bluuebunny/crossref_metadata_2025 huggingface dataset:
A B cell-restricted activation antigen (B8.7) functionally related to the low molecular weight B cell growth factor receptor.
Authors: Leprince C , Richard Y , Krief P , Treton D , Boucheix C , Galanaud P
Published: December 2022

A novel monoclonal antibody (anti-B8.7) is reported which recognizes an epitope expressed either on in vitro activated B cells or on a fraction of fresh large B cells (putatively in vivo preactivated). B8.7 antigen is also present on two out of eight B cell lines tested and is characterized as a membrane component displaying an approximate molecular weight of 55,000 to 60,000. By contrast, B8.7 is absent from resting B cells, monocytes, resting or activated T cells, and from the eight non-B cell lines tested. After in vitro activation, B8.7 antigen appears later than the transferrin receptor and its expression increases until day 3. The anti-B8.7 monoclonal antibody induces a dose-related inhibition of the low molecular weight B cell growth factor-dependent proliferation of activated B cells, whereas it does not affect their response to interleukin 2. This strongly suggests that the B8.7 epitope is present on a molecule selectively involved in the interaction between B cells and a B cell growth factor.
In vitro anti-human immunodeficiency virus activities of tumor necrosis factor-alpha and interferon-gamma.
Authors: Wong G H , Krowka J F , Stites D P , Goeddel D V
Published: December 2022

Treatment of cells with tumor necrosis factor-alpha and interferon-gamma greatly reduces their susceptibility to infection with human immunodeficiency virus and suppresses the production of human immunodeficiency virus (HIV) mRNA, core protein p24, and infectious HIV. The combination treatment is cytotoxic for HIV-infected cells and reduces HIV RNA levels in chronically infected cells.
Qa-2 antigen encoded by Q7b is biochemically indistinguishable from Qa-2 expressed on the surface of C57BL/10 mouse spleen cells.
Authors: Sherman D H , Waneck G L , Flavell R A
Published: December 2022

Qa-2 was immunoprecipitated from the surface of 125I-labeled C57BL/10 (B10) mouse spleen cells and compared with Qa-2 immunoprecipitated from the surface of R1.1 thymoma cells transfected with Q7b. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that Qa-2 glycoproteins from both of these sources have a relative molecular mass of approximately 37 kDa. After treatment with endoglycosidase F, the Qa-2 polypeptide chains derived from C57BL/10 spleen and Q7b-transfected R1.1 cells displayed identical mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis because of removal of N-linked oligosaccharide residues. Furthermore, treatment of Qa-2 proteins from both sources with cyanogen bromide or alpha-chymotrypsin resulted in identical peptide fragmentation patterns. These results therefore provide a biochemical correlation between a cloned Qa-region gene produce expressed on the surface of transfected cells, and the Qa-2 glycoprotein on spleen cells that was described a decade ago by serologic methods.
Phorbol diesters and dioctanoylglycerol stimulate accumulation of both diacylglycerols and alkylacylglycerols in human neutrophils.
Authors: Rider L G , Dougherty R W , Niedel J E
Published: December 2022

Human neutrophils treated with phorbol 12-myristate 13-acetate (PMA) or dioctanoylglycerol exhibited a large (10-fold), sustained accumulation of the mass of diradylglycerol, beginning 1 min after stimulation and continuing for 30 to 60 min. Phorbol dibutyrate was less potent than PMA in stimulating diradylglycerol accumulation, whereas the 4-alpha analogs of PMA and phorbol dibutyrate were inactive. Submaximal concentrations of PMA (0.5 to 2.5 nM) plus the calcium ionophore, ionomycin (15 to 60 nM), led to synergistic accumulation of diradylglycerols. Chlorpromazine and sphingosine, inhibitors of protein kinase C, blocked PMA-stimulated accumulation of diradylglycerol with IC50 concentrations of 32 and 9 microM, respectively, paralleling their inhibition of PMA-stimulated O2- production. These compounds also inhibited the ionomycin-stimulated accumulation of diradylglycerols. A third protein kinase C inhibitor, H-7, was less effective, inhibiting PMA-stimulated accumulation of diradylglycerol by 25% at 100 microM. Differential sensitivity to alkaline hydrolysis suggests that diradylglycerols that accumulate in response to PMA or ionomycin stimulation are composed of a mixture of two distinct diglyceride species, diacylglycerols and alkylacylglycerols. Whereas diacylglycerol may activate cellular protein kinase C, the importance of the production of alkylacylglycerols is uncertain.
Potentiation of lymphokine-activated killer cell differentiation and lymphocyte proliferation by stimulation of protein kinase C or inhibition of adenylate cyclase.
Authors: Beckner S K , Farrar W L
Published: December 2022

Interleukin 2 (IL-2) stimulated the differentiation of human peripheral blood leukocytes into lymphokine-activated killer cells, as well as DNA synthesis of human T lymphocytes. Both effects of IL-2 could be inhibited by prostaglandin E2, a potent stimulator of adenylate cyclase; however, the inhibitory effect of prostaglandin E2 could be overcome by increased concentrations of IL-2. The opposite effects of IL-2 and prostaglandin E2 were paralleled by their respective abilities to inhibit and stimulate cAMP production in intact cells. Other agents, which inhibit adenylate cyclase directly (somatostatin, beta-endorphin, UK 14.3041) or indirectly by activation of protein kinase C (phenylephrine), could stimulate both differentiation and proliferation. None of these agents alone or in combination were as effective as maximal concentrations of IL-2. However, all agents potentiated differentiation and proliferation induced by submaximal and maximal concentrations of IL-2. Additionally, combinations of agents which stimulated protein kinase C with those that inhibited adenylate cyclase were additive in the potentiation of IL-2-induced differentiation. Neither inhibition nor potentiation of IL-2-induced lymphokine-activated killer cell differentiation was accompanied by changes in Tac expression or gamma-interferon production. The data indicate that the stimulation of lymphokine-activated killer cell differentiation and lymphocyte proliferation in human cells share a common initial biochemical signal. Although the inhibition of adenylate cyclase is not sufficient to maximally stimulate either process and cannot bypass the requirement for IL-2, modulation of this enzyme complex, positively or negatively, can regulate the ultimate physiologic response to IL-2.
Heart cross-reactive antigens of mutans streptococci share epitopes with group A streptococci and myosin.
Authors: Ayakawa G Y , Bleiweis A S , Crowley P J , Cunningham M W
Published: December 2022

