bluuebunny/crossref_metadata_2025 huggingface dataset:
Maturation of CD4+ Lymphocytes in the Aged Microenvironment Results in a Memory-Enriched Population
Authors: Timm Jenna A. , Thoman Marilyn L.
Published: December 2022

With advancing age the CD4+ T lymphocyte compartment becomes enriched for memory cells in both humans and experimental animals. Although it has been assumed that the shift from a naive to a memory-dominant population is due to a lifetime of antigenic exposure and selection as well as a loss of naive cell input due to reduced thymopoiesis, the present data suggest that the aged microenvironment influences the maturation of newly produced CD4+ T cells. In two models, aged and young mice were compared for the ability to reconstitute their peripheral CD4+ T cell pools following depletion, and both age groups were found to be competent to renew this population. However, the phenotype and lymphokine profile of populations arising in aged animals were distinctly different from those in the young mice. In contrast to the expectation that depletion and reconstitution might give rise to a naive-dominant T cell pool, aged mice reconstituted a population nearly indistinguishable from that found in control age-matched individuals. The majority of the CD4+ pool were CD44high CD45RBlow Mel-14low and upon activation with anti-CD3 these CD4+ T cells produced mRNA for IL-2, IL-4, IL-5, and IFN-γ. In aged bone marrow-transplanted mice, the same phenotypic profile and cytokine mRNA pattern were found in CD4+ T cells of host and donor origin. In contrast, the majority of CD4+ T cells in young reconstituted mice were CD44low CD45RBhigh Mel-14high. These lymphocytes, when activated, produced high levels of mRNA for IL-2, with little or no IL-4, IL-5, or IFN-γ mRNA.
Critical Requirement for Aspartic Acid at Position 82 of Myelin Basic Protein 73–86 for Recruitment of Vβ8.2+ T Cells and Encephalitogenicity in the Lewis Rat
Authors: Smeltz Ronald B. , Wauben Marca H. M. , Wolf Norbert A. , Swanborg Robert H.
Published: December 2022

We synthesized single amino acid-substituted peptide analogues of guinea pig myelin basic protein (MBP) 73–86 to study the importance of aspartic acid at residue 82 (QKSQRSQDENPV), which previous reports have suggested is a critical TCR contact residue. Whereas the wild-type 73–86 peptide elicited severe experimental autoimmune encephalomyelitis (EAE) in the Lewis rat, none of the peptide analogues with substitutions at position 82 were capable of inducing EAE. The inability to cause EAE was not due to a failure to bind MHC or to elicit T cell proliferation and cytokine secretion. T cells specific for MBP73–86 did not cross-react with any of the analogues tested, further indicating the importance of this residue in T cell responses to 73–86. Analysis by flow cytometry showed that only the wild-type 73–86 peptide was capable of recruiting Vβ8.2+ T cells, which have been shown previously to be important for disease induction. Reduced expression of the Vβ8.2 TCR was also seen in Lewis rats protected from EAE by coimmunization of MBP73–86 with 73–86(82D→A), despite an increase in cytokine production when both peptides were present during in vitro culture. The data indicate that aspartic acid 82 is a critical TCR contact residue and is required for the recruitment of Vβ8.2+ T cells and the encephalitogenic activity of MBP73–86.
Conserved Residues Amino-Terminal of Cytoplasmic Tyrosines Contribute to the SHP-1-Mediated Inhibitory Function of Killer Cell Ig-Like Receptors
Authors: Burshtyn Deborah N. , Lam Alan S. , Weston Margaret , Gupta Neetu , Warmerdam Petra A. M. , Long Eric O.
Published: December 2022

The sequence I/VxYxxL, often referred to as an immunoreceptor tyrosine-based inhibition motif (ITIM), binds to the C-terminal Src homology 2 domain of the tyrosine phosphatase SHP-1. Conserved residues N-terminal of the tyrosine are not ordinarily found in other Src homology 2 domain binding motifs. The inhibitory forms of killer cell Ig-like receptors (KIR) contain two ITIMs. The role of each ITIM, and of the conserved residues upstream of the tyrosine, in the inhibition of NK cells was tested by vaccinia virus-mediated expression of mutant KIRs. Substitution of the tyrosine in the membrane-proximal ITIM abrogated the ability of KIR to block Ab-dependent cellular cytotoxicity, whereas mutation of the membrane-distal ITIM tyrosine had little effect. Substitution of the conserved hydrophobic amino acid that was located two residues N-terminal to the tyrosine weakened, but did not eliminate, the function of the receptor. In contrast, these substitutions drastically reduced the amount of SHP-1 immunoprecipitated with KIR, suggesting that weak interactions with SHP-1 may be sufficient for inhibition.
IL-4 Selectively Inhibits IL-2-Triggered Stat5 Activation, But Not Proliferation, in Human T Cells
Authors: Castro Aaron , Sengupta Tapas K. , Ruiz Donna C. , Yang Edward , Ivashkiv Lionel B.
Published: December 2022

