bluuebunny/crossref_metadata_2025 huggingface dataset:
CD40 expression in malignant plasma cells. Role in stimulation of autocrine IL-6 secretion by a human myeloma cell line.
Authors: Westendorf J J , Ahmann G J , Armitage R J , Spriggs M K , Lust J A , Greipp P R , Katzmann J A , Jelinek D F
Published: December 2022

Myeloma is a neoplasia characterized by the accumulation of malignant plasma cells in the bone marrow. In these studies, we have demonstrated that CD40 is expressed in human myeloma cells and have used a recently established IL-6-dependent myeloma cell line, ANBL-6, to examine the potential function of CD40 expression in myeloma cells. In addition to its expression on the ANBL-6 cells, we show that CD40 is expressed on freshly isolated myeloma cells from seven of seven patients tested. To address the role of CD40 expression in myeloma cells, we have examined the responsiveness of the ANBL-6 cell line to a CD40-specific mAb, G28-5. This cell line has previously been shown to proliferate only in response to IL-6. Of interest in this study, G28-5 also induced proliferation of the ANBL-6 cells. This proliferation was substantially inhibited by an IL-6-neutralizing mAb. Analysis of ANBL-6 cell culture supernatants by ELISA demonstrated that G28-5-stimulated cells secreted significant levels of IL-6, whereas unstimulated cell culture supernatants contained undetectable levels of IL-6. Furthermore, CV-1/EBNA cells expressing the human CD40 ligand also induced the proliferation of the ANBL-6 cell line, an effect that was inhibited by the anti-IL-6 mAb. Lastly, RNA blot analysis demonstrated an increase in IL-6 message in G28-5-stimulated ANBL-6 cells over unstimulated cells. These results indicate that the primary mechanism of anti-CD40-stimulated proliferation of the ANBL-6 cells is the induction of autocrine IL-6 production. Moreover, these data suggest that the expression of CD40 in malignant plasma cells may play a role in tumor cell expansion, possibly by stimulating the induction of autocrine IL-6 secretion.
Human epidermal Langerhans cells secrete a soluble receptor for IgG (Fc gamma RII/CD32) that inhibits the binding of immune complexes to Fc gamma R+ cells.
Authors: Astier A , de la Salle H , de la Salle C , Bieber T , Esposito-Farese M E , Freund M , Cazenave J P , Fridman W H , Teillaud J L , Hanau D
Published: December 2022

Langerhans cells (LC) express Fc gamma RII on their cell surface. In this paper, we demonstrate that these cells also release soluble Fc gamma RII (sFc gamma RII) molecules. LC express transcripts encoding a membrane-associated receptor and a transmembrane-deleted Fc gamma RIIA. The latter form was identified in LC culture supernatants using specific antibodies. CHO cells, transfected with LC-derived cDNA encoding the transmembrane-deleted Fc gamma RIIA, secrete sFc gamma RIIA that include the intracellular domain and exhibit the same backbone as the protein identified in LC supernatants. Secreted sFc gamma RIIA exhibits the same pattern of binding to human and mouse IgG subclasses as do membrane Fc gamma RII and inhibits the binding of immune complexes to Fc gamma RII+ cells. In addition, CHO cells expressing the membrane-associated Fc gamma RIIA release truncated and unstable Fc gamma RIIA molecules that lack the intracellular domain. Thus, sFc gamma RII can result from shedding of membrane molecules and/or from secretion of soluble receptors lacking the transmembrane domain.
A novel granulocyte-derived peptide with lipopolysaccharide-neutralizing activity.
Authors: Larrick J W , Hirata M , Zheng H , Zhong J , Bolin D , Cavaillon J M , Warren H S , Wright S C
Published: December 2022

Rabbit CAP18 (cationic antimicrobial protein, 18 kDa) is a leukocyte protein identified and purified using as an assay its capacity to bind and inhibit various activities of LPS. Oligonucleotide probes designed from the putative N-terminal protein sequence were used to obtain the corresponding cDNA from a rabbit bone marrow cDNA library. Examination of the cDNA sequence revealed that the protein fragment of the putative N-terminus was actually a 37-amino-acid C-terminal fragment. This fragment, designated CAP18(106-142), inhibits many activities of LPS. In the present studies, synthetic CAP18(106-142) is shown to: 1) bind to erythrocytes coated with diverse strains of LPS; 2) inhibit LPS-induced release of cytokines (TNF, IL-1, IL-6) and nitric oxide from macrophages; 3) inhibit LPS-induced LAL coagulation and 4) protect mice from LPS lethality. CAP18(106-142) may have therapeutic utility for conditions associated with elevated concentrations of LPS.
Differential requirement for autoantibody-producing B cells for induction of lymphocytic versus granulomatous experimental autoimmune thyroiditis.
Authors: Braley-Mullen H , Sharp G C , Kyriakos M
Published: December 2022

Mouse thyroglobulin (MTg)-sensitized spleen cells activated in vitro with MTG transfer experimental autoimmune thyroiditis (EAT) in which the thyroid cellular infiltrate consists primarily of mononuclear cells (lymphocytic EAT). Addition of anti-IL2R antibody to cultures with MTg leads to activation of cells that induce granulomatous EAT, accompanied by high serum anti-MTg autoantibody responses, in recipient mice. CD4+ T cells are required to induce both forms of EAT; whether B cells and/or autoantibodies produced by MTg-sensitized B cells also contribute to disease severity or the type of thyroid histopathology is unknown. In our study, B cells and autoantibody responses produced in recipient mice were reduced either by column removal of B cells from donor spleen cells or by treatment of recipient mice with anti-I-AK mAb at the time of cell transfer. These maneuvers only slightly reduced the severity of lymphocytic EAT but markedly reduced the severity and incidence of granulomatous EAT developing in recipient mice. Delaying the initiation of anti-I-AK treatment until 6 days after cell transfer was less effective in reducing anti-MTg autoantibody responses or granulomatous EAT. These studies all suggested that anti-MTg autoantibodies were required for development of granulomatous but not lymphocytic EAT. However anti-I-AK-treated recipients receiving injections of anti-MTg antibody or having serum antibody induced by prior immunization with MTg and LPS also developed less severe granulomatous EAT than controls. These results suggest that sensitized CD4+ T cells and circulating anti-MTg autoantibody are not sufficient for development of granulomatous thyroid lesions. It is possible that antibodies having a unique function or specificity are produced in mice developing granulomatous EAT or thyroid-infiltrating B cells may directly contribute to the granulomatous inflammatory response.
Both phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase products are increased by antigen receptor signaling in B cells.
Authors: Gold M R , Aebersold R
Published: December 2022

