bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism of cyclosporin-induced tolerance to primarily vascularized allografts in miniature swine. Effect of administration of exogenous IL-2.
Authors: Gianello P R , Blancho G , Fishbein J F , Lorf T , Nickeleit V , Vitiello D , Sachs D H
Published: December 2022

Indefinite tolerance to kidney allografts across a two-haplotype class I disparity can be induced in miniature swine in 100% of cases by a short course of cyclosporin A (CyA). Without CyA, all recipients reject kidney allografts within 2 wk. These animals therefore provide a unique opportunity to study the mechanisms of induction and maintenance of tolerance in a large animal model. Previous studies of cellular and humoral immunity suggested that a T cell help deficit at the time of the first exposure of the host's immune system to alloantigens was involved in tolerance induction. We have now studied the effect of exogenous T cell help in the form of an IL-2 infusion during both the induction and maintenance phases of tolerance. Lymphoid infiltrates were seen in class I mismatched renal allografts by day 8 in all animals whether treated with CyA or not. Administration of i.v. IL-2 on postoperative days 8, 9, and 10 to animals receiving the full CyA tolerizing regimen led to acute rejection in four of four animals. These rejecting animals showed induction of IL-2R expression on graft infiltrating cells in the kidney, suggesting that the infiltrates present before IL-2 administration were capable of causing rejection once T cell help was provided. Treatment with IL-2 did not abrogate long-term tolerance. Thus, limitation of T cell help at the time of first exposure to Ag in this model appears to be required to prevent rejection during the time required for active tolerance to develop. Once established, this tolerance does not appear to require continuous limitation of T cell help to be maintained, suggesting loss, inactivation, or suppression of the cells capable of causing rejection.
Specific T cell receptor gene rearrangements at the site of muscle degeneration in Duchenne muscular dystrophy.
Authors: Gussoni E , Pavlath G K , Miller R G , Panzara M A , Powell M , Blau H M , Steinman L
Published: December 2022

Mononuclear cells infiltrate degenerating muscles of Duchenne muscular dystrophy (DMD) patients. Using a quantitative PCR, we first characterized the T cells infiltrating muscle biopsies from six DMD patients. High levels of TCR V beta 2 transcripts were observed in DMD muscle tissue. TCR V beta 2 transcripts from seven DMD patients and five controls were sequenced, and the VDJ junctional region analyzed in 166 clones. One specific amino acid motif, RVSG, was found in the third complementary determining region (CDR3) of TCR V beta 2 chains in samples from five DMD patients, but not in controls. A specific immune reaction at the site of tissue degeneration may play an important role in the pathogenesis of DMD.
Characterization of a granule-independent lytic mechanism used by CTL hybridomas.
Authors: Garner R , Helgason C D , Atkinson E A , Pinkoski M J , Ostergaard H L , Sorensen O , Fu A , Lapchak P H , Rabinovitch A , McElhaney J E
Published: December 2022

The mechanism(s) by which CTL induce target cell lysis have not been clearly elucidated. Perforin and the cytotoxic cell proteinases (granzymes) contained within the granules of CTL and NK, have been implicated, but abundant evidence for the existence of alternate lytic pathways has accumulated. In this report we characterize the mechanism of killing used by two cytolytic hybridomas (PMM-1 and MD90) that express neither perforin nor the granzymes. These characteristics are compared with results obtained by using a representative Ag-dependent, granule-containing T cell clone in cytolysis assays. The major differences were that the granule-negative hybridomas could lyse a variety of target cells in the presence of cyclosporin and the absence of calcium. All the effectors could kill in the presence of protein synthesis inhibitors (cycloheximide and emetine) and induced DNA fragmentation in the target cells. The cytolytic hybridomas had to be stimulated to be cytolytic and this activation required the presence of calcium, was dependent on protein synthesis, and inhibited by the addition of cyclosporin. Although TNF was shown not be involved, the sensitivity of the target cells to lysis by the granule-negative killers correlated with the level of expression of Fas Ag. With the use of L1210 and an L1210 cell line transfected with Fas cDNA we demonstrated that these MD90 and PMM-1 kill the latter much more effectively and that this increase was effectively inhibited with anti-Fas Ab. Furthermore the lack of sensitivity to cyclosporin, cycloheximide, emetine, and EGTA was confirmed with these targets. We conclude that these two cytolytic hybridomas use the Fas lytic pathway to induce lysis in target cells.
CD27 is a signal-transducing molecule involved in CD45RA+ naive T cell costimulation.
Authors: Kobata T , Agematsu K , Kameoka J , Schlossman S F , Morimoto C
Published: December 2022

CD27 is a 120-kDa transmembrane homodimeric molecule expressed on the majority of T cells, B cells, and NK cells that belongs to the TNFR/nerve growth factor receptor family. The interaction between CD27 and its ligand, CD70, is thought to play an important role in T cell activation. In this paper we have examined the signal-transducing potential of CD27 in T cell costimulation. Anti-CD27 mAb, anti-1A4, induced substantial proliferation of peripheral blood T cells in the presence of a suboptimal dose of PMA, phytohemagglutinin, anti-CD2, or anti-CD3 together with a second Ab to cross-link the CD27 molecule. This T cell proliferation was also observed by using CD70 transfectant cells. CD27 cross-linking maximally induced proliferation of CD45RA+CD4 T cells but only slightly induced proliferation of CD45RO+CD4 T cells. CD27-mediated T cell proliferation did not seem to be dependent on the IL-2/IL-2R system because no detectable level of IL-2 was secreted, and only a partial inhibition was observed with anti-IL-2 and anti-IL-2R Abs. Furthermore, an increase in intracellular Ca2+ was observed in PMA-treated T cells when the CD27 molecule was cross-linked. More importantly, CD27 ligation induced protein tyrosine phosphorylation, especially 70 kDa of cellular substrate, including ZAP-70, in T cells. Herbimycin A, a protein tyrosine kinase inhibitor, and staurosporine, a protein kinase C inhibitor, blocked T cell proliferation induced by CD27 ligation, suggesting the possibility that the activation of protein tyrosine kinase and protein kinase C is required for CD27-mediated T cell costimulation. These results clearly demonstrate that the CD27/CD70 interaction induces costimulatory signals in T cells, especially CD45RA+ naive T cells, indicating that CD27 serves as a T cell signal-transducing molecule.
A soluble form of the HLA-G antigen is encoded by a messenger ribonucleic acid containing intron 4.
Authors: Fujii T , Ishitani A , Geraghty D E
Published: December 2022

