bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of TGF-β1 on the IgE-Dependent Anaphylaxis Reaction
Authors: Kim Hyung-Min , Lee Young-Mi
Published: January 2023

TGF-β1 is a member of a family of polypeptide factors that control proliferation, differentiation, chemotaxis, and other functions in many cell types. TGF-β1 has been shown to inhibit many immunologic functions. However, here we report that TGF-β1 has an important role in the elicitation of IgE-dependent allergic reactions. The synthetic antisense TGF-β1 oligonucleotides dose-dependently inhibit passive cutaneous anaphylaxis (PCA) reaction and histamine release from the mast cells activated by anti-DNP IgE in rats. The level of cAMP in mast cells, when antisense TGF-β1 oligonucleotides was added, significantly increased ∼7-fold compared with that of basal cells. The antisense TGF-β1 oligonucleotides also had a significant inhibitory effect on anti-DNP IgE-induced TNF-α release from mast cells. In situ hybridization analysis showed that the PCA reaction sites treated with antisense TGF-β1 oligonucleotides exhibited no detectable levels of TGF-β1 and l-histidine decarboxylase mRNA after anti-DNP IgE stimulation, whereas the PCA reaction sites treated with sense TGF-β1 oligonucleotides possessed significant amounts of their mRNA. Additionally, neutralizing Ab to TGF-β1 blocked the PCA reaction significantly, but its Ab did not inhibit peritoneal mast cell-released histamine upon treatment with anti-DNP IgE. Our results suggest that TGF-β1 is critical to the development of IgE-dependent anaphylaxis reactions.
Cutting Edge: Proteasome Involvement in the Degradation of Unassembled Ig Light Chains
Authors: O’Hare Thomas , Wiens Gregory D. , Whitcomb Elizabeth A. , Enns Caroline A. , Rittenberg Marvin B.
Published: January 2023

Several studies on disposal of nonsecreted Ig L chains have identified the endoplasmic reticulum as the site of degradation. Here, we examine degradation of a nonsecreted Ig L chain, T15L, and an experimentally endoplasmic reticulum-retained secretion-competent L chain, D16L, in the absence of H chains. We demonstrate that 1) degradation is specifically impaired by the proteasome-specific inhibitors carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone (Z-L3VS) and lactacystin, 2) L chain degradation occurs early in the biosynthetic pathway, and 3) degradation does not require vesicular transport. Our findings indicate that previous assertions of L chain disposal within the endoplasmic reticulum must be modified. To our knowledge, we provide the first direct evidence supporting a new paradigm for removal of nonsecreted Ig L chains via dislocation to cytosolic proteasomes.
IFN-γ- and Cell-to-Cell Contact-Dependent Cytotoxicity of Allograft-Induced Macrophages Against Syngeneic Tumor Cells and Cell Lines: An Application of Allografting to Cancer Treatment
Authors: Yoshida Ryotaro , Yoneda Yukio , Kuriyama Manabu , Kubota Takahiro
Published: January 2023

In allogeneic tumor or skin transplantation, the rejection process that destroys the allogeneic cells leaves syngeneic cells intact by discrimination between self and nonself. Here, we examined whether the cells infiltrating into the allografts could be cytotoxic against syngeneic immortal cells in vitro and in vivo. The leukocytes (i.e., macrophages (Mφ; 55–65% of bulk infiltrates), granulocytes (20–25%), and lymphocytes (15–20%)) infiltrating into allografts, but not into autografts, in C57BL/6 mice were cytotoxic against syngeneic tumor cells and cell lines, whereas the cytotoxic activity was hardly induced in allografted, IFN-γ−/− C57BL/6 mice. Among the leukocytes, Mφ were the major population of cytotoxic cells; and the cytotoxic activity appeared to be cell-to-cell contact dependent. When syngeneic tumor cells were s.c. injected into normal C57BL/6 mice simultaneously with the Mφ-rich population or allogeneic, but not syngeneic, fibroblastic cells, tumor growth was suppressed in a cell number-dependent manner, and tumor cells were rejected either with a Mφ:tumor ratio of about 30 or with an allograft:tumor ratio of ∼200. In the case of IFN-γ−/− C57BL/6 mice, however, the s.c. injection of the allograft simultaneously with tumor cells had no effect on the tumor growth. These results suggest that allograft or allograft-induced Mφ may be applicable for use in cancer treatment and that IFN-γ induction by the allograft may be crucial for the treatment.
Transfection of Immature Murine Bone Marrow-Derived Dendritic Cells with the Granulocyte-Macrophage Colony-Stimulating Factor Gene Potently Enhances Their In Vivo Antigen-Presenting Capacity
Authors: Curiel-Lewandrowski Clara , Mahnke Karsten , Labeur Marta , Roters Berthold , Schmidt Walter , Granstein Richard D. , Luger Thomas A. , Schwarz Thomas , Grabbe Stephan
Published: January 2023

Ag presentation by dendritic cells (DC) is crucial for induction of primary T cell-mediated immune responses in vivo. Because DC culture from blood or bone marrow-derived progenitors is now clinically applicable, this study investigated the effectiveness of in vitro-generated murine bone marrow-derived DC (Bm-DC) for in vivo immunization protocols. Previous studies demonstrated that GM-CSF is an essential growth and differentiation factor for DC in culture and that in vivo administration of GM-CSF augments primary immune responses, which renders GM-CSF an attractive candidate to further enhance the effectiveness of DC-based immunotherapy protocols. Therefore, immature Bm-DC were transiently transfected with the GM-CSF gene and tested for differentiation, migration, and Ag-presenting capacity in vitro and in vivo. In vitro, GM-CSF gene-transfected Bm-DC were largely unaltered with regard to MHC and costimulatory molecule expression as well as alloantigen or peptide Ag-presenting capacity. When used for in vivo immunizations, however, the Ag-presenting capacity of GM-CSF gene-transfected Bm-DC was greatly enhanced compared with mock-transfected or untransfected cells, as determined by their effectiveness to induce primary immune reactions against hapten, protein Ag, and tumor Ag, respectively. Increased effectiveness in vivo correlated with the better migratory capacity of GM-CSF gene-transfected Bm-DC. These results show that GM-CSF gene transfection significantly enhances the capacity of DC to induce primary immune responses in vivo, which might also improve DC-based vaccines currently under clinical investigation.
A Model for CD8+ CTL Tumor Immunosurveillance and Regulation of Tumor Escape by CD4 T Cells Through an Effect on Quality of CTL
Authors: Matsui So , Ahlers Jeffrey D. , Vortmeyer Alex O. , Terabe Masaki , Tsukui Taku , Carbone David P. , Liotta Lance A. , Berzofsky Jay A.
Published: January 2023