The effects of Ag binding on B cell development and activation are mediated by intracellular signals initiated by the B cell AgR. In this report, we show that the B cell AgR regulates the production of inositol phospholipids involved in two different signal transduction pathways, the phosphatidylinositol 3-kinase (PtdIns 3-kinase) pathway and the phospholipase C (PLC) pathway. Phosphatidylinositol 3-phosphate (PtdIns3P), phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2], and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] are produced by PtdIns 3-kinase, an enzyme that appears to be an essential component of tyrosine kinase-mediated signaling. Both PtdIns(3,4)P2 and PtdIns(3,4,5)P3 are likely to function as second messengers in vivo because they can activate the zeta isoform of protein kinase C (PKC) in vitro. We show that cross-linking of the B cell AgR with anti-Ig antibodies caused a five- to sixfold increase in the levels of PtdIns(3,4)P2 in both the mature B cell line BAL 17 and the immature B cell line WEHI-231. PtdIns(3,4)P2 levels increased within 15 s of anti-Ig addition and remained elevated for at least 5 min. AgR cross-linking also caused a slower increase in PtdIns3P levels (approximately 50% over control) and a small, transient increase in PtdIns(3,4,5)P3 levels. Thus, the B cell AgR activates the PtdIns 3-kinase pathway. The other inositol phospholipid signaling pathway involves PLC, which cleaves phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], yielding second messengers that increase intracellular calcium and activate other isoforms of PKC. We analyzed the effects of AgR signaling on PtdIns(4,5)P2 and its precursor, phosphatidylinositol 4-phosphate (PtdIns4P). Consistent with its ability to activate PLC, AgR ligation decreased the levels of PtdIns(4,5)P2. In contrast, AgR cross-linking increased the levels of PtdIns4P. Increased synthesis of PtdIns4P followed by phosphorylation at the D-5 position may prevent depletion of PtdIns(4,5)P2. Thus, signaling by the B cell AgR increases the levels of PtdIns 4-kinase products and PtdIns 3-kinase products. The simplest interpretation of our results is that the B cell AgR activates both PtdIns 3-kinase and PtdIns 4-kinase.
Recovery of T cell populations after acute graft-vs-host reaction.
Authors: Hakim F T , Payne S , Shearer G M
Published: December 2022

We previously have observed that T dependent immune functions were deficient for several months after induction of acute suppressive graft-vs-host-reaction (GVHR) by injection of parental C57BL/10 donor spleen cells into unirradiated (B10 x B10.BR) F, hosts. We therefore investigated whether new T cells matured after acute GVHR, and whether these were tolerant of host Ag. By 8 to 17 mo after GVHR, the frequencies of splenic CD4 and CD8 T cells were found to be comparable to age-matched untreated hosts, although the lymphoid organ size and hence the total number of T cells was significantly reduced. When GVHR was induced with a combination of C57BL/6 (Thy-1.2) mature lymphocytes and B6.PL (Thy-1.1) bone marrow stem cells, the mature donor Thy-1.2 T cells initially predominated during the acute GVHR. After several months, however, 75% of the CD4 and 50% of the CD8 T cell population was derived from donor Thy-1.1% pre-T cells that had matured in the host. Long term GVHR spleen cells were unresponsive to host Ag in CTL assays, but did not suppress anti-host CTL responses. Finally, host-reactive V beta 11 TCR expressing cells were found to be clonally deleted from splenic CD4 and CD8 populations, consistent with intrathymic negative selection. This evidence suggests that the post-GVHR thymus has the capacity to produce and negatively select phenotypically mature CD4 and CD8 T cells and that a failure to clonally delete self-reactive populations is not a contributing factor to the development of chronic GVHR in this system.
B cell IL-7. Human B cell lines constitutively secrete IL-7 and express IL-7 receptors.
Authors: Benjamin D , Sharma V , Knobloch T J , Armitage R J , Dayton M A , Goodwin R G
Published: December 2022

