bluuebunny/crossref_metadata_2025 huggingface dataset:
TAT-mediated transcellular activation of HIV-1 long terminal repeat directed gene expression by HIV-1-infected peripheral blood mononuclear cells.
Authors: Thomas C A , Dobkin J , Weinberger O K
Published: December 2022

We have previously shown evidence of Tat-mediated transcellular activation of the HIV-1 long terminal repeat (LTR) in vitro by using a laboratory-adapted strain of HIV-1. To examine the biologic significance of this observation, we asked whether primary PBMCs from HIV-1-infected individuals will transactivate the HIV-1 LTR transcellularly in suitable indicator cells. In cultures of PBMCs isolated from HIV-1-infected patients at various clinical stages, with either HIV-1 LTR-transfected Jurkat T cells or nonfusigenic HIV-1 LTR-transfected murine fibroblasts, transcellular activation was readily detected. Transactivation of the LTR in cocultured cells with HIV-1-infected PBMCs is detectable 1 to 2 wk before the onset of significant virus production and at ratios as low as 1 infected cell to 10(6) surrounding cells. Addition of the Tat inhibitor RO5-3335 substantially decreases transcellular activation, even at low concentrations (0.01 microM) that do not affect virus levels. In contrast, addition of the antiretroviral agent zidovudine has no effect on transcellular activation. These data suggest that Tat-mediated transcellular activation of the HIV-1 LTR occurs independently of cellular infection, and provides a mechanism that can promote the spread of HIV-1 in susceptible cell populations.
Formation of high affinity IL-2 receptors is dependent on a nonligand binding region of the alpha subunit.
Authors: Gutgsell N S , Malek T R
Published: December 2022

The IL-2R is composed of three subunits, alpha, beta, and gamma, each of which individually contributes to binding IL-2 in the high affinity IL-2R. The molecular mechanism by which the high affinity IL-2R assembles has not been established. Previous studies have shown that human IL-2R subunits form high affinity hybrid IL-2R when mixed with appropriate mouse IL-2R subunits, but the efficiency of this process is not known. In this study, we produced stable cell lines that expressed hybrid human/mouse IL-2R, comprised of heterologous alpha- and beta-chains, to determine the contribution of individual receptor subunits in stabilizing the high affinity IL-2R. Quantitative ligand binding studies and FACS analysis were performed for EL4J cells that expressed hybrid mouse/human IL-2R, and these data were compared with those of cells that expressed homologous mouse IL-2R subunits. EL4J cells that expressed human beta/mouse alpha formed high affinity IL-2R, comparable with cells that express homologous mouse alpha and beta subunits. In contrast, EL4J cells that expressed homologous mouse IL-2R contained an approximately 10-fold higher level of high affinity IL-2R than did EL4J cells that expressed heterologous human alpha/mouse beta IL-2R. This difference was not accounted for by lower levels of human alpha-chains in these cell lines or lower expression of the mouse IL-2R beta or gamma subunits. These results suggest that amino acid sequences within the alpha subunit, distinct from the IL-2 binding site, are important in the assembly and stabilization of the high affinity IL-2R.
In vivo CD4+ lymph node T cells from lpr mice generate CD4-CD8-B220+TCR-beta low cells.
Authors: Laouar Y , Ezine S
Published: December 2022

Double-negative CD4-CD8-T cells (DNT) have been shown to be the major population of T cells responsible for the massive lymphadenopathy associated with the early onset of the lupus-like syndrome in mice bearing the lpr gene. Previously, we demonstrated that these cells do not proliferate in the peripheral lymphoid organs that they invade; furthermore, we showed that a wide range of CD4 Ag expression was observed on lymph node CD4+ T cells. In this study, we used an in vivo transfer system to analyze the progeny of CD4+ T cells from B6-lpr/lpr mice. Purified CD4+ T cells injected into B6 nude mice are able to generate DNT cells; furthermore, phenotypic and functional characterizations of the DNT cells generated in vivo show that they share the same properties as DNT cells from B6-lpr/lpr mice. We also show that, after in vitro bromodeoxyuridine incorporation, only CD4+ cells cycle. From these studies, we conclude that the lymphoproliferation occurs at the CD4+ stage and that down-regulation of this Ag probably is followed by arrest of the cell cycle.
CD7 is associated with CD3 and CD45 on human T cells.
Authors: Lazarovits A I , Osman N , Le Feuvre C E , Ley S C , Crumpton M J
Published: December 2022

The CD7 cluster of mAb identifies a 40-kDa glycopolypeptide that is present on a major subset of human T cells. CD7 Ag mediates an accessory pathway of T cell activation in that cross-linked CD7 mAb are mitogenic, and signals delivered via CD7 Ag stimulate integrin-mediated adhesion. We have found that the CD7 molecule is associated with a tyrosine kinase whose major substrate is CD45. In vitro phosphorylation of CD7, CD3, or CD45 immunoprecipitates prepared from lysates of human T cells showed a similar pattern of multiple phosphorylated polypeptides; in addition, these immunoprecipitates phosphorylated a tyrosine kinase-specific peptide. Surface-iodinated T cells were lysed and immunoprecipitated with CD7, CD3, and CD45 mAb. Bands characteristic of CD45 and CD3 were identified in CD7 immunoprecipitates. Confirmation of an association of CD7 with CD3 and CD45 was obtained from Western blotting and fluorescence resonance energy transfer experiments. Furthermore, we provide evidence using immunoprecipitation and Western blotting that CD7 exists as a homodimer. These data support the hypothesis that CD7 exists in an oligomeric complex with CD3/TCR, the protein tyrosine phosphatase CD45, and a tyrosine kinase, thereby providing a physical basis for the accessory role of the CD7 molecule in T cell activation.
Systemic cytokine profiles in BALB/c mice immunized with trivalent influenza vaccine containing MF59 oil emulsion and other advanced adjuvants.
Authors: Valensi J P , Carlson J R , Van Nest G A
Published: December 2022