Understanding immune mechanisms influencing cancer regression, recurrence, and metastasis may be critical to developing effective immunotherapy. Using a tumor expressing HIV gp160 as a model viral tumor Ag, we found a growth-regression-recurrence pattern, and used this to investigate mechanisms of immunosurveillance. Regression was dependent on CD8 T cells, and recurrent tumors were resistant to CTL, had substantially reduced expression of epitope mRNA, but retained the gp160 gene, MHC, and processing apparatus. Increasing CTL numbers by advance priming with vaccinia virus expressing gp160 prevented only the initial tumor growth but not the later appearance of escape variants. Unexpectedly, CD4 cell depletion protected mice from tumor recurrence, whereas IL-4 knockout mice, deficient in Th2 cells, did not show this protection, and IFN-γ knockout mice were more susceptible. Purified CD8 T cells from CD4-depleted mice following tumor regression had more IFN-γ mRNA and lysed tumor cells without stimulation ex vivo, in contrast to CD4-intact mice. Thus, the quality as well as quantity of CD8+ CTL determines the completeness of immunosurveillance and is controlled by CD4 T cells but not solely Th2 cytokines. This model of immunosurveillance may indicate ways to enhance the efficacy of surveillance and improve immunotherapy.
Genes Encoding Three New Members of the Leukocyte Antigen 6 Superfamily and a Novel Member of Ig Superfamily, Together with Genes Encoding the Regulatory Nuclear Chloride Ion Channel Protein (hRNCC) and aNω-Nω-Dimethylarginine Dimethylaminohydrolase Homologue, Are Found in a 30-kb Segment of the MHC Class III Region
Authors: Ribas Gloria , Neville Matt , Wixon Joanne L. , Cheng Jianhua , Campbell R. Duncan
Published: January 2023

Many of the genes in the class III region of the human MHC encode proteins involved in the immune and inflammatory responses. We have sequenced a 30-kb segment of the MHC class III region lying between the heat shock protein 70 and TNF genes as part of a program aimed at identifying genes that could be involved in autoimmune disease susceptibility. The sequence analysis has revealed the localization of seven genes, whose precise position and order is cen-G7-G6-G6A-G6B-G6C-G6D-G6E-tel, five of which are fully encoded in the sequence, allowing their genomic structures to be defined. Three of them (G6C, G6D, and G6E) encode putative proteins that belong to the Ly-6 superfamily, known to be GPI-anchored proteins attached to the cell surface. Members of the family are specifically expressed and are important in leukocyte maturation. A fourth gene, G6B, encodes a novel member of the Ig superfamily containing a single Ig V-like domain and a cytoplasmic tail with several signal transduction features. The G6 gene encodes a regulatory nuclear chloride ion channel protein, while the G6A gene encodes a putative homologue of the enzyme Nω,Nω-dimethylarginine dimethylaminohydrolase, which is thought to be involved in regulating nitric oxide synthesis. In addition, three microsatellite markers, 9N-1, 82-2, and D6S273 are contained within the sequence, the last two of which have been reported to be strongly associated with the autoimmune disease ankylosing spondylitis.
Differential Cytokine Modulation and T Cell Activation by Two Distinct Classes of Thalidomide Analogues That Are Potent Inhibitors of TNF-α
Authors: Corral Laura G. , Haslett Patrick A. J. , Muller George W. , Chen Roger , Wong Lu-Min , Ocampo Christopher J. , Patterson Rebecca T. , Stirling David I. , Kaplan Gilla
Published: January 2023

TNF-α mediates both protective and detrimental manifestations of the host immune response. Our previous work has shown thalidomide to be a relatively selective inhibitor of TNF-α production in vivo and in vitro. Additionally, we have recently reported that thalidomide exerts a costimulatory effect on T cell responses. To develop thalidomide analogues with increased anti-TNF-α activity and reduced or absent toxicities, novel TNF-α inhibitors were designed and synthesized. When a selected group of these compounds was examined for their immunomodulatory activities, different patterns of cytokine modulation were revealed. The tested compounds segregated into two distinct classes: one class of compounds, shown to be potent phosphodiesterase 4 inhibitors, inhibited TNF-α production, increased IL-10 production by LPS-induced PBMC, and had little effect on T cell activation; the other class of compounds, similar to thalidomide, were not phosphodiesterase 4 inhibitors and markedly stimulated T cell proliferation and IL-2 and IFN-γ production. These compounds inhibited TNF-α, IL-1β, and IL-6 and greatly increased IL-10 production by LPS-induced PBMC. Similar to thalidomide, the effect of these agents on IL-12 production was dichotomous; IL-12 was inhibited when PBMC were stimulated with LPS but increased when cells were stimulated by cross-linking the TCR. The latter effect was associated with increased T cell CD40 ligand expression. The distinct immunomodulatory activities of these classes of thalidomide analogues may potentially allow them to be used in the clinic for the treatment of different immunopathological disorders.
Myelin Oligodendrocyte Glycoprotein Induces Experimental Autoimmune Encephalomyelitis in the “Resistant” Brown Norway Rat: Disease Susceptibility Is Determined by MHC and MHC-Linked Effects on the B Cell Response
Authors: Stefferl Andreas , Brehm Uschi , Storch Maria , Lambracht-Washington Doris , Bourquin Carole , Wonigeit Kurt , Lassmann Hans , Linington Christopher
Published: January 2023