IL-7 was originally reported as a cytokine produced by stromal cells which supports pre-B cell proliferation in vitro. To determine whether human B cells secrete IL-7 and express IL-7R we studied a wide panel of B cell lines (CLS). In Northern blot analysis we detected 2.4 kb IL-7 mRNA and by quantitative PCR we demonstrated IL-7 expression in 5 of 6 B CLS derived from patients with AIDS-associated Burkitt's lymphoma (AABCL), 3 of 3 CLS derived from patients with American Burkitt's lymphoma and 5 of 6 normal lymphoblastoid CLS. Only 1 of 5 African Burkitt's lymphoma CLS and 2 of 7 EBV- CLS expressed IL-7. A total of 484-bp amplicons was cloned and sequenced and found to correspond to the original IL-7 sequence. Constitutive IL-7 secretion was detected in 5 of 6 AABCL and in 6 of 7 normal lymphoblastoid CLS but in none of the 7 EBV- CLS. IL-7R expression was demonstrated in 8 of 26 CLS, none of which secreted IL-7. Our data suggest that 1) IL-7 mRNA is expressed in malignant B cell phenotypes, which correspond to a narrow window in the B cell differentiation pathway (pre-B, early B, intermediate B), as well as in normal lymphoblastoid B CLS. 2) IL-7 mRNA is expressed in both EBV+ and EBV- CLS, but only the EBV+ CLS secrete IL-7. 3) B cells activated by both EBV and HIV-1 (AABCL) secrete the greatest amount of IL-7. 4) IL-7 autocrine loops are not evident since IL-7R were detected on on CLS, which do not secrete IL-7. Our data provide the first direct evidence of IL-7 secretion by human cells and it is yet to be determined whether IL-7 is secreted by other cell types.
Differential expression of ZAP-70 and Syk protein tyrosine kinases, and the role of this family of protein tyrosine kinases in TCR signaling.
Authors: Chan A C , van Oers N S , Tran A , Turka L , Law C L , Ryan J C , Clark E A , Weiss A
Published: December 2022

TCR stimulation results in the tyrosine phosphorylation of a number of cellular substrates. We have recently identified a 70-kDa protein tyrosine kinase, ZAP-70, which associates with the human TCR zeta-chain after TCR stimulation. We report here the isolation and sequence of a cDNA clone that encodes murine ZAP-70. Murine and human ZAP-70 share 93% amino acid identity and are homologous to the 72-kDa protein tyrosine kinase Syk. Syk has been implicated in the signal transduction pathways of the B cell membrane Ig and high affinity IgE receptors, Fc epsilon RI. In addition, we examined the tissue distribution of ZAP-70 and Syk in human and murine thymocyte subsets, B cells, and peripheral T cell subsets. ZAP-70 protein is expressed in all major thymocyte populations, with the level of expression being comparable to that found in both CD4+ and CD8+ peripheral T cells. Although Syk protein is also present in all thymocyte subsets, expression of Syk protein is down-regulated threefold to fourfold in peripheral T cells. In contrast to ZAP-70, expression of Syk is 12- to 15-fold higher in peripheral B cells when compared with peripheral T cells. In addition, whereas T cell stimulation results in down-regulation of Lck, no significant change in ZAP-70 or Syk protein is detected. Finally, we provide evidence that both ZAP-70 and Syk can associate with the TCR after TCR stimulation. With the use of a heterologous expression system, we show that, like ZAP-70, Syk is dependent upon a Src-family protein tyrosine kinase for association with the phosphorylated zeta-chain. Thus, the differential expression of these kinases suggests the possibility of different roles for ZAP-70 and Syk in TCR signaling and thymic development.
In vitro maturation of human neonatal CD4 T lymphocytes. II. Cytokines present at priming modulate the development of lymphokine production.
Authors: Demeure C E , Wu C Y , Shu U , Schneider P V , Heusser C , Yssel H , Delespesse G
Published: December 2022

The development of naive CD4 T cells into type 1 or type 2 Th cells has been extensively analyzed in the mouse. Using neonatal CD4 T lymphocytes as a source of human naive cells, we report that these cells may be induced to differentiate into effector cells producing predominantly Th1 or Th2 cytokines. After 3 days of stimulation with anti-CD3 mAb immobilized on CD32 transfected mouse fibroblasts, followed by 3 days of culture in the presence of IL-2, neonatal cells acquire the phenotypic and functional characteristics of effector cells. Primed cells are enriched in CD45R0hi and CD31- cells, and upon stimulation with PMA+ ionomycin they release significant amounts of IL-2, IFN-gamma, IL-4, IL-5, and IL-10. Addition of exogenous cytokines during the period of activation with anti-CD3 markedly alters the profile of cytokine production by primed cells: 1) IL-2 uniformly enhances Th1 and Th2 cytokine production; 2) IL-4 markedly enhances the release of IL-4, IL-5, and IL-10 and suppresses that of IFN-gamma; 3) IFN-gamma strongly inhibits IL-4 and IL-5 production but slightly enhances IFN-gamma release; 4) IFN-alpha markedly inhibits IL-4 and IL-5 production and increases the production of both IFN-gamma and of IL-10; 5) TGF-beta suppresses IL-4 and IL-5 (and to a lesser extent IL-2) production but has inconsistent effect on IL-10 and IFN-gamma production. These effects of exogenous cytokines are not associated with an alteration of CD31 expression on primed cells.
TCR V beta 3+ and V beta 6+ CTL recognize tumor-associated antigens related to HER2/neu expression in HLA-A2+ ovarian cancers.
Authors: Peoples G E , Yoshino I , Douville C C , Andrews J V , Goedegebuure P S , Eberlein T J
Published: December 2022

We have recently shown that HLA-A2-restricted, tumor-specific CTL can be isolated from tumor-infiltrating lymphocytes (TIL) in ovarian cancer, and that the sensitivity of ovarian tumors to these CTL is correlated with HER2/neu expression. Furthermore, utilizing PCR, we have documented previously that V beta 2, V beta 3, V beta 6, and V beta 7 are represented in increased proportions in ovarian tumor-specific CTL lines. Therefore, to correlate the interaction of these specific TCR V beta segments with the HLA-A2 molecule and potential tumor-associated Ags (TAA) related to HER2/neu expression, we have utilized available mAbs to V beta 2, V beta 3, and V beta 6. We found that V beta 2+, V beta 3+, and V beta 6+ CTL mediate antitumor activity, and a combination of these mAbs resulted in 83 to 95% inhibition of the cytotoxicity against autologous tumor from three separate patients. These mAbs also were capable of blocking HLA-A2-matched allogeneic cytotoxicity, suggesting that all three V beta families recognize TAA in the context of HLA-A2. An HLA-A2+ melanoma was transfected with the HER2/neu gene and became sensitive to HLA-A2+ ovarian cancer-specific CTL lysis. This cytotoxicity was mediated by V beta 3+ and V beta 6+ CTL, as demonstrated by mAb-blocking studies. FACS-depletion studies confirmed that CTL populations depleted of V beta 3 or V beta 6 no longer could recognize the HER2/neu transfectant. We conclude that V beta 3 and V beta 6 recognize some TAA that are either derived from the HER2/neu protein or induced by the expression of the HER2/neu gene and presented in the context of HLA-A2. Furthermore, V beta 2 seems to recognize an HER2/neu-unrelated Ag system also presented by HLA-A2.
Coronary atherosclerosis in transplanted mouse hearts. II. Importance of humoral immunity.
Authors: Russell P S , Chase C M , Winn H J , Colvin R B
Published: December 2022

