bluuebunny/crossref_metadata_2025 huggingface dataset:
Glucocorticoids Inhibit Bioactive IL-12p70 Production by In Vitro-Generated Human Dendritic Cells Without Affecting Their T Cell Stimulatory Potential
Authors: Vieira Pedro L. , Kaliński Paweł , Wierenga Eddy A. , Kapsenberg Martien L. , de Jong Esther C.
Published: December 2022

Glucocorticoids (GC) are known to affect the immune response at several stages. However, little is known about how GC influence the initiation of the specific immune response at the level of dendritic cells (DC), the highly professional APC for T cells. Therefore, we studied whether GC modulate the cytokine production and T cell stimulatory function of DC. In LPS-stimulated DC, GC strongly reduced the secretion of the Th1-skewing factor IL-12p70 and, to a lesser extent, the production of the proinflammatory cytokines IL-6 and TNF-α. Regarding the T cell stimulatory function of DC, GC did not influence the cell surface expression of HLA-DR or the costimulatory molecules CD40 and CD80 and did not influence the ability of DC to take up Ag. Consequently, GC pretreatment of DC indeed did not affect their ability to stimulate CD4+ Th cell proliferation in response to superantigen. However, as a result of their defective production of bioactive IL-12, GC-pretreated DC have a reduced ability to promote the production of IFN-γ in CD4+ Th lymphocytes, as shown by the observation that IFN-γ production could be restored by exogenous IL-12. In contrast, GC treatment of DC enhanced the secretion of the antiinflammatory cytokine IL-10 and the type 2 cytokine IL-5 by the T cells. It is concluded that, in addition to their role as potent inhibitors of inflammation via the direct suppression of cytokine production in T cells, GC may further inhibit T cell-mediated inflammation indirectly via the suppression of IL-12 production by DC.
HLA-B27 Subtype Polymorphism and CTL Epitope Choice: Studies with EBV Peptides Link Immunogenicity with Stability of the B27:Peptide Complex
Authors: Brooks Jill M. , Colbert Robert A. , Mear John P. , Leese Alison M. , Rickinson Alan B.
Published: December 2022

HLA-B27-restricted CTL responses to EBV are principally directed against two of the EBV nuclear Ags, EBNAs 3B and 3C. We have previously described a target epitope derived from EBNA 3C (residues 258–266, sequence RRIYDLIEL) that is immunodominant in the context of at least three different B27 subtypes, including B*2705 and B*2702. In this study, we show that this peptide binds well to B*2705 and B*2702 in a cell surface binding assay, and that the two B27:peptide complexes are relatively stable, with t1/2 of 20 and 37 h, respectively. We now identify another B27-restricted epitope derived from EBNA 3B (residues 243–253, sequence RRARSLSAERY), which again accords well with the B*2705/B*2702 consensus motifs, having an arginine residue at position 2 and a tyrosine residue at the carboxyl terminus. In this case, five of five B*2702-positive donors respond to the epitope, whereas there was no response in any B*2705-positive donor studied. This peptide binds at least as well to B*2705 as to its restriction element B*2702; however, the two class I:peptide complexes show marked differences in stability, with t1/2 of 9 and 42 h, respectively. Thus, the stability of B27:peptide complexes can vary markedly between different B27 subtypes in ways that may not be apparent from cell surface binding assays and cannot be predicted from currently known peptide consensus motifs, yet which may critically influence CTL epitope choice.
B7.1 Is a Quantitatively Stronger Costimulus Than B7.2 in the Activation of Naive CD8+ TCR-Transgenic T Cells
Authors: Fields Patrick E. , Finch Rosalynde J. , Gray Gary S. , Zollner Richard , Thomas Jenifer L. , Sturmhoefel Knut , Lee Kwan , Wolf Stanley , Gajewski Thomas F. , Fitch Frank W.
Published: December 2022

Using a TCR transgenic mouse bred onto a recombinase-activating gene-2-deficient background, we have examined the influence of B7.1 and B7.2 on activation of naive, CD8+ T cells in vitro. We found that B7.1 was a more potent costimulus than B7.2 for induction of proliferation and IL-2 production by naive CD8+ T cells. This difference appeared to be quantitative in nature, as determined using transfectants expressing various defined levels of B7.1 or B7.2, or using purified B7.1 or B7.2 fusion proteins. In contrast to the quantitative differences seen in stimulation of naive T cells, B7.1 and B7.2 were comparable in their ability to costimulate responses in T cells previously primed in vitro. In addition, primed, but not naive, T cells were capable of proliferating and producing IL-2 in response to a TCR stimulus alone, apparently in the absence of B7 costimulation. Lastly, we found that B7.1 and B7.2 were equivalently capable of driving differentiation of naive CD8+ T cells into an IL-4-producing phenotype when exogenous IL-4 was added to the primary culture or to an IFN-γ-producing phenotype in the presence of IL-12. These results indicate that signals generated by B7.1 and B7.2 are qualitatively similar, but that B7.1 is quantitatively stronger than B7.2. Further, our results indicate that the activation state of the responding T cell may influence the efficiency with which the T cell can respond to a costimulatory signal provided by either B7.1 or B7.2.
Normal Human CD4+ Memory T Cells Display Broad Heterogeneity in Their Activation Threshold for Cytokine Synthesis
Authors: Waldrop Shar L. , Davis Kenneth A. , Maino Vernon C. , Picker Louis J.
Published: December 2022

