bluuebunny/crossref_metadata_2025 huggingface dataset:
An absolute and restricted requirement for IL-12 in natural killer cell IFN-gamma production and antiviral defense. Studies of natural killer and T cell responses in contrasting viral infections.
Authors: Orange J S , Biron C A
Published: December 2022

IL-12 is a potent induce of IFN-gamma and is associated with a wide variety of immunoregulatory activities. Using murine CMV (MCMV) and lymphocytic choriomeningitis virus (LCMV) models of acute viral infection, we show here that IL-12 is integrally involved in certain aspects of antiviral immunity. Experiments evaluating the production and function of IL-12 demonstrated the induction of biologically active factor early during Smith strain MCMV, but not Armstrong strain LCMV, infection. The MCMV-induced IL-12 was responsible for early NK cell IFN-gamma production and viral control, as in vivo IL-12 neutralization by Ab treatment blocked both of these. In contrast, NK cell IFN-gamma production could not be detected during LCMV infection, and IL-12 neutralization had no effect on early LCMV replication. In both infections, treatments neutralizing IL-12 failed to alter either early NK cell cytotoxicity or later T cell responses, including IFN-gamma production and viral clearance on days 7 and 9 after infection. These data demonstrate the induction of IL-12 production during certain viral infections and the function of IL-12 for eliciting NK cell IFN-gamma production and antiviral defense. Furthermore, they suggest the existence of IL-12-independent mechanisms for IFN-gamma induction and viral control by T cells.
IL-12, as an adjuvant, promotes a T helper 1 cell, but does not suppress a T helper 2 cell recall response.
Authors: Bliss J , Van Cleave V , Murray K , Wiencis A , Ketchum M , Maylor R , Haire T , Resmini C , Abbas A K , Wolf S F
Published: December 2022

IL-12 is a potent inducer of NK and cytolytic T cell activity, IFN-gamma production, and T cell proliferation, and is necessary for differentiation of naive T cells to the Th1 subset. We have previously shown that IL-12 promotes a primary Th1 response and suppresses a primary Th2 response in lymph nodes of mice primed with a model hapten-protein conjugate, 2,4,6-trinitrophenyl (TNP)-keyhole limpet hemocyanin (KLH). We have now extended these studies to determine the Th phenotype of the recall response following immunization with soluble Ag and IL-12. For these experiments, mice were primed with TNP-KLH with or without treatment with IL-12, allowed to progress beyond the primary immune response, and challenged by i.p. injection of TNP-KLH. The phenotype of the recall response was monitored by measuring ex vivo production of IFN-gamma and IL-4 in Ag-stimulated lymph node and spleen cell cultures. Titer and isotype of TNP-specific serum Abs were also evaluated. Mice primed with Ag+IL-12 developed a Th1 recall response, as detected by KLH-specific IFN-gamma production from cultured spleen cells and the presence of TNP-specific IgG2a Ab in serum. However, they also developed an Ag-specific Th2 recall response, as characterized by Ag-induced IL-4 production from spleen cells and the presence of high titers of anti-TNP IgG1 in the serum. Studies of the cytokine profile during the primary response revealed that IL-12 induced in spleen cells the capacity to express both IL-4 and IFN-gamma. CD4+ T cells are necessary for production of IL-4 in the spleens of IL-12-treated mice, and most likely account for the Th2 recall response detected in mice primed with Ag+IL-12. These results indicate that the Th1 phenotype induced by immunization with IL-12 and Ag is maintained so that a Th1 recall response is expressed upon subsequent challenge with Ag. However, immunization with IL-12 also supports the development of a Th2 recall response, indicating that the Th1-inducing effect of IL-12 in vivo is not accompanied by a long lasting suppression of Th2 development.
Roles of IFN-gamma and IFN-alpha in IL-12-induced T helper cell-1 development.
Authors: Wenner C A , Güler M L , Macatonia S E , O'Garra A , Murphy K M
Published: December 2022

IL-12 and IL-4 direct T cell development toward Th1 and Th2 phenotypes, respectively. While IFN-gamma and IFN-alpha have been reported to regulate Th1 development as well, the mechanism and cellular locus of their effects are unclear. In this study, we use a TCR-transgenic system to examine the actions of these cytokines on CD4+ T cell phenotype development. We find that neither IFN-gamma nor IFN-alpha can induce Th1 development alone. However, IFN-gamma can significantly augment IL-12 priming for subsequent IFN-gamma production by T cells. Interestingly, lymphocyte endothelial cell adhesion molecule-1bright (naive) T cells require IFN-gamma during primary activation for maximal IL-12-induced Th1 development, whereas lymphocyte endothelial cell adhesion molecule-1dull (memory) T cells do not. IFN-alpha only partially substitutes for IFN-gamma in promoting IL-12-induced Th1 development. When the endogenous IFN-gamma present in primary T cell cultures is neutralized, IFN-alpha treatment augments IL-12-induced effects on inhibition of subsequent IL-4 production, but fails to significantly enhance IL-12 priming for subsequent IFN-gamma production. Thus, our data suggest that IFN-gamma provides a direct costimulatory signal to T cells to up-regulate IL-12-induced Th1 development and may operate by inducing IL-12 responsiveness in naive T cells.
Glycosylphosphatidylinositol toxin of Plasmodium induces nitric oxide synthase expression in macrophages and vascular endothelial cells by a protein tyrosine kinase-dependent and protein kinase C-dependent signaling pathway.
Authors: Tachado S D , Gerold P , McConville M J , Baldwin T , Quilici D , Schwarz R T , Schofield L
Published: December 2022