Monoclonal antibodies (mAb) generated by immunization of mice with membranes of Streptococcus pyogenes Manfredo (M type 5) were tested for their reactivity with sodium dodecyl sulfate extracts of Streptococcus mutans GS5, Streptococcus rattus BHT (formerly S. mutans, serotype b), and S. mutans Ingbritt 175 in the Western blot. The reaction of the sodium dodecyl sulfate-extracted proteins of whole S. mutans, serotype b), and S. rattus with the mAb revealed that the shared cross-reactive antigens were near m.w. 62,000 to 67,000. Several higher m.w. proteins in S. mutans Ingbritt 175 reacted with the mAb but were not observed in the other two strains of streptococci. Cytoplasmic membranes of S. rattus BHT reacted with these mAb in the enzyme-linked immunosorbent assay and Western blot. The most reactive BHT membrane component detected by these mAb was a 62-kDa polypeptide that was similar in m.w. to the membrane component recognized by these antibodies in purified S. pyogenes membranes. The reactivity of the mAb with BHT membranes in the enzyme-linked immunosorbent assay was absorbed by rabbit skeletal muscle myosin. The patterns of reactivity observed in the BHT membrane immunoblots in this study closely resemble those previously obtained using polyclonal rabbit anti-human heart sera.
Inhibition of transformed T cell growth in vitro by monoclonal antibodies directed against distinct activating molecules.
Authors: Merćep M , Bluestone J A , Noguchi P D , Ashwell J D
Published: December 2022

Stimulatory of antigen-specific murine T cell hybridomas with the appropriate antigen has been shown to cause lymphokine secretion and inhibition of spontaneous cell growth. In this study, the effect of cellular activation on the growth of transformed T cells, of known or unknown antigen specificity, was explored with stimulatory monoclonal antibodies (mAb) that recognize nonclonally distributed T cell surface molecules. Anti-CD3 antibodies stimulated interleukin 2 (IL-2) secretion while they inhibited murine and human T cell tumor growth in vitro. Both responses required external cross-linking of the anti-CD3 antibodies. Activation via two molecules that are not physically associated with the T cell antigen receptor, Thy-1 and Ly-6, also inhibited transformed T cell growth while inducing IL-2 secretion. Notably, an anti-Thy-1 mAb that did not cause the transformed T cells to secrete lymphokines failed to affect their growth, and in fact blocked the growth inhibition induced by the stimulatory mAb. Regardless of which stimulating mAb was used, IL-2 production and cell growth were inversely proportional, manifesting similar antibody dose-response curves. Activation of a T cell hybridoma with stimulatory mAb resulted in rapid lysis, as evidenced by the release of 51Cr and lactate dehydrogenase. Cell cycle analysis demonstrated that cellular activation was accompanied by a cell cycle block between the G1 and S phases, and probably a slowing of the transit of cells already in S. These results demonstrate that the growth of a spectrum of neoplastic T cells, murine and human, can be inhibited by what are normally growth-promoting signals for non-transformed T cells.
Anti-interleukin 2 receptor antibody suppresses murine diabetic insulitis and lupus nephritis.
Authors: Kelley V E , Gaulton G N , Hattori M , Ikegami H , Eisenbarth G , Strom T B
Published: December 2022

In order to assess the importance of interleukin 2 receptor (IL-2R)-positive activated lymphocytes and macrophages in the pathogenesis of autoimmunity, we tested the prophylactic therapeutic efficacy of an anti-IL-2R (M7/20) monoclonal antibody, which recognizes the 55-kDa subunit of the heterodimeric IL-2R in two distinct models: the nonobese diabetic mouse and the NZB x NZW F1 hybrid with lupus. Treatment with anti-IL-2R monoclonal antibody suppressed autoimmune insulitis in nonobese diabetic mice and lupus nephritis in the NZB x NZW F1 hybrid. These studies indicate that highly selective targeting to activated lymphocytes and macrophages expressing the IL-2R provides a discrete method of dampening autoimmunity.
Human T cells can be directed to lyse tumor targets through the alternative activation/T11-E rosette receptor pathway.
Authors: Scott C F , Lambert J M , Kalish R S , Morimoto C , Schlossman S F
Published: December 2022

