bluuebunny/crossref_metadata_2025 huggingface dataset:
Monoclonal Antibody Therapy of B Cell Lymphoma: Signaling Activity on Tumor Cells Appears More Important Than Recruitment of Effectors
Authors: Tutt Alison L. , French Ruth R. , Illidge Timothy M. , Honeychurch Jamie , McBride Harry M. , Penfold Christine A. , Fearon Douglas T. , Parkhouse R. Michael E. , Klaus Gerry G. B. , Glennie Martin J.
Published: December 2022

Despite the recent success of mAb in the treatment of certain malignancies, there is still considerable uncertainty about the mechanism of action of anti-cancer Abs. Here, a panel of rat anti-mouse B cell mAb, including Ab directed at surface IgM Id, CD19, CD22, CD40, CD74, and MHC class II, has been investigated in the treatment of two syngeneic mouse B cell lymphomas, BCL1 and A31. Only three mAb were therapeutically active in vivo, anti-Id, anti-CD19, and anti-CD40. mAb to the other Ags showed little or no therapeutic activity in either model despite giving good levels of surface binding and activity in Ag-dependent cellular cytotoxicity and complement assays, and in some cases inhibiting cell growth in vitro. We conclude that the activity of mAb in vitro does not predict therapeutic performance in vivo. Furthermore, in vivo tracking experiments using fluorescently tagged cells showed that anti-Id and anti-CD40 mAb probably operate via different mechanisms: the anti-Id mAb cause growth arrest that is almost immediate and does not eliminate cells over a period of 5 or 6 days, and the anti-CD40 mAb have a delayed effect that allows tumor to grow normally for 3 days, but then abruptly eradicates lymphoma cells. This work supports the belief that mAb specificity is critical to therapeutic success in lymphoma and that, in addition to any effector-recruiting activity they may possess, in vivo mAb operate via mechanisms that involve cross-linking and signaling of key cellular receptors.
Generation of Human Cytolytic T Lymphocyte Lines Directed Against Prostate-Specific Antigen (PSA) Employing a PSA Oligoepitope Peptide
Authors: Correale Pierpaolo , Walmsley Konstantin , Zaremba Sam , Zhu MingZhu , Schlom Jeffrey , Tsang Kwong Y.
Published: December 2022

Prostate-specific Ag (PSA), which is expressed in a majority of prostate cancers, is a potential target for specific immunotherapy. Previous studies have shown that two 10-mer PSA peptides (designated PSA-1 and PSA-3) selected to conform to human HLA class I-A2 motifs can elicit CTL responses in vitro. A longer PSA peptide (30-mer) designated PSA-OP (oligoepitope peptide), which contains both the PSA-1 and PSA-3 HLA-A2 epitopes and an additional potential CTL epitope (designated PSA-9) for the HLA-class I-A3 allele, was investigated for the ability to induce cytotoxic T cell activity. T cell lines from different HLA-A2 and HLA-A3 donors were established by in vitro stimulation with PSA-OP; the CTL lines lysed PSA-OP as well as PSA-1- or PSA-3-pulsed C1R-A2 cells, and PSA-OP and PSA-9-pulsed C1R-A3 cells, respectively. The CTL lines derived from the PSA-OP peptide also lysed PSA-positive prostate cancer cells. PSA-OP-derived T cell lines also lysed recombinant vaccinia-PSA-infected targets but not targets infected with wild-type vaccinia. PSA-OP did not bind HLA-A2 and HLA-A3 molecules. The decrease in cytotoxicity in the presence of protease inhibitors suggests that the PSA-OP is cleaved into shorter peptides, which in turn can interact with HLA-class I molecules and, as a consequence, induce CTL-mediated lysis. We have also demonstrated that it is possible to induce CTL responses in HLA-A2.1/Kb transgenic mice by immunization with PSA-OP with adjuvant. These studies thus provide evidence that oligopeptides such as PSA-OP may be useful candidates for peptide-based cancer vaccines.
Cutting Edge: Differential Responsiveness of the IL-5 and IL-4 Genes to Transcription Factor GATA-3
Authors: Zhang Dong-Hong , Yang Liyan , Ray Anuradha
Published: December 2022

The cytokines IL-4 and IL-5 are often coordinately produced by Th2 cells as in asthma. However, it is unclear whether similar molecular mechanisms underlie transcription of the two genes. We have previously shown that the transcription factor GATA-3 is expressed in Th2 but not Th1 cells and is crucial for activation of the IL-5 promoter by different stimuli. In a different study, GATA-3 was shown to be sufficient for the expression of IL-4 and other Th2 cytokine genes. Here, we show that ectopic expression of GATA-3 is sufficient to drive IL-5 but not IL-4 gene expression. Also, in Th2 cells, antisense GATA-3 RNA inhibits IL-5 but not IL-4 promoter activation. The induction of IL-5 gene expression by GATA-3 involves high affinity binding of GATA-3 to an inverted GATA repeat in the IL-5 promoter.
High Antigen Density and IL-2 Are Required for Generation of CD4 Effectors Secreting Th1 Rather Than Th0 Cytokines
Authors: Rogers Paul R. , Huston Gail , Swain Susan L.
Published: December 2022

We reevaluated the effects of Ag dose on the polarization of CD4 effectors generated in vitro from naive pigeon cytochrome c-specific TCR transgenic T cells under conditions in which we could eliminate contaminating non-naive CD4 cells and the effects of heterogeneous Ag-presenting populations. When the possibility of contaminating non-naive T cells was reduced by using T cells from transgenic mice on a RAG-2−/− background, Ag dose did not have a significant effect in Th1 and Th2 polarization unless exogenous IL-2 was initially added to cultures. Effectors generated were uniformly Th0 but produced only IL-2 in substantial amounts. When exogenous IL-2 was added to priming cultures, T cells secreting a Th0 phenotype (large quantities of IL-2, IL-4, IL-5, and IFN-γ) developed, except at very high doses of Ag, where there was a striking reduction in IL-4 and IL-5 secretion. Our results imply that Ag dose does not have a direct effect on Th1/Th2 polarization, except under conditions that include a high level of TCR ligation and in the presence of high levels of IL-2, where production of Th2 cytokines may be down-regulated by a mechanism that is not yet clear.
Murine Langerhans Cells Cultured Under Serum-Free Conditions Mature into Potent Stimulators of Primary Immune Responses In Vitro and In Vivo
Authors: Kočiková Alena , Kolesarić Andrea , Koszik Frieder , Stingl Georg , Elbe-Bürger Adelheid
Published: December 2022

