bluuebunny/crossref_metadata_2025 huggingface dataset:
Thymic Expression of the Transcription Factor Nur77 Rescues the T Cell But Not the B Cell Abnormality of gld/gld Mice
Authors: Chan Francis Ka-Ming , Astar Winoto Ann Chen
Published: December 2022

Fas and Fas ligand are critical regulators of lymphocyte homeostasis. Disruption of this pathway in the spontaneous mouse mutant gld leads to autoimmunity characterized by the appearance of a population of CD4−8−B220+ T cells and the production of autoantibodies. Nur77 is a transcription factor that is induced upon TCR signaling. Constitutive thymic expression of Nur77 leads to apoptosis. We have previously shown that introduction of this Nur77 transgene can eliminate the accumulation of abnormal T cells in the periphery of gld/gld mice. In this report, we further characterized the effects of the Nur77 transgene on the gld phenotype. Nur77-mediated apoptosis is evident in the thymuses of mice with either a gld/gld homozygous or gld/+ heterozygous background. Consequently, few mature T cells are generated in these mice. In addition, mature T cells exhibit a diminished response to proliferative signals through CD3. Interestingly, the Nur77 transgene failed to reduce serum levels of Igs and anti-DNA Abs to wild-type levels. These data suggest that the rescue of the T cell lymphoproliferative syndrome in gld/gld mice by the Nur77 transgene is mediated by events in the thymus and that B cell autoimmune disease associated with the gld mutation can develop independently of the T cell abnormality.
Interaction with Secretory Component Stimulates Effector Functions of Human Eosinophils But Not of Neutrophils
Authors: Motegi Youichi , Kita Hirohito
Published: December 2022

Eosinophils and their products are important in the pathophysiology of allergic inflammation in mucosal tissues. Secretory component bound to IgA mediates transepithelial transport of IgA and confers increased stability on the resultant secretory IgA; however, the effect of secretory component on the biologic activity of IgA is unknown. Here, we report that secretory IgA and secretory component preferentially activate human eosinophils. When eosinophils were stimulated with immobilized secretory IgA, degranulation and superoxide production were two- to threefold greater than when stimulated with serum IgA. In contrast, neutrophils responded similarly to secretory IgA and serum IgA. Flow cytometric analysis showed that eosinophils bound to purified secretory component. The binding of 125I-labeled secretory component was inhibited by unlabeled secretory component or secretory IgA but not by serum IgA. Superoxide production by eosinophils stimulated with cytokines or IgG was enhanced synergistically by immobilized secretory component; secretory component showed no effect on neutrophil activation. Finally, anti-CD18 mAb abolished eosinophil superoxide production stimulated with secretory IgA or secretory component but not with serum IgA, suggesting a crucial role for β2 integrins in eosinophil interactions with secretory IgA or secretory component. Thus, secretory component plays important roles in activating eosinophil functions but not neutrophil functions. This preferential interaction between secretory component and eosinophils may provide a novel mechanism to regulate mucosal tissue inflammation.
Oxidized α2-Macroglobulin (α2M) Differentially Regulates Receptor Binding by Cytokines/Growth Factors: Implications for Tissue Injury and Repair Mechanisms in Inflammation
Authors: Wu Sean M. , Patel Dhavalkumar D. , Pizzo Salvatore V.
Published: December 2022

α2M binds specifically to TNF-α, IL-1β, IL-2, IL-6, IL-8, basic fibroblast growth factor (bFGF), β-nerve growth factor (β-NGF), platelet-derived growth factor (PDGF), and TGF-β. Since many of these cytokines are released along with neutrophil-derived oxidants during acute inflammation, we hypothesize that oxidation alters the ability of α2M to bind to these cytokines, resulting in differentially regulated cytokine functions. Using hypochlorite, a neutrophil-derived oxidant, we show that oxidized α2M exhibits increased binding to TNF-α, IL-2, and IL-6 and decreased binding to β-NGF, PDGF-BB, TGF-β1, and TGF-β2. Hypochlorite oxidation of methylamine-treated α2M (α2M*), an analogue of the proteinase/α2M complex, also results in decreased binding to bFGF, β-NGF, PDGF-BB, TGF-β1, and TGF-β2. Concomitantly, we observed decreased ability to inhibit TGF-β binding and regulation of cells by oxidized α2M and α2M*. We then isolated α2M from human rheumatoid arthritis synovial fluid and showed that the protein is extensively oxidized and has significantly decreased ability to bind to TGF-β compared with α2M derived from plasma and osteoarthritis synovial fluid. We, therefore, propose that oxidation serves as a switch mechanism that down-regulates the progression of acute inflammation by sequestering TNF-α, IL-2, and IL-6, while up-regulating the development of tissue repair processes by releasing bFGF, β-NGF, PDGF, and TGF-β from binding to α2M.
Complement-Induced Expression of Chemokine Genes in Endothelium: Regulation by IL-1-Dependent and -Independent Mechanisms
Authors: Selvan Rathinam S. , Kapadia Homi B. , Platt Jeffrey L.
Published: December 2022