We investigated the ability of human T cells to be directed to lyse murine and human tumor targets by antibodies (Ab) to the T11-E rosette (CD2) receptor. We found that the human cytotoxic T lymphocyte clone TBI-6, which is specific for the Epstein-Barr virus-transformed cell line, CM-EBV, could be directed to lyse the Fc receptor-positive murine tumor P388D1, by the combination of anti-T11(2) plus anti-T11(3) Ab. This activation and lysis was demonstrable only with an Fc receptor expressing tumor target and only with those Ab or with anti-T3 (CD3) Ab but not with other anti-T11 Ab or other Ab directed against surface structures on the clone. We therefore constructed heterodimeric Ab consisting of anti-T11(2) or anti-T11(3) Ab and the J5 anti-common acute lymphoblastic leukemic antigen (anti-CALLA) Ab. The purity and retained functional properties of the dimers were demonstrated by sodium dodecyl sulfate-polyacrylamide gels, fluorescence-activated cell sorter analysis on relevant cells, and by the ability of these conjugates to activate human peripheral blood lymphocytes to proliferate. These heterodimeric Ab conjugates were shown to be able to direct the lysis of CALLA+ targets by TBI-6. The specificity of this lysis was demonstrated by the inability of these heterodimers to direct the lysis of CALLA- targets by the cytotoxic T lymphocyte clone, and by the ability of excess free J5, but not an irrelevant Ab of the same isotype, to block this type of lysis. The potential clinical significance of these reagents is discussed.
Antigen-driven differentiation of naive Ig-transgenic B cells in vitro.
Authors: Dustin L B , Bullock E D , Hamada Y , Azuma T , Loh D Y
Published: December 2022

We have established a culture system in which naive B cells bearing a transgenic, chicken OVA (cOVA)-specific Ig differentiate to plasma cells in vitro after interaction with cOVA plus cOVA-specific helper T cells. B cell-enriched populations from Ig-transgenic mice, but not from nontransgenic mice, proliferated after presenting nanomolar concentrations of cross-linked cOVA to DO11.10 (cOVA plus IAd-specific) T cells. After 6 to 9 days of culture with Ag and specific T cells, the B cells acquired a plasma cell phenotype and secreted the transgene-derived Ig at high levels. Engagement of B cell surface Ig was not essential for primary B cell differentiation. Differentiating B cells enlarged, clustered, and acquired two plasma cell markers, Syndecan and CD43. B cell CD45 isoform expression changed: the B220 isoform was lost in a T cell-dependent manner, whereas the CD45RB isoform was gained in a T-independent manner. Although unstimulated B cells survived less than 72 h in vitro, those in Ag-stimulated cultures showed reduced early death, a surge of proliferation at 3 to 5 days, and increased death late in the culture. Using a large population of naive B cells of defined antigenic specificity permits us to study a primary immune response to an Ag, rather than to less physiologic polyclonal stimuli. Because all steps of differentiation occurred in vitro, they are easily accessible for study. This coculture system provides an opportunity to observe Ag-specific T cell-B cell collaboration.
The CD40 ligand expressed by human B cells costimulates B cell responses.
Authors: Grammer A C , Bergman M C , Miura Y , Fujita K , Davis L S , Lipsky P E
Published: December 2022

The possibility that activated B cells might express a ligand for CD40 that was of functional importance for B cell responses was examined by using highly purified human peripheral blood B cells, as well as a variety of B lymphoblastoid cell lines and hybridomas. Following stimulation with the combination of a calcium ionophore and a phorbol ester, human B cells bound a soluble fusion protein containing the extracellular portion of CD40 and the Fc region of IgG1 (CD40.Ig). A variety of B cell lines and hybridomas also bound CD40.Ig, either constitutively or after activation. In addition, CD40.Ig specifically immunoprecipitated a 33-kDa glycoprotein from surface 125I-labeled activated B cells. The nucleotide sequence of the coding region of the CD40 ligand mRNA amplified by RT-PCR from activated T cells and B cell lines was identical. The CD40 ligand expressed on human B cells was important functionally because homotypic aggregation of CD40 ligand-expressing B cells was inhibited by the CD40.Ig construct. Additionally, RNA and DNA synthesis as well as Ig production by polyclonally activated, highly purified peripheral B cells and a variety of B cell lines were inhibited significantly by the CD40.Ig construct. Finally, B cell lines expressing the CD40 ligand induced Ig production from resting normal B cells in a CD40-dependent manner. These results indicate that human B cells express a ligand for CD40 that is identical with that expressed by activated T cells and that the B cell-expressed CD40 ligand plays an important role in facilitating responses of activated B cells.
Up-regulation of recombination activating gene expression by signal transduction through the surface Ig receptor.
Authors: Verkoczy L K , Stiernhdm B J , Berinstein N L
Published: December 2022

Recent evidence has demonstrated that a signal transduced through the lymphocyte Ag receptor may regulate the expression of the recombination activating genes (RAG). Although several groups have shown that such a signal may be required to down-regulate RAG-1 and RAG-2 after a functional Ag receptor has been generated in early mature T or B cells, recently it has been suggested that under some circumstances, cross-linking the B cell Ag receptor may result in up-regulation of RAG expression. To study this possibility, we used a unique set of cell variants isolated from a human mature B cell line, which differ in their expression of both the surface Ig receptor (sIg) and RAG-1 and RAG-2 genes. Two forms of stimulation were employed to generate a signal; either a soluble F(ab')2 anti-mu fragment or the combination of PMA and ionomycin. Northern blot analysis demonstrated that RAG-1 mRNA levels were increased in sIg+ variants after cross-linking with anti-mu. Increases were also observed in all variants after stimulation with PMA and ionomycin. Further analysis of cross-linked sIg+ variants suggests that the observed up-regulation in RAG expression was a reversible event. Furthermore, we have determined that both increased transcription and transcript stabilization contributed to this inducible up-regulation. We thus describe a mature B cell line in which RAG expression is up-regulated after sIg cross-linking. This finding is discussed in the context of its potential role in situations where sIg+ B cells may undergo secondary rearrangements for the purpose of "editing" their sIg receptors.
The role of c-Myb or a related factor in regulating the T cell receptor gamma gene enhancer.
Authors: Hsiang Y H , Goldman J P , Raulet D H
Published: December 2022