The HLA-G primary transcript is alternatively spliced to yield mRNAs encoding three alternative membrane bound proteins. In addition to these forms, a soluble HLA-G protein has been described which is not encoded directly by any of the three alternative mRNAs. To explain the process which might lead to the expression of a soluble HLA-G Ag, we investigated the potential roles proteolytic processing and additional alternative splicing of HLA-G RNA might play. By generating transfected cells with HLA-G cDNA expression driven by a retroviral promoter, it was possible to rule out proteolytic processing of the membrane-bound HLA-G as a mechanism of generating soluble HLA-G, resulting in our focus on alternative splicing as an explanation. Analysis of PCR-amplified cDNA revealed a relatively abundant transcript present in all samples examined which consisted of the full length HLA-G mRNA sequence interrupted by intron 4 sequence. The open reading frame in this mRNA continues into intron 4 terminating 21 amino acids after the alpha 3 domain, thus excluding the transmembrane encoding region and yielding a protein with a highly charged carboxyl terminus. Transfection of the intron 4 containing cDNA, inserted into a retroviral expression vector, into LCL .221 followed by comparison of the class I protein to native soluble G by two dimensional isoelectric focusing/SDS-PAGE analysis, demonstrated this message encoded the soluble HLA-G protein. In addition, a similar intron containing message derived from the HLA-G2 mRNA was found, suggesting the existence of a soluble form of this alternative HLA-G protein. These findings are discussed in relation to other soluble class I molecules and with regard to potential functions of the soluble HLA-G Ag.
Regulation of the human IgE receptor (Fc epsilon RII/CD23) by EBV. Localization of an intron EBV-responsive enhancer and characterization of its cognate GC-box binding factors.
Authors: Lacy J , Roth G , Shieh B
Published: December 2022

EBV infection of human B lymphocytes induces expression of the low affinity IgE receptor, Fc epsilon RII/CD23. CD23 is constitutively expressed in EBV-immortalized B cells and may play an essential role in immortalization. We previously explored the regulation of CD23 by EBV, showing that induction results from transcriptional activation that is mediated, in part, by an EBV-responsive transcriptional regulatory element in the 5' region of CD23 (-229 to +305 relative to the type a promoter). We now report the localization of the regulatory element and characterization of its cognate DNA-binding proteins. Reporter gene assays in EBV-positive and -negative lines localized a functional EBV-responsive enhancer to a 37-bp fragment (+248 to +284) that contains a GC-rich sequence (GC box) within intron I of type a CD23. This fragment was shown by mobility shift assays to specifically bind nuclear protein(s) from EBV-positive lines, but not EBV-negative lines. Mutation of the GC box resulted in a loss of protein-binding activity, implicating involvement of a GC box-binding protein in the DNA/protein interaction. Supershift assays suggested that the ubiquitous GC box-binding transcription factor, Sp1, is not a part of the complex, and UV-crosslinking studies demonstrated that the DNA/protein complex contains at least two proteins that differ in size from other known GC box-binding proteins. Binding of these proteins to the enhancer element requires phosphorylation, because phosphatase treatment of nuclear extracts abolished formation of the DNA/protein complex. These studies reveal the presence of an EBV-responsive enhancer element in intron I of type a CD23 and implicate a GC box-binding transcription factor in the activation of CD23 by EBV.
One gene encodes the heavy chains for three different forms of IgY in the duck.
Authors: Magor K E , Higgins D A , Middleton D L , Warr G W
Published: December 2022

IgY, the major Ab of the duck (Anas platyrhynchos), exists in two secreted forms and a transmembrane (TM) form. To investigate the genetic relationships of the multiple IgY we cloned the gene encoding the IgY (upsilon) heavy chains. The heavy chain of the smaller (5.7S) IgY, which lacks the third and fourth constant domains, results from the use of a unique terminal exon found in the intron between the second and third C region exons. Alternate pre-mRNA processing pathways also produce a full-length upsilon-chain and a TM form, each having four C region domains. Although the number of secretory exons and the inferred positions of intramolecular disulfide bonds indicate structural similarity between IgY and IgE, the TM exons of duck IgY share high sequence identity and a similar pattern of RNA processing with those of IgG. These results suggest that IgG and IgE may have diverged from an ancestral molecule resembling IgY.
Candidacidal mechanisms in the human neonate. Impaired IFN-gamma activation of macrophages in newborn infants.
Authors: Maródi L , Káposzta R , Campbell D E , Polin R A , Csongor J , Johnston R B
Published: December 2022

