bluuebunny/crossref_metadata_2025 huggingface dataset:
Anti-HLA class I antibodies inhibit the T cell-independent proliferation of human B lymphocytes.
Authors: Taylor D S , Nowell P C , Kornbluth J
Published: December 2022

The role of major histocompatibility complex-encoded class I molecules in the proliferation of human B lymphocytes is presently unclear. This question was addressed by investigating the effect of three individually derived anti-HLA class I monoclonal antibodies (mAb) on purified human B cells (less than 1.5% T cells) stimulated by either the T-independent mitogen Staphylococcus aureus or the phorbol ester, phorbol-12-myristate-13-acetate. The three anti-HLA class I antibodies, whether specific for gene products of the HLA-A locus (mAb 131), HLA-B locus (mAb 4E), or HLA-A, -B, and -C locus (mAb W6/32), inhibited S. aureus-induced proliferation by 70 to 90%. This inhibition was significant over a 5-day culture period, was not altered by the addition of exogenous interleukin 2 or B cell growth factor, and was not due to nonspecific cytotoxicity. In addition, the inhibition of proliferation was unchanged when the mAb were added 12 hr after the initiation of culture. The proliferative response was not affected by either of the control antibodies OKB7 and R3-367. In contrast with S. aureus-stimulated B cells, phorbol-12-myristate-13-acetate-induced proliferation was resistant to the inhibitory activity of HLA class I-specific antibodies. These results suggest that HLA class I molecules are involved in human B lymphocyte proliferation and may regulate a critical event preceding the upregulation of protein kinase C activity.
The phenotype of thymocytes derived from a single clonogenic precursor.
Authors: Ezine S , Jerabek L , Weissman I
Published: December 2022

Clonogenic repopulation of the thymus of thymus-homing bone marrow cells leads to intrathymic populations representing all four major Lyt-2- and L3T4-defined phenotypes. Although all four phenotypes may be represented in a single clone, quite often a striking bias in the proportion of L3T4 single positive to Lyt-2 single positive cells may exist within a clone, but not in the host thymocytes in general. Because at any one time these clones may be located in specific subregions of the thymus (specifically cortex only, medulla only, or cortex and medulla), we propose the hypothesis that different microenvironments in the thymus might, in fact, be responsible for the predominant maturation of different single positive mature thymic subsets.
Monoclonal antibodies to adenosine receptor by an auto-anti-idiotypic approach.
Authors: Ku H H , Cleveland W L , Erlanger B F
Published: December 2022

Monoclonal antiadenosine receptor antibodies have been raised by an auto-anti-idiotypic approach. BALB/c mice were immunized with adenosine 6-aminocaproyl-bovine serum albumin. Hybridoma cell lines were raised and lines that secreted antibodies that bound to rabbit antiadenosine antibodies were obtained. Two such monoclonal antibodies, AA18 and AA21, were studied in detail and found to be directed at adenosine receptors by the following criteria. They inhibited the binding of [3H] adenosine to rabbit antiadenosine antibodies that had binding characteristics similar to those of adenosine receptors. They bound to rat brain membranes and binding could be inhibited by N6-cyclohexyladenosine and L-N6-phenylisopropyladenosine, both adenosine receptor agonists. They also inhibited the binding of [3H]L-N6-phenylisopropyladenosine to rat brain membranes. In functional assays, they inhibited adenylate cyclase of rat brain membranes, but had no effect on adenylate cyclase of rat hepatic membranes, indicating that they mimic agonists of the A1 receptor, therefore, carrying an "internal image" of the adenosine molecule. When adenosine receptors of rat brain membranes were solubilized with 1% cholic acid, partially purified on an adenosine 6-aminocaproyl AH-Sepharose column and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, both AA18 and AA21 recognized a 62,000 band under nonreducing conditions, and a major band of 36,000 under reducing conditions. We conclude that the auto-anti-idiotypic route has yielded specific antibodies that recognize the A1 adenosine receptor.
Mutant human B cell lines deficient in class II major histocompatibility complex transcription.
Authors: Calman A F , Peterlin B M
Published: December 2022

By mutagenesis and selection, two independent Ia- mutant cell lines derived from the Ia+ human B cell line Raji were isolated. One mutant failed to express detectable surface HLA-DR and HLA-DQ molecules, while the other expressed very low levels of these molecules. mRNA encoding Ia molecules was correspondingly absent or reduced in these cell lines. In contrast, levels of class I major histocompatibility complex (MHC) mRNA and protein were normal in these cells. The class II MHC genes of the mutant cells were grossly intact, suggesting that the defective expression of these genes was due to an absent or abnormal trans-acting regulatory factor. In vitro nuclear run-on transcription assays and measurement of class II MHC mRNA stability suggested that the specific defect in class II MHC gene expression in both mutant cell lines was at the level of transcription initiation.
Interleukin 1 stimulates proliferation of a nontransformed T lymphocyte line in the absence of a co-mitogen.
Authors: Lacey D L , Chappel J C , Teitelbaum S L
Published: December 2022