An enhancer has been localized 3 kb downstream of the C gamma 1 gene segment of the murine TCR-gamma locus. One element, the gamma 3 site, has been shown to be critical for its functional activity. Here we have determined that Myb-related transcription factors bind to the gamma 3 site and appear to be critical for the full activity of the TCR-gamma enhancer. c-myb products can transactivate the gamma 3 site in cell lines that do not ordinarily support enhancer activity of the gamma 3 site. Mutations in the myb site or an adjacent site for core-binding factor(s) prevent transactivation. c-myb expression in various cell lines is consistent with their capacity to activate the gamma 3 enhancer element using transient transfection assays. Therefore, c-Myb or a related factor appears to play an important role in regulating the murine TCR-gamma enhancer.
Cyclic AMP inhibits expression of the IL-2 gene through the nuclear factor of activated T cells (NF-AT) site, and transfection of NF-AT cDNAs abrogates the sensitivity of EL-4 cells to cyclic AMP.
Authors: Tsuruta L , Lee H J , Masuda E S , Koyano-Nakagawa N , Arai N , Arai K , Yokota T
Published: December 2022

cAMP inhibits PMA-induced IL-2 production at the transcriptional level in EL-4, a mouse lymphoma line. The region of the mouse IL-2 promoter covering positions from -321 to +46 relative to the transcription initiation site is required for activation by PMA and inhibition by cAMP. This region contains the nuclear factor of activated T cells (NF-AT), nuclear factor-kappa B (NF-kappa B), AP-1, and Oct binding sites, and the role of each element in responding to PMA and/or cAMP signals was characterized. The IL-2 promoter carrying mutations in each element reduced response to PMA while it retained sensitivity to cAMP, thereby suggesting that multiple elements contribute to positive and negative responses to PMA and cAMP, respectively. Using reporter plasmid carrying multiple copies of each element, we then found that the NF-AT construct was most effective in responding to PMA activation and to cAMP inhibition. Electrophoretic mobility shift assay revealed that, after exposure of cells to Bt2cAMP, NF-AT binding complex changed in amount or in mobility as a function of time. Furthermore, overexpression of the cytoplasmic component of NF-AT abrogated the inhibitory action of cAMP. These results indicate that the NF-AT site is a target of the inhibitory action of cAMP. In addition, binding of the NF-kappa B (p50/p65) heterodimer to the NF-kappa B site was inhibited by cAMP. Taken together, our data show that cAMP in EL-4 cells inhibits mouse IL-2 gene transcription through cis regulatory elements that include the NF-AT site as well as the NF-kappa B site.
Growth inhibition of Candida albicans hyphae by CD8+ lymphocytes.
Authors: Beno D W , Stöver A G , Mathews H L
Published: December 2022

We have shown previously that IL-2-activated splenocytes can inhibit the growth of Candida albicans hyphae in vitro. Herein we demonstrate that plastic nonadherent lymphocytes that are CD8+ mediate the antifungal activity. Enrichment for CD8+ cells markedly enhanced the antifungal activity of the IL-2-activated lymphocyte population for C. albicans and the cytotoxic activity of the lymphocytes for an NK-resistant cell line. Depletion of CD8+ cells reduced the lymphocyte population's antifungal activity and cytotoxic activity for the NK-resistant cell line. Enrichment for NK1.1+ cells markedly reduced the antifungal activity of the lymphocyte population for C. albicans and increased the cytotoxic activity of the lymphocytes for an NK-sensitive cell line. Depletion of NK1.1+ cells increased the lymphocyte population's antifungal activity and cytotoxic activity for the NK-resistant cell line. Generation of the antifungal lymphocytes in culture required IL-2 and was not replaced with IFN-gamma. These data show that IL-2-activated CD8+ T lymphocytes exert the greatest amount of antifungal effect against the hyphal form of C. albicans, whereas IL-2- or IFN-gamma-activated NK cells have little or no effect against the hyphae.
5-Fluorocytosine-induced eradication of murine adenocarcinomas engineered to express the cytosine deaminase suicide gene requires host immune competence and leaves an efficient memory.
Authors: Consalvo M , Mullen C A , Modesti A , Musiani P , Allione A , Cavallo F , Giovarelli M , Forni G
Published: December 2022

The nonmammalian cytosine deaminase (CD) enzyme converts the nontoxic prodrug 5-fluorocytosine (5-FC) to the toxic metabolite 5-fluorouracil. Parental cells of a mammary adenocarcinoma (TSA-pc) of BALB/c mice were transfected with the CD gene (TSA-CD), and the ability of 5-FC to hamper their growth was evaluated. A quantity amounting to 0.5 mg of 5-FC/0.3 ml of medium inhibits the proliferation of TSA-CD cells, but not that of TSA-pc, nor that of TSA-pc transfected with neomycin-resistance gene only (TSA-neo). In BALB/c mice, 800 mg 5-FC/kg of body weight injected daily i.p. for 30 days causes total regression of incipient (1-day-old), and established (3- and 7-day-old) TSA-CD tumors, and of 3-day-old experimental lung metastases, but does not impair TSA-pc nor TSA-neo cell growth. Because in CD8+ T lymphocyte- and granulocyte-depleted mice 5-FC no longer impairs TSA-CD growth, immune mechanisms appear to play an important role in this regression. Following, regression, all mice are resistant to subsequent s.c. or i.v. lethal challenges with TSA-pc. The induction of this immune memory is dependent on CD4+ lymphocytes, whereas its effector phase depends on both CD4+ and CD8+ lymphocytes. The memory elicited in tumor-bearing mice by the 5-FC-dependent regression of TSA-CD tumors cures a significant number of mice with 4-day-old TSA-pc metastases, but does not impair the growth of 4-day-old solid s.c. tumors. The reliability of this regression and the subsequent establishment of an efficient immune memory against poorly immunogenic TSA-pc offer the prospect that CD-transduced tumor cells and 5-FC can be used as components of a live antitumor vaccine.
Morphologic and functional alterations of mucosal T cells by cholera toxin and its B subunit.
Authors: Elson C O , Holland S P , Dertzbaugh M T , Cuff C F , Anderson A O
Published: December 2022