In this study, we demonstrate that glycosylphosphatidylinositol (GPI) is a major toxin of Plasmodium falciparum origin responsible for nitric oxide (NO) production in host cells. Purified malarial GPI is sufficient to induce NO release in a time- and dose-dependent manner in macrophages and vascular endothelial cells, and regulates inducible NO synthase expression in macrophages. GPI-induced NO production was blocked by the NO synthase-specific inhibitor L-N-monomethylarginine. GPI also synergizes with IFN-gamma in regulating NO production. The structurally related molecules dipalmitoylphosphatidylinositol and iM4 glycoinositolphospholipid from Leishmania mexicana had no such activity, and the latter antagonized IFN-gamma-induced NO output. GPI activates macrophages by initiating an early onset tyrosine kinase-mediated signaling process, similar to that induced by total parasite extracts. The tyrosine kinase antagonists tyrphostin and genistein inhibited the release of NO by parasite extracts and by GPI, alone or in combination with IFN-gamma, demonstrating the involvement of one or more tyrosine kinases in the signaling cascade. GPI-induced NO release was also blocked by the protein kinase C inhibitor calphostin C, demonstrating a role for protein kinase C in GPI-mediated cell signaling, and by pyrrolidine dithiocarbamate, indicating the involvement of the NF-kappa B/c-rel family of transcription factors in cell activation. A neutralizing mAb to malarial GPI inhibited NO production induced by GPI and total malarial parasite extracts in human vascular endothelial cells and murine macrophages, indicating that GPI is a necessary agent of parasite origin in parasite-induced NO output. Thus, in contrast to dipalmitoylphosphatidylinositol and glycoinositolphospholipids of Leishmania, malarial GPI initiates a protein tyrosine kinase- and protein kinase C-mediated signal transduction pathway, regulating inducible NO synthase expression with the participation of NF-kappa B/c-rel, which leads to macrophage and vascular endothelial cell activation and downstream production of NO. These events may play a role in the etiology of severe malaria.
Role of IL-7 in the shaping of the pulmonary gamma delta T cell repertoire.
Authors: Hayes S M , Sirr A , Jacob S , Sim G K , Augustin A
Published: December 2022

Gamma delta T cells bearing the canonical fetal-type V gamma 6/V delta 1 rearrangements are the predominant gamma delta T cells in the lungs of adult mice. In contrast, these V gamma 6/V delta 1 T cells are virtually absent in the pulmonary epithelia of nude mice. The intraepithelial dominance of gamma delta T cells that express this particular TCR is thought to result from a preferred thymic pathway of gene rearrangement and not from TCR-mediated positive selection. We now show that gamma delta T cell precursors in the lung epithelium of both euthymic and athymic neonatal mice generate this rearrangement in situ. In athymic mice, these clonotypes do not survive, but can be rescued in vitro and in vivo by the lymphokine IL-7.
Differential dysregulation of nitric oxide production in macrophages with targeted disruptions in IFN regulatory factor-1 and -2 genes.
Authors: Salkowski C A , Barber S A , Detore G R , Vogel S N
Published: December 2022

Recent studies have reported that IFN regulatory factor-1 (IRF-1) regulates nitric oxide (NO.) production in murine macrophages (M phi). Since IRF-2 recognizes the same consensus sequence as IRF-1, we postulated that IRF-2 may also regulate NO.. Therefore, the ability of M phi from INF-2 homozygous (IRF-2-/-) and heterozygous (IRF-2+/-) knockout mice to produce NO. following LPS and/or IFN-gamma stimulation was compared with the responses of IRF-1-/-, IRF-1+/-, and C57BL/6+/+ M phi. IRF-2-/- M phi produced less LPS-induced NO2- than IRF-2+/- or C57BL/6 M phi. LPS and IFN-gamma synergized to increase NO2- production from IRF-2-/- M phi to approximately 50% of IRF-2+/- and C57BL/6 levels. Unexpectedly, IRF-2-/-, IRF-2+/- and C57BL/6 M phi produced comparable levels of inducible NO synthase mRNA in response to treatment with LPS and IFN-gamma. IRF-1-/- M phi produced barely detectable NO2- and low, but detectable, inducible NO. synthase mRNA in response to IFN-gamma and LPS. These results indicate that IRF-1 and IRF-2 differ in their mechanism of NO. regulation and that IRF-2 regulates inducible NO. synthase post-transcriptionally.
Differential expression of homing molecules on recirculating lymphocytes from sheep gut, peripheral, and lung lymph.
Authors: Abitorabi M A , Mackay C R , Jerome E H , Osorio O , Butcher E C , Erle D J
Published: December 2022

The adhesion molecules integrin alpha 4 beta 7 and L-selectin have been hypothesized to help direct selective migration (homing) of lymphocytes to the gut and peripheral lymph nodes, respectively. An important prediction of current models is that lymphocytes selectively recirculating through an organ will preferentially express adhesion receptors for organ-specific endothelial ligands. To test this prediction, we directly examined the expression of cell adhesion molecules on lymphocytes recirculating through the gut, the periphery, and the lung. Integrin beta 7 was highly expressed on CD4+CD45R- "memory/effector" T cells recirculating through the gut (mesenteric efferent and lower thoracic duct lymph). In contrast, cells recirculating through the periphery (prescapular efferent lymph) and the lung (caudal mediastinal efferent lymph) had much less beta 7 expression. A similar pattern of organ-specific beta 7 expression was seen on B cells. Beta 7 expression corresponded with adhesion to the gut mucosal addressin, MAdCAM-1, in vitro. The peripheral lymph node homing receptor, L-selectin, was expressed at higher levels on CD4+CD45R- T cells from peripheral lymph than on cells from gut or lung lymph. These results provide additional strong support for alpha 4 beta 7 and L-selectin involvement in lymphocyte homing to the gut and to peripheral lymph nodes, respectively. Lymphocytes emigrating from the lung expressed low levels of both homing receptors and likely utilize molecules other than alpha 4 beta 7 and L-selectin for migration to the lung and associated lymphoid tissue.
Herpesvirus saimiri open reading frame 14, a protein encoded by T lymphotropic herpesvirus, binds to MHC class II molecules and stimulates T cell proliferation.
Authors: Yao Z , Maraskovsky E , Spriggs M K , Cohen J I , Armitage R J , Alderson M R
Published: December 2022