Obstructive lesions in arterial vessels of transplanted organs constitute an important factor in the late failure of these organs, especially for the heart. The absence of obstructive lesions in syngeneic donor-recipient combinations, suggests that they depend upon recipient immune responsiveness. It is controversial whether humoral or cellular aspects of the immune response predominate in the process. The present experiments employed hearts transplanted between inbred mice. After brief preoperative immunosuppression of recipients with mAbs to CD4 and CD8 determinants, hearts transplanted between mice incompatible for histocompatibility Ags survived for prolonged periods and most developed typical, obstructive coronary lesions. Our quantitative scoring of vascular changes on tissue sections of excised hearts, grades both their severity and prevalence. Transplants between strains that produced Abs to donor cells (B10.A to B10.BR), developed coronary lesions exceeding those in the reverse combination in which no detectable Ab was formed (B10.BR to B10.A; p < 0.00001), even though their histoincompatibility was similar. Treatment of B10.A recipients of B10.BR hearts with an antiserum against the donor significantly increased coronary lesions (p < 0.0003) in a dose-dependent fashion. Coronary arteries of B10.BR hearts transplanted to C.B-17 SCID mice remained largely free of lesions, whereas transplants to SCID recipients that received continuing injections of an antiserum directed to Ags of the donor developed striking, obstructive coronary lesions. We conclude that humoral immunity can be the prime instigator of atheromatous changes that occur in transplanted hearts.
Identification of CD7 glycoprotein as an accessory molecule in HIV-1-mediated syncytium formation and cellfree infection.
Authors: Sato A I , Balamuth F B , Ugen K E , Williams W V , Weiner D B
Published: December 2022

A major cytopathic effect seen upon in vitro infection of CD4+ human T cells by the HIV is cell-to-cell fusion that results in giant cell (or syncytium) formation. Membrane fusion is required for infection by cellfree virions and in syncytium formation. We report here that the human T cell surface molecule, CD7, is important for the HIV-1 fusion process. CD7 is a roughly 40-kDa glycoprotein member of the Ig supergene family that is expressed early in the ontogeny of thymocytes and on the majority of peripheral blood T cells, as well as on NK cells and a small subpopulation of B cells. Anti-CD7 mAbs inhibited HIV-1-induced cell-cell fusion and prevented cellfree infection of SupT1 cells. The antisyncytial activity of the CD7 Abs is not because of cross-reactivity with CD4 or with viral proteins. Epitope mapping revealed at least two regions of the molecule that are important for preventing membrane fusion. Cells rendered CD7- are poorly infectable by cellfree virus. Additionally, cells rendered CD7- are more easily inhibited from fusing in syncytium formation assays. The collective results support a central role for human CD7 in the process of HIV infection.
Role of CD44 in the development of natural killer cells from precursors in long-term cultures of mouse bone marrow.
Authors: Delfino D V , Patrene K D , DeLeo A B , DeLeo R , Herberman R B , Boggs S S
Published: December 2022

The role of the adhesion molecule CD44 in the development of NK cells was analyzed in a mouse long-term bone marrow culture system. After 4 wk of culture (day 0), recombinant human IL-2 was added and 13 days later the cells generated were shown to have substantial cytotoxic activity against YAC-1 and to be enriched for NK cells, as assessed for NK-1.1 phenotype by flow cytometric analysis. Physical separation between stroma and precursors partially inhibited proliferation and, consequently, a lower number of cytotoxic cells were produced. Similar results were obtained when an anti-CD44 mAb was added together with IL-2 at day 0. The disruption of hyaluronic acid (HA), one of the ligands of CD44, by hyaluronidase or the competition for the binding of CD44 by soluble HA added with IL-2 on day 0 inhibited both proliferation and development of cytotoxicity to a greater degree than did anti-CD44. These results indicate that interaction of CD44 with HA plays an important role in the development of pre-NK cells into cytotoxic effector cells.
Keratinocyte-derived T cell costimulation induces preferential production of IL-2 and IL-4 but not IFN-gamma.
Authors: Goodman R E , Nestle F , Naidu Y M , Green J M , Thompson C B , Nickoloff B J , Turka L A
Published: December 2022