Although interleukin (IL) 2-responsive T cell lines provide an opportunity to study the cellular effects of this lymphokine on homogeneous T lymphocyte populations, T cell clones which proliferate in response to IL-1 alone have not been available. We have isolated from cultures of the nontransformed murine T helper cell line, D10 . G4 . 1, a variant (MD10 cells) which proliferates (no lectin or antigen needed) in response to IL-1 alone. The MD10 cells are markedly sensitive to either murine or human recombinant IL-alpha (HrIL-1 alpha) with half-maximal responses observed at monokine concentrations as low as 0.4 X 10(-12) M or 0.8 U/ml, respectively. MD10 cells show the maximal IL-1 effect at 72 hr where the response exceeds the base line by 100-fold (approximately 3,000----300,000 cpm of [3H]thymidine). Whereas both HrIL-2 and purified murine B cell-stimulatory factor 1 (MpBSF-1) induce MD10 proliferation, the maximal response to either is much lower (HrIL-2: 50X baseline; MpBSF-1: less than 20X base line) than to IL-1. Conditioned media from control, concanavalin A-, or IL-1-treated MD10 cells fail to stimulate CTLL or HT-2 cell proliferation alone or inhibit CTLL mitogenesis in the presence of added HrIL-2. Furthermore, monoclonal antibodies to BSF-1 fail to inhibit IL-1-stimulated MD10 replication, and neither HT-2 nor CTLL cells proliferate despite direct cell-to-cell contact with IL-1-treated MD10 cells. When combined, IL-1 (10(-13), 10(-12) M) and IL-2 (10(-13) to 10(-10) M) act synergistically in their MD10 cell growth-promoting effects. MD10 proliferation induced by either IL-1 or IL-2 is relatively resistant to cyclosporine A, with the ID50 of cyclosporine for both IL-1- and IL-2-exposed MD10 cells (ID50 5000 ng/ml) exceeding that for concanavalin A-activated splenocytes (ID50 20 ng/ml) by 2 to 3 orders of magnitude. Finally, MD10 cells bear the L3T4 antigen, IL-2 receptors, and the same clonotypic antigen receptor as the parent clone as recognized by monoclonal antibody 3D3. These data suggest that, in respect to this particular T cell line, IL-1 is directly growth-promoting or, alternatively, induces the production of undetectable, intermediate growth factor(s) resistant to inhibition by cyclosporine A.
Interleukin 5 and interleukin 4 produced by Peyer's patch T cells selectively enhance immunoglobulin A expression.
Authors: Murray P D , McKenzie D T , Swain S L , Kagnoff M F
Published: December 2022

Considerable evidence suggests that the high frequency of B cells committed to the IgA isotype in Peyer's patches is regulated by T lymphocytes. To understand more accurately the mechanism of this immunoregulation, an autoreactive T cell line from Peyer's patches was generated by culturing L3T4+ Peyer's patches T cells with syngeneic B cell blasts. The resulting T cell line, designated PT-1, and a clone derived from this line, PT-1.14, stimulated immunoglobulin secretion in spleen B cells with a preferential enhancement of IgA and IgG1 isotypes. Supernatant derived from concanavalin A-stimulated PT-1 or PT-1.14 cells could also enhance IgA secretion if spleen B cells were preactivated with lipopolysaccharide. Peyer's patches T cell supernatant did not contain IgA-specific binding factors. PT-1 supernatant scored positive in lymphokine assays for interleukin (IL)-2, IL-4 (B cell stimulatory factor 1), IL-5 (B cell growth factor II), and interferon-gamma, whereas PT-1.14 supernatant was positive for IL-4 and IL-5 and negative for IL-2 and interferon-gamma. Only IL-5 enhanced IgA secretion in lipopolysaccharide-activated B cells and this response was increased two- to three-fold by IL-4. These results suggest that the type 2 T helper subset which produces both IL-5 and IL-4 plays a primary role in regulating IgA expression.
Cooperation between CD16(Leu-11b)+ NK cells and HLA-DR+ cells in natural killing of herpesvirus-infected fibroblasts.
Authors: Oh S H , Bandyopadhyay S , Miller D S , Starr S E
Published: December 2022

We reported previously that natural killer (NK) cell-mediated lysis of cytomegalovirus-infected targets requires the presence of HLA-DR+ nonadherent peripheral blood mononuclear cells (NPBMC). In the present study we determined whether NK cell-mediated lysis of other herpesvirus-infected targets also requires HLA-DR+ cells. Depletion of either cluster designation (CD)16+ NK cells or HLA-DR+ cells from NPBMC significantly reduced their ability to lyse herpes simplex virus (HSV)- and varicella-zoster virus (VZV)-infected fibroblasts. When CD16- and HLA-DR- populations were mixed, lysis of both HSV- and VZV-infected targets was virtually completely restored, indicating that NK cells and HLA-DR+ cells were required for lysis to occur. Cell-free supernatants, obtained by incubating NPBMC or CD16- cells with HSV-o-VZV-infected targets, contained antiviral activity and stimulated HLA-DR-cells to mediate lysis of corresponding virus-infected targets. The addition of anti-interferon-alpha to supernatants abolished their ability to stimulate lysis. Thus, secretion of interferon-alpha by HLA-DR+ cells contributes to NK cell-mediated lysis of herpesvirus-infected targets.
Inverse Ir gene control of the antibody and T cell proliferative responses to human basement membrane collagen.
Authors: Tite J P , Foellmer H G , Madri J A , Janeway C A
Published: December 2022