CD4+ memory T cells coordinate immune responses against viruses and other pathogens via the Ag-induced secretion of potent effector cytokines. The efficacy of these responses depends on both the overall number of pathogen-specific memory T cells and the particular array of cytokines that these cells are programmed to secrete. Here, we provide evidence that heterogeneity in Ag triggering thresholds constitutes an additional critical determinant of memory T cell function. Using a novel assay that allows single-cell detection of Ag-specific T cell cytokine production, we demonstrate that CMV-specific CD4+ memory cells from human peripheral blood display pronounced differences in their costimulatory requirements for Ag-induced triggering of IFN-γ and IL-2 secretion, ranging from cells that trigger with little costimulation (e.g., resting APC alone) to cells requiring potent costimulation through multiple pathways (resting APC plus multiple costimulatory mAbs, or activated APC). These differences in costimulatory requirements are independent of clonal differences in TCR signaling intensity, consistent with an intrinsic activation-threshold heterogeneity that is “downstream” from the TCR. Thus, “effective” frequencies of Ag-specific CD4+ memory T cells appear to depend on the activation status of available APC, a dependence that would allow the immune system to rapidly adjust the number of functional Ag-specific memory T cells in a particular effector site according to local conditions.
Oral Tolerance to Low Dose β2-Glycoprotein I: Immunomodulation of Experimental Antiphospholipid Syndrome
Authors: Blank Miri , George Jacob , Barak Vivian , Tincani Angela , Koike Takao , Shoenfeld Yehuda
Published: December 2022

Oral tolerance was induced in BALB/c mice by feeding low dose β2-glycoprotein I (β2GPI). The β2GPI-fed mice did not develop serologic and clinical markers of experimental antiphospholipid syndrome (APS) upon immunization with the autoantigen. The treated group was characterized by low titers of serum anti-β2GPI and anticardiolipin Abs in the serum, lack of fetal resorptions, low incidence of thrombocytopenia, and normal aPTT (activated partial thromboplastin time) values. β2GPI given orally before priming with β2GPI resulted in complete prevention of experimental APS development; β2GPI given at an early stage of the disease reduced clinical manifestations. However, administration of β2GPI 70 days postimmunization had a less significant effect on disease expression. Tolerized mice exhibited a diminished T lymphocyte proliferation response to β2GPI in comparison with β2GPI-immunized mice fed with OVA. When nontolerant β2GPI-primed T lymphocytes were mixed with T lymphocytes derived from tolerized mice, a significant inhibition of proliferation upon exposure to β2GPI was observed. The induction of suppression was β2GPI specific and driven, as well as TGF-β mediated. The β2GPI-specific response of T lymphocytes from the β2GPI-fed mice was reversed by anti-TGF-β Abs. The tolerance was adoptively transferred by CD8+ T cells from the tolerized mice into naive mice. Those CD8+ cells were MHC class I restricted, found to secrete TGF-β, and had no cytolytic activity. Oral administration of β2GPI suppressed priming of CTLs in the recipient mice. In sum, β2GPI-induced oral tolerance has an immunomodulatory effect in experimental APS, demonstrating the importance of β2GPI in the pathogenesis of the disease.
Mutational Analysis of CD28-Mediated Costimulation of Jun-N-Terminal Kinase and IL-2 Production
Authors: Barz Corinna , Nagel Thomas , Truitt Kenneth E. , Imboden John B.
Published: December 2022

The accessory molecule CD28 delivers a costimulus that acts in concert with TCR signals to promote T cell activation. Activation of Jun-N-terminal kinases (JNK) requires simultaneous stimulation of the TCR and CD28 and, therefore, likely plays an important role in signal integration during costimulation. We investigated the effects of mutations in the 41-amino acid cytoplasmic domain of murine CD28 on its ability to deliver costimuli for JNK activation and IL-2 production when expressed in Jurkat T cells. Our results indicate that the costimulus for JNK activation requires the membrane-proximal 24 amino acids of the CD28 cytoplasmic domain and is not mediated by the tyrosine-based recruitment of signaling molecules, including phosphatidylinositol 3-kinase. Deletion of the carboxyl-terminal 17 amino acids does not affect the ability of CD28 to augment JNK activation but impairs its ability to enhance TCR-mediated production of IL-2, demonstrating that optimal costimulation of IL-2 production requires CD28 signals in addition to the activation of JNK.
Antigen Drives Very Low Affinity B Cells to Become Plasmacytes and Enter Germinal Centers
Authors: Dal Porto Joseph M. , Haberman Ann M. , Shlomchik Mark J. , Kelsoe Garnett
Published: December 2022

In the first week of the primary immune response to the (4-hydroxy-3-nitrophenyl)acetyl (NP) hapten, plasmacytic foci and germinal centers (GCs) in C57BL/6 mice are comprised of polyclonal populations of B lymphocytes bearing the λ1 L-chain (λ1+). The Ig H-chains of these early populations of B cells are encoded by a variety of VH and D exons undiversified by hypermutation while later, oligoclonal populations are dominated by mutated rearrangements of the VH186.2 and DFL16.1 gene segments. To assess directly Ab affinities within these defined splenic microenvironments, representative VDJ rearrangements were recovered from B cells participating in the early immune response to NP, inserted into Ig H-chain expression cassettes, and transfected into J558L (H−; λ1+) myeloma cells. These transfectoma Abs expressed a remarkably wide range of measured affinities (Ka = 5 × 104-1.3 × 106 M−1) for NP. VDJs recovered from both foci and early GCs generated comparable affinities, suggesting that initial differentiation into these compartments occurs stochastically. We conclude that Ag normally activates B cells bearing an unexpectedly wide spectrum of Ab affinities and that this initial, promiscuous clonal activation is followed by affinity-driven competition to determine survival and clonal expansion within GCs and entry into the memory and bone marrow plasmacyte compartments.
Generation of Melanoma-Specific Cytotoxic T Lymphocytes by Dendritic Cells Transduced with a MART-1 Adenovirus
Authors: Butterfield Lisa H. , Jilani Syed M. , Chakraborty Nitya G. , Bui Lynne A. , Ribas Antoni , Dissette Vivian Beck , Lau Roy , Gamradt Seth C. , Glaspy John A. , McBride William H. , Mukherji Bijay , Economou James S.
Published: December 2022