We have studied serum cytokine profiles in BALB/c mice after immunization with influenza vaccine alone or combined with the following adjuvants: alum; MF59 emulsion; MF59 containing the muramyl peptide N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2- dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy)) ethylamide (MTP-PE); MF59 plus the lipid A analogue monophosphoryl lipid A; MF59 plus the Quil A saponin fraction LTC; or LTC alone. Pooled mouse sera were analyzed by ELISA at various times after immunization for IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IFN-gamma, and TNF-alpha. In naive mice, vaccine alone induced low levels of IL-3 and IL-5 only; vaccine plus alum induced a low IL-6 response as well. The MF59-based adjuvants significantly increased the IL-5 and IL-6 levels, whereas Quil A LTC induced strong IFN-gamma and measurable IL-2 responses, in addition to moderate IL-5 and IL-6. In previously infected mice, MF59 and MF59/MTP-PE were capable of generating IFN-gamma responses, as well as IL-5 and IL-6. All of the cytokine responses were rapid (peaking 3 to 12 h postimmunization) and short lived. In naive mice, the MF59 adjuvants induced serum cytokine profiles that are consistent with a primarily Th2-type response, whereas the Quil A LTC induced cytokines associated with both Th1 and Th2 responses. Ab analyses indicated that, although the adjuvants strongly affected the magnitude of the humoral response, there was no obvious correlation between the cytokine profile observed and the subclasses of Ab induced.
Human vascular smooth muscle cells poorly co-stimulate and actively inhibit allogeneic CD4+ T cell proliferation in vitro.
Authors: Murray A G , Libby P , Pober J S
Published: December 2022

We compared immunologic functions of human vascular smooth muscle cells (VSMC) with those of endothelial cells (EC) cultured from saphenous vein. Both cell types can express comparable levels of MHC class II molecules. However, class II-positive VSMCs, unlike ECs, do not stimulate resting CD4+ T cell proliferation. Limiting dilution analyses revealed IL-2-producing cells alloreactive to class II-positive ECs but not VSMCs. Class II molecules on VSMCs are functional, inducing CD25 expression on resting CD4+ T cells and stimulating proliferation of CD4+ T cells that have been pre-activated by ECs. VSMC expression of the co-stimulator molecules CD44, CD54, CD58, and CD59 is comparable to EC expression, and neither VCAM-1 nor B7 are expressed on either cell. However, VSMCs are less efficient than ECs at co-stimulating IL-2 production by PHA-stimulated PBL. VSMCs but not ECs cultured across a Transwell inhibit CD4+ T cell proliferation to allogeneic ECs and, to a lesser extent, IL-2 production in the same assay. Inhibition of proliferation cannot be transferred by VSMC-conditioned media, nor reversed by inhibitors of prostaglandin synthesis, TGF-beta 1, or nitric oxide synthesis. CD4+ T cells cocultured with class II-positive VSMCs proliferate to a subsequent challenge with ECs from the same donor as well as freshly isolated T cells. We conclude that VSMCs express functional MHC class II molecules and stimulate pre-activated T cells. However, VSMCs lack adequate costimulators to fully stimulate resting T cells, and VSMCs inhibit T cell proliferation.
Analysis of antigen receptor signaling in B cells from mice with a retrovirus-induced acquired immunodeficiency syndrome.
Authors: Selvey L A , Morse H C , June C H , Hodes R J
Published: December 2022

MAIDS is a retrovirus-induced immunodeficiency syndrome in mice that has similarities to human AIDS. Because the functional defects in B cells from retroviral immunodeficiency syndromes have not been characterized in detail, we examined early and late parameters of B cell responses to IgM cross-linking in B cells from MAIDS and normal mice. Splenic B cells from mice with MAIDS have defective in vitro proliferative responses to LPS and anti-IgM-mediated stimuli, as well as to PMA plus calcium ionophore, indicating a generalized defect in proliferative response potential independent of specific receptor-mediated signaling. When early signaling parameters were analyzed in response to IgM cross-linking, it was found that calcium flux in B cells from MAIDS mice was significantly reduced; this reduction was not accounted for by quantitative differences in cell-surface IgM expression and therefore indicates a defect in early signal transduction through the IgM receptor. The tyrosine phosphorylation response to IgM cross-linking was also markedly deficient; tyrosine phosphorylation of Ig-alpha, Ig-beta, and an undefined protein of 80 kDa was detected in MAIDS B cells after anti-IgM stimulation, at levels substantially less than those observed in normal B cells. Multiple other tyrosine phosphorylation events observed in normal B cells, including phosphorylation of GTPase-activating protein, P13-kinase, and syk kinase, were not detected in MAIDS B cells in response to IgM cross-linking. The defect in tyrosine phosphorylation seemed to correlate with reduced surface IgM levels on a subpopulation of MAIDS B cells. B cells from mice expressing the MAIDS retrovirus-induced immunodeficiency thus reflect defects in early signaling through the Ag-specific IgM receptor as well as a generalized defect in proliferative responsiveness.
Inhibition of neutrophil adhesion by adenosine and an adenosine kinase inhibitor. The role of selectins.
Authors: Firestein G S , Bullough D A , Erion M D , Jimenez R , Ramirez-Weinhouse M , Barankiewicz J , Smith C W , Gruber H E , Mullane K M
Published: December 2022

