bluuebunny/crossref_metadata_2025 huggingface dataset:
Bacille Calmette-Guérin Vaccination Enhances Human γδ T Cell Responsiveness to Mycobacteria Suggestive of a Memory-Like Phenotype
Authors: Hoft Daniel F. , Brown Robin M. , Roodman Stanford T.
Published: December 2022

Bacille Calmette-Guérin (BCG) immunity can be studied as one experimental model for mycobacterial protective immunity. We have used flow cytometry to investigate human T cell subsets induced by BCG vaccination. PBMC harvested from BCG-vaccinated individuals and controls were stimulated with mycobacterial Ags, and the T cell subsets present after 7 days of in vitro expansion were characterized. The most dramatic expansions induced by mycobacterial Ags were detected in γδ T cells. The γδ T cell expansions measured after in vitro stimulation with mycobacterial Ags were significantly greater in BCG responders compared with nonsensitized controls, indicating that BCG vaccination induced γδ T cell activation associated with enhanced secondary responses. The majority of γδ T cells induced by BCG vaccination were γ9+δ2+ T cells reactive with isoprenyl pyrophosphates. Coculture with CD4+ T cells induced optimal γδ T cell expansion, although IL-2 alone could provide this helper function in the absence of CD4+ T cells. γδ T cells were found to provide helper functions for mycobacterial specific CD4+ and CD8+ T cells as well, demonstrating reciprocal stimulatory interactions between γδ T cells and other T cell subsets. Finally, prominent mycobacterial specific γδ T cell expansions were detected in a subset of unvaccinated controls with evidence for prior sensitization to mycobacterial lysates (elevated mycobacterial specific lymphoproliferative responses). These latter findings are consistent with the hypothesis that exposure to atypical mycobacteria or related environmental Ags may induce γδ T cells cross-reactive with Ags present in the Mycobacterium tuberculosis complex. Our results suggest that γδ T cells may be capable of developing a memory immune-like phenotype, and therefore might be important targets for new vaccines.
The Clonal Composition of a Peptide-Specific Oligoclonal CTL Repertoire Selected in Response to Persistent EBV Infection Is Stable Over Time
Authors: Levitsky Victor , de Campos-Lima Pedro-Otavio , Frisan Teresa , Masucci Maria G.
Published: December 2022

The TCR repertoire of a peptide-specific HLA A11-restricted CTL response to persistent infection with EBV was followed for a period of 57 mo. Sequencing of TCR Vα and Vβ chains and alanine scanning mutagenesis analysis of 83 CTL clones isolated in five reactivation experiments demonstrated that this repertoire is composed of at least four distinct CTL clonotypes that are constantly reactivated from donor’s blood and express structurally heterogeneous TCRs. Target cell recognition and CD8 blocking experiments indicate that the four clonotypes possess different avidity and TCR affinity for the specific Ag. This demonstrates that at least in some individuals a heterogeneous peptide-specific memory CTL repertoire selected by a persistent Ag can be remarkably stable in time and accommodate a range of TCR affinities and T cell avidities. Our results suggest that competition for the specific Ag may be not the major force driving the maintenance of memory CTLs and that the nature of the first antigenic challenge may largely determine the clonal composition of memory.
Potentiation by Thyroid Hormone of Human IFN-γ-Induced HLA-DR Expression
Authors: Lin Hung-Yun , Martino Leon J. , Wilcox Brian D. , Davis Faith B. , Gordinier Jennifer K. , Davis Paul J.
Published: December 2022