We studied the interaction between Candida albicans and mononuclear phagocytes derived from cord blood. In the presence of normal serum, the extent of phagocytosis and killing of candida by monocyte-derived macrophages was equivalent for newborns and adults. In the absence of serum both phagocytosis and killing by macrophages were reduced by half, but cord and adult cells were still equivalent. Mannosylated BSA and mannan inhibited ingestion of unopsonized candida by macrophages, suggesting a role for the mannose receptor. Exposure of cord and adult macrophages to IFN-gamma (10-500 U/ml) gave quantitatively different results in Candida killing, as well as in release of superoxide anion (O2-). Maximal increase in these functions with adult macrophages was achieved with 100 U/ml IFN-gamma. No enhancement with cord macrophages could be detected after treatment with 100 U/ml, and at 500 U/ml there was still significantly lower killing and O2- release compared with adult cells. Defective up-regulation of O2- release was also present in cord monocytes exposed to IFN-gamma on day 0. Studies of the surface expression of IFN-gamma receptors using a "nonblocking" mAb against the IFN-gamma receptor revealed a comparable number of receptors on cord and adult monocytes. When blocking Abs were used, however, there was a three times higher number of positive cells in cord monocytes. Specific binding of 125I-IFN-gamma to cord monocytes and macrophages was also higher compared with adult cells. These data suggest that neonatal macrophages have a normal capacity to ingest and kill both opsonized and unopsonized Candida but cannot be fully activated by IFN-gamma, a finding that could not be attributed to lower expression of IFN-gamma receptors on the neonatal cells.
Transfection of vitamin D receptor cDNA into the monoblastoid cell line U937. The role of vitamin D3 in homotypic macrophage adhesion.
Authors: Hewison M , Dabrowski M , Faulkner L , Hughson E , Vadher S , Rut A , Brickell P M , O'Riordan J L , Katz D R
Published: December 2022

A 2-kB cDNA for the vitamin D receptor (VDR) was cloned in sense orientation into the plasmid pMEP4 (containing a cadmium-inducible metallothionein II promoter and a hygromycin-resistance selection gene) and transfected into monoblastoid U937 cells. The resultant cell line, DH39, expressed two species of VDR mRNA: 4.6-kb wild-type mRNA (present in native U937 cells or cells transfected with pMEP4 alone) and 2-kb transfected mRNA, which increased with cadmium treatment. Binding studies (using the active vitamin D metabolite, 1,25-dihydroxycholecalciferol (1,25-DHCC)) showed that DH39 cells contained five times more VDR per cell than controls, and ten times more after cadmium treatment. DH39 were sensitive to 1,25-DHCC: adding cadmium with 100 nM 1,25-DHCC for 72 h completely inhibited proliferation and induced concomitant differentiation. Unlike control cells, differentiation of DH39 by 1,25-DHCC led to homotypic cell-cell adhesion and formation of macrophage clusters. FACS analysis showed that 1,25-DHCC increased the number of cells expressing CD11b in both DH39 and controls, and the number of cells expressing CD11c in DH39. There was a quantitative increase in mean fluorescence intensity of expression of CD11a and CD18 in DH39. Northern blotting showed increased CD11a and CD18 mRNA in DH39. Ab inhibition of 1,25-DHCC-induced homotypic adhesion showed that CD11a/18 mediated the cell-cell clustering. CD50 expression was decreased on DH39, but the CD11a/18 ligand implicated was CD54. DH39 provides a model system not only for investigating the VDR role in 1,25-DHCC anti-proliferative effects, but also for regulation of homotypic macrophage adhesion mechanisms that are important in disease pathogenesis.
Lipopolysaccharide induces activation of the Raf-1/MAP kinase pathway. A putative role for Raf-1 in the induction of the IL-1 beta and the TNF-alpha genes.
Authors: Reimann T , Büscher D , Hipskind R A , Krautwald S , Lohmann-Matthes M L , Baccarini M
Published: December 2022

Bacterial LPS is a potent macrophage activator. The early steps in LPS signal transduction involve the tyrosine phosphorylation and activation of a number of kinases of the src family, and inhibition of this pathway causes a severe impairment in the production of the cytokines TNF-alpha and IL-1 beta. We find that LPS-induced macrophages activation also involves the Raf-1 kinase, a key component in mitogenic signal transduction. Treatment of BAC-1.2F5 macrophages with LPS causes phosphorylation and activation of Raf-1. This is paralleled by the stimulation of MEK-1 and MAP-kinase activity and by the phosphorylation of the transcription factor Elk-1, a nuclear target of MAP-kinase. Activation of the Raf/MAP-kinase pathway was inhibited upon pretreatment of the cells with genistein, a tyrosine kinase inhibitor. Raf-1 must thus lie downstream of tyrosine kinase in LPS signal transduction. However, Raf-1 is not a direct substrate of a LPS-induced tyrosine kinase, because Raf-1 immunoisolated from LPS-induced cells contains only phosphoserine. This resembles the situation after CSF-1-stimulation of macrophages, in which Raf-1 clearly transduces a signal generated by the CSF-1 receptor kinase, but is phosphorylated exclusively in serine. Phosphopeptide maps of Raf-1 immunoprecipitated from LPS- or CSF-1-treated cells are indistinguishable, suggesting that these agents activate Raf-1 by similar mechanisms. Finally, v-raf-infected BAC-1.2F5 macrophages were found to constitutively express low levels of IL-1 beta and TNF-alpha. These data argue that Raf-1 functions downstream of tyrosine kinases in LPS-mediated macrophage activation and cytokine production.
Immunosuppression to cardiac allografts and soluble antigens by anti-vascular cellular adhesion molecule-1 and anti-very late antigen-4 monoclonal antibodies.
Authors: Isobe M , Suzuki J , Yagita H , Okumura K , Yamazaki S , Nagai R , Yazaki Y , Sekiguchi M
Published: December 2022