Despite the mucosal immunogenicity and adjuvanticity in vivo of cholera toxin (CT), both CT and CT B subunit are strong inhibitors of T cell activation in vitro. This study asked whether such T cell inhibition is relevant to the mucosal effects of CT in vivo. The activation of T cells pulsed in vitro for only 15 to 120 min with CT or CT B subunit, respectively, was inhibited, consistent with the expected short exposure times in vivo. Although both CD8+ and CD4+ T cells were inhibited in vitro, CD8+ T cells bound more toxin and were inhibited to a greater degree than were CD4+ T cells. Intestinal gavage of mice with 10 micrograms CT did not alter the overall composition of Peyer's Patch, mesenteric lymph node, or spleen but did cause a marked depletion of intraepithelial lymphocytes, mainly CD8+ T cells, and of lymphocytes in the dome epithelium over Peyer's Patch. To determine whether such inhibition of T cells was functionally relevant in vivo, T cells from mice fed keyhole limpet hemocyanin (KLH) were adoptively transferred into naive recipients, who were then parenterally immunized. T cells from mice fed KLH alone inhibited both the systemic IgG and secretory IgA anti-KLH response, but T cells from mice fed KLH plus CT did not, indicating that mucosally applied CT was able to abrogate the induction of this suppressor T cell. We conclude that one of the mechanisms of CT's mucosal effects in vivo is the inhibition of certain mucosal T cell functions and alteration of the regulatory T cell environment in gut-associated lymphoid tissue.
Genetic evidence for difference between intracellular and extracellular peptides in influenza A matrix peptide-specific CTL recognition.
Authors: Matsui M , Moots R J , Warburton R J , Peace-Brewer A L , Tussey L G , Quinn D G , McMichael A J , Frelinger J A
Published: December 2022

During the course of extensive mutagenesis of HLA-A2.1, we examined influenza A matrix peptide (FMP)-specific CTL recognition of HMy2.C1R (C1R) cells expressing mutant HLA-A2.1 molecules, sensitized with synthetic peptide, FMP 58-66, (exogenous peptide), or infected with influenza A virus (endogenous peptide). Most mutants showed equivalent presentation of exogenous and endogenous peptides to FMP-specific CTL. However, five of the mutants differed in this property. Two of the five mutants, F9L and T134K, present exogenous peptide to FMP-specific CTL, but fail to present endogenous peptide to CTL. Western blot analysis using anti-matrix protein Ab indicates that the matrix protein is expressed in these mutants after infection with virus. Interestingly, transfection of these two mutants with a minigene encoding FMP 58-66 results in efficient lysis by FMP-specific CTL. Peptide-binding assays demonstrate that the two mutations dramatically decrease the binding of FMP. However, these mutants bind FMP as well as wild type in the presence of exogenously added human beta 2-m, suggesting that the lower affinity for beta 2-m leads to the inability to present endogenous peptide. The remaining three mutants, Y27N, Q32K, and S132C, fail to present exogenous peptide, but present endogenous peptide to FMP-specific CTL. Pulse-chase analyses followed by endoglycosidase-H treatment show that the rate of maturation and processing of the five mutant HLA-A2 molecules in C1R cells is identical to that of wild type. Overall, this study suggests that the assembly and subsequent recognition of endogenous peptide differs from that of exogenous peptide.
Split tolerance in spleen chimeras.
Authors: Sprent J , Hurd M , Schaefer M , Heath W
Published: December 2022

Transferring small doses of T cells to heavily irradiated F1 mice expressing isolated MHC class I or class II differences invariably leads to rapid death from graft-vs-host disease (GVHD). Paradoxically, GVHD is mild or absent when irradiated F1 mice are reconstituted with large doses of unseparated parental strain spleen cells. This applies when bulk populations of B6 spleen cells are transferred to irradiated class II-different (B6 x bm12)F1 mice or class I-different (B6 x bm1)F1 mice. In this study, we examined whether the donor T cells in long-term spleen chimeras become tolerant to host MHC Ags. On the basis of skin-allograft rejection and induction of GVHD on adoptive transfer, the results show that the donor T cells display strong tolerance to host antigens; this applies to CD4+ cells in class II-different chimeras and to CD8+ cells in class I-different chimeras. In marked contrast to the profound tolerance seen by these in vivo parameters, little or no tolerance is observed in standard in vitro assays. The results illustrate that typical in vitro tests for alloreactivity are an imprecise guide to physiologic tolerance of T cells in vivo.
Antigen receptor cross-linking differentially regulates germ-line CH ribonucleic acid expression in murine B cells.
Authors: Zelazowski P , Collins J T , Dunnick W , Snapper C M
Published: December 2022