We have investigated the mechanism by which thyroid hormone potentiates IFN-γ-induced HLA-DR expression. IFN-γ-induced HLA-DR expression requires activation of STAT1α and induction of the Class II trans-activator, CIITA. HeLa and CV-1 cells treated only with l-thyroxine (T4) demonstrated increased tyrosine phosphorylation and nuclear translocation (= activation) of STAT1α; this hormone effect on signal transduction, and T4 potentiation of IFN-γ-induced HLA-DR expression, were blocked by the inhibitors CGP 41251 (PKC) and genistein (tyrosine kinase). Treatment of cells with T4-agarose also caused activation of STAT1α. In the presence of IFN-γ, T4 enhanced cytokine-induced STAT1α activation. Potentiation by T4 of IFN-γ action was associated with increased mRNA for both CIITA and HLA-DR, with peak enhancement at 16 h (CIITA), and 2 d (HLA-DR). T4 increased IFN-γ-induced HLA-DR protein 2.2-fold and HLA-DR mRNA fourfold after 2 d. Treatment with actinomycin D after induction of HLA-DR mRNA with IFN-γ, with or without T4, showed that thyroid hormone decreased the t1/2 of mRNA from 2.4 to 1.1 h. HeLa and CV-1 cells lack functional nuclear thyroid hormone receptor. Tetraiodothyroacetic acid (tetrac) and 3,5,3′-triiodo-thyroacetic acid (triac) blocked T4 potentiation of IFN-γ-induced HLA-DR expression and T4 activation of STAT1α. These studies define an early hormone recognition step at the cell surface that is novel, distinct from nuclear thyroid hormone receptor, and blocked by tetrac and triac. Thus, thyroid hormone potentiation of IFN-γ-induced HLA-DR transcription is mediated by a cell membrane hormone binding site, enhanced activation of STAT1α, and increased CIITA induction.
Regulation of Apoptosis by Tyrosine-Containing Domains of IL-4Rα: Y497 and Y713, But Not the STAT6-Docking Tyrosines, Signal Protection from Apoptosis
Authors: Zamorano José , Keegan Achsah D.
Published: December 2022

IL-4 is a cytokine with important antiapoptotic activity. We have analyzed the role that tyrosine-containing domains within the cytoplasmic tail of IL-4Rα play in IL-4-mediated protection from apoptosis. 32D cells expressing a wt huIL-4Rα or one truncated at aa 557 were protected by huIL-4 from apoptosis while cells expressing a receptor truncated at aa 657 were not, suggesting that the carboxyl-terminal domain signals protection from apoptosis. However, changing Y713 within this region to phenylalanine had no effect. To analyze the contribution of tyrosine-containing domains independently, we transplanted regions of the huIL-4Rα to a truncated form of the huIL-2Rβ that could not signal protection from apoptosis. Transplantation of the huIL-4Rα domains containing Y497 or Y713 partially prevented cell death and together signaled protection from apoptosis in response to IL-2 as well as the wt IL-2Rβ. Mutation of Y497 and Y713 to phenylalanine inhibited protection. In contrast, transplantation of the domain containing the potential STAT6-docking tyrosines alone had no effect, yet it inhibited the protection mediated by the other domains. Although IL-4Rα signals Shc and SH2-containing inositol phosphatase (SHIP) phosphorylation, we could not establish an association between their activation and protection from apoptosis. Taken together, this study suggests that the domains of the huIL-4Rα containing Y497 and Y713 positively regulate protection from apoptosis while the domain containing the STAT6 docking sites suppresses this protection, and that additional signaling molecules other than insulin receptor substrate-1 (IRS1), Shc, or SHIP may be involved in antiapoptotic signaling.
Cutting Edge: Induction of Antigen-Specific Hyporesponsiveness by Transplantation of Hemopoietic Cells Containing an MHC Class I Transgene Regulated by a Lymphocyte-Specific Promoter
Authors: Hansal Susan A. , Morris Diane I. , Sechler Joan M. G. , Love Paul E. , Rosenberg Amy S.
Published: December 2022

We explored a novel approach to tolerance induction by the transplantation of bone marrow (BM) cells (BMCs) that themselves do not express a foreign histocompatibility Ag, but which give rise to mature lymphocytes that do so. Lines of transgenic (FVB) mice were generated that contained an MHC class I Dd cDNA regulated by a CD2 promoter. Because the CD2 promoter is lymphocyte-specific and activated relatively late in lymphocyte ontogeny, Dd is expressed on most mature lymphocytes in the periphery but only on developing B cells in the BM of transgenic mice. Transgenic BMCs are tolerogenic and reproducibly engraft in nontransgenic mice using a conditioning regimen that is nonpermissive for the engraftment of conventional (MHC promoter) Dd-transgenic BMCs. Engrafted BMCs generate transgene-expressing lymphocytes and confer a state of Ag-specific hyporesponsiveness on the host that is primarily attributable to a peripheral mechanism. The strategies by which tolerance can be optimized in this system are discussed.
Novel Diversity in Th1, Th2 Type Differentiation of Hemagglutinin-Specific T Cell Clones Elicited by Natural Influenza Virus Infection in Three Major Haplotypes (H-2b,d,k)
Authors: Graham Christine M. , Smith Claire A. , Thomas D. Brian
Published: December 2022