Adenosine and adenosine analogues exhibit anti-inflammatory effects in vitro and in vivo, but their usefulness is limited by profound cardiovascular side effects. Therefore, we synthesized inhibitors of an enzyme involved in adenosine metabolism, adenosine kinase (AK) (EC 2.7.1.20), to enhance endogenous adenosine concentrations at sites of inflammation. GP-1-515 (4-amino-1-(5-amino-5-deoxy-1-beta-D- ribofuranosyl)-3-bromo-pyrazolo[3,4-d]pyrimidine), a novel AK inhibitor, decreased adhesion of activated human neutrophils to cultured endothelial cell monolayers by increasing local adenosine levels. The mechanism of inhibition in this assay seemed to involve selectin blockade and was independent of the beta 2 integrins. GP-1-515 and 2-chloroadenosine (a nonmetabolizable adenosine analogue) had no effect on the surface expression or shedding of adhesion molecules. An agent that disrupts the cytoskeleton, cytochalasin B, mimicked the effect of adenosine on cell adhesion. Interactions between L-selectin and the neutrophil cytoskeleton might be altered by adenosine and could contribute to adenosine-mediated adhesion inhibition.
HIV-1-specific cytotoxic T lymphocyte responses in the first year of life.
Authors: Luzuriaga K , Holmes D , Hereema A , Wong J , Panicali D L , Sullivan J L
Published: December 2022

HIV-1-specific CTL responses were prospectively evaluated in infants born to HIV-1-seropositive women to assess the capability of the young infant to generate HIV-1-specific CTL and to examine the potential role of HIV-1-specific CTL in the pathogenesis of vertical infection. Our results indicate that some young infants, and even the fetus, seem to be capable of generating virus-specific CTL responses. The detection of HIV-1-specific CTL responses varied among infants, however, with respect to timing, HIV-1 gene product recognition, and the magnitude of detectable responses; HIV-1-specific CTL responses were uncommonly detected in the first few months of life. The less consistent detection of HIV-specific CTL in early infancy contrasts with reports of the detection of HIV-1-specific CTL soon after primary infection in adults. HIV-1-specific CTL were not detected in any uninfected infants born to HIV-1-seropositive women. This description of HIV-1-specific CTL in infants may have important implications for understanding the pathogenesis of vertical HIV-1 infection and for the development of a vaccine to interrupt vertical infection.
Regulation of cAMP-responsive enhancer binding proteins during cell cycle progression in T lymphocytes stimulated by IL-2.
Authors: Feuerstein N , Huang D , Hinrichs S H , Orten D J , Aiyar N , Prystowsky M B
Published: December 2022

IL-2 stimulates the proliferative response of various lymphoid cells. Previous studies showed an increase in intracellular levels of cAMP concomitant with an increase in phosphorylation of discrete proteins by protein kinase A at late G1 phase in mitogen-stimulated lymphocytes. Thus, experiments were undertaken to study nuclear proteins that bind to the cAMP-responsive enhancer (CRE) in cloned T lymphocytes stimulated with IL-2. With the use of a 32P-labeled CRE consensus sequence in a DNA binding gel mobility shift assay, we showed that IL-2 stimulation resulted in the induction of two major DNA-protein complexes at late G1/S during the cell cycle. This binding was competed in a dose-dependent manner by a nonlabeled CRE oligonucleotide but was not competed by a nonlabeled AP-1 oligonucleotide. Rapamycin, a potent immunosuppressant, which arrests IL-2-stimulated T lymphocytes at G1/S, inhibited the IL-2-induced CRE binding activities concomitantly with inhibition of DNA synthesis. By using specific Abs in a gel mobility shift assay, we identified two known CREB/ATF transcription factors in the IL-2-induced CRE complexes: the CRE binding factor (CREB), and ATF1. The induction of CREB binding by IL-2 was not associated with an increase in its abundance but was associated with a major increase in CREB phosphorylation that was particularly prominent at late G1/S. However, we found that G1/S progression induced by IL-2 was not associated with an increase in the intracellular levels of cAMP. These results suggest that 1) the transcription factors CREB and ATF1 and possibly other CRE binding proteins may have an important role in the modulation of specific gene expression at G1/S during cell cycle progression induced by IL-2. 2) The involvement of these CRE binding transcription factors in IL-2-stimulated cells is regulated via a mechanism that is not cAMP dependent.
Selective loss of NK cytotoxicity in antisense NK-TR1 rat LGL cell lines. Abrogation of antibody-independent tumor and virus-infected target cell killing.
Authors: Giardina S L , Anderson S K , Sayers T J , Chambers W H , Palumbo G A , Young H A , Ortaldo J R
Published: December 2022

We have shown that NK-TR1, a protein containing a cyclophilin-like domain, is associated with a receptor/triggering molecule on the surface of human large granular lymphocytes (1). In the present study, we have further defined the role of NK-TR1 in target cell recognition/killing by generating antisense NK-TR1 transfectants in the rat LGL cell line, RNK-16. Stable transfectants were identified by analyzing permeabilized cells with the anti-NK-TR1 mAb, 4F9. Transfectants with low levels of 4F9 staining showed drastically reduced levels of killing against three NK-susceptible target cell lines. Lytic activity against vaccinia virus-infected cell lines also was dramatically reduced. In contrast, transfected cells showing normal levels of NK-TR1 expression demonstrated normal killing of all target cells. The ability of all transfectants to form conjugates was identical to that observed with the wild-type RNK cell line. Lectin-dependent cytotoxicity, reverse ADCC via NKR-PI, and ADCC-mediated killing were comparable in both high or low NK-TR1 expressing clones, demonstrating that the lytic machinery was still intact. BLT-esterase activity, PF levels, and surface marker phenotype were not significantly affected. These results provide strong evidence that NK-TR1 is an essential element in a signaling pathway leading to MHC unrestricted killing of tumor and virus-infected cells.
IL-12 activates IFN-gamma production through the preferential activation of CD30+ T cells.
Authors: Alzona M , Jäck H M , Fisher R I , Ellis T M
Published: December 2022