Experimental autoimmune encephalomyelitis (EAE) induced by active immunization with the myelin oligodendrocyte glycoprotein (MOG) is an Ab-mediated, T cell-dependent autoimmune disease that replicates the inflammatory demyelinating pathology of multiple sclerosis. We report that disease susceptibility and severity are determined by MHC and MHC-linked effects on the MOG-specific B cell response that mediate severe clinical EAE in the EAE-resistant Brown Norway (BN) rat. Immunization with the extracellular domain of MOG in CFA induced fulminant clinical disease associated with widespread demyelination and with an inflammatory infiltrate containing large numbers of polymorphonuclear cells and eosinophils within 10 days of immunization. To analyze the effects of the MHC (RT1 system) we compared BN (RT1 n) rats with Lewis (LEW) (RT1 l) and two reciprocal MHC congenic strains, LEW.1N (RT1n) and BN.1L (RT1 l). This comparison revealed that disease severity and clinical course were strongly influenced by the MHC haplotype that modulated the pathogenic MOG-specific autoantibody response. The intra-MHC recombinant congenic strain LEW.1R38 demonstrated that gene loci located both within the centromeric segment of the MHC containing classical class I and class II genes and within the telomeric RT1.M region containing the MOG gene are involved in determining Ab production and disease susceptibility. This study indicates that the current T cell-centered interpretation of MHC-mediated effects on disease susceptibility must be reassessed in multiple sclerosis and other autoimmune diseases in which autoantibody is involved in disease pathogenesis.
The Murine Antiapoptotic Protein A1 Is Induced in Inflammatory Macrophages and Constitutively Expressed in Neutrophils
Authors: Orlofsky Amos , Somogyi Robert D. , Weiss Louis M. , Prystowsky Michael B.
Published: January 2023

Myeloid leukocytes are thought to regulate their susceptibility to apoptosis upon migration to a site of inflammation. However, factors that determine survival have not been well characterized in these cells. We have examined the expression of murine A1, an antiapoptotic Bcl-2 relative found in activated myeloid cells, during the course of an acute inflammatory response. Intraperitoneal infection of mice with the virulent RH strain of Toxoplasma gondii led to a 5- to 10-fold increase in A1 mRNA levels in peritoneal cells after several days. Bcl-2 expression was unchanged. The increase in A1 expression depended on the dose of the organism and coincided with a sharp increase in peritoneal cellularity. A1 protein levels were also increased as determined by Western blot analysis and immunohistochemical studies. All neutrophils and approximately half of the macrophages in the inflammatory exudate contained high levels of A1 in cytoplasm. A1 expression did not correlate with intracellular parasitization. Peripheral blood neutrophils from normal mice strongly expressed A1 protein, whereas normal monocytes showed only weak staining. Bax mRNA was induced in parallel with A1 in macrophages. Exudate macrophages and granulocytes that were apoptotic by TUNEL staining occasionally appeared to display A1 throughout the cell nucleus. These studies identify A1 as a potential regulator of apoptosis during acute inflammation.
Repeated Administration of Adenoviral Vectors in Lungs of Human CD4 Transgenic Mice Treated with a Nondepleting CD4 Antibody
Authors: Chirmule Narendra , Truneh Alemseged , Haecker Sarah Ehlen , Tazelaar John , Gao Guang-ping , Raper Steven E. , Hughes Joseph V. , Wilson James M.
Published: January 2023

The central role of CD4+ T cells in regulation of adenovirus vector-mediated immune responses has been documented previously in murine models. We analyzed the effects of a nondepleting mAb to human CD4 (CD4 mAb; Clenoliximab) on immune functions following intratracheal administration of adenoviral vectors in murine CD4-deficient mice (muCD4KO) expressing a human CD4 transgene (HuCD4 mice). Treatment of HuCD4 mice with Clenoliximab inhibited both cell-mediated and humoral immune responses to adenoviral Ags. Chronic treatment of HuCD4 mice with Clenoliximab permitted successful readministration of adenoviral vectors at least four times. The ability to readminister these vectors is associated with marked suppression of neutralizing Ab responses to viral capsid proteins. Clenoliximab also inhibited CTL and prolonged expression of the transgene. T or B cell responses to adenovirus did not emerge after the effects of a short course of Clenoliximab diminished. These data illustrate the potential utility of a nondepleting CD4 Ab in facilitating gene therapy using adenoviral vectors.
Differential Role for p38 Mitogen-Activated Protein Kinase in Regulating CD40-Induced Gene Expression in Dendritic Cells and B Cells
Authors: Aicher Alexandra , Shu Geraldine L. , Magaletti Dario , Mulvania Thera , Pezzutto Antonio , Craxton Andrew , Clark Edward A.
Published: January 2023

We investigated whether human monocyte-derived dendritic cells (DCs) differed from tonsillar B cells in the set of cell fate genes they express constitutively and in the way these genes are affected after CD40 ligation. In particular, Bcl-2, TNF receptor-associated factor-2 (TRAF2), and TRAF4 were clearly inducible via CD40 in B cells but not in DCs. DCs, unlike B cells, were induced to increase expression of IL-1β, IL-1Ra, IL-8, IL-12 p40, RANTES, macrophage inflammatory protein-1α, and monocyte chemoattractant protein-1 after CD40 ligation. We next tested whether CD40-induced signaling pathways were different in DCs vs B cells. In DCs, as in B cells, CD40 ligation activated p38 mitogen-activated protein kinase (MAPK), its downstream target, MAPKAPK-2, and the c-Jun N-terminal kinase. The p38 MAPK-specific inhibitor, SB203580, blocked CD40-induced MAPKAPK-2 activation, but did not affect activation of c-Jun N-terminal kinase. Furthermore, unlike in B cells, extracellular signal-regulated kinase-1 and -2 were activated after CD40 ligation in DCs. SB203580 strongly blocked CD40-induced IL-12 p40 production in DCs at both mRNA and protein levels, while having minimal effect on CD40-induced expression of the chemokine RANTES. In contrast, no detectable IL-12 p40 protein was secreted in CD40-stimulated B cells. Furthermore, CD40-induced mRNA expression of cellular inhibitor of apoptosis protein-2 was also dependent on the p38 MAPK pathway in DCs and differed compared with that in B cells. In conclusion, CD40 induces distinct programs in DCs and B cells, and the set of p38 MAPK-dependent genes in DCs (IL-12 p40 and cellular inhibitor of apoptosis protein-2) is different from that in B cells (IL-10 and IL-1β).
Phosphorothioate Oligodeoxynucleotides Promote the In Vitro Development of Human Allergen-Specific CD4+ T Cells into Th1 Effectors
Authors: Parronchi Paola , Brugnolo Francesca , Annunziato Francesco , Manuelli Cinzia , Sampognaro Salvatore , Mavilia Carmelo , Romagnani Sergio , Maggi Enrico
Published: January 2023