We report novel diversity in the lymphokine (LK) secretion profile of hemagglutinin-specific, CD4+ T cell clones elicited by influenza virus infection in three major haplotypes: I-Ad- or I-Ed-restricted T cell clones obtained from individual BALB/c donors, and specific for three distinct antigenic peptides (p56–76, or p186–205 or p177–199), were uniformly Th1 type, releasing only IFN-γ on activation. In contrast, extensive diversity was evident for the C57BL/10 or CBA/Ca repertoire. Sibling T cell clones, established from the same C57BL/10 donor and expressing identical TCR β-chains in their recognition of p186–205, released either (IFN-γ and IL-5) or (IFN-γ and IL-4 and IL-5) or (IL-4 and IL-5 and IL-10) following Ag-specific or nonspecific stimulation. Similarly, I-Ak-restricted T cell clones, specific for p120–139 secreted either (IFN-γ only) or (IFN-γ and IL-5) or (IFN-γ and IL-2 and IL-5) on activation. Despite such phenotypic diversity within the individual’s repertoire, all clones had been maintained under identical in vitro culture conditions. Moreover, sequence analyses of TCR β gene usage indicated that in most instances clones from the same donor expressed identical (VDJ)β rearrangements, indicative of a common progenitor cell. FACS analysis of cytoplasmic cytokine production confirmed that for the novel phenotype (IFN-γ and IL-5), both LKs were synthesized at the single cell level. Sibling families of T cell clones, established from a common donor following viral infection but differing in LK secretion, may offer a suitable model system for further studies of signal transduction mechanisms that discriminate between Th1- and Th2-specific responses to a well defined protective Ag.
A Pivotal Role of Cyclin D3 and Cyclin-Dependent Kinase Inhibitor p27 in the Regulation of IL-2-, IL-4-, or IL-10-Mediated Human B Cell Proliferation
Authors: Wagner Eric F. , Hleb Marija , Hanna Nazeeh , Sharma Surendra
Published: December 2022

The functional differences between IgDhighCD38− naive and IgD−CD38− memory (M) or IgDlowCD38+ germinal center (GC) B cells may stem from their variable response to signals that regulate activation, proliferation, and differentiation. In this report, we provide evidence for differential induction of cell cycle regulators in tonsillar human B cell subpopulations that were activated with anti-IgM and anti-CD40 in the presence or absence of IL-2, IL-4, or IL-10. Naive (IgDhigh) B cells exhibited a significant proliferative response to IL-4, but not to IL-2 or IL-10, whereas these cytokines triggered variable levels of growth in the combined GC/M subpopulation (referred to as IgDlow), as measured by [3H]thymidine incorporation. Induction of growth by cytokines in B cell subpopulations strictly correlated with the increased levels of cyclin D3 and cyclin-dependent protein kinase (cdk) 6. Moreover, only cyclin D3/cdk6 complexes were functional as observed in both naive and GC/M B cells stimulated in the presence of IL-4. In addition, active growth was associated with cytokine-mediated elimination of the cell cycle inhibitor p27. The significance of p27 in human B cell cycle was further demonstrated by rapamycin-mediated growth inhibition of IL-4-dependent proliferation, which resulted in strikingly increased p27 levels. Taken together, our findings suggest that cyclin D3, cdk6, and p27 play key roles in IL-2-, IL-4-, and IL-10-mediated human B cell proliferation. Furthermore, these results may provide a molecular basis for different cycling characteristics of naive and GC/M B cell subpopulations.
Surrogate Light Chain Production During B Cell Differentiation: Differential Intracellular Versus Cell Surface Expression
Authors: Wang Yui-Hsi , Nomura Jun , Faye-Petersen Ona Marie , Cooper Max D.
Published: December 2022