The immune response to pepsin-soluble human basement membrane-derived type IV collagen in mice has been characterized. Both T cell proliferative and antibody responses have been shown to be under major histocompatibility complex (MHC)-linked Ir gene control in inbred and MHC congenic mice. However, unlike previous examples studied, this response shows a separation of these two types of immunologic responsiveness. Only mice having I-As give potent in vitro T cell proliferative responses to type IV collagen whereas all mice except those having I-As give high antibody responses to this antigen. In (I-As X I-Anon-s) F1 mice, the T cell proliferative response was dominant, whereas antibody responses were markedly reduced compared with the responder parent. Given the recent demonstration that class II MHC-restricted, L3T4+ T cells can be divided into two sets, one of which helps for antibody responses and the other of which produces interleukin 2 and can also suppress such responses, it seems likely that these data can be accounted for on the basis of differential activation by this antigen of these two cell sets in mice of different MHC genotypes.
Immune regulation by platelet-activating factor. I. Induction of suppressor cell activity in human monocytes and CD8+ T cells and of helper cell activity in CD4+ T cells.
Authors: Rola-Pleszczynski M , Pouliot C , Turcotte S , Pignol B , Braquet P , Bouvrette L
Published: December 2022

Platelet-activating factor (PAF) is a powerful mediator of inflammation. We have recently described a potential role for PAF in immune reactions, as it inhibits T cell proliferation and IL-2 production in response to mitogens. To further define the mechanism through which this inhibition is exerted, we used a coculture system in which PBML are preincubated with increasing concentrations of PAF for 24 h, followed by washing, treatment with mitomycin C and addition to fresh autologous PBML stimulated with PHA. In this context, a significant (40 to 60%) inhibition of proliferation was observed. In parallel, PAF-pre-treated cells induced a reduction (30 to 50%) of IL-2 production by PHA-stimulated lymphocytes. The PAF receptor antagonist BN52021 could partially block the PAF-induced suppressor cell activity, but also showed some suppressor cell-inducing properties of its own (20 to 30%). The expression of suppressor cell function during the co-culture could be partially abrogated by the inclusion of indomethacin, suggesting that cycloxygenase metabolites of arachidonic acid were involved in this phase of suppression. When PBML were fractionated into monocytes, lymphocytes, or T cell subsets before pre-incubation with PAF, indomethacin-sensitive suppressor cell function was generated in the monocyte population. Monocyte-depleted lymphocytes showed slight helper effect, whereas CD8+ T cells were induced to become indomethacin-resistant suppressor cells. CD4+ T cells, in contrast, were activated to exert very marked helper effect. When incubated with PAF for 24 h, monocyte-depleted lymphocytes showed a 30% decrease in CD4+ T cell numbers and a 50% increase in CD8+ T cell numbers. Our data suggest a novel immunoregulatory role for PAF and potentially important interactions of this lipid mediator of inflammation with lymphocyte and monocyte functions.
Impaired clonal expansion in athymic nude CD8+CD4- T cells.
Authors: Kung J T
Published: December 2022

A comparative study of the phenotype and immune functions of highly purified CD8+CD4- T cells obtained from the spleen and thymus of normal mice and from the spleen of athymic nude mice was conducted. Of seven individual normal and nude mice examined, the range of V beta 8+ cells among CD8+ T cells was a heterogeneous 4.3 to 30.5% for athymic nude mice and a much more uniform spread from 14.7 to 18.5% for normal mice. In six of the seven nude mice examined, the fraction of V beta 8+ cells was below the lower limit of the V beta 8 distribution in normal mice. However, one of the seven nude mice contained nearly twice the percentage of normal V beta 8+ cells. A reduction in the density of V beta 8 as well as CD3 Ag expression was also observed in athymic CD8+CD4- cells although an Ly-6-linked Ag, B4B2 displayed a highly increased expression. Considering the battery of Ag analyzed in entirety, athymic CD8+CD4- T cells were clearly distinct from their "counterpart" CD8+CD4- T cells isolated from either thymus or spleen of normal (euthymic) mice. Anti-CD3-mediated triggering of the TCR:CD3 complex caused extensive clonal proliferation in cultures to which single responding CD8+ T cells had been deposited. Under identical conditions, however, anti-CD3 caused little, if any clonal expansion in CD8+ cells from athymic nude mice. Highly purified athymic CD8+CD4- cells produced readily detectable IL-2R expression and IL-2 synthesis and secretion upon stimulation by anti-CD3 and by Con A. Production of IL-2 by purified athymic CD8+CD4- cells was due to CD8+CD4- cells and not due to a minor population of contaminating CD8- cells as anti-CD8 + C treatment completely abrogated the ability of athymic CD8+CD4- cells to produce IL-2. Despite IL-2 production and IL-2R expression by athymic nude CD8+CD4- T cells in response to anti-CD3 and to Con A, an impaired proliferative response followed.
Dual regulation of resistance against Toxoplasma gondii infection by Lyt-2+ and Lyt-1+, L3T4+ T cells in mice.
Authors: Suzuki Y , Remington J S
Published: December 2022

Studies were performed to attempt to define the T cell subset responsible for resistance to Toxoplasma gondii. A temperature-sensitive mutant (ts-4) strain of T. gondii was used for immunization because it causes infection but does not persist in the host. Immunization with this strain induced marked resistance against lethal challenge infection with virulent strains of T. gondii in mice. The resistance could be transferred to normal recipient mice by i.v. injection of spleen cells from ts-4-immunized mice. Marked inhibition of cyst formation in the recipient mice was also noted. The protective activity of immune spleen cells was removed by pretreatment of the spleen cells with anti-Thy-1.2 and C, indicating that T cells are responsible for the observed protection. Pretreatment of immune spleen cells with anti-Lyt-2.2 and C completely ablated their protective effect; pretreatment with anti-Lyt-1.2 or anti-L3T4 and C had lesser effects on their ability to transfer resistance. The effect of anti-Lyt-1.2 was the same as that obtained with anti-L3T4. This suggested that one T cell subset that is partially responsible for protection has both Lyt-1.2 and L3T4 markers on the cell surface. These results indicate that there are substantial roles for both the Lyt-2+ and Lyt-1+, L3T4 T cell subsets in dual regulation of resistance against toxoplasma infection and that Lyt-2+ T cells are the principal mediator of the resistance.
Thymus-dependent and thymus-independent developmental pathways for peripheral T cell receptor-gamma delta-bearing lymphocytes.
Authors: Pardoll D M , Fowlkes B J , Lew A M , Maloy W L , Weston M A , Bluestone J A , Schwartz R H , Coligan J E , Kruisbeek A M
Published: December 2022