By using superantigens, we have found previously that keratinocytes activated by IFN-gamma could serve as accessory cells, providing costimulatory signals needed to induce T cell proliferation. Here, we compared the profile of cytokines produced by T cells stimulated in the presence of activated keratinocytes with the response seen using professional APCs. When keratinocytes are used as accessory cells there is a specific defect in T cell IFN-gamma production, whereas IL-2 and IL-4 are induced at levels comparable with those seen when professional APCs are used as accessory cells. Because keratinocytes express BB-1, a CD28-ligand distinct from B7-1 or B7-2 (which are found on professional APCs), we examined the possibility that the defect in IFN-gamma production might be a result of nonproductive CD28 engagement. However, even when the CD28 pathway is directly activated by a stimulatory mAb, there is no induction of IFN-gamma production in keratinocyte-supported cultures. In these same cultures IL-2 production is increased 10-fold, thus demonstrating a specific deficiency in the induction of IFN-gamma rather than a failure to respond to CD28 stimulation. Analysis by reverse transcriptase-PCR and ELISA for the inducible p40 chain of IL-12 reveals that keratinocytes produce little if any messenger RNA and no protein for IL-12 p40 compared with professional APCs. Addition of rIL-12 to keratinocyte-supported cultures restores IFN-gamma levels to those seen when professional APCs are present. Finally, when T cells are restimulated and analyzed at later time points (10 to 14 days) we find a refinement in cytokine profiles: T cells stimulated in the presence of professional APCs produced the Th1 cytokines IL-2 and IFN-gamma, whereas T cells stimulated in the presence of activated keratinocytes produced only the Th2 cytokine IL-4. The specific ability of keratinocytes to induce a Th2 response seems most closely linked to their absence of IL-12 production, and may be important in the maintenance of peripheral tolerance to self-Ags or in the immune response to exogenous Ags, pathogens, or haptens encountered in skin.
Activation of Ca2+ current in Jurkat T cells following the depletion of Ca2+ stores by microsomal Ca(2+)-ATPase inhibitors.
Authors: Premack B A , McDonald T V , Gardner P
Published: December 2022

The mechanism of TCR-stimulated Ca2+ influx was studied in the Jurkat human T cell line using Ca2+ indicator dyes and whole-cell patch clamp. Ca2+ influx induced by inositol 1,4,5-triphosphate (IP3)-coupled surface receptors (either the TCR or a heterologous muscarinic receptor) was compared with Ca2+ influx induced by inhibitors of the microsomal Ca(2+)-ATPase (thapsigargin, cyclopiazonic acid, di-tert-butylhydroquinone), which release stored Ca2+ without production of IP3. The same Ca2+ influx pathway could be activated by IP3-dependent or IP3-independent means, and therefore appeared to be regulated by the fullness of the microsomal Ca2+ stores rather than by the direct action of IP3. Depletion of stored Ca2+ by either receptor stimulation or microsomal Ca(2+)-ATPase inhibition activated a low conductance, Ca(2+)-selective, non-voltage-activated membrane current. Ca2+ currents induced by receptor stimulation and Ca(2+)-ATPase inhibition were not additive. Several properties of the depletion-activated Ca2+ current suggest that it is carried by a novel type of Ca2+ channel rather than an electrogenic carrier or pump. The conductance saturated when external Ca2+ was raised (Kd approximately 2 mM) and became highly permeable to monovalent cations when external Ca2+ was lowered to below 100 nM, much as has been observed for some voltage-gated Ca2+ channels. The Ca2+ current was reversibly blocked by > 90% with 0.3 mM Cd2+, whereas the same concentration of Ni2+ or Co2+ blocked only 50 to 60% of the current. However, the absence of voltage-dependent activation, relative conductance sequence for divalent cations (Ca2+ > Ba2+ approximately Sr2+ > Mn2+), and lack of inhibition by nifedipine, D600, diltiazem, delta-conotoxin, or aga-IVa were unlike that of voltage-gated Ca2+ channels.
Unique antigen recognition by a herpesvirus-specific TCR-gamma delta cell.
Authors: Sciammas R , Johnson R M , Sperling A I , Brady W , Linsley P S , Spear P G , Fitch F W , Bluestone J A
Published: December 2022

TCR-gamma delta cells, a T cell subset present in the epithelial and lymphoid tissues, have been implicated in viral and bacterial infections. We have identified a TCR-gamma delta clone (TgI4.4) that, unlike TCR-alpha beta cells, recognizes a herpes simplex virus type 1 transmembrane glycoprotein, gI, in an MHC class I- and class II-independent fashion. The TCR of TgI4.4 is composed of rearranged V delta 8 (a V alpha 2 family member) and V gamma 1.2 variable genes, a heterodimeric pair not previously described. Furthermore, anti-V alpha 2 mAbs are sufficient to block recognition of the gI ligand. Strikingly, anti-gI Abs also are capable of blocking recognition, a phenomena that is very rare in TCR-alpha beta Ag recognition. Therefore, to dissect the mechanism involved in this unique form of Ag recognition, we constructed a mutant of gI, gIt, that lacks cell surface expression upon transfection into APCs. This form of gI was not sufficient for Ag presentation. In contrast, wild-type gI expressed in the Ag-processing mutant cell, RMA-S, is recognized by TgI4.4, suggesting that gI presentation occurs independently of classical Ag-processing pathways. In fact, through the use of a soluble recombinant gI molecule, gI-Ig, we show that TgI4.4 can recognize whole, unprocessed gI protein in the absence of any APCs. These results suggest that there exist alternate and novel forms of TCR Ag recognition, and that the TCR-gamma delta clone, TgI4.4, may represent a novel T cell subset that, during pathogenic challenge, may respond directly to Ags on the surfaces of bacteria and viruses.
Both naive and memory CD4 T cell subsets become anergic during MAIDS and each subset can sustain disease.
Authors: Koch S , Muralidhar G , Swain S L
Published: December 2022