DNA vaccination is an effective approach in inducing the switch of murine immune responses from a Th2 to a Th1 profile of cytokine production that has been related to the activity of unmethylated CpG motifs present in bacterial, but not mammalian, DNA. We report here that some synthetic phosphorothioate, but not phosphodiester, oligodeoxynucleotides (ODNs) were able to induce B cell proliferation and to shift the in vitro differentiation of Dermatophagoides pteronyssinus group 1-specific human CD4+ T cells from atopic donors into Th cell effectors showing a prevalent Th1, instead of Th2, cytokine profile. This latter effect was completely blocked by the neutralization of IL-12 and IFN (α and γ) in bulk culture, suggesting that the Th1-inducing activity of phosphorothioate ODNs was mediated by their ability to stimulate the production of these cytokines by monocytes, dendritic, and NK cells. Cytosine methylation abolished the Th1-inducing activity of ODNs; however, CpG dinucleotide-containing ODNs exhibited the Th1-shifting effect independently of the presence or the absence of CpG motifs (5′-pur-pur-CpG-pyr-pyr-3′). Moreover, the inversion of CpG to GpC resulted only in a partial reduction of this activity, suggesting that the motif responsible for the Th1-skewing effect in humans is at least partially different from that previously defined in mice. These results support the concept that the injection of allergens mixed to, or conjugated with, appropriate ODNs may provide a novel allergen-specific immunotherapeutic regimen for the treatment of allergic disorders.
8B4/20, A Private CD43 Epitope on Developing Human Thymocytes, Is Involved in Thymocyte Maturation
Authors: Fabbi Marina , Geginat Jens , Tiso Micaela , Ramarli Dunia , Parent David , Bargellesi Antonio , Remold-O’Donnell Eileen
Published: January 2023

The 8B4/20 Ag is a 120-kDa molecule whose expression on human thymocytes varies according to the differentiation stage: high density on immature CD3−/low thymocytes, reduced density on CD3medium and double-positive thymocytes, and absent on CD3high and single-positive thymocytes and on circulating T cells. In this paper we present immunological and biochemical evidence demonstrating that 8B4/20 Ag is a variant of CD43. We show that 8B4/20-expressing molecules, which are a subset of the CD43 molecules on thymocytes, are heterogeneous in charge, suggesting varying sialylation levels. The 8B4/20 epitope was mapped to the peripherally exposed N-terminal region of CD43, and the 8B4/20 antigenic determinant was characterized by requirement for the sialic acid exocyclic polyhydroxyl side chain, a feature shared with ligands of CD22. Altogether, 8B4/20-CD43 expression pattern and biochemical characteristics suggest its participation in carbohydrate-based interactions in the thymus. We therefore used specific Ab to mimic putative 8B4/20 interactions with natural ligand and examined the effect on isolated thymocytes. Treatment with 8B4/20 had no effect on in vitro apoptosis of isolated thymocytes. In contrast, 8B4/20 ligation enhanced the conversion of isolated thymocytes to differentiated phenotypes. Increased numbers were found in 8B4/20-treated cultures of CD3high and single-positive thymocytes and decreased numbers of CD3−/low and double-positive thymocytes, strongly suggesting that engagement of 8B4/20 delivers a positive signal that favors completion of the thymocyte maturation program. The ability of 8B4/20 mAb to drive thymocyte maturation in vitro suggests that CD43 molecules bearing the 8B4/20 epitope participate in early events of thymic selection.
IκBε-Deficient Mice: Reduction of One T Cell Precursor Subspecies and Enhanced Ig Isotype Switching and Cytokine Synthesis
Authors: Mémet Sylvie , Laouini Dhafer , Epinat Jean-Charles , Whiteside Simon T. , Goudeau Bertrand , Philpott Dana , Kayal Samer , Sansonetti Philippe J. , Berche Patrick , Kanellopoulos Jean , Israël Alain
Published: January 2023

Three major inhibitors of the NF-κB/Rel family of transcription factors, IκBα, IκBβ, and IκBε, have been described. To examine the in vivo role of the most recently discovered member of the IκB family, IκBε, we generated a null allele of the murine IκBε gene by replacement of all coding sequences with nlslacZ. Unlike IκBα nullizygous mice, mice lacking IκBε are viable, fertile, and indistinguishable from wild-type animals in appearance and histology. Analysis of β-galactosidase expression pattern revealed that IκBε is mainly expressed in T cells in the thymus, spleen, and lymph nodes. Flow cytometric analysis of immune cell populations from the bone marrow, thymus, spleen, and lymph nodes did not show any specific differences between the wild-type and the mutant mice, with the exception of a reproducible 50% reduction of the CD44−CD25+ T cell subspecies. The IκBε-null mice present constitutive up-regulation of IgM and IgG1 Ig isotypes together with a further increased synthesis of these two isotypes after immunization against T cell-dependent or independent Ags. The failure of observable augmentation of constitutive nuclear NF-κB/Rel-binding activity is probably due to compensatory mechanisms involving IκBα and IκBβ, which are up-regulated in several organs. RNase-mapping analysis indicated that IL-1α, IL-1β, IL-1Ra, and IL-6 mRNA levels are constitutively elevated in thioglycolate-elicited IκBε-null macrophages in contrast to GM-CSF, G-CSF, and IFN-γ, which remain undetectable.
Enhancement of Fcγ Receptor-Mediated Phagocytosis by Transforming Mutants of Cbl
Authors: Sato Norihito , Kim Moo-Kyung , Schreiber Alan D.
Published: January 2023

