bluuebunny/crossref_metadata_2025 huggingface dataset:
New Immunosuppressive Drug PNU156804 Blocks IL-2-Dependent Proliferation and NF-κB and AP-1 Activation
Authors: Mortellaro Alessandra , Songia Simona , Gnocchi Paola , Ferrari Mario , Fornasiero Chiara , D’Alessio Roberto , Isetta Anna , Colotta Francesco , Golay Josée
Published: December 2022

We had previously shown that the drug undecylprodigiosin (UP) blocks human lymphocyte proliferation in vitro. We have now investigated the mechanism of action of a new analogue of UP, PNU156804, which shows a more favorable activity profile than UP in mice. We demonstrate here that the biological effect of PNU156804 in vitro is indistinguishable from UP: PNU156804 blocks human T cell proliferation in mid-late G1, as determined by cell cycle analysis, expression of cyclins, and cyclin-dependent kinases and retinoblastoma phosphorylation. In addition, we show that PNU156804 does not block significantly the induction of either IL-2 or IL-2R α- and γ-chains but inhibits IL-2-dependent T cell proliferation. We have investigated several molecular pathways that are known to be activated by IL-2 in T cells. We show that PNU156804 does not inhibit c-myc and bcl-2 mRNA induction. On the other hand, PNU156804 efficiently inhibits the activation of the NF-κB and AP-1 transcription factors. PNU156804 inhibition of NF-κB activation is due to the inhibition of the degradation of IκB-α and IκB-β. PNU156804 action is restricted to some signaling pathways; it does not affect NF-κB activation by PMA in T cells but blocks that induced by CD40 cross-linking in B lymphocytes. We conclude that the prodigiosin family of immunosuppressants is a new family of molecules that show a novel target specificity clearly distinct from that of other immunosuppressive drugs such as cyclosporin A, FK506, and rapamycin.
p38α Mitogen-Activated Protein Kinase Is Activated by CD28-Mediated Signaling and Is Required for IL-4 Production by Human CD4+CD45RO+ T Cells and Th2 Effector Cells
Authors: Schafer Peter H. , Wadsworth Scott A. , Wang Liwen , Siekierka John J.
Published: December 2022

T cell proliferation and cytokine production usually require stimulation via both the TCR/CD3 complex and the CD28 costimulatory receptor. Using purified human CD4+ peripheral blood T cells, we show that CD28 stimulation alone activates p38α mitogen-activated protein kinase (p38α). Cell proliferation induced by CD28 stimulation alone, a response attributed to CD4+CD45RO+ memory T cells, was blocked by the highly specific p38 inhibitors SB 203580 (IC50 = 10–80 nM) and RWJ 67657 (IC50 = 0.5–4 nM). In contrast, proliferation induced by anti-CD3 plus anti-CD28 mAbs was not blocked. Inhibitors of p38 also blocked CD4+ T cell production of IL-4 (SB 203580 IC50 = 20–100 nM), but not IL-2, in response to CD3 and CD28 stimulation. IL-5, TNF-α, and IFN-γ production were also inhibited, but to a lesser degree than IL-4. IL-4 production was attributed to CD4+CD45RO+ T cells, and its induction was suppressed by p38 inhibitors at the mRNA level. In polarized Th1 and Th2 cell lines, SB 203580 strongly inhibited IL-4 production by Th2 cells (IC50 = 10–80 nM), but only partially inhibited IFN-γ and IL-2 production by Th1 cells (<50% inhibition at 1 μM). In both Th1 and Th2 cells, CD28 signaling activated p38α and was required for cytokine production. These results show that p38α plays an important role in some, but not all, CD28-dependent cellular responses. Its preferential involvement in IL-4 production by CD4+CD45RO+ T cells and Th2 effector cells suggests that p38α may be important in the generation of Th2-type responses in humans.
Perturbed Regulation of ZAP-70 and Sustained Tyrosine Phosphorylation of LAT and SLP-76 in c-Cbl-Deficient Thymocytes
Authors: Thien Christine B. F. , Bowtell David D. L. , Langdon Wallace Y.
Published: December 2022

Recent studies indicate that c-Cbl and its oncogenic variants can modulate the activity of protein tyrosine kinases. This finding is supported by studies showing that c-Cbl interacts directly with a negative regulatory tyrosine in ZAP-70, and that the levels of tyrosine-phosphorylated ZAP-70 and numerous other proteins are increased in TCR-stimulated thymocytes from c-Cbl-deficient mice. Here, we demonstrate that this enhanced phosphorylation of ZAP-70 and that of two substrates, LAT and SLP-76, is not due to altered protein levels but is the consequence of two separate events. First, we find increased expression of tyrosine-phosphorylated TCRζ chain in c-Cbl-deficient thymocytes, which results in a higher level of ζ-chain-associated ZAP-70 that is initially accessible for activation. Thus, more ZAP-70 is activated and more of its substrates (LAT and SLP-76) become tyrosine-phosphorylated after TCR stimulation. However, an additional mechanism of ZAP-70 regulation is evident at a later time poststimulation. At this time, ZAP-70 from both normal and c-Cbl−/− thymocytes becomes hyperphosphorylated; however, only in normal thymocytes does this correlate with ZAP-70 down-regulation and a diminished ability to phosphorylate LAT and SLP-76. In contrast, c-Cbl-deficient thymocytes display altered phosphorylation kinetics, for which LAT phosphorylation is increased and SLP-76 phosphorylation is sustained. Thus, the ability to down-regulate the phosphorylation of two ZAP-70 substrates is impaired in c-Cbl−/− thymocytes. These findings provide evidence that c-Cbl is involved in the negative regulation of the phosphorylation of LAT and SLP-76 by ZAP-70.
Protection Against CD95-Mediated Apoptosis by Inorganic Mercury in Jurkat T Cells
Authors: Whitekus Michael J. , Santini Ronald P. , Rosenspire Allen J. , McCabe Michael J.
Published: December 2022