The ability of Ag-pulsed dendritic cells (DC) to induce primary immune responses has led them to be used for vaccination purposes. However, irrelevant Ags (e.g., FCS) can also be taken up by DC during their isolation and culture and then presented in vivo. To circumvent this, we have established a serum-free (SF) culture system. Murine epidermal cell (EC) suspensions were prepared with and without FCS and cultured for 3 days either in SF or FCS-containing medium. In spite of the lower Langerhans cell (LC) yields under SF conditions, both SF- and FCS-cultured LC (SF-cLC, FCS-cLC) underwent a similar maturation process, as evidenced by a similar increase in the cell surface expression of MHC class II and of costimulatory molecules. The further observation that SF-EC cultures elaborated comparable amounts of granulocyte-macrophage (GM)-CSF as FCS-cultured EC, but were relatively impaired in their IL-1α and TNF-α production, supports the role of GM-CSF in LC maturation and, less so, in LC survival. Functionally, freshly isolated SF-LC compared with FCS-LC in their Ag-processing capacity. Three-day-cultured SF-LC were as potent stimulators of polyclonal T cell responses and of the primary allogeneic MLR as FCS-cLC, but were relatively poor activators of naive, syngeneic CD4+ T cells. In vivo, hapten-modified SF-cLC induced a contact hypersensitivity response similar in magnitude and kinetics to that evoked by FCS-cLC. Our data show that, in the absence of serum and exogenous cytokines, LC mature into potent activators of T cell responses and could thus be a valuable cellular source for DC-based immunotherapy.
Inhibitory and Stimulatory Functions of Paired Ig-Like Receptor (PIR) Family in RBL-2H3 Cells
Authors: Yamashita Yumi , Ono Masao , Takai Toshiyuki
Published: December 2022

In this study, we demonstrate potent regulatory function of the murine killer cell inhibitory receptor-like molecules, paired Ig-like receptors (PIRs) or p91, using chimeric receptors expressed on the rat basophilic leukemia cell line RBL-2H3. One of the chimeras, which has the transmembrane and cytoplasmic domain of PIR-B fused to the extracellular portion of type IIB receptor for IgG, was able to inhibit the type I receptor for IgE-mediated degranulation response upon coaggregation. This chimera also suppressed cytoplasmic Ca2+ mobilization in the presence and absence of calcium ion in the extracellular medium. Tyrosine to phenylalanine point mutations at the third and fourth immunoreceptor tyrosine-based inhibitory motif-like sequences of PIR-B attenuated the inhibitory effects on degranulation and on cytoplasmic Ca2+ mobilization, indicating the important role of these tyrosines for the delivery of negative signal. In contrast, the cross-linking of another chimeric receptor composed of the type IIB receptor for IgG extracellular portion and the transmembrane and short cytoplasmic sequence of PIR-A elicited Ca2+ mobilization and degranulation. These results indicate that PIR molecules may regulate cellular functions both positively and negatively.
The Schistosome Granuloma: Characterization of Lymphocyte Migration, Activation, and Cytokine Production
Authors: Rumbley Catherine A. , Zekavat S. Ali , Sugaya Hiroko , Perrin Peter J. , Ramadan Mohamad Ali , Phillips S. Michael
Published: December 2022

Granuloma formation and its regulation are dependent on lymphocytes. Therefore, we compared the characteristics of lymphocytes derived from the spleens and granulomas of Schistosoma mansoni-infected mice during the course of their disease. We examined lymphocyte cell cycle kinetics, migration, expression of activation Ags (CD69 and IL-2R), cytokine production (IL-2, IL-4, IFN-γ), and apoptosis. Lymphocytes in the G2/M phase of the cell cycle and high levels of lymphocyte intracellular IL-2 were found in the spleen but not in the granuloma. Cell trafficking experiments showed Ag-specific recruitment of schistosomal egg Ag (SEA)-reactive lymphoblasts into granulomas in vivo, as well as recruitment to, residence within, and egress from granulomas in vitro. Granuloma-derived lymphocytes were more highly activated than splenic lymphocytes based on higher levels of CD69 and IL-2R expression. While the granuloma microenvironment was rich in Th2 cytokines, during peak granuloma formation, the lymphocytes per se from the spleen and granuloma did not exhibit a dominant Th1 or Th2 cytokine profile, producing low but similar levels of IL-4 and IFN-γ. The discrepancy between high IL-2R expression and low levels of IL-2 protein production by granuloma lymphocytes was associated with increased apoptosis in the granuloma compared with the spleen. These findings support the hypothesis that granulomas may play a role in the regulation of systemic pathology in schistosomiasis by adversely affecting the survival of SEA-reactive, immunopathogenic T lymphocytes.
CC-3052: A Water-Soluble Analog of Thalidomide and Potent Inhibitor of Activation-Induced TNF-α Production
Authors: Marriott J. Blake , Westby Michael , Cookson Sharon , Guckian Mary , Goodbourn Steve , Muller George , Shire Mary G. , Stirling David , Dalgleish Angus G.
Published: December 2022