CD30 is normally expressed by a subset (15 to 20%) of CD45RO+ T cells after activation by a variety of T cell stimuli and defines the principal IFN-gamma-producing T cells subset. Inasmuch as the production of IFN-gamma is regulated by IL-2 and IL-12, we have examined the effects of these cytokines on the proliferation, induction, and function of CD30+ and CD30- T cell subsets. Upon isolation, CD30+CD25+ T cells exhibit high levels of baseline proliferation compared with CD30-CD25+ T cells. Neutralizing Abs specific for IL-2, IL-4, IL-6, or IL-12 had no effect on basal levels of proliferation in CD30+CD25+ T cells, whereas anti-IL-2 inhibited the basal proliferative activity of CD30-CD25+ T cells. The addition of exogenous rIL-12 to purified CD30+CD25+ and CD30-CD25+ T cells subsets induced significantly higher maximal levels of proliferation in the CD30+ subset, whereas rIL-2 supported comparable levels of maximal proliferation in each subset. The addition of rIL-2 to anti-CD3-activated PBMC increased the relative numbers of CD30+ T cells by expansion of CD30+ T cells, as opposed to recruitment of CD30+ T cells from the CD30- population. Furthermore, the development of the CD30+ T cell subset was inhibited by either anti-IL-2, anti-IL-12, or rIL-10. Inhibition by anti-IL-2 and rIL-10 was overcome by the addition of rIL-12, but not IL-2, to the cultures. Finally, rIL-12 increased IFN-gamma production by CD30+ T cells, yet had little effect on IFN-gamma production by CD30- T cells. Thus, CD30+ T cells are preferentially regulated by IL-12, and the effects of IL-12 on T cell IFN-gamma production are mediated largely through its effects on the CD30+ subset.
Differences in intraepithelial lymphocyte T cell subsets isolated from murine small versus large intestine.
Authors: Beagley K W , Fujihashi K , Lagoo A S , Lagoo-Deenadaylan S , Black C A , Murray A M , Sharmanov A T , Yamamoto M , McGhee J R , Elson C O
Published: December 2022

Intraepithelial lymphocytes (IELs) have been extensively studied in the murine small intestine. However, to date no studies have assessed IEL in the large intestine, despite the marked differences in function and lumenal environment. In the present study, we isolated IEL from both small and large intestine of three mouse strains (BALB/c, C3H/HeN, C57BL/6) and determined the frequency of CD2, CD4, and CD8 expression on CD3+ IEL, as well as the frequency of alpha beta and gamma delta TCR usage and V beta distribution. Higher numbers of IEL/unit length were always isolated from the small intestine (20-30 x 10(6)/5 mice) compared with large intestine (1.1-2.5 x 10(6)/5 mice). Interestingly, IEL from the large intestine of all strains were predominantly alpha beta TCR+ whereas gamma delta TCR+ IELs predominated in small intestine. Large intestinal IELs were mainly CD4+, in both BALB/c and C3H/HeN mouse strains. IELs from large intestine of C57BL/6 mice were mainly CD8+; however, the CD4+ subset was fourfold higher when compared with small intestine IEL. Potential functional differences between IEL subsets was assessed by determining the relative levels of mRNA for IL-1, 2, 4, 5, 10, IFN-gamma, TGF-beta, and TNF-gamma. Similar patterns of IL-1, IFN-gamma and TNF-alpha were seen while more IL-2, IL-4, IL-5, and IL-10 mRNA was noted in large intestinal IEL. Stimulation of C3H/HeJ IEL with anti-CD3 also resulted in higher levels of IL-3/GM-CSF, IL-4, and IL-6 by IEL from large intestine. These results show that marked differences occur among the T cell subsets present in IELs from mouse small and large intestine.
Deletion of a C-terminal sequence of the class II-associated invariant chain abrogates invariant chains oligomer formation and class II antigen presentation.
Authors: Bertolino P , Staschewski M , Trescol-Biémont M C , Freisewinkel I M , Schenck K , Chrétien I , Forquet F , Gerlier D , Rabourdin-Combe C , Koch N
Published: December 2022

The MHC class II-associated invariant chain (Ii) is involved in Ag processing and presentation. Physical association of MHC class II molecules with Ii and an effect of Ii on peptide loading to class II have been demonstrated, but to date these functions have not been related to a particular region of Ii. We investigated luminal deletion mutants of Ii and their role in Ag processing and presentation. IAk-expressing L cells were transfected with deletion mutants of the Ii gene and assayed for their ability to present hen egg lysozyme to three different T cell hybridomas. It is shown that the sequence aa 131-191 of Ii is important for the presentation of native hen egg lysozyme. In addition, this C terminal region is shown to be responsible for Ii oligomer formation. It is therefore conceivable that oligomer formation of Ii is a prerequisite for class II-restricted Ag processing and presentation.
Antigen-driven induction of CD11c on intestinal intraepithelial lymphocytes and CD8+ T cells in vivo.
Authors: Huleatt J W , Lefrançois L
Published: December 2022