Dysregulation of CD95/Fas-mediated apoptosis has been implicated as a contributing factor in autoimmune disorders. Animal studies clearly have established a connection between mercury exposure and autoimmune disease in rodents, while case reports have suggested a link between accidental mercury contamination and autoimmune disease in humans. The mechanism(s) for these associations are poorly understood. Using the Jurkat cell model, we have found that low levels (≤10 μM) of inorganic mercury (i.e., HgCl2) attenuated anti-CD95-mediated growth arrest and markedly enhanced cell survival. Several biochemical assays for apoptosis, including DNA degradation, poly(ADP-ribose) polymerase degradation, and phosphatidylserine externalization, directly verified that HgCl2 attenuated anti-CD95-mediated apoptosis. In an attempt to further characterize the effect of mercury on CD95-mediated apoptosis, several signaling components of the CD95 death pathway were analyzed to determine whether HgCl2 could modulate them. HgCl2 did not modulate CD95 expression; however, it did block CD95-induced caspase-3 activation. HgCl2 was not able to attenuate TNF-α-mediated apoptosis in U-937 cells, or ceramide-C6-mediated apoptosis in Jurkat cells, suggesting that mercury acts upstream of, or does not involve, these signals. Thus, inorganic mercury specifically attenuates CD95-mediated apoptosis likely by targeting a signaling component that is upstream of caspase-3 activation and downstream of CD95.
MHC Class II Antigen Processing in B Cells: Accelerated Intracellular Targeting of Antigens
Authors: Cheng Paul C. , Steele Carrie R. , Gu Lin , Song Wenxia , Pierce Susan K.
Published: December 2022

Processing and presentation by Ag-specific B cells is initiated by Ag binding to the B cell Ag receptor (BCR). Cross-linking of the BCR by Ag results in a rapid targeting of the BCR and bound Ag to the MHC class II peptide loading compartment (IIPLC). This accelerated delivery of Ag may be essential in vivo during periods of rapid Ag-driven B cell expansion and T cell-dependent selection. Here, we use both immunoelectron microscopy and a nondisruptive protein chemical polymerization method to define the intracellular pathway of the targeting of Ags by the BCR. We show that following cross-linking, the BCR is rapidly transported through transferrin receptor-containing early endosomes to a LAMP-1+, β-hexosaminadase+, multivesicular compartment that is an active site of peptide-class II complex assembly, containing both class II-invariant chain complexes in the process of invariant chain proteolytic removal as well as mature peptide-class II complexes. The BCR enters the class II-containing compartment as an intact mIg/Igα/Igβ complex bound to Ag. The pathway by which the BCR targets Ag to the IIPLC appears not to be identical to that by which Ags taken up by fluid phase pinocytosis traffick, suggesting that the accelerated BCR pathway may be specialized and potentially independently regulated.
CD94/NKG2-A Inhibitory Complex Blocks CD16-Triggered Syk and Extracellular Regulated Kinase Activation, Leading to Cytotoxic Function of Human NK Cells
Authors: Palmieri Gabriella , Tullio Valentino , Zingoni Alessandra , Piccoli Mario , Frati Luigi , Lopez-Botet Miguel , Santoni Angela
Published: December 2022

The CD94/NKG2-A complex is the inhibitory receptor for the nonclassical MHC class I molecule HLA-E on human NK cells. Here we studied the molecular mechanisms underlying the inhibitory activity of CD94/NKG2-A on NK cell functions by analyzing its interference on CD16-initiated signaling pathways involved in the control of cytolytic activity. Both tyrosine phosphorylation and activation of Syk kinase together with tyrosine phosphorylation of CD16 receptor ζ subunit are markedly inhibited by the coengagement of CD94/NKG2-A complex. As a downstream consequence, CD94/NKG2-A cross-linking impairs the CD16-induced activation of extracellular regulated kinases (ERKs), a pathway involved in NK cytotoxic function. The block of ERK activation is exerted at an early, PTK-dependent stage in the events leading to p21ras activation, as the CD16-induced tyrosine phosphorylation of Shc adaptor protein and the formation of Shc/Grb-2 complex are abrogated by CD94/NKG2-A simultaneous engagement. Our observations indicate that CD94/NKG2-A inhibits the CD16-triggered activation of two signaling pathways involved in the cytotoxic activity of NK cells. They thus provide molecular evidence to explain the inhibitory function of CD94/NKG2-A receptor on NK effector functions.
TNF-α-Mediated Apoptosis Is Initiated in Caveolae-Like Domains
Authors: Ko Young-Gyu , Lee Jae-Seon , Kang Young-Sun , Ahn Jung-Hyuck , Seo Jeong-Sun
Published: December 2022

Caveolae-like domains (CLDs) have been hypothesized to mediate apoptosis, since they contain sphingomyelin and initiate the conversion of sphingomyelin to ceramide. To address whether CLDs are directly involved in apoptosis, CLDs from U937 cells were isolated, taking advantage of their detergent insolubility and low density. The CLDs contained alkaline phosphatase as well as many signaling molecules, including Fyn, protein kinase Cα, Raf-1, phospholipase Cγ1, and tyrosine phosphoproteins. Immunoblotting and immunofluorescent data showed that TNF receptor 1 colocalized with CD36 in CLDs, suggesting that TNF-α-initiated apoptosis occurs in CLDs. When cells were incubated with lipoprotein-deficient medium, the cholesterol concentration was greatly decreased in CLDs but not in other fractions, implying that the CLDs were selectively disrupted. In the CLD-disrupted cells, the surface expression of TNF receptor 1 and CD36 was significantly reduced. Analysis of cellular morphology, percent DNA fragmentation, DNA laddering, and caspase-3 activity showed that TNF-α-mediated apoptosis was blocked in CLD-disrupted cells, whereas anti-Fas-mediated apoptosis was not. Since Fas was not found in CLDs of Jurkat cells, apoptosis by Fas ligation might not require CLDs. Taken together, these data strongly imply that TNF-α-mediated apoptosis is initiated in CLDs.
IL-12 Induces IFN Regulating Factor-1 (IRF-1) Gene Expression in Human NK and T Cells
Authors: Galon Jérôme , Sudarshan Chitra , Ito Satochi , Finbloom David , O’Shea John J.
Published: December 2022

