bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Eotaxin Induces α4 and β2 Integrin-Dependent Eosinophil Accumulation in Rat Skin In Vivo: Delayed Generation of Eotaxin in Response to IL-4
Authors: Sanz Maria-Jesus , Ponath Paul D. , Mackay Charles R. , Newman Walter , Miyasaka Masayuki , Tamatani Tayuka , Flanagan Brian F. , Lobb Roy R. , Williams Timothy J. , Nourshargh Sussan , Jose Peter J.
Published: December 2022

The CC chemokine eotaxin, originally purified from bronchoalveolar lavage fluid of sensitized guinea pigs following allergen challenge, is a potent eosinophil-selective chemoattractant. In the present study, we have used 111In-eosinophils and human eotaxin to characterize the profile of chemokine-induced eosinophil accumulation in vivo in rat skin. Intradermally injected eotaxin caused a dose-dependent accumulation of 111In-eosinophils. Time course studies indicated that the response was rapid, since all the accumulation occurred within the first 1 to 2 h of eotaxin injection. The i.v. administration of anti-intercellular adhesion molecule-1, anti-vascular cell adhesion molecule-1, or anti-α4 integrin mAbs significantly inhibited the eosinophil accumulation induced by 100 pmol of human eotaxin by 73, 43, and 67%, respectively. Further, when 111In-eosinophils were pretreated in vitro with anti-α4 integrin or anti-β2 integrin mAbs, or with a combination of both mAbs, eotaxin-induced responses in vivo were reduced by 52, 49, and 68%, respectively. Eosinophil accumulation induced by intradermal IL-4, but not that induced by TNF-α or leukotriene B4, appeared to be mediated in part by endogenously generated eotaxin. Anti-eotaxin Abs significantly inhibited (54%) the later phases (24–28 h) but not the early phase (0–4 h) of the response to IL-4. This was consistent with eotaxin mRNA expression peaking at 18 h after IL-4 injection. Our findings show that human eotaxin is a potent inducer of eosinophil accumulation in vivo, this response being dependent on α4 integrin/vascular cell adhesion molecule-1 and β2 integrin/intercellular adhesion molecule-1 adhesion pathways. Further, the eosinophil accumulation in response to IL-4 is partly mediated by endogenously generated eotaxin.
Cardiac Allografts from IL-4 Knockout Recipients: Assessment of Transplant Arteriosclerosis and Peripheral Tolerance
Authors: Mottram Patricia L. , Räisänen-Sokolowski Anne , Glysing-Jensen Troels , Stein-Oakley Alicia N. , Russell2 Mary E.
Published: December 2022

To study the role of IL-4 in tolerance induction and transplant arteriosclerosis, BALB/c hearts were transplanted into C57BL/6J wild-type or IL-4 knockout (IL-4−/−) recipients. A 30-day course of anti-CD4/8 mAb was used to induce long term graft survival. Primary graft survival was 50% (5 of 10) in IL-4−/− recipients comparable to 63% (5 of 8) in wild-type recipients. Mice with allografts surviving >80 days were tested for tolerance by challenge with a second donor or third party (CBA) heart. Secondary donor-strain heart grafts survived >30 days, but showed histologic evidence of ongoing alloimmune response. Third party hearts rejected rapidly. Although immunostaining and 32P RT-PCR assays showed no differences in the mononuclear cell infiltration and T cell activation between IL-4−/− and wild-type tolerant recipients, some monokines (IL-12, TNF-α, and allograft inflammatory factor-1) were up-regulated in grafts from IL-4−/− recipients. Computer-assisted analysis of elastin-stained vessels revealed that the severity of vascular thickening (percentage of luminal occlusion, mean ± SD, n = 329) was similar in grafts from IL-4−/− (63.7 ± 16.9%) and wild-type (69.5 ± 17.6%) recipients. Thus, IL-4 deficiency did not alter primary or secondary graft survival, infiltration, or vascular thickening. The selective alterations in monokine expression suggests that alternative pathways are activated and may compensate in IL-4−/− mice.
Autoreactivity of Human VH Domains from cDNA Libraries: Analysis with a Bacterial Expression System
Authors: Lecerf Jean-Michel , Chen Yan , Richalet-Sécordel Pascale , Wang Xiaowei , Stollar B. David
Published: December 2022

Previous studies showed that VH domains of several anti-DNA Abs can bind DNA in the absence of VL. In the current work, we tested the VH autoreactive potential more generally, examining VH domains that did not come from known autoantibodies. Using a bacterial expression system, we produced 11 fusion proteins, each containing a VH domain and a B domain of staphylococcal protein A. The VH domains were coded in cDNA libraries from circulating B cells of healthy young adult humans. Thus, binding properties of the Ig molecules from which they came were unknown. The B cells had not been stimulated in vitro. Seven cDNA clones combined the frequently expressed VH3-23 gene segment with varied DH and JH segments. The other clones contained unmutated VH3-7, VH3-9, VH3-53, and VH4-39 segments. We compared these bacterial expression products with single-chain Fv, VH and VL domains of IgM mAb 18/2, a VH3-23-encoded, DNA-binding autoantibody. Submicromolar concentrations of 5 of the 11 VH domains bound to ssDNA. Those and one more also bound to immobilized poly(dT), and two bound to circular plasmid dsDNA. Soluble poly(dT) was the most potent inhibitor in competitive ELISA. Seven of the VH domains also bound to immobilized nuclear ribonucleoprotein, four to histone and none to thyroglobulin. Two interacted with the matrix of a Sephacryl S-100 column. The polyreactive autoantigen-binding properties of these VH domains raise the question of whether these properties may play a role in the formation of the VH repertoire of circulating B cells.
Efficient Recombination of a Switch Substrate Retrovector in CD40-Activated B Lymphocytes: Implications for the Control of CH Gene Switch Recombination
Authors: Ballantyne Jack , Henry Diane L. , Muller Jurgen R. , Briere Francine , Snapper Clifford M. , Kehry Marilyn , Marcu Kenneth B.
Published: December 2022