Intraepithelial lymphocytes (IEL) of the intestinal epithelium represent a phenotypically and functionally distinct subpopulation of peripheral T cells. In this study, we report the production of a mAb, designated HL3, which exhibits reactivity with a subset of IEL. In differential screening assays HL3 reacted with 30 to 50% of IEL, but not with T cells of the thymus, spleen, or lymph nodes. Biochemical characterization revealed that the HL3 mAb recognized p150,95 (CD11c/CD18; CR4), a member of the beta 2-integrin family. Fluorescence flow cytometric analyses showed that p150,95 was expressed by TCR-alpha beta or TCR-gamma delta CD4-8+ IEL but not by CD4+8- IEL. Induction of graft-vs-host (GVH) disease resulted in up-regulation of p150,95 expression on donor-derived CD8+ T cells in the intestinal epithelium, as well as in the spleen and lymph nodes. GVH also induced MAC-1 (CD11b) expression on a subset of CD8+ lymph node T cells, but MAC-1 was not up-regulated on CD8+ IEL in this situation. In contrast, activation of identical T cell responders in vitro resulted in weak induction of p150,95 and MAC-1 expression. This result suggested that activation alone was insufficient for p150,95 up-regulation and that additional factors available in vivo were essential in this process. In the intestine, induction of p150,95 required the presence of intestinal flora as IEL from germfree mice lacked p150,95. Interestingly, gamma delta IEL expressing a non-IEL type transgenic TCR were also p150,95-, but exposure to Ag in vivo, but not in vitro, resulted in p150,95 induction. This result indicated that the constitutive expression of p150,95 on IEL is likely due to Ag stimulation via the TCR and not a bystander phenomenon. Overall, the results demonstrated p150,95 to be a hallmark of T cell activation in vivo and an indicator of ongoing antigen-specific T cell activation in the intestinal epithelium.
Efficient maturation of alpha beta lineage thymocytes to the CD4+CD8+ stage in the absence of TCR-beta rearrangement.
Authors: Kersh G J , Hooshmand F F , Hedrick S M
Published: December 2022

We have produced transgenic mice that express rearranged T cell Ag receptor gamma and delta transgenes in the alpha beta lineage of thymocytes. Thymi in these mice contain normal numbers of CD4+CD8+ cells that express low levels of the TCR-gamma delta. Analysis of the delta locus in these thymi indicates that these cells are in the alpha beta lineage even though they express the TCR-gamma delta. This shows that expression of the TCR-gamma delta in early thymocytes can lead to all of the consequences that are normally mediated by the beta-chain. These consequences include maturation to the CD4+CD8+ stage, entry into the cell cycle, and cessation of beta rearrangement. Therefore, the data support a model in which formation of a functional CD3 complex on immature CD4-CD8- thymocytes leads to further development in the absence of extracellular ligand recognition. The data also show that the gamma delta vs alpha beta lineage decision is made in a manner that is independent of gamma and beta gene expression.
MHC class II compartments and the kinetics of antigen presentation in activated mouse spleen dendritic cells.
Authors: Kleijmeer M J , Ossevoort M A , van Veen C J , van Hellemond J J , Neefjes J J , Kast W M , Melief C J , Geuze H J
Published: December 2022

MHC class II (MHC-II) molecules bind fragments of exogenous Ags in an intracellular endocytotic compartment. In view of divergent data on the MHC-II distribution in different cell lines, it was of interest to localize MHC-II molecules in a natural and the most potent APC type, the dendritic cell (DC). By using immunogold labeling of ultrathin cryosections of cultured mouse spleen DC, we found that MHC-II molecules were present abundantly at the plasma membrane and in intracellular compartments containing internal membrane vesicles and/or membrane sheets. The majority of these compartments was situated late in the endocytotic route, as demonstrated by the late appearance (after a lag of 30 min) of internalized exogenous tracer. These compartments contained the lysosomal enzymes cathepsin D and beta-hexosaminidase, but lacked the late endosomal marker cation-dependent mannose-6-phosphate receptor. We conclude that most of the intracellular MHC-II molecules in cultured spleen DC reside in a compartment with (pre)lysosomal characteristics, resembling the so-called MHC-II-enriched compartments (MIIC), originally described in B cells. We also investigated whether the presence of MHC-II molecules in endocytotic compartments was related to the kinetics of Ag processing and presentation by these cells. Pulse-chase endocytosis experiments with hen egg lysozyme (HEL) as a model Ag showed that activated spleen DC were able to efficiently process and present this Ag to an HEL-specific T hybridoma cell line. However, presentation started only after a lag of 2 h and was maximal after 6 h. The difference in time between the arrival of Ag in proteolytic endocytotic compartments, in particular MIIC, and effective Ag presentation is discussed in the context of DC maturation.
Influence of TGF-beta on murine thymocyte development in fetal thymus organ culture.
Authors: Plum J , De Smedt M , Leclercq G , Vandekerckhove B
Published: December 2022

