bluuebunny/crossref_metadata_2025_split huggingface dataset:
Heterogeneity of Mouse Spleen Dendritic Cells: In Vivo Phagocytic Activity, Expression of Macrophage Markers, and Subpopulation Turnover
Authors: Leenen Pieter J. M. , Radošević Katarina , Voerman Jane S. A. , Salomon Benoı̂t , van Rooijen Nico , Klatzmann David , van Ewijk Willem 
Published: January 2023

AbstractIn the normal mouse spleen, two distinct populations of dendritic cells (DC) are present that differ in microanatomical location. The major population of marginal DC is found in the “marginal zone bridging channels” and extends into the red pulp. The interdigitating cells (IDC) are localized in the T cell areas in the white pulp. The aim of the present study was to characterize these two splenic DC populations with regard to their phenotype, in vivo phagocytic function, and turnover. Both marginal DC and IDC are CD11c+ and CD13+, but only IDC are NLDC-145+ and CD8α+. Notably, both populations, when freshly isolated, express the macrophage markers F4/80, BM8, and Mac-1. To study the phagocytic capacity of these cells, we employed the macrophage “suicide” technique by injecting liposomes loaded with clodronate i.v. Marginal DC, but not IDC, were eliminated by this treatment. Phagocytosis of DiI-labeled liposomes by DC confirmed this finding. The two DC populations differed significantly with regard to their turnover rates, as studied in a transgenic mouse model of conditional depletion of DC populations with high turnover. In these mice, marginal DC were completely eliminated, but the IDC population remained virtually intact. From these data we conclude that the marginal DC population has a high turnover, in contrast to the IDC population. Taken together, the present results indicate that marginal DC and IDC represent two essentially distinct populations of DC in the mouse spleen. They differ not only in location, but also in phenotype, phagocytic ability, and turnover.
IL-4 Enhances Long-Term Survival of CD28-Deficient T Cells
Authors: Stack Risa M. , Thompson Craig B. , Fitch Frank W. 
Published: January 2023

Abstract
CD28 signaling is critical for IL-2 production by established Th1 clones, but CD28 does not appear to play a role in the activation of established Th2 clones. To determine the role of CD28 in the generation of polarized T cells, clones were derived using cells from CD28-deficient (CD28−/− mice, which had been bred with mice that express the DO11.10 transgene, a CD4+ TCR-αβ receptor that recognizes OVA peptide 323–339 bound to I-Ad. Most T cell clones derived from CD28+/+ mice survived multiple stimulations, while T cell clones derived from CD28−/− mice survived only if they were derived initially in the presence of IL-4 or both IL-2 and IL-4. Signaling through the CD28 molecule did not appear to be important in the initial activation of T cell clones, as the precursor frequency of clones derived from normal (CD28+/+) and CD28−/− mice was similar. Primary stimulation in the presence of IL-4 increased cell number and viability of both CD28+/+ and CD28−/− T cells in primary culture. However, the survival of CD28−/− cells is more dependent on IL-4 than is the survival of CD28+/+ cells. The continued presence of anti-IL-4 mAb dramatically decreased the number of viable cells in the CD28−/− cultures but had little effect on the viability of the CD28+/+ clones. Thus, initial culture with IL-4 allows the isolation of CD28−/− T cell clones that produce IL-4. In these clones, IL-4 acts as both an autocrine growth and survival factor.
Identification of an IL-7-Associated Pre-Pro-B Cell Growth-Stimulating Factor (PPBSF). II. PPBSF Is a Covalently Linked Heterodimer of IL-7 and aMr 30,000 Cofactor
Authors: Lai Laijun , Chen Fangqi , McKenna Sean , Goldschneider Irving 
Published: January 2023