Effective activation of T cells in immune responses depends on two signals, one from the CD3/TCR complex and another from accessory cell surface proteins. Very late Ag-4 (VLA-4) can exert a costimulatory function upon binding with vascular cellular adhesion molecule-1 (VCAM-1). We investigated the effects of mAbs against VCAM-1 and VLA-4 on cardiac allograft survival and humoral response to soluble Ags. BALB/c hearts were transplanted into C3H/He recipients. mAbs (100 micrograms/day) were administered i.p. for the first 6 days. Graft survival in mice treated with M/K-2 (anti-VCAM-1; median survival: 20 days, n = 6) and those treated with PS/2 (anti-VLA-4; 30 days, n = 6) was greater than that in control mice (8 days, n = 7). Eight of 18 mice treated with both M/K-2 and PS/2 accepted the grafts over 65 days, and five of them accepted the grafts over 100 days. Allografts treated with the two mAbs showed scattered infiltration of leukocytes without evidence of active rejection at 65 days. Mice with long-surviving cardiac grafts were challenged with skin grafts from donor and third-party (C57BL/6) strains. Survival of the donor-type skin was significantly greater than that of the third-party skin. One mouse specifically accepted the donor-type skin indefinitely (> 150 days). FACS analysis of splenocytes at 55 days after transplantation showed complete recovery of VLA-4 expression from a down-regulation observed at day 7. In addition, mice immunized with heat-aggregated human gamma-globulin did not produce specific Ab, even after boost immunization, if PS/2 was administered at the time of the first immunization. This unresponsiveness to xenogeneic protein lasted for more than 50 days. These results indicate that in vivo administration of anti-VCAM-1 and anti-VLA-4 mAbs induces specific immunosuppression to cardiac allografts and soluble Ags.
Defective T cells from gld mice play a pivotal role in development of Thy-1.2+B220+ cells and autoimmunity.
Authors: Yasutomo K , Maeda K , Nagata S , Nagasawa H , Okada K , Good R A , Kuroda Y , Himeno K
Published: December 2022

The gld mouse represents a fascinating animal model of autoimmune disease, which is characterized by massive development of Thy-1.2+B220+ CD4-CD8- cells. These cells thus have double positive markers for T and B cells, but are double negative for CD4 and CD8 markers and are thus designated DN cells in the present context. An additional important feature in gld mice is a defect in expression of Fas ligand. To investigate the regulatory role of bone marrow-derived cells for the development of these DN cells and of gld autoimmunity, we constructed chimeric mice transplanted with fetal liver cells or fetal thymus from gld mice into nonirradiated severe combined immunodeficient (SCID) mice. These chimeric mice regenerated, developed both these DN cells and the gld autoimmune syndrome and also generalized lymphoproliferative disorders. However, when fetal liver cells from both gld and non-gld mice (C57BL/10 Thy-1.1 mice) were co-transplanted into SCID mice, the development of DN cells was apparently inhibited. Further, this inhibition was also seen in SCID mice that had been grafted with both gld and non-gld fetal thymus revealing the pivotal role played by T cells in development of DN cells. When B cells purified from non-gld (C3H+/+) mice were transplanted into SCID mice grafted with gld fetal thymus, the development of DN cells was not inhibited. Taken together, these findings indicate that T cells from non-gld mice inhibit the expression of gld features, e.g., lymphoproliferation, immune-based nephritic disease, and autoantibody production. These findings also suggest that the Fas ligand is selectively expressed on T cells.
Expression and function of the MAdCAM-1 receptor, integrin alpha 4 beta 7, on human leukocytes.
Authors: Erle D J , Briskin M J , Butcher E C , Garcia-Pardo A , Lazarovits A I , Tidswell M
Published: December 2022

Recirculation of mouse lymphocytes to the gut involves binding of the lymphocyte integrin alpha 4 beta 7 to the mucosal vascular addressin, MAdCAM-1. In humans, indirect evidence suggests that CD4+ T cells that express high levels of alpha 4 beta 7 migrate selectively to the gut. We now report that human adult blood CD8+ T cells and B cells, like CD4+ T cells, have heterogeneous expression of alpha 4 beta 7. In contrast, NK cells, eosinophils, and newborn blood T and B cells have relatively homogeneous expression of alpha 4 beta 7. CD4+ and CD8+ T cell expression of alpha 4 beta 7 was related to age, CD45RA expression, and integrin beta 1 (CD29) expression, suggesting that alpha 4 beta 7 expression changes after primary activation of CD4+ and CD8+ T cells in vivo. To directly determine whether human alpha 4 beta 7 mediates adhesion to MAdCAM-1, we performed in vitro adhesion assays with two alpha 4 beta 7+ human lymphoma cell lines. The results indicate that human alpha 4 beta 7 is a receptor for MAdCAM-1, whereas alpha 4 beta 1 is not. Adhesion of HUT 78 cells to MAdCAM-1 required Mn2+, whereas adhesion of RPMI 8866 cells did not, suggesting that alpha 4 beta 7 may have at least two distinct functional states. The ability of lymphocytes to bind to MAdCAM-1 and recirculate to mucosal organs is likely to be influenced both by the level of alpha 4 beta 7 expression and by the functional state of the alpha 4 beta 7 molecule.
Induction of T cell differentiation and lymphomagenesis in the thymus of mice with severe combined immune deficiency (SCID).
Authors: Murphy W J , Durum S K , Anver M R , Ferris D K , McVicar D W , O'Shea J J , Ruscetti S K , Smith M R , Young H A , Longo D L
Published: December 2022