Expression of the surrogate light (ψL) chain genes encoding the VpreB and λ5/14.1 proteins is restricted to B-lineage cells. Pro-B and pre-B cells produce ψL chains, but whether both employ these as cell surface receptor components remains enigmatic. Recombinant human VpreB protein was used to generate a large panel of monoclonal anti-VpreB Abs to examine this issue. Native ψL chain proteins within pro-B cells as well as those serving as receptor components on pre-B cells were precipitated by 16 of the 26 anti-VpreB Abs. Surrogate light chains were easily detected on pre-B cell lines, whereas these anti-VpreB Abs reacted with pro-B cell lines only after plasma membrane permeabilization. The subpopulation of normal bone marrow cells bearing pre-B receptors included large and small pre-B cells exclusively, although pro-B cells also contained intracellular VpreB. VpreB proteins were not detected on or within B cells in bone marrow or the circulation, but a subpopulation of B cells in germinal centers was found to express the VpreB proteins intracellularly. Surrogate L chains are thus intermittently produced during human B-lineage differentiation, while their role as receptor components appears limited to the pre-B cell stage.
Monocyte-Dependent Death of Freshly Isolated T Lymphocytes: Induction by Phorbolester and Mitogens and Differential Effects of Catalase
Authors: Wesch Daniela , Marx Sibylle , Kabelitz Dieter
Published: December 2022

Resting T cells are resistant to anti-Fas (CD95) mAb-mediated apoptosis but undergo apoptosis when triggered by anti-CD3 mAb or phorbolester PMA in the presence of PMA-activated monocytes. In this study, PMA, as well as the mitogens PHA and Con A, was found to induce death of resting T cells in the presence of autologous or allogeneic monocytes, while PWM was ineffective. Although several established monocytic and myelocytic cell lines were potent accessory cells for the mitogen-induced expansion of T lymphocytes, they all failed to replace plastic-adherent monocytes in the induction of monocyte-dependent cell death (MDCD) by PMA or PHA. CD45RA-positive cord blood T cells were as susceptible as peripheral blood T cells from adult donors to PMA-stimulated induction of MDCD. Using optimal concentrations of phorbolester, MDCD was inhibited neither by Fas-Fc fusion protein or neutralizing anti-Fas mAb, nor by inhibitors of IL-1β-converting enzyme (ICE)-like proteases. In striking contrast, the H2O2 scavenger catalase completely prevented the PMA-stimulated T cell death, thereby revealing a potent mitogenic activity of PMA for human T cells in the presence of monocytes. Taken together, our results demonstrate that the accessory cell activity of monocytes/macrophages can be separated into “T cell death” and “T cell expansion” costimulatory functions, of which only the latter is mediated by established cell lines. Moreover, our results point to a pivotal role of reactive oxygen intermediates in the execution of MDCD triggered by PMA.
A Subset of CD4+ T Cells Expressing Early Activation Antigen CD69 in Murine Lupus: Possible Abnormal Regulatory Role for Cytokine Imbalance
Authors: Ishikawa Sho , Akakura Shin , Abe Masaaki , Terashima Kazuo , Chijiiwa Karina , Nishimura Hiroyuki , Hirose Sachiko , Shirai Toshikazu
Published: December 2022