Herpesvirus saimiri (HVS) is an oncogenic, lymphotropic, gamma-herpesvirus that transforms human and simian T cells in vitro and causes lymphomas and leukemias in various species of New World primates. Nucleotide sequence analysis of the HVS genome revealed an open reading frame with 22% amino acid identity to the mouse mammary tumor virus 7 superantigen. In this study, we demonstrate that this open reading frame, HVS14, encodes a heavily glycosylated protein that is secreted. Both the HVS14 present in the supernatant of transfected cells and a chimeric HVS14.Fc fusion protein were found to bind to heterodimeric MHC class II HLA-DR molecules. The supernatant from HVS14-transfected cells induced the proliferation of human PBMC, which could be specifically inhibited by HVS14-specific mAbs. Purified peripheral blood T cells were induced to proliferate in the presence of accessory cells and HVS14-containing supernatant. Whereas the HVS14 protein stimulated T cell proliferation, the HVS14.Fc fusion protein blocked proliferative responses to soluble Ags in vitro. Collectively, these data indicate that HVS14 can function as an immunomodulator that may contribute to the immunopathology of HVS infection.
Autoimmune lpr/lpr mice deficient in CD40 ligand: spontaneous Ig class switching with dichotomy of autoantibody responses.
Authors: Ma J , Xu J , Madaio M P , Peng Q , Zhang J , Grewal I S , Flavell R A , Craft J
Published: December 2022

Fas-deficient MRL/Mp-lpr/lpr mice develop a syndrome that resembles human systemic lupus erythematosus, including production of IgG autoantibodies against small nuclear ribonucleoproteins (snRNPs), dsDNA, and self IgG (rheumatoid factor). To investigate the necessity for T-B cell contact in MRL autoimmunity, mice deficient in CD40 ligand (CD40L) were backcrossed onto this background, and Ab synthesis was assessed. In comparison to their CD40L-intact lpr/lpr counterparts, CD40L-deficient lpr/lpr mice had elevated levels of serum IgM and lower levels of IgG; however, a subset of animals had IgG2a, and to a lesser extent, IgG2b levels similar to those found in wild-type lpr/lpr mice. Levels of both isotypes in CD40L-deficient lpr/lpr mice were significantly greater than those found in nonautoimmune CD40L-deficient animals. IgG autoantibodies, including those directed against small nuclear ribonucleoproteins, also arose in CD40L-deficient lpr/lpr mice; however, they did not develop IgG rheumatoid factors or anti-dsDNA, and lacked histologic evidence of overt glomerulonephritis at age 3 mo, in contrast to CD40L-intact lpr/lpr animals. These results indicate that isotype switching occurs in lpr/lpr mice deficient in CD40L, and that production of IgG autoantibodies to ribonucleoproteins is at least partially preserved. They also suggest that different mechanisms may be responsible for eliciting autoantibody responses in lpr/lpr mice.
Peripheral V gamma 9/V delta 2 T cell deletion and anergy to nonpeptidic mycobacterial antigens in asymptomatic HIV-1-infected persons.
Authors: Poccia F , Boullier S , Lecoeur H , Cochet M , Poquet Y , Colizzi V , Fournie J J , Gougeon M L
Published: December 2022

Gamma delta T cells represent a minor population of human peripheral lymphocytes, the majority of them expressing the V delta 2/V gamma 9 TCR. Their accumulation in infectious disease lesions and their reactivity toward mycobacterial Ags suggest that V gamma 9/V delta 2 T cells play a role during infectious diseases. We have shown previously a significant expansion of the V delta 1 subset parallel to a dramatic decrease of the V delta 2 subset in PBMC from HIV-infected persons. To understand the mechanisms involved in the deletion of V delta 2 T cells, we analyzed their ability to respond in vitro to several V gamma 9/V delta 2 t cell-specific ligands. We observed that in 60% of asymptomatic HIV-infected persons, V delta 2 T cells exhibited a functional anergy to Daudi and to Mycobacterium tuberculosis stimulations. These observations were supported by the defective expansion of this subset to the recently described nonpeptidic phosphorylated Ag, TUBAg-1. Since V delta 2 responsiveness to mycobacterial Ags was shown to be normally dependent on IL-2 secretion by Th1-type CD4 T cells, the ability of IL-2 to restore V delta 2 T cells' responsiveness to TUBAg-1 was tested. V delta 2 T cell anergy persisted in spite of the presence of IL-2, and was frequently correlated with a defect in CD25 expression on stimulated V delta 2 T cells. Since V delta 2 anergy was associated with an in vivo depletion of this subset, we studied whether programmed cell death could be involved in this process, particularly because of their activated phenotype. Although peripheral V delta 2 T cells from some HIV-infected persons showed an increased susceptibility to spontaneous and activation-induced apoptosis, statistical comparison between HIV+ and HIV- donors indicated that there was no difference between both groups in the rate of V delta 2 apoptosis. Finally, V delta 2 complementarity-determining region 3 TCR analysis indicated that, in vivo, the remaining V delta 2 T cells were still polyclonal. All together these results suggest that the qualitative and quantitative alterations of the V delta 2 subset in the course of HIV infection are the consequence of a chronic antigenic stimulation, and raise the question of the contribution of a cellular ligand induced or modified by chronic HIV infection.
Acceleration of spontaneous diabetes in TCR-beta-transgenic nonobese diabetic mice by beta-cell cytotoxic CD8+ T cells expressing identical endogenous TCR-alpha chains.
Authors: Verdaguer J , Yoon J W , Anderson B , Averill N , Utsugi T , Park B J , Santamaria P
Published: December 2022