Dendritic cells (DC) are potent stimulators of primary T cell responses. In this study, we demonstrate that DC, genetically engineered to express the MART-1/Melan-A (MART-1) tumor-associated Ag, express MART-1 mRNA and protein, correctly process and present the HLA-A2.1-restricted immunodominant MART-1 peptide (MART-127–35), and serve as potent stimulators of MART-1-specific CTL in vitro. A replication-defective E1-deleted adenovirus (AdV) was constructed that expresses MART-1 (AdVMART1). Transduced DC produce full length MART-1 mRNA as well as MART-1 protein. AdVMART1 does not significantly down-regulate cell surface class I expression despite having an intact E3 region. Transduction of an HLA-A2-positive/MART-1-negative cell line with AdVMART1 renders these cells sensitive to lysis by CTL specific for the MART-127–35 immunodominant peptide. In addition, DC transduced with AdVMART1 stimulated MART-127–35-specific tumor-infiltrating lymphocytes to synthesize IFN-γ. Finally, AdVMART1-transduced DC were able to generate MART-127–35 peptide-specific, class I-restricted CTL in PBL cultures from normal donors. This study supports the use of tumor Ag-engineered DC in genetic immunotherapy.
Human Complement Component C1q Inhibits the Infectivity of Cell-Free HTLV-I
Authors: Ikeda Fumihiro , Haraguchi Yuji , Jinno Atsushi , Iino Yuichi , Morishita Yasuo , Shiraki Hiroshi , Hoshino Hiroo
Published: December 2022

Human T cell leukemia virus type I (HTLV-I) is a retrovirus that is not lysed by human serum or complement. It has not been determined, however, whether HTLV-I directly binds to complement components or whether it retains infectivity after incubation with human serum. We investigated the effects of human serum on the infectivity of cell-free HTLV-I produced by human and animal cells. Plating of vesicular stomatitis virus (HTLV-I) pseudotypes prepared in cat or human cells and formation of HTLV-I DNA after infection of cell-free HTLV-I produced by cat or human cells were markedly inhibited by treatment with fresh human serum, but not by heat-inactivated serum. HTLV-I infection was also inhibited by treatment with C2-, C3-, C6-, or C9-deficient serum, but not by C1q-deficient serum. Inhibitory activities of normal human serum against HTLV-I were neutralized by anti-C1q serum. Furthermore, purified C1q inhibited HTLV-I infection. The direct binding of C1q to HTLV-I was confirmed by comigration of C1q with HTLV-I virion upon sucrose density gradient ultracentrifugation of HTLV-I virion treated with C1q. Binding assay using synthetic envelope peptides indicated that C1q bound to an extramembrane region of the gp21 transmembrane protein. These findings indicate that the human complement component C1q inactivates HTLV-I infectivity.
ICAM-1 Signaling Pathways Associated with Rho Activation in Microvascular Brain Endothelial Cells
Authors: Etienne Sandrine , Adamson Peter , Greenwood John , Strosberg A. Donny , Cazaubon Sylvie , Couraud Pierre-Olivier
Published: December 2022

Endothelium of the cerebral blood vessels, which constitutes the blood-brain barrier, controls leukocyte adhesion and trafficking to the brain. Investigating signaling pathways triggered by the engagement of adhesion molecules expressed on brain endothelial cells, we report here that ICAM-1 cross-linking induces tyrosine phosphorylation of three cytoskeleton-associated proteins: focal adhesion kinase, paxillin, and p130Cas (Cas), which are found to associate as complexes. Tyrosine-phosphorylated Cas associates with the adaptor protein Crk and the GTP exchange factor C3G. In the same conditions the small G protein Rho was activated, as shown by the increase in its GTP loading. In addition, tyrosine phosphorylation of focal adhesion kinase, paxillin, and Cas as well as triggering of the Crk signaling pathway are blocked by pretreatment of the cells with the exoenzyme C3, a specific Rho inhibitor. C3-sensitive activation of the c-Jun N-terminal kinase in response to ICAM-1 cross-linking is also observed, whereas no significant activation of Ras or of the extracellular signal-regulated kinase was detected. In conclusion, these results suggest that through coupling to Rho activation and phosphorylation of cytoskeletal proteins and transcription factors, ICAM-1 cross-linking participates in the cell shape changes and gene regulation that may accompany lymphocyte migration through the blood-brain barrier.
Monocytes from Wiskott-Aldrich Patients Display Reduced Chemotaxis and Lack of Cell Polarization in Response to Monocyte Chemoattractant Protein-1 and Formyl-Methionyl-Leucyl-Phenylalanine
Authors: Badolato Raffaele , Sozzani Silvano , Malacarne Fabio , Bresciani Susanna , Fiorini Maurilia , Borsatti Alessandro , Albertini Alberto , Mantovani Alberto , Ugazio Alberto G. , Notarangelo Luigi D.
Published: December 2022

Wiskott-Aldrich syndrome (WAS) is an X-linked disorder characterized by trombocytopenia, eczema, and progressive decline of the immune function. In addition, lymphocytes and platelets from WAS patients have morphologic abnormalities. Since chemokines may induce morphologic changes and migration of leukocytes, we investigated the monocyte response to chemoattractants in cells from WAS patients with an identified mutation in the WAS protein gene. Here, we report that monocytes derived from four patients with molecularly defined typical WAS have a severely impaired migration in response to FMLP and to the chemokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1α compared with normal donors. Conversely, neither MCP-1 binding to monocytes nor induction of the respiratory burst by MCP-1 and FMLP is significantly different between WAS patients and normal donors. Within a few minutes of stimulation, monocytes respond to chemokines with increased expression of adhesion molecules and with morphologic changes such as cell polarization. Although up-regulation of CD11b/CD18 expression following stimulation with FMLP or MCP-1 is preserved in WAS patients, cell polarization is dramatically decreased. Staining of F-actin by FITC-phalloidin in monocytes stimulated with chemoattractants shows F-actin to have a rounded shape in WAS patients, as opposed to the polymorphic distribution of F-actin in the polarized monocytes from healthy donors. These results suggest that WAS protein is involved in the monocyte response to the chemokines MCP-1 and macrophage inflammatory protein-1α.
Immunity to T Cell Receptor Peptides in Multiple Sclerosis. III. Preferential Immunogenicity of Complementarity-Determining Region 2 Peptides from Disease-Associated T Cell Receptor BV Genes*
Authors: Bourdette Dennis N. , Chou Yuan K. , Whitham Ruth H. , Buckner Jane , Kwon Hi Jong , Nepom Gerald T. , Buenafe Abigail , Cooper Shelley A. , Allegretta Mark , Hashim George A. , Offner Halina , Vandenbark Arthur A.
Published: December 2022