IL-12 is a critical immunoregulatory cytokine that promotes cell-mediated immune responses and the differentiation of naive CD4+ cells to Th1 cells; however, relatively few IL-12 target genes have been identified. To better clarify the molecular basis of IL-12 action, we set out to characterize genes up-regulated by IL-12, first by contrasting IL-12- and IFN-α-inducible genes. We identified several genes up-regulated by IL-12, namely, MIP-1α, MIP-1β, IL-1RA, and IFN regulatory factor-1 (IRF-1). IRF-1 is a transcription factor regulated by IFNs that is also essential for Th1 responses. We demonstrated that IL-12 directly up-regulates IRF-1 to the same extent as IFN-α in normal human T cells and in NK cells. We showed that IL-12 had a direct effect on IRF-1, an effect not mediated indirectly by the induction of IFN-γ production. Furthermore, IL-2 and IL-12 synergistically induced IRF-1, whereas IFN-α and IL-12 did not. The participation of STAT4 in the regulation of IRF-1 was demonstrated in two ways. First, STAT4 was required for the IL-12-dependent transactivation of an IRF-1 reporter construct, and second, STAT4 binding to the IRF-1 promoter was shown using EMSA. In contrast to IL-12, no up-regulation of IRF-1 was found in IL-4-stimulated cells, and IL-4 did not block IL-12-dependent up-regulation of IRF-1. Therefore, IRF-1 may be an important contributor to IL-12 signaling, and we speculate that the defective IL-12 responses seen in IRF-1−/− mice might be attributable, in part, to the absence of this transcription factor.
Virus Infection Activates IL-1β and IL-18 Production in Human Macrophages by a Caspase-1-Dependent Pathway
Authors: Pirhonen Jaana , Sareneva Timo , Kurimoto Masashi , Julkunen Ilkka , Matikainen Sampsa
Published: December 2022

Monocytes and macrophages play a significant role in host’s defense system, since they produce a number of cytokines in response to microbial infections. We have studied IL-1β, IL-18, IFN-α/β, and TNF-α gene expression and protein production in human primary monocytes and GM-CSF-differentiated macrophages during influenza A and Sendai virus infections. Virus-infected monocytes released only small amounts of IL-1β or IL-18 protein, whereas 7- and 14-day-old GM-CSF-differentiated macrophages readily produced these cytokines. Constitutive expression of proIL-18 was seen in monocytes and macrophages, and the expression of it was enhanced during monocyte/macrophage differentiation. Expression of IL-18 mRNA was clearly induced only by Sendai virus, whereas both influenza A and Sendai viruses induced IL-1β mRNA expression. Since caspase-1 is known to cleave proIL-1β and proIL-18 into their mature, active forms, we analyzed the effect of a specific caspase-1 inhibitor on virus-induced IL-1β and IL-18 production. The release of IL-1β and IL-18, but not that of IFN-α/β or TNF-α, was clearly blocked by the inhibitor. Our results suggest that the cellular differentiation is a crucial factor that affects the capacity of monocytes/macrophages to produce IL-1β and IL-18 in response to virus infections. Furthermore, the virus-induced activation of caspase-1 is required for the efficient production of biologically active IL-1β and IL-18.
Induction of Antitumor Immunity with Fas/APO-1 Ligand (CD95L)-Transfected Neuroblastoma Neuro-2a Cells
Authors: Shimizu Motomu , Fontana Adriano , Takeda Yasutaka , Yagita Hideki , Yoshimoto Takayuki , Matsuzawa Akio
Published: December 2022

Fas/Apo-1 (CD95)-Fas ligand (FasL) system has been implicated in the suppression and stimulation of immune responses. We examined the induction of antitumor immunity with neuroblastoma Neuro-2a cells transfected with FasL cDNA (Neuro-2a+FasL). Neuro-2a+FasL cells expressed FasL on the cell surface and secreted soluble FasL. Histologic and flow cytometric analyses revealed that Neuro-2a+FasL cells caused neutrophils to infiltrate into the injected site, resulting in strong inflammation. Neutrophil infiltration was inhibited by treatment with anti-FasL mAb and did not occur in Fas-deficient lpr mice. Normal syngeneic mice rejected Neuro-2a+FasL cells after the inflammation and acquired tumor-specific protective immunity. CD8+ T cells were responsible for the antitumor immunity. Neuro-2a+FasL cells formed tumors after far longer latency compared with mock-transfected Neuro-2a+Neo cells in nude mice, and immune competent mice rejected Neuro-2a cells but not sarcoma S713a cells when they were injected with Neuro-2a+FasL cells in a mixture. These results suggest that neutrophils attracted through the Fas-FasL system may impair tumor cells by inflammation at the initial step, followed by development of CD8+ T cell-dependent tumor-specific antitumor immunity, leading to complete eradication of tumor cells. Importantly, the treatment with Neuro-2a+FasL cells exhibited therapeutic efficacy against growing tumors.
Plasticity of Immune Responses Suppressing Parasitemia During AcutePlasmodium chabaudiMalaria
Authors: Weidanz William P. , Kemp Justin R. , Batchelder Joan M. , Cigel Francine K. , Sandor Matyas , Heyde Henri C. van der
Published: December 2022