The role of target cell autoantigens and their repertoire vs those of foreign Ags, superantigens, or non-Ag-specific stimuli in the activation and recruitment of effector T cells in most spontaneous models of autoimmune diseases remains elusive. Here we report on the use of single TCR-beta transgenic mice to study the mechanisms that drive the accumulation of pathogenic T cells in the pancreatic islets of nonobese diabetic (NOD) mice, a model for insulin-dependent diabetes mellitus. Expression of the V(beta)8.1+ TCR-beta rearrangement of a diabetogenic H-2Kd-restricted beta cell cytotoxic CD8+ T cell (beta-CTL) clone in NOD mice caused a 10-fold increase in the peripheral precursor frequency of beta-CTL and a selective acceleration of the recruitment of CD8+ T cells to the pancreatic islets of prediabetic animals. This resulted in an earlier onset and a faster progression of beta cell depletion, and led to a dramatic acceleration of the onset of diabetes. Most islet-derived beta-CTL from diabetic transgenic NOD mice expressed an endogenously-derived TCR-alpha sequence identical to that of the clonotype donating the TCR-beta transgene, and a TCR-alpha-CDR3 sequence homologous to those expressed by most islet-derived beta-CTL from nontransgenic NOD mice. TCR-beta transgene expression did not change the peripheral frequency of beta cell-specific CD4+ T cells, the rate at which these cells accumulated in the pancreatic islets, or the incidence of diabetes. Taken together, our data indicate that retention of CD8+ and CD4+ T cells in the pancreatic islets of NOD mice is driven by beta cell autoantigens, rather than by local superantigens or non-Ag-specific stimuli, and that beta-CTL are major effectors of beta cell damage in spontaneous insulin-dependent diabetes mellitus.
A dominant negative mutant of an IFN regulatory factor family protein inhibits both type I and type II IFN-stimulated gene expression and antiproliferative activity of IFNs.
Authors: Thornton A M , Ogryzko V V , Dent A , Sharf R , Levi B Z , Kanno Y , Staudt L M , Howard B H , Ozato K
Published: December 2022

Type I (alpha,beta) and type II (gamma) IFNs elicit antiproliferative and antiviral activities through two distinct transcription pathways involving 1) IRF family proteins and ISGF3, and 2) STAT1. We have employed a dominant negative strategy to study the role of IRF family proteins in eliciting the biologic activities of IFN. A truncated IRF protein retaining the DNA-binding domain (DBD) of ICSBP (a member of the IRF family) was stably transfected into U937 monocytic cells. Clones expressing DBD had markedly reduced ISRE-binding activity and were defective in expressing several type I IFN-inducible genes. STAT1 was one such type I IFN-inducible gene whose expression was also inhibited in DBD clones. As a result, the expression of several IFN-gamma-inducible genes was also inhibited in these clones, indicating functional coupling of the type I and type II IFN transcription pathways. Furthermore, DBD clones grew more slowly than control clones and were refractory to antiproliferative effects of both types of IFNs. We found that IFN treatment of U937 cells leads to a G1 arrest and an increase in underphosphorylated retinoblastoma gene product. However, IFN treatment did not change the cell cycle profile, nor retinoblastoma gene product phosphorylation state in DBD clones. These data indicate that expression of DBD disrupts cell cycle regulatory mechanisms. Combined with the previously noted failure of DBD clones to elicit antiviral activity, the present work shows that IRF family proteins play an integral part in growth control activities of IFNs.
The immune response to Trichophyton tonsurans: distinct T cell cytokine profiles to a single protein among subjects with immediate and delayed hypersensitivity.
Authors: Slunt J B , Taketomi E A , Woodfolk J A , Hayden M L , Platts-Mills T A
Published: December 2022

Skin testing with an extract from the dermatophyte fungus Trichophyton tonsurans can result in either immediate (IH) or delayed hypersensitivity (DH). These experiments were designed to examine in vitro T cell cytokine production in response to purified proteins from T. tonsurans in subjects with different skin test reactivities. Peripheral blood mononuclear cells were obtained from subjects with immediate, delayed, or negative skin tests, and cellular proliferation was studied. Subjects with either IH or DH had positive proliferative responses to crude extracts and two purified proteins, protein IV (83 kDa) and Tri t 1 (30 kDa). Nine cell lines were established from 5 IH subjects, which produced a cytokine profile characteristic of Th2/Th0 cells, i.e., ratio of IFN-gamma to IL-4 or IL-5 <2:1. By contrast 8 of 10 cell lines from DH subjects had a Th1 profile, i.e., IFN-gamma to IL-4 or IL-5 >20:1. Lymphocytes from subjects with negative skin tests show very poor proliferative responses; however, 6 cell lines derived from these individuals showed a cytokine profile characteristic of Th1 cells. Levels of IL-5 were significantly different when comparing the IH group with the DH group (p < 0.001). The results demonstrate that a single defined protein from T. tonsurans can produce distinct T cell cytokine profiles that correspond to in vivo skin test reactivities and serum Ab levels.
Activation of nuclear factor-kappaB via T cell receptor requires a Raf kinase and Ca2+ influx. Functional synergy between Raf and calcineurin.
Authors: Kanno T , Siebenlist U
Published: December 2022