To elucidate the developmental pathways of T cells that bear TCR gamma delta, we have analyzed the kinetics of expression and biochemical characteristics of gamma delta receptors in the thymus and spleen of normal and athymic (nude) mice, as well as nude mice engrafted with neonatal thymuses. TCR gamma delta-bearing thymocytes and splenocytes have a CD4-8- phenotype, and both populations express products of the C gamma 1 locus. TCR gamma delta-bearing cells develop in the thymus before their appearance in the spleen. Young nude mice have no detectable TCR gamma delta-bearing cells in their spleens. When young nude mice are given thymus grafts, TCR gamma delta-bearing cells of host origin first develop in the engrafted thymus, followed by their appearance in the spleen. In the absence of a thymus graft, the spleens of old nude mice eventually develop small numbers of TCR gamma delta + cells, as well as TCR alpha beta + cells. These results demonstrate that there is a major thymic-dependent pathway for TCR gamma delta expression, as well as a minor thymic-independent pathway seen in older nude mice. The development of TCR gamma delta + cells in the thymus before their appearance in the spleen, both in normal ontogeny as well as in the thymus-engrafted nude mouse model, suggests that thymic TCR gamma delta + cells are precursors of the thymus-dependent population of peripheral TCR gamma delta + cells.
Immunomodulating properties of a novel series of protein kinase C activators. The bryostatins.
Authors: Trenn G , Pettit G R , Takayama H , Hu-Li J , Sitkovsky M V
Published: December 2022

Low concentrations of the protein kinase C activators, bryostatins 1 and 2 synergized with recombinant B cell stimulatory factor-1 in triggering differentiation (granule enzyme expression) and cytotoxic T lymphocyte (CTL) development in naive, resting lymph node T cells. Bryostatin greatly enhances efficiency of recombinant interleukin-2 in triggering development of in vivo primed CTL during in vitro incubation, thereby providing experimental evidence for the efficacious use of lower concentrations of recombinant interleukin-2 for in vivo tumor rejection studies. Both bryostatins 1 and 2 were able to trigger cytotoxicity of CTL clones against antigen-nonbearing target cells and inhibited CTL cytotoxicity against Ag-specific target cells. Bryostatin 1 and 2 synergize with Ca2+ ionophores in triggering the exocytosis of cytolytic granules from CTL at very low concentrations. In view of the lack of tumor promoting activity of the bryostatins, the possible use of these agents in vivo is discussed.
The structure of the murine Fc receptor for IgG. Assignment of intrachain disulfide bonds, identification of N-linked glycosylation sites, and evidence for a fourth form of Fc receptor.
Authors: Hibbs M L , Classon B J , Walker I D , McKenzie I F , Hogarth P M
Published: December 2022

The Fc receptor (Fc gamma R) of the murine macrophage cell line, J774, was purified by immunoaffinity chromatography then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino-terminal sequencing. FcR material judged to be pure by these criteria was digested with a number of enzymes to identify the cysteine residues engaged in disulfide bonds within the native structure. The results clearly establish that the mouse macrophage Fc gamma R contains two intrachain disulfide bonds, each of which connects adjacent cysteine residues within the two putative extracellular domains of the molecule. In addition, each disulfide-bonded domain was shown to contain two authentic sites of N-linked glycosylation. Extensive peptide sequencing resulted in the unexpected identification of peptide fragments from a fourth Fc gamma R whose sequences were highly homologous to sequences surrounding the two Cys residues in the amino-terminal domain of both alpha and beta 1 Fc gamma R. The fourth Fc gamma R contains a disulfide-bonded amino-terminal domain similar to beta 1 Fc gamma R.
Herpesvirus ateles immortalizes in vitro a CD3+CD4+CD8+ marmoset lymphocyte with NK function.
Authors: Kiyotaki M , Solomon K R , Letvin N L
Published: December 2022

Herpesvirus ateles, nonpathogenic in its natural host, the spider monkey, induces a fatal lymphoproliferative syndrome in a variety of New World primate species. Whereas the closely related New World primate virus Herpesvirus saimiri immortalizes in vitro common marmoset lymphocytes that express a TCR and phenotypically are CD4-CD8+NKH1+, we now show that H. ateles-immortalized marmoset lymphocytes are CD3+ and CD4+. Furthermore, these CD4+ lymphocytes coexpress CD8 and NKH1. The NK function of cloned H. ateles-immortalized lymphocyte populations is proportional to the extent to which they express the CD8 antigen. These studies illustrate an interesting example of restricted viral tropism and may indicate a potentially useful means of generating phenotypically stable, functionally competent, cloned lymphocyte populations for study.
Immunoglobulins and complement in demyelination induced in mice by Theiler's virus.
Authors: Rodriguez M , Lucchinetti C F , Clark R J , Yakash T L , Markowitz H , Lennon V A
Published: December 2022