IL-2 activates several distinct signaling pathways that are important for T cell activation, proliferation, and differentiation into both Th1 and Th2 phenotypes. IL-4, the major cytokine that promotes differentiation of Th2 cells, has been shown to block signaling of the Th1-promoting cytokine IL-12. As IL-2 synergizes with IL-12 in promoting Th1 differentiation, the effects of IL-4 on IL-2 signal transduction were investigated. IL-4 suppressed activation of DNA binding and tyrosine phosphorylation of the transcription factor Stat5 by IL-2, and suppressed the expression of the IL-2-inducible genes CD25, CIS, the PGE2 receptor, and cytokine responsive (CR) genes CR1 and CR8. Activation of Stat5 by cytokines that share a common γ receptor subunit, IL-2, IL-7, and IL-15, was suppressed by preculture in IL-4. Activation of the Jak1 and Jak3 kinases that are proximal to Stat5 in the IL-2-Jak-STAT signaling pathway was suppressed, and this correlated with inhibition of IL-2Rβ subunit expression. In contrast to suppression of Stat5, proliferative responses to IL-2 were augmented in IL-4-cultured cells, and activation of proliferative pathways leading to activation of mitogen activated protein kinases, induction of expression of Myc, Fos, Pim-1, and cyclin D3, and decreased levels of the cyclin-dependent kinase inhibitor p27 were intact. These results identify molecular mechanisms underlying interactions between IL-4 and IL-2 in T cells and demonstrate that one mechanism of regulation of IL-2 activity is selective and differential modulation of signaling pathways.
Comparison of Lung Dendritic Cells and B Cells in Stimulating Naive Antigen-Specific T Cells
Authors: Masten Barbara J. , Lipscomb Mary F.
Published: December 2022

Dendritic cells (DCs) are specialized APCs that are important in priming naive T cells and can be manipulated in vitro and in vivo to enhance immunizations against microorganisms and tumors. A limitation in the development of suitable immunotherapeutic vaccines for the lung is incomplete information on the role of DCs and other potential APCs in the lung in priming naive T cells. In the current study, we analyzed the relative contributions of murine lung DCs and B cells to process and present OVA to naive CD4+ OVA323–339-specific (DO11.10) T cells in vitro. We also examined their expression of MHC class II and accessory molecules before and after maturation in culture. Similar to DCs from other sites, freshly isolated lung DCs can process OVA, spontaneously up-regulate MHC class II and accessory molecules during overnight culture, and stimulate naive T cells in an Ag-specific manner. In contrast, freshly isolated lung B cells were unable to both process and present native OVA. Furthermore, under conditions of limited OVA323–339 peptide exposure, B cells had a significantly diminished capacity to stimulate T cells, and this correlated with a decreased density of both MHC class II and important costimulatory molecules as compared with lung DCs.
Specificity of the SH2 Domains of SHP-1 in the Interaction with the Immunoreceptor Tyrosine-Based Inhibitory Motif-Bearing Receptor gp49B
Authors: Wang Lawrence L. , Blasioli Julie , Plas David R. , Thomas Matthew L. , Yokoyama Wayne M.
Published: December 2022

Inhibitory receptors on hemopoietic cells critically regulate cellular function. Despite their expression on a variety of cell types, these inhibitory receptors signal through a common mechanism involving tyrosine phosphorylation of the immunoreceptor tyrosine-based inhibitory motif (ITIM), which engages Src homology 2 (SH2) domain-containing cytoplasmic tyrosine or inositol phosphatases. In this study, we have investigated the proximal signal-transduction pathway of an ITIM-bearing receptor, gp49B, a member of a newly described family of murine NK and mast cell receptors. We demonstrate that the tyrosine residues within the ITIMs are phosphorylated and serve for the association and activation of the cytoplasmic tyrosine phosphatase SHP-1. Furthermore, we demonstrate a physiologic association between gp49B and SHP-1 by coimmunoprecipitation studies from NK cells. To address the mechanism of binding between gp49B and SHP-1, binding studies involving glutathione S-transferase SHP-1 mutants were performed. Utilizing the tandem SH2 domains of SHP-1, we show that either SH2 domain can interact with phosphorylated gp49B. Full-length SHP-1, with an inactivated amino SH2 domain, also retained gp49B binding. However, binding to gp49B was disrupted by inactivation of the carboxyl SH2 domain of full-length SHP-1, suggesting that in the presence of the phosphatase domain, the carboxyl SH2 domain is required for the recruitment of phosphorylated gp49B. Thus, gp49B signaling involves SHP-1, and this association is dependent on tyrosine phosphorylation of the gp49B ITIMs, and an intact SHP-1 carboxyl SH2 domain.
Tpm1,a Locus Controlling IL-12 Responsiveness, Acts by a Cell-Autonomous Mechanism
Authors: Guler Mehmet L. , Gorham James D. , Dietrich William F. , Murphy Theresa L. , Steen Robert G. , Parvin Curtis A. , Fenoglio Dominic , Grupe Andrew , Peltz Gary , Murphy Kenneth M.
Published: December 2022

Th phenotype development is controlled not only by cytokines but also by other parameters including genetic background. One site of genetic variation between murine strains that has direct impact on Th development is the expression of the IL-12 receptor. T cells from B10.D2 and BALB/c mice show distinct control of IL-12 receptor expression. When activated by Ag, B10.D2 T cells express functional IL-12 receptors and maintain IL-12 responsiveness. In contrast, under the same conditions, BALB/c T cells fail to express IL-12 receptors and become unresponsive to IL-12, precluding any Th1-inducing effects if subsequently exposed to IL-12. Previously, we identified a locus, which we termed T cell phenotype modifier 1 (Tpm1), on murine chromosome 11 that controls this differential maintenance of IL-12 responsiveness. In this study, we have produced a higher resolution map around Tpm1. We produced and analyzed a series of recombinants from a first-generation backcross that significantly narrows the genetic boundaries of Tpm1. This allowed us to exclude from consideration certain previous candidates for Tpm1, including IFN-regulatory factor-1. Also, cellular analysis of F1(B10.D2 × BALB/c) T cells demonstrates that Tpm1 exerts its effect on IL-12 receptor expression in a cell-autonomous manner, rather than through influencing the extracellular milieu. This result strongly implies that despite the proximity of our locus to the IL-13/IL-4 gene cluster, these cytokines are not candidates for Tpm1.
Evidence for Immune Regulation in the Induction of Transplantation Tolerance: A Conditional but Limited Role for IL-4
Authors: Bushell Andrew , Niimi Masanori , Morris Peter J. , Wood Kathryn J.
Published: December 2022