Signals transduced via the TCR activate the transcription factor nuclear factor-kappaB (NF-kappaB), which, in turn, is critical to the transcriptional induction of many genes important for the proliferation and expression of a differentiated phenotype. Treatment of T cells with the protein kinase C activator PMA in combination with Ca2+ ionophores mimics this process, and the two agents are often substituted for TCR stimulation, bypassing the TCR. Here we identify intracellular signaling components involved in activation of NF-kappaB following TCR stimulation. TCR signaling was triggered by treating Jurkat T cells with PHA or anti-CD3 Abs, and NF-kappaB activation was monitored by electrophoretic mobility shift assays and/or by kappaB-dependent reporter assays. Contrary to the idea that protein kinase C is involved in TCR-mediated activation of NF-kappaB, high doses of staurosporine did not interfere with activation of NF-kappaB by PHA, while the same dose of staurosporine completely blocked activation by PMA. PHA-induced kappaB-dependent reporter activity was, however, effectively blocked by a dominant negative form of Raf-1, suggesting a critical role for a Raf kinase. The TCR-mediated activation of NF-kappaB was also dependent on a Ca2+ influx, because the Ca2+ channel blocker, SK&F 96365, as well as other agents that prevented the Ca2+ influx, inhibited NF-kappaB activation. Cotransfection of a constitutively active form of calcineurin largely substituted for the Ca2+ requirement and reversed the blockade by SK&F 96365. Consistent with these observations, coexpression of constitutively active forms of Raf-1 and calcineurin synergistically induced kappaB-dependent reporter activity, suggesting a physiologically relevant functional interaction between the kinase and the phosphatase.
IL-2 induces beta2-integrin adhesion via a wortmannin/LY294002-sensitive, rapamycin-resistant pathway. Phosphorylation of a 125-kilodalton protein correlates with induction of adhesion, but not mitogenesis.
Authors: Nielsen M , Svejgaard A , Skov S , Dobson P , Bendtzen K , Geisler C , Odum N
Published: December 2022

Besides its function as a growth factor, IL-2 induces beta2-integrin-dependent, homotypic adhesion of IL-2R-positive T cells. In this study, we investigated how IL-2R are functionally and biochemically linked to the beta2-integrin adhesion pathway. After a lag period of 15 to 20 min, IL-2 induces beta2-integrin-dependent, homotypic adhesion in Ag-specific, human T cell lines. The IL-2 adhesion response is blocked by wortmannin and LY294002, inhibitors of phosphatidylinositol-3 (PI-3) kinase activity. In contrast, rapamycin strongly inhibits IL-2-induced proliferation without inhibiting IL-2-induced adhesion. Herbimycin A and genestein, inhibitors of protein tyrosine kinases, inhibit cytokine-induced adhesion and mitogenesis in parallel, whereas cytochalasin E, an inhibitor of actin polymerization, almost completely blocks the adhesion response at concentrations that have little effect on mitogenesis. IL-2R ligation rapidly (<5 min) induces tyrosine phosphorylation of several proteins, the most prominent being signal transducer and activator of transcription (Stat) proteins, the p85 subunit of the PI-3 kinase, and an as yet unidentified 125-kDa protein (p125). Wortmannin, LY294002, and cytochalasin E almost completely inhibit cytokine-induced tyrosine phosphorylation of p125, whereas tyrosine phosphorylation of PI-3 kinase, Janus kinases, Stat3, Stat5, and other proteins is unaffected. In contrast, rapamycin has little effect on IL-2-induced phosphorylation of p125. Taken together, these data suggest that 1) IL-2R ligation induces homotypic adhesion through a wortmannin/LY294002-sensitive, rapamycin-resistant pathway, 2) tyrosine kinases play a critical role in cytokine-induced adhesion, and 3) adhesion, but not mitogenesis, correlates with enhanced tyrosine phosphorylation of an as yet unidentified protein of 125 kDa.
A major role for the Fas pathway in acute graft-versus-host disease.
Authors: Via C S , Nguyen P , Shustov A , Drappa J , Elkon K B
Published: December 2022

Recent studies have suggested a role for the Fas pathway in the wasting syndrome associated with lpr-->wild-type bone marrow transplants. To directly examine whether Fas ligand has a major role in the development of acute graft-vs-host disease (GVHD), Fas ligand-deficient (gld) mice were used as donors and C3H/HeJ x C57BL/6F1 as recipients in the parent-into-F1 model of acute GVHD. Transplantation of C3H/gld spleen cells induced significantly less host lymphoid depletion and was associated with less antihost cytotoxic activity in vitro when compared with wild-type C3H donor cells. The reduced depletion of host lymphocytes was explained by both impaired antihost T cell cytolytic activity and by reduced expansion of gld donor T cells in F1 recipients. These findings not only indicate that the Fas ligand is an important effector molecule in acute GVHD, but also provide in vivo evidence supporting a role for Fas/Fas ligand interactions in T cell expansion and maturation.
Determinants of polyreactivity in a large panel of recombinant human antibodies from HIV-1 infection.
Authors: Ditzel H J , Itoh K , Burton D R
Published: December 2022