Maturing B lymphocytes possess a recombination activity that switches the class of heavy chain Ig. The nature of the recombination activity, its molecular requirements and regulation remain elusive questions about B lymphocyte biology and development. Class switch recombination is controlled by cytokine response elements that are required to differentially activate CH gene transcription before their subsequent recombination. Here, we show that cultures of purified murine and human B cells, stimulated only by CD40 receptor engagement, possess a potent switch recombination activity. CD40 ligand-stimulated murine and human B lymphocytes were infected with recombinant retroviruses containing Sμ and Sγ2b sequences. Chromosomally integrated switch substrate retrovectors (SSRs), harboring constitutively transcribed S sequences, underwent extensive recombinations restricted to their S sequences with structural features akin to endogenous switching. SSR recombination commenced 4 days postinfection (5 days poststimulation) with extensive switch sequence recombination over the next 2 to 3 days. In contrast, endogenous Sγ2b and Sγ1 sequences did not undergo appreciable switch recombination upon CD40 signaling alone. As expected, IL-4 induced endogenous Sμ to Sγ1 switching, while endogenous Sμ to Sγ2b fusions remained undetectable. Surprisingly, IL-4 enhanced the onset of SSR recombination in CD40-stimulated murine B cells, with S-S products appearing only 2 days postinfection and reaching a maximum within 2 to 3 days. The efficiency of switch recombination with SSRs ressembles that seen for endogenous CH class switching.
Mouse MD-1, a Molecule That Is Physically Associated with RP105 and Positively Regulates Its Expression
Authors: Miyake Kensuke , Shimazu Rintaro , Kondo Jun , Niki Tamotsu , Akashi Sachiko , Ogata Hirotaka , Yamashita Yoshio , Miura Yoshihiro , Kimoto Masao
Published: December 2022

RP105 is a leucine-rich repeat molecule that is expressed on mouse B cells and transmits a growth-promoting signal. An anti-RP105 Ab precipitated additional molecules as well as RP105. These molecules were found to be a mouse homologue of chicken MD-1. Chicken MD-1 was previously isolated as a v-myb-regulated gene, since its transcription increases rapidly after v-myb induction. Mouse MD-1, when transiently expressed as an epitope-tagged protein, is secreted in culture fluid but tethered to the cell surface by coexpressed RP105. An association of these molecules was confirmed by immunoprecipitation with the anti-RP105 Ab and subsequent probing of the epitope tag on MD-1. Moreover, MD-1 has an effect on the expression of RP105. In transient transfection of RP105, the percentage of RP105-positive cells increased more than twice with the coexpression of MD-1. The stable expression of MD-1 conferred approximately a sevenfold increase in cell surface RP105 on a cell line that expresses RP105 alone. Thus, MD-1 is physically associated with RP105 and is important for efficient cell surface expression.
Cutting Edge: Identification of an Additional Isoform of STAT5 Expressed in Immature Macrophages
Authors: Lokuta Mary A. , McDowell Mary Ann , Paulnock Donna M.
Published: December 2022

We are interested in understanding the molecular basis of macrophage (Mφ) differentiation and activation by cytokines. Recent reports have suggested that the transcription factor STAT5 may play a role in Mφ differentiation. In the experiments described here, we assessed the expression of STAT5-related molecules in three Mφ cell lines, RAW 264.7, WEHI-3, and WEHI-3D+, which represent different stages of Mφ maturation, and also in primary peritoneal and bone marrow Mφ from BALB/c mice. The studies revealed that the previously characterized STAT5a and STAT5b isoforms are detectable at both the mRNA and protein levels in these Mφ populations. Additional STAT5-related proteins were detected by immunoblot analysis and were preferentially expressed in both the immature WEHI-3 cell population and the adherent bone marrow population containing immature Mφ. These results identify new isoforms of STAT5 and demonstrate that distinct patterns of expression of STAT5-related proteins are observed in Mφ at different stages of maturation.
Transcription Factor E2F Controls the Reversible γδ T Cell Growth Arrest Mediated Through WC1
Authors: Kirkham Paul A. , Lam Eric W.-F. , Takamatsu Haru-Hisa , Michael R. , Parkhouse E.
Published: December 2022

IL-2-stimulated expansion of T cells requires continued and sequential passage of the dividing cells through a major cell cycle check point in the G1 phase. We have previously shown that a γδ T cell-specific surface receptor, WC1, induces G0/G1 growth arrest, reversible with Con A, in proliferating IL-2-dependent γδ T cells. We now show that this reversible WC1-induced cell cycle arrest is correlated with induction of the cyclin kinase inhibitor p27kip1 and an associated down-regulation in cyclins A, D2, and D3 expression, along with dephosphorylation of pocket proteins p107, p130, and pRb. Together with diminished pocket protein phosphorylation, p107 expression levels are significantly down-regulated in response to WC1 stimulation. This coordinated sequence of signaling events is focused on E2F regulation so that, downstream of the pocket proteins, WC1 stimulation results in a diminished DNA binding activity for free E2F as a consequence of reduced E2F1 expression, whereas E2F4 expression is unaffected. Consistent with this interpretation, overexpression of E2F1 overcomes the growth-arresting effects induced by WC1 stimulation. Finally, in accordance with our previous observations at both the cellular and molecular level, subsequent mitogen stimulation can reverse all the above changes induced by WC1. These results, focused on E2F regulation, therefore provide a first insight into the effects of both positive (mitogen) and negative (anti-WC1) stimuli on cell cycle control in IL-2-dependent γδ T cells.
Low Dose TGF-β Attenuates IL-12 Responsiveness in Murine Th Cells
Authors: Gorham James D. , Güler Mehmet L. , Fenoglio Domenic , Gubler Ueli , Murphy Kenneth M.
Published: December 2022