The immunomodulatory drug thalidomide has been shown to be clinically useful in a number of situations due to its ability to inhibit TNF-α synthesis. However, its use is restricted by potentially serious side effects, including teratogenicity and neuorotoxicity; furthermore, insolubility may present problems in terms of systemic bioavailability. Recently, structural modifications of thalidomide have been designed enabling greatly enhanced anti-TNF-α activity in LPS-treated mice. In contrast to thalidomide (LPS-induced TNF-α IC50 ∼200 μM in DMSO) and other analogs tested, one of these compounds, CC-3052 (IC50 ∼1 μM in water), is water soluble. Furthermore, this analog exhibits increased stability in human plasma (t1/2 ∼17.5 vs 1.5 h for thalidomide) and appears to be nontoxic, nonmutagenic, and nonteratogenic. At pharmacologically active levels, cellular proliferation and LPS-induced IL-6 mRNA and IL-12p40 mRNA (as well as IL-1β and IL-6 protein levels) in whole blood cultures were not affected; apparent inhibition of NK activity by CC-3052 was reversed upon addition of exogenous rTNF-α. In addition, IL-10 mRNA and protein levels were increased. These properties are consistent with results indicating inhibition of phosphodiesterase type IV activity by CC-3052. Furthermore, CC-3052 did not increase the degradation rate of macrophage TNF-α transcripts nor inhibit LPS-induced primary macrophage NF-κB activation. Taken together, the potency of selective TNF-α inhibition, water solubility, and increased plasma stability make CC-3052 an excellent candidate for further development and clinical evaluation for the treatment of TNF-α-mediated disease.
HIV-1 Envelope gp120 Inhibits the Monocyte Response to Chemokines Through CD4 Signal-Dependent Chemokine Receptor Down-Regulation
Authors: Wang Ji Ming , Ueda Hirotsugu , Howard O. M. Zack , Grimm Michael C. , Chertov Oleg , Gong Xiaoqi , Gong Wanghua , Resau James H. , Broder Christopher C. , Evans Gerald , Arthur Larry O. , Ruscetti Francis W. , Oppenheim Joost J.
Published: December 2022

Since HIV-1 infection results in severe immunosuppression, and the envelope protein gp120 has been reported to interact with some of the chemokine receptors on human T lymphocytes, we postulated that gp120 may also affect monocyte activation by a variety of chemokines. This study shows that human peripheral blood monocytes when preincubated with gp120 either purified from laboratory-adapted strains or as recombinant proteins exhibited markedly reduced binding, calcium mobilization, and chemotactic response to chemokines. The gp-120-pretreated monocytes also showed a decreased response to FMLP. This broad inhibition of monocyte activation by chemoattractants required interaction of gp120 with CD4, since the effect of gp120 was only observed in CD4+ monocytes and in HEK 293 cells only if cotransfected with both chemokine receptors and an intact CD4, but not a CD4 lacking its cytoplasmic domain. Anti-CD4 mAbs mimicked the effect of gp120, and both anti-CD4 Ab and gp120 caused internalization of CXCR4 in HEK 293 cells provided they also expressed CD4. Staurosporine blocked the inhibitory effect of gp120 on monocytes, suggesting that cellular signaling was required for gp120 to inhibit the response of CD4+ cells to chemoattractants. Our study demonstrates a broad suppressive effect of gp120 on monocyte activation by chemoattractants through the down-regulation of cell surface receptors. Thus, gp120 may be used by HIV-1 to disarm the monocyte response to inflammatory stimulation.
A Chondroitin Sulfate Proteoglycan on Human Neutrophils Specifically Binds Platelet Factor 4 and Is Involved in Cell Activation
Authors: Petersen Frank , Bock Lothar , Flad Hans-Dieter , Brandt Ernst
Published: December 2022

Platelet factor 4 (PF-4), a member of the α-chemokine subfamily of cytokines, activates human neutrophils independently of intracellular free calcium mobilization or binding to IL-8R. In the present study, we have identified and partially characterized a receptor for PF-4 on human neutrophils, which displays weak cross-reactivity with the IFN-γ-inducible protein 10, but not with other α-chemokines such as IL-8, neutrophil-activating peptide 2, or melanoma growth-stimulatory activity (GROα). Binding studies revealed that human neutrophils express a high number of receptors (Bmax ∼ 7.6 × 106 sites/cell) of moderate affinity (Kd ≈ 650 nM). The kinetics of PF-4-binding correlates with the proportion of PF-4 tetramers in solution and with the activation of neutrophils for exocytosis. Reduction of PF-4 binding and PF-4-induced exocytosis in the presence of various glycosaminoglycans or following treatment of cells with chondroitinase ABC (but not other glycosaminoglycan-degrading enzymes) altogether demonstrates that the PF-4 receptor is a proteoglycan of the chondroitin sulfate class. Cross-linking experiments with radiolabeled PF-4 revealed a receptor-ligand complex of ∼250 kDa. Taken together, our data show that a distinct chondroitin sulfate proteoglycan represents specific receptors for tetrameric PF-4 on human neutrophils.
Selective Accumulation of Related CD4+ T Cell Clones in the Synovial Fluid of Patients with Rheumatoid Arthritis
Authors: Striebich Christopher C. , Falta Michael T. , Wang Yi , Bill Jerry , Kotzin Brian L.
Published: December 2022

The role of T cells in the pathogenesis of rheumatoid arthritis (RA), especially in the perpetuation of advanced disease, remains unclear. Previous studies have focused on the TCR repertoire of synovial T cells in an attempt to determine whether the pattern of expression is characteristic of Ag-stimulated populations. However, the results of past studies have been conflicting. In the present work, we have undertaken an extensive analysis of the TCRs expressed by CD4+ T cells freshly isolated from synovial fluid of different joints and blood in three patients with established RA. Despite marked heterogeneity of synovial TCR expression, the results showed that 20 to 30% of the TCR β-chain gene (TCRB) sequences found in one joint were also expressed in a second joint, but not in peripheral blood T cells of the same individual. Analysis of expressed TCRB complementarity-determining region 3 sequences showed the presence of multiple expanded clonal populations that were not predicted by quantitation of β-chain variable region (Vβ) expression by immunofluorescence staining. These studies also demonstrated sets of related, but different, complementarity-determining region 3 nucleotide sequences that encoded identical or highly homologous β-chain amino acid sequences. Analysis of matching T cell clones derived from the joint by limiting dilution culture confirmed coexpression of highly homologous TCR α-chain gene (TCRA) and TCRB sequences. Together, these studies suggest that a significant proportion of synovial CD4+ T cells has been selected and expanded by conventional Ag(s) in this disease.
The IL-2 Receptor Promotes Proliferation,bcl-2andbcl-xInduction, But Not Cell Viability Through the Adapter Molecule Shc
Authors: Lord James D. , McIntosh Bryan C. , Greenberg Philip D. , Nelson Brad H.
Published: December 2022