A considerable part of the Ab repertoire is given over to polyreactive Abs capable of interacting with multiple antigenic species. Neither the function of these Abs nor the molecular basis for their activity is known. To address the latter problem, we have compared the amino acid sequences of a large panel (n = 70) of polyreactive human monoclonal Fab fragments and conducted a series of engineering experiments on a prototype polyreactive Fab. The Fab fragments were retrieved from combinatorial IgG libraries prepared from the bone marrow of long term asymptomatic HIV-1 seropositive donors. The general features displayed by the panel of IgG polyreactive Abs include 1) skewed VH family usage with a predominance of VH1 and VH4 clones and a paucity of the normally prevalent VH3 family; 2) use of a variety of different VH germ-line genes within the context of the family usage and no restriction in D or JH gene usage; 3) skewed VL gene usage: 75% of Fabs used one of two germ lines; and 4) extensive somatic modification of both heavy and light chains. The importance of the heavy chain, in particular the heavy chain CDR3 (HCDR3), in dictating the polyreactive phenotype was demonstrated for the prototype Fab by chain shuffling and CDR transplantation experiments. in addition, and most strikingly, a constrained peptide based on the HCDR3 sequence was shown to be polyreactive and to inhibit binding of the parent Ab to a panel of Ags. A role for conformational flexibility in polyreactivity was suggested by a marked temperature dependence of Ab recognition of Ag. One Ab was shown to be polyreactive at 37 degrees C, but was apparently monoreactive at 4 degrees C. We hypothesize that Ab polyreactivity is associated with conformationally flexible HCDR3 regions in the context of certain favorable framework configurations.
Somatically mutated B cell pool provides precursors for insulin antibodies.
Authors: Thomas J W , Hulbert C
Published: December 2022

Antibodies to insulin are products of autoreactive B lymphocytes that escape inactivation or clonal deletion and are examples of "clonal ignorance." To understand the genetic origin of Abs from clonally ignorant B cells, the roles of somatic mutation and germ-line V(H) structures were examined for two murine IgG1 mAb that bind human and rodent insulin. Engineered mAb constructs that express germ-line or mutated V(H) genes show that somatic mutations introducing aspartic acid in or adjacent to CDRH2 play a key role in insulin binding. When either of the two anti-insulin V(H) regions is returned to its germ-line (unmutated) sequence, neither mAb binds insulin and the germ-line-encoded mAb are not polyreactive. Reconstruction of the somatic evolution of insulin binding in both mAbs shows that a single mutation in CDRH2 is sufficient to generate anti-insulin activity from a nonbinding precursor. When the role of somatic mutation in the binding of rodent insulin is examined, autoreactivity is associated with single mutations in both Abs. Together these findings indicate that, despite a low mutation frequency, IgG insulin Abs may not be derived directly from germ-line (unmutated) precursors. The requirement for somatic mutation as a prerequisite for measurable insulin binding suggests these Abs have their origin in a previously mutated B cell pool as a consequence of the individual's immune history. Low avidity interaction with endogenous insulin may play a role in selection of these B cells and contribute to the origin of clonal ignorance.
Enhanced growth of murine melanoma in ultraviolet-irradiated skin is associated with local inhibition of immune effector mechanisms.
Authors: Donawho C K , Muller H K , Bucana C D , Kripke M L
Published: December 2022

We have developed a model for studying the role of local immunologic mechanisms in tumor development, in which injection of K1735 melanoma cells into the UV-irradiated ears of C3H mice results in a significantly higher incidence of tumors than injection into unirradiated ears. This effect of UV irradiation is immunologically mediated. We hypothesized that UV blocks the efferent arm of the immune response, thereby facilitating tumor development within the irradiated site. We demonstrate that elicitation of a delayed type hypersensitivity response to alloantigen is diminished in UV-irradiated ears. in addition, tumor rejection is impaired in melanoma-immune mice challenged in UV-irradiated ears, even though such mice exhibit systemic immunity when challenged in a nonirradiated site. The ability of immune lymphoid cells to inhibit melanoma growth when mixed with tumor cells and injected into the ears was inhibited by prior UV irradiation of the ears, indicating that the activity of immune effector cells is abrogated in the UV-irradiated microenvironment. Analysis of lymphoid cells in growing tumors indicated that the number of CD8+ T lymphocytes was reduced in the UV-irradiated site. We conclude that efferent immune responses are impaired in UV-irradiated tissue and suggest that the impairment may involve reductions in both the number and the activity of immune effector cells. These studies illustrate that conditions in the local microenvironment during the early stages of tumor growth may profoundly influence the outcome of the host-tumor interaction.
Recombinant polyepitope vaccines for the delivery of multiple CD8 cytotoxic T cell epitopes.
Authors: Thomson S A , Elliott S L , Sherritt M A , Sproat K W , Coupar B E , Scalzo A A , Forbes C A , Ladhams A M , Mo X Y , Tripp R A , Doherty P C , Moss D J , Suhrbier A
Published: December 2022

Development of epitope-based CD8 alpha beta CTL vaccines requires effective strategies for codelivery of large numbers of individual epitopes. We have designed an artificial "polyepitope" protein containing 10 contiguous minimal CTL epitopes, which were restricted by five MHC alleles and derived from five viruses, a parasite, and a tumor model. A recombinant vaccinia virus coding for this protein was capable of inducing MHC-restricted primary CTL responses to all 10 epitopes. Mice immunized with this recombinant vaccinia showed protection against murine cytomegalovirus, Sendai virus, and a tumor model. This simple generic approach to multiepitope delivery should find application in CTL-based vaccine design.
Mice lacking the TNF receptor p55 fail to resolve lesions caused by infection with Leishmania major, but control parasite replication.
Authors: Vieira L Q , Goldschmidt M , Nashleanas M , Pfeffer K , Mak T , Scott P
Published: December 2022