Intracerebral inoculation of Theiler's murine encephalomyelitis virus (TMEV) produces chronic demyelination and persistent infection in the central nervous system (CNS) of susceptible SJL mice. This series of experiments examined the contribution of humoral immunity and C to myelin destruction. As in multiple sclerosis, mice persistently infected with TMEV had elevated levels of IgG and oligoclonal bands in the cerebrospinal fluid (CSF). Immunoblot studies revealed that even in animals exhibiting profound demyelination, IgG in the serum and CSF was directed primarily at virus antigen rather than at normal myelin components. Inflammatory cells positive for Ig were distributed mainly around blood vessels, but occasionally they infiltrated the spinal cord parenchyma. Rare examples of myelin sheaths positive for IgG were found by immunoelectron microscopy in spinal cord sections from infected mice; the third component of complement (C3) was commonly found in the walls of CNS blood vessels but not on myelin. Neither serum nor CSF IgG from infected mice bound to myelin sheaths or other CNS components in sections of normal syngeneic spinal cord. There were significantly more demyelinating lesions in infected mice depleted of C components with cobra venom factor. These data do not support a humoral autoimmune basis for the CNS demyelination that occurs in association with persistent TMEV infection. However, the humoral immune response directed at TMEV antigens may either limit virus spread or promote virus persistence.
Keratinocyte-derived granulocyte/macrophage colony-stimulating factor induces DNA synthesis by peritoneal macrophages.
Authors: Chodakewitz J A , Kupper T S , Coleman D L
Published: December 2022

Keratinocytes have been demonstrated to produce a number of cytokines, including growth factors such as the CSF IL-3. Circulating blood monocytes and some elicited macrophages retain a significant proliferative potential in response to colony-stimulating activity. Because a macrophage response is prominent in a variety of cutaneous immune reactions, we have studied the ability of conditioned media (CM) from a transformed murine keratinocyte cell line (PAM 212) and from normal murine keratinocytes to induce growth of peritoneal macrophages. CM from both normal and transformed keratinocyte cultures induces [3H]thymidine incorporation by thioglycollate-elicited, but not resident, peritoneal macrophages. IEF of PAM 212 CM reveals peaks of activity at pI 4.8 and less than or equal to 4.2. Analysis of CM by reversed-phase HPLC demonstrates active fractions that elute at 46 to 48% and 53 to 55% acetonitrile. The Mr of the 46 to 48% acetonitrile factor is 25 to 30 kDa by gel filtration HPLC. Polyclonal anti-granulocyte/macrophage (GM) CSF antibody blocks the induction of macrophage [3H]thymidine incorporation by factors with pI 4.8 and eluting at 46 to 48% acetonitrile but does not reduce the activity of crude CM or the factor eluting at 53 to 55% acetonitrile. Based on both physiochemical criteria and antibody neutralization, keratinocytes produce GM-CSF. Keratinocyte-derived factors, including GM-CSF, may play an important role in regulating cutaneous macrophage responses.
Transcriptional regulation of the interferon-beta 2/B cell differentiation factor BSF-2/hepatocyte-stimulating factor gene in human fibroblasts by other cytokines.
Authors: Walther Z , May L T , Sehgal P B
Published: December 2022

Levels of mRNA for IFN-beta 2/B cell differentiation factor2/hepatocyte-stimulating factor (IFN-beta 2) in confluent quiescent cultures of human diploid fibroblasts (FS-4 strain) are enhanced by TNF, IL-1 alpha and beta, platelet-derived growth factor (PDGF) and IFN-beta 1. Of these cytokines, IL-1 alpha and beta cause a particularly strong increase in the accumulation of IFN-beta 2 mRNA in fibroblasts. We have evaluated whether the IFN-beta 2 gene is regulated at the transcriptional level by using nuclear run-on transcription assays. We observed that the IFN-beta 2 gene is transcribed at a low level in uninduced FS-4 cells and that this transcriptional activity is increased 2- to 3-fold in cycloheximide-treated cells, 20- to 35-fold in IL-1 alpha-treated cells, and 5- to 15-fold in TNF-treated cells. PDGF and IFN-beta 1 enhance transcription across the IFN-beta 2 gene 2- to 3-fold. The enhancing effect of IL-1 alpha on IFN-beta 2 gene transcription, but not that of TNF, PDGF, or IFN-beta 1, is inhibited by cycloheximide, suggesting that newly-synthesized protein is involved in the increase in IFN-beta 2 transcription in response to IL-1 alpha but not in the response to the other stimuli. Furthermore, the enhancement of IFN-beta 2 transcription is sustained for up to 14 h after IL-1 alpha induction but is transient and declines to base line levels within 6 h after TNF addition. These observations suggest that there are important differences in the mechanisms by which IL-1 alpha and TNF increase IFN-beta 2 gene transcription in fibroblasts.
In vivo effects of recombinant IL-2. I. Isolation of circulating Leu-19+ lymphokine-activated killer effector cells from cancer patients receiving recombinant IL-2.
Authors: McMannis J D , Fisher R I , Creekmore S P , Braun D P , Harris J E , Ellis T M
Published: December 2022