IL-2, the principal mitogenic factor for activated T cells, delivers a proliferative signal through ligation of the heterotrimeric IL-2R. This proliferative signal is critically dependent upon cytoplasmic tyrosines on the β-chain of this receptor (IL-2Rβ) becoming phosphorylated in response to ligand. We found that at least one of these tyrosines (Y338) also mediates cell survival and induction of bcl-2, bcl-x, and c-myc in the murine T cell line CTLL-2. Since the adapter molecule Shc binds to phosphorylated Y338, the specific contribution of Shc to these events was evaluated. An IL-2Rβ/Shc fusion protein, in which Shc was covalently tethered to a truncated version of IL-2Rβ lacking all cytoplasmic tyrosines, revealed a robust proliferative signal mediated through Shc. This Shc-mediated signal induced expression of c-myc as well as the antiapoptotic genes bcl-2 and bcl-x with normal magnitude and kinetics. Nonetheless, signals from this fusion protein failed to sustain the long-term viability of CTLL-2 cells. Thus, induction of bcl family genes and delivery of a competent proliferative signal are not sufficient to promote cell survival and mediate the antiapoptotic effects associated with a complete IL-2 signal.
Stat3 Activation Is Responsible for IL-6-Dependent T Cell Proliferation Through Preventing Apoptosis: Generation and Characterization of T Cell-Specific Stat3-Deficient Mice
Authors: Takeda Kiyoshi , Kaisho Tsuneyasu , Yoshida Nobuaki , Takeda Junji , Kishimoto Tadamitsu , Akira Shizuo
Published: December 2022

Stat3, a member of STAT, is activated by a variety of cytokines such as IL-6 family of cytokines, granulocyte CSF, epidermal growth factor, and leptin. A recent study with mice genetically deficient in the Stat3 gene has revealed its important role in the early embryogenesis. To assess the function of Stat3 in adult tissues, we disrupted the Stat3 gene specifically in T cells by conditional gene targeting using Cre-loxP system. In Stat3-deficient T cells, IL-6-induced proliferation was severely impaired. IL-6 did not enhance cell cycle progression, but prevented apoptosis of normal T cells. In contrast, IL-6 did not prevent apoptosis of Stat3-deficient T cells. Antiapoptotic protein, Bcl-2, was normally up-regulated in response to IL-6 even in Stat3-deficient T cells. These results demonstrate that Stat3 activation is involved in IL-6-dependent T cell proliferation through prevention of apoptosis independently of Bcl-2.
The Role of Protein Kinase C Signaling in Activated DRA Transcription
Authors: Setterblad Niclas , Onyango Isaac , Pihlgren Ulla , Rask Lars , Andersson Göran
Published: December 2022

Expression of human MHC HLA-DRA class II gene can be up-regulated in B cells by Ig cross-linking as well as by phorbol esters such as 12-O-tetradecanoyl phorbol 13-acetate (TPA). Induced DRA expression involves activation of restricted protein kinase C (PKC) isoforms, resulting in activated activator protein-1-dependent transcription. In this report expression profiles and activation of PKC were analyzed in human Raji B lymphoblastoid cells. Transient transfection analysis with target plasmids containing either DRA promoter (wild-type or mutated) or TPA response elements demonstrated that pretreatment with the selective PKC inhibitor GF 109203X repressed TPA-mediated activation. Western analysis performed on cellular fractions of resting cells and of TPA-activated cells revealed abundant expression of classical PKC-α (cPKC-α), cPKC-βII, and atypical PKC-ζ isoforms and identified a sustained translocation of cPKC-α and cPKC-βII from the cytosolic compartment to membranes. As expected, the distribution of atypical PKC-ζ was unaffected by TPA treatment and displayed an even distribution between cytosol and membranes. This finding was confirmed by immunofluorescence microscopy. The TPA-mediated translocation of cPKC-α and cPKC-βII was not influenced by pretreatment with GF 109203X. Finally, functional activation and translocation of PKC were investigated with a selective in vitro kinase assay. Together, these results show that activated HLA-DRA expression in response to TPA treatment is strictly dependent on PKC activation acting on the X2 box of the DRA promoter and that selective inhibition of PKC enzymatic activity does not influence subcellular localization of expressed PKC isoenzymes. Thus, the translocation event per se occurs independently of PKC activation in these cells.
Mycobacterium avium-intracellulare complexActivates Nuclear Transcription Factor-κB in Different Cell Types Through Reactive Oxygen Intermediates
Authors: Giri Dipak K. , Mehta Reeta T. , Kansal Rita G. , Aggarwal Bharat B.
Published: December 2022

Mycobacterium avium-intracellulare complex (MAC) is one of the most common opportunistic pathogens in HIV-infected patients. Their synergistic interaction leads to a rapid deterioration of the host defense. In vivo, MAC manifests as a disseminated granulomatous disease that produces a massive inflammatory tissue response perhaps through its activation of inflammatory cytokines. The intracellular signaling following interaction of the mycobacterium with host cells is incompletely understood. Because the response is dependent, in part, on the activation of NF-κB, we investigated the effect of MAC on this nuclear transcription factor in cells of macrophage and nonmacrophage lineage. We demonstrate that both high and low virulence strains of MAC potently and rapidly activated NF-κB. In supershift assays, using specific Abs against the NF-κB subunits, we identified a p50/p65 heterodimer that was formed within 5 min after incubation with the bacterium too rapidly for cytokines to be involved in the activation. This activation was instead mediated through the generation of reactive oxygen intermediates, inasmuch as preincubation of cells with a variety of antioxidants inhibited NF-κB activation. Likewise, the transfection of cells with Mn-superoxide dismutase blocked the NF-κB activation induced by the bacterium. These data suggest that NF-κB activation is a consequence of interaction of host cells with the bacterium and that the interaction may play a pivotal role in the pathogenesis of the disease.
Gut-Derived Intraepithelial Lymphocytes Induce Long Term Immunity AgainstToxoplasma gondii
Authors: Lepage Anne C. , Buzoni-Gatel Dominique , Bout Daniel T. , Kasper Lloyd H.
Published: December 2022