Vaccination with synthetic TCR peptides from the BV5S2 complementarity-determining region 2 (CDR2) can boost significantly the frequency of circulating CD4+ peptide-specific Th2 cells in multiple sclerosis (MS) patients, with an associated decrease in the frequency of myelin basic protein (MBP)-reactive Th1 cells and possible clinical benefit. To evaluate the immunogenicity of CDR2 vs other regions of the TCR, we vaccinated seven MS patients with overlapping BV5S2 peptides spanning amino acids 1–94. Six patients responded to at least one of three overlapping or substituted CDR2 peptides possessing a core epitope of residues 44–52, and one patient also responded to a CDR1 peptide. Of the CDR2 peptides, the substituted (Y49T)BV5S2-38–58 peptide was the most immunogenic but cross-reacted with the native sequence and had the strongest binding affinity for MS-associated HLA-DR2 alleles, suggesting that position 49 is an MHC rather than a TCR contact residue. Two MS patients who did not respond to BV5S2 peptides were immunized successfully with CDR2 peptides from different BV gene families overexpressed by their MBP-specific T cells. Taken together, these results suggest that a widely active vaccine for MS might well involve a limited set of slightly modified CDR2 peptides from BV genes involved in T cell recognition of MBP.
Cutting Edge: The Chemokine Receptor CCR8 Is Preferentially Expressed in Th2 But Not Th1 Cells
Authors: Zingoni Alessandra , Soto Hortensia , Hedrick Joseph A. , Stoppacciaro Antonella , Storlazzi Clelia T. , Sinigaglia Francesco , D’Ambrosio Daniele , O’Garra Anne , Robinson Douglas , Rocchi Mariano , Santoni Angela , Zlotnik Albert , Napolitano Monica
Published: December 2022

In this paper we report on the cloning and characterization of mouse CCR8. Like its human homologue, it is predominantly expressed in the thymus. In the periphery, murine CCR8 mRNA was found most abundantly expressed in activated Th2-polarized cells and in NK1.1+CD4+ T cells. Human CCR8 is also preferentially expressed in human Th2-polarized cells and clones. This pattern of expression suggests that CCR8 is part of a Th2-specific gene expression program. The CCR8 ligands I-309 and its mouse homologue T cell activation gene 3 (TCA-3) are potent chemoattractants for Th2-polarized cells. Taken together, these observations strongly suggest that CCR8 plays a role in the control of Th2 responses, and may represent a potential target for treatment of allergic diseases.
Loss of Endogenous Mouse Mammary Tumor Virus Superantigen Increases Tumor Resistance
Authors: Schirrmacher Volker , Beutner Ulrich , Bucur Marianna , Umansky Victor , Rocha Marian , von Hoegen Paul
Published: December 2022

From a cross between a tumor-susceptible mouse strain (DBA/2; D) and a tumor-resistant MHC-identical strain (B10.D2; D2) new recombinant inbred mouse strains were established over many generations of inbreeding and tumor resistance selection. Since resistance to the highly metastatic DBA/2 lymphoma variant ESb had an immunologic basis, and the two parental strains differed in endogenous viral superantigens (vSAGs), DNA of three D2×D recombinant inbred mouse lines was typed for endogenous mouse mammary tumor viruses using mouse mammary tumor virus long terminal repeat- and env gene-specific probes. The resistant D2×D mice were very similar to the susceptible parental strain D in their Mtv Southern blots, except for the lack of a single band corresponding to Mtv-7, the provirus coding for the strong DBA/2 superantigen Mls-1a. A backcross analysis revealed that Mtv-7-negative F2 mice were significantly more resistant than Mtv-7-positive F2 mice. When Mtv-7 was reintroduced into the resistant lines by crossing them with either CBA/J or BALB/D2.Mls-1a, the mice became again more tumor susceptible. Finally, we demonstrate the ability to transfer immunoresistance and graft-vs-leukemia reactivity from tumor-resistant to tumor-susceptible mice.
Relationship Between Peptide Selectivities of Human Transporters Associated with Antigen Processing and HLA Class I Molecules
Authors: Daniel Soizic , Brusic Vladimir , Caillat-Zucman Sophie , Petrovsky Nicolai , Harrison Leonard , Riganelli Daniela , Sinigaglia Francesco , Gallazzi Fabio , Hammer Jürgen , van Endert Peter M.
Published: December 2022

Efficiency of presentation of a peptide epitope by a MHC class I molecule depends on two parameters: its binding to the MHC molecule and its generation by intracellular Ag processing. In contrast to the former parameter, the mechanisms underlying peptide selection in Ag processing are poorly understood. Peptide translocation by the TAP transporter is required for presentation of most epitopes and may modulate peptide supply to MHC class I molecules. To study the role of human TAP for peptide presentation by individual HLA class I molecules, we generated artificial neural networks capable of predicting the affinity of TAP for random sequence 9-mer peptides. Using neural network-based predictions of TAP affinity, we found that peptides eluted from three different HLA class I molecules had higher TAP affinities than control peptides with equal binding affinities for the same HLA class I molecules, suggesting that human TAP may contribute to epitope selection. In simulated TAP binding experiments with 408 HLA class I binding peptides, HLA class I molecules differed significantly with respect to TAP affinities of their ligands. As a result, some class I molecules, especially HLA-B27, may be particularly efficient in presentation of cytosolic peptides with low concentrations, while most class I molecules may predominantly present abundant cytosolic peptides.
The Role of IL-7 in Thymic and Extrathymic Development of TCRγδ Cells
Authors: Laky K. , Lefrançois L. , von Freeden-Jeffry U. , Murray R. , Puddington L.
Published: December 2022