Cytokines are believed to regulate Ig class switching, in part, through selective modulation of germ-line constant heavy (CH) gene transcription. B cell activators such as LPS or activated T cell membranes also influence germ-line CH RNA expression in the absence of exogenous cytokines. In this report we determined whether multivalent Ag receptor cross-linking, utilizing dextran-conjugated anti-IgD Abs (alpha delta-dex), could also regulate germ-line CH RNA expression. We demonstrated that alpha delta-dex markedly inhibited germ-line epsilon RNA expression, but strongly augmented germ-line gamma 1 RNA, in LPS + IL-4-stimulated cultures. This was correlated with > 90% alpha delta-dex-mediated suppression in the secretion of IgE and generation of membrane (m)IgE+ cells, and a more modest 50% reduction in IgG1 synthesis and mIgG1+ cells. Furthermore, alpha delta-dex inhibited the LPS induction of both gamma 3 and gamma 2b germ-line RNA and the associated secretion of IgG3 and IgG2b. A similar alpha delta-dex-mediated suppression of germ-line gamma 2a RNA and IgG2a secretion in LPS + IFN-gamma-stimulated cultures was observed. By contrast, activation of resting B cells with alpha delta-dex alone led to induction of germ-line gamma 3, gamma 1, and gamma 2b RNA but did not stimulate detectable expression of RNA specific for gamma 2a or epsilon. These studies demonstrate that: 1) germ-line gamma 1 gene expression is regulated uniquely, 2) germ-line transcription and switch recombination can be dissociated, 3) the germ-line transcription of each IgG isotype has an independent pattern of regulation, and 4) cross-linking of the Ag receptor, by itself, can stimulate small amounts of germ-line CH RNA.
Dengue virus-specific, HLA-B35-restricted, human CD8+ cytotoxic T lymphocyte (CTL) clones. Recognition of NS3 amino acids 500 to 508 by CTL clones of two different serotype specificities.
Authors: Livingston P G , Kurane I , Dai L C , Okamoto Y , Lai C J , Men R , Karaki S , Takiguchi M , Ennis F A
Published: December 2022

Dengue virus infections are a major cause of morbidity and mortality in tropical and subtropical areas of the world. We analyzed dengue virus-specific CD8+ CD4- CTL at the clonal level to further understand the role of CD8+ CTL in dengue virus infections. Dengue virus-specific CD8+ CTL clones were established from lymphocytes of a dengue 4-immune adult. Three patterns of dengue serotype specificities were identified: 1) specific for dengue 4, 2) cross-reactive for dengue 2 and dengue 4 (subcomplex-specific); and 3) cross-reactive for all four dengue virus serotypes. Three dengue 4-specific clones and one dengue 2/dengue 4 cross-reactive clone were further analyzed. All four of the clones were HLA-B35 restricted and recognized NS3. The epitopes were mapped to amino acids (aa) 483 to 618 of NS3. The epitope was then defined by using synthetic peptides. Three dengue 4-specific clones and one dengue 2/dengue 4 cross-reactive clone recognized the same peptide (TPEGIIPTL) encompassing aa 500 to 508 of dengue 4 NS3. The peptide encompassing aa 500-508 of dengue 2 NS3 was recognized by a dengue 2/dengue 4 cross-reactive clone but was not recognized by the dengue 4-specific clones. Dengue 4-specific and dengue 2/dengue 4 cross-reactive clones used different TCR. These results indicate that CD8+ CTL clones that use different TCR and demonstrate two distinct serotype specificities recognize the same 9-mer peptide in the context of HLA-B35.
Chemotactic agonists induce cytokine generation in eosinophils.
Authors: Miyamasu M , Hirai K , Takahashi Y , Iida M , Yamaguchi M , Koshino T , Takaishi T , Morita Y , Ohta K , Kasahara T
Published: December 2022

Recent studies have shown that eosinophils are capable of generating and releasing cytokines, providing a novel biologic aspect of eosinophils for regulating allergic inflammation by an autocrine or paracrine mechanism. Eosinophils synthesize various cytokines; however, the physiologic stimuli that trigger eosinophils to generate cytokines have not been fully elucidated. We examined the effect of chemotactic agonists on eosinophil cytokine generation by employing the determination of IL-8 as the main parameter. Both C5a and FMLP stimulated eosinophils to release IL-8, whereas platelet-activating factor and C-C chemokines did not exert any significant effects. On a molar basis, C5a was two orders of magnitude more potent than FMLP. The generation of IL-8 by chemoattractants was absolutely dependent on the presence of cytochalasin B. Pertussis toxin completely attenuated C5a- and FMLP-induced IL-8 production, indicating the involvement of pertussis toxin-sensitive G-proteins in the signal-transduction process leading to these responses. Experiments of in situ hybridization and PCR amplification revealed that both C5a and FMLP promoted eosinophil IL-8 production through transcriptional gene activation. Pyrrolidine dithiocarbamate completely abrogated chemoattractant-induced IL-8 production, indicating the involvement of NF-kappa B in the cytoplasmic/nuclear signal-transduction process. Furthermore, chemoattractant-induced cytokine production was not limited to IL-8; C5a and FMLP but not platelet-activating factor induced significant secretion of granulocyte-macrophage-CSF from eosinophils. These results indicate that C5a and FMLP stimulate eosinophils to elaborate cytokines, which could be an important mechanism in the regulation of allergic inflammation.
Selection of oligoclonal V beta-specific T cells in the intradermal response to Kveim-Siltzbach reagent in individuals with sarcoidosis.
Authors: Klein J T , Horn T D , Forman J D , Silver R F , Teirstein A S , Moller D R
Published: December 2022