Severe combined immune deficiency (SCID) mice have a defect in their recombinase system and cannot productively rearrange their immune receptor genes. Thus, SCID thymocytes are arrested at the immature "triple negative" phase, not expressing CD3, CD4, or CD8 surface markers. Whole body irradiation of SCID mice induced maturation of their thymocytes to the CD4+/CD8+ double positive, CD3+low stage of differentiation, and resulted in the generation of a thymic cortical region on histologic examination. No mature single positive T cells were detected in the thymus or the periphery. VDJ rearrangements of TCR-beta with restricted clonality were observed in the double positive cells from a given individual. The CD3 complex was expressed on some of these cells, but the cells failed to mobilize intracellular calcium after cross-linking with CD3 Abs. The double positive cells appeared several weeks after irradiation, persisted for many months in the thymus, and by 6 mo generally developed into metastatic lymphoma. Retroviral activation was undetectable in both the preneoplastic and transformed thymocytes. Thus, it appears that the earliest steps in T cell development can be induced in SCID mice by inducing DNA breaks with radiation. This system represents a model of early thymic development, preneoplasia, and neoplasia.
Inhibition of T cell activation by a monoclonal antibody reactive against the alpha 3 domain of human MHC class I molecules.
Authors: Smith D M , Bluestone J A , Jeyarajah D R , Newberg M H , Engelhard V H , Thistlethwaite J R , Woodle E S
Published: December 2022

A long term goal of investigators working in our laboratory has been to develop new mAbs for use as immunosuppressive agents. As a means toward achieving this goal, several new mAbs (hybridomas) have been developed by screening fusions for supernatants that possess T cell-inhibitory properties. Of these new mAbs, one mAb, designated 5H7, has been shown to possess both a unique combination of specificity for a monomorphic determinant of the alpha 3 domain of human class I MHC heavy chains and highly potent T cell inhibitory properties. mAb 5H7 profoundly inhibited T cell proliferation in response to anti-CD3 mAb in primary or secondary allogenic MLR or in primary human anti-mouse xenogenic MLR. mAb 5H7 inhibited expression of an early T cell activation marker, Leu23 (CD69); expression of IL-2Rs; and IL-2 production by both CD4+ and CD8+ T cells. mAb 5H7 inhibited IL-2 release by the Jurkat (E6-1) human T cell leukemia line in response to immobilized anti-CD3 mAb, thus, providing further evidence that 5H7 can inhibit activation directly at the level of the T cell. 5H7 profoundly blocked CD3-dependent (anti-CD3, anti-CD3 plus PMA, or anti-CD3 plus anti-CD28) pathways, but only partially blocked a CD3-independent (anti-CD28 plus PMA) pathway of T cell activation. In conclusion, class I MHC molecules that are expressed on the T cell may regulate early TCR/CD3-dependent signaling events. In addition, 5H7 mAb may provide a reagent for suppression of cellular immunity in vivo.
Structure-immunogenicity relationship of melittin, its transposed analogues, and D-melittin.
Authors: King T P , Wade D , Coscia M R , Mitchell S , Kochoumian L , Merrifield B
Published: December 2022

Melittin, a 26-residue bee venom peptide, is known to induce murine Abs specific for its hydrophilic C-terminus of residues 20-26 and T cell responses specific for its hydrophobic mid-region of residue 11-19. Synthetic melittin analogues with transposed sequences of Ac(21-26) (1-20) and Ac(26-21) (1-20) are found to induce murine Abs specific for the transposed peptide segment and to induce T cell responses that are cross-reactive with melittin. Compared with melittin, the transposed melittin analogues are weaker immunogens and have lower hemolytic activities, lower helical contents, and a lower degree of association in micelles. A melittin analogue with a lactoside group at its C-terminus was found to induce lactoside-specific murine Abs. Present studies show that another analogue with a lactoside group at its N-terminus induces only Abs specific for the C-terminal region of melittin, and no lactoside-specific Abs are detected. These immunochemical observations suggest that the immunogenicity of melittin or its analogues is a consequence of its binding to cell membranes with subsequent oligomer formation in lipid bilayers. Apparently melittin or its analogues bind to cell membrane in an asymmetric manner with the exposed and the buried segments functioning as B and T cell epitopes, respectively. D-melittin is non-immunogenic in mice, although D-melittin has the same hemolytic activity as melittin. This finding may be correlated with the known resistance of D-melittin to proteolysis and hence to processing for Ag presentation to T lymphocytes.
Kappa light chain rearrangement in mouse fetal liver.
Authors: Ramsden D A , Paige C J , Wu G E
Published: December 2022

Ig variable domains are generated by the recombination of V, D, and J segments (V(D)J rearrangement). V(D)J rearrangement is capable of generating a vast repertoire of different variable domains. In this report, we quantify and characterize the repertoire of kappa rearrangements in fetal liver ontogeny. VJ kappa rearrangements are first observable at approximately day 14 of gestation. Characterization of these rearrangements indicates that only 33% are in a productive reading frame, which supports the argument that they have been generated recently and have not as yet undergone significant Ag-driven selection. Further analysis of rearrangements from a pool of 133 cloned VJ kappa junctions (from both day 14 and day 16 of gestation) indicates that the repertoire is fairly diverse with respect to the V kappa gene families used, as well as the number of members from each gene family. The frequency of V kappa 4 family use in rearrangements to J kappa 5, however, was approximately twice that of the frequency of V kappa 4 family use in rearrangement to other J kappa s. The fine structure of fetal VJ kappa junctions was also diverse, which indicates that precise deletion of sequence identities shared between rearranging V kappa J kappa pairs does not significantly reduce junctional diversity, as has been observed in DJH rearrangements. Lastly, a number of junctions contained P nucleotides, in contrast to the repertoire of expressed VJ kappa junctions.
Structure-function studies of anti-3-fucosyllactosamine (Le(x)) and galactosylgloboside antibodies.
Authors: Snyder J G , Dinh Q , Morrison S L , Padlan E A , Mitchell M , Yu-Lee L Y , Marcus D M
Published: December 2022