Infection of C57BL/6 mice with the LP-BM5 mixture of retroviruses causes a severe murine acquired immunodeficiency syndrome (MAIDS). After infection, the entire CD4 T cell population shifts to a memory/activated phenotype and the CD4 cells become anergic to further stimulation. This drastic shift in phenotype could result either from a response of naive cells to the virus or its products and/or from the selective expansion of cells already having a memory phenotype. We have investigated whether both naive and memory CD4 T cells can be anergized during BM5 infection and whether each subset is sufficient for disease development. We show that V beta 3/V alpha 11 TCR transgenic mice that have > 98% CD4 T cells with naive phenotype develop MAIDS. Their spleens and lymph nodes become enlarged, their CD4 population expressing the TCR transgene shifts to a memory/activated phenotype, and the CD4 cells neither proliferate nor secrete IL-2 in response to specific Ag, a fragment of pigeon cytochrome C, or to mitogens. Adult thymectomized mice, having memory but not naive CD4 T cells, also developed MAIDS and anergy of the CD4 T cell population at a rate and extent comparable to normal control mice. Furthermore, CD4 T cells from mice primed with keyhole limpet hemocyanin 6 wk before infection with BM5 also became unresponsive to KLH during MAIDS. Thus, both naive and memory CD4 T cells are rendered anergic by the BM5 virus infection and it is likely that each subset is sufficient to sustain the development of MAIDS.
Clonal expansion and persistence of human T cells specific for an immunodominant myelin basic protein peptide.
Authors: Wucherpfennig K W , Zhang J , Witek C , Matsui M , Modabber Y , Ota K , Hafler D A
Published: December 2022

TCR rearrangements were used to probe the clonal origin of myelin basic protein (MBP)-reactive T cells from patients with multiple sclerosis (n = 7) and normal subjects (n = 3). The majority of MBP-specific T cell lines were specific for the immunodominant MBP(84-102) and MBP(143-168) peptides and were restricted by HLA-DR molecules. In two patients with the DR2 haplotype, the T cell response to MBP was focused on the MBP(84-102) peptide. In both patients, in vivo expanded population(s) (three expanded populations in the first patient, one expanded population in the second patient) dominated the response to the MBP(84-102) peptide. Two MBP(84-102)-specific T cell clones from a normal subject with the DR2 haplotype were also found to have identical TCR sequences. Clonality was proven by demonstrating that independent clones had identical TCR alpha- and TCR beta-chain sequences as well as identical sequences of a TCR gamma-chain or of a second TCR alpha-chain rearrangement. Repeated analysis of one patient after 13 mo demonstrated that the three expanded clones had persisted in vivo. A representative of one of the expanded clones was again obtained after 31 mo by IL-2 stimulation suggesting that this clone was activated in vivo. These data suggest that the response to human MBP is dominated in at least some subjects by expanded clones that may persist in vivo for relatively long periods of time.
Thiol-mediated redox regulation of lymphocyte proliferation. Possible involvement of adult T cell leukemia-derived factor and glutathione in transferrin receptor expression.
Authors: Iwata S , Hori T , Sato N , Ueda-Taniguchi Y , Yamabe T , Nakamura H , Masutani H , Yodoi J
Published: December 2022

The proliferative response of PBMC to PHA, Con A, OKT3 mAb and IL-2-dependent proliferation of PHA-blasts was examined in a thiol-free environment (cultured in a L-cystine- and GSH-free medium). [3H]TdR incorporation assay and cell cycle analysis revealed that stimulated PBMC could not enter the S phase when deprived of these thiol compounds. In thiol-free cultures, an increase in intracellular free Ca2+ concentration and IL-2R alpha-chain/p 55 (Tac) induction was still observed, whereas transferrin receptor induction was markedly reduced, suggesting that the proliferative response of mitogenically stimulated PBMC was arrested in the late G1 phase in which transferrin receptor is induced. In GSH-depleted cultures, a similar reduction of the proliferative response of PBMC and PHA-blasts was observed when the concentration of L-cystine was lowered, in a dose-dependent manner. Each reduction or loss of proliferative response was partially restored by supplementation of 2-ME or adult T cell leukemia-derived factor (ADF)/human thioredoxin which is considered to be an endogenous dithiol-reducing factor. L-Cystine transport analysis showed that mitogenically stimulated PBMC and PHA blasts incorporated L-cystine, whereas resting PBMC did not. Furthermore, ADF as well as 2-ME exhibited an enhancing activity on the L-cystine transport in PHA blasts. Together with the fact that L-cystine transport is a limiting step in glutathione synthesis, these findings suggest that GSH and ADF might cooperate in the thiol-mediated redox regulation process and might also play key roles in cell cycle (late G1 to S) progression of activated lymphocytes.
TCR-dependent and -independent signaling mechanisms differentially regulate lymphokine gene expression in the murine T helper clone D10.G4.1.
Authors: Naora H , Altin J G , Young I G
Published: December 2022

The signaling mechanisms that regulate lymphokine gene expression in the murine Th2 clone D10.G4.1 were investigated by comparing the steady state mRNA levels of six lymphokine genes in response to cellular treatment with various activators and inhibitors of several key signaling pathways. A surprising degree of differential regulation was found. All of the genes studied (IL-3, IL-4, IL-5, IL-6, IL-10, and granulocyte-macrophage (GM)-CSF) were induced by the lectin Con A and the TCR idiotype-specific mAb 3D3. However, the induction of the IL-3, IL-4, and GM-CSF genes, but not the IL-5, IL-6, and IL-10 genes, was strongly inhibited by cyclosporin A. Furthermore, IL-5, IL-6, and IL-10 genes were independently induced by IL-1 alpha, the phorbol ester PMA, and by forskolin, an activator of adenylate cyclase. Results of studies performed with use of the Ca2+ ionophore A23187 indicated that elevation of intracellular Ca2+ levels is sufficient to fully induce IL-3 and IL-4 gene expression. Protein kinase C activation was also required for full induction of the GM-CSF gene and seemed to be obligatory for maximal IL-5 gene expression. The patterns of mRNA induction by the different stimuli broadly correlated with increased rates of transcription. In addition to their induction by IL-1 alpha, the IL-5, IL-6, and IL-10 genes were also induced by mAbs to CD2 and to CD45. In contrast, adding CD45 mAb strongly inhibited the induction of IL-3, IL-4, and GM-CSF genes through TCR stimulation. These results indicate that distinct groups of lymphokine genes may be differentially regulated by signaling pathways that are activated by stimulation of the TCR and other cell surface molecules.
Inhibition of the transendothelial migration of human T lymphocytes by prostaglandin E2.
Authors: Oppenheimer-Marks N , Kavanaugh A F , Lipsky P E
Published: December 2022