γδ T cells have a crucial role in cell-mediated immunity (CMI) against P. chabaudi malaria, but δ-chain knockout (KO) (δo/o) mice and mice depleted of γδ T cells with mAb cure this infection. To address the question of why mice deficient in γδ T cells resolve P. chabaudi infections, we immunized δo/o mice by infection with viable blood-stage parasites. Sera from infection-immunized mice were tested for their ability to protect JHo/o, δo/o double KO mice passively against P. chabaudi challenge infection. The onset of parasitemia was significantly delayed in mice receiving immune sera, compared with saline or uninfected serum controls. Immune sera were then fractionated into Ig-rich and Ig-depleted fractions by HPLC on a protein G column. Double KO mice were passively immunized with either fraction and challenged with P. chabaudi. The onset of parasitemia was significantly delayed in recipients of the Ig-rich fraction compared with recipients of the Ig-poor fraction of immune sera. We conclude that δo/o mice, which are unable to activate CMI against the parasite, suppress P. chabaudi infection by a redundant Ab-mediated process.
Alcohol (Ethanol) Inhibits IL-8 and TNF: Role of the p38 Pathway
Authors: Arbabi Saman , Garcia Iris , Bauer Gregory J. , Maier Ronald V.
Published: December 2022

Acute ethanol (EtOH) intoxication has been identified as a risk factor for infectious complications in trauma and burn victims. However, the mechanism of this immune dysfunction has yet to be elucidated. The monocyte/macrophage production of cytokines, in particular IL-8 and TNF-α, is critical in the regulation of the acute inflammatory response to infectious challenge. IL-8 is a potent chemoattractant and activator of neutrophils. TNF-α, a proinflammatory cytokine, initiates expression of endothelial cell surface adhesion molecules and neutrophil migration. p38, a member of the mitogen-activated protein kinases, plays an important role in mediating intracellular signal transduction in endotoxin-induced inflammatory responses. We examined the effects of LPS and ethanol on p38 activation and the corresponding IL-8 and TNF-α production in human mononuclear cells. LPS-induced IL-8 and TNF-α production was inhibited in a similar pattern by pretreatment with either EtOH or SB202190 (1 μM), a specific inhibitor of p38 kinase. Western blot analysis, using a dual phospho-specific p38 mitogen-activated protein kinase Ab, demonstrated that EtOH pretreatment inhibited LPS-induced p38 activation. These results demonstrate that alcohol suppresses the normal host immune inflammatory response to LPS. This dysregulation appears to be mediated in part via inhibition of p38 activation. Inhibition of IL-8 and TNF-α production by acute EtOH intoxication may inhibit inflammatory focused neutrophil migration and activation and may be a mechanism explaining the increased risk of trauma- and burn-related infections.
IL-12, Independently of IFN-γ, Plays a Crucial Role in the Pathogenesis of a Murine Psoriasis-Like Skin Disorder
Authors: Hong Kenneth , Chu Alvina , Lúdvíksson Björn R. , Berg Ellen L. , Ehrhardt Rolf O.
Published: December 2022

The onset of acute psoriasis and the exacerbation of chronic psoriasis are often associated with a history of bacterial infection. We demonstrate that while only few scid/scid mice develop disease when CD4+CD45Rbhigh T cells are transferred alone, coadministration of LPS plus IL-12 or staphylococcal enterotoxin B into scid/scid mice 1 day after CD4+CD45Rbhigh T cell transfer greatly enhances disease penetrance and severity. Most importantly, the skin lesions induced by this method exhibit many of the histologic hallmarks observed in human psoriasis. Skin infiltrating CD4+ T cells were predominantly memory/effector cells (CD45Rblow) and exhibited a highly polarized Th1 phenotype. To test whether the development of pathogenic T cells was dependent on their production of IFN-γ, we transferred IFN-γ−/− CD4+CD45Rbhigh T cells into scid/scid or into T, B and NK cell-deficient scid/beige mice. Surprisingly, the incidence of psoriasis was similar to scid/scid animals that received IFN-γ+/+ T cells, although acanthosis of the skin was attenuated. In contrast, the development of psoriasis was abolished if anti-IL-12 mAb was administered on day 7 and 35 after T cell transfer. Skin-derived IFN-γ−/− inflammatory cells, but not cells from anti-IL-12-treated animals, secreted substantial amounts of TNF-α, suggesting that the inflammatory effect of IFN-γ−/− T cells may be partly exerted by TNF-α and that the therapeutic effect of anti-IL-12 may depend on its ability to down-regulate both TNF-α and IFN-γ. Overall, these results suggest that IL-12, independently of IFN-γ, is able to induce pathogenic, inflammatory T cells that are able to induce psoriasiform lesions in mice.
The Role of an Epithelial Neutrophil-Activating Peptide-78-Like Protein in Rat Adjuvant-Induced Arthritis
Authors: Halloran Margaret M. , Woods James M. , Strieter Robert M. , Szekanecz Zoltan , Volin Michael V. , Hosaka Shigeru , Haines G. Kenneth , Kunkel Steven L. , Burdick Marie D. , Walz Alfred , Koch Alisa E.
Published: December 2022

The chemokine, epithelial neutrophil-activating peptide-78 (ENA-78), is a potent neutrophil chemotaxin whose expression is increased in inflamed synovial tissue and fluid in human rheumatoid arthritis compared with osteoarthritis. Since ENA-78 has been implicated in the pathogenesis of RA, we examined the expression of an ENA-78-like protein during the development of rat adjuvant-induced arthritis (AIA). Using an ELISA assay, we found increased levels of antigenic ENA-78-like protein in the sera of AIA animals compared with control normal animals by day 7 postadjuvant injection. ENA-78-like protein levels continued to increase as AIA developed. ENA-78-like protein levels in joint homogenates were increased in AIA animals later in the development of the disease, by day 18 during maximal arthritis, compared with control animals. Expression of ENA-78-like protein in both the AIA serum and joint correlated with the progression of inflammation of the joints. Anti-human ENA-78 administered before disease onset modified the severity of AIA, while administration of anti-ENA-78 after clinical onset of AIA did not modify the disease. These data support a role for an ENA-78-like protein as an important chemokine in the progression and maintenance of AIA.
T Cell Autoimmunity in Ig Transgenic Mice
Authors: Shinde Sanjay , Gee Renelle , Santulli-Marotto Sandra , Bockenstedt Linda K. , Clarke Stephen H. , Mamula Mark J.
Published: December 2022