Most experimental models of allograft tolerance depend on manipulation of immune responses at the time of transplant. In such systems, the graft itself probably plays an important role in the induction of unresponsiveness but as a consequence may suffer immune mediated damage. Ideally, recipients would be made specifically unresponsive before transplant such that the graft is protected from the outset. In this report, we demonstrate that CBA mice pretreated with donor-specific transfusion plus anti-CD4 Ab 28 days before transplant accept cardiac allografts indefinitely without further intervention. Adoptive transfer of spleen cells from mice with long term surviving grafts results in donor-specific graft acceptance in naive secondary recipients, indicating that tolerance in this system involves immuneregulation. Regulation develops as a result of the pretreatment protocol alone, since transfer of cells from pretreated but untransplanted mice to naive recipients also leads to prolonged allograft survival without additional therapy. Neutralizing IL-4 at the time of tolerance induction had no effect on graft outcome in primary recipients. However, removal of IL-4 from the adoptive transfer donors at the time of tolerance induction prevented long term engraftment in the majority of secondary recipients. Our data demonstrate that pretreatment of transplant recipients can establish immune regulation powerful enough to override the responses of an intact immune repertoire and that under stringent conditions at least, development of this regulatory population may in part be dependent on IL-4.
Induction of Specific T Cell Tolerance by Fas Ligand- Expressing Antigen-Presenting Cells
Authors: Zhang Huang-ge , Su Xiao , Liu Di , Liu Weimin , Yang Pingar , Wang Zheng , Edwards Carl K. , Bluethmann Horst , Mountz John D. , Zhou Tong
Published: December 2022

Autocrine interaction of Fas and Fas ligand leads to apoptosis of activated T cells, a process that is critical for the maintenance of peripheral T cell tolerance. Paracrine interactions of Fas ligand with T cells also may play an important role in the maintenance of tolerance, as Fas ligand can create immune-privileged sites and prevent graft rejection by inducing apoptosis in T cells. We surmised that APCs that express Fas ligand might directly induce apoptosis of T cells during presentation of Ag to the T cells, thus inducing Ag-specific, systemic T cell tolerance. Here, we show that profound, specific T cell unresponsiveness to alloantigen was induced by treatment of H-2k mice with H-2b APCs that expressed Fas ligand and that profound T cell unresponsiveness specific for the H-Y Ag was induced by treatment of H-2Db/H-Y TCR transgenic female mice with H-2Db/H-Y APCs that expressed Fas ligand. The induction of this systemic T cell tolerance required the expression of Fas ligand on the APCs as well as the expression of Fas on the T cells. The tolerance was restricted to the Ag presented by the APCs. The rapid and profound clonal deletion of the Ag-specific, peripheral T cells mediated by the Fas ligand-expressing APCs contributed to the induction of tolerance. These findings demonstrate that Ag-specific T cell tolerance can be induced by APCs that express Fas ligand and suggest a novel function for APCs in the induction of T cell apoptosis. Furthermore, they indicate a novel immunointervention strategy for treatment of graft rejection and autoantigen-specific autoimmune diseases.
Evidence for Repression of IL-2 Gene Activation in Anergic T Cells
Authors: Telander David G. , Malvey Erika-Nell , Mueller Daniel L.
Published: December 2022

The induction of clonal anergy in a T cell inhibits IL-2 secretion because of the development of a proximal signal transduction defect. Fusion of anergic murine T cells to human Jurkat T leukemia cells and formation of heterokaryons failed to result in a complementation of this signaling defect and restoration of murine IL-2 mRNA inducibility. Instead, signal transduction to the human IL-2 gene became disrupted. Heterokaryons formed by the fusion of anergic murine T cells to normal murine T cells also failed to accumulate intracellular IL-2 protein in response to stimulation either with the combination of CD3 and CD28 mAbs or with ionomycin plus a protein kinase C-activating phorbol ester. The results argue against a loss-of-function signaling defect as the sole basis for clonal anergy induction and document the presence of a dominant-acting repressor molecule that inhibits signal transduction to the IL-2 gene within viable anergic T cells.
Structure and Genomic Organization of a Second Cluster of Immunoglobulin Heavy Chain Gene Segments in the Channel Catfish
Authors: Ghaffari Seyed H. , Lobb Craig J.
Published: December 2022

The structure, organization, and partial sequence of a 25-kb genomic region containing a second cluster of H chain gene segments in the channel catfish (Ictalurus punctatus) has been determined. Multiple VH gene segments, representing different VH families, are located upstream of a germline-joined VDJ. The VDJ segment has a split leader sequence and a single open reading consistent with that expressed in members of the VH1 family. Downstream of the germline-joined VDJ is a single JH segment and two pseudogene exons structurally similar to the Cμ1 and Cμ2 exons of the functional gene. Both pseudogene exons are multiply crippled with RNA splice sites destroyed, and open reading frames are interrupted by termination codons, insertions, and/or deletions. Sequence alignment of a 10.8-kb region within the second H chain cluster with the genomic sequence of the nine JH segments and the functional Cμ within the first H chain gene cluster indicates that the second H chain gene cluster probably arose by a massive duplication event. The JH region of the VDJ, the coding and flanking regions of the single JH segment, and the pseudogene Cμ exons were readily aligned with homologous segments in the first gene cluster. This duplication event may have extended to include the upstream VH segments. A member of the Tc1 mariner family of transposable elements is located downstream of the pseudogene Cμ2, which suggests that the transposition may have contributed to the evolution of the duplicated Cμ.
MHC Class II-Dependent NK1.1+ γδ T Cells Are Induced in Mice bySalmonellaInfection
Authors: Nishimura Hitoshi , Washizu Junji , Naiki Yoshikazu , Hara Toru , Fukui Yoshinori , Sasazuki Takehiko , Yoshikai Yasunobu
Published: December 2022