TGF-beta is a multifunctional growth regulator that can either inhibit or stimulate the growth and differentiation of lymphocytes. For several cell types the effect of TGF-beta was found to correlate with the differentiation stage of the cells. We have studied the influence of TGF-beta on the differentiation of murine thymocytes by evaluating the effect of TGF-beta on the generation of thymocyte subpopulations in fetal thymus organ culture. TGF-beta inhibited the growth and differentiation of CD4-CD8- double-negative thymocytes. In the CD4-CD8- double-negative cell population, most cells remained CD44+CD25-, with CD44+CD25+ and CD44-CD25- subpopulations dramatically decreased in cell numbers. The accumulation of cells with a phenotype characteristic of cells in early stage of differentiation suggests a block at very early transition steps. These observations were confirmed in experiments with precursor cells from fetal liver transferred to 2-deoxyguanosine-treated alymphoid thymic lobes, inasmuch as addition of TGF-beta caused a complete inhibition of T cell development. Differentiation into CD4+CD8+ double-positive thymocytes and CD4+ single-positive thymocytes was impaired because these cell numbers were greatly reduced. In contrast, the CD8+ single-positive subpopulation retained normal cell numbers. This CD8+ population had characteristics of a mature subset as the cells expressed CD8 beta and high levels of TCR-alpha beta and CD3. This TCR-alpha beta + cell population was not actively dividing, suggesting that these cells arise de novo by differentiation.
Induction and reversal of long-lived specific unresponsiveness to a T-dependent antigen following CTLA4Ig treatment.
Authors: Wallace P M , Rodgers J N , Leytze G M , Johnson J S , Linsley P S
Published: December 2022

We have examined the ability of CTLA4Ig to induce long-lasting Ag-specific tolerance to a T-dependent Ag. Treatment of mice with murine CTLA4Ig following immunization with SRBC induced immune unresponsiveness to SRBC that was only reversible upon repeated Ag exposure. This unresponsiveness was Ag specific, lasted > 90 days, was observed in thymectomized mice, and was maintained even during challenge with another strong T-dependent Ag. A third challenge with SRBC, however, led to an Ab response. Splenic T cells and B cells from unresponsive mice were functional upon transfer to irradiated hosts, indicating that they had been neither depleted nor rendered permanently "tolerant" by CTLA4Ig. The mechanism of immunosuppression by CTLA4Ig was investigated by measuring cytokine transcripts in spleens of immunized mice. CTLA4Ig treatment following primary immunization blocked the induction of IL-2 transcripts in splenic T cells and IL-4 transcripts in both T cells and non-B, non-T cells. Splenocytes from CTLA4Ig-treated, SRBC-unresponsive mice showed altered induction of IL-2 and IL-4 transcripts, but T cells nonetheless became primed for IL-4 mRNA synthesis despite the lack of a measurable Ab response. Anti-IL-4 mAb and human CTLA4Ig were synergistic in their ability to induce unresponsiveness, indicating that incomplete suppression of IL-4 production by CTLA4Ig limited its effectiveness.
Prediction of peptide affinity to HLA DRB1*0401.
Authors: Marshall K W , Wilson K J , Liang J , Woods A , Zaller D , Rothbard J B
Published: December 2022

A method to predict quantitatively peptide binding to HLA DRB1*0401 has been developed using a data set of the relative contributions of each of the naturally occurring amino acids in the context of a simplified peptide back-bone. The prediction assumed that the relative role of each of the peptide side chains could be treated independently and could be measured by assaying each of the 20 naturally occurring amino acids at the central 11 positions of a 13-residue peptide previously shown to contain the minimal requirements for high-affinity binding to HLA-DR proteins. The resultant database was shown to have predictive value when tested on a set of 13 unrelated peptides known to bind DRB1*0401 with a wide range of apparent affinity. The database was tested further by analyzing myelin basic protein. All 13 amino acid peptides containing a hydrophobic amino acid at the third position were synthesized and assayed for binding purified DRB1*0401. In every case, the measured affinity correlated with the predictive values within the experimental error of the assays. Finally, the ability to predict peptide binding to MHC class II molecules was shown to help in identifying T cell determinants. The specificity of DRB1*0401-restricted T cell hybridomas against human serum albumin corresponded to two peptides, predicted and shown to bind the class II protein with high affinity.
Novel isoforms of murine intercellular adhesion molecule-1 generated by alternative RNA splicing.
Authors: King P D , Sandberg E T , Selvakumar A , Fang P , Beaudet A L , Dupont B
Published: December 2022

Intercellular adhesion molecule-1 (ICAM-1)-deficient mice, produced by homologous recombination and previously recognized to be devoid of the common form of ICAM-1, are shown to express residual amounts of ICAM-1 Ag in thymus and lung. We demonstrate that this expression of ICAM-1 is possible because the mutated exon 5 in these animals has been skipped by alternative splicing of RNA. Three different alternative isoforms of ICAM-1 are expressed in mutant mice. Moreover, two of these isoforms are expressed in wild-type mice together with two additional alternative isoforms that cannot be produced in mutant mice. All alternatively spliced isoforms of ICAM-1 detected are missing complete extracellular Ig domains. In both mutant and wild-type mice, expression of alternatively spliced isoforms is up-regulated following stimulation of animals with LPS. Furthermore, all alternative isoforms of ICAM-1, except one, retain the ability to bind to the well-recognized ICAM-1 counter-receptor LFA-1. These findings, along with the restricted tissue distribution in mutant mice, indicate that alternative isoforms of ICAM-1 are significant physiologic adhesion structures which could play a distinct role in the functioning of the immune system of intact animals.
A monoclonal antibody that blocks the complement regulatory activity of guinea pig erythrocytes and characterization of the antigen involved as guinea pig decay-accelerating factor.
Authors: Okada N , Tanaka H , Takizawa H , Okada H
Published: December 2022