Autoantibodies directed at a diverse group of proteins of the U1/Sm ribonucleoprotein (snRNP) are characteristic of systemic lupus erythematosus and are found in the MRL murine model of this disease. This study examines the role of transgenic B lymphocytes in the regulation of autoreactive T cells to the snRNP autoantigen. Transgenic mice were developed bearing an Ig heavy chain gene specific for the D protein component of murine snRNP. B lymphocytes in these mice are neither deleted nor anergic and are of an immature (heat-stable Aghigh) phenotype. T lymphocytes from anti-snRNP transgenic mice were examined using a recombinant form of the D protein of the murine snRNP complex. Our results revealed that transgenic anti-snRNP B cell APCs stimulated CD4 T cells from wild-type C57BL/6 and MRL lpr/lpr mice, while nonspecific APCs failed to stimulate CD4 T cells. This study demonstrates that autoreactive T cells are not deleted from wild-type mice, although their activation is facilitated by autoantigen-specific APCs. The snRNP-reactive T cells in C57BL/6 transgenic mice are tolerized, in contrast to those T cells from MRL lpr/lpr transgenic mice. These studies implicate a role for autoreactive B lymphocytes in the in vivo activation and/or diversification of autoreactive T cells.
Induction of MHC Class I Expression on Immature Thymocytes in HIV-1-Infected SCID-hu Thy/Liv Mice: Evidence of Indirect Mechanisms
Authors: Kovalev Grigoriy , Duus Karen , Wang Liping , Lee Robert , Bonyhadi Mark , Ho David , McCune Joseph M. , Kaneshima Hideto , Su Lishan
Published: December 2022

The SCID-hu Thy/Liv mouse and human fetal thymic organ culture (HF-TOC) models have been used to explore the pathophysiologic mechanisms of HIV-1 infection in the thymus. We report here that HIV-1 infection of the SCID-hu Thy/Liv mouse leads to the induction of MHC class I (MHCI) expression on CD4+CD8+ (DP) thymocytes, which normally express low levels of MHCI. Induction of MHCI on DP thymocytes in HIV-1-infected Thy/Liv organs precedes their depletion and correlates with the pathogenic activity of the HIV-1 isolates. Both MHCI protein and mRNA are induced in thymocytes from HIV-1-infected Thy/Liv organs, indicating induction of MHCI gene expression. Indirect mechanisms are involved, because only a fraction (<10%) of the DP thymocytes were directly infected by HIV-1, although the majority of DP thymocytes are induced to express high levels of MHCI. We further demonstrate that IL-10 is induced in HIV-1-infected thymus organs. Similar HIV-1-mediated induction of MHCI expression was observed in HF-TOC assays. Exogenous IL-10 in HF-TOC induces MHCI expression on DP thymocytes. Therefore, HIV-1 infection of the thymus organ leads to induction of MHCI expression on immature thymocytes via indirect mechanisms involving IL-10. Overexpression of MHCI on DP thymocytes can interfere with thymocyte maturation and may contribute to HIV-1-induced thymocyte depletion.
IL-2 Plasmid Therapy of Murine Ovarian Carcinoma Inhibits the Growth of Tumor Ascites and Alters Its Cytokine Profile
Authors: Horton Holly M. , Dorigo Oliver , Hernandez Pepe , Anderson Deborah , Berek Jonathan S. , Parker Suezanne E.
Published: December 2022

We have evaluated whether i.p. murine ovarian tumors could be treated with an IL-2 plasmid DNA complexed with the cationic lipid, (±)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide/dioleoylphosphatidylethanolamine (DMRIE/DOPE). Reporter gene studies were initially conducted in which mice bearing i.p. murine ovarian teratocarcinoma (MOT) were injected i.p. with reporter gene plasmid DNA (pDNA):DMRIE/DOPE. Histochemical analyses revealed that transfection occurred primarily in the tumor cells of the ascites, with only a minority of other ascitic cells or surrounding tissues transfected. IL-2 levels in the MOT ascites were determined after i.p. injection of either IL-2 pDNA:DMRIE/DOPE or recombinant IL-2 protein. IL-2 was detected in tumor ascites for up to 10 days after a single i.p. injection of IL-2 pDNA:DMRIE/DOPE, but was undetectable 24 h after a single i.p. injection of IL-2 protein. In an antitumor efficacy study, MOT tumor-bearing mice injected i.p. with IL-2 pDNA:DMRIE/DOPE on days 5, 8, and 11 after tumor cell implant had a significant inhibition of tumor ascites (p = 0.001) as well as a significant increase in survival (p = 0.008). A cytokine profile of the MOT tumor ascites revealed that mice treated with IL-2 pDNA:DMRIE/DOPE had an IL-2-specific increase in the levels of IFN-γ and GM-CSF. Taken together, these findings indicate that i.p. treatment of ovarian tumors with IL-2 pDNA:DMRIE/DOPE can lead to an increase in local IL-2 levels, a change in the cytokine profile of the tumor ascites, and a significant antitumor effect.
Role of APC in the Selection of Immunodominant T Cell Epitopes
Authors: Ma Chenglie , Whiteley Phyllis E. , Cameron Patricia M. , Freed Daniel C. , Pressey Alison , Chen Shiow-Ling , Garni-Wagner Beth , Fang Carol , Zaller Dennis M. , Wicker Linda S. , Blum Janice S.
Published: December 2022