We observed the emergence of a novel population of γδ T cells expressing NK1.1 Ag in the peritoneal cavity of mice infected with Salmonella choleraesuis. The NK1.1+γδ T cells accounted for approximately 20% of all γδ T cells emerging in the peritoneal cavity of C57BL/6 mice and expressed preferentially rearranged Vγ4-Jγ1 and Vδ6.3-Dδ1-Dδ2-Jδ1 genes with N diversity. The γδ T cells proliferated vigorously in response to PHA-treated spleen cells and produced IFN-γ in the culture supernatant. However, spleen cells from Aβb-deficient mice were unable to stimulate the γδ T cells. Furthermore, the NK1.1+γδ T cells were stimulated not only by Chinese hamster ovary (CHO) cells expressing wild-type IAb but also by those expressing IAb/Eα52-68 or IAb/pigeon cytochrome c-derived analogue peptide complex. These proliferation activities were inhibited by mAb specific for IAb chain. Consistent with these findings, the emergence of NK1.1+γδ T cells was reduced in the peritoneal cavity of Aβb-deficient mice after Salmonella infection, whereas NK1.1+γδ T cells were rather abundant in the peritoneal cavity of Salmonella-infected β2m-deficient mice. Moreover, the NK1.1+γδ T cells were easily identified in the thymus of β2m-deficient but not Aβb-deficient mice. Our results indicated that MHC class II expression is essential for development and activation of NK1.1+γδ T cells in the thymus and the periphery.
Decreased Leukotriene C4 Synthesis Accompanies Adherence-Dependent Nuclear Import of 5-Lipoxygenase in Human Blood Eosinophils
Authors: Brock Thomas G. , Anderson James A. , Fries Francine P. , Peters-Golden Marc , Sporn Peter H. S.
Published: December 2022

The enzyme 5-lipoxygenase (5-LO) catalyzes the synthesis of leukotrienes (LTs) from arachidonic acid (AA). Adherence or recruitment of polymorphonuclear neutrophils (PMN) induces nuclear import of 5-LO from the cytosol, which is associated with enhanced LTB4 synthesis upon subsequent cell stimulation. In this study, we asked whether adherence of human eosinophils (EOS) causes a similar redistribution of 5-LO and an increase in LTC4 synthesis. Purified blood EOS examined either in suspension or after adherence to fibronectin for 5 min contained only cytosolic 5-LO. Cell stimulation resulted in activation of 5-LO, as evidenced by its translocation to membranes and LTC4 synthesis. As with PMN, adherence of EOS to fibronectin for 120 min caused nuclear import of 5-LO. Unexpectedly, however, adherence also caused a time-dependent decrease in LTC4 synthesis: EOS adhered for 120 min produced 90% less LTC4 than did cells adhered for 5 min. Adherence did not diminish the release of [3H]AA from prelabeled EOS or reduce the synthesis of the prostanoids thromboxane and PGE2. Also, inhibition of LTC4 production caused by adherence could not be overcome by the addition of exogenous AA. Adherence increased, rather than decreased, LTC4 synthase activity. However, the stimulation of adherent EOS failed to induce translocation of 5-LO from the nucleoplasm to the nuclear envelope. This resistance to activation of the nuclear pool of 5-LO with diminished LT production represents a novel mode of regulation of the enzyme, distinct from the paradigm of up-regulated LT synthesis associated with intranuclear localization of 5-LO observed in PMN and other cell types.
Identification of Autoimmune T Cells Among In Vivo Expanded CD25+ T Cells in Multiple Sclerosis
Authors: Illés Zsolt , Kondo Takayuki , Yokoyama Kazumasa , Ohashi Takashi , Tabira Takeshi , Yamamura Takashi
Published: December 2022

Although clonal expansion of autoimmune T cells has been reported in multiple sclerosis (MS), very limited information is available on specificities, clonal size, or activation state of the expanded clones. Here we address the issue of clonal expansion by using a novel technique demonstrating clonotypes defined by single-strand conformation polymorphism of TCR β-chain cDNAs. Examination of activated T cells (CD3+CD25+) isolated from the peripheral blood of MS revealed limited numbers (20∼82) of expanded clones defined by single-strand conformation polymorphism clonotype. To estimate the Ag specificities of dominant clonotypes in the activated T cells, these samples were examined in parallel with Th1 T cell clones specific for myelin basic protein or proteolipid protein (PLP) derived from the same patients. Analysis of two patients demonstrated that the dominant clonotypes would contain those specific for myelin basic protein or PLP. Although the majority of the clonotypes could be detected only transiently, a PLP95–116-specific clonotype was found to persist for over 1 yr. Thus, single-strand conformation polymorphism clonotype analysis allows us to monitor the kinetics of given T cell clones in vivo and could provide useful information for designing clonotype (Id)-specific manipulation of human diseases such as MS.
B7.2 Has Opposing Roles During the Activation Versus Effector Stages of Experimental Autoimmune Thyroiditis
Authors: Peterson Karin E. , Sharp Gordon C. , Tang Haiwen , Braley-Mullen Helen
Published: December 2022