IL-7-deficient (IL-7−/−) mice have reduced numbers of B and TCRαβ cells, but lack mature TCRγδ cells. Although most T cell development occurs in the thymus, some intestinal intraepithelial lymphocytes (IEL), including TCRγδ cells, can develop extrathymically. Epithelial cells in both thymus and intestine synthesize IL-7, suggesting that TCRγδ cell development could occur in either site. To evaluate the role of thymic IL-7 in development of TCRγδ cells, newborn TCRβ-deficient (TCRβ−/−) thymi were grafted to IL-7−/− mice. Donor- and host-derived TCRγδ cells were recovered from thymus grafts, spleen, and IEL. However, when IL-7−/− thymi were grafted to TCRβ−/− mice, no development of graft-derived TCRγδ cells occurred, indicating that extrathymic IL-7 did not support TCRγδ IEL generation from newborn thymic precursors. In contrast, TCRγδ IEL development occurred efficiently in adult, thymectomized, irradiated C57BL/6J mice reconstituted with IL-7−/− bone marrow. This demonstrated that extrathymic development of TCRγδ IEL required extrathymic IL-7 production. Thus, intrathymic IL-7 was required for development of thymic TCRγδ cells, while peripheral IL-7 was sufficient for development of extrathymic TCRγδ IEL.
Identification of CD68+lin− Peripheral Blood Cells with Dendritic Precursor Characteristics
Authors: Strobl Herbert , Scheinecker Clemens , Riedl Elisabeth , Csmarits Bettina , Bello-Fernandez Concha , Pickl Winfried F. , Majdic Otto , Knapp Walter
Published: December 2022

Expression of CD68 (macrosialin) in the absence of surface and lysosomal lineage marker molecules is a characteristic feature of T zone-associated plasmacytoid monocytes, which were recently shown to represent precursors of dendritic cells (DC). We demonstrate here a minor population of strongly CD68-positive (CD68bright) blood cells that lack all analyzed myeloid surface (CD14−, CD33−, CD13−, CD11b−, CD11c−) and lysosomal (myeloperoxidase, MPO− and lysozyme, LZ−) marker molecules (0.4 ± 2% of the total mononuclear cells). These CD68bright, lineage marker-negative (lin−) cells can be induced to proliferate in the presence of IL-3. They do not acquire myeloid features even upon stimulation with granulocyte-macrophage CSF plus IL-1, IL-3, and IL-6. Instead, these cells develop typical DC characteristics upon culture. Furthermore, these CD68brightlin− DC precursors acquire mature DC characteristics (CD86+, CD83+, CD54bright) upon stimulation with CD40 ligand plus IL-3. A second subset of DC precursor-like blood cells was found to weakly express CD68 (0.3 ± 0.2% of the total mononuclear cells) and to coexpress several myeloid lineage associated molecules (LZ+, CD11c+, CD33+, CD13+). Cells of this second subset resemble both previously described myeloid-related peripheral blood DC and germinal center DC. Analysis of peripheral blood leukocytes for CD68 thus revealed the existence of two cell subsets that phenotypically resemble lymphoid tissue-associated DC. The unique phenotype CD68brightlin− is highly reminiscent of T zone-associated plasmacytoid monocytes. CD68brightlin− blood leukocytes also functionally resemble plasmacytoid monocytes. The lack of all analyzed myeloid features by CD68brightlin− blood leukocytes suggests that these cells arise from a novel nonmyeloid human DC differentiation pathway.
Anti-MUC1 Antibodies React Directly with MUC1 Peptides Presented by Class I H2 and HLA Molecules
Authors: Apostolopoulos Vasso , Chelvanayagam Gareth , Xing Pei-Xiang , McKenzie Ian F. C.
Published: December 2022

Peptides bound in the groove of MHC class I molecules and detected by CTLs are not normally accessible to Ab. We now report that MUC1 peptides that are bound within the groove of MHC class I molecules (H2 and HLA) and that can be detected by CTLs can also be detected by anti-MUC1 Abs. mAbs to the middle and C-terminal regions of the class I-associated peptides but not to the N terminus were able to react with MUC1 peptides bound to H2Kb and HLA-A*0201, and only to the mid-region for H2Db, by flow cytometry and also to block CTL activity. Molecular modeling showed that the N terminus is buried (and not accessible), whereas the midpeptide residues form a loop and the C terminus is free, making these two regions accessible to Ab. The findings demonstrate for the first time that peptides associated with class I molecules can be detected by anti-peptide Abs.
HLA Class II Expression in Uninducible Hepatocarcinoma Cells After Transfection ofAIR-1Gene Product CIITA: Acquisition of Antigen Processing and Presentation Capacity
Authors: Sartoris Silvia , Valle Maria Teresa , Barbaro Andrea De Lerma , Tosi Giovanna , Cestari Tiziana , D’Agostino Antonella , Megiovanni Anna Maria , Manca Fabrizio , Accolla2 Roberto S.
Published: December 2022