Systemic lupus erythematosus (SLE), which spontaneously develops in (NZB (New Zealand Black) × NZW (New Zealand White)) F1 mice, is strictly dependent on CD4+ T cells. We found that in these mice with overt SLE, CD4+ T cells expressing CD69 molecules, an early activation Ag, are dramatically increased in peripheral lymphoid tissues and inflammatory infiltrates in the kidney and lung, but not in peripheral blood, while CD8+ and NK1.1+ T cells were virtually CD69−. Various adhesion molecules, including LFA-1, ICAM-1, CD43, CD44, P-selectin, and E-selectin, were up-regulated. Analysis of the TCR repertoire showed no skewed TCR Vβ usage. Studies on in vitro cytokine production of spleen cells on TCR cross-linking indicated that compared with findings in young mice, the aged mice showed severely impaired production of IL-2, IL-3, and IL-4, whereas the levels of IL-10 and IFN-γ remained relatively intact. FACS-sorted CD69−CD4+ T cells from aged mice produced substantial amounts of these cytokines, including IL-2, IL-3, and IL-4, whereas CD69+CD4+ T cells were poor producers. Intriguingly, when cocultured, CD69+CD4+ T cells significantly inhibited the production of IL-2 by CD69−CD4+ T cells. IL-2 production by spleen cells from young mice was also markedly inhibited in the presence of CD69+CD4+ T cells obtained from aged mice. We propose that CD69+CD4+ T cells that are continuously activated by self peptides bound to MHC class II molecules in (NZB × NZW)F1 mice may be involved in the pathogenesis of SLE through abnormal regulatory effects on cytokine balance.
Zinc Bound to the Killer Cell-Inhibitory Receptor Modulates the Negative Signal in Human NK Cells
Authors: Rajagopalan Sumati , Long Eric O.
Published: December 2022

The lysis of target cells by human NK cells is inhibited by several kinds of receptors with varying specificities for the MHC class I molecules of target cells. The requirements for complete inhibition of NK cytotoxicity appear to be complex and not well defined. The HLA-C-specific members of the killer cell-inhibitory receptor (KIR) family, carrying two Ig domains (KIR2D), are unusual among Ig superfamily members in their ability to bind zinc. A role for the zinc-binding site in KIR-mediated inhibition was demonstrated in this study using a functional reconstitution system in NK cells. Replacement of six histidines by alanine residues in putative zinc binding sites of a KIR2D ablated zinc binding and markedly impaired its inhibitory function, but left intact its ability to bind HLA-C and to transduce a positive signal through an immunoreceptor tyrosine-based activation motif grafted onto its cytoplasmic tail. Thus, zinc modulates specifically the negative signal transmitted by this KIR molecule. Mutation of an exposed amino-terminal zinc-binding motif alone was sufficient to impair the inhibitory function of KIR. The data suggest that complete inhibition of HLA-C-specific NK cells requires a zinc-dependent protein-protein interaction via the amino-terminal end of KIR2D.
Pig MHC Mediates Positive Selection of Mouse CD4+ T Cells with a Mouse MHC-Restricted TCR in Pig Thymus Grafts
Authors: Zhao Yong , Swenson Kirsten , Sergio Justin J. , Sykes Megan
Published: December 2022

Remarkably normal immune function and specific T cell tolerance to discordant xenogeneic donors can be achieved by grafting fetal pig thymus and liver (FP THY/LIV) tissue to T cell and NK cell-depleted, thymectomized (ATX) mice. To determine whether or not host class II MHC molecules participate in the positive selection of mouse CD4+ T cells in FP THY/LIV grafts, we compared their development in ATX “AND” TCR-transgenic mice with positive selecting or nonselecting host MHC genotypes. Mouse TCR-transgenic CD4 single positive T cells repopulated the periphery significantly and to a similar extent in both T/NK cell-depleted, ATX AND mice with positive-selecting or nonselecting MHC backgrounds after grafting with FP THY/LIV. Therefore, MHC molecules from a widely disparate xenogeneic species can positively select T cells bearing a host class II MHC-restricted TCR without a contribution from the host MHC. These results, in combination with previous studies performed in this model, suggest that the T cell repertoire that is generated by the combination of positive selection on xenogeneic MHC and negative selection on both recipient and xenogeneic porcine MHC is tolerant of both donor and recipient and has sufficient cross-reactivity with host MHC/foreign peptide complexes to confer a high level of immunocompetence. The results have implications for the potential clinical applicability of xenogeneic thymic transplantation and also suggest a predominant role for the TCR recognition of species-conserved MHC residues in positive selection.
Origin and Characteristics of Ultraviolet-B Radiation-Induced Suppressor T Lymphocytes
Authors: Shreedhar Vijay K. , Pride Michael W. , Sun Yan , Kripke Margaret L. , Strickland Faith M.
Published: December 2022