We are studying murine mAbs against two carbohydrate epitopes, 3-fucosyllactosamine (Le(x), CD15) and galactosylgloboside. The VH domains of both panels of Ab are encoded by VH441, a member of the X24 family of Ig genes. To evaluate the contribution of the heavy chain CDR3 to the affinity of the anti-3-fucosyllactosamine Ab, CDR3-H of PMN6, a low affinity Ab, was replaced by the CDR3 of PM81, a higher affinity Ab. The affinity of the chimeric 6/81 Ab was increased when the heavy chain was paired with the PM81 light chain, but not when paired with another light chain (M5), which differs from PM81 light chain by three amino acids. To evaluate the contribution of somatic mutations to the binding of GalGb4, the 3A9 VH sequence, which contains three amino acid substitutions, was replaced by a germ-line sequence encoded by either VH441 or VHX24. The chimeric Ab, 441/3A9 and X24/3A9, bound Ag as well as the wild-type 3A9 Ab. Computer models of the Fv fragments of PM81 and 3A9 were compared with the crystal structure of the Fv fragment of J539, a galactan-binding myeloma protein that is encoded by the same VH and VK genes as 3A9. The surfaces of 3A9 and J539 have shallow pockets that are potential Ag-binding sites. Replacement of CDR3-H Tyr99, which is a prominent component of the pocket, by Ala abolished the binding of Ag. In contrast, the Fv surface of PM81 contains a large cleft rather than a pocket. These models indicate how the same VH gene segment can be used to encode Abs that exhibit different specificities.
Follicular dendritic cells retain HIV-1 particles on their plasma membrane, but are not productively infected in asymptomatic patients with follicular hyperplasia.
Authors: Schmitz J , van Lunzen J , Tenner-Racz K , Grossschupff G , Racz P , Schmitz H , Dietrich M , Hufert F T
Published: December 2022

During HIV-1 infection, extensive alterations can be observed in the germinal centers (GCs) of lymphatic tissue. The initial hyperplasia of GCs is followed by disintegration and involution of these structures. The process is accompanied by progressive destruction of the network of follicular dendritic cells (FDCs). We investigated whether or not the destruction of GCs is caused by HIV-1 infection of FDCs in vivo. FDCs were purified by FACS or magnetic cell sorter (MACS) from enlarged lymph nodes (LNs) of four asymptomatic HIV-1-infected patients with persistent generalized lymphadenopathy. In situ hybridization demonstrated positive signals for HIV-1 RNA on the cell membrane of FDCs. Virus particles attached to the dendritic network could be demonstrated by electron microscopy, but budding was not seen. Furthermore, proviral DNA was detected by nested PCR (NPCR) in limiting dilution assays of FACS-purified FDCs (up to 1.0 x 10(4) cells) in only one cell fraction. In comparison, proviral DNA could be found in 5.0 x 10(3) to 2.5 x 10(5) FACS-purified Th cells obtained from peripheral blood and in 7.5 x 10(5) to 2.5 x 10(4) Th cells from LN of the same patients. In conclusion, FDCs retain HIV-1 particles on their cell membrane during HIV-1 infection, but the progressive destruction of FDC in vivo is probably not caused by a productive infection of FDCs with HIV-1.
Dual stimulatory and inhibitory effect of NK cell stimulatory factor/IL-12 on human hematopoiesis.
Authors: Bellone G , Trinchieri G
Published: December 2022

NK cell stimulatory factor, or IL-12 (NKSF/IL-12), is a heterodimeric cytokine produced by monocyte-macrophages, B cells, and possibly other accessory cell types. Although the major biologic effects of NKSF/IL-12 have been demonstrated on mature T and NK cells, in which it induces cytokine secretion, increased cytotoxicity, and proliferation, recent evidence in the murine system has suggested that NKSF/IL-12 may play a role in the differentiation of early lymphohematopoietic progenitor cells and thymocytes. In this paper, we have analyzed the effect of human rNKSF/IL-12 on the formation of colonies by highly enriched hematopoietic progenitor cells from human peripheral blood and bone marrow. At concentrations between 1 and 10 ng/ml, NKSF/IL-12 synergizes with a combination of steel factor and IL-3 to induce formation of mixed, erythroid, and myeloid colonies. Therefore, human NKSF/IL-12, like murine NKSF/IL-12, seems to belong to a small group of early acting cytokines, including IL-6, granulocyte-CSF, leukemia-inhibitory factor, and IL-11, which are able to synergize with steel factor and IL-3 to induce proliferation and differentiation of very early hematopoietic progenitor cells. However, in the presence of enriched preparations of NK cells cultured together with the progenitor cells, NKSF/IL-12 inhibits formation of hematopoietic colonies supported by IL-3 and granulocyte-macrophage CSF, by inducing production of IFN-gamma and TNF-alpha, two cytokines with synergistic inhibitory effects on hematopoietic colony formation. Because cell types that are able to produce NKSF/IL-12 are present in normal bone marrow and NKSF/IL-12 production in vivo and can be stimulated during bacterial or parasitic infection, it is possible that the direct stimulatory effect of NKSF/IL-12 on hematopoietic progenitor cells and the indirect inhibitory effect mediated by secondary cytokine production by lymphoid cells may play a role in the regulation of physiologic hematopoiesis and in its alterations during infection.
Identification and characterization of rat gamma/delta T lymphocytes in peripheral lymphoid organs, small intestine, and skin with a monoclonal antibody to a constant determinant of the gamma/delta T cell receptor.
Authors: Kühnlein P , Park J H , Herrmann T , Elbe A , Hünig T
Published: December 2022