Activation of complement in the vicinity of endothelium is thought to contribute to the tissue manifestations of inflammatory and immune responses. Endothelial cells contribute to these processes in part by the elaboration of chemokines that activate various leukocytes and direct their migration into tissues. We investigated the mechanisms by which activation of complement on endothelial cell surfaces might influence the expression of chemokine genes in endothelial cells. In a model for the immune reaction occurring in a xenograft, human serum, as a source of xenoreactive anti-endothelial Abs and complement, induced expression of the monocyte chemotactic protein-1 (MCP-1), IL-8, and RANTES genes. The MCP-1 and IL-8 genes were expressed within 3 h as a first phase and at >12 h as a second phase. The RANTES gene was expressed in porcine endothelial cells only 12 h after exposure to human serum. The expression of these genes required activation of complement and assembly of membrane attack complex, as it was inhibited by soluble CR1 and did not occur in the absence of C8. The early phase of MCP-1 and IL-8 gene expression did not require de novo protein synthesis. The late phase of MCP-1, IL-8, and RANTES gene expression predominantly required the production of IL-1α as an intermediate step. The results indicate that the expression of chemokine genes in endothelial cells occurs as a function of differential responses to complement and may in part be conditioned by the availability of IL-1α.
Detection of HIV-1-Specific CTLs in the Semen of HIV-Infected Individuals
Authors: Quayle Alison J. , Coston Wanda M. P. , K. Trocha Alicja , Kalams Spyros A. , Mayer Kenneth H. , Anderson Deborah J.
Published: December 2022

CTLs play an important role in controlling cell-associated HIV. Since the majority of HIV infections are acquired through sexual transmission, we investigated whether antiviral CTLs were present in the male urogenital tract using semen as a source of T cells. We were able to establish anti-HIV cytolytic lines in five of five HIV-infected men with CD4 counts of >500/μl, although cloning efficiencies were lower than with peripheral blood-derived T cells. CTLs generated from the semen of three men were analyzed in detail and showed a broadly active response, recognizing gag, env, and pol proteins. Detailed analysis of two gag-specific clones from one of the individuals demonstrated HLA class I restriction and recognition of the same p24 epitope (EQASQEVKNWMT). In summary, our results demonstrate the presence of a broad CTL response to HIV in the urogenital tract and provide a rationale for further studies of local enhancement of genital mucosal responses by anti-HIV immunization.
IL-10 Impacts Autoimmune Diabetes Via a CD8+ T Cell Pathway Circumventing the Requirement for CD4+ T and B Lymphocytes
Authors: Balasa Balaji , Davies Joanna D. , Lee Jae , Good Augusta , Yeung Brian T. , Sarvetnick Nora
Published: December 2022

IL-10 is essential for an early phase of diabetes in nonobese diabetic (NOD) mice, but later becomes protective against its development. The mechanism by which IL-10 mediates the pathway to diabetes in these mice is unknown. Herein, we dissected the cellular and costimulation requirements for diabetes in transgenic (tg) NOD mice that expressed IL-10 in their pancreatic islets (IL-10-NOD mice). We found that IL-10 alone did not cause diabetes because the offspring (IL-10-NOD-scid mice) from backcrosses of IL-10-NOD mice with NOD-scid mice had no diabetes. Moreover, these IL-10-NOD-scid mice were free of lymphocytic infiltration. Treatment of IL-10-NOD mice with depleting anti-CD4 mAb or control mAb had no effect on diabetes. Surprisingly, depletion of CD8+ T cells by treatment with the corresponding mAb inhibited diabetes without attenuating insulitis, demonstrating a critical role for CD8+ T cells in the disease process. Interestingly, B cell-deficient IL-10-NOD mice readily developed diabetes with kinetics and incidence similar to those observed in wild-type mice, demonstrating that B lymphocytes as APCs were not required in the disease process. Administration of anti-CD40 ligand (CD40L) mAb did not prevent disease, indicating that CD40/CD40L costimulation is not required for diabetes in IL-10-NOD mice. Immunization of IL-10-NOD mice with CFA or heat-shock protein 65, known to block diabetes in NOD mice, had no effect on their diabetes. We demonstrate that IL-10 contributes early to the pathology of diabetes via a CD8+ T cell pathway, eliminating the requirement for B lymphocytes and CD40-CD40L costimulation. Our findings provide a mechanism for the participation of IL-10 in the early development of diabetes.
Cutting Edge: Senescent BALB/c Mice Exhibit Decreased Expression of λ5 Surrogate Light Chains and Reduced Development Within the Pre-B Cell Compartment
Authors: Sherwood Erin M. , Blomberg Bonnie B. , Xu Wei , Warner Cynthia A. , Riley Richard L.
Published: December 2022

Although senescent BALB/c mice (∼2 years old) have reduced numbers of small pre-B cells, early pre-B cells (CD43+CD25+B220+) are present in comparable numbers within the bone marrow of both young (3–6-month-old) and senescent BALB/c mice. The transition of CD43+ pre-B cells to the CD43− pre-B cell compartments is dependent on proliferation and clonal maturation dictated by the pre-B cell receptor (μ/λ5/VpreB). In vivo, senescent CD43+B220+ pro-B/early pre-B cells demonstrated reduction of λ5 mRNA, by RT-PCR analysis, and of both surface and cytoplasmic λ5 protein. Decreased λ5 protein expression was also seen among pro-B/pre-B cells derived from senescent bone marrow after stimulation in vitro with IL-7. We propose that diminished expression of the λ5 surrogate light chain results in decreased pre-B cell receptor formation and contributes to reduced recruitment of nascent CD43+ pre-B cells into the CD43− large and small pre-B cell compartments.
Cutting Edge: Thymic Positive Selection and Peripheral Activation of Islet Antigen-Specific T Cells: Separation of Two Diabetogenic Steps by an I-Ag7 Class II MHC β-Chain Mutant
Authors: Kanagawa Osami , Vaupel Barbara A. , Xu Guan , Unanue Emil R. , Katz Jonathan D.
Published: December 2022