Expression of IL-12Rs is one important checkpoint for Th1 development. BALB/c DO11.10 CD4+ T cells stimulated by Ag in neutral conditions lose expression of the IL-12R β2 subunit and become unresponsive to IL-12. In contrast, B10.D2 or F1 (BALB/c × B10.D2) DO11.10 CD4+ T cells maintain IL-12Rβ2 expression when stimulated similarly. Here we show that the loss of IL-12 responsiveness by BALB/c T cells involves the action of endogenous TGF-β. BALB/c T cells stimulated in the presence of anti-TGF-β specifically maintain IL-12 responsiveness, express IL-12Rβ2 mRNA, and can stimulate nitric oxide production in peritoneal exudate cells. Low concentrations of TGF-β added exogenously during primary activation of B10.D2 or F1 T cells significantly inhibit their development of IL-12 responsiveness. These effects of anti-TGF-β are dependent on endogenous IFN-γ and are inhibited by exogenously added IL-4. Thus, at least one effect of TGF-β on Th1/Th2 development may be the attenuation of IL-12Rβ2 expression.
Aspects of Antigen Mimicry Revealed by Immunization with a Peptide Mimetic of Cryptococcus neoformans Polysaccharide
Authors: Valadon Philippe , Nussbaum Gabriel , Oh Jin , Scharff Matthew D.
Published: December 2022

We have recently identified peptide mimetics of the Cryptococcus neoformans capsular polysaccharide by screening phage display peptide libraries. 2H1, one of a large family of mAbs against the glucuronoxylomannan fraction (GXM), is highly protective and binds several peptide motifs. This study analyzes the immunologic properties of P601E (SYSWMYE), a peptide from the low affinity motif (W/YXWM/LYE) that has an extended cross-reactivity among anti-GXM mAbs and whose binding correlates with the protective potential of mAbs in experimental infection. P601E is a mimetic, since it competes for GXM binding to 2H1, but not a mimotope, since it does not elicit an anti-GXM response. Sequence analysis of 14 anti-P601E mAbs indicates that anti-P601E mAbs elicited in BALB/c mice have an order of homology with 2H1 of Vκ > Jκ ≫ VH > JH > D. Further screening of a peptide library with anti-P601E mAbs isolated peptides having a motif almost identical to the peptide motif selected by 2H1. When these results are compared to the crystal structure of a related peptide in complex with 2H1, there is a clear correlation between the ability to elicit V region components of 2H1 Ab and peptide association with the V region, suggesting that the completeness of the fit in the binding site is an important driving force for mimicry. As a consequence, improving affinity of a mimetic for the Ab binding site seems to be the most logical way to insure that all of the appropriate V region segments are elicited and that useful mimotopes are created.
Type I IFNs Enhance the Terminal Differentiation of Dendritic Cells
Authors: Luft Thomas , Pang Ken C. , Thomas Elisabeth , Hertzog Paul , Hart Derek N. J. , Trapani Joseph , Cebon Jonathan
Published: December 2022

This study identifies type I IFNs as activating cytokines in a serum-free system in which human dendritic cells (DC) were generated from CD34+ progenitor cells. After 14 days of culture in GM-CSF, TNF-α, and IL-4, CD34+ progenitors gave rise to a population of large, immature DC expressing CD1a and CD11b but lacking CD14, CD80, CD83, CD86, and CMRF44. During the next 2 wk, this population spontaneously matured into nonadherent, CD1alow/−, CD11blow/−, CD14−, CD80+, CD83+, CD86+, CMRF44+ DC with high allostimulatory activity in the MLR. To examine which factors influenced this maturation, 25 different cytokines or factors were added to the immature DC culture. Only type I IFNs (α or β) accelerated this maturation in a dose-dependent manner, so that after only 3 days the majority of large cells acquired the morphology, phenotype, and function characteristics of mature DC. Furthermore, supernatants from cultures containing spontaneously maturing DC revealed low levels of endogenous IFN production. Because of the similarity of the activation of DC in our culture system with the phenotypic and functional changes observed during Langerhans cells activation and migration in vivo, we investigated the effect of IFN-α on human Langerhans cell migration. IFN-α also activated the migration of human split skin-derived DC, demonstrating that this effect was not limited to DC derived in vitro from hemopoietic progenitor cells. DC activation by type I IFNs represents a novel mechanism of immunomodulation by these cytokines, which could be important during antiviral responses and autoimmune reactions.
HLA-DQ6 and HLA-DQ8 Transgenic Mice Respond to Ragweed Allergens and Recognize a Distinct Set of Epitopes on Short and Giant Ragweed Group 5 Antigens
Authors: Chapoval Svetlana P. , Neeno Teresa , Krco Christopher J. , Marietta Eric V. , Harders Jerry , David Chella S.
Published: December 2022