A mAb called V65 was raised to a CD3+, TCR-alpha/beta- rat/mouse T cell hybrid that selectively reacts with all CD3+, TCR-alpha/beta- rat lymphocytes. Both anti-CD3 and V65 precipitate a 48- to 50-kDa heterodimeric protein from digitonin-lysed surface-iodinated cells. V65+ but not V65- T cells and T cell hybridoma cells express TCR-gamma mRNA. Together, these results show that V65 detects a constant determinant of the rat TCR-gamma/delta. In the presence of either IL-2 or IL-4, V65 stimulates proliferation in peripheral rat gamma/delta T cells. Approximately 90% of gamma/delta T cells from peripheral lymphoid organs have the same cell surface phenotype as thymus-derived MHC class I-restricted alpha/beta T cells, i.e., they are CD4- but express the CD8 alpha/beta heterodimer together with CD2 and CD5. In contrast, gamma/delta T cells from the epithelium of the small intestine lack CD2, CD4, and CD5 and express CD8 alpha only. Finally, V65 directly identifies a dense network of dendritic cells in the epidermis as gamma/delta T cells. These dendritic epidermal T cells are absent from athymic rats, indicating that like their mouse counterparts, they are thymus dependent.
Stage-specific expression of activation antigens on NK cells at uterine implantation sites in mice.
Authors: Linnemeyer P A , Pollack S B
Published: December 2022

The differentiation of the cellular components of the uterine decidua, in particular the life history of NK cells, is poorly understood. With the use of two mAbs that recognize stage-specific activation Ags on NK cells, we investigated the development of NK cells known as granulated metrial gland (GMG) cells. Immunohistochemical analysis demonstrated that mAb 3C2, but not mAb 4H12, recognized numerous cells throughout the uterine decidua basalis during early gestation. Isolated (panned) 3C2+ decidual cells from day 7 of pregnancy co-expressed the NK1.1 Ag, displayed NK cytolytic activity, and proliferated in IL-2-containing media. A small percentage of those cells expressed the GMG-associated Ag 4H12. Immunohistochemical analysis of serial sections at midgestation demonstrated that most of the 3C2+ NK cells co-expressed 4H12 Ag. During the later part of pregnancy, however, 3C2 expression in the decidua was down-regulated, and the cells expressed high levels of 4H12 Ag. When 3C2+ NK cells were isolated from cell suspensions of decidua from 7-day pregnant mice, and cultured in IL-2-containing medium, the cells developed the large and granular morphology characteristics of GMG cells, and acquired 4H12 Ag. These results demonstrate that 4H12+ GMG cells differentiate from 3C2+, NK1.1+, cytolytic precursors that reside in the decidua during early gestation.
Invariant chain prevents the HLA-DR-restricted presentation of a cytosolic peptide.
Authors: Long E O , LaVaute T , Pinet V , Jaraquemada D
Published: December 2022

Three properties of the HLA-DR-associated invariant chain (Ii) may contribute to the distinction between the class I and class II Ag presentation pathways. First, Ii prevents peptide binding to alpha beta 1 Ii complexes. Second, Ii promotes assembly of class II alpha beta heterodimers and their transport out of the endoplasmic reticulum. Third, Ii provides a targeting signal for the transport of class II molecules to endocytic compartments. However, it is not known whether Ii can prevent class II-restricted T cell recognition of endogenous peptides transported into the endoplasmic reticulum. In addition, recent evidence has indicated that, in the absence of Ii, newly synthesized class II molecules cannot form stable complexes with peptides. In this study, transfected human fibroblast cells expressing HLA-DR1 alone or with an excess of Ii were tested for their ability to present a DR1-restricted epitope of the influenza virus matrix protein produced as a short cytosolic peptide by use of an episomal expression vector. Presentation to a DR1-restricted T cell clone was very efficient in cells expressing class II molecules without Ii, but not in cells expressing class II and Ii. The inhibition by Ii was specific for the endogenous cytosolic peptide, because the same epitope processed from exogenous influenza virus particles was presented only by cells expressing class II with Ii. Ii did not inhibit the HLA-A2-restricted presentation of another cytosolic peptide. Thus, T cells can detect a cytosolic peptide loaded onto class II alpha beta heterodimers, and Ii prevents such endogenous peptide presentation.
Helper T cell-independent proliferation of CD8+ cytotoxic T lymphocytes transduced with an IL-1 receptor retrovirus.
Authors: Nagoya S , Greenberg P D , Yee C , Weisser K E , Sugawara H , Widmer M B , Slack J , Dower S K , Lupton S D , Overell R W
Published: December 2022