MCA44 is a mAb with the capacity to sensitize neuraminidase-treated guinea pig E for hemolysis by homologous guinea pig C, and the Fab fragments of this mAb could also sensitize guinea pig E interfering with the function of a membrane inhibitor of C on guinea pig E. Using an immunosorbent column to which MCA44 was coupled, the antigenic molecule termed 44Ag was purified from the glycoprotein fraction extracted from E membranes. C intermediate sheep E treated with guinea pig C1 and C4 after sensitization with Ab (EAC14b cells) lost the ability to generate C3 convertase with C2 after incubation with 44Ag. Treatment of guinea pig E and PBL with phosphatidyl-inositol specific phospholipase C (PIPLC) partially removed 44Ag, as determined by flow cytometric analysis after immunofluorescence staining with MCA44. However, 125I-labeled 44Ag adsorbed to human E was efficiently removed by PIPLC treatment with a slight reduction in M(r). The 44Ag purified on an immunosorbent column showed three bands on SDS-PAGE. However, partial N-terminal amino acid sequences of the 55-kDa, 70-kDa, and 88-kDa bands under nonreducing conditions were identical and the sequence was 55% homologous to the N-terminal sequence of human decay accelerating factor (CD55). Intracutaneous administration of MCA44 or its F(ab')2 fragment resulted in increased capillary permeability, even after 3 days, as determined by the appearance of Evans blue spots after i.v. administration of the dye. Because control Abs including anti-class I-MHC did not cause such increased capillary permeability, the increase in permeability caused by MCA44 was likely induced by blocking the function of 44Ag in vivo, indicating a crucial role for these molecules in preventing over-activation of C at the site.
Loss of cytotoxic T lymphocyte function in Chediak-Higashi syndrome arises from a secretory defect that prevents lytic granule exocytosis.
Authors: Baetz K , Isaaz S , Griffiths G M
Published: December 2022

CTLs from patients with Chediak-Higashi syndrome (CHS) are unable to destroy target cells recognized via the TCR. To determine the mechanism responsible for the loss of cytotoxicity, CD8+ CTL clones have been derived from a patient with CHS. Individual CTL clones show poor killing that can be increased in longer assays. However, in the presence of cycloheximide, the small amount of killing observed is abolished, indicating killing arises from newly synthesized proteins, rather than from proteins stored in granules. In this study, we show that the CHS CTL clones express normal levels of the lytic proteins granzyme A, granzyme B, and perforin, which are processed properly during biosynthesis and targeted correctly to giant lytic granules. Despite the difference in size, CHS and normal lytic granules are similar, in that both contain the lysosomal enzyme cathepsin D and the lytic protein granzyme A, and lack the mannose-6-phosphate receptor (MPR). However, unlike normal CTL clones, the CHS CTL clones are unable to secrete their giant granules in which the lytic proteins are stored. After cross-linking the TCR, CHS CTL clones fail to secrete granzyme A, as assayed by both enzyme release and confocal microscopy. We suggest that the defect in CHS lies in a protein that is involved in membrane fusion and is essential for the secretion of lysosomal compartments in certain hemopoietic cells.
An epitope-selective, transporter associated with antigen presentation (TAP)-1/2-independent pathway and a more general TAP-1/2-dependent antigen-processing pathway allow recognition of the HIV-1 envelope glycoprotein by CD8+ CTL.
Authors: Hammond S A , Johnson R P , Kalams S A , Walker B D , Takiguchi M , Safrit J T , Koup R A , Siliciano R F
Published: December 2022

The lysis of virally infected cells by CTLs requires the recognition of processed fragments of viral proteins presented in association with class I MHC molecules on the surfaces of infected cells. Processing begins in the cytosol with the degradation of viral proteins into peptides that are then transported into the endoplasmic reticulum (ER) for association with newly synthesized class I molecules. Transport is mediated by a heterodimer of the MHC-encoded proteins, transporter associated with Ag presentation (TAP)-1 and TAP-2. Uncertainty exists over the site of processing of viral envelope (env) proteins. The extracellular domains of env proteins are not present in the cytosol, the site in which the class I-restricted Ag-processing pathway begins. Rather, the ecto-domains of env proteins are cotranslationally translocated into the ER during biosynthesis. We have analyzed the processing of the HIV-1 env protein by using a large series of env-specific human CD8+ CTL clones. These studies have led to the delineation of two distinct processing pathways. The first pathway permits a subset of class I-restricted epitopes in the ecto-domain of the env protein to be generated efficiently by a TAP-1/2-independent mechanism localized to the ER or a premedial Golgi compartment. A second, more general pathway that is capable of generating all env epitopes uses as a substrate env protein mislocalized to the cytosol and produces peptides that are transported from the cytoplasm to the ER in a TAP-1/2-dependent fashion.
Concomitant infection with exogenous mouse mammary tumor virus encoding I-E-dependent superantigen in I-E-negative mouse strain.
Authors: Ando Y , Wajjwalku W , Niimi N , Hiromatsu K , Morishima T , Yoshikai Y
Published: December 2022

We found that milk from II TES mice contained two species of exogenous mouse mammary tumor viruses (MMTV). Sequence analysis of the open reading frame (ORF) in the MMTV 3' long terminal repeat indicated that the two MMTV, MMTV (II TES2) and MMTV (II TES14), encode superantigens specific for V beta 2+ T cells and V beta 14+ T cells, respectively. In an experiment of subcutaneous injection of II TES milk, both T cells bearing TCR V beta 2 and V beta 14 proliferated vigorously in the draining lymph node from BALB/c mice (H-2d I-E+), whereas only V beta 14+ T cells showed significant proliferation in C57BL/6 mouse (B6 H-2b I-E-) lymph nodes. These findings indicated that the superantigen encoded by MMTV (II TES2) required MHC class II I-E molecules exclusively for Ag presentation, but MMTV (II TES14) stimulated V beta 14+ T cells even in the absence of I-E molecules. Semiquantitative analysis of MMTV proviruses using PCR revealed that B6 mice were not infected with MMTV (II TES2) by injection of this MMTV alone. However, injection of II TES milk containing both MMTV (II TES14) and MMTV (II TES2) induced infection of B6 mice with MMTV (II TES2) besides MMTV (II TES14), in spite of no expansion of V beta 2+ T cells in this mouse strain. These results suggested that I-E-negative mice were concomitantly infected with MMTV (II TES2) with the help of I-E independent T cell activation mediated by MMTV (II TES14).
Superantigen-induced anergy in cytotoxic CD8+ T cells.
Authors: Sundstedt A , Höidén I , Hansson J , Hedlund G , Kalland T , Dohlsten M
Published: December 2022