We have investigated the genetic and molecular basis of immune responsiveness to short ragweed (SRW) (Ambrosia artemisiifolia) extract, and group 5 allergens from short and giant (Ambrosia trifida) ragweed using transgenic mice expressing DQ6 (HLA-DQA1*0103, HLA-DQB1*0601) and DQ8 (HLA-DQA1*0301, HLA-DQB1*0302) genes in class II knockout (Aβ0) mice. Panels of overlapping peptides spanning the Amb a 5 and Amb t 5 Ags were synthesized. Mice were immunized with whole SRW extract or individual peptides s.c. and lymph node cells (LNC) were challenged in vitro. Strong T cell responses to SRW extract were measured in both HLA-DQ transgenic mice, while control, HLA-DQ6−/DQ8−/H-2Aβ0, mice were unresponsive. IL-5 and IL-10 were the primary cytokines produced by in vitro challenged LNC of SRW-primed transgenic mice. HLA-DQ6-restricted T cell responses were detected to all three peptides of Amb t 5 and two determinants (residues 1–20 and 11–30) on Amb a 5. In contrast, LNC of HLA-DQ8 mice did not recognize peptide 11–30 of Amb t 5 Ag, but recognized several Amb a 5 determinants. The immune response in transgenic mice was dependent upon CD4+ T cells and was HLA-DQ restricted. Primed with purified Amb t 5, both transgenics recognized peptide 21–40, and an additional DQ6-restricted epitope was found within residue 1–20. SRW-immunized HLA-DQ6 mice respond to peptide 11–30 of Amb a 5, while HLA-DQ8 mice strongly recognize peptide 1–20. These results demonstrate the specificity of HLA class II polymorphism in allergen sensitivity and pave the way for developing antagonistic peptides for desensitization.
Human Endothelial Cells Effectively Costimulate Cytokine Production by, But Not Differentiation of, Naive CD4+ T Cells
Authors: Ma Weilie , Pober Jordan S.
Published: December 2022

We compared costimulatory signals provided by human endothelial cells (ECs) to those provided by conventional bone marrow-derived APCs, i.e., peripheral blood-adherent mononuclear cells (PBAMCs), by measuring their effects on cytokine production by naive or memory CD4+ T cells stimulated by PHA. In these assays, ECs effectively costimulate secretion of IL-2, IFN-γ, and IL-4 from both naive and memory CD4+ T cells, quantified by ELISA or intracellular cytokine staining. ECs, which lack B7 molecules, use predominantly leukocyte-function associated Ag 3 (LFA-3) to provide costimulation. ECs are comparable to or better than PBAMCs, which use both the LFA-3 and B7 molecules, at costimulating IL-2 and IL-4 production. ECs are less effective than PBAMCs at costimulating IFN-γ production by naive T cells. ECs do not secrete IL-12, and addition of exogenous IL-12 enables ECs to costimulate IFN-γ at a level comparable to that observed with PBAMCs. ECs do not promote differentiation of naive T cells to Th1-like cells, whereas PBAMCs do. Again, addition of exogenous IL-12 enables ECs to do so. Transfection of ECs to express B7-1 or B7-2 is less effective than IL-12 supplementation for restoring these responses. These experiments suggest that a deficiency in costimulation due to lack of B7 molecule expression does not fully explain the inability of ECs to activate resting naive CD4+ T cells.
Th2-Type CD4+ Cells Neither Enhance nor Suppress Antitumor CTL Activity in a Mouse Tumor Model
Authors: Fernando Germain J. P. , Stewart Trina J. , Tindle Robert W. , Frazer Ian H.
Published: December 2022

Many cervical cancers express the E7 protein of human papillomavirus 16 as a tumor-specific Ag (TSA). To establish the role of E7-specific T cell help in CD8+ CTL-mediated tumor regression, C57BL/6J mice were immunized with E7 protein or with a peptide (GF001) comprising a minimal CTL epitope of E7, together with different adjuvants. Immunized mice were challenged with an E7-expressing tumor cell line, EL4.E7. Growth of EL4.E7 was reduced following immunization with E7 and Quil-A (an adjuvant that induced a Th1-type response to E7) or with GF001 and Quil-A. Depletion of CD8+ cells, but not CD4+ cells, from an immunized animal abrogated protection, confirming that E7-specific CTL are necessary and sufficient for TSA-specific protection in this model. Immunization with E7 and Algammulin (an alum-based adjuvant) induced a Th2-like response and provided no tumor protection. To investigate whether a Th2 T helper response to E7 could prevent the development of an E7-specific CTL-mediated protection, mice were simultaneously immunized with E7/Algammulin and GF001/Quil-A or, alternatively, were immunized with GF001/Quil-A 8 wk after immunization with E7/Algammulin. Tumor protection was observed in each case. We conclude that an established Th2 response to a TSA does not prevent the development of TSA-specific tumor protective CTL.
Advanced Oxidation Protein Products as Novel Mediators of Inflammation and Monocyte Activation in Chronic Renal Failure1, 2
Authors: Witko-Sarsat Véronique , Friedlander Miriam , Khoa Thao Nguyen , Capeillère-Blandin Chantal , Nguyen Anh Thu , Canteloup Sandrine , Dayer Jean-Michel , Jungers Paul , Drüeke Tilman , Descamps-Latscha Béatrice
Published: December 2022

We previously demonstrated the presence of advanced oxidation protein products (AOPP), a novel marker of oxidative stress in the plasma of uremic patients receiving maintenance dialysis. The present study in a cohort of 162 uremic patients showed that plasma concentrations of AOPP increased with progression of chronic renal failure and were closely related to advanced glycation end products (AGE)-pentosidine (r = 0.52, p < 0.001), taken as a marker of AGE. In vivo, the relevance of AOPP and AGE-pentosidine in monocyte-mediated inflammatory syndrome associated with uremia was evidenced by close correlations between AOPP or AGE-pentosidine and monocyte activation markers, including neopterin, IL-1R antagonist, TNF-α, and TNF soluble receptors (TNF-sR55 and TNF-sR75). To determine the mechanisms by which AOPP and AGE could be directly involved in monocyte activation, AOPP-human serum albumin (HSA) and AGE-HSA were produced in vitro by treating HSA with oxidants or glucose, respectively. Spectroscopic analysis confirmed that AOPP-HSA contains carbonyls and dityrosine. Both AOPP-HSA and AGE-HSA, but not purified dityrosine, were capable of triggering the oxidative burst of human monocytes in cultures. The AOPP-HSA-induced respiratory burst was dependent on the chlorinated nature of the oxidant and on the molar ratio HSA/HOCl. Collectively, these data first demonstrate that AOPP act as a mediator of oxidative stress and monocyte respiratory burst, which points to monocytes as both target and actor in the immune dysregulation associated with chronic uremia.
Isolation, Structural Characterization, and Chromosomal Mapping of the Mouse Vascular Adhesion Protein-1 Gene and Promoter
Authors: Bono Petri , Salmi Marko , Smith David J. , Leppänen Ilona , Horelli-Kuitunen Nina , Palotie Aarno , Jalkanen Sirpa
Published: December 2022