Intraepithelial lymphocytes (IEL) of the intestine represent an important barrier in the prevention of infection against orally acquired pathogens. Adoptive transfer of Ag-primed IEL into a naive host can protect against challenge. Using a murine model, we demonstrate in two genetically distinct mouse strains (C57BL/6 and CBA/J) that protective IEL can be isolated at specific times after oral infection with cysts containing bradyzoites. Adoptive transfer of IEL obtained from the intestine of infected mice at these specific times can provide long term protection, as determined by mortality and cyst number against challenge. The protective IEL appear to be CD8+, TCR-α/β and are at least partially dependent upon the presence of TCR-γ/δ T cells in the host. Endogenous production of the pivotal cytokine, IFN-γ, is essential for host immunity. These findings demonstrate that gut-derived IEL represent a potentially important mechanism to provide long term immunity to the host.
Accessibility of SSA/Ro and SSB/La Antigens to Maternal Autoantibodies in Apoptotic Human Fetal Cardiac Myocytes
Authors: Miranda M. Eugenia , Tseng Chung-E , Rashbaum William , Ochs Robert L. , Casiano Carlos A. , Di Donato Francis , Chan Edward K. L. , Buyon Jill P.
Published: December 2022

Access of intracellular Ags SSA/Ro and SSB/La to cognate maternal autoantibodies is unexplained despite their strong association with congenital heart block. To investigate the hypothesis that apoptosis facilitates surface accessibility of these Ags, human fetal cardiac myocytes from 16- to 22-wk abortuses were established in culture using a novel technique in which cells were isolated after perfusing the aorta with collagenase. Confirmation of cardiac myocytes included positive staining with antisarcomeric α-actinin and contractility induced by 1.8 mM calcium. Incubation with 0.5 μM staurosporine or 0.3 mM 2,3-dimethoxy-1,4-naphthoquinone induced the characteristic morphologic and biochemical changes of apoptosis. The cellular topology of Ro and La was evaluated with confocal microscopy and determined in nonapoptotic and apoptotic cardiocytes by indirect immunofluorescence. In permeabilized nonapoptotic cardiocytes, Ro and La were predominantly nuclear, and propidium iodide (PI) stained the nucleus. In early apoptotic cardiocytes, condensation of the PI- and Ro- or La-stained nucleus was observed, accompanied by Ro/La fluorescence around the cell periphery. In later stages of apoptosis, nuclear Ro and La staining became weaker, and PI demonstrated nuclear fragmentation. Ro/La-stained blebs emerged from the cell membrane, a finding observed in nonpermeabilized cells, supporting an Ab-Ag interaction at the cell surface. In summary, induction of apoptosis in cultured cardiocytes results in surface translocation of Ro/La and recognition by Abs. Although apoptotic cells are programmed to die and do not characteristically evoke inflammation, binding of maternal Abs and subsequent influx of leukocytes could damage surrounding healthy fetal cardiocytes.
Antibody Repertoire Development in Fetal and Neonatal Piglets. I. Four VH Genes Account for 80 Percent of VH Usage During 84 Days of Fetal Life
Authors: Sun J. , Hayward C. , Shinde R. , Christenson R. , Ford S. P. , Butler J. E.
Published: December 2022

VDJ rearrangement and VH gene usage during fetal development in 35 outbred piglets was examined by PCR amplification of VDJs; VDJs were subsequently characterized by hybridization with VH-specific gene probes and by sequencing. VDJ rearrangement was first seen in the fetal liver on day 30 of a 114-day gestation. Four VH genes (VHA, VHB, VHC, and VHE) accounted for ~80% of all VH gene usage regardless of gestational age, choice of piglet, or lymphoid tissue tested; DHA and DHB were used in >90% of the fetal VDJs examined. Evidence of somatic hypermutation during fetal development was not found. The proportion of the four prominent fetal VH genes did not differ significantly between cDNA and DNA, suggesting the absence of selective B cell differentiation. A comparison of recombination signal sequences, flanking sequences, and framework sequences of these fetal genes with other germline VH genes of swine offered no clue as to their selective usage. N-region additions were prominent on day 40 but not on day 30, suggesting that the onset of terminal deoxynucleotidyltransferase activity occurs after 30 days of fetal development. These collective findings indicate that the preimmune, “natural Ab” repertoire of the fetal piglet is largely restricted to the use of four nonpolymorphic and nonmutated VH genes and two nonmutated DH segments. This suggests that the preimmune repertoire of swine is either highly restricted or almost entirely determined by junctional diversity in complementarity-determining region-3.
In Vivo Treatment with a MHC Class I-Restricted Blocking Peptide Can Prevent Virus-Induced Autoimmune Diabetes
Authors: von Herrath Matthias G. , Coon Bryan , Lewicki Hanna , Mazarguil Honore , Gairin Jean Edouard , Oldstone Michael B. A.
Published: December 2022