Following antigenic challenge, MHC-restricted T cell responses are directed against a few dominant antigenic epitopes. Here, evidence is provided demonstrating the importance of APC in modulating the hierarchy of MHC class II-restricted T cell responses. Biochemical analysis of class II:peptide complexes in B cells revealed the presentation of a hierarchy of peptides derived from the Ig self Ag. Functional studies of κ peptide:class II complexes from these cells indicated that nearly 20-fold more of an immunodominant epitope derived from κ L chains was bound to class II DR4 compared with a subdominant epitope from this same Ag. In vivo, T cell responses were preferentially directed against the dominant κ epitope as shown using Ig-primed DR4 transgenic mice. The bias in κ epitope presentation was not linked to differences in class II:κ peptide-binding affinity or epitope editing by HLA-DM. Rather, changes in native Ag structure were found to disrupt presentation of the immunodominant but not the subdominant κ epitope; Ag refolding restored κ epitope presentation. Thus, Ag tertiary conformation along with processing reactions within APC contribute to the selective presentation of a hierarchy of epitopes by MHC class II molecules.
Regulated Association Between the Tyrosine Kinase Emt/Itk/Tsk and Phospholipase-Cγ1 in Human T Lymphocytes
Authors: Perez-Villar Juan J. , Kanner Steven B.
Published: December 2022

The Emt/Itk/Tsk tyrosine kinase is involved in intracellular signaling events induced by several lymphocyte surface receptors. Modulation of TCR/CD3-induced phospholipase-Cγ1 (PLCγ1) activity by the tyrosine kinase Emt/Itk/Tsk has been demonstrated based on studies of Itk-deficient murine T lymphocytes. Here we report a TCR/CD3-regulated association between Emt and PLCγ1 in both normal and leukemic T cells. In addition, this association was enhanced following independent ligation of the CD2, CD4, or CD28 costimulatory molecules, but not of CD5 or CD6 surface receptors, correlating to the induced tyrosine phosphorylation of Emt. Before Ab-induced T cell activation, we found that the Emt-SH3 domain was crucial for the constitutive Emt/PLCγ1 association; however, upon TCR/CD3 engagement, the Emt-SH2 domain was more efficient in mediating the enhanced Emt/PLCγ1 interaction. Furthermore, the PLCγ1-SH3 domain, but not the two PLCγ1-SH2 domains, contributed to formation of the protein complex. Thus, we describe a regulated interaction between Emt and PLCγ1, and based on our studies with individual Emt and PLCγ1 SH2/SH3 domains, we propose a mechanism for this association.
Aluminium Hydroxide Adjuvant Initiates Strong Antigen-Specific Th2 Responses in the Absence of IL-4- or IL-13-Mediated Signaling
Authors: Brewer James M. , Conacher Margaret , Hunter Christopher A. , Mohrs Markus , Brombacher Frank , Alexander James
Published: December 2022

Previous studies demonstrate that aluminium hydroxide adjuvant (alum) produces increased Th1 responses in IL-4-deficient mice compared with wild-type animals, although the continued production of IL-5 by spleen cells from these mice also indicates that Th2 responses are induced. In the present study, we demonstrate that alum can induce Th2-associated IL-4 and IL-5 production in the absence of IL-4 signaling in mice deficient in either IL-4Rα or Stat6. The Th2 responses observed could not be due to IL-13 as IL-13 responses are also impaired in IL-4Rα- and Stat6-deficient mice. We also detected higher levels of IL-4 in IL-4Rα gene-deficient, though not Stat6-deficient, mice compared with their wild-type counterparts. The increased levels of IL-4 could be explained by the IL-4R being unavailable to neutralize this cytokine in IL-4Rα-deficient mice. While levels of IL-5 production in IL-4Rα- or Stat6-deficient mice were similar to IL-4-deficient and wild-type mice, other type 2-associated responses, which are largely or wholly IL-4 dependent, such as the production of IgG1 or IgE Abs, were either reduced or absent. We conclude that alum adjuvants can induce IL-4 production and Th2 responses independently of IL-4 or IL-13, negating the requirement for an early source of IL-4 in the Th2 response induced by this adjuvant.
The In Vivo Fate of APCs Displaying Minor H Antigen and/or MHC Differences Is Regulated by CTLs Specific for Immunodominant Class I-Associated Epitopes
Authors: Loyer Véronique , Fontaine Pierre , Pion Stéphane , Hétu Francis , Roy Denis-Claude , Perreault Claude
Published: December 2022

The goal of this work was to evaluate the fate of APCs following interactions with T cells in unprimed mice with a normal T cell repertoire. We elaborated a model in which male adherent peritoneal mononuclear cells were injected into the foreleg footpads of naive female recipients mismatched for either minor or major histocompatibility Ags. At various times after injection, APC numbers in the draining (axillary and brachial) lymph nodes were assessed using a Ube1y gene-specific PCR assay. Our experimental model was designed so that the number of APCs expressing the priming epitope was similar to what is observed under real life conditions. Thus, early after injection, the frequency of afferent lymph-derived APCs expressing the priming epitope was in the range of 101–102/106 lymph node cells. We found that APCs presenting some, but not all, nonself epitopes were killed rapidly after entrance into the lymph nodes. Rapid elimination of APCs occurred following interactions with MHC class I-restricted, but not class II-restricted, T cells and was observed when APCs presented an immunodominant (B6dom1/H7a), but not a nondominant (HY), epitope. Killing of APCs was mediated partly, but not exclusively, by perforin-dependent process. We propose that killing of APCs by CTLs specific for immunodominant MHC class I-restricted epitopes may be instrumental in regulating the intensity, duration, and diversity of T cell responses.
MHC Class I-Restricted Cytotoxic Lymphocyte Responses Induced by Enterotoxin-Based Mucosal Adjuvants
Authors: Simmons Cameron P. , Mastroeni Pietro , Fowler Ray , Ghaem-maghami Marjan , Lycke Nils , Pizza Mariagrazia , Rappuoli Rino , Dougan Gordon
Published: December 2022