Vascular adhesion protein-1 (VAP-1) is an endothelial cell adhesion molecule which mediates lymphocyte binding to endothelial cells. The cloning of a mouse VAP-1 (mVAP-1) cDNA revealed that mVAP-1 is a novel 110/220 kDa transmembrane molecule with significant identity to copper-containing amine oxidases. In this work the nucleotide sequence and primary structure of the mVAP-1 gene was determined and the promoter region was structurally characterized. The isolated approximately 14.4-kb mVAP-1 gene consists of 4 exons and 3 introns. Primer extension analysis and 5′ rapid amplification of cDNA ends revealed multiple transcription initiation sites in different tissues suggesting that the mVAP-1 transcription is differently regulated in different tissues. Analysis of the sequence immediately upstream of the detected transcription initiation sites showed no canonical TATA or CCAAT elements, but putative regulatory elements were found close to the detected transcription start sites. The cloning of the mVAP-1 gene reveals the first insight into the genomic organization of murine amine oxidases and will, by targeted disruption of the gene, allow us to understand better the importance of VAP-1 in leukocyte trafficking and monoamine oxidase activity for the function of the immune system.
Astrocyte-Targeted Expression of IFN-α1 Protects Mice from Acute Ocular Herpes Simplex Virus Type 1 Infection
Authors: Carr Daniel J. J. , Veress Livia A. , Noisakran Sansanee , Campbell Iain L.
Published: December 2022

Type I IFNs (i.e., IFN-α and IFN-β) play a key role in the host’s innate defense against viral pathogens. To examine the biologic relevance of IFN-α to a viral pathogen within the confines of the nervous system, IFN-α1 transgenic mice whose transgene is under the control of the glial fibrillary acidic protein promoter (GFAP-IFN-α, astrocyte specific) were examined for resistance to an ocular herpes simplex virus type 1 (HSV-1) infection. GFAP-IFN-α mice expressed significantly higher levels of IFN-αβ (533 U) in the trigeminal ganglion compared with nontransgenic mice (70 U) 72 h postinfection that corresponded with a significant reduction in the mRNA expression of the HSV-1 immediate early gene infected cell polypeptide 27 and late gene VP16, as well as the chemokines monocyte-chemoattractant protein-1 and cytokine response gene-2 in the eye and trigeminal ganglion. Six days postinfection, the viral load and the expression of infected cell polypeptide 27, CD8, RANTES, IFN-γ, and IFN-α mRNA levels were reduced in the trigeminal ganglion of GFAP-IFN-α mice compared with the wild-type mice. Following the establishment of HSV-1 latency (i.e., 30 days postinfection), only one of nine (11%) GFAP-IFN-α mice was found to be latent compared with seven of eight (88%) of the wild-type mice, as determined by the expression of the latency-associated transcript RNAs. Likewise, only three of nine GFAP-IFN-α mice screened showed seroconversion by day 30 postinfection compared with nine of ten wild-type mice screened. Collectively, the results show that the IFN-α1 transgenic mice are less susceptible to acute HSV-1 infection and the establishment of viral latency.
Identification of a Conserved Universal Th Epitope in HIV-1 Reverse Transcriptase That Is Processed and Presented to HIV-Specific CD4+ T Cells by at Least Four Unrelated HLA-DR Molecules
Authors: van der Burg Sjoerd H. , Kwappenberg Kitty M. C. , Geluk Annemieke , van der Kruk Marjolein , Pontesilli Oscar , Hovenkamp Egbert , Franken Kees L. M. C. , van Meijgaarden Krista E. , Drijfhout Jan-Wouter , Ottenhoff Tom H. M. , Melief Cornelis J. M. , Offringa Rienk
Published: December 2022

CD4+ Th cells play an important role in the induction and maintenance of specific T cell immunity. Indications for a protective role of CD4+ T cells against HIV-1 infection were found in subjects who were able to control HIV-1 viremia as well as in highly HIV-1-exposed, yet seronegative, individuals. This study describes the identification of an HIV-1-specific Th epitope that exhibits high affinity binding as well as high immunogenicity in the context of at least four different HLA-DR molecules that together cover 50–60% of the Caucasian, Oriental, and Negroid populations. This HIV-1 reverse transcriptase-derived peptide (RT171–190) is highly conserved among different HIV-1 isolates. Importantly, stimulation of PBL cultures from HIV-1 seronegative donors with this peptide resulted in Th1-type lymphocytes capable of efficient recognition of HIV-1-pulsed APCs. Taken together, these data indicate that peptide RT171–190 constitutes an attractive component of vaccines aiming at induction or enhancement of HIV-1-specific T cell immunity.
CD1 Expression by Dendritic Cells in Human Leprosy Lesions: Correlation with Effective Host Immunity
Authors: Sieling Peter A. , Jullien Denis , Dahlem Monica , Tedder Thomas F. , Rea Thomas H. , Modlin Robert L. , Porcelli Steven A.
Published: December 2022