The AIR-1-encoded CIITA transcriptional activator is crucial for both constitutive and IFN-γ-induced MHC class II gene transcription. We show here that the MHC class II negative phenotype of the human hepatocarcinoma cell lines Alexander and HepG2 remains unmodified after treatment with IFN-γ, although MHC class I expression is up-modulated. This correlates with absence of CIITA mature transcripts. Transfection of an expressible CIITA cDNA in Alexander cells resulted in a very high cell surface expression of all three human class II subsets, HLA-DR, -DP and -DQ, indicating that normally observed induction of CIITA expression by IFN-γ is probably blocked, in the hepatocarcinoma cell lines, at the level of CIITA transcription and not at the level of IFN-γ receptor binding and signal transduction mechanisms. To assess whether MHC class II expression on CIITA-transfected Alexander cells could have functional relevance, we tested their capacity to present antigenic peptides to an HLA-DR-restricted T cell line specific for a peptide of Mycobacterium tuberculosis Ag85 protein. It was found that the transfected cells could not only present the exogenously supplemented peptide but also process Ag85 protein to generate the specific epitope recognized by the HLA-DR-restricted T cell line. Similar results were obtained with CIITA-transfected CFPAC-1 pancreatic adenocarcinoma cells, which differed from Alexander cells in that they were inducible by IFN-γ. These results suggest new strategies to act on CIITA for increasing the potential of a tumor cell to present putative tumor Ags to the immune system.
Mapping Quantitative Trait Loci for Immune Capacity in the Pig
Authors: Edfors-Lilja Inger , Wattrang Eva , Marklund Lena , Moller Maria , Andersson-Eklund Lena , Andersson Leif , Fossum Caroline
Published: December 2022

Immune capacity traits show considerable genetic variation in outbred populations. To identify quantitative trait loci (QTLs) for immune capacity in the pig, various measures of immune function (total and differential leukocyte counts, neutrophil phagocytosis, mitogen-induced proliferation, IL-2 production, and virus induced IFN-α production in whole blood cultures, and Ab responses to two Escherichia coli antigens) were determined in 200 F2 animals from a wild pig–Swedish Yorkshire intercross. The pedigree has been typed for 236 genetic markers covering all autosomes, the X chromosome and the X/Y pseudoautosomal region. Through interval mapping using a least-squares method, four QTLs with significant effects were identified; one for total leukocyte counts, one for mitogen-induced proliferation, one for prevaccination levels of Abs to E. coli Ag K88, and one for Ab response to the O149 Ag. In addition, several putative QTLs were indicated. The results from the present study conclusively show that it is possible to identify QTLs for immune capacity traits in outbred pig populations by genome analysis.
Molecular Regulation of the Interaction Between Leukocyte Function-Associated Antigen-1 and Soluble ICAM-1 by Divalent Metal Cations
Authors: Labadia Mark E. , Jeanfavre Deborah Durham , Caviness Gary O. , Morelock Maurice M.
Published: December 2022

Surface plasmon resonance (SPR) was used to investigate and characterize the interaction between LFA-1 and sICAM-1 (a soluble form of ICAM-1). Full-length LFA-1 was immobilized on a hydrophobic surface, and sICAM-1 binding was monitored in a flow cell format. The binding of sICAM-1 to LFA-1 was specific and dependent upon Mg2+; Abs to both sICAM-1 and LFA-1 blocked the interaction, and EDTA abolished all binding. Association and dissociation rate constants (ka and kd, respectively) for sICAM-1 were 2.24 × 105 M−1 s−1 and 2.98 × 10−2 s−1, respectively, giving a calculated KICAM of 133 nM. Since the LFA-1/ICAM-1 interaction is highly sensitive to the presence of metal cations, SPR was also used to probe the affinity of the metal binding sites. The KMg values were 160 and 12 μM in the absence (EGTA) and the presence of Ca2+ (100 μM), respectively; in addition, KMn was 2 μM in the presence of Ca2+ (100 μM). Increasing Ca2+ into the millimolar concentration range, however, resulted in a competitive displacement of Mg2+/Mn2+ and decreased sICAM-1 binding. Based on these data, a synergistic model for the molecular regulation of LFA-1 by divalent metal cations is proposed, and implications to cellular adhesion are discussed.
Human Dendritic Cell (DC)-Based Anti-Infective Therapy: Engineering DCs to Secrete Functional IFN-γ and IL-12
Authors: Ahuja Seema S. , Mummidi Srinivas , Malech Harry L. , Ahuja Sunil K.
Published: December 2022

An imbalance in the Th1- and Th2-type cytokine responses may allow certain microbes to modify the host response to favor their own persistence. We now show that infection/pulsing of human CD34+ peripheral blood hemopoietic progenitor cell-derived dendritic cells (DCs) with Leishmania donovani promastigotes, Histoplasma capsulatum, and Mycobacterium kansasii impairs the constitutive production of IL-12 from these cells. Thus, strategies aimed at modulating a dysregulated Th1/Th2 response to infection would be of great interest. To both augment the host immune response and deliver potent immunomodulatory cytokines such as IL-12 and IFN-γ, our goal is to develop a therapeutic strategy using genetically modified, microbial Ag-pulsed DCs. Toward developing such immunotherapies, we used retrovirus-mediated somatic gene transfer techniques to engineer human DCs to secrete biologically active IL-12 and IFN-γ. DCs pulsed with microbial antigens (e.g., leishmania and histoplasma Ags) were capable of inducing proliferative responses in autologous CD4+ lymphocytes. CD4+ lymphocytes cocultured with IL-12-transduced autologous DCs had enhanced Ag-specific proliferative responses compared with CD4+ lymphocytes cocultured with nontransduced or IFN-γ- transduced DCs. In this cell culture model system we demonstrate that IL-12 has a negative effect on IL-4 secretion that is independent of its ability to induce IFN-γ secretion. Taken together, these results indicate that IL-12-transduced DCs may be specifically suited in inducing or down-modulating Ag-specific Th1 or Th2 responses, respectively, and thus may be useful as adjunctive therapy in those intracellular infections in which a dominant Th1 response is critical for the resolution of infection.
T Cell-Mediated, IFN-γ-Facilitated Rejection of Murine B16 Melanomas
Authors: Böhm Waltraud , Thoma Stefan , Leithäuser Frank , Möller Peter , Schirmbeck Reinhold , Reimann Jörg
Published: December 2022