The diabetes-susceptible class II MHC genes (in human and mouse) share unique nonaspartic acid residues at position 57 of the class II β-chain. Transgenic expression of a mutant I-Ag7, substituting histidine and serine at position 56 and 57 of β-chain with proline and aspartic acid (I-Ag7PD), respectively, inhibits diabetes development in the nonobese diabetic mouse model. Here, we demonstrate that immature thymocytes expressing a diabetogenic islet Ag-specific transgenic TCR are positively selected by I-Ag7PD class II MHC to give rise to mature CD4+ T cells. However, splenic APCs expressing the same I-Ag7PD fail to present pancreatic islet Ag to mature T cells bearing this diabetogenic TCR. These results indicate that nonaspartic acid residues at position 57 of class II MHC β-chain is important for diabetogenic CD4+ T cell activation in the periphery but is not essential for the formation of a diabetogenic T cell repertoire in the thymus.
Cutting Edge: Rapid Induction of Mitogen-Activated Protein Kinases by Immune Stimulatory CpG DNA
Authors: Yi Ae-Kyung , Krieg Arthur M.
Published: December 2022

Unmethylated CpG motifs in bacterial DNA or synthetic oligodeoxynucleotides (CpG DNA) rapidly activate B cells and monocyte-derived cells; however, the intracellular signaling pathways involved in this process are unclear. Here, we demonstrate that CpG DNA induces the activation of c-Jun NH2-terminal kinase and p38 but does not activate the extracellular receptor kinase in murine B and monocyte-like cell lines. CpG DNA also induces the phosphorylation of activating transcription factor-2, c-Jun, and mitogen-activated protein kinase (MAPK)-activated protein kinase 2 as well as the activation of activator protein-1 (AP-1) DNA binding. Inhibition of p38 led to the suppression of CpG DNA-induced AP-1 DNA-binding activity and cytokine production, indicating that the p38 pathway is required for mediating these immune stimulatory effects of CpG DNA. Chloroquine, an endosomal acidification inhibitor, selectively abolished CpG DNA-mediated MAPK activation. Our results indicate that CpG DNA activates the p38 and c-Jun NH2-terminal kinase MAPK and leads to the activation of AP-1 via a pathway which is sensitive to chloroquine.
Dendritic Cell Immunization Breaks Cytotoxic T Lymphocyte Tolerance in Hepatitis B Virus Transgenic Mice
Authors: Shimizu Yukihiro , Guidotti Luca G. , Fowler Patricia , Chisari Francis V.
Published: December 2022

Hepatitis B virus (HBV) transgenic mice that are immunologically tolerant to HBV-encoded Ags represent a model of chronic HBV infection suitable for the development of therapeutic immunization strategies before testing in humans. Five lineages of HBV transgenic mice were immunized with plasmid DNA that encodes hepatitis B surface Ag (HBsAg) or with cytokine-activated bone marrow-derived dendritic cells (DCs) in an attempt to break tolerance to HBsAg at the B and T cell levels. DNA immunization stimulated an Ab response but not a cytotoxic T lymphocyte response to HBsAg in two of the five transgenic lineages studied. In contrast, infusion of activated transgenic or nontransgenic DCs stimulated a splenic CTL response in all three transgenic lineages immunized in this manner at precursor frequencies comparable to those in nontransgenic mice, indicating that DC function is normal, and HBsAg-specific CTLs are present but functionally silent in these transgenic animals. Importantly, none of the animals developed hepatitis or displayed suppressed viral gene expression or replication following either DNA immunization or DC administration even in the presence of anti-hepatitis B surface (HBs) Abs and HBs-specific CTLs. These results indicate that Ag presentation by cytokine-activated DCs can break tolerance and trigger an anti-viral CTL response in HBV transgenic mice, and they suggest that this strategy is more efficient than DNA immunization in this setting. Nonetheless, more efficient immunization strategies are needed to stimulate an immune response of sufficient quality and magnitude to achieve an immunotherapeutic antiviral effect.
Longitudinal Analysis of the Acute Sendai Virus-Specific CD4+ T Cell Response and Memory
Authors: Topham David J. , Doherty Peter C.
Published: December 2022

The development and persistence of Sendai virus-specific CD4+ T cell memory has been analyzed following respiratory infection of C57BL/6J mice by determining the prevalence of IL-2-producing Th cell precursors (Thp). Frequencies as high as 1:40 virus-specific CD4+ T cells were found in the regional lymph nodes and spleen during the acute phase of the host response and persisted at levels ≥1:500 for 2 to 3 mo. Thereafter, these CD4+ T cells tended to distribute more to the spleen than to the lymph nodes, a pattern that persisted for the life of the animals. From 3 to 12 mo after infection, virus-specific Thp were always detectable, although the numbers were diminished relative to those measured during the acute phase. Thereafter, however, in both contemporary and cumulative assays, there was a progressive increase in both the frequency and number of Thp. These increases were especially apparent for mice more than 2 years of age. This may reflect enrichment of the CD4+CD44high memory set due to the gradual diminution of the naive CD4+CD62LhighCD44low component. Analysis of DNA staining profiles for the CD4+ T cells showed high levels of cycling for the acute phase of the response, whereas the rate of T cell turnover measured for the CD4+CD44high population by bromodeoxyuridine incorporation indicated a pattern of stable, continuing proliferation throughout life. Virus-specific CD4+ T cell memory resulting from a single exposure to a readily eliminated RNA virus is thus maintained indefinitely in laboratory mice.
Autoimmunity Develops in Lupus-Prone NZB Mice Despite Normal T Cell Tolerance
Authors: Wither Joan , Vukusic Brian
Published: December 2022