A potential role for the CD1 family of lipid Ag-presenting molecules in antimicrobial immunity in vivo was investigated in human leprosy skin lesions. Strong induction of three CD1 proteins (CD1a, -b, and -c) was observed in dermal granulomas in biopsy samples of involved skin from patients with the tuberculoid form of leprosy or with reversal reactions, which represent clinical patterns of disease associated with active cellular immunity to Mycobacterium leprae. In contrast, lesions from patients with the lepromatous form of the disease who lack effective cell-mediated immunity to the pathogen did not show induction of CD1 proteins. Thus, expression of CD1 correlated directly with effective immunity to M. leprae, as assessed by the clinical course of infection. CD1a, -b, and -c could be induced to similar levels on monocytes from the blood of either tuberculoid or lepromatous leprosy patients. This suggested that the absence of expression in lepromatous lesions was most likely due to local factors at the site of infection as opposed to a primary defect of the CD1 system itself. The majority of cells expressing CD1 in leprosy lesions were identified as a population of CD83+ dendritic cells. Initial in vitro studies of the Ag-presenting function of CD1+CD83+ monocyte-derived dendritic cells showed that such cells were highly efficient APCs for CD1-restricted T cells. These results indicate that the CD1 system can be up-regulated in human infectious diseases in vivo, and may play a role in augmenting host defense against microbial pathogens.
Cutting Edge: Secondary Lymphoid-Tissue Chemokine (SLC) and CC Chemokine Receptor 7 (CCR7) Participate in the Emigration Pathway of Mature Dendritic Cells from the Skin to Regional Lymph Nodes
Authors: Saeki Hidehisa , Moore Adrienne M. , Brown Martin J. , Hwang Sam T.
Published: December 2022

Dendritic cells (DCs) emigrate to regional lymph nodes (LNs) during immune responses via afferent lymphatic channels. Secondary lymphoid-tissue chemokine (SLC), a CC chemokine, is expressed in secondary lymphoid organs and mediates the chemotaxis of lymphocytes and DCs via its receptor, CC chemokine receptor 7 (CCR7). By dual-label fluorescence confocal microscopy, we showed MHC class II-positive cells within SLC-staining lymphatic channels in the mouse dermis. SLC was a potent in vitro chemoattractant for cultured, migratory skin DCs, and it enhanced the emigration of MHC class II-positive DCs from mouse skin explants by an average of 2.5-fold. Mature or cytokine-activated, but not resting, Langerhans cells expressed CCR7 mRNA by RT-PCR. Anti-SLC Abs, but not control or anti-eotaxin Abs, blocked the in vivo migration of 51Cr-labeled, skin-derived DCs from footpads to draining LNs by 50% (n = 9, p < 0.005). Thus, we provide direct evidence that SLC and CCR7 participate in the emigration of DCs from peripheral tissue to LNs via lymphatics.
Ig Heavy Chain Expression and Class Switching In Vitro from an Allele Lacking the 3′ Enhancers DNase I-Hypersensitive hs3A and hs1,2
Authors: Saleque Shireen , Singh Mallika , Birshtein Barbara K.
Published: December 2022

The murine Ig heavy chain (IgH) 3′ regulatory region contains four enhancers: hs3A, hs1,2, hs3B, and hs4. Various studies have suggested a role for these enhancers in regulating IgH expression and class switching. Here we assess the role of hs3A and hs1,2 in these processes by exploiting a naturally occurring deletion of these enhancers from the expressed, C57BL/6 allele of the F1 pre-B cell line, 70Z/3. Equivalent μ expression in 70Z/3 and 18-81 (which has an intact 3′ region) indicated that hs3A and hs1,2 were not essential for μ expression at the pre-B cell stage. To further examine the role of hs3A and hs1,2 in IgH function at the plasma cell stage, we fused 70Z/3 with the plasmacytoma NSO. Electromobility shift assay analysis of the 70Z/3-NSO hybrids revealed a transcription factor complement conducive to the activation of the 3′ enhancers. Despite the lack of enhancers, hs3A and hs1,2, the level of μ RNA and protein in the 70Z/3-NSO fusion hybrids was substantially elevated relative to its pre-B parent and comparable with that observed in a number of μ-producing spleen cell hybridomas. Additionally, ELISAspot assays showed that the 70Z/3-NSO hybrid underwent spontaneous class switching in culture to IgG1 at a frequency comparable with that of most hybridomas. These results indicate that hs3A and hs1,2 are not essential for high levels of IgH expression or for spontaneous class switching in a plasma cell line.
Genetic Evidence for Functional Redundancy of Platelet/Endothelial Cell Adhesion Molecule-1 (PECAM-1): CD31-Deficient Mice Reveal PECAM-1-Dependent and PECAM-1-Independent Functions
Authors: Duncan Gordon S. , Andrew David P. , Takimoto Hiroaki , Kaufman Stephen A. , Yoshida Hiroki , Spellberg Jason , Luis de la Pompa José , Elia Andrew , Wakeham Andrew , Karan-Tamir Barbara , Muller William A. , Senaldi Giorgio , Zukowski Mark M. , Mak Tak W.
Published: December 2022

Platelet/endothelial cell adhesion molecule-1 (PECAM-1; CD31), a member of the Ig superfamily, is expressed strongly at endothelial cell-cell junctions, on platelets, and on most leukocytes. CD31 has been postulated to play a role in vasculogenesis and angiogenesis, and has been implicated as a key mediator of the transendothelial migration of leukocytes. To further define the physiologic role of CD31, we used targeted gene disruption of the CD31 gene in embryonic stem cells to generate CD31-deficient mice. CD31-deficient mice (CD31KO) are viable and born at the expected Mendelian frequency, remain healthy, and exhibit no obvious vascular developmental defects. In response to inflammatory challenge, polymorphonuclear leukocytes of CD31KO mice are arrested between the vascular endothelium and the basement membrane of inflammatory site mesenteric microvessels, confirming a role for CD31 in the migration of neutrophils through the subendothelial extracellular matrix. Normal numbers of leukocytes are recovered from inflammatory sites in CD31KO mice, however, suggesting that the defect in leukocyte migration across basal lamina observed in the absence of CD31 may be compensated for by the use of other adhesion molecules, or possibly an increased rate of migration. Homing of T lymphocytes in vivo is normal, and CD31KO mice are able to mount a cutaneous hypersensitivity response normally. In addition, CD31-mediated homophilic adhesion does not appear to play a role in platelet aggregation in vitro. This study provides genetic evidence that CD31 is involved in transbasement membrane migration, but does not play an obligatory role in either vascular development or leukocyte migration.
Plasma Cell Development in Synovial Germinal Centers in Patients with Rheumatoid and Reactive Arthritis
Authors: Kim Hye-Jung , Krenn Veit , Steinhauser Gudrun , Berek Claudia
Published: December 2022