We tested the in vivo potential of a MHC class I-restricted blocking peptide to sufficiently lower an anti-viral CTL response for preventing virus-induced CTL-mediated autoimmune diabetes (insulin-dependent diabetes mellitus (IDDM)) in vivo without affecting systemic viral clearance. By designing and screening several peptides with high binding affinities to MHC class I H-2Db for best efficiency in blocking killing of target cells by lymphocytic choriomeningitis virus (LCMV) and other viral CTL, we identified the peptide for this study. In vitro, it selectively lowered CTL killing restricted to the Db allele, which correlated directly with the affinity of the respective epitopes. Expression of the blocking peptide in the target cell lowered recognition of all Db-restricted LCMV epitopes. In addition, in vitro expansion of LCMV memory CTL was prevented, resulting in decreased IFN-γ secretion. In vivo, a 2-wk treatment with this peptide lowered the LCMV Db-restricted CTL response by over threefold without affecting viral clearance. However, the CTL reduction by the peptide treatment was sufficient to prevent LCMV-induced IDDM in rat insulin promoter-LCMV-glycoprotein transgenic mice. Following LCMV infection, these mice develop IDDM, which depends on Db-restricted anti-self (viral) CTL. Precursor numbers of splenic LCMV-CTL in peptide-treated mice were reduced, but their cytokine profile was not altered, indicating that the peptide did not induce regulatory cells. Further, non-LCMV-CTL recognizing the blocking peptide secreted IFN-γ and did not protect from IDDM. This study demonstrates that in vivo treatment with a MHC class I blocking peptide can prevent autoimmune disease by directly affecting expansion of autoreactive CTL.
Signaling Through the EBV/C3d Receptor (CR2, CD21) in Human B Lymphocytes: Activation of Phosphatidylinositol 3-Kinase via a CD19-Independent Pathway
Authors: Bouillie Sylvie , Barel Monique , Frade Raymond
Published: December 2022

We herein analyzed the regulation of phosphatidylinositol 3-kinase (PI 3-kinase) activity by CR2 activated on B lymphocyte cell surface. We demonstrated that CR2 activation triggered in vivo PI 3-kinase activity and interaction of PI 3-kinase p85 subunit with a tyrosine-phosphorylated p95 component. The specificity of PI 3-kinase activity was controlled using wortmannin and LY294002. CR2 activation did not trigger tyrosine phosphorylation of PI 3-kinase p85 subunit, but induced direct interaction of tyrosine phosphorylated p95 with the Src homology 2 domain of p85 subunit, as shown using glutathione-S-transferase fusion proteins. Despite identical molecular masses, immunoblotting analysis demonstrated that tyrosine-phosphorylated p95 that interacted in vivo and in vitro with p85 was neither CD19, the 95-kDa proto-oncogene vav, nor Gab1 (a 95-kDa adaptor molecule). Furthermore, p95 tyrosine phosphoprotein also expressed in K562A cells (CR2+ CD19− cells) interacted with Src homology 2 domain of PI 3-kinase p85 subunit after CR2 activation. Activated CR2 did not interact directly with p85 subunit or tyrosine-phosphorylated p95. This suggests the presence of an intermediate molecule between activated CR2 and tyrosine-phosphorylated p95, which may be 3BP2. In addition, in contrast to CD19 activation, CR2 activation did not trigger interaction of CD19 or Vav with PI 3-kinase p85 subunit or coprecipitation of PI 3-kinase activity with CD19. Together, these data clearly demonstrated that CR2 activation triggered in vivo PI 3-kinase activation through a pathway distinct from that triggered through CD19 activation.
Immunosuppressive Activities of Recombinant Glycosylation -Inhibiting Factor Mutants
Authors: Tomura Takafumi , Watarai Hiroshi , Honma Nakayuki , Sato Masahiro , Iwamatsu Akihiro , Kato Yoichi , Kuroki Ryota , Nakano Tatsumi , Mikayama Toshifumi , Ishizaka Kimishige
Published: December 2022

We have shown previously that glycosylation-inhibiting factor (GIF) in culture supernatants of suppressor T cell (Ts) hybridomas had bioactivity, while the same cells contained a substantial quantity of inactive GIF in cytosol. Mass-spectrometric analysis of GIF in the culture supernatant and cytosol of a Ts hybridoma provided direct evidence that GIF protein was posttranslationally modified in the Ts cells, and that the GIF bioactivity is associated with the posttranslationally modified species. Assuming that conformational changes induced by the posttranslational modifications are responsible for generation of bioactivity, we constructed cysteine mutants of human rGIF (rhGIF) in which cysteine at position 57, 60, or 81 was replaced with Ala, and the mutants were expressed in Escherichia coli. Replacement of Cys57 or Cys60 with Ala resulted in generation of bioactivity, while replacement of Cys81 with Ala failed to do so. It was also found that replacement of Cys57 with Ala and carboxymethylation of a sulfhydryl group in Cys60 synergistically increased the GIF bioactivity of the GIF derivatives. A mutated GIF protein, in which Cys57 and Asn106 in the rhGIF were replaced with Ala and Ser, respectively, had immunosuppressive effects on the IgE and IgG1 Ab responses of BDF1 mice to DNP-OVA, while wild-type rhGIF did not. Evidence was obtained that the mutated GIF suppressed Ag priming of Th cells for the Ab responses and proliferative response.
IL-4 Enhances IL-10 Gene Expression in Murine Th2 Cells in the Absence of TCR Engagement
Authors: Schmidt-Weber C. B. , Alexander S. I. , Henault L. E. , James L. , Lichtman A. H.
Published: December 2022

Both IL-4 and IL-10 are regulatory cytokines produced by Th2 cells that can down-regulate cell-mediated immune responses. The studies reported here examine the influence of various cytokines in the regulation of T cell IL-10 production. The results indicate that IL-10 gene expression by TCR transgenic Th2 cells is significantly up-regulated by IL-4 in the absence of TCR signals. IL-4 enhances both IL-10 mRNA levels and secreted protein, and this effect is not related to enhanced mRNA stability. TCR-mediated IL-10 gene expression is inhibited by cyclosporin A, but IL-4-mediated IL-10 expression is not. IL-4 also enhances IL-13 mRNA levels, to a lesser extent than IL-10, but does not significantly effect the expression of other cytokine mRNAs. Furthermore, IL-4 does not significantly enhance IL-10 expression in Th1 cells. IL-2 also enhances effector cytokine production in the absence of TCR signals, but in a subset nonspecific manner, increasing both Th2 IL-4 mRNA and Th1 IFN-γ mRNA. These data suggest that Th2 IL-4 production may contribute to the down-regulation of immune responses by directly enhancing Th2 IL-10 production. In addition, the data clearly demonstrate that exogenous cytokines can significantly influence effector cytokine production by effector T cells without the requirement for TCR signals.
IL-2 Induces STAT4 Activation in Primary NK Cells and NK Cell Lines, But Not in T Cells
Authors: Wang Kathy S. , Ritz Jerome , Frank David A.
Published: December 2022