AbstractEvidence is provided in a companion paper for an IL-7-associated molecular complex that selectively stimulates the proliferation and presumptive differentiation of pre-pro-B cells in our long-term bone marrow culture system and “primes” them to proliferate in response to monomeric IL-7. Here, Western immunoblot analysis reveals that this pre-pro-B cell growth-stimulating factor (PPBSF) is a self-assembling heterodimer of IL-7 and a cofactor with a Mr of 30,000. Thus, when developed with anti-IL-7 mAb, PPBSF migrates electrophoretically as a covalently bound ∼55-kDa molecule under nonreducing conditions but dissociates under reducing conditions. Furthermore, the addition of rIL-7 or native IL-7 to medium conditioned by stromal cells from IL-7 gene-deleted (−/−) mice results in the formation of active 45-kDa and 55-kDa molecular complexes, respectively. Antiserum prepared in IL-7(−/−) mice against affinity-purified PPBSF contained separable reactivities for IL-7 and the non-IL-7 component of PPBSF. The PPBSF cofactor detected by this antiserum migrates as an ∼30-kDa molecule and is able to maintain the viability, but not the proliferation, of pre-pro-B cells. Furthermore, the cofactor is produced constitutively by IL-7(−/−) and IL-7(+/+) bone marrow stromal cells under pro-B- but not pre-B-type culture conditions. Consequently, IL-7 appears to exist almost entirely as a heterodimer (i.e., PPBSF) in pro-B-type cultures, whereas it exists almost entirely as a monomer in pre-B-type cultures. Although the identity of the PPBSF cofactor remains to be determined, it does not appear to be stem cell factor, insulin-like growth factor-1, thymic stromal-derived lymphopoietin, flt3, stromal cell-derived factor-1, or IL-7R.
Characterization of the Interactions Between MHC Class I Subunits: A Systematic Approach for the Engineering of Higher Affinity Variants of β2-Microglobulin
Authors: Shields Michael J. , Assefi Nassim , Hodgson Wesley , Kim Ellen J. , Ribaudo Randall K. 
Published: January 2023

AbstractHuman β2m (hβ2m) binds to murine MHC I molecules with higher affinity than does murine β2m and therefore can be used as a model system to define and dissect the interactions between β2m and MHC I heavy chains that promote the stability of the complex. In the present study we compare three-dimensional crystal structures of human and murine MHC I molecules and use functional studies of chimeric human:murine β2m variants to define a region of β2m that is involved in the higher affinity of hβ2m for murine MHC I heavy chains. Further examination of the three-dimensional structure in this region revealed conformational differences between human and murine β2m that affect the ability of an aspartic acid residue at position 53 (D53) conserved in both β2ms to form an ionic bond with arginine residues at positions 35 and 48 of the heavy chain. Mutation of residue D53 to either asparagine (D53N) or valine (D53V) largely abrogated the stabilizing effects of hβ2m on murine MHC I expression in a predictable manner. Based on this observation a variant of hβ2m was engineered to create an ionic bond between the heavy chain and β2m. This variant stabilizes cell surface H-2Dd heavy chains to a greater extent than wild-type hβ2m. Studying these interactions in light of the growing database of MHC I crystal structures should allow the rational design of higher affinity hβ2m variants for use in novel peptide-based vaccines capable of inducing cell-mediated immune responses to viruses and tumors.
Induction of Nuclear Factor-κB During Primary B Cell Differentiation
Authors: Kistler Barbara , Rolink Antonius , Marienfeld Ralf , Neumann Manfred , Wirth Thomas 
Published: January 2023

Abstract
We have investigated activation of nuclear factor-κB (NF-κB) in the process of primary B cell differentiation in vitro. In this system, NF-κB is strongly induced when B cells develop from the pre-B cell to the immature B cell stage. Unlike the typical NF-κB activation in response to exogenous stimuli, induction proceeds with a slow time course. NF-κB induction is only observed in B cells that undergo differentiation, not in Rag2-deficient cells. Nuclear DNA binding complexes predominantly comprise p50/RelA heterodimers and, to a lesser extent, c-Rel-containing dimers. The increase in NF-κB binding activity is accompanied by a slow and steady decrease in IκBβ protein levels. Interestingly, absolute RelA protein levels remain unaffected, whereas RelB and c-Rel synthesis is induced. The reason for preferential nuclear translocation of RelA complexes appears to be selective inhibition by the IκBβ protein. IκBβ can efficiently inhibit p50/RelA complexes, but has a much reduced ability to interfere with p50/c-Rel DNA binding both in vitro and in vivo. Interestingly, p50/RelB complexes are not at all targeted by IκBβ, and coimmunoprecipitation experiments show no evidence for an association of IκBβ and RelB in vivo. Consistent with these observations, IκBβ cotransfection can inhibit p50/RelA-mediated trans-activation, but barely affects p50/RelB mediated trans-activation.
Characterization of cis-Regulatory Elements and Nuclear Factors Conferring Th2-Specific Expression of the IL-5 Gene: A Role for a GATA-Binding Protein
Authors: Lee Hyun Jun , O’Garra Anne , Arai Ken-ichi , Arai Naoko 
Published: January 2023