TNF is involved in host resistance to several pathogens. Recently it was found that mice lacking the p55 receptor for TNF (TNFRp55 -/-) do not control growth of the intracellular bacteria, Listeria monocytogenes and Mycobacterium tuberculosis. Here we report that the course of infection in TNFRp55 -/- mice with another intracellular pathogen, the protozoan parasite Leishmania major, is also quite different from normal mice. TNFRp55 -/- mice developed larger lesions than control mice and failed to resolve these lesions. However, they were able to eliminate parasites within the lesions. Histologic analysis indicated that at late stages lesions from TNFRp55 -/- mice appeared similar to lesions associated with cutaneous graft-vs-host disease. Both TNFRp55 -/- and control mice developed a normal Th1-type response during infection. We also found that IFN-gamma-activated macrophages from TNFRp55 -/- mice produced nitric oxide and killed L. major in vitro, which correlated with the ability of TNFRp55 -/- mice to eliminate the parasites in vivo. The production of nitric oxide by macrophages from TNFRp55 -/- mice required the presence of the parasites, however, since in their absence TNF could only synergize with IFN-gamma for nitric oxide production when added to normal, but not TNFRp55 -/-, macrophages. These results indicate that neither macrophage microbicidal activity nor nitric oxide production is absolutely dependent on the p55 receptor for TNF. Furthermore, they uncover a previously undefined role for TNFRp55 in resolution of parasite-induced inflammatory lesions.
Chicken monoclonal antibody isolated by a phage display system.
Authors: Yamanaka H I , Inoue T , Ikeda-Tanaka O
Published: December 2022

A method for obtaining chicken mAb using a phage display system was established. Chickens were hyperimmunized with murine serum albumin, and polyadenylated RNA was extracted from their spleen cells. cDNA of V regions of heavy or lambda-chain genes were obtained by reverse transcription-PCR using a pair of newly designed primers. A phage display library of 1.4 x10(7) clones expressing the chicken Ab variable region as a single chain was constructed. The library was panned three times against the Ag, then screened by ELISA. Of a number of Ag-binding clones, five mAb were identified by DNA sequencing. The specificity of these mAb for serum albumin of various species was examined by ELISA. The results showed different degrees of cross-reactivity to rat serum albumin, but not to other albumin. This method provides a procedure for efficiently generating specific chicken mAb against murine proteins and against the proteins of other mammals, whose amino acid sequences are highly conserved in mammalian species. The nucleotide sequences of lambda-chains revealed components to develop diversification of chicken Ab molecules. Four to seven pseudogene sequences were found per lambda-chain, which are regarded as donors in gene conversion for preimmune repertoire formation. A total of 28 nucleotide substitutions, which may be somatic hypermutations in Ag-driven affinity maturation but could also be errors introduced during gene conversion or polymorphic residues, were found in five lambda-chains.
B cells selected for apoptosis in the sheep ileal Peyer's patch have enhanced mutational diversity in the Ig V lambda light chain.
Authors: Maybaum T A , Reynolds J D
Published: December 2022

To investigate the molecular events associated with B cell apoptosis, we analyzed follicular B cells from the large Peyer's patch (PP) in the sheep ileum. Over 95% of B cells generated in the ileal PP are rapidly destroyed by apoptosis. Ig V lambda sequences from apoptotic B cells were compared with sequence from B cells about to emigrate from the PP. The sequences originated from two germline genes, V lambda 5.1 and V lambda 5.3. Only V lambda 5.1 was rearranged in apoptotic cells, whereas both V lambda 5.1 and V lambda 5.3 were rearranged in B cells about to emigrate. Apoptotic B cells had evidence of increased Ig sequence diversity based on: 1) significantly greater replacement to silent mutation ratios in the complementarity determining regions, 2) the more random distribution of mutations, and 3) the lack of mutational specificity compared with the mutational bias favoring transitions and purines in B cells about to emigrate. Based on this analysis, we propose that the continual proliferation of B cells in the PP follicle might increase their affinity to local Ags. Those Ags that are sequestered in this environment might be expected to stimulate the production of B cells with such high-affinity receptors that ligation would trigger apoptosis. This could account for the deletion of B cells with specificity for self-antigens, selecting ligands as well as gut-derived food and microbial Ags. This process could contribute to the elimination of self-reactive B cells, the expansion of the antibody repertoire, and the generation of oral tolerance.
The rat MHC haplotype RT1c expresses two classical class I molecules.
Authors: Joly E , Leong L , Coadwell W J , Clarkson C , Butcher G W
Published: December 2022

Cloning and characterization of classical MHC class I coding sequences of the laboratory rat Rattus norvegicus has been reported so far for only four haplotypes, RT1a, RT1(1), RT1n, and RT1u. In all four cases, only one RT1.A classical class I molecule was found. Here we report that, in contrast, the RT1c haplotype expresses two different classical class I molecules. Using recombinant rat strains, we find that allotypic serologic determinants carried by the two molecules map to the RT1.A region, and so we have named them RT1.A1c and RT1.A2c. Multiple clones of functional cDNAs for each of these two molecules were isolated using a recently developed PCR-based expression-cloning method. Using a panel of 20 RT1.Ac-reactive mAb, we find that six recognize RT1.A1c, seven recognize RT1.A2c, and seven recognize both. We also show that both molecules are recognized and distinguished by primary alloreactive cytotoxic T lymphocytes, and that they correspond to identifiable and distinct molecular species in cells that express RT1c naturally. These data all concur to demonstrate that the RT1.Ac region carries two different loci, each of which encodes a functional classical class I molecule.
Perforin, Fas ligand, and tumor necrosis factor are the major cytotoxic molecules used by lymphokine-activated killer cells.
Authors: Lee R K , Spielman J , Zhao D Y , Olsen K J , Podack E R
Published: December 2022