Plasma cells are found surrounding the inflammatory infiltrates of macrophages, T, and B cells in the synovial tissue of patients with rheumatoid and reactive arthritis. This characteristic arrangement suggests that in the synovial tissue CD20+ B cells differentiate into plasma cells. To examine clonal relationships, we have used micromanipulation to separately isolate CD20+ B cells and plasma cells from single infiltrates. DNA was extracted, and from both populations the VH/VL gene repertoires was determined. The data show that in the inflamed synovial tissue activated B cells are clonally expanded. During proliferation in the network of follicular dendritic cells, V gene variants are generated by the hypermutation mechanism. Surprisingly, we do not find identical rearrangements between CD20+ B cells and plasma cells. Nevertheless, the finding of clonally related plasma cells within single infiltrates suggests that these cells underwent terminal differentiation in the synovial tissue. These results indicate that B cell differentiation in the synovial tissue is a dynamic process. Whereas CD20+ B cells may turnover rapidly, plasma cells may well be long lived and thus accumulate in the synovial tissue. The analysis of individual B cells recovered from synovial tissue opens a new way to determine the specificity of those cells that take part in the local immune reaction. This will provide new insights into the pathogenesis of chronic inflammatory diseases like rheumatoid or reactive arthritis.
IL-4 and IFN-γ Increase Steady State Levels of Polymeric Ig Receptor mRNA in Human Airway and Intestinal Epithelial Cells
Authors: Ackermann Laynez W. , Wollenweber Laura A. , Denning Gerene M.
Published: December 2022

Delivery of IgA to the mucosal surface occurs via transcytosis of polymeric IgA (pIgA) across the epithelium, a process mediated by the pIgR. Several factors increase pIgR expression in human epithelial cells, including IL-4 and IFN-γ. Using an RNase protection assay, we found that IL-4 and IFN-γ increase steady state levels of pIgR mRNA in both human intestinal (HT29) and airway (Calu-3) epithelial cells. Time course studies in HT29 clone 19A cells showed that with each cytokine alone and with both together: 1) there was a significant lag before mRNA levels increased; 2) maximal levels were not reached until 48–72 h after the addition of cytokines; 3) mRNA levels remained elevated in the continued presence of cytokines; and 4) addition of actinomycin D or removal of cytokines led to decreases in mRNA levels with a half-life of ∼20–28 h. Cytokine-dependent increases in steady state levels of pIgR mRNA were inhibited by cycloheximide and by protein tyrosine kinase inhibitors but not by inhibitors of protein kinase C or cAMP-dependent protein kinase A. Both IFN-γ and IL-4 increased expression of the inducible transcription factor IFN regulatory factor-1 (IRF-1), but levels of IRF-1 only weakly correlated with levels of pIgR mRNA, suggesting that additional transcription factors are required. These studies provide additional insights into the mechanisms by which cytokines regulate expression of the pIgR, a central player in mucosal immunity.
Dendritic Cells Undergo Rapid Apoptosis In Vitro During Antigen-Specific Interaction with CD4+ T Cells
Authors: Matsue Hiroyuki , Edelbaum Dale , Hartmann Aubrey C. , Morita Akimichi , Bergstresser Paul R. , Yagita Hideo , Okumura Ko , Takashima Akira
Published: December 2022

The terminal fate of dendritic cells (DC) remains relatively uncertain. In this study, we tested the hypothesis that DC undergo apoptosis after Ag-specific interaction with T cells. When splenic DC isolated from BALB/c mice were cocultured with HDK-1 T cells (a keyhole limpet hemocyanin (KLH)-specific CD4+ Th1 clone) in the presence of KLH, they showed conspicuous cell death as measured by propidium iodide (PI) uptake and chromatin condensation, whereas they remained relatively intact when incubated with either T cells or KLH alone. Likewise, the long term DC line XS52, which was established from BALB/c mouse epidermis, also died rapidly (within 2 h), and they exhibited characteristic DNA laddering when cocultured with HDK-1 T cells in the presence of KLH. RT-PCR and FACS analyses revealed the expression of CD95 (Fas) by XS52 DC and of CD95 ligand (CD95L) (Fas ligand) by activated HDK-1 T cells, suggesting a functional role for these molecules. In fact, anti-CD95L mAb inhibited partially (50%) T cell-mediated XS52 cell death, and coupling of surface CD95 with anti-CD95 mAb triggered significant XS52 cell death, but only in the presence of cycloheximide. Thus, ligation of CD95 (on DC) with CD95L (on T cells) is one, but not the only, mechanism by which T cells induce DC death. Finally, DC isolated from the CD95-deficient mice were found to be significantly more efficient than DC from control mice in their capacity to induce delayed type hypersensitivity responses in vivo. We propose that T cell-induced DC apoptosis serves as a unique down-regulatory mechanism that prevents the interminable activation of T cells by Ag-bearing DC.
Naive, Effector, and Memory CD8 T Cells in Protection Against Pulmonary Influenza Virus Infection: Homing Properties Rather Than Initial Frequencies Are Crucial
Authors: Cerwenka Adelheid , Morgan Tammy M. , Dutton Richard W.
Published: December 2022