APCs provide costimulatory and down-regulatory signals to Ag-activated T cells through interactions between B7.1 and B7.2 on APCs with either CD28 or CTL Ag-4 expressed on T cells. Recipients of mouse thyroglobulin (MTg)-primed spleen cells activated in the presence of anti-B7.2 had decreased experimental autoimmune thyroiditis (EAT) severity compared with recipients of cells cultured with control rat Ig or anti-B7.1. Blocking B7.2 during in vivo priming also suppressed the ability of MTg-primed spleen cells to transfer EAT, implicating a role for B7.2 for priming and in vitro activation of EAT effector cells. In contrast, administration of anti-B7.2 or anti-B7.2 Fab to recipients of MTg-activated spleen cells increased the severity of EAT compared with recipients receiving control Ig. Thyroids from anti-B7.2-treated recipients had increased expression of IL-4 mRNA compared with thyroids from rat Ig-treated controls. Both B7.1 and B7.2 molecules were expressed in the thyroids of mice with EAT, although B7.2 was more prevalent than B7.1. Administration of both anti-B7.1 and anti-B7.2 to recipient mice suppressed the development of EAT, while anti-B7.1 treatment alone had no effect on EAT severity. The suppression of EAT was not observed when anti-B7.1 and anti-B7.2 treatment was delayed until 7 days after cell transfer, suggesting a requirement for B7 in the initiation of EAT in recipient mice. These results suggest that costimulation is required during the effector phase of EAT and that B7.2 may have opposing roles in the activation versus effector stages of autoreactive T cells.
Antigen Receptor-Induced Signal Transduction Imbalances Associated with the Negative Selection of Immature B Cells
Authors: King Leslie B. , Norvell Amanda , Monroe John G.
Published: December 2022

Signals transduced through the B cell Ag receptor (BCR) drive B cell development. However, BCR-induced responses are developmentally regulated; immature B cells are tolerized following antigenic exposure while mature B cells are triggered to proliferate and differentiate. This differential responsiveness allows for the negative selection of self-reactive immature B cells while simultaneously allowing for clonal expansion of mature B cells in response to foreign Ags. Intrinsic differences in BCR-induced signal transduction at various stages of development may account for this functional dichotomy. We had previously demonstrated that the BCR-induced proliferation of mature B cells is accompanied by an increase in intracellular calcium levels and polyphosphoinositide bis phosphate (PIP2) hydrolysis. In contrast, immature B cells that undergo BCR-induced apoptosis increase intracellular calcium in the relative absence of PIP2 hydrolysis. Since PIP2 hydrolysis leads to the generation of diacylglycerol, a cofactor for protein kinase C (PKC) activation, these data suggested that an “imbalance” in BCR-induced signal transduction resulting from a relative inability to activate PKC may play a role in the susceptibility of immature B cells to BCR-induced apoptosis. In support of this hypothesis, we demonstrate that PKC activation can rescue immature B cells from BCR-induced apoptosis. Furthermore, the susceptibility of immature B cells to BCR-induced apoptosis is recapitulated in mature B cells that are either PKC depleted or are stimulated in the presence of PKC inhibitors, suggesting that an uncoupling of PKC activation from BCR-induced signaling is responsible for the apoptotic response of immature B cells.
Soluble Class I MHC with β2-Microglobulin Covalently Linked Peptides: Specific Binding to a T Cell Hybridoma
Authors: White Janice , Crawford Frances , Fremont Daved , Marrack Philippa , Kappler John
Published: December 2022

Soluble forms of the mouse MHC class I molecule, Dd, were produced in which the peptide binding groove was uniformly occupied by peptides attached via a covalent flexible peptide linker to the N terminus of the associated β2-microglobulin. The MHC heavy chain and β2-microglobulin were firmly associated, and the molecules displayed an Ab epitope requiring proper occupancy of the peptide binding groove. Soluble Dd containing a covalent version of a well-characterized Dd-binding peptide from HIV stimulated a T cell hybridoma specific for this combination. Furthermore, a tetravalent version of this molecule bound specifically with apparent high avidity to this hybridoma.
Regulators of G Protein Signaling Exhibit Distinct Patterns of Gene Expression and Target G Protein Specificity in Human Lymphocytes
Authors: Beadling Carol , Druey Kirk M. , Richter Gunther , Kehrl John H. , Smith Kendall A.
Published: December 2022

The newly recognized regulators of G protein signaling (RGS) attenuate heterotrimeric G protein signaling pathways. We have cloned an IL-2-induced gene from human T cells, cytokine-responsive gene 1, which encodes a member of the RGS family, RGS16. The RGS16 protein binds Giα and Gqα proteins present in T cells, and inhibits Gi- and Gq-mediated signaling pathways. By comparison, the mitogen-induced RGS2 inhibits Gq but not Gi signaling. Moreover, the two RGS genes exhibit marked differences in expression patterns. The IL-2-induced expression of the RGS16 gene in T cells is suppressed by elevated cAMP, whereas the RGS2 gene shows a reciprocal pattern of regulation by these stimuli. Because the mitogen and cytokine receptors that trigger expression of RGS2 and RGS16 in T cells do not activate heterotrimeric G proteins, these RGS proteins and the G proteins that they regulate may play a heretofore unrecognized role in T cell functional responses to Ag and cytokine activation.
IL-4 Preferentially Activates a Novel STAT6 Isoform in Mast Cells
Authors: Sherman Melanie A. , Secor Virginia H. , Brown Melissa A.
Published: December 2022

IL-4 is a pleiotropic cytokine that signals through STAT6 to direct the transactivation of multiple gene targets. In this study, we demonstrate that mast cells express a distinct STAT6 isoform. This “mast cell STAT” is a product of the STAT6 gene, but is only 65 kDa in size and appears to lack the defined C-terminal transactivation domain. Despite the presence of the conventional 94-kDa STAT6 molecule, it is the smaller isoform that associates with a consensus STAT6 binding site in extracts from IL-4-treated mast cells. This is the first evidence that STAT6 isoforms can be preferentially activated and bind to DNA in a cell-specific manner. These results imply that an additional level of specificity in the IL-4R signaling mechanism exists and may partially explain the diverse effects that IL-4 exerts on different cell types.
Dissecting NK Cell Development Using a Novel Alymphoid Mouse Model: Investigating the Role of the c-abl Proto-Oncogene in Murine NK Cell Differentiation
Authors: Colucci Francesco , Soudais Claire , Rosmaraki Eleftheria , Vanes Lesley , Tybulewicz Victor L. J. , Di Santo James P.
Published: December 2022

