the survival of individually cultured primitive Lin- Sca-1+ hematopoietic progenitors, in that stem cell factor (SCF)-, granulocyte-CSF-, IL-6-, and IL-11-induced survival is potently counteracted by TNF-alpha (42-86%), whereas TNF-alpha synergistically enhances IL-1alpha-induced survival up to threefold. The bidirectional effects of TNF-alpha on hematopoietic growth factor-induced survival of hematopoietic progenitors were reflected in that TNF-alpha enhanced apoptosis of Lin- Sca-1+ cells when combined with SCF, whereas TNF-alpha synergistically suppressed apoptosis in response to IL-1alpha.