This study was designed to isolate and phenotypically characterize lymphokine-activated killer (LAK) cells generated in vivo during administration of high dose rIL-2 to cancer patients. The development of circulating LAK effector cells in these patients was demonstrated by the ability of fresh PBL to exhibit lytic activity against the NK-resistant Daudi cell line and fresh tumor cells without prior in vitro culture with rIL-2. Kinetic studies demonstrated that circulating LAK effector cells are detectable 4 to 6 wk after the initiation of rIL-2 therapy. Cells isolated by FACS revealed that circulating LAK cells are Leu-19+, Leu-17+ but CD5-. We have previously reported that circulating Leu-19+ cells are heterogeneous with regard to the expression of CD16 and CD8. Since sorting of cells expressing Leu-19 and either low quantities of CD8 or CD16 resulted in cytolytic activity in both the positive and negative fractions, these latter two markers do not identify subpopulations of Leu-19+ cells with or without LAK cytolytic activity. Although all LAK cells generated in vivo were Leu-19+, we generated LAK cells from the Leu-19- subpopulation after in vitro culture with rIL-2, suggesting that at least some of in vitro generated LAK cells are derived from Leu-19- precursor cells. These LAK cells did not, however, express the Leu-19 surface marker. Based on the functional data reported in this paper, we conclude that circulating LAK effector cells are a phenotypically heterogeneous population that express surface Ag in association with NK cells and not T lymphocytes.
Subclass of individual IgA-secreting human lymphocytes. Investigation of in vivo pneumococcal polysaccharide-induced and in vitro mitogen-induced blood B cells by monolayer plaque-forming cell assays.
Authors: Heilmann C , Barington T , Sigsgaard T
Published: December 2022

The subclass of individual human IgA B cells was investigated by means of monolayer plaque-forming cell assays permitting analysis of all IgA-secreting cells as well as of cells secreting IgA anti-pneumococcal polysaccharide antibody. Center cells were examined by indirect immunofluorescence staining with mouse mAb against either of the two IgA subclasses as primary antibodies and FITC-conjugated rabbit anti-mouse Ig as the second antibody. Blood lymphocytes spontaneously secreting IgA (mean 399/10(6) mononuclear cells) produced mainly IgA1 (73%). A similar distribution of subclasses was recorded among IgA-secreting blood cells in PWM- and EBV-stimulated cultures. In contrast, a predominance of IgA2 (54%) was found among IgA-secreting cells (2531/10(6)) isolated from the blood 7 days after in vivo stimulation with pneumococcal polysaccharides, and a similar proportion (51%) of IgA2 producing cells was found among IgA anti-pneumococcal polysaccharide-secreting cells. It was thus confirmed that IgA1 is the predominant subclass of blood IgA-secreting cells in general. However, the high percentage of IgA2-secreting cells found after vaccination with pneumococcal polysaccharides suggests that these Ag have an unusually high ability to activate IgA2 B cells, or that the B cells stimulated originate from lymphatic tissues with a high frequency of IgA2 committed cells.
Structure-function relationships for the IL-2 receptor system. V. Structure-activity analysis of modified and truncated forms of the Tac receptor protein: site-specific mutagenesis of cysteine residues.
Authors: Rusk C M , Neeper M P , Kuo L M , Kutny R M , Robb R J
Published: December 2022

IL-2R on activated lymphocytes contain the Tac protein. As part of an effort to characterize this molecule, we examined the structure-activity relationship for each of its 12 Cys residues. A preliminary map of intramolecular disulfide bonding was derived by analysis of cystine-linked enzymatic fragments of the Tac protein. The results indicated that disulfide bonds linked Cys-3 with Cys-147, Cys-131 with Cys-163, and Cys-28,30 with Cys-59,61. The contribution of the Cys residues to an active protein conformation was tested by site-specific mutagenesis, followed by expression of the modified molecules in murine L cells. The results indicated that Cys-192 and -225 could be replaced without affecting ligand binding. In contrast, modification of any of the other 10 Cys residues, either singly or in combinations corresponding to the predicted disulfide bonds, greatly reduced the ability of the corresponding protein to bind IL-2 or either of two mAb (anti-Tac and 7G7/B6) which recognize the Tac protein. Each of the latter mutations also interfered with the molecule's post-translational modification and cell-surface expression. Consistent with these findings, transfection of the L cells with vectors containing truncated Tac cDNA inserts resulted in secretion of Tac fragments capable of ligand binding when the polypeptide chains terminated after Cys-163 (the 10th Cys residue in the full length molecule), but resulted in inactive fragments of Tac which were poorly secreted when they terminated before Cys-163. These findings emphasize the remarkable sensitivity of the active conformation of the Tac molecule to each of the postulated intramolecular disulfide bonds.
Monoclonal antibodies specific for the amastigote stage of Leishmania pifanoi. I. Characterization of antigens associated with stage- and species-specific determinants.
Authors: Pan A A , McMahon-Pratt D
Published: December 2022

Eight mAb were produced against membrane-enriched preparations of Leishmania pifanoi amastigotes either grown in axenic culture (P-1 through P-6) or isolated from macrophage cell culture (P-7 and P-8). Two mAb produced against promastigote membranes (P-9 and P-10) were found to be specific against this stage. Antibodies P-1 through P-8 on analysis by radioimmune binding only reacted with determinants present on amastigotes. mAb P-2, P-4, and P-8 also reacted with Leishmania amazonensis amastigotes but not promastigotes. No cross-reactions were found on any other species of Leishmania or with membranes of Trypanosoma cruzi epimastigotes or amastigotes. An indirect immunofluorescence assay using mAb P-1 through P-8 confirmed the stage specificity and binding to L. pifanoi axenically grown amastigotes, amastigotes within infected hamster tissue, and amastigotes within J774.1 macrophages. When Western blot analysis of amastigote membranes was conducted, one distinct group of molecules associated with L. pifanoi-specific determinants was identified. mAb P-1, P-3, P-5, P-7, and P-8 bound to molecules Mr 43 and 34 kDa. Promastigote-specific mAb P-9 recognized a diffuse pattern from 88 to greater than 200 kDa, and mAb P-10 localized a second class of proteins with Mr53 kDa. On immunoprecipitation of solubilized [35S]methionine-labeled amastigotes, mAb P-2 recognized a doublet of Mr 35 and 33 kDa and another doublet at Mr 17.5 and 13.5 kDa. mAb P-4 and P-7 each precipitated a band at Mr 34 kDa. These studies indicate that antigenically the axenically cultured amastigote is closely related to macrophage-derived amastigote. These mAb and/or purified protein Ag may be useful in studying stage differentiation, monitoring transformation, and for further taxonomic, epidemiologic, and immunologic studies of New World leishmaniasis.
G protein coupling of antigen receptor-stimulated polyphosphoinositide hydrolysis in B cells.
Authors: Harnett M M , Klaus G G
Published: December 2022