The murine melanoma cell line B16.F10 (H-2b) was used to study specific T cell responses that reject tumors. Stable B16 transfectants were established that express viral Ags, either the hepatitis B surface Ag (HBsAg) or the large tumor Ag (T-Ag) of SV40. B16 cells and their transfected sublines were CD40+CD44+ but expressed no (or low levels of the) costimulator molecules CD154 (CD40L), CD48, CD54, CD80, and CD86. Surface expression of MHC class I (Kb, Db) and class II (I-Ab) molecules by B16 cells was low, but strikingly up-regulated by IFN-γ. CD95 (Fas) and CD95 ligand (CD95L (FasL)) were “spontaneously” expressed by B16 cells growing in vitro in serum-free medium; these markers were strikingly up-regulated by IFN-γ. B16 cells coexpressing CD95 and CD95L were irreversibly programed for apoptosis. In vitro, noninduced B16 transfectants stimulated a specific IFN-γ release response, but no cytolytic response (in a 4-h assay) in MHC class I-restricted CTL; in contrast, IFN-γ-induced B16 targets were efficiently and specifically lysed by CTL. In vivo, B16 transfectants were specifically rejected by DNA-vaccinated syngeneic hosts through a T-dependent immune effector mechanism. The tumors showed evidence of massive apoptosis in vivo during the rejection process. The data suggest that CTL-derived IFN-γ enhances an intrinsic suicide mechanism of these tumor cells in addition to facilitating lytic interactions of effectors with tumor targets.
Secondary Lymphoid-Tissue Chemokine (SLC) Stimulates Integrin α4β7-Mediated Adhesion of Lymphocytes to Mucosal Addressin Cell Adhesion Molecule-1 (MAdCAM-1) Under Flow
Authors: Pachynski Russell K. , Wu Steve W. , Gunn Michael D. , Erle David J.
Published: December 2022

The attachment of leukocytes to the endothelium is a multistep process that depends upon a very rapid increase in the adhesive activity of leukocyte integrins. A pertussis toxin-sensitive pathway stimulates integrin-dependent lymphocyte adhesion to Peyer’s patch high endothelial venules in vivo, but the factors responsible for activating this pathway have not been identified previously. We now report that secondary lymphoid-tissue chemokine (SLC) (also known as 6Ckine, Exodus-2, and thymus-derived chemotactic agent 4), a recently described CC chemokine that is expressed in Peyer’s patches and lymph nodes, rapidly activates integrin-mediated lymphocyte adhesion. Immobilized SLC increased the adhesion of HUT-78 T cells and human PBLs to mucosal addressin cell adhesion molecule-1, a protein that is expressed on Peyer’s patch and mesenteric lymph node high endothelial venules. This effect of SLC was seen in both static and flow chamber adhesion assays, was mediated by integrin α4β7, and was inhibited by pertussis toxin. The other CC chemokines tested did not increase adhesion to mucosal addressin cell adhesion molecule-1. SLC had a greater effect on naive CD4+ T cells than on memory CD4+ T cells; CD8+ T cells, B cells, and NK cells were also responsive to SLC. SLC is likely to play an important role in regulating the recruitment of lymphocytes to Peyer’s patches and lymph nodes.
Cutting Edge: Curative Treatment of an Experimentally Induced Colitis by a CD44 Variant V7-Specific Antibody
Authors: Wittig Bianca , Schwärzler Christoph , Föhr Nicole , Günthert Ursula , Zöller Margot
Published: December 2022

Inflammatory bowel disease is a quite severe chronic inflammation, treated mainly by immunosuppression, which often has serious side effects. As CD44 is important in lymphocyte activation and migration, we asked whether Abs against CD44 isoforms influence trinitrobenzenesulfonic acid (TNBS)-induced colitis in mice. A lethal colitis (73/111 mice) could be prevented in 69 of 97 mice by anti-CD44v7 (CD44 variant isoform v7), whereas anti-CD44s (CD44 standard isoform) and anti-CD44v6 had no effect. Upon receiving anti-CD44v7 after the disease had been fully exacerbated, >90% of the mice recovered. TNBS plus anti-CD44v7-treated mice developed early signs of inflammation, with infiltration of leukocytes in the lamina propria and increased IFN-γ production. However, while control mice developed a severe pancolitis, the intestine fully regenerated in anti-CD44v7-treated mice. Locally and systemically, a strong increase in IL-10 production was noted. Thus, anti-CD44v7 can be regarded as a highly efficient and specific therapeutic reagent in chronic colitis, which probably functions by regulating an overshooting Th1 reaction.
Cutting Edge: Differential Regulation of Chemokine Receptors During Dendritic Cell Maturation: A Model for Their Trafficking Properties
Authors: Sozzani Silvano , Allavena Paola , D’Amico Giovanna , Luini Walter , Bianchi Giancarlo , Kataura Motoji , Imai Toshio , Yoshie Osamu , Bonecchi Raffaella , Mantovani Alberto
Published: December 2022

Upon exposure to immune or inflammatory stimuli, dendritic cells (DC) migrate from peripheral tissues to lymphoid organs, where they present Ag. CC chemokines induce chemotactic and transendothelial migration of immature DC, in vitro. Maturation of DC by CD40L, or by LPS, IL-1, and TNF, induces down-regulation of the two main CC chemokine receptors expressed by these cells, CCR1 and CCR5, and abrogates chemotaxis to their ligands. Inhibition was rapid (<1 h) and included the unrelated agent FMLP. Concomitantly, the expression of CCR7 and the migration to its ligand EBI1 ligand chemokine (ELC)/macrophage inflammatory protein (MIP)-3β, a chemokine expressed in lymphoid organs, were strongly up-regulated, though with slower kinetics (24–48 h). Rapid inhibition of responsiveness to chemoattractants present at sites of inflammation and immune reaction may be permissive for leaving peripheral tissues. Conversely, the slower acquisition of responsiveness to ELC/MIP-3β may guide subsequent localization of DC in lymphoid organs.
Dendritic Cell Heterogeneity In Vivo: Two Functionally Different Dendritic Cell Populations in Rat Intestinal Lymph Can Be Distinguished by CD4 Expression
Authors: Liu LiMing , Zhang ManHua , Jenkins Chris , MacPherson G. Gordon
Published: December 2022