IL-2 exerts potent but distinct functional effects on two critical cell populations of the immune system, T cells and NK cells. Whereas IL-2 leads to proliferation in both cell types, it enhances cytotoxicity primarily in NK cells. In both T cells and NK cells, IL-2 induces the activation of STAT1, STAT3, and STAT5. Given this similarity in intracellular signaling, the mechanism underlying the distinct response to IL-2 in T cells and NK cells is not clear. In this study, we show that in primary NK cells and NK cell lines, in addition to the activation of STAT1 and STAT5, IL-2 induces tyrosine phosphorylation of STAT4, a STAT previously reported to be activated only in response to IL-12 and IFN-α. This activation of STAT4 in response to IL-2 is not due to the autocrine production of IL-12 or IFN-α. STAT4 activated in response to IL-2 is able to bind to a STAT-binding DNA sequence, suggesting that in NK cells IL-2 is capable of activating target genes through phosphorylation of STAT4. IL-2 induces the activation of Jak2 uniquely in NK cells, which may underlie the ability of IL-2 to activate STAT4 only in these cells. Although the activation of STAT4 in response to IL-2 occurs in primary resting and activated NK cells, it does not occur in primary resting T cells or mitogen-activated T cells. The unique activation of the STAT4-signaling pathway in NK cells may underlie the distinct functional effect of IL-2 on this cell population.
A Selective Defect of IFN-γ- But Not of IFN-α-Induced JAK/STAT Pathway in a Subset of U937 Clones Prevents the Antiretroviral Effect of IFN-γ Against HIV-1
Authors: Bovolenta Chiara , Lorini Alessandro L. , Mantelli Barbara , Camorali Laura , Novelli Francesco , Biswas Priscilla , Poli Guido
Published: December 2022

IFN-γ induces transcription of several IFN-stimulated genes (ISGs). Recently, the IFN-γ-dependent Janus kinase (JAK)/STAT pathway has been shown to mediate the activation of some ISGs, by the sequential phosphorylation of two JAK kinases (JAK1 and JAK2) and of STAT1. Given that the JAK/STAT is the major, but not the only pathway linked to the IFN-γR, aim of our work was to investigate the signal-transduction pathway(s) by which IFN-γ exerts its effects on acute replication of HIV in monocytic cells. To this end, we utilized clones previously derived from the U937 promonocytic cell line, differing for their efficient (plus clones) or inefficient (minus clones) abilities of supporting HIV replication. Unlike IFN-α, IFN-γ did not inhibit HIV replication in plus clones, whereas virus production in minus cells was efficiently inhibited by both types of IFN. Plus clones generated a JAK/STAT signal-transduction pathway in response to IFN-α, but not IFN-γ. In contrast, minus clones responded to either cytokines. The functional defect of plus clones in response to IFN-γ was correlated to a selective defect of IFN-γR2, but not IFN-γR1, membrane expression. Surprisingly enough, IFN-γ stimulation of plus clones induced IFN-stimulated gene factor 3 (ISGF3γ). These results strongly support the hypothesis that the JAK/STAT pathway is responsible for the antiretroviral effect of IFN-γ, and further provide evidence for a potential second pathway triggered by IFN-γ in the absence of IFN-γR2 chain cell surface expression and involving ISGF3γ.
Activation of Human CD8+ αβ TCR+ Cells byMycobacterium tuberculosisVia an Alternate Class I MHC Antigen-Processing Pathway
Authors: Canaday David H. , Ziebold Christine , Noss Erika H. , Chervenak Keith A. , Harding Clifford V. , Boom W. Henry
Published: December 2022

Human immune responses to M. tuberculosis are characterized by activation of multiple T cell subsets including CD4+, CD8+, and γδ T cells, and the role of CD8+ αβ TCR+ T cells in this response is poorly understood. Stimulation of T cells from healthy tuberculin skin test-positive persons with live M. tuberculosis-H37Ra or soluble M. tuberculosis Ags readily up-regulated IL-2Rα (CD25) expression on CD8+ T cells. Purified resting and activated CD8+ T cells produced IFN-γ and proliferated to both M. tuberculosis bacilli and soluble mycobacterial Ags with monocytes as APC. Precursor frequency of mycobacterial Ag-specific CD8+ T cells by IFN-γ enzyme-linked immunospot was 5–10-fold lower than the precursor frequency of CD4+ T cells, and IFN-γ secretion by CD8+ T cells was 50–100-fold lower. CD8+ T cells secreted ∼10-fold less IFN-γ per cell than CD4+ T cells in response to mycobacterial Ags. CD8+ T cell responses to M. tuberculosis bacilli were blocked by anti-MHC class I antibody and required Ag processing. Processing of M. tuberculosis bacilli by monocytes for presentation to MHC class I-restricted CD8+ T cells was insensitive to brefeldin A treatment, which blocks the conventional MHC class I Ag-processing pathway. These results represent the first demonstration that human cells can process pathogen Ags via an alternate Ag-processing pathway for MHC class I and suggest a mechanism for participation of IFN-γ-secreting CD8+ T cells in the human immune responses to M. tuberculosis.
Evaluation of the Role of Mitogen-Activated Protein Kinases in the Expression of Inducible Nitric Oxide Synthase by IFN-γ and TNF-α in Mouse Macrophages
Authors: Chan Edward D. , Winston Brent W. , Uh Soo-Taek , Wynes Murry W. , Rose David M. , Riches David W. H.
Published: December 2022