Cross-linking of the IgM and IgD Ag-R on mature B lymphocytes provokes the rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate. We show here that in permeabilized, [3H]inositol-labeled mouse B cells the nonhydrolyzable GTP analogue GTP gamma S induces release of inositol phosphates, including inositol trisphosphate. The response is markedly augmented by the addition of polyclonal anti-Ig or anti-mu or anti-delta mAb. Inositol phosphate release provoked in intact B cells by any of the anti-receptor antibodies was not inhibited by pertussis toxin and only partially inhibited by cholera toxin. The results therefore indicate that both IgM- and IgD-R on B cells are coupled to the polyphosphoinositide-specific phosphodiesterase by one or more G proteins, which have yet to be identified.
C5 convertase of the alternative complement pathway: covalent linkage between two C3b molecules within the trimolecular complex enzyme.
Authors: Kinoshita T , Takata Y , Kozono H , Takeda J , Hong K S , Inoue K
Published: December 2022

C5 convertase of the alternative C pathway is a complex enzyme consisting of three C fragments--one molecule of a major fragment of factor B (Bb) and two molecules of a major fragment of C3 (C3b). Within this C3bBbC3b complex, the first C3b binds covalently to the target surface, and Bb, which bears a catalytic site, binds noncovalently to the first C3b. In the present investigation, we studied the nature of the convertase that is assembled on E surfaces and obtained evidence that the second C3b binds directly to the alpha'-chain of the first through an ester bond rather than to the target surface. Thus, the alternative pathway C5 convertase could be described as a trimolecular complex in which Bb binds noncovalently to a covalently linked C3b dimer. We also obtained evidence that not only the second C3b but also the first C3b participates in binding C5, that is, covalently-linked C3b dimer acts as a substrate-binding site. Because of this two-site binding, the convertase has a much higher affinity for C5 than the surrounding monomeric C3b molecules. Based on this evidence, a new model of the alternative pathway C5 convertase is proposed. Covalent association of two subunits and the bivalent binding of the substrate are then common properties of the alternative and classical pathway C5 convertases.
A novel T cell-activating molecule (THAM) highly expressed on CD4-CD8- murine thymocytes.
Authors: Naquet P , MacDonald H R , Brekelmans P , Barbet J , Marchetto S , Van Ewijk W , Pierres M
Published: December 2022

Recent studies have focused on the potential role of accessory molecules such as CD2, CD28, Thy-1, or TAP in the delivery of activating signals to thymocytes through antigen-independent pathways. To better understand the molecular interactions involved in the expansion of early thymic immigrants, rat mAb were raised against murine thymocyte-surface molecules and screened for their capacity to trigger thymocyte proliferation. One of these mAb (H194-112, IgG2a) was found to recognize a novel heterodimeric thymocyte-activating molecule (THAM) of Mr = 110,000 to 128,000. Flow cytometric analyses and staining patterns on frozen thymus sections subdivided adult thymocytes in three subsets expressing THAM at either low (10%), moderate (80%), or high (5 to 8%) cell-surface density; these cell groups were found to correspond, respectively, to the medullary, the cortical, and the immature CD4-CD8-, J11d+ thymocytes, in which the T cell precursor pool is included. Moreover, most (90%) day 16 fetal thymocytes were also found to upregulate THAM cell-surface expression. The THAMhigh cells were localized in the subcapsular area of the neonatal thymus and scattered throughout the adult organ. Cross-linked mAb H194-112 induced the proliferation of both immature and mature thymocytes in the presence of either PMA or IL-1 and IL-2. The observation that early thymocytes up-regulate THAM along with the IL-2R suggests that this molecule might be involved in an important activation pathway during thymocyte differentiation.
The activation of pigeon cytochrome c-specific T cell hybridomas by antigenic peptides is influenced by non-native sequences at the amino terminus of the determinant.
Authors: Bhayani H , Carbone F R , Paterson Y
Published: December 2022

We recently demonstrated that the sequence 95-104 contains all the residues necessary for direct recognition of the I-Ek restricted pigeon cytochrome c determinant but that residues located in sequences to the amino-terminal side of residue 95 improve the ability of peptides containing the sequence 95-104 to stimulate Ag-specific T cell clones. In this study we use synthetic peptides with amino-terminal leader sequences containing residues that differ with respect to their conformational stabilizing effects, charge, and hydrophilicity to examine the mechanism by which they modulate T cell recognition. Our findings indicate that the role of these residues in T cell stimulation is not related to their ability to stabilize alpha-helical secondary structure, nor do they appear to be processed differently. The leader sequences do not differentially influence the ability of the peptides to be presented by APC displaying Ia molecules of related haplotype, i.e., E alpha kE beta k, E alpha kE beta b, and E alpha kE beta s, to T cells which recognize the pigeon cytochrome c determinant on such presenting cells. Because antigenic potency correlates with the inclusion of hydrophobic residues and positively charged residues in the leader sequences, we discuss our findings with reference to the possibility that they non-specifically enhance the interaction of the antigenic peptides with the APC membrane.
Antigenic variation in Giardia lamblia.
Authors: Nash T E , Aggarwal A , Adam R D , Conrad J T , Merritt J W
Published: December 2022