DC derived from rat pseudo-afferent lymph (L-DC) vary in CD4, CD11b/c, Thy1, and OX41 expression. CD4 and OX41 are expressed by the same subpopulation (50–60%) of L-DC. CD4+/OX41+ L-DC express short fine processes and low nonspecific esterase, whereas CD4− DC/OX41− express long pseudopodia, high nonspecific esterase, and many cytoplasmic inclusions. These differences are stable in culture. Both populations express similar amounts of MHC class II, ICAM-1, CD11b/c and OX62. Most CD4−/OX41− L-DC are strongly positive for B7, but CD4+ L-DC express less B7, and some may be negative. Both populations express invariant chain, but both the absolute numbers and levels of expression were higher for CD4− DC. Surprisingly, CD4+ L-DC are more potent APC than CD4− cells in MLRs, for sensitized T cells in vitro and for naive T cells in vivo. Cultured CD4+/OX41+ DC can still process and present native Ag. Cultured CD4−/OX41− cells cannot present native Ag but can stimulate strong MLRs. CD4− DC invariant chain expression decreases in culture, whereas expression by CD4+ DC is stable for 48 h. CD4+ and CD4− L-DC have similar turnover times in vivo, suggesting that one population is not the precursor of the other. Thus, two separate DC populations that differ functionally and phenotypically migrate from intestine to mesenteric nodes. This may reflect distinct DC lineages or differentiation modulated by different microenvironmental stimuli.
Down-Modulation of a Novel Bax-Associated Protein During Apoptosis in Normal Mature B Lymphocytes
Authors: He Huiling , Hershberger Pamela A. , McCarthy Susan A.
Published: December 2022

We have recently characterized a novel 16-kDa Bax-associated protein. In this study, we investigate the regulation of this protein’s expression during in vitro induction of apoptosis in mature splenic B cells. A panel of biochemically distinct apoptotic stimuli induced the dramatic down-modulation of the 16-kDa protein in B cells; this down-modulation was rapid, and did not require DNA fragmentation. Reciprocally, stimuli that induced protection from apoptosis prevented down-modulation of the 16-kDa protein. These regulatory effects were specific, since Bcl-2 and Bax protein levels were not similarly modulated. Stimuli that reduce expression of the 16-kDa protein may therefore act indirectly to increase the proapoptotic activity of Bax, perhaps by altering Bax binding to other cellular proteins.
Induction of Acute Inflammation In Vivo by Staphylococcal Superantigens. II. Critical Role for Chemokines, ICAM-1, and TNF-α
Authors: Tessier Philippe A. , Naccache Paul H. , Diener Kerrilyn R. , Gladue Ronald P. , Neote Kuldeep S. , Clark-Lewis Ian , McColl Shaun R.
Published: December 2022

Superantigens such as staphylococcal enterotoxin A and B (SEA and SEB) activate the immune system by stimulating a large proportion of T lymphocytes through specific Vβ regions of the TCR and activating macrophages by binding to MHC class II molecules. While the mechanisms by which superantigens activate T lymphocytes have been elucidated, their role in the generation of local immune responses to bacterial invasion is still unclear. In this study we have examined the ability of the superantigens SEA and SEB to elicit an inflammatory reaction in vivo, in s.c. air pouches in the mouse. Upon injection into the s.c. air pouch, the two superantigens stimulated a time-dependent increase in the number of leukocytes appearing in the pouch exudate. The leukocytes migrating into the pouch exudate were predominantly neutrophils, with some mononuclear phagocytes and eosinophils present. No T lymphocytes were detected either in the pouch lining tissue or in the exudate cells. Injection of SEA resulted in increased ICAM-1 expression, as detected by immunohistochemistry, on endothelial cells in the tissue surrounding the air pouch and accumulation of TNF-α and the chemokines macrophage inflammatory protein-2 (MIP-2), MIP-1α, and JE in the pouch exudate. In addition, pretreatment of mice with Abs raised against ICAM-1, TNF-α, MIP-2, MIP-1α, KC, or JE inhibited leukocyte accumulation induced by SEA. These data demonstrate that bacterial superantigens may promote inflammation at extravascular sites in vivo, and that this response is secondary to the generation of inflammatory mediators, including chemokines.
IL-4-Producing NK1.1+ T Cells Are Resistant to Glucocorticoid-Induced Apoptosis: Implications for the Th1/Th2 Balance
Authors: Tamada Koji , Harada Mamoru , Abe Koichiro , Li Tieli , Nomoto Kikuo
Published: December 2022

To elucidate the mechanisms by which glucocorticoids promote Th2-type responses, we investigated the influence of dexamethasone (DEX) on both cytokine production and viability of NK1.1+ T cells. The in vivo administration of DEX enhanced the IL-4 production of spleen cells and liver mononuclear cells in wild-type mice, but not in β2m-deficient mice. DEX reduced the cellularity of conventional T cells, but not that of NK1.1+ T cells, in both spleen and liver, suggesting an increased proportion of NK1.1+ T cells. Moreover, the proportion of IL-4-producing NK1.1+ T cells increased in the DEX-injected mice. These results suggest that DEX induced IL-4 production through the preferential survival of IL-4-producing NK1.1+ T cells. In investigating the reason for the preferential survival of NK1.1+ T cells, we found that NK1.1+ T cells were resistant to DEX-induced apoptosis and expressed a higher level of intracellular Bcl-2 compared with conventional NK1.1− T cells. In addition, splenic and hepatic NK1.1+ T cells were resistant to radiation-induced apoptosis. Collectively, our findings revealed an important role for NK1.1+ T cells in the regulation of Th1/Th2 balance by glucocorticoids and their possible functions under various apoptotic stimuli.