Although the proliferation of CD8+ CTL typically requires cytokine support provided by helper T cells, a subset of naturally occurring CD8+ CTL are capable of proliferating independently of T cell help. Such helper-independent CTL have previously been shown to possess IL-1 receptors (IL-1R) and to proliferate in response to IL-1 through endogenous production of IL-2. In this study, we have transduced conventional helper-dependent CTL clones with a retroviral vector encoding the murine type I IL-1R. Transduced CTL selected in G418 expressed vector-derived transcripts encoding IL-1R and displayed approximately 1000 cell surface receptors with an IL-1 affinity typical for the type I IL-1R. In contrast to parental cells, transduced CTL proliferated in response to IL-1 in the presence of Ag, without a requirement for helper T cells, IL-2, or other cytokine support. Stimulation with both IL-1 and Ag was necessary for the proliferative response. No endogenous synthesis of IL-2 could be detected in the IL-1R transduced cells in response to IL-1 stimulation, in the presence or absence of Ag. The IL-1R-induced phenotype was demonstrated in two independent T cell clones, both of which retained Ag-specific cytolytic activity. No such conversion to a helper-independent phenotype was induced by a retroviral vector encoding only the neo gene. The behavior of the IL-1R-transduced CTL in proliferation assays thus resembled that of the naturally occurring helper-independent CTL.
Developmental regulation of the TCR zeta-chain. Differential expression and tyrosine phosphorylation of the TCR zeta-chain in resting immature and mature T lymphocytes.
Authors: Rozdzial M M , Kubo R T , Turner S L , Finkel T H
Published: December 2022

The zeta subunit of the TCR complex targets receptor surface expression, is phosphorylated on tyrosine residues upon T cell activation, and is implicated in signal transduction after TCR ligation. Here we show that, although intrathymic expression of the murine TCR-associated zeta-chain relative to the other chains of the Ag receptor complex remains unchanged during early thymocyte development, there is a doubling of TCR-associated zeta-chain surface expression upon thymocyte maturation. The ratio of tyrosine-phosphorylated relative to nonphosphorylated TCR-associated zeta-chain also changes with thymocyte development. This ratio was quantified after the purification and detergent extraction of receptor complexes from freshly isolated immature or mature thymocytes. Immunoprecipitation of the zeta-chain released from the complex allowed for the isolation of the tyrosine-phosphorylated and nonphosphorylated forms of TCR-associated zeta-chain. Intracellular free zeta-chain was characterized by immunoprecipitation after clearing the cell lysate of intact TCR complexes. Densitometric analysis of immunoblots indicated that surface phosphorylated zeta-chain is more abundant in immature relative to mature T cell populations, whereas the inverse is true of intracellular phosphorylated zeta-chain. Surface phosphorylated zeta-chain also migrated at a higher m.w. than its cytoplasmic counterpart, suggesting that it is more highly modified on some or all of its available tyrosines. These findings demonstrate that the stoichiometry and post-translational modification of the TCR complex are regulated, in vivo, and may determine the functional maturation of T cell signaling, selection, and activation.
Cells infected with Yersinia present an epitope to class I MHC-restricted CTL.
Authors: Starnbach M N , Bevan M J
Published: December 2022

Bacteria of the genus Yersinia cause disease in humans ranging from enteritis and lymphadenitis to plaque. These organisms have the ability to bind to host cells and, in a complex interaction, are able to deliver a specific subset of proteins into the host cell. These proteins alter host structures and functions, and in the case of macrophages, prevent phagocytosis of the organism. In Yersinia enterocolitica, one of these proteins, Yop51, is known to be a protein tyrosine phosphatase. By using a directed approach, we have raised murine CTL that recognize a peptide epitope from Yop51 in the context of the MHC class I molecule H-2 Db. This epitope, Yop51 249-257, seems to be presented both by cells that produce Yop51 endogenously and by epithelial cells infected with virulent Y. enterocolitica. In vivo, Y. enterocolitica is believed to remain mostly in an extracellular niche. Therefore, detection of this organism by cytotoxic T cells and, possibly, resistance to disease may depend on recognition of epitopes from a subset of virulence determinants delivered into the cytoplasm of host cells by surface-bound organisms.
Ig heavy chain protein controls B cell development by regulating germ-line transcription and retargeting V(D)J recombination.
Authors: Schlissel M S , Morrow T
Published: December 2022

The membrane-associated form of Ig heavy chain (mu) protein has been implicated as a critical regulator of B cell development. Mutant mice unable to produce the membrane form of mu protein fail to produce mature B cells. Splenic B cells from mice transgenic for a functionally rearranged Ig mu gene show a marked decrease in endogenous heavy chain gene rearrangement. We have analyzed the effects of a human mu transgene on the regulation of V(D)J recombination during B cell development in the mouse fetal liver. We found that mu transgenic and wild-type littermate mice begin kappa light chain gene rearrangement at the same time during development but the transgenic mice show a striking increase in the frequency of kappa gene rearrangement. The transgenic mice also show an increase in the levels of a germ-line kappa gene transcript known to be associated with kappa gene rearrangement. D-to-JH heavy chain gene rearrangement is unaffected throughout development by the presence of the mu transgene. Endogenous heavy chain gene V-to-DJH rearrangement occurs with similar frequency in transgenic and nontransgenic fetal livers during midgestation but is reduced in late gestation mu transgenic fetal liver. We show that this decrease in rearrangement is associated with a decrease in unrearranged VH gene transcription. Furthermore, we show that changes in the frequencies of rearranged kappa and mu genes are accompanied by changes in the frequencies of dsDNA breaks, a V(D)J recombination reaction intermediate associated with each of these loci. We propose that membrane-associated mu protein regulates B cell development by signaling a change in the pattern of unrearranged Ig gene transcriptional activity, thereby retargeting the V(D)J recombinase.