NZB mice spontaneously develop an autoimmune disease characterized by production of anti-RBC, -lymphocyte, and -ssDNA Abs. Evidence suggests that the NZB mouse strain has all of the immunologic defects required to produce lupus nephritis but lacks an MHC locus that allows pathogenic anti-dsDNA Ab production. The capacity to produce diverse autoantibodies in these mice raises the possibility that they possess a generalized defect in self-tolerance. To determine whether this defect is found within the T cell subset, we backcrossed a transgene encoding bovine insulin (BI) onto the NZB background. In nonautoimmune BALB/c mice, the BI transgene induces a profound but incomplete state of T cell tolerance mediated predominantly by clonal anergy. Comparison of tolerance in NZB and BALB/c BI-transgenic mice clearly demonstrated that NZB T cells were at least as tolerant to BI as BALB/c T cells. NZB BI-transgenic mice did not spontaneously produce anti-BI Abs, and following antigenic challenge, BI-specific Ab production was comparably reduced in both BI-transgenic NZB and BALB/c mice. Further, in vitro BI-specific T cell proliferation and cytokine secretion were appropriately decreased for primed lymph node and splenic T cells derived from NZB BI-transgenic relative to their nontransgenic counterparts. These data indicate that a generalized T cell tolerance defect does not underlie the autoimmune disease in NZB mice. Instead, we propose that the T cell-dependent production of pathogenic IgG autoantibodies in these mice arises from abnormal activation of T cells in the setting of normal but incomplete tolerance.
A Mouse Carrying Genetic Defect in the Choice Between T and B Lymphocytes
Authors: Tokoro Yayoi , Sugawara Takehiko , Yaginuma Hiroyuki , Nakauchi Hiromitsu , Terhorst Cox , Wang Baoping , Takahama Yousuke
Published: December 2022

Transgenic mice with human CD3ε gene have been shown to exhibit early arrest of T cell development in the thymus. The present study shows that, instead of T cells, B cells are generated in the thymus of a line, tgε26, of the human CD3ε transgenic mice. The accumulation of mature B cells in the thymus was found only in tgε26 mice, not in other human CD3ε transgenic mouse lines or other T cell-deficient mice, including CD3-ε knockout mice and TCR-β/TCR-δ double knockout mice. Hanging drop-mediated transfer into 2-deoxyguanosine-treated thymus lobes showed that lymphoid progenitor cells rather than thymus stromal cells were responsible for abnormal B cell development in tgε26 thymus, and that tgε26 fetal liver cells were destined to become B cells in normal thymus even in the presence of normal progenitor cells undergoing T cell development. These results indicate that lymphoid progenitor cells in tgε26 mice are genetically defective in thymic choice between T cells and B cells, generating B cells even in normal thymus environment. Interestingly, tgε26 thymocytes expressed GATA-3 and TCF-1, but not LEF-1 and PEBP-2α, among T cell-specific transcription factors that are involved in early T cell development, indicating that GATA-3 and TCF-1 expressed during thymocyte development do not necessarily determine the cell fate into T cell lineage. Thus, tgε26 mice provide a novel mouse model in that lineage choice between T and B lymphocytes is genetically defective.
B Cell Deletion, Anergy, and Receptor Editing in “Knock In” Mice Targeted with a Germline-Encoded or Somatically Mutated Anti-DNA Heavy Chain
Authors: Pewzner-Jung Yael , Friedmann Dinorah , Sonoda Eiichiro , Jung Steffen , Rajewsky Klaus , Eilat Dan
Published: December 2022

To study the relative contributions of clonal deletion, clonal anergy, and receptor editing to tolerance induction in autoreactive B cells and their dependence on B cell receptor affinity, we have constructed “knock in” mice in which germline encoded or somatically mutated, rearranged anti-DNA heavy (H) chains were targeted to the H chain locus of the mouse. The targeted H chains were expressed on the vast majority of bone marrow (BM) and splenic B cells and were capable of Ig class switching and the acquisition of somatic mutations. A quantitative analysis of B cell populations in the BM as well as of Jκ utilization and DNA binding of hybridoma Abs suggested that immature B cell deletion and light (L) chain editing were the major mechanisms affecting tolerance. Unexpectedly, these mechanisms were less effective in targeted mice expressing the somatically mutated, anti-DNA H chain than in mice expressing the germline-encoded H chain, possibly due to the greater abundance of high affinity, anti-DNA immature B cells in the BM. Consequently, autoreactive B cells that showed features of clonal anergy could be recovered in the periphery of these mice. Our results suggest that clonal deletion and receptor editing are interrelated mechanisms that act in concert to eliminate autoreactive B cells from the immune system. Clonal anergy may serve as a back-up mechanism for central tolerance, or it may represent an intermediate step in clonal deletion.
IL-4 in Combination with TGF-β Favors an Alternative Pathway of Th1 Development Independent of IL-12
Authors: Lingnau Karen , Hoehn Petra , Kerdine Saadia , Koelsch Stephan , Neudoerfl Christine , Palm Norbert , Ruede Erwin , Schmitt Edgar
Published: December 2022

IL-4 was found to be the essential differentiation factor for Th2 cells and simultaneously to be a potent inhibitor of Th1 development that is induced by IFN-γ and IL-12. Furthermore, it was demonstrated that TGF-β can also inhibit Th1 development. In this work, we demonstrate that polyclonal activation of Mel-14highCD4+ T cells by immobilized anti-αβTCR mAb together with a mixture of IL-4 and TGF-β can lead to the development of both Th1 and Th2 cells, depending on the concentration of these cytokines. Additional experiments revealed that Th1 induction by a combination of IL-4 and TGF-β depends on the presence of endogenous IFN-γ, and that this alternative Th1 development is further enhanced by IL-12, but is not dependent on this cytokine. Moreover, naive OVA323–339-specific Th cells that were stimulated by APCs and OVA323–339 peptide differentiated toward Th1 cells after priming in the presence of IL-4 in combination with TGF-β. Hence, this finding confirmed the results obtained by polyclonal activation of naive CD4+ Th cells and implicates that this alternative Th1 development may also occur in vivo under the influence of TGF-β and IL-4 independently of the Th1-promoting effect of IL-12.
The Importance of Pairwise Interactions Between Peptide Residues in the Delineation of TCR Specificity
Authors: Leggatt Graham R. , Hosmalin Anne , Pendleton C. David , Kumar Anita , Hoffman Stephen , Berzofsky Jay A.
Published: December 2022