Phagocytosis mediated by FcγR plays an important role in host defense. The molecular events involved in this process have not been completely defined. The adapter protein Cbl has been implicated in FcγR signaling, but the function of Cbl in phagocytosis is unknown. Here we show that overexpression of the transforming mutants of Cbl, Cbl-70Z, and v-Cbl, but not wild-type (wt) Cbl, enhance phagocytosis mediated by FcγR in COS cells. Cbl-70Z, but not Cbl-wt, also enhanced FcγR-mediated phagocytosis in P388D1 murine macrophage cells. Cbl-70Z did not affect tyrosine phosphorylation or in vitro kinase activity of Syk, indicating that Syk may not be the direct target of Cbl-70Z in the enhancement of phagocytosis. A point mutation (G306E) in the phosphotyrosine domain of Cbl-70Z, as well as a C-terminal 67-aa deletion, partially abolished the enhancing effect on FcγR-mediated phagocytosis. A double mutant of Cbl-70Z containing both the G306E mutation and the C-terminal deletion completely lacked the ability to enhance phagocytosis. Thus, both the phosphotyrosine binding domain and the carboxyl-terminal tail were required for optimal enhancement of phagocytosis by Cbl-70Z. Functional phosphatidylinositol 3-kinase was required for Cbl-70Z to enhance phagocytosis, since wortmannin, a phosphatidylinositol 3-kinase inhibitor, inhibited FcγR-mediated phagocytosis in the presence of Cbl-70Z. These studies demonstrate that mutants of Cbl can modulate the phagocytic pathway mediated by FcγR and imply a functional involvement of c-Cbl in Fcγ receptor-mediated phagocytosis.
15-Deoxy-Δ12,1412,14-prostaglandin J2 Inhibits the β2 Integrin-Dependent Oxidative Burst: Involvement of a Mechanism Distinct from Peroxisome Proliferator-Activated Receptor γ Ligation
Authors: Vaidya Sanskruti , Somers Elizabeth P. , Wright Samuel D. , Detmers Patricia A. , Bansal Vinay S.
Published: January 2023

15-Deoxy-Δ12,14-PGJ2 (dPGJ2) is a bioactive metabolite of the J2 series that has been identified as a ligand for peroxisome proliferator-activated receptor γ (PPARγ) and has received attention for its potential antiinflammatory effects. Because neutrophils express cell-surface receptors for PGs, the effect of dPGJ2 was tested on an inflammatory response that should not require PPARγ, the oxidative burst made by adherent human neutrophils. dPGJ2 inhibited adhesion-dependent H2O2 production with an IC50 of 1.5 μM when neutrophils were stimulated with TNF, N-formylnorleucylleucylphenylalanine, or LPS. Inhibition by dPGJ2 occurred during the lag phase, before generation of peroxide, suggesting blockade of an early signaling step. Indeed, dPGJ2 blocked adhesion of neutrophils to fibrinogen in response to TNF or LPS with an IC50 of 3–5 μM. dPGJ2 was more potent at inhibiting the adhesion-dependent oxidative burst than several other PGs tested. Further, dPGJ2 did not appear to act through either the DP receptor or receptors for PGE2. PG receptors modulate cAMP levels, and the inhibition of adhesion and oxidative burst by dPGJ2 was enhanced in the presence of 3-isobutyl-1-methylxanthine, a cAMP phosphodiesterase inhibitor. A potent PPARγ agonist (AD-5075) did not inhibit peroxide production or adhesion, nor did it change the IC50 for dPGJ2 inhibition. These studies suggest that dPGJ2 may interact with an unknown receptor on neutrophils, distinct from PPARγ, to modulate the production of reactive oxygen intermediates.
The Reaction of the White Blood Cells to Specific Precipitates
Authors: Cromwell H. W. , Centeno J. A.
Published: January 2023

When fresh normal citrated blood is mixed with a protein and its specific antibody, the polymorphonuclears, lymphocytes and mononuclears become strikingly vacuolated. This vacuolation is definitely associated with the product of the antigen-antibody reaction since (a) it does not occur if the precipitate is removed before the whole blood is added, and (b) the reaction does occur when the whole blood is added to the thoroughly washed precipitate suspension.

Non-specific particulate matter, as heat-coagulated protein, collodion particles and charcoal particles, does not stimulate the reaction.

Phagocytosis of sensitized B. typhosus and pneumococcus by the same types of cells has been observed with marked vacuolation of the polymorphonuclears and mononuclears. The vacuolation appears more slowly than with the specific precipitate and seems not to occur in the lymphocytes at all.

It is considered that the specific precipitate is probably phagocyted and that the vacuoles are associated in some manner with its subsequent digestion.