Abstract
Expression of the IL-5 gene is restricted to the Th2 subset of helper T cells. We have previously defined four cis-regulatory elements of the IL-5 promoter responding to PMA and cAMP in EL-4 cells. We now report that the 1.2-kb region of the IL-5 promoter directs expression of the IL-5 gene in a Th2 clone but not a Th1 clone, indicating that transcription from the IL-5 promoter is Th2 specific. For the functioning of the IL-5 promoter in a Th2 clone, IL-5C and IL-5CLE0 were critical. IL-5CLE0 interacted with both constitutive and inducible nuclear factors (designated NFIL-5CLE0), which existed in both Th1 and Th2 clones, whereas IL-5C interacted with a constitutive nuclear factor (designated NFIL-5C), which was found only in Th2 but not in Th1 clones. Th2 specificity of NFIL-5C was also confirmed using in vitro-differentiated Th1 and Th2 cells derived from TCR-transgenic mice. The sequence for NFIL-5C binding bears homology with GATA-binding sites. The NFIL-5C complex was supershifted by an anti-GATA-3 Ab and inhibited by an oligonucleotide containing GATA-binding sites. We showed preferential expression of GATA-3 in Th2 cells. Finally, we demonstrated that in vitro-translated GATA-3 bound to IL-5C and overexpression of GATA-3 augmented stimulation-dependent IL-5 promoter activity in EL-4 cells. Taken together, our results provide evidence that GATA-related factors may be involved in Th2-specific expression of the IL-5 gene.
Contribution of Cells at the Site of DNA Vaccination to the Generation of Antigen-Specific Immunity and Memory
Authors: Klinman Dennis M. , Sechler Joan M. G. , Conover Jacqueline , Gu Mili , Rosenberg Amy S. 
Published: January 2023

Abstract
Gene gun-mediated DNA vaccination stimulates an immune response characterized by the activation of IgG-secreting B cells and IFN-γ-secreting T cells. To monitor the contribution of cells at the site of vaccination to this process, transfected skin was periodically removed and grafted onto naive recipients. Immediate removal of vaccinated skin abrogated the development of an immune response. Low-level IgG production was stimulated when the vaccination site was left in place for ≥5 h, with the strength of this response increasing the longer the site remained intact (for up to 2 wk). Measurable primary T cell responses were observed in animals whose vaccination site remained in place for ≥1 day. Skin grafts transferred 0 to 24 h postvaccination stimulated a primary immune response in naive recipients. Memory B and T cells were generated in animals whose site of vaccination remained intact for 5 to 12 h. Skin transferred within 12 h of vaccination triggered memory B and T cell development in graft recipients, while the removal of skin &gt;12 h postvaccination did not reduce memory in vaccinated mice. These findings suggest that 1) primary immunity is induced by cells that migrate rapidly from the site of immunization, 2) nonmigratory cells influence the magnitude of this primary response, and 3) migratory cells alone are responsible for the induction of immunologic memory.
Granulocyte-Macrophage Colony-Stimulating Factor Enhances EBV-Induced Synthesis of Chemotactic Factors in Human Neutrophils
Authors: Roberge Charles J. , McColl Shaun R. , Larochelle Bernard , Gosselin Jean 
Published: January 2023

Abstract
We have recently demonstrated that EBV binds to human neutrophils and stimulates a wide range of activities, including homeotypic aggregation, total RNA synthesis, and expression of the chemokines IL-8 and macrophage inflammatory protein-1α (MIP-1α). Neutrophil function is also known to be modulated by priming with granulocyte-macrophage colony-stimulating factor (GM-CSF). We have therefore investigated the modulation of EBV-induced activation of human neutrophils by GM-CSF. Treatment of neutrophils with GM-CSF before EBV activation enhanced the production of both MIP-1α and IL-8. The IL-8 produced under these conditions was biologically active as determined in the calcium mobilization assay. GM-CSF was also found to increase the ability of EBV to prime neutrophils for increased leukotriene B4 (LTB4) synthesis. Prior treatment of GM-CSF with neutralizing Abs inhibited these effects. GM-CSF also increased the specific binding of FITC-EBV to the neutrophil surface, as evaluated by fluorocytometry. Local production of GM-CSF in tissues invaded by EBV could therefore serve to potentiate a host defense mechanism directed toward the destruction of the infectious virus via increased production of chemotactic factors. Since both IL-8 and MIP-1α are reported to be chemoattractants in vitro for T cells and T and B cells, respectively, the ability of EBV to induce their production by neutrophils may enhance its ability to infect B and T lymphocytes via increased recruitment to sites of infection.
Mapping IgG Epitopes Bound by Rheumatoid Factors from Immunized Controls Identifies Disease-Specific Rheumatoid Factors Produced by Patients with Rheumatoid Arthritis
Authors: Bonagura Vincent R. , Agostino Nick , Børretzen Marie , Thompson Keith M. , Natvig Jacob B. , Morrison Sherie L. 
Published: January 2023