A minimal, nonamer epitope (TEMEKEGKI) from the reverse transcriptase protein of HIV-1, restricted by H-2Kk, was identified and the function of individual residues determined. Besides classical anchor residues at positions 2 and 9, methionine at position 3 was identified as an important MHC anchor and improved binding of a different (malarial) nonamer epitope to H-2Kk, albeit while also abolishing CTL recognition. Lysine at position 5 was replaceable by alanine for CTL raised against wild-type peptide but abolished recognition for CTL raised against the variant 5ALA peptide, indicating a unidirectional cross-reactivity. Interestingly, one CTL line raised against the 5ALA substituted peptide was permissive for a double substitution at positions 5 and 6, in which lysine was permissive at position 5 only if the adjacent glutamic acid was replaced by alanine. Extensive analysis revealed three distinct patterns of responses with peptides doubly substituted in this region: recognition of both single substitutions but not the double substitution, recognition of only one single substitution but also the double substitution, or recognition of both single substitutions and the double substitution. A second complementary substitution can therefore restore function lost through a first substitution. Thus, no residue acts independently of its neighbors, and pairs of substitutions may give results not predictable from the effects of each taken singly. This finding may have bearing on viral infections (such as HIV), in which the accumulation of two mutations in the epitope may lead to the reengagement of memory CTL previously silenced by the initial mutation.
Dexamethasone But Not Indomethacin Inhibits Human Phagocyte Nicotinamide Adenine Dinucleotide Phosphate Oxidase Activity by Down-Regulating Expression of Genes Encoding Oxidase Components
Authors: Condino-Neto Antonio , Whitney Constance , Newburger Peter E.
Published: December 2022

We investigated the effects of dexamethasone or indomethacin on the NADPH oxidase activity, cytochrome b558 content, and expression of genes encoding the components gp91-phox and p47-phox of the NADPH oxidase system in the human monocytic THP-1 cell line, differentiated with IFN-γ and TNF-α, alone or in combination, for up to 7 days. IFN-γ and TNF-α, alone or in combination, caused a significant up-regulation of the NADPH oxidase system as reflected by an enhancement of the PMA-stimulated superoxide release, cytochrome b558 content, and expression of gp91-phox and p47-phox genes on both days 2 and 7 of cell culture. Noteworthy was the tremendous synergism between IFN-γ and TNF-α for all studied parameters. Dexamethasone down-regulated the NADPH oxidase system of cytokine-differentiated THP-1 cells as assessed by an inhibition on the PMA-stimulated superoxide release, cytochrome b558 content, and expression of the gp91-phox and p47-phox genes. The nuclear run-on assays indicated that dexamethasone down-regulated the NADPH oxidase system at least in part by inhibiting the transcription of gp91-phox and p47-phox genes. Indomethacin inhibited only the PMA-stimulated superoxide release of THP-1 cells differentiated with IFN-γ and TNF-α during 7 days. None of the other parameters was affected by indomethacin. We conclude that dexamethasone down-regulates the NADPH oxidase system at least in part by inhibiting the expression of genes encoding the gp91-phox and p47-phox components of the NADPH oxidase system.
Nicotinamide-Adenine Dinucleotide Phosphate Oxidase Assembly and Activation in EBV-Transformed B Lymphoblastoid Cell Lines of Normal and Chronic Granulomatous Disease Patients
Authors: Dusi Stefano , Nadalini Katyuscia A. , Donini Marta , Zentilin Lorena , Wientjes Frans B. , Roos Dirk , Giacca Mauro , Rossi Filippo
Published: December 2022

This paper deals with the mechanisms of activation of NADPH oxidase investigated using EBV-transformed human B lymphoblastoid cell lines (B cells) from normal subjects and from patients affected by X-linked chronic granulomatous disease (CGD). The results reported are as follows. 1) In normal B cells, the NADPH oxidase components p67phox, p40phox, p22phox, and gp91phox were less expressed than in polymorphonuclear neutrophils. 2) In normal B cells stimulated with PMA, p47phox, p67phox, and p40phox translocated to the membranes as occurs in polymorphonuclear neutrophils. 3) In CGD, B cells expressing p22phox in the absence of gp91phox, p47phox, p67phox, and p40phox did not translocate to the membranes after stimulation with PMA. 4) In PMA-stimulated B cells from an X91+ CGD patient in which p22phox was normally expressed and gp91phox was present but lacked five amino acids, translocation of p47phox to the membranes was unaffected, but p67phox and p40phox were poorly translocated, and the production of O2− was greatly reduced with respect to that by normal B cells. Taken together, these findings indicate that 1) a low expression of some NADPH oxidase components may represent the molecular basis of the low production of O2− in B lymphocytes; 2) the cytosolic components of NADPH oxidase cannot bind to p22phox on the membranes in the absence of gp91phox; 3) p47phox can translocate to the membranes independently of p67phox and p40phox; and 4) gp91phox may have a role in mediating and/or stabilizing the binding of p67phox and p40phox to the membranes of activated cells.
Identification of Basophilic Cells that Express Mast Cell Granule Proteases in the Peripheral Blood of Asthma, Allergy, and Drug-Reactive Patients
Authors: Li Lixin , Li Yong , Reddel Stephen W. , Cherrian Maya , Friend Daniel S. , Stevens Richard L. , Krilis Steven A.
Published: December 2022