These observations are significant in pointing out the probable importance of phagocytosis in determining the fate of the specific precipitate in vivo.
Cellular immune response to Hantaan virus nucleocapsid protein during the acute phase of hemorrhagic fever with renal syndrome: negative correlation with disease severity (128.13)
Authors: Wang Meiliang , Wang Jiuping , Zhu Yong , Xu Zhuwei , Yang Kun , Yang Angang , Jin Boquan
Published: January 2023

The cellular immune response to Hantaan virus (HTNV) is incompletely understood, especially in humans. To investigate the cellular immunity during acute HTNV infection, the magnitude of T cell response to HTNV nucleocapsid protein was quantitated by direct ex vivo IFN-γ ELISPOT using overlapping peptides. We found that the combined frequencies of HTNV-specific T cells at early time point were significantly higher in patients with mild or moderate hemorrhagic fever with renal syndrome (HFRS) than in those with severe or critical HFRS (P = .006). Moreover, these frequencies were higher in patients with subsequent mild renal failure (maximum serum creatinine level, =707 μmol/L) than in those with subsequent severe renal failure (maximum serum creatinine level, >707 μmol/L) (P = .006). Kinetic analysis showed that a decrease in the serum creatinine level during the acute phase of illness was often accompanied by an increase in the magnitude of IFN-γ-producing T cells. These data suggest that IFN-γ-producing T cells may help reduce the risk of progression to acute renal failure caused by HTNV infection. This work was supported by grants from the National Natural Science Foundation of China (No.30471625).
MHC class I Dk gene expression licenses Ly49G2+ NK cell function and rescues MCMV susceptibility (128.19)
Authors: Xie Xuefang , Brown Michael G
Published: January 2023

NK cell-mediated murine CMV (MCMV) resistance is under H-2k genetic control in MA/My mice, however the underlying gene(s) is unclear. We generated a panel of intra-H-2 recombinant congenic mouse strains to map a 0.3-Mb critical interval in the H-2 class I D subregion. The interval spans 30 genes including the class I Dk gene. Because Ly49 NK cell receptors are licensed by and responsive to MHC class I molecules, Dk is a positional candidate. Thus, a 10-kb Dk genomic DNA was cloned and injected into MCMV susceptible (Cmvs) (MA/My.L-H2b x C57L)F1 blastocysts. A transgenic founder (Tg3-Dk) mouse was healthy and competent in germline transgene transmission. We separately backcrossed Tg3-Dk to both C57L and MA/My.L-H2b Cmvs genetic backgrounds and found that Dk was expressed at the cell surface of leukocytes only in Tg3-Dk offspring. Importantly, Tg3-Dk gene expression conferred virus resistance; Tg3-Dk offspring spleens had ~1000-fold lower MCMV at 90 h after infection than non-transgenic littermate controls. MCMV resistance in this system may need Dk-licensed Ly49G2+ NK cells. In support, we have found that Ly49G2+ NK cells are selectively licensed in, and preferentially expand and become activated during MCMV infection in Dk-expressing C57L.M-H2k(R7) congenic mice. This is under study in Tg3-Dk mice. We conclude that Dk gene expression is essential in H-2k MCMV resistance. Supported by NIH R01 AI50072.
Assessment of Ebola Specific Antibody Responses in Vaccinated Macaques (128.20)
Authors: Lear Calli  Marie , Olinger Gene G.
Published: January 2023

Ebola virus (EBOV) infection causes severe disease accompanied by high mortality rates. Several vaccine platforms have shown promise in protecting against Ebola exposure; however, there are no licensed vaccines or therapies approved and available for human use. A vaccine approach utilizing Venezuelan equine encephalitis virus (VEE) replicon particles (VRP) expressing filovirus protein(s) was the first vaccine approach that provided complete protection against Marburg infection in non-human primates (NHP). We evaluated the ability of VEE replicons expressing the glycoprotein (GP), nucleoprotein (NP), VP24, VP30, VP35, and VP40 to protect against Ebola Zaire (ZEBOV) exposure in NHP by various routes of administration, intramuscular (IM) versus subcutaneous (SC). To further evaluate protective efficacy, NHP were vaccinated IM or SC with VEE replicon expressing the viral glycoprotein or expressing all six proteins. Protective efficacy was demonstrated in the both cynomolgus macaques and rhesus macaques. Enzyme-Linked ImmunoSorbent Assays (ELISA) were developed using the whole antigen or recombinant Ebola GP to assess vaccine induced immunity against EBOV. The ELISA based assay assessing humoral responses did not accurately predict protection. The protection generated by the VEE replicon expression of Ebola proteins demonstrates the efficacy of the vaccines platform and warrants further evaluation as a human use vaccine. Further studies are needed to develop accurate correlate of immunity assays.
Impact of TLR deletion on brain pathology in a mouse model of encephalitis (128.22)
Authors: Drescher Kristen M. , Schemmel Andrew , Leland Korey
Published: January 2023

The innate immune response represents the host's first line of defense to infection. The toll-like receptors (TLRs) are a family of pattern recognition receptors triggered by specific pathogen-associated molecular patterns (PAMPs). To examine the role of TLRs in a mouse model of viral encephalitis, mice with defects in TLR-2, TLR-3 or TLR-4 were intracerebrally infected with Theiler's murine encephalomyelitis virus (TMEV) to determine the impact of TLR deletion on the extent of disease. At day 16 post-infection, a timepoint when limited pathology is observed in TMEV-infected wild-type control animals, extensive brain pathology was observed in TLR-2 and TLR-3 knockout mice. While pathology was observed in TLR-4 knockout mice following TMEV infection, the pathology was less than that observed in the TLR-2 and TLR-3 knockout animals. TLR-4 knockout mice mounted more robust antibody responses to TMEV the other strains of mice, suggesting that this mechanism of virus clearance may account for the reduced level of pathology observed in these animals.

Support: National Multiple Sclerosis Society (RG3870A3); Department of Defense (PR064574)
HIV infection inhibits granulysin expression by memory CD8+ T cells (128.26)
Authors: Hogg Alison E , Ji Jiaxiang , Lancaster Katrina L , Cloyd Miles W , Endsley Janice J
Published: January 2023