Sarcoidosis is a multiorgan granulomatous disorder of unknown etiology characterized by noncaseating granulomas in involved tissues. A positive Kveim-Siltzbach reaction is a granulomatous response to an intradermal injection of a suspension of sarcoid tissue extract in individuals with sarcoidosis. The protracted time course and granulomatous features of this reaction have a striking resemblance to the Mitsuda reaction in tuberculous leprosy, which suggests that the Kveim-Siltzbach reaction is a response to an unknown Ag(s). To evaluate whether this reaction is Ag-driven, an analysis of the TCR V beta repertoire in 15 Kveim-Siltzbach reaction sites was performed using a PCR technique and primers specific for 20 V beta gene families. Results of this analysis demonstrated a pattern of V beta expression dominated by expression of V beta 2, V beta 3, V beta 6, or V beta 8 to levels > 20% of total V beta gene expression in nine of 15 individuals. Analysis of paired biopsy and blood specimens revealed a preferential expression of specific V beta genes, such as V beta 3, V beta 5, and V beta 8, at sites of Kveim-Siltzbach reactions to levels four to seven times that of the corresponding peripheral blood. Sequence analysis demonstrated that preferential expression of specific V beta genes at Kveim-Siltzbach reaction sites is oligoclonal. Furthermore, the dominant V beta 8 sequence present at one of the reaction sites contained a sequence motif in the variable-diversity-joining junctional region previously identified in sarcoid lung and blood T cell populations. These results suggest that the Kveim-Siltzbach reaction is characterized by a limited TCR beta-chain repertoire consistent with an Ag-driven T cell immune response.
Interaction between CD40 and its ligand gp39 in the development of murine lupus nephritis.
Authors: Mohan C , Shi Y , Laman J D , Datta S K
Published: December 2022

We investigated the role of gp39-CD40 interaction in the development of glomerulonephritis in lupus mice. In contrast to normal mice, lupus mice had much higher percentages of intensely gp39+ T cells in their spleens even at the preautoimmune age of 1 mo, and the further increase in gp39 expression by anti-CD3 Ab stimulation was markedly greater in lupus T cells. The pathogenic autoantibody-inducing ability of Th clones and splenic Th cells from lupus mice could be blocked in vitro by anti-gp39 Ab. Acceleration of lupus nephritis by the transfer of pathogenic autoantibody-inducing Th clones in vivo could also be completely blocked by anti-gp39 Ab. Surprisingly, a brief treatment of lupus mice with anti-gp39 Ab had a sustained beneficial effect on their spontaneous disease long after the Ab had been cleared from their systems. Only three injections of anti-gp39 Ab given to prenephritic lupus mice at 3 mo of age markedly delayed and reduced the incidence of lupus nephritis up to 12 mo of age by which time almost all the control mice had developed severe glomerulonephritis. Remarkably, pathogenic Th cells were left intact in these anti-gp39-treated mice but their B cells could not produce pathogenic autoantibodies even 9 mo after the therapy. Our studies suggest that blocking the interaction between gp39 on pathogenic Th cells and CD40 on lupus B cells at a crucial window of time delays the expansion autoimmune memory B cells resulting in long-term therapeutic benefits.
Intracellular signaling events during positive and negative selection of CD4+CD8+ thymocytes in vitro.
Authors: Anderson G , Anderson K L , Conroy L A , Hallam T J , Moore N C , Owen J J , Jenkinson E J
Published: December 2022

We have used in vitro models of thymocyte positive and negative selection in conjunction with selective inhibitors of the TCR-mediated signaling cascade to investigate the intracellular signaling events that mediate these processes. We report that Ro 31.8425, a potent and selective inhibitor of protein kinase C, which blocks the activation of mature T cells in a dose-dependent fashion, has no effect on either positive or negative selection of CD4+8+ thymocytes. In contrast, cyclosporin A fails to prevent negative selection, but inhibits positive selection through a direct effect on developing thymocytes, rather than through the perturbation of stromal cell support. Thus, our data suggest that positive and negative selection may operate via distinct intracellular signaling pathways.
IL-8-induced signal transduction in T lymphocytes involves receptor-mediated activation of phospholipases C and D.
Authors: Bacon K B , Flores-Romo L , Life P F , Taub D D , Premack B A , Arkinstall S J , Wells T N , Schall T J , Power C A
Published: December 2022

We have characterized the IL-8-induced signal transduction processes in T lymphocytes. A basal level of IL-8 receptor expression was shown on mixed PBL, as identified by using phycoerythrin (PE)-coupled IL-8, and this expression was increased following IL-2 stimulation. Scatchard analysis of T cells revealed competitive binding of IL-8 with a Kd of 0.55 nM, with approximately 1200 receptors per cell, on freshly isolated T cells. After 24 h in culture following purification, reverse transcriptase PCR (RT-PCR) analyses show the mRNA for only the type B IL-8R on these cultured T lymphocytes and the cell line MOLT-4. Stimulation of T lymphocytes or T cell clones with IL-8 led to generation of inositol trisphosphate and calcium flux. In addition, when T cells were prelabeled with [3H]oleic acid, IL-8 caused a long lasting, time- and dose-related increase in [3H]phosphatidylethanol (PtE), indicating activation of phospholipase D (PLD). By contrast, this IL-8-dependent PLD activity was undetectable in IL-8-stimulated neutrophils. PLD activation appeared to be downstream of protein kinase C, because several inhibitors abrogated the increase in [3H]PtE, whereas guanosine-5'-O-(3-thiotriphosphate (GTP(gamma)S) and inositol trisphosphorothioate (IP3S3) both increased the generation of [3H]PtE. Together, these results demonstrate that the IL-8RB receptor is sufficient to mediate phospholipase C (PLC) and PLD activation in T lymphocytes, but not in neutrophils, and indicate an important difference in receptor usage and signal transduction pathways between IL-8-stimulated lymphocytes and neutrophils.
Distinct sequence motifs within the cytoplasmic domain of the human IL-4 receptor differentially regulate apoptosis inhibition and cell growth.
Authors: Deutsch H H , Koettnitz K , Chung J , Kalthoff F S
Published: December 2022