Metachromatic cells in the peripheral blood of patients with asthma, allergy, or an allergic drug reaction were evaluated for their nuclear morphology, surface expression of the mast cell (MC) marker c-kit, surface expression of the basophil marker Bsp-1, and granule expression of MC proteases. Consistent with previous findings by others, Bsp-1+/metachromatic cells represented <1% of the cells in the peripheral blood of normal individuals. These cells generally contained segmented nuclei. Very little, if any, tryptase (Try), chymase (Chy), or carboxypeptidase A (CPA) was found in their granules, and very little, if any, c-kit was observed on their surfaces. The number of metachromatic cells increased in the peripheral blood of the three groups of patients. Like the basophils in normal individuals, most of these metachromatic cells contained segmented nuclei and expressed Bsp-1. However, in contrast to the basophils in normal individuals, many of the metachromatic cells in the three patient groups expressed c-kit, Try, Chy, and/or CPA. That the metachromatic cells in the blood of our patients have some features of MCs and some features of basophils suggests that human basophils and MCs are derived from a common progenitor. As assessed by the chloroacetate esterase cytochemical assay, the immunoreactive Chy in the peripheral blood of these patients is enzymatically active. Because MC proteases regulate numerous immunologic and other biologic systems, the expression of Try, Chy, and/or CPA in a peripheral blood-localized cell in an individual having asthma, allergy, or an allergic drug reaction has important clinical implications.
Diverse TCRs Recognize Murine CD1
Authors: Behar Samuel M. , Podrebarac T. A. , Roy C. J. , Wang C. R. , Brenner M. B.
Published: December 2022

Human and murine T cells that specifically recognize CD1d and produce IL-4 and IFN-γ play a role in immunoregulation and tumor rejection. In the mouse, most CD1d1-reactive T cells described express an invariant Vα14-Jα281 TCR associated with TCR β-chains of limited diversity. Similarly, human CD1d-reactive T cells express a highly restricted TCR repertoire. Here we report the unexpected result that in mice immunized with CD1d1-bearing transfectant cells, a diverse repertoire of TCRs was expressed by CD1d1-reactive T cell clones isolated by limiting dilution without preselection for NK1 expression. Only 3 of 10 CD1d1-reactive T cell clones expressed the invariant Vα14-Jα281 TCRα rearrangement. T cells expressing Vα10, -11, -15, and -17, and having non-germline-encoded nucleotides resulting in diverse V-J junctions were identified. Like CD1d1-reactive T cells expressing the invariant Vα14-Jα281 TCR α-chain, CD1d1-reactive clones with diverse TCRs produced both Type 1 (IFN-γ) and Type 2 (IL-4, IL-10) cytokines. This establishes the existence of significant diversity in the TCRs directly reactive to the CD1d1 protein. Our findings reveal that CD1d interacts with a broad array of TCRs, suggesting substantial redundancy and flexibility of the immune system in providing T cells serving the role(s) mediated by CD1d reactivity.
Lyn Dissociation from Phosphorylated FcεRI Subunits: A New Regulatory Step in the FcεRI Signaling Cascade Revealed by Studies of FcεRI Dimer Signaling Activity
Authors: Ortega Enrique , Lara Martha , Lee Irene , Santana Carla , Martinez A. Marina , Pfeiffer Janet R. , Lee Rebecca J. , Wilson Bridget S. , Oliver Janet M.
Published: December 2022

Cross-linking the heterotrimeric (αβγ2) IgE receptor, FcεRI, of mast cells activates two tyrosine kinases: Lyn, which phosphorylates β and γ subunit immunoreceptor tyrosine-based activation motifs, and Syk, which binds γ-phospho-immunoreceptor tyrosine-based activation motifs and initiates cellular responses. We studied three FcεRI-dimerizing mAbs that maintain similar dispersed distributions over the surface of RBL-2H3 mast cells but elicit very different signaling responses. Specifically, mAb H10 receptor dimers induce very little inositol 1,4,5-trisphosphate synthesis, Ca2+ mobilization, secretion, spreading, ruffling, and actin plaque assembly, whereas dimers generated with the other anti-FcεRI mAbs induce responses that are only modestly lower than that to multivalent Ag. H10 receptor dimers activate Lyn and support FcεRI β and γ subunit phosphorylation but are poor Syk activators compared with Ag and the other anti-FcεRI mAbs. H10 receptor dimers have two other distinguishing features. First, they induce stable complexes between activated Lyn and receptor subunits. Second, the predominant Lyn-binding phospho-β isoform found in mAb H10-treated cells is a less tyrosine phosphorylated, more electrophoretically mobile species than the predominant isoform in Ag-treated cells that does not coprecipitate with Lyn. These studies implicate Lyn dissociation from highly phosphorylated receptor subunits as a new regulatory step in the FcεRI signaling cascade required for Syk activation and signal progression.
Alloreactivity as a Source of High Avidity Peptide-Specific Human CTL
Authors: Münz Christian , Obst Reinhard , Osen Wolfram , Stevanović Stefan , Rammensee Hans-Georg
Published: December 2022

PBL from HLA-A2− or HLA-A3− donors were stimulated with synthetic peptide libraries fitting HLA-A2 or HLA-A3 motifs and presented on HLA-A2- or HLA-A3-expressing TAP− cells. Peptide library-specific allorestricted CTL were found to constitute up to half the alloreactive CTL response and occurred at twofold lower frequency than autologous peptide library-specific CTL. This indicates that positive selection by one particular MHC class I molecule is not absolutely essential for the generation of CTL restricted to the same molecule. However, positive selection increases their frequency. The CTL obtained differed greatly both with respect to peptide dependency and peptide specificity. Determination of the peptide avidity for one representative CTL clone, 10F4, proved that the method described here allows the stimulation of high avidity cytotoxic T cells. This approach involving in vitro stimulation of T cells restricted toward a MHC molecule that was not present during their negative selection might therefore offer the possibility of isolating CTL against self and foreign peptides with varying avidities. Such T cells might indeed be useful for tumor immunotherapy.
Fas/Fas Ligand-Driven T Cell Apoptosis as a Consequence of Ineffective Thyroid Immunoprivilege in Hashimoto’s Thyroiditis
Authors: Stassi Giorgio , Todaro Matilde , Bucchieri Fabio , Stoppacciaro Antonella , Farina Felicia , Zummo Giovanni , Testi Roberto , Maria Ruggero De
Published: December 2022