The ability of enterotoxin-based mucosal adjuvants to induce CD8+ MHC class I-restricted CTL responses to a codelivered bystander Ag was examined. Escherichia coli heat-labile toxin (LT), or derivatives of LT carrying mutations in the A subunit (LTR72, LTK63), were tested in parallel with cholera toxin (CT) or a fusion protein consisting of the A1 subunit of CT fused to the Ig binding domain of Staphylococcus aureus protein A (called CTA1-DD). Intranasal (i.n.) immunization of C57BL/6 mice with CT, CTA1-DD, LT, LTR72, LTK63, but not rLT-B, elicited MHC class I-restricted CD8+ T cell responses to coadministered OVA or the OVA CTL peptide SIINFEKL (OVA257–264). CT, LT, and LTR72 also induced CTL responses to OVA after s.c. or oral coimmunization whereas LTK63 only activated responses after s.c. coimmunization. rLT-B was unable to adjuvant CTL responses to OVA or OVA257–264 administered by any route. Mice treated with an anti-CD4 mAb to deplete CD4+ T cells mounted significant OVA-specific CTL responses after i.n. coadministration of LT with OVA or OVA257–264. Both 51Cr release assays and IFN-γ enzyme-linked immunospot assays indicated that IFN-γ−/− and IL-12 p40−/− gene knockout mice developed CTL responses equivalent to those detected in normal C57BL/6 mice. The results highlight the versatility of toxin-based adjuvants and suggest that LT potentiates CTL responses independently of IL-12 and IFN-γ and probably by a mechanism unrelated to cross-priming.
Central Nervous System-Targeted Expression of the Complement Inhibitor sCrry Prevents Experimental Allergic Encephalomyelitis
Authors: Davoust Nathalie , Nataf Serge , Reiman Rachael , V. Holers Michael , Campbell Iain L. , Barnum Scott R.
Published: December 2022

Although generally thought of as a T cell-driven autoimmune disease, recent studies in experimental allergic encephalomyelitis (EAE), the animal model of multiple sclerosis, suggest a significant role for innate immune mechanisms. To address the possibility that the complement system plays a central role in these diseases, we developed a transgenic mouse with astrocyte-targeted production of a soluble inhibitor of complement activation, complement receptor-related protein y (sCrry). Here, we show that sCrry transgenic mice are either fully protected against EAE or develop significantly delayed clinical signs. These results indicate that complement activation may have an essential role in the pathogenesis of the disease and that complement-mediated events may occur early during the effector phase of EAE. Furthermore, this work underscores the importance of humoral immunity in amplifying a T cell-initiated pathogenic process.
TNF and Lymphotoxin β Cooperate in the Maintenance of Secondary Lymphoid Tissue Microarchitecture But Not in the Development of Lymph Nodes
Authors: Kuprash23 Dmitry V. , Alimzhanov2 Marat B. , Tumanov2 Alexei V. , Anderson Arthur O. , Pfeffer Klaus , Nedospasov Sergei A.
Published: December 2022

Inactivation of genes encoding members of TNF and TNF receptor families reveal their divergent roles in the formation and function of secondary lymphoid organs. Most lymphotoxin α (ltα)- and all lymphotoxin β receptor (ltβr)-deficient mice are completely devoid of lymph nodes (LNs); however, most lymphotoxin β (ltβ)-deficient mice develop mesenteric LNs. Tnf- and tnfrp55-deficient mice develop a complete set of LNs, while ltβ/tnfrp55 double-deficient mice lack all LNs, demonstrating cooperation between LTβ and TNFRp55 in LN development. Now we report that ltβ/tnf double-deficient mice develop the same set of mucosal LNs as do ltβ-deficient mice, suggesting that ligands other than TNF signal through TNFRp55 during LN development. These LNs retain distinct T and B cells areas; however, they lack follicular dendritic cell networks. Structures resembling germinal centers can be found in the LNs from immunized ltβ-deficient mice but not in ltβ/tnf double-deficient mice. Additionally, stromal components of the spleen and LNs appear to be more severely disturbed in ltβ/tnf double-deficient mice as compared with ltβ-deficient mice. We conclude that LTβ and TNF cooperate in the establishment of the correct microarchitecture of lymphoid organs.
Commitment to the CD4 Lineage Mediated by Extracellular Signal-Related Kinase Mitogen-Activated Protein Kinase and Lck Signaling
Authors: Sharp Leslie L. , Hedrick Stephen M.
Published: December 2022

The development of T cells results in a concordance between the specificity of the TCR for MHC class I and class II molecules and the expression of CD8 and CD4 coreceptors. Based on analogy to simple metazoan models of organ development and lineage commitment, we sought to determine whether extracellular signal-related kinase (Erk) mitogen-activated protein (MAP) kinase pathway signaling acts as an inductive signal for the CD4 lineage. Here, we show that, by altering the intracellular signaling involving the Erk/MAP kinase pathway, T cells with specificity for MHC class I can be diverted to express CD4, and, conversely, T cells with specificity for MHC class II can be diverted to express CD8. Furthermore, we find that activation of the src-family tyrosine kinase, p56lck is an upstream mediator of lineage commitment. These results suggest a simple mechanism for lineage commitment in T cell development.
Analysis of the Defect in IFN-γ Induction of MHC Class II Genes in G1B Cells: Identification of a Novel and Functionally Critical Leucine-Rich Motif (62-LYLYLQL-68) in the Regulatory Factor X 5 Transcription Factor
Authors: Brickey W. June , Wright Kenneth L. , Zhu Xin-Sheng , P.-Y. Ting Jenny
Published: December 2022