Cutaneous exposure to low dose (2 kJ/m2) ultraviolet B radiation impairs the induction of contact hypersensitivity (CHS) responses to haptens applied to UV-irradiated skin and induces hapten-specific suppressor T lymphocytes (Ts). Cells collected from the draining lymph nodes of UV-irradiated, FITC-sensitized mice have impaired Ag-presenting activity and induce Ts cells upon injection into syngeneic recipients. This study investigates whether Ts cells originate in the UV-irradiated donor mice or are induced in lymph node cell recipients and the mechanism of suppression. Using congenic mice, we determined that the Ts cells in recipient animals were derived from T cells in the draining lymph nodes of the UV-irradiated donors. Cell lines and clones established from unirradiated and UV-irradiated, FITC-sensitized mice were CD4+, CD8−, TCR-α/β+, MHC restricted, and hapten specific. The T cells proliferated in response to APC sensitized in vivo, but not to APC coupled in vitro with FITC. Cell lines from unirradiated mice were Th1 like, producing large amounts of IFN-γ, but little IL-4 or IL-10, whereas cloned Ts cells from UV-irradiated mice produced IL-10, but no IL-4 or IFN-γ. Ts cells blocked APC functions and IL-12 production in vitro. Injection of 5 × 104 cloned Ts cells into untreated recipients suppressed the induction of CHS. These results suggest that UV radiation can induce a distinct T regulatory type 1-like Ts population that may block the activation of Th1 cell-mediated immune responses.
The Mitogen-Activated Protein Kinase Pathway Inhibits Ceramide-Induced Terminal Differentiation of a Human Monoblastic Leukemia Cell Line, U937
Authors: Ragg Scott J. , Kaga Shuji , Berg Karen A. , Ochi Atsuo
Published: December 2022

This communication describes an extracellular signal-regulated kinase kinase (MEK)-dependent signal transduction pathway that prevents the terminal differentiation of a hemopoietic cell line. Both PMA and the cell-permeable ceramide, C2-ceramide, caused differentiation of U937 cells, but with distinct cell morphology and CD11b/CD14 surface expression. While PMA activated extracellular signal-regulated kinase (ERK), a downstream kinase of Raf-MEK signaling, C2-ceramide activated c-Jun NH2-terminal kinase (JNK), an anchor kinase of stress-induced signaling. Furthermore, only C2-ceramide stimulated an induction of cell cycle arrest that was associated with stable expression of p21CIP1 and retinoblastoma nuclear phosphoprotein dephosphorylation. Expression of p21CIP1 and JNK activation were also observed in sphingosine-treated cells, whereas sphingosine did not induce detectable differentiation. Concomitant stimulation with C2-ceramide and PMA resulted in the PMA phenotype, and cell cycle arrest was absent. ERK activation was enhanced by C2-ceramide plus PMA stimulation, whereas the activation of JNK was aborted. Strikingly, the inhibition of MEK with PD98059 altered the phenotype of C2-ceramide- and PMA-stimulated U937 cells to that of cells treated with C2-ceramide alone. Thus, ERK and JNK pathways deliver distinct signals, and the ERK pathway is dominant to the JNK cascade. Furthermore, differentiation and cell cycle arrest caused by C2-ceramide rely on independent signaling pathways, and JNK is an unlikely signaling element for this differentiation. Importantly, during C2-ceramide and PMA costimulation, the JNK pathway is not simply blocked by ERK activation; rather, cross-talk between these MAP kinase pathways acts to simultaneously augment ERK activity and down-regulate JNK activity.
Covalent Cross-Linking of Immune Complexes by Oxygen Radicals and Nitrite
Authors: Uesugi Masaaki , Hayashi Takeshi , Jasin Hugo E.
Published: December 2022