Clones of the WB isolate of Giardia lamblia were exposed to cytotoxic mAb 6E7 which reacts with a 170-kDa surface Ag. Surviving progeny occurred at a frequency of about 1 in 1000 and were resistant to the effects of mAb 6E7. Analysis of progeny and clones of these progeny by surface radiolabeling, surface immunofluorescence, and Western blotting failed to detect the 170-kDa Ag. Loss of this Ag was associated with the appearance of a series of new surface Ag. A cytotoxic mAb (5C1) was produced to one of the newly appearing antigens (approximately equal to 64 kDa) and Giardia resistant to the cytotoxic effects of 5C1 isolated. Neither the approximately equal to 64 kDa nor the 170 kDa Ag were present and were replaced by a second series of new Ag. These studies clearly establish the loss and subsequent replacement of two antigenically distinct epitopes on Giardia derived from a single organism.
Immunoregulatory role of Ig isotypes. II. Activation of cells that block induction of contact sensitivity responses by antibodies of IgG2a and IgG2b isotypes.
Authors: Ptak W , Janeway C A , Flood P M
Published: December 2022

The influence that the isotype of Ag-specific antibody has on the induction of contact hypersensitivity (CS) has been investigated. Injection (i.v.) of mice with haptenated peritoneal exudate cells (PEC) pretreated with anti-hapten mAb of the IgG2a and IgG2b isotypes results in the activation of Ag-specific afferent acting Ts cells (Ts-aff). These suppressor cells are not generated when animals are injected with anti-hapten antibodies of other isotypes. The Ts-aff cells function to inhibit the generation of CS responses when injected into naive animals. Suppression is due to the induction of both Lyt-1+,2- I-J+ and Lyt-1-,2+ I-J+ T cells, both of which adhere to the lectin Vicia villosa. Attachment of both TNP and 4-ethoxymethylene-2-phenyloxazolone haptens to the same PEC, followed by treatment with an IgG2a anti-TNP antibody, generates Ts-aff cells specific for both 4-ethoxymethylene-2-phenyloxazolone and TNP. The MHC haplotype of the PEC is irrelevant, as allogeneic PEC will also induce Ts-aff cells when injected by using an identical protocol. Ts-aff cells cannot be generated in B cell-depleted mice, nor does the Ts-aff cells generated in normal mice suppress CS responses in B cell-depleted mice. These results show that Ag-antibody complexes bound on the surface of a PEC can induce potent afferent suppression in vivo. A possible general role for antibody isotypes in directing regulatory activities is discussed.
HLA-E. A novel HLA class I gene expressed in resting T lymphocytes.
Authors: Koller B H , Geraghty D E , Shimizu Y , DeMars R , Orr H T
Published: December 2022

A MHC class I gene, HLA-6.2, has been identified. Examination of its primary structure indicates that HLA-6.2 is the most divergent member of the class I gene family characterized to date. Tissue distribution of HLA-6.2 RNA differs from other class I genes in that the highest level of RNA is detected in resting T lymphocytes. After transfer of the HLA-6.2 gene into a class I null human lymphoblastoid cell line, a protein associated with beta 2-microglobulin is detected. Analysis of HLA deletion mutant cell lines places the HLA-6.2 gene between the HLA-C and HLA-A loci on the short arm of chromosome 6. These data support the fact that the HLA-6.2 gene is a distinct expressible HLA class I gene, which we propose be designated HLA-E.
Genetic analysis of the presentation of minor lymphocyte stimulating determinants. I. Combined importance of MHC and non-MHC influences.
Authors: Ryan J J , Mond J J , Finkelman F D
Published: December 2022

In the course of studying the MHC restriction of minor lymphocyte stimulating (Mls) determinants, we observed that variation in the ability to present Mlsc determinants occurred with stimulator cells from different mouse strains that express the same class II MHC restricting elements; for example, one Iad-bearing strain, C3H.HTG, presented this non-MHC moiety, whereas another, C3H.OH, could not. As another example, the prototype Mlsb nonstimulatory H-2d stimulator cell, BALB/c, was shown to encode Mlsc even though it failed to trigger proliferation across this non-MHC barrier. In contrast, H-2d-compatible DBA/2 stimulator cells were capable of eliciting detectable levels of unprimed responder T cell proliferation across an Mlsc difference. Even when the BALB/c H-2d haplotype was replaced with the fully permissive H-2K halplotype, these BALB.K stimulator cells presented Mlsc (but not MHC) less effectively than H-2K-compatible C3H/HeJ stimulator cells. Analysis of the Mlsc-presenting capacity of stimulator cells obtained from (BALB.K x C3H) F1 x BALB.K first backcross and (BALB.K x C3H)F2 animals indicated that non-MHC-control influencing stimulatory ability of this non-H-2 Ag was multigenic. In addition, the capacity of DBA/2 to present Mlsa determinants more effectively than MHC-identical LT/ChReSv stimulator cells may indicate that the presentation of this Mls specificity is also influenced by non-MHC Ir genes. Thus the Mls phenotype of an animal should be considered the combined result of an Mls structural gene, the MHC haplotype, and multiple non-H-2 regulatory influences.