Hematopoietin receptors generally function as multimeric complexes composed of a unique ligand-binding chain and a second component often shared between several members of this receptor family. To better understand the signal transduction of the human IL-4 receptor (hIL4R), we analyzed the functionality of targeted mutations in two cytoplasmic regions of the ligand-binding hIL4R chain that we previously identified to be necessary for growth mediation in factor-dependent murine Ba/F3 cells. Here, we provide evidence that transient inhibition of apoptotic death of Ba/F3 cells and the competence to proliferate indefinitely depend on separated and distinct sequence motifs of the hIL4R. In particular, hIL4R constructs with a truncation of the recently described gp130 box1 from P242 to K264, or a deletion of the acidic region between S330 and S365, fail to stimulate growth or to mediate the inhibition of apoptosis. hIL4R bearing a point mutation within the gp130 box1 (P242S) is defective for growth stimulation but still signals the transient inhibition of apoptotic cell death and the induction of c-myc RNA. A third region required for IL4-mediated cell growth is localized between T462 and S476 and includes the sequence NPAY previously described to serve as interaction motif in signaling of epidermal growth factor and insulin receptors. Conversion of Y472 into F472 within the latter hIL4R motif affects the competence of stably transfected BA/F3 cells to proliferate indefinitely in the presence of hIL4. Sequences C-terminal of S476 are not essential for growth stimulation of BA/F3 transfectants.
Functional maturation of human naive T helper cells in the absence of accessory cells. Generation of IL-4-producing T helper cells does not require exogenous IL-4.
Authors: Kaliński P , Hilkens C M , Wierenga E A , van der Pouw-Kraan T C , van Lier R A , Bos J D , Kapsenberg M L , Snijdewint F G
Published: December 2022

In the human model, requirements for the primary onset of IFN-gamma and IL-4 production in maturing T helper lymphocytes were compared. Stimulation of freshly isolated CD4+CD45RA+ naive Th cells with immobilized CD3 mAb in the presence of exogenous IL-2 resulted in the proliferative response of this subset, which was equal to or higher than CD4+CD45R0+ memory Th cells. Throughout the first 6 days after this mode of stimulation, naive Th cells did not secrete IL-4 and produced only small amounts of IFN-gamma, whereas high amounts of both lymphokines were secreted by stimulated autologous memory Th cells. Under these conditions, naive Th cells acquired the CD45RA-CD45R0+ memory phenotype. After restimulation, such in vitro-generated CD45R0+ cells produced high amounts of IFN-gamma but, despite the full phenotype conversion, they produced only trace amounts of IL-4. In contrast, when the primary stimulation and the expansion of cells proceeded in the presence of IL-1 beta or CD28 mAb, both IFN-gamma and IL-4 were produced after restimulation, in similar amounts compared with those produced by memory Th cells. The effect of IL-1 beta and CD28 signaling could not be obtained by the administration of exogenous IL-4 nor could the onset of IL-4 production be prevented by the presence of a neutralizing anti-IL-4 Ab in primary cultures. These data show that the development of human IL-4-producing Th cells can proceed in the absence of any pre-existing source of IL-4 and can be driven solely by the APC-related signals.
The human anti-murine xenogeneic cytotoxic response. II. Activated murine antigen-presenting cells directly stimulate human T helper cells.
Authors: Lucas P J , Bare C V , Gress R E
Published: December 2022

Generation of a human T cell anti-murine xenogeneic response has previously been shown to be dependent on presentation of murine Ag by human APC. We have undertaken a series of experiments to better delineate the cellular defects that prevent effective production of IL-2 by human T cells upon direct exposure to murine stimulator populations. It was found that although resting human T cells cannot respond effectively to resting murine APC, they can respond to activated murine stimulator populations. Such APC activation could be mediated by murine granulocyte-macrophage-CSF or LPS that were associated with increased expression of B7-2 on the xenogeneic stimulating cell populations. Blocking studies with Ab provided further evidence that costimulation through CD28 played a critical role in the stimulation of human T cells by activated murine-stimulator cells in the production of IL-2. These results demonstrate the usefulness of this xenogeneic system in understanding human T cell-APC interactions and defining minimally sufficient T cell activation requirements. They further delineate the cellular level of deficient activation in the xenogeneic stimulation of human T cells by murine cell populations, and identify the potential importance of CD28/CTLA4 and its ligands in xenogeneic responses. These observations and concepts have implications for clinical efforts in xenografting.
Human placental HLA-G expression is restricted to differentiated cytotrophoblasts.
Authors: McMaster M T , Librach C L , Zhou Y , Lim K H , Janatpour M J , DeMars R , Kovats S , Damsky C , Fisher S J
Published: December 2022

Human placental trophoblasts lie at the maternal-fetal interface, a position in which they could play an important role in maternal tolerance of the fetal semi-allograft. Central to this hypothesis is their unusual MHC class I expression: they suppress class Ia production while expressing HLA-G, a class Ib molecule. We investigated human trophoblast HLA-G protein production in vivo and in vitro. We first used a synthetic peptide corresponding to the variable sequence of the alpha 1 domain to produce mAbs that recognized HLA-G. Ab specificity was demonstrated by immunoaffinity purification of a single protein with the same molecular mass (38 kDa) as HLA-G from choriocarcinoma cells. Use of these Abs to stain tissue sections of the maternal-fetal interface containing cytotrophoblasts in all stages of differentiation showed that HLA-G is expressed only by cytotrophoblasts that invade the uterus. Our previous in vitro studies showed that when early-gestation cytotrophoblast stem cells are cultured, they differentiate rapidly along the invasive pathway, as demonstrated by their expression of stage-specific markers. Here we show they also up-regulate HLA-G production. Cytotrophoblasts from term placentas, which have reduced invasive capacity in vitro, also had decreased ability to up-regulate HLA-G protein expression. We detected high levels of HLA-G mRNA in cytotrophoblasts isolated from first- and second-trimester placentas, but only trace amounts in term cells. Taken together, these results suggest that HLA-G production is a critical component of cytotrophoblast differentiation along the invasive pathway.