The hallmark of HIV infection is severe immuno-suppression and an increased susceptibility to many infectious agents including M. tuberculosis. Granulysin is expressed by cytotoxic T cells (CTL) and is strongly lytic against susceptible and drug resistant strains of M. tuberculosis. We hypothesized that the reduced CTL activity observed following HIV infection may, in part, be due to suppression of granulysin. We and others have shown that granulysin expression is regulated by common γc cytokines IL-2, IL-15 and IL-21. We used an in vitro model, in which we infected PBMC from healthy donors with HIV. We found that induction of granulysin in CD8+ T cells following stimulation with IL-2, IL-15 or IL-21 was reduced in PBMC infected with HIV. Moreover, we observed that phosphorylation of STAT5, a downstream signaling molecule crucial for the induction of granulysin by IL-2 and IL-15 was also reduced by HIV infection. In an antigen-specific memory response, using PBMC from PPD+ donors, we found that expression of granulysin by memory CD8+ T cells following PPD stimulation was inhibited in HIV-infected PBMC. Our results suggest that suppression of granulysin in response to γc cytokines and specific TB antigen by HIV may be one mechanism for the increased susceptibility of HIV+ patients to TB infection and could represent an important avenue for immune intervention.
Identification of CD8 T lymphocyte responses to HHV-8/KSHV Proteins (128.28)
Authors: Lepone Lauren , Rappocciolo Giovanna , Knowlton Emilee , Piazza Paolo , Jais Mariel , Jenkins Frank , Rinaldo Charles
Published: January 2023

Objectives: CD8 T cells producing more than one cytokine or cytotoxicity factor, i.e. polyfunctional, have been shown to relate to control of HIV-1 infection. As it is currently not clear if polyfunctional CD8 T cells have a role in controlling HHV-8 infection, we investigated CD8 T cell cytokine responses to multiple lytic and latency viral proteins.

Methods: T cells were stimulated with dendritic cells from HHV-8 seropositive subjects that were loaded with overlapping peptides derived from gB and K8.1 lytic proteins, and K12 and LANA-1 latency proteins, and screened for IFNγ production by extended culture ELISPOT. Positive peptides were assessed for production of IFNγ, IL2, TNFα, MIP1β and CD107a by flow cytometry.

Results: ELISPOT and flow cytometry revealed new, potential HLA A*0201 9mer epitopes for each protein. In HHV-8 seropositive healthy donors controlling infection, the predominant CD8 T cell response to protein epitopes was monofunctional, with a portion of CD8 T cells being polyfunctional.

Conclusions: This demonstrates that HHV-8 proteins predominantly induce single factors in CD8 T cells from healthy, HHV-8 seropositive individuals, with a portion of T cells producing 2 or more of these factors. These immunogenic regions of the viral proteins could be important in immunopathogenesis of KSHV/HHV-8 and progression to Kaposi's sarcoma. (U01-AI35041, R01 CA82053, T32 AI065380-04)
An Altered T-cell Repertoire Mediates Heterologous Immunity and Protection from Epstein-Barr Virus Infection (128.32)
Authors: Watkin Levi , Quigley Maire , Aslan Nuray , Sullivan John L , Luzuriaga Katherine , Selin Liisa K
Published: January 2023

Previously we have reported that cross-reactive memory CD8 T cells specific for the Flu-M158-66 epitope (GILGFVFTL) respond to the EBV-BMLF1280-288 epitope (GLCTLVAML) during the course of EBV infection and contribute to the onset of infectious mononucleosis. Recently we have found that the memory CD8 T cells specific for the Flu-M158-66 are also cross-reactive to an additional EBV epitope, EBV-BRLF1109-117 (YVLDHLIVV). Here we sought to determine the cross-reactive properties of the Flu-M158-66 specific cells in EBV sero-negative individuals. CD8 T cells cultured in the presence of the cross-reactive EBV epitopes preferentially expanded the Flu-M158-66 specific cells. Although these expanded cells did not stain with EBV pentamers they were able to produce cytokine in response to EBV peptides. Additionally these cells were capable of killing autologous EBV infected targets. Interestingly the T cell repertoire demonstrated unique characteristics including nearly complete domination by a single Vβ17 and Vα27clonotype with a unique CDR3 motif. Taken together these data suggest that an altered T cell repertoire could mediate protection against viral infection.
γδT Cell Promotes Maturation of Dendritic Cell During West Nile Virus Infection (128.4)
Authors: Fang Hao , Welte Thomas , Zheng Xin , Wang Tian
Published: January 2023

γδ T cells are important for early control of West Nile virus (WNV) dissemination. TCRδ-/-(γ δT Cell Deficient) mice also have reduced CD8+ T cell memory response and are more susceptible to secondary WNV infection. Here, we investigated the role of γδ T cells in regulation of CD4+ T cell response following WNV challenge. We found that dendritic cells (DCs) of WNV infected TCRδ-/-mice had reduced levels of CD40, CD80, CD86 and major compatibility complex (MHC) II expression and IL-12 production than wild-type mice. We also detected a significant viral load in WNV infected γδ T cells. Infected γδ T cells produced high levels of IFN-γ and proinflammatory cytokines, including TNF-α and IL-6, which were known to promote DC maturation. Nevertheless, levels of Toll-like receptors (TLR) 2, 3, 4 and 7 in these cells were not different from those of non-infected, suggesting a TLR-independent activation following infection. Naïve DCs co-cultured with γδ T cells from WNV infected mice or from in vitro infection had higher levels of costimulating molecules, MHC class II expression and IL-12 production. Co-culture of CD4+ T cells purified from OT II transgenic mice and DCs from WNV infected TCRδ-/-mice induced less IFN-γ and IL-2 than those of wild-type mice. Overall, these data suggest that WNV induced γδ T cell activation might promote DC maturation and initiate CD4+ T cell priming in a TLR independent manner. (Supported by NIH grant R01 AI072060)
Enhancing potency of anti-HIV chimeric immune receptor (CIR) (128.5)
Authors: Selliah Nithianandan , Skowron Gail , Lu Li , Yang Wen , Junghans Richard P
Published: January 2023