To determine whether part of the anti-inflammatory effects of prostaglandin E2 (PGE2) was related to inhibition of T cell interactions with endothelial cells (EC), the effects of PGE2 and other cAMP-elevating agents on the transendothelial migration of human T cells was examined. Although PGE2 did not effect T cell binding to EC, concentration-dependent inhibition of the transendothelial migration of T cells through unstimulated or IL-1-activated EC was observed. PGE2 inhibited the function of both T cells and EC, with maximal inhibition observed when both T cells and EC were treated with PGE2. However, the inhibitory action of PGE2 could not be ascribed to an effect on the adhesion receptor pair, CD11a/CD18-CD54. The inhibitory effect of PGE2 seemed to relate to its capacity to elevate cellular cAMP levels, because 3-isobutyl-1-methylxanthine enhanced PGE2 activity and dibutyryl cAMP and forskolin also inhibited transendothelial migration. The inhibitory effect of PGE2 and the other cAMP-elevating agents on the function of T cells related in part to suppression of their intrinsic locomotory behavior as random migration in the absence of EC was blocked. In EC, PGE2 and the other cAMP-elevating agents increased the barrier function of EC as evidenced by a decrease in the diffusion of [3H]mannitol through the endothelium. These results indicate that part of the anti-inflammatory action of PGE2 relates to its capacity to suppress the transendothelial migration of T cells by cAMP-mediated alterations in the function of both T cells and EC.
Control of T lymphocyte activation and IL-2 receptor expression by endogenously secreted lymphokines.
Authors: Pimentel-Muiños F X , Muñoz-Fernández M A , Fresno M
Published: December 2022

The relative contribution of endogenously secreted lymphokines, TNF-alpha and IL-2, to T cell activation has been studied. Interestingly, a neutralizing anti-TNF-alpha mAb strongly inhibited the proliferation, IL-2R alpha expression, and activation of the nuclear factor (NF)-kappa B induced on human purified T lymphocytes by an immobilized anti-CD3 mAb (imm.anti-CD3). Furthermore, the addition of exogenous TNF-alpha to T cells activated by imm.anti-CD3 or phorbol esters strongly stimulated all those activities. Similarly, neutralizing anti-IL-2R alpha Abs inhibited the cell proliferation and the IL-2R alpha expression induced by imm.anti-CD3, whereas exogenous addition of IL-2 enhanced both activities. However, exogenous addition of IL-2 did not affect NF-kappa B activation. Cyclosporin A, which prevented lymphokine mRNA transcription, inhibited more than 90% of the IL-2R alpha and NF-kappa B levels induced by TCR/CD3-mediated activation. Exogenous TNF-alpha and IL-2 were equally important to the partial recovery of this IL-2R alpha inhibition, and high doses of both lymphokines together completely abolished the inhibitory effect of cyclosporin A. However, only TNF-alpha (but not IL-2) efficiently recovered the nuclear NF-kappa B levels. Those results indicate that the autocrine secretion of TNF-alpha and IL-2, and not the primary stimulus itself, contribute mostly to the regulation of IL-2R; thereby playing a very important role in quantitative control of T cell proliferation and leaving to the specifically TCR/CD3-derived signals the triggering role in T cell activation.
Identification of a novel 33-kDa C1q-binding site on human blood platelets.
Authors: Peerschke E I , Reid K B , Ghebrehiwet B
Published: December 2022

The constitutive expression of a 60-kDa platelet membrane protein (cC1qR) recognizing the collagen-like amino terminal of C1q was previously described. Recently, a novel 33-kDa C1q receptor (gC1qR) that interacts with the globular head region of C1q was identified on Raji cells, as well as PBLs, neutrophils, and eosinophils. The present study demonstrates that polyclonal Abs directed against this novel C1q-binding protein also recognize a 33-kDa platelet membrane constituent on Western blots. Interestingly, Ab reactivity with platelets in suspension was minimal, but increased nearly 10-fold after platelet adhesion to collagen, fibrinogen, or fibronectin-coated surfaces. Similar increases in Ab reactivity were not achieved after platelet stimulation in suspension, even with strong agonists such as thrombin or A23187. Platelet function studies, however, demonstrated that both the globular C-terminal domain of C1q and the collagen-like N-terminal region participate in platelet aggregation in response to C1q multimers. Moreover, a synthetic 18 amino acid peptide (X18) corresponding to the amino terminal sequence of the cloned Raji cell gC1qR inhibited both platelet adhesion to immobilized C1q and aggregated C1q-induced platelet aggregation. Aggregated C1q-induced platelet aggregation was also inhibited by a mAb (1B4) directed against the recombinant gC1qR. The data support the involvement of both carboxy- and amino-terminal regions of C1q in platelet-C1q interactions, and suggest a role for the gC1qR in this process.
High expression of V gamma 8 is a shared feature of human gamma delta T cells in the epithelium of the gut and in the inflamed synovial tissue.
Authors: Söderström K , Bucht A , Halapi E , Lundqvist C , Grönberg A , Nilsson E , Orsini D L , van de Wal Y , Koning F , Hammarström M L
Published: December 2022