MHC class II deficiency found in bare lymphocyte syndrome patients results from the absence or dysfunction of MHC class II transcriptional regulators, such as regulatory factor X (RFX) and class II transactivator (CIITA). Understanding the roles of these factors has been greatly facilitated by the study of genetic defects in cell lines of bare lymphocyte syndrome patients, as well as in cell lines that have been generated by chemical mutagenesis in vitro. The latter group includes MHC class II-deficient lines that are no longer responsive to induction by IFN-γ. Here, we show that the defect in G1B, one such cell line, is attributed to the lack of functional RFX5, the largest subunit of RFX. The RFX5 gene isolated from G1B cells contains two separate single-base pair mutations. One alteration does not exhibit a phenotype, whereas a leucine-to-histidine mutation eliminates DNA-binding and transactivating functions. This mutation lies outside of previously defined functional domains of RFX5 but within an unusual, leucine-rich region (62-LYLYLQL-68). To further investigate the significance of the leucine-rich region, we targeted all neighboring leucine residues for mutagenesis. These mutants were also unable to transactivate a MHC class II reporter gene, confirming that these leucine residues play an essential role in RFX activity and characterize a novel leucine-rich motif.
Staphylococcal Enterotoxin H Displays Unique MHC Class II-Binding Properties
Authors: Nilsson Helen , Björk Per , Dohlsten Mikael , Antonsson Per
Published: December 2022

Staphylococcal enterotoxin H (SEH) has been described as a superantigen by sequence homology with the SEA subfamily and briefly characterized for its in vivo activity. In this study, we demonstrate that SEH is a potent T cell mitogen and inducer of T cell cytotoxicity that possesses unique MHC class II-binding properties. The apparent affinity of SEH for MHC class II molecules is the highest affinity ever measured for a staphylococcal enterotoxin (Bmax1/2 ∼ 0.5 nM for MHC class II expressed on Raji cells). An excess of SEA or SEAF47A, which has reduced binding to the MHC class II α-chain, is able to compete for binding of SEH to MHC class II, indicating an overlap in the binding sites at the MHC class II β-chain. The binding of SEH to MHC class II is like SEA, SED, and SEE dependent on the presence of zinc ions. However, SEH, in contrast to SEA, binds to the alanine-substituted DR1 molecule, βH81A, believed to have impaired zinc-bridging capacity. Furthermore, alanine substitution of residues D167, D203, and D208 in SEH decreases the affinity for MHC class II as well as its in vitro potency. Together, this indicates an MHC class II binding site on SEH with a different topology as compared with SEA. These unique binding properties will be beneficial for SEH to overcome MHC class II isotype variability and polymorphism as well as to allow an effective presentation on APCs also at low MHC class II surface expression.
Mutational Analysis of Avidity and Fine Specificity of Anti-Levan Antibodies
Authors: Brorson Kurt , Thompson Cynthia , Wei George , Krasnokutsky Michael , Stein Kathryn E.
Published: December 2022

Using the polyfructose, bacterial levan, as a model polysaccharide, we analyzed how V regions affect binding in anti-polysaccharide mAbs. Previously, panels of mAb were constructed from bacterial levan-immunized BALB/c and CBA/Ca mice. The BALB/c mAb were mostly germline VHJ606:Vκ11, and a subset contained presumed somatic mutations in the complementarity-determining regions (CDRs) that correlated with increases in avidity for the β(2→1) inulin linkage of levan. The CBA/Ca mAb were more heterogeneous in V gene usage, but a subset of inulin-nonreactive mAb were VHJ606:Vλ and had VH sequence differences in the CDRs from the VHJ606 regions of the BALB/c mAb. In this report, VHJ606 Abs containing various combinations of specifically mutated H and L chains were produced by engineered transfectants and tested for inulin avidity and levan binding. Two presumed somatic mutations seen in CDRs of the BALB/c hybridomas were shown to directly cause marked increases in avidity for inulin (VH N53H, 9-fold; VL N53I, 20-fold; together, 46-fold) but not for β(2→6) levan. Exchange of either positions 50 or 53 in VH or the H3 loop between the BALB/c and CBA/Ca mAb resulted in either fine specificity shift or total loss of bacterial levan binding. Three-dimensional models of the V regions suggested that residues that affect binding to inulin alone are near the edge of the CDR surface, while residues involved with binding both forms of levan and affecting fine specificity are in the VH:VL junctional area.
Induction of Th2 Responses and IgE Is Largely Due to Carbohydrates Functioning as Adjuvants onSchistosoma mansoniEgg Antigens
Authors: Okano Mitsuhiro , Satoskar Abhay R. , Nishizaki Kazunori , Abe Mie , Harn Donald A.
Published: December 2022

Infection with the helminth parasite Schistosoma mansoni induces a pronounced Th2-type response that is associated with significant IgE production. To better understand how the parasite drives these responses, we investigated the relative roles of proteins and carbohydrates in driving Th2-type and/or IgE responses using a murine model of intranasal sensitization with soluble egg Ags (SEA) of Schistosoma mansoni. We found that repeated intranasal sensitization with soluble egg Ags led to the induction of both total and specific IgE production and nasal eosinophilia. By comparing the responses of mice sensitized with SEA or metaperiodate-treated SEA we were able to demonstrate that carbohydrates on SEA are the major inducers of IgE production and nasal recruitment of eosinophils. Mice sensitized with periodate-treated SEA displayed a significant decrease in both total and specific IgE levels in comparison to mice sensitized with native SEA. Furthermore, sensitization of mice with periodate-treated SEA significantly reduced levels of Ag-specific IgG1, but had no effect on IgG2a production. Nasal lymphocytes from mice sensitized with native SEA, but not with periodate-treated SEA, produced IL-4, IL-5, and IL-10 when restimulated with native SEA in vitro. On the other hand, lymphocytes from mice sensitized with periodate-treated SEA did not produce any of these same cytokines following in vitro restimulation, suggesting that carbohydrates were required for in vivo induction of Th2 response and for that of associated cytokine responses in this model. Lastly, competitive inhibition ELISA showed that although carbohydrates are required for SEA-specific IgE induction, they are not targets of the induced IgE response.