Immunotherapies that have the potential to eradicate reservoirs of HIV infected cells may be useful to suppress or cure HIV/AIDS. in combination with current antiviral drug therapies. T cells expressing chimeric immune receptors (CIR) through gene modification ("designer T cells") can be redirected to kill HIV-infected cells. Previously, an anti-HIV CIR, CD4-ζ (1st generation), was created to bind to HIV-1 gp120 antigen through its CD4 extracellular domain and induction of signaling through cytoplasmic CD3ζ chain. However, the adoptive transfer of autologous CD4-ζ T cells to HIV/AIDS patients failed to control the viral infection in clinical trials. To improve potency, our laboratory has created 2nd generation anti-HIV designer T cells that incorporate CD28 costimulation (CD4-CD28-ζ). 2nd generation designer T cells kill HIV-Env expressing Jurkat cells and HIV-1 infected CEM-SS cells as well or better than 1st generation designer T cells. Stimulation with anti-CD4 mAb induces more IL2 production in 2nd generation than 1st generation designer T cells. In addition, 2nd generation designer T cells showed significantly better proliferative response upon culture with HIV-Env expressing HeLa cells or HIV-1 infected CEM-SS cells. These data suggest that gene modified CD8+ T cells could be an effective therapy to eradicate HIV+ cells during and after anti-retroviral therapy. We hypothesize that CD28 domain in the CIR will confer a benefit of longer T cell survival after infusion into HIV patients.

Research supported by NIH, R21.
ESAT-6 inhibits IFN-γ secretion by Mycobacterium tuberculosis responsive-human T cells (129.12)
Authors: Wang Xisheng , Barnes Peter F , Townsend James C , Chung Yoon-tae , Shams Homayoun , Samten Buka
Published: January 2023

Early secreted antigenic target of 6 kD (ESAT-6) of Mycobacterium tuberculosis (M. tb) is a potent antigen with vaccine potential, but is also a virulence factor. We studied the effect of recombinant His-tagged ESAT-6 on IFN-γ secretion by human primary T cells, using the 10 kD culture filtrate protein (CFP10) as a control. ESAT-6 but not CFP10 started to inhibit IFN-γ secretion by M-tb stimulated PBMC at 1.6 μM and this reached 90% inhibition, compared to that with CFP10 (12.5 + 5.4 vs. 1.3 + 1.2 ng/ml) at 3.3 μM (20 μg/ml). Dimerization of ESAT-6 with CFP10 did not affect inhibition. ESAT-6 inhibited IFN-γ production of α-CD3/CD28 mAb-stimulated T cells by 89% (14.5 + 1.8 vs. 1.6 + 0.4 ng/ml), and proliferation, as measured by CFSE assay. Depleting ESAT-6 with a nickel resin abrogated the inhibition. The inhibitory effect of ESAT-6 was not due to T cell toxicity or apoptosis, as measured by MTT assay and Annexin V staining. ESAT-6 inhibited expression of M. tb-induced IFN-γ mRNA (> 90%) and the transcription factors, ATF-2 and c-Jun, which regulate IFN-γ transcription. ESAT-6 bound to T cells and inhibited expression of CD69 and CD25, without affecting proximal TCR signaling, ZAP70 activation. We conclude that high concentrations of ESAT-6 inhibit T cell responses to M. tb by affecting signaling pathways downstream of ZAP70, and may be a mechanism by which M. tb inhibits effective immunity. (NIH AI063514)
Host response to Tick-bone encephalitis and Omsk hemorrhagic fever (129.22)
Authors: Tigabu Bersabeh , Holbrook Michael
Published: January 2023

Omsk hemorrhagic fever virus (OHFV) and Russian spring-summer encephalitis virus (RSSEV) are tick-borne flaviviruses of the genus Flavivirus, Family Flaviviridae. Although OHFV and RSSEV have 90% homology at their amino acid level, they have different disease outcomes. OHFV infection mainly causes hemorrhagic fever, however RSSEV infection results in a severe form of CNS disorder. Understanding of the pathogenesis of tick-borne encephalitis and hemorrhagic fever is of major importance due to significant public health problem and the lack of treatment. Dendritic cells (DC) play a critical role for both innate and adaptive immune responses. DC function as the best antigen presenting cells to T-cells by upregulating expression of costimulatory and MHC molecules, and cytokine production. In these experiments we examined interactions between OHFV/RSSEV and murine DC to identify responses that contribute to the pathogenesis of tick-borne flaviviruses. The DC surface markers were analyzed using flow cytometry, and cytokines and chemokines were quantified by Bioplex assay. OHFV/RSSEV infection of DC resulted in the secretion of a broad array of cytokines and chemokines. OHFV/RSSEV exposure did not activate DC maturation markers as compared to LPS stimulated DC. Infected DC had similar levels of CD80 and CD86 expression relative to the mock infected DC. Our data demonstrate that the functional maturation of DC in response to OHFV/RSSEV infection is modified leading to a reduction of the capacity of DC antigen presentation and consequently T-cell activation.

This research was supported by the Western Regional Center of Excellence (U54AI056157)
HIV-1 compromises CD8+T cell effector function in Mtb- infected lung (129.26)
Authors: Lancaster Katrina  L , Hogg Alison E , Clement Cecilia G , Aronson Judith F , Endsley Janice J
Published: January 2023

Tuberculosis (TB) remains a global health problem with more than 8 million new cases and 1 million deaths per year worldwide. HIV-1 infected persons have a greatly increased risk of Mycobacterium tuberculosis (M. tb) co-infection. A key feature of M.tb infection is the formation of granulomas, cellular accumulations composed of macrophages, epithelial cells, and a surrounding mantle of T cells, which are important for the containment of infection. The cytolytic T cell effector molecules, perforin and granulysin, play a critical role in protective immunity to M.tb. It has been shown that decreased expression of perforin and granulysin at the site of pulmonary disease is linked to chronic TB. However, the effects of HIV-1 on CD8+T cells numbers and expression of cytolytic effector molecules such as perforin and granulysin in M. tb induced granulomas has not been explored. In this study, we used immunohistological techniques to assess the differences in T cell populations, granulysin expression, and pathology in tissue sections from Mtb- and Mtb/HIV-1-infected human lung. Our results indicate that granulomas from persons with TB/HIV-1 are characterized by dysregulated T cell organization and poor expression of granulysin. We propose that defective granulysin expression by M.tb-specific CD8+T cells contributes to the development of disease in TB/HIV-1 co-infected persons.