NK lymphocytes participate in both innate and adaptive immunity by their prompt secretion of cytokines including IFN-γ, which activates macrophages, and by their ability to lyse virally infected cells and tumor cells without prior sensitization. Although these characteristics of NK cells are well documented, little is known about the genetic program that orchestrates NK development or about the signaling pathways that trigger NK effector functions. By crossing NK-deficient common γ-chain (γc) and recombinase activating gene (RAG)-2 mutant mice, we have generated a novel alymphoid (B−, T−, and NK−) mouse strain (RAG2/γc) suitable for NK complementation in vivo. The role of the c-abl proto-oncogene in murine NK cell differentiation has been addressed in hemopoietic chimeras generated using RAG2/γc mice reconstituted with c-abl−/− fetal liver cells. The phenotypically mature NK cells that developed in the absence of c-abl were capable of lysing tumor targets, recognizing “missing self,” and performing Ab-dependent cellular cytotoxicity. Taken together, these results exclude any essential role for c-abl in murine NK cell differentiation in vivo. The RAG2/γc model thereby provides a novel approach to establish a genetic map of NK cell development.
IL-5 Induces IgG1 Isotype Switch Recombination in Mouse CD38-Activated sIgD-Positive B Lymphocytes
Authors: Mizoguchi Chieko , Uehara Shoji , Akira Shizuo , Takatsu Kiyoshi
Published: December 2022

Mouse B cells express CD38, whose ligation by anti-CD38 Ab induces their proliferation and protection from apoptosis. We previously showed that stimulation of mouse splenic B cells with IL-5 together with CS/2, an anti-mouse CD38 mAb, induces production of IgG1 and IgM. Here we examined the role of IL-5 and CS/2 in the expression of germline γ1 transcripts and the generation of reciprocal products forming DNA circles as byproducts of μ-γ1 switch recombination. By itself, CS/2 induced significant expression of germline γ1 transcripts in splenic naive B cells, whereas IL-5 neither induced nor enhanced germline γ1 expression. Increased cellular content of reciprocal product, which is characteristic of μ-γ1 recombination, was not observed after culturing B cells with CS/2, but increased reciprocal product, along with high levels of lgG1 secretion, was found when B cells were cultured with CS/2 plus IL-5. Although IL-4 did not, by itself, induce μ-γ1 recombination in B cells stimulated with CS/2, in conjunction with CS/2 plus IL-5, IL-4 dramatically enhanced sterile γ1 transcription and IgG1 production. These results demonstrate that CD38 ligation induces only germline γ1 transcription and that IL-5 promotes both μ-γ1 switch recombination and lgG1 secretion in an IL-4-independent manner.
NF-ATc Isoforms Are Differentially Expressed and Regulated in Murine T and Mast Cells
Authors: Sherman Melanie A. , Powell Doris R. , Weiss Deborah L. , Brown Melissa A.
Published: December 2022

NF of activated T cells (NF-AT) denotes a family of transcription factors that regulate the activation-dependent expression of many immunologically important proteins. At least four distinct genes encode the various family members, and several isoforms of these have been identified as well. The overlapping expression patterns and similar in vitro binding and trans-activation activities on various promoter elements of NF-AT-regulated genes suggest some redundancy in the function of these proteins. However, the phenotypic analysis of NF-AT-deficient mice supports the idea that there are tissue- and gene-specific functions as well. In this study we have characterized the expression of NF-AT cDNAs in murine mast cells. The majority of clones identified correspond to two NF-ATc isoforms that differ only in their amino-terminal sequence. Despite minimal discrepancies in the coding region, there are striking tissue- and cell type-specific differences in isoform expression patterns. Detection of NF-ATc.α mRNA is strictly dependent on cell activation signals in both T and mast cell lines. In contrast, the β isoform is expressed at very low constitutive levels in both cell types but is only up-regulated in response to mast cell activation signals delivered through the FcεRI or via calcium ionophores. These results demonstrate another level of regulation within the NF-AT family that can contribute to cell type-specific gene expression.
Cancer Dormancy. VII. A Regulatory Role for CD8+ T Cells and IFN-γ in Establishing and Maintaining the Tumor-Dormant State
Authors: Farrar J. David , Katz Kathy H. , Windsor Jana , Thrush Gerald , Scheuermann Richard H. , Uhr Jonathan W. , Street Nancy E.
Published: December 2022

Dormant tumor cells resistant to ablative cancer therapy represent a significant clinical obstacle due to later relapse. Experimentally, the murine B cell lymphoma (BCL1) is used as a model of tumor dormancy in mice vaccinated with the BCL1 Ig. Here, we used this model to explore the cellular mechanisms underlying dormancy. Our previous studies have demonstrated that T cell-mediated immunity is an important component in the regulation of tumor dormancy because Id-immune T cells adoptively transferred into passively immunized SCID mice challenged with BCL1 cells significantly increased the incidence and duration of the dormant state. We have extended these observations and demonstrate that CD8+, but not CD4+, T cells are required for the maintenance of dormancy in BCL1 Ig-immunized BALB/c mice. In parallel studies, the transfer of Id-immune CD8+ cells, but not Id-immune CD4+ cells, conferred significant protection to SCID mice passively immunized with nonprotective levels of polyclonal anti-Id and then challenged with BCL1 cells. Furthermore, the ability of CD8+ T cells to induce a state of dormancy in passively immunized SCID mice was completely abrogated by treatment with neutralizing α-IFN-γ mAbs in vivo. In vitro studies demonstrated that IFN-γ alone or in combination with reagents to cross-link the surface Ig induced both cell cycle arrest and apoptosis in a BCL1 cell line. Collectively, these data demonstrate a role for CD8+ T cells via endogenous production of IFN-γ in collaboration with humoral immunity to both induce and maintain a state of tumor dormancy.
IFN-γ and CD8+ T Cells Restore Host Defenses AgainstPneumocystis cariniiin Mice Depleted of CD4+ T Cells
Authors: Kolls Jay K. , Habetz Scott , Shean Mary K. , Vazquez Carlos , Brown Julie A. , Lei Dinghua , Schwarzenberger Paul , Ye Peng , Nelson Steve , Summer Warren R. , Shellito Judd E.
Published: December 2022