Hashimoto’s thyroiditis (HT) is a chronic autoimmune disease resulting from Fas-mediated thyrocyte destruction. Although autocrine/paracrine Fas-Fas ligand (FasL) interaction is responsible for thyrocyte cell death during the active phases of HT, the role of infiltrating T lymphocytes (ITL) in this process is still unknown. Therefore, we investigated the expression and function of Fas and FasL in ITL. All ITL expressed high levels of Fas and CD69, an early marker of T cell activation associated with functional Fas expression in T cells in vivo. In contrast to thyrocytes that were found to produce high levels of FasL, ITL did not express significant amounts of FasL, suggesting that ITL are not directly involved in thyrocyte destruction. The analysis of ITL purified from HT thyroids showed that ITL were massively killed by Fas crosslinking and that a considerable number (24–36%) underwent spontaneous apoptosis within 36 h of culture. Accordingly, in situ TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining revealed that a significant number (10–15%) of ITL in proximity to FasL-producing thyroid follicles were apoptotic. Moreover, virtually all ITL in proximity to thyroid follicles were preapoptotic, as they expressed high levels of GD3 ganglioside, a killer glycolipid responsible for the generation of irreversible apoptotic signals that accumulate in hematopoietic cells shortly after Fas crosslinking. These data demonstrate that ITL are not directly involved in thyrocyte cell death during HT, suggesting that autocrine/paracrine Fas-FasL interaction is a major mechanism in autoimmune thyrocyte destruction.
Extensive Alanine Substitutions Increase Binding Affinity of an Influenza Nucleoprotein Peptide to HLA-Aw68 and Do Not Abrogate Peptide-Specific CTL Recognition
Authors: Collins Edward J. , Booth Bruce L. , Cerundolo Vincenzo
Published: December 2022

Class I MHC molecules bind peptides in the endoplasmic reticulum and present them at the cell surface to circulating CD8+ T cells for analysis. We have examined binding of peptides and stabilization of HLA-Aw68 class I molecules using synthetic peptide variants of an influenza virus nucleoprotein peptide, NP91-99 (KTGGPIYKR). We have demonstrated that insertion of increasing numbers of alanines in the center of the peptide (between P and I), to increase a natural bulging out of the peptide-binding cleft, results in a large decrease in thermal stability. Although there is a great decrease in the t1/2 of the MHC/peptide complex for NP-1A compared with NP91-99, a T cell line, stimulated by NP91-99, recognizes NP-1A efficiently. Peptide variants with three or more alanines do not show saturable binding to HLA-Aw68 and also are not recognized by the T cell line. Thermal studies show that polyalanine peptides with minimal anchors and nearly all TCR contact residues exchanged stabilized HLA-Aw68 to high temperatures. Additionally, some of these polyalanine peptides are recognized by T cell lines generated against NP91-99. Analysis of the peptide sequences show that the stabilization effects are not due to the hydrophobicity of the peptide. These data suggest that the strength of binding of peptides to HLA-Aw68 is not only dictated by the presence of anchor residues but also by the lack of unfavorable contacts between the peptide ligand and class I MHC-binding cleft.
IL-10 Is a Major Mediator of Sepsis-Induced Impairment in Lung Antibacterial Host Defense
Authors: Steinhauser Matthew L. , Hogaboam Cory M. , Kunkel Steven L. , Lukacs Nicholas W. , Strieter Robert M. , Standiford Theodore J.
Published: December 2022

To explore the mechanism of immunosuppression associated with sepsis, we developed a murine model of sepsis-induced Pseudomonas aeruginosa pneumonia. CD-1 mice underwent either cecal ligation and 26-gauge needle puncture (CLP) or sham surgery, followed by the intratracheal (i.t.) administration of P. aeruginosa or saline. Survival in mice undergoing CLP followed 24 h later by the i.t. administration of saline or P. aeruginosa was 58% and 10%, respectively, whereas 95% of animals undergoing sham surgery followed by P. aeruginosa administration survived. Increased mortality in the CLP/P. aeruginosa group was attributable to markedly impaired lung bacterial clearance and the early development of P. aeruginosa bacteremia. The i.t. administration of bacteria to CLP-, but not sham-, operated mice resulted in an impressive intrapulmonary accumulation of neutrophils. Furthermore, P. aeruginosa challenge in septic mice resulted in a relative shift toward enhanced lung IL-10 production concomitant with a trend toward decreased IL-12. The i.p., but not i.t., administration of IL-10 Abs given just before P. aeruginosa challenge in septic mice significantly improved both survival and clearance of bacteria from the lungs of septic animals administered P. aeruginosa. Finally, alveolar macrophages isolated from animals undergoing CLP displayed a marked impairment in the ability to ingest and kill P. aeruginosa ex vivo, and this defect was partially reversed by the in vivo neutralization of IL-10. Collectively, these observations indicate that the septic response substantially impairs lung innate immunity to P. aeruginosa, and this effect is mediated primarily by endogenously produced IL-10.
Up-Regulation of CCR1 and CCR3 and Induction of Chemotaxis to CC Chemokines by IFN-γ in Human Neutrophils
Authors: Bonecchi Raffaella , Polentarutti Nadia , Luini Walter , Borsatti Alessandro , Bernasconi Sergio , Locati Massimo , Power Christine , Proudfoot Amanda , Wells Timothy N. C. , Mackay Charles , Mantovani Alberto , Sozzani Silvano
Published: December 2022