We have shown that polymorphonuclear neutrophils mediate the covalent cross-linking of immune complexes (ICs) using H2O2 and myeloperoxidase (MPO). Moreover, activated superficial chondrocytes produce large amounts of nitric oxide (NO), suggesting that high concentrations of these radicals may interact at the cartilage surface in rheumatoid arthritis. We describe the effects of the interaction of NO and its decay product, NO2, with H2O2 and MPO on IC cross-linking. Cross-linking was measured by resistance to the guanidine extraction of plastic-bound ICs. The combination of H2O2, MPO, and NO in the absence of O2 did not alter the magnitude of cross-linking. The addition of O2 resulted in a significant enhancement of cross-linking (p < 0.004), suggesting that nitrite was responsible for the increase observed. Indeed, NaNO2 greatly increased H2O2-dependent cross-linking (control: 29.2 ± 3.8; 1 mM NaNO2: 58.4 ± 9.9; 10 mM: 60.4 ± 4.2% cross-linking, p < 0.0002). Sodium azide, which is an inhibitor of MPO, completely inhibited cross-linking. These results indicated that the product of interaction of H2O2 and NO2 mediated by MPO may be responsible for the increase in cross-linking. The generation of nitrotyrosine was demonstrated when NO2 was added to the cross-linking system. Cross-linking was also shown with an O2−-generating system and NO. Peroxynitrite alone mediated cross-linking (100 μM ONOO−: 40.3 ± 1.9% cross-linking; p < 0.002), and the addition of MPO significantly enhanced this effect (100 μM: 57.7 ± 6.0%; p < 0.0002 with respect to no nitrite control). Oxygen radicals and NO are likely to interact at the cartilage surface in inflammatory arthritis, resulting in an increase in oxidative damage within the joint cavity.
Extracellular Signal-Regulated Kinase-2, But Not c-Jun NH2-Terminal Kinase, Activation Correlates with Surface IgM-Mediated Apoptosis in the WEHI 231 B Cell Line
Authors: Lee Jong Ran , Koretzky Gary A.
Published: December 2022

Both extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) have been implicated in mediating the signaling events that precede apoptosis. We studied the activation of these kinases during apoptosis of WEHI 231 B cells. Surface IgM ligation induces apoptosis of WEHI 231 cells. This effect is augmented by simultaneous engagement of CD95 and is inhibited by costimulation with either CD40 or IL-4R. We determined that surface IgM ligation activates ERK2 to a much greater level than JNK, and that IgM-mediated ERK2 activation is enhanced by costimulation with anti-CD95. Costimulation with either IL-4 or anti-CD40 interferes with anti-IgM-stimulated ERK2 activation. Transient expression of mitogen-activated protein kinase phosphatase-1 (MKP-1) inhibits both ERK2 activation and cell death following stimulation with anti-IgM and the combination of anti-IgM plus anti-CD95. CD40 engagement alone activates JNK, but IL-4 stimulation does not. N-acetyl-l-cysteine pretreatment, which blocks CD40-mediated JNK activation, does not affect the ability of CD40 to inhibit anti-IgM-mediated ERK2 activation and apoptosis. Together, these data suggest that JNK activation is not required for CD40 inhibition of surface IgM-induced cell death and that ERK2 plays an active role in mediating anti-IgM-induced apoptosis of WEHI 231 B cells.
TCR Ligation on CD8+ T Cells Creates Double-Negative Cells In Vivo
Authors: Mehal Wajahat Z. , Crispe I. Nicholas
Published: December 2022

The lack of CD95 in mice is associated with an accumulation of TCRαβ+ CD4−CD8− (double-negative (DN)) cells in the lymph nodes (LNs) and other organs. To test the hypothesis that these DN cells arise from TCRαβ+CD8+ cells after activation via the TCR, we have crossed an MHC class I-restricted TCR transgene (tg) onto the lpr genotype to generate two TCR-transgenic experimental groups, TCRtg+ lpr/+ (CD95-intact) and TCRtg+ lpr/lpr (CD95-deficient). Specific peptide administration resulted in peripheral deletion of TCRαβ cells from the LNs of CD95-intact and CD95-deficient mice. On day 3 after peptide administration in the CD95-deficient but not the CD95-intact mice, there was a ninefold increase in the percentage of DN cells in the LN; this increase returned to control levels by day 10. Peripheral deletion was associated with an accumulation of TCRαβ+CD8high cells in the livers of mice of both genotypes by day 3, which returned to control levels by day 10 without an increase in the percentage or total number of DN cells. Our data show that the in vivo stimulation of TCRαβ+CD8+ cells in the absence of CD95 results in an initial accumulation and an eventual loss of DN cells. This identifies a role for CD95 after TCRαβ stimulation in the efficient removal of TCRαβ+CD8+ cells after the down-regulation of CD8. CD95 is not essential for this process, because other mechanisms can compensate, but such mechanisms are less efficient in the LN.
β2-Microglobulin-Dependent T Cells Are Not Necessary for Alloantigen-Induced Th2 Responses After Neonatal Induction of Lymphoid Chimerism in Mice
Authors: Foucras Gilles , Coureau Christiane , Beijleveld Leo , Druet Philippe , Saoudi Abdelhadi , Guéry Jean-Charles
Published: December 2022