Host defenses against infection are profoundly compromised in HIV-infected hosts due to progressive depletion of CD4+ T lymphocytes and defective cell-mediated immunity. Although recent advances in antiretroviral therapy can dramatically lower HIV viral load, blood CD4+ T lymphocytes are not restored to normal levels. Therefore, we investigated mechanisms of host defense other than those involving CD4+ T lymphocytes against a common HIV-related opportunistic infection, Pneumocystis carinii (PC) pneumonia. Using CD4-depleted mice, which are permissive for chronic PC infection, we show that up-regulation of murine IFN-γ by gene transfer into the lung tissue results in clearance of PC from the lungs in the absence of CD4+ lymphocytes. This resolution of infection was associated with a >4-fold increase in recruited CD8+ T lymphocytes and NK cells into the lungs. The role of CD8+ T cells as effector cells in this model was further confirmed by a lack of an effect of IFN-γ gene transfer in scid mice or mice depleted of both CD4+ and CD8+ T cells. Cytokine mRNA analysis revealed that recruited, lung-derived CD8+ T cells had greater expression of IFN-γ message in animals treated with the IFN-γ gene. These results indicate that CD8+ T cells are capable of clearing PC pneumonia in the absence of CD4+ T cells and that this host defense function of CD8+ T cells, as well as their cytokine repertoire, can be up-regulated through cytokine gene transfer.
The Human T Cell Leukemia Virus Type I-tax Gene Is Responsible for the Development of Both Inflammatory Polyarthropathy Resembling Rheumatoid Arthritis and Noninflammatory Ankylotic Arthropathy in Transgenic Mice
Authors: Habu Kiyoshi , Nakayama-Yamada Junko , Asano Masahide , Saijo Shinobu , Itagaki Keiko , Horai Reiko , Yamamoto Hiroaki , Sekiguchi Toyozo , Nosaka Tetsuya , Hatanaka Masakazu , Iwakura Yoichiro
Published: December 2022

We previously reported that inflammatory arthropathy resembling rheumatoid arthritis (RA) develops among transgenic mice carrying the long terminal repeat (LTR)-env-pX-LTR region of human T cell leukemia virus type I (LTR-pX-Tg mice). Because four genes are encoded in this region, we produced transgenic mice that only express the tax gene to examine its role in the development of arthritis. Transgenic mice were produced by constructing DNAs that express the tax gene alone under the control of either its own LTR or CD4 enhancer/promoter and by microinjecting them into C3H/HeN-fertilized ova. We produced seven transgenic mice carrying the LTR-tax gene and nine mice carrying the CD4-tax and found that one of the LTR-tax-Tg mice and five of CD4-tax-Tg mice developed RA-like inflammatory arthropathy similar to LTR-pX-Tg mice, indicating that the tax gene is arthritogenic. On the other hand, the other two LTR-tax-Tg mice had ankylotic changes caused by new bone formation without inflammation. In these ankylotic mice, tax mRNA, inflammatory cytokine mRNA, and autoantibody levels except for TGF-β1 level were lower than those in LTR-pX- or CD4-tax-Tg mice. These results show that Tax is responsible for the development of inflammatory arthropathy resembling RA and that this protein also causes ankylotic arthropathy.
Lymphocyte Migration Through Brain Endothelial Cell Monolayers Involves Signaling Through Endothelial ICAM-1 Via a Rho-Dependent Pathway
Authors: Adamson Peter , Etienne Sandrine , Couraud Pierre-Olivier , Calder Virginia , Greenwood John
Published: December 2022

Lymphocyte extravasation into the brain is mediated largely by the Ig superfamily molecule ICAM-1. Several lines of evidence indicate that at the tight vascular barriers of the central nervous system (CNS), endothelial cell (EC) ICAM-1 not only acts as a docking molecule for circulating lymphocytes, but is also involved in transducing signals to the EC. In this paper, we examine the signaling pathways in brain EC following Ab ligation of endothelial ICAM-1, which mimics adhesion of lymphocytes to CNS endothelia. ICAM-1 cross-linking results in a reorganization of the endothelial actin cytoskeleton to form stress fibers and activation of the small guanosine triphosphate (GTP)-binding protein Rho. ICAM-1-stimulated tyrosine phosphorylation of the actin-associated molecule cortactin and ICAM-1-mediated, Ag/IL-2-stimulated T lymphocyte migration through EC monolayers were inhibited following pretreatment of EC with cytochalasin D. Pretreatment of EC with C3 transferase, a specific inhibitor of Rho proteins, significantly inhibited the transmonolayer migration of T lymphocytes, endothelial Rho-GTP loading, and endothelial actin reorganization, without affecting either lymphocyte adhesion to EC or cortactin phosphorylation. These data show that brain vascular EC are actively involved in facilitating T lymphocyte migration through the tight blood-brain barrier of the CNS and that this process involves ICAM-1-stimulated rearrangement of the endothelial actin cytoskeleton and functional EC Rho proteins.
Cutting Edge: Adenovirus E19 Has Two Mechanisms for Affecting Class I MHC Expression1
Authors: Bennett Elizabeth M. , Bennink Jack R. , Yewdell Jonathan W. , Brodsky Frances M.
Published: December 2022