Lymphokine-activated killer (LAK) cells generated from perforin knockout mice possess significantly reduced cytotoxicity against a panel of tumor target cell lines, with some tumor cells being lysed exclusively by the perforin pathway. LAK cells are also capable of Fas ligand-mediated cytotoxicity. LAK cells generated from mice deficient in both perforin and Fas ligand (PKO/gld) were not cytolytic in short term cytotoxicity assays, demonstrating that perforin and Fas ligand are required for acute target cell lysis. However, PKO/gld LAK cells were cytotoxic in long term cytotoxicity assays against TNF-sensitive tumor lines, and this cytotoxicity was completely inhibited by neutralizing TNF Abs. This potent TNF cytotoxicity has not been fully appreciated previously because of the presence of dominant-acting perforin and Fas ligand in acute tumor cell lysis. TNF-based cytotoxicity by PKO/gld LAK was both soluble and membrane bound, and both forms of TNF were constitutively expressed. Thus, LAK cells are armed with at least three cytotoxic molecules: perforin, Fas ligand, and TNF.
Improved induction of melanoma-reactive CTL with peptides from the melanoma antigen gp100 modified at HLA-A*0201-binding residues.
Authors: Parkhurst M R , Salgaller M L , Southwood S , Robbins P F , Sette A , Rosenberg S A , Kawakami Y
Published: December 2022

Recognition of the melanoma Ag gp100 by tumor-infiltrating lymphocytes (TIL) in vitro has been correlated with tumor regression in patients with metastatic melanoma treated with the adoptive transfer of TIL plus IL-2. Three common gp100 epitopes have been identified that are recognized in the context of HLA-A2 by TIL from different patients: G9154 (KTWGQYWQV), G9209 (ITDQVPFSV), and G9280 (YLEPGPVTA). Upon stimulation with these peptides, melanoma-reactive CTL could be induced in vitro from PBL of some HLA-A2+ melanoma patients. However, numerous restimulations were required, and specific reactivity could not be generated in many patients. Therefore, to enhance the immunogenicity of gp100 peptides, amino acid substitutions were introduced into G9154, G9209, and G9280 at HLA-A*0201-binding anchor positions, but not at TCR contact residues, to increase peptide class I MHC-binding affinity. Several modified gp100 peptides bound with greater affinity to HLA-A*0201 than unmodified peptides and were recognized by TIL specific for the natural epitopes. These peptides were used to sensitize PBL from HLA-A2+ melanoma patients in vitro using peptide-pulsed autologous PBMC as stimulators. After five weekly restimulations with either the native G9209 or G9280 peptide, melanoma-reactive CTL could only be induced from two of seven patients. However, amino acid substitutions in these peptides enabled the induction of melanoma-reactive CTL from all seven patients. These results suggest that modified gp100 peptides may be more immunogenic than the native epitopes, and may be useful in immunotherapy protocols for patients with melanoma.
Phenotypic and functional characterization of long-lived NK cell lines of different maturational status obtained from mouse fetal liver.
Authors: Manoussaka M , Georgiou A , Rossiter B , Shrestha S , Toomey J A , Sivakumar P V , Bennett M , Kumar V , Brooks C G
Published: December 2022

Culture of day 14 mouse fetal liver (FL) cells in high dose IL-2, together with appropriate combinations of IL-4 and PMA, resulted in the generation of cell lines, termed FL-A lines, that were phenotypically and functionally indistinguishable from cultured adult splenic NK cell populations with the single important exception that no Ly49-expressing cells were present. By contrast, when FL cells were cultured in low-dose IL-2 alone, a second population of slow-growing NK-like cells, termed FL-B cells, emerged. These cells expressed the NK markers asialoGM1, 10A7, 2B4, and Fc gammaRII/III but differed from FL-A and splenic NK cells in expressing IL-2R alpha and stem cell factor receptor (SCFR) but no B220. Most lines derived in this manner had minimal or no cytolytic activity and only very low levels of NK1.1. However, they could secrete substantial quantities of several lymphokines including IL-3, granulocyte-macrophage (GM)-CSF, TNF-alpha, and, most surprisingly, IL-2. A minority of FL-B lines, typified by line 903, displayed marked cytolytic activity, moderate levels of NK1.1, reduced production of IL-2, and the capacity for accelerated growth in high-dose IL-2. FL-B lines generally expressed mRNA for CD3gamma but not for other CD3 chains, whereas FL-A and fetal thymic (FT) NK lines often expressed mRNA for all four CD3 chains. Despite many similarities to pro-T cells, FL-B cells showed no capacity to differentiate into mature T cells. Taken together, our results suggest that NK lines of different maturity can be obtained from fetal liver, with FL-B lines being the most immature, FL-A lines the most mature, and lines such as FL-B 903 representing an intermediate state of differentiation.
Astrocyte-specific but not neuron-specific transmembrane TNF triggers inflammation and degeneration in the central nervous system of transgenic mice.
Authors: Akassoglou K , Probert L , Kontogeorgos G , Kollias G
Published: December 2022

To study the involvement of TNF in cerebral pathology in vivo and to define cellular interactions within the central nervous system (CNS) that promote TNF signaling, we have expressed this cytokine either as a wild-type or a mutant transmembrane form in astrocytes or neurons of transgenic mice. Mice expressing wild-type human TNF in either of these cell types spontaneously develop a neurologic disorder manifested by ataxia, seizures, and paralysis and bear histologic evidence of chronic CNS inflammation and degeneration. Moreover, astrocyte-specific expression of transmembrane TNF triggers a similar neurologic phenotype. Interestingly, transgenic mice producing a high level of transmembrane TNF in their neurons develop no apparent phenotypic abnormalities, suggesting that appropriate cellular interactions should form to allow for contact-dependent TNF signals to induce CNS pathology. These results demonstrate that target cells mediating the neuroinflammatory activities of TNF localize in the vicinity of astrocytes rather than neurons.