AbstractWe have mapped the specificity of 28 monoclonal IgM rheumatoid factors (RFs) produced by heterohybridomas derived from five healthy blood donors immunized with mismatched human red blood cells (HID). The HID-RFs did not differ in their binding specificity for IgG epitopes from RFs that we previously analyzed from patients with Waldenström’s macroglobulinemia. However, IgM RFs produced by HID differed in their specificity for IgG compared with RFs expressed by patients with rheumatoid arthritis (RA-RFs). Only 1 of 28 HID-RFs bound all IgG subclasses (pan binding pattern) compared with 7 of 19 RA-RFs (p = 0.006). Three HID-RFs bound IgG3 compared with 9 RA-RFs (p = 0.007). Fine specificity differences were also identified between HID- and RA-RFs. Therefore, some RA-RFs show novel specificities for IgG not found among RFs from HID or individuals with Waldenström’s macroglobulinemia who do not have joint disease. These Abs with unique specificities may represent disease-specific autoantibodies in patients with RA. Nine of the HID-RFs from the same individual were clonally related, and several contained somatic mutations. Even when the clonally related HID-RFs were considered as one RF for comparison, the reactivity of the HID-RFs differed significantly from RA-RFs in their inability to recognize all IgG subclasses (p = 0.044) and recognize IgG3 (p = 0.041). Interestingly, among the clonally related RFs, considerable differences in the specificity for IgG were also observed, with the RF containing the most somatic mutations in VH and VL showing the most distinctive specificity changes. Therefore, these studies also demonstrate a correlation between somatic mutation and binding specificity.
Cutting Edge: Liposomal Formulation of a Self Lymphoma Antigen Induces Potent Protective Antitumor Immunity
Authors: Kwak Larry W. , Pennington Robin , Boni Larry , Ochoa Augusto C. , Robb Richard J. , Popescu Mircea C. 
Published: January 2023

Abstract
We developed a liposome carrier for a model nonimmunogenic, self Ag. This carrier reproducibly converted lymphoma Ig into a potent tumor rejection Ag in mice. A single immunization induced protection against challenges representing 20 to 100 times the minimum lethal dose of parental tumor. This protective effect required minimal amounts of incorporated Ag and IL-2 and elicited specific Abs (compared with free Ag or liposomal control Ig which did not elicit any specific Abs); depletion experiments demonstrated a requirement for effector CD4+ and CD8+ T cells. Head-to-head comparisons, indicating superior potency and induction of specific T cell activation, distinguished liposomal from prototype, carrier-conjugated Ag. These results provide a strategy for formulating weak tumor or other clinically important Ags into vaccines.
T:T Antigen Presentation by Activated Murine CD8+ T Cells Induces Anergy and Apoptosis
Authors: Chai Jian-Guo , Bartok Istvan , Scott Diane , Dyson Julian , Lechler Robert 
Published: January 2023

AbstractUsing an IL-2-secreting, noncytolytic, H-Y-specific, CD8+ T cell clone, the functional consequences of Ag presentation by T cells to T cells were investigated. Incubation of the T cells with H-Y-soluble peptide led to nonresponsiveness to Ag rechallenge. This was due to the simultaneous induction of apoptosis, involving approximately 40% of the T cells, and of anergy in the surviving cells. These effects were strictly dependent upon bidirectional T:T presentation, in that exposure of C6 cells to peptide-pulsed T cells from the same clone induced proliferation but not apoptosis or anergy. The inhibitory effects of T:T presentation were not due to a lack of costimulation, since the T cells expressed levels of CD80 and CD86 higher than those detected on cultured dendritic cells and equipped them to function as efficient APCs for primary CD8+ T cell responses. Following incubation with soluble peptide, CD80 expression increased, and high levels of CTLA-4 (CD152) expression were induced. Although addition of anti-CTLA-4 Ab augmented proliferation in response to soluble peptide, no protection from apoptosis or anergy was observed. Neither Fas nor TNF-α was expressed/produced by the C6 cells, and coligation of MHC class I molecules and TCR failed to reproduce the effects of T:T presentation. Taken together, these data suggest that T:T Ag presentation induces anergy and apoptosis in murine CD8+ T cells and may reflect the regulatory consequences of T:T interactions in the course of clonal expansion in vivo.
Identification of a Developmentally Regulated Phase of Postselection Expansion Driven by Thymic Epithelium
Authors: Hare Katherine J. , Wilkinson Robert W. , Jenkinson Eric J. , Anderson Graham 
Published: January 2023