Viral strategies for immune evasion include inhibition of various steps in the class I MHC assembly pathway. Here, we demonstrate that adenovirus produces one gene product with a dual function in this regard. It is well established that adenovirus E19 binds class I molecules and retains them in the endoplasmic reticulum (ER). However, E19 also delays the expression of class I alleles to which it cannot tightly bind. Here, we show that E19 binds TAP and acts as a tapasin inhibitor, preventing class I/TAP association. ΔE19, an E19 mutant lacking the ER-retention signal, delays maturation of class I molecules, indicating that E19’s inhibition of class I/TAP interaction is sufficient to delay class I expression. These data identify tapasin inhibition as a novel mechanism of viral immune evasion and suggest that, through this secondary mechanism, adenovirus can affect Ag presentation by MHC alleles that it can only weakly affect by direct retention.
Orally Induced Peripheral Nonresponsiveness Is Maintained in the Absence of Functional Th1 or Th2 Cells
Authors: Shi Hai Ning , Grusby Michael J. , Nagler-Anderson Cathryn
Published: December 2022

Intragastric administration of soluble protein Ags results in peripheral tolerance to the fed Ag. To examine the role of cytokine regulation in the induction of oral tolerance, we fed OVA to mice deficient in Th1 (Stat 4−/−) and Th2 (Stat 6−/−) cells and compared their response to that of normal BALB/c controls. We found that, in spite of these deficiencies, OVA-specific peripheral cell-mediated and humoral nonresponsiveness was maintained in both Stat 4−/− and Stat 6−/− mice. In the mucosa, both Peyer’s patch T cell proliferative responses and OVA-specific fecal IgA were reduced in Stat 4−/− and Stat 6−/− mice fed OVA but not in normal BALB/c controls. Mucosal, but not peripheral, nonresponsiveness was abrogated by the inclusion of a neutralizing Ab to TGF-β in the culture medium. Our results show that, in the periphery, tolerance to oral Ag can be induced in both a Th1- or Th2-deficient environment. In the mucosa, however, the absence of Th1 and Th2 cytokines can markedly affect this response, perhaps by regulation of TGF-β-secreting cells.
Signaling Through CD5 Involves Acidic Sphingomyelinase, Protein Kinase C-ζ, Mitogen-Activated Protein Kinase Kinase, and c-Jun NH2-Terminal Kinase
Authors: Simarro María , Calvo Javier , Vilà Josep M. , Places Lourdes , Padilla Olga , Alberola-Ila José , Vives Jordi , Lozano Francisco
Published: December 2022

The CD5 lymphocyte surface glycoprotein is a coreceptor involved in the modulation of Ag-specific receptor-mediated activation and differentiation signals. The molecular basis for its modulatory properties is not yet well understood. In the present study we describe early biochemical events triggered by CD5 stimulation, which include the phosphatidylcholine-specific phospholipase C (PC-PLC)-dependent activation of acidic sphingomyelinase (A-SMase) in normal and lymphoblastoid T and B cells. The functional coupling of PC-PLC and A-SMase is demonstrated by the abrogation of A-SMase activation by 1) xanthogenate tricyclodecan-9-yl (D609), a selective inhibitor of PC-PLC, and 2) replacement of several C-terminal serine residues (S458, S459, and S461) present in the cytoplasmic tail of CD5 that are known to be critical for PC-PLC activation. Additionally, we demonstrate that activation of protein kinase C-ζ (PKC-ζ) and members of the mitogen-activated protein kinase (MAPK) cascade (MAPK kinase and c-Jun NH2-terminal kinase), but not the NF-κB, are downstream events of the CD5 signaling pathway. A-SMase, PKC-ζ, and MAPK family members are key mediators of cell responses as diverse as proliferation, differentiation, and growth arrest and may contribute to CD5-mediated modulation of TCR or BCR signaling.
Splenic NK1.1-Negative, TCRαβ Intermediate CD4+ T Cells Exist in Naive NK1.1 Allelic Positive and Negative Mice, with the Capacity to Rapidly Secrete Large Amounts of IL-4 and IFN-γ Upon Primary TCR Stimulation
Authors: Moodycliffe Angus M. , Maiti Sourindra , Ullrich Stephen E.
Published: December 2022

Splenic NK1.1+CD4+ T cells that express intermediate levels of TCRαβ molecules (TCRint) and the DX5 Ag (believed to identify an equivalent population in NK1.1 allelic negative mice) possess the ability to rapidly produce high quantities of immunomodulatory cytokines, notably IL-4 and IFN-γ, upon primary TCR activation in vivo. Indeed, only T cells expressing the NK1.1 Ag appear to be capable of this function. In this study, we demonstrate that splenic NK1.1-negative TCRintCD4+ T cells, identified on the basis of FcγR expression, exist in naive NK1.1 allelic positive (C57BL/6) and negative (C3H/HeN) mice with the capacity to produce large amounts of IL-4 and IFN-γ after only 8 h of primary CD3 stimulation in vitro. Furthermore, a comparison of the amounts of early cytokines produced by FcγR+CD4+TCRint T cells with NK1.1+CD4+ or DX5+CD4+TCRint T cells, simultaneously isolated from C57BL/6 or C3H/HeN mice, revealed strain and population differences. Thus, FcγR defines another subpopulation of splenic CD4+TCRint cells that can rapidly produce large concentrations of immunomodulatory cytokines, suggesting that CD4+TCRint T cells themselves may represent a unique family of immunoregulatory CD4+ T cells whose members include FcγR+CD4+ and NK1.1/DX5+CD4+ T cells.