This study describes the use of bacterial superantigens to investigate the mechanisms governing peripheral tolerance in CD8+ T cells. Administration of superantigens to mice activates T cells to proliferation, cytokine production, and cytotoxicity, but responding cells subsequently enter a state of hyporesponsiveness or are deleted. Superantigen-induced inactivation has so far mainly been demonstrated for CD4+ T cells. Injection of amounts of the superantigen staphylococcal enterotoxin A (SEA) that are optimal for T cell activation and which induce anergy in CD4+ T cells result in preserved responsiveness in CD8+ CTLs. In contrast, we found that intravenous injection of low concentrations of SEA induced a profound down-regulation of the cytotoxic function in SEA-reactive CD8+ TCR V beta 11+ T cells. No reduction in the number of CD8+V beta 11+ T cells was found, suggesting that anergy and not deletion is the main mechanism for the observed cytotoxic hyporesponsiveness. The cytotoxic anergy was evident 2 days after low-dose priming and remained present 4 wk later, indicating a rapid induction phase and long-lasting persistence. The anergized CD8+ T cell subset expressed lower levels of the alpha-(CD11a) chain of the cell adhesion molecule lymphocyte function-associated Ag 1 (LFA-1) and failed to mediate cytotoxicity, but retained the capacity to proliferate, express IL-2R, produce IFN-gamma, and express granzyme mRNA, which imply a partial defect in TCR-transduced signals. Taken together, these findings suggest that there is a biphasic stimulus-dependent threshold for acquiring responsiveness or anergy in CD8+ T cells.
CD4-positive suppressor cells block allotransplant rejection.
Authors: Yin D , Fathman C G
Published: December 2022

Mechanisms of maintenance of transplantation tolerance induced in adult rats by depletive regimens of anti-CD4 before transplantation of vascularized heart allografts were studied. Despite the fact that there has been little evidence that tolerant lymphocytes could prevent allograft rejection after adoptive transfer, we demonstrated a suppressive role for lymphocytes from tolerant animals in vivo. These experiments analyzed the ability of lymphocytes from tolerant rats to protect passenger leukocyte-depleted Lewis heart grafts that had been "parked" in ACI rats (treated with pretransplant anti-CD4 and maintained for > 100 days) compared with their ability to protect transplantation of fresh Lewis heart grafts in naive ACI rats. Although parked Lewis heart grafts were rejected in unmanipulated ACI recipients, parked hearts (but not naive Lewis heart allografts), were permanently accepted by naive ACI rats when syngeneic tolerant spleen cells were adoptively transferred at the time of transplantation. Further, we demonstrated that the suppressor cells in the tolerant spleen cells were CD4+. These results suggest that CD4+ spleen cells from tolerant rats inhibit allograft recognition and may maintain allograft tolerance by blocking the indirect pathway of allorecognition.
Neutralizing murine monoclonal antibodies to human IL-5 isolated from hybridomas and a filamentous phage Fab display library.
Authors: Ames R S , Tornetta M A , McMillan L J , Kaiser K F , Holmes S D , Appelbaum E , Cusimano D M , Theisen T W , Gross M S , Jones C S
Published: December 2022

Conventional hybridomas and combinatorial Ab libraries were used to develop neutralizing murine mAbs to human IL-5. Mice were immunized with rIL-5. Spleens from two mice were used to generate hybridomas. Spleens from an additional three mice were used to construct a combinatorial library. In both instances, Abs were identified and selected by ELISA using 96-well plates coated with rIL-5. These Abs were tested for the ability to block binding of iodinated rIL-5 to the alpha-chain of the human IL-5 receptor (IL-5R alpha) and to inhibit proliferation of IL-5-dependent cells. By hybridoma technology, 16 mAbs were obtained, 11 of which blocked binding to IL-5R alpha, including three that inhibited proliferation. Quantitative binding assays and sequence analysis revealed that these latter three mAbs were closely related. Combinatorial cloning and selection by phage display was used to isolate 24 bacterial colonies secreting Fabs that bound to 125I-rIL-5 and to rIL-5-coated plates. Sequencing of 10 of the Fabs indicated that four unique Abs were obtained, comprising one predominant VH paired with one of two different VL. The sequence of the Fabs was distinct from the sequences of the neutralizing mAbs. In contrast to the mAbs, none of the Fabs blocked binding of 125I-IL-5 to IL-5R alpha or neutralized the biologic activity of IL-5. The inability to identify neutralizing Fabs was shown not to result from their monovalency, because a Fab derived from one of the neutralizing mAbs, by cloning and expression of its Fd and kappa light chains, retained neutralizing activity. By chain shuffling, pairing of the Fd fragment of the heavy chain of one of the neutralizing mAbs (2B6), with the light chain library derived from the IL-5-immunized mice, neutralizing Fabs were obtained. These Fabs contained light chain sequences closely related to the original light chain of 2B6. Hence, chain shuffling allowed detection of a light chain sequence that was not evident upon two-chain combinatorial selection. The results reveal differences in the Abs obtained from a combinatorial library vs hybridomas and demonstrate how these approaches can be used in concert to select mAbs with neutralizing activity.