The expression of inducible nitric oxide synthase (iNOS) by macrophages is stimulated by coexposure to IFN-γ and a number of stimuli, including TNF-α. Recent work has shown that TNF-α activates members of the mitogen-activated protein kinase family that subsequently trans-activate transcription factors implicated in the regulation of iNOS expression. The objective of this study was to systematically evaluate the role of: 1) p42mapk/erk2, 2) p46 c-Jun NH2-terminal kinase/stress-activated protein kinase (p46 JNK/SAPK), and 3) p38mapk in the induction of iNOS expression during costimulation of mouse macrophages with IFN-γ and TNF-α. All three kinases were activated during costimulation with IFN-γ and TNF-α. However, specific antagonism of the p42mapk/erk2 and p38mapk with PD98059 and SKF86002, respectively, had no effect on the induction of iNOS expression. In contrast, blockade of all three kinases with N-acetylcysteine completely blocked the induction of iNOS expression. In addition, specific antagonism of the JNK/SAPK upstream kinases MEKK (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase) and MKK4 (mitogen-activated protein kinase kinase 4) with dominant inhibitory mutants blocked transcriptional activation of the iNOS promoter in response to costimulation with IFN-γ and TNF-α. Collectively, these findings support the involvement of p46 JNK/SAPK and its upstream kinases in regulating the induction of iNOS following ligation of the TNF-α receptor CD120a (p55) in the presence of IFN-γ.
Regulation of T Cell Activation In Vitro and In Vivo by Targeting the OX40-OX40 Ligand Interaction: Amelioration of Ongoing Inflammatory Bowel Disease with an OX40-IgG Fusion Protein, But Not with an OX40 Ligand-IgG Fusion Protein
Authors: Higgins Lisa M. , McDonald Stuart A. C. , Whittle Nigel , Crockett Nigel , Shields John G. , MacDonald Thomas T.
Published: December 2022

OX40 is a member of the TNFR superfamily, and is found predominantly on activated CD4-positive T cells. In vitro an OX40-IgG fusion protein inhibits mitogen- and Ag-driven proliferation and cytokine release by splenocytes and lymph node T cells. In contrast, an OX40 ligand-IgG fusion protein enhanced proliferative responses. In normal mice, OX40-positive cells are observed only in lymphoid tissues, including Peyer’s patches of the gut. In mice with hapten-induced colitis or IL-2 knockout mice with spontaneous colitis, OX40-positive cells are found infiltrating the lamina propria. Administration of the OX40-IgG fusion protein to mice with ongoing colitis (but not the OX40 ligand-IgG) ameliorated disease in both mouse models of inflammatory bowel disease. This was evidenced by a reduction in tissue myeloperoxidase; reduced transcripts for TNF-α, IL-1, IL-12, and IFN-γ; and a reduction in the T cell infiltrate. Targeting OX40 therefore shows considerable promise as a new strategy to inhibit ongoing T cell reactions in the gut.
IL-17 Is Produced by Nickel-Specific T Lymphocytes and Regulates ICAM-1 Expression and Chemokine Production in Human Keratinocytes: Synergistic or Antagonist Effects with IFN-γ and TNF-α
Authors: Albanesi Cristina , Cavani Andrea , Girolomoni Giampiero
Published: December 2022

IL-17 is a novel T cell-derived cytokine that can regulate the functions of a variety of cell types. In this study, we investigated whether hapten-specific T cells isolated from patients with allergic contact dermatitis (ACD) to nickel produce IL-17 and the effects of IL-17 alone or in combination with IFN-γ or TNF-α on the immune activation of keratinocytes. Skin affected with ACD to nickel and skin-derived, nickel-specific CD4+ T cell lines expressed IFN-γ, TNF-α, and IL-17 mRNAs. Four of seven nickel-specific CD4+ T cell clones positive for the skin-homing receptor, cutaneous lymphocyte-associated Ag, were shown to corelease IL-17, IFN-γ, and TNF-α. In contrast, two nickel-specific CD8+ T cell clones failed to synthesize IL-17. Normal human keratinocytes were found to express constitutively the IL-17 receptor gene. IL-17 specifically and dose-dependently augmented IFN-γ-induced ICAM-1 expression on keratinocytes at both the mRNA and the protein level, whereas HLA-DR, MHC class I, and CD40 levels were not modulated by IL-17. On the other hand, IL-17 alone did not affect ICAM-1 or enhance TNF-α-induced ICAM-1. In addition, IL-17, both directly and in synergism with IFN-γ and/or TNF-α, stimulated synthesis and release of IL-8 by keratinocytes. In contrast, IFN-γ- and TNF-α-induced production of RANTES was markedly inhibited by IL-17, and the synthesis of macrophage chemotactic protein 1 was not changed. Taken together, the results suggest that IL-17 is an important player of T cell-mediated skin immune responses, with synergistic or antagonist effects on IFN-γ- and TNF-α-stimulated keratinocyte activation.
Cutting Edge: Human Myeloid Cells Express an Activating ILT Receptor (ILT1) That Associates with Fc Receptor γ-Chain
Authors: Nakajima Hideo , Samaridis Jacqueline , Angman Lena , Colonna Marco
Published: December 2022

Ig-like transcripts (ILTs) encode cell surface receptors expressed on myeloid and lymphoid cells that are structurally and functionally related to killer cell inhibitory receptors. One ILT, designated ILT1, contains a short cytoplasmic domain that lacks sequence motifs implicated in signal transduction. Its function is unknown. Similar short cytoplasmic domains have been observed in activating NK cell receptors and FcαR, which transduce stimulatory signals via associated DAP12 and FcεRIγ proteins, respectively. Here we show that ILT1 receptor is selectively expressed on myeloid cells, functions as an activating receptor, and associates with FcεRIγ rather than DAP12.