Abstract
To investigate events following the initiation of positive selection, we have used reaggregate organ cultures to follow the maturation of purified CD4+8+69+ thymocytes; these thymocytes represent a subpopulation of thymocytes which have already received positive selection signals. Using a dilution analysis of an FITC-based membrane-binding dye, 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester, to allow a quantitative measure of proliferation, we show that while newly selected CD4+ and CD8+ cells are nondividing, both subsets subsequently undergo a wave of postpositive selection proliferation involving multiple cell divisions. Moreover, in the presence of fetal stromal cells, postselection expansion is more extensive in newborn thymocytes compared with adult thymocytes, suggesting that this phase of expansion is developmentally regulated. We also show that proliferation of CD4+ and CD8+ cells is seen in reaggregates of purified MHC class II+ thymic epithelial cells, while CD4+ and CD8+ cells generated from bcl-2 transgenic CD4+8+69+ thymocytes in the absence of stromal cell support survive but do not proliferate; this observation indicates that MHC class II+ thymic epithelial cells are both necessary and sufficient to mediate this wave of cell division. Finally, the maturation of CD4+8+69+ thymocytes and the subsequent proliferation of CD4+ and CD8+ cells occur in the presence of MHC-mismatched thymic stromal cells, suggesting that the later stages of positive selection and the associated postselection events do not depend on interactions with the same peptide/MHC complexes responsible for initiation.
Reprogramming of Lipopolysaccharide-Primed Macrophages Is Controlled by a Counterbalanced Production of IL-10 and IL-12
Authors: Shnyra Alexander , Brewington Ryan , Alipio Arlene , Amura Claudia , Morrison David C. 
Published: January 2023

AbstractWe studied the potential role of a cytokine regulatory mechanism(s) in LPS-dependent reprogramming and modulation of TNF-α and nitric oxide (NO) responses in mouse peritoneal macrophages. Reciprocal regulation of TNF-α and NO production by LPS-primed and LPS-stimulated macrophages was found to be dependent on the presence of soluble secretory products released by the cells during the initial LPS priming interaction. Pretreatment of naïve macrophages with different mouse recombinant cytokines such as rIL-10, rIL-12, and rIFN-γ dose dependently and differentially regulated subsequent LPS-induced production of TNF-α, IL-6, and NO by cytokine-primed cells. Analysis of IL-12 and IL-10 levels present in culture supernatants of LPS-primed and LPS-stimulated macrophages revealed a high degree of correlation between the profiles of TNF-α and IL-12 as well as NO and IL-10. Furthermore, LPS priming of macrophages in the presence of anti-IL-12-neutralizing mAb attenuated TNF-α responses while at the same time up-regulated NO production. In contrast, neutralization of endogenous IL-10 with anti-IL-10 mAb resulted in considerable TNF-α response at LPS priming doses under conditions that would otherwise strongly inhibit TNF-α production. We also found that the initial LPS priming of naïve macrophages differentially and dose dependently regulates expression of mRNAs for IL-10, IL-12, and IFN-γ in LPS-primed macrophages. Collectively, our data provide experimental support for the hypothesis that a cytokine regulatory network, most probably autocrine, tightly controls the reciprocal modulation of TNF-α and NO responses in LPS-primed macrophages.
BcmdDecreases the Life Span of B-2 But Not B-1 Cells in A/WySnJ Mice
Authors: Lentz Vicky M. , Hayes Colleen E. , Cancro Michael P. 
Published: January 2023

AbstractPeripheral B cells are divided into two subpopulations, B-1 and B-2, the relationship of which remains obscure. We recently showed that the Bcmd mutation in A/WySnJ mice reduces average B cell life span, yielding 90% fewer peripheral B cells. Despite this defect, A/WySnJ mice have an elevated proportion of peritoneal CD5+ B cells, suggesting that Bcmd may be the first B-cell-intrinsic gene to differentially affect the B-1 and B-2 subpopulations. To test this hypothesis in detail, we have used in vivo BrdU labeling and four-color cytofluorometry to examine the numbers and turnover rates of sIgM+CD23−CD43+ (B-1) and sIgM+CD23+CD43− (B-2) splenocytes in A/WySnJ and A/J mice. The results show the expected 90% reduction of splenic B-2 cells among A/WySnJ mice, but a normal splenic B-1 cell pool. Increased B-1 cell renewal cannot explain this undiminished pool, because BrdU labeling kinetics reveals an identical splenic B-1 subset turnover rate of ∼4%/day in both A/WySnJ and A/J strains. Thus, B-1 cells are Bcmd-independent but B-2 cells are Bcmd-dependent, suggesting Bcmd functions in a positive signaling pathway that imparts longevity to quiescent B cells, but that is not required for cycling B cells. Moreover these results show that the requisites for maturation and longevity differ between the B-1 and B-2 subsets.