We have analyzed the V-gene usage in gamma delta T cells of the human gut and joint by using a new mAb (B18) specific for V gamma 8 of human TCR-gamma delta+ T cells. The B18+ population constituted a minor subset of the gamma delta T cells in peripheral blood (PB) of healthy persons (6 +/- 5%) and only 1 of 35 gamma delta T cell clones analyzed was positive. In contrast, the B18+ subset was a dominant gamma delta T cell population among intraepithelial lymphocytes (IEL) derived from the human intestine (74 +/- 29, p < 0.002), and two of three IEL clones from patients with coeliac disease were B18+. Interestingly, a higher proportion of B18+ gamma delta T cells was found in the synovial fluid of patients with rheumatoid arthritis (RA) (21 +/- 18%, 0.02 < p < 0.05) compared with normal PB. Furthermore, the B18+ subset was more frequent among IL-2-expanded gamma delta T cells (42 +/- 20%) derived from synovial tissue than among IL-2-expanded cells derived from synovial fluid (p < 0.002) and PB from RA patients (p < 0.02) as well as normal PB (p < 0.002). The V-gene usage of 13 gamma delta T cell clones from the synovial fluid of arthritic patients was analyzed. All B18+ clones (n = 7) expressed mRNA for V gamma 8 together with mRNA for V delta 1 (n = 5) or mRNA for V delta 3 (n = 2). None of the B18- clones expressed V gamma 8 (n = 6). We conclude that the gamma delta T cell that expresses V gamma 8, together with mainly V delta 1, is a major gamma delta T cell subset among the IEL of the gut and a highly frequent subset in the synovial tissue of patients with RA. This subset may correspond to the mouse V gamma 7+ IEL, which has a high degree of amino acid sequence homology with the human V gamma 8 protein.
Extrathymic expression of the intracellular hepatitis B core antigen results in T cell tolerance in transgenic mice.
Authors: Milich D R , Jones J E , Hughes J L , Maruyama T , Price J , Melhado I , Jirik F
Published: December 2022

We previously developed a transgenic (Tg) murine lineage (B10.S-Tg31e), which secretes the hepatitis B e Ag (HBeAg) into the serum at a concentration of 10 ng/ml. This serum concentration was sufficient to render B10.S-Tg31e mice functionally tolerant at the T cell but not B cell level. To determine the tolerogenic potential of an intracellular form of this Ag, namely the hepatitis B core Ag (HBcAg), expressed outside the thymus, the B10.S-Tg10c lineage was developed. In B10.S-Tg10c mice the HBcAg is expressed as an intracellular "self"-Ag predominantly in the liver, and cannot be detected in the serum, the thymus or in nonthymic lymphoid tissue. Despite the liver-specific and intracellular location of this transgenic self-protein, B10-STg10c mice demonstrate a significant degree of HBcAg-specific T cell tolerance at the level of T cell proliferation. Similarly, in vivo anti-HBc antibody production after HBcAg immunization is significantly reduced as compared with non-Tg littermate controls. No spontaneous anti-HBc antibody is produced in B10.S-Tg10c mice, however, adoptive transfer of HBcAg-specific T cells from non-Tg B10.S mice elicits anti-HBc specific "autoantibody" production. Interestingly, antibodies with specificity for the HBeAg as well as the HBcAg are produced. Antibody production in B10.S-Tg10c mice adoptively transferred with T cells indicates that sufficient native HBcAg can gain access to the extracellular compartment to engage HBcAg-specific B cells that are clearly not tolerant in this model. No liver injury was observed as a consequence of HBcAg expression, even in B10.S-Tg10c mice adoptively transferred with HBcAg-specific T cells. Unless HBcAg is unique in this regard, these results suggest that organ-specific, intracellular self-Ag may be released during normal cell turnover in sufficient concentrations to elicit systemic T cell tolerance. B10.S-Tg10c mice also serve as an immunologic model system for chronic infection with the HBeAg-negative mutant of the hepatitis B virus.
Urokinase receptor. An activation antigen in human T lymphocytes.
Authors: Nykjaer A , Møller B , Todd R F , Christensen T , Andreasen P A , Gliemann J , Petersen C M
Published: December 2022

The ability of activated T lymphocytes to extravasate and reach inflammatory and malignant foci in the tissues is a basic function of cellular immunity. Recent evidence strongly suggests that the urokinase receptor (uPAR) holds a central position in the development of human two-chain urokinase-mediated pericellular proteolysis and matrix degradation, an important element in cell migration. In this report we establish uPAR as a pan T cell activation Ag. As determined by FACS analysis, CD3+ lymphocytes from healthy donors exhibited no significant uPAR expression. In contrast, patients (e.g., HIV-positive donors) showed distinct uPAR expression, confined to HLA-DR+ cells, in up to 80% of all T cells. In vitro activation by PMA caused a rapid up-regulation of membrane uPAR in all healthy donor T cells and was accompanied by enhanced receptor synthesis and elevated uPAR mRNA levels. A similar induction resulted from activation via the TCR/CD3 complex using mitogens (PHA, and Con A), anti-CD3 antibodies, and alloantigen. Receptor expression at single cell level was also modulated by a number of cytokines. IL-2, IL-4 and IL-7 increased uPAR presentation on 20 to 50% of the T cell population, and combined stimulation of bulk cultures demonstrated an additive effect of IL-2 and IL-7, whereas the response to each of the two was inhibited by IL-4. In addition, TGF-beta 1 substantially reduced the uPAR expression in T cell cultures responding to PHA, IL-2, and IL-7. Irrespective of the activating reagent, the T cells appeared to produce the same molecular uPAR species, but the affinity of uPAR expressed in PMA blasts was decreased, presumably because of a differential location at the cell surface. All activated cultures showed co-expression of uPAR and CD25. The finding that the urokinase receptor is an activation Ag may suggest that cell-associated plasminogen activation is involved in extravasation and migration of activated T cells.