The goal of adoptive immunotherapy is to target a high number of persisting effector cells to the site of a virus infection or tumor. In this study, we compared the protective value of hemagglutinin peptide-specific CD8 T cells generated from the clone-4 TCR-transgenic mice, defined by different stages of their differentiation, against lethal pulmonary influenza infection. We show that the adoptive transfer of high numbers of Ag-specific unprimed, naive CD8 T cells failed to clear the pulmonary virus titer and to promote host survival. The same numbers of in vitro generated primary Ag-specific Tc1 effector cells, producing high amounts of IFN-γ, or resting Tc1 memory cells, generated from these effectors, were protective. Highly activated CD62Llow Tc1 effectors accumulated in the lung with rapid kinetics and most efficiently reduced the pulmonary viral titer early during infection. The resting CD62Lhigh naive and memory populations first increased in cell numbers in the draining lymph nodes. Subsequently, memory cells accumulated more rapidly and to a greater extent in the lung lavage as compared with naive cells. Thus, effector cells are most effective against a localized virus infection, which correlates with their ability to rapidly distribute at the infected tissue site. The finding that similar numbers of naive Ag-specific CD8 T cells are not protective supports the view that qualitative differences between the two resting populations, the naive and the memory population, may play a major role in their protective value against disease.
Th Cells and Th2 Responses Can Develop in the Absence of MHC Class II-CD4 Interactions
Authors: Wack Andreas , Corbella Paola , Harker Nicola , Roderick Kathleen , Norton Trisha , Williams Keith , Williams Owen , Kioussis Dimitris
Published: December 2022

In this paper, we address the question whether CD4 and MHC class II expression are necessary for the development of the T helper lineage during thymocyte maturation and for activation-induced Th2 responses. To bypass the CD4-MHC class II interaction requirements for positive selection and activation, we used mice that are doubly transgenic for CD8 and for the MHC class I-restricted TCR F5. This transgene combination leads to MHC class I-dependent maturation of CD4 lineage cells. Upon activation, these CD4 lineage T cells secrete IL-4 and give help to B cells but show no cytotoxic activity. Remarkably, neither MHC class II nor CD4 expression are necessary for the generation and helper functions of these cells. This suggests that under normal conditions, coreceptor-MHC interactions are necessary to ensure the canonical combinations of coreceptor and function in developing thymocytes, but that they do not determine functional commitment. Our results also imply that expression of the CD4 gene does not influence, but is merely associated with the decision to establish the T helper program. In addition, we show that activation through TCR-MHC class I interactions can induce Th2 responses independently of CD4 and MHC class II expression.
Involvement of the SHP-1 Tyrosine Phosphatase in Regulation of T Cell Selection
Authors: Zhang Jinyi , Somani Ally-Khan , Yuen Darren , Yang Ye , Love Paul E. , Siminovitch Katherine A.
Published: December 2022

The selection events shaping T cell development in the thymus represent the outcome of TCR-driven intracellular signaling cascades evoked by Ag receptor interaction with cognate ligand. In view of data indicating TCR-evoked thymocyte proliferation to be negatively modulated by the SHP-1 tyrosine phosphatase, a potential role for SHP-1 in regulating selection processes was investigated by analysis of T cell development in H-Y TCR transgenic mice rendered SHP-1 deficient by introduction of the viable motheaten mutation or a dominant negative SHP-1-encoding transgene. Characterization of thymocyte and peripheral T cell populations in H-Y TCR-viable motheaten mice revealed TCR-evoked proliferation as well as the positive and negative selection of H-Y-specific thymocytes to be enhanced in these mice, thus implicating SHP-1 in the negative regulation of each of these processes. T cell selection processes were also augmented in H-Y TCR mice carrying a transgene driving lymphoid-restricted expression of a catalytically inert, dominant-negative form of SHP-1. SHP-1-negative effects on thymocyte TCR signaling were not influenced by co-cross-linking of the CD28 costimulatory and/or CTLA-4 inhibitory receptors and appear, accordingly, to be realized independently of these comodulators. These observations indicate that SHP-1 raises the signaling threshold required for both positive and negative selection and reveal the inhibitory effects of SHP-1 on TCR signaling to be cell autonomous. The demonstrated capacity for SHP-1 to inhibit TCR-evoked proliferation and selection indicate SHP-1 modulatory effects on the magnitude of TCR-generated signal to be a key factor in determining the cellular consequences of TCR-ligand interaction.
Alloreactive and Syngeneic CTL Are Comparably Dependent on Interaction with MHC Class I α-Helical Residues
Authors: Hornell Tara M. C. , Solheim Joyce C. , Myers Nancy B. , Gillanders William E. , Balendiran Ganesaratnam K. , Hansen Ted H. , Connolly Janet M.
Published: December 2022

The molecular basis for the difference in the strength of T cell responses to self vs alloantigens is unknown, but may reflect how T cells are selected in the thymus. Because T cells with a high affinity for foreign as opposed to self MHC molecules are able to mature, it has been proposed that alloreactive T cells may be more strongly dependent upon interaction with MHC residues than are self-restricted T cells. This study was undertaken to rigorously address this hypothesis. Whereas other studies have compared self vs alloantigen recognition of different MHC alleles by a single T cell clone, we have compared self vs alloantigen recognition of a single MHC allele, H-2Ld, by a large panel of self-restricted and alloreactive T cell clones. Target cells expressing Ld molecules mutated at several different potential TCR contact residues were analyzed to determine which residues are important for recognition by self-restricted vs alloreactive T cells. We unequivocally demonstrate that self-restricted and alloreactive T cells do not differ, but rather are comparably dependent on interaction with MHC residues. Importantly, both self-restricted and alloreactive T cells are dependent upon the same MHC residues as primary contacts and, in addition, share a common recognition pattern of Ld. Furthermore, our analysis enables us to provide a model for allotype-specific T cell recognition of Ld vs Kb class I molecules.