Human neutrophils (polymorphonuclear leukocytes; PMN) respond to some CXC chemokines but do not migrate to CC chemokines. Recent work has shown that chemokine receptors can be modulated by inflammatory cytokines. In this study, the effect of IFN-γ, a prototypic Th1 cytokine, on chemokine receptor expression in PMN was investigated. IFN-γ caused a rapid (∼1 h) and concentration-dependent increase of CCR1 and CCR3 mRNA. The expression of CCR2, CCR5, and CXCR1–4 was not augmented. IFN-γ-treated PMN, but not control cells, expressed specific binding sites for labeled monocyte-chemotactic protein (MCP)-3 and migrated to macrophage-inflammatory protein (MIP)-1α, RANTES, MCP-3, MIP-5/HCC2, and eotaxin. 7B11, a mAb for CCR3, inhibited the chemotactic response of IFN-γ-treated PMN to eotaxin, and aminoxypentane-RANTES blocked PMN migration to RANTES. These results suggest that the selectivity of certain chemokines for their target cells may be altered by cytokines produced within an inflammatory context. Since PMN may play a role in orienting immunity toward Th1 responses, it is possible to speculate that IFN-γ not only promotes Th1 differentiation directly, but also reorients the functional significance of Th2 effector cytokines by broadening the spectrum of their action to include PMN.
Comparison of Primary Sensitization of Naive Human T Cells to Varicella-Zoster Virus Peptides by Dendritic Cells In Vitro with Responses Elicited In Vivo by Varicella Vaccination
Authors: Jenkins Darlene E. , Yasukawa Linda L. , Bergen Randy , Benike Claudia , Engleman Edgar G. , Arvin Ann M.
Published: December 2022

Dendritic cells (DC) are potent APC during primary and secondary immune responses. The first objective of this study was to determine whether human DC mediate in vitro sensitization of naive CD4+ T cells to epitopes of the immediate early 62 (IE62) protein of varicella zoster virus (VZV). The induction of CD4+ T cell proliferative responses to eight synthetic peptides representing amino acid sequences of the VZV IE62 protein was assessed using T cells and DC from VZV-susceptible donors. The second objective was to compare in vitro responses of naive T cells with responses to VZV peptides induced in vivo after immunization with varicella vaccine. T cell proliferation was induced by three peptides, P1, P4, and P7, in 71–100% of the donors tested before and after vaccination using DC as APC. Monocytes were effective APC for VZV peptides only after immunization. Two peptides, P2 and P8, induced naive T cell proliferation less effectively and were also less immunogenic for T cells from vaccinated or naturally immune donors. T cell recognition of specific peptides was concordant between naive, DC-mediated responses, and postvaccine responses using monocytes as APC in 69% of comparisons (p = 0.05; χ2); the predictive value of a positive response to an IE62 peptide before immunization for T cell sensitization in vivo was 82%. These observations indicate that primary T cell responses detected in vitro using DC as APC may be useful to characterize the potential immunogenicity of viral protein epitopes in vivo.
T Cell Recognition of Hypervariable Region-1 from Hepatitis C Virus Envelope Protein with Multiple Class II MHC Molecules in Mice and Humans: Preferential Help for Induction of Antibodies to the Hypervariable Region
Authors: Shirai Mutsunori , Arichi Tatsumi , Chen Ming , Nishioka Mikio , Ikeda Kazumasa , Takahashi Hidemi , Enomoto Nobuyuki , Saito Takafumi , Major Marian E. , Nakazawa Teruko , Akatsuka Toshitaka , Feinstone Stephen M. , Berzofsky Jay A.
Published: December 2022

Hypervariable region-1 (HVR1) from the hepatitis C virus (HCV) envelope protein is thought to be a target for neutralizing Abs. To explore HVR1 recognition by helper T cells, and their role in Ab responses, we attempted to generate helper T cells specific for HVR1 in mice of three MHC types, and with PBMC from HCV-infected HLA-diverse humans. In both species, HVR1 was presented by >1 class II MHC molecule to CD4+ helper T cells and showed surprising interisolate cross-reactivity. The epitope for two DR4+ patients was mapped to a more conserved C-terminal sequence containing a DR4 binding motif, possibly accounting for cross-reactivity. Strikingly, Abs to patients’ own HVR1 sequences were found only in patients with T cell responses to HVR1, even though all had Abs to envelope protein, suggesting that induction of Abs to HVR1 depends on helper T cells specific for a sequence proximal to the Ab epitope. Thus, helper T cells specific for HVR1 may be functionally important in inducing neutralizing Abs to HCV. These results may be the first example of “T-B reciprocity,” in which proximity of a helper T cell epitope determines Ab epitope specificity, in a human disease setting.
Gene Therapy of Experimental Autoimmune Thyroiditis by In Vivo Administration of Plasmid DNA Coding for Fas Ligand
Authors: Batteux Frédéric , Tourneur Léa , Trebeden Hélène , Charreire Jeannine , Chiocchia Gilles
Published: December 2022

Fas-Fas ligand (FasL) interaction is required for the maintenance of immune homeostasis and seems to be responsible for the privileged immune status of some tissues. Experimental autoimmune thyroiditis (EAT), which is characterized by autoreactive T and B cell responses and a marked lymphocytic infiltration of the thyroid, is a model of choice to study the therapeutic effects of FasL. Here, we provide evidence that direct injection of DNA expression vectors encoding FasL into the inflamed thyroid inhibited development of lymphocytic infiltration of the thyroid and induced death of infiltrating T cells. These results were paralleled by a total abrogation of anti-Tg cytotoxic T cell response in FasL-treated animals vs controls. In summary, our results show that FasL expression on thyrocytes may have a curative effect on ongoing EAT by inducing death of pathogenic autoreactive infiltrating T lymphocytes.