We have analyzed the requirement for β2-microglobulin (β2m)-dependent T cells in the generation of allogeneic Th2 responses in vivo. A neonatal injection of semiallogeneic cells in BALB/c mice induces a state of chimerism that promotes the differentiation of donor-specific CD4+ T cells toward the Th2 phenotype. Polyclonal T-B cell interactions occur in this model between host Th2 and donor B cells, resulting in the production of IgE Abs. IgE production and Th2-priming are critically dependent upon the early production of IL-4. Our data in the present paper demonstrate that: 1) IgE synthesis and the up-regulation of MHC class II and CD23 molecules on B cells are independent of β2m expression in the host, 2) no difference in the induction of CD4 alloreactive Th2 cells could be observed between β2m−/− and their wild-type control littermates when Th2-priming was measured in adult mice, and 3) the Th2 response and IgE production is induced in the complete absence of β2m-dependent T cells both in the host and in the inoculum. Therefore, using a variety of assays, we could not demonstrate diminished responses in mice with a disrupted β2m gene in this model of Th2-mediated allogeneic interaction, indicating that β2m-dependent NK1.1+ and CD8+ T cells are not required for the generation of alloreactive Th2 responses in vivo.
Granulysin-Induced Apoptosis. I. Involvement of at Least Two Distinct Pathways
Authors: Gamen Susana , Hanson Dennis A. , Kaspar Allan , Naval Javier , Krensky Alan M. , Anel Alberto
Published: December 2022

Granulysin is a newly described cytolytic molecule released by CTL and NK cells via granule-mediated exocytosis. It shares homology with saposin-like proteins, including NK-lysin and amoebapores, and has been implicated in the lysis of tumor cells and microbes. In the present study we show that recombinant granulysin alone induces apoptosis of Jurkat cells. This apoptosis is associated with a sixfold increase in the ceramide/sphingomyelin ratio, implicating the activation of sphingomyelinases. Granulysin- and ceramide-induced apoptosis are similar in that they both are only minimally inhibited by the more selective cysteine protease p32 (caspase 3)-like caspase inhibitor N-acetyl-Asp-Glu-Val-Asp aldehyde, while they are significantly inhibited by the more general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk). Nevertheless, while Z-VAD-fmk almost completely inhibits ceramide-induced apoptosis, a Z-VAD-fmk-resistant component was observed using granulysin. Granulysin also causes apoptosis in cells depleted of sphingomyelin by prolonged treatment with the ceramide synthase inhibitor fumonisin B1. These data indicate that granulysin induces target cell death by both ceramide- and caspase-dependent and -independent pathways.
The Invariant Chain Gene Intronic Enhancer Shows Homology to Class II Promoter Elements
Authors: Beilue Moore Bethany , Cao Zhu Alexander , McRae Theresa L. , Woo Colleen H. , Conley Sarah , Jones Patricia P.
Published: December 2022

Coordinate expression of MHC class II proteins and the class II-associated invariant chain (Ii) is important for proper MHC class II functioning in Ag processing and presentation. The coordinate regulation of these genes results, in part, from the sharing of transcriptional regulatory regions between MHC class II and Ii genes; the Ii has previously been shown to have an upstream enhancer closely related to the essential class II promoter elements. We report here the characterization of a second enhancer in the Ii gene, located within the first intron. This intronic enhancer is contained within a 155-bp region, enhances transcription from the Ii minimal promoter, and also contains elements that are homologous to class II promoter elements X1, X2, and Y boxes.
Cutting Edge: Dendritic Cells Require Maturation via CD40 to Generate Protective Antitumor Immunity
Authors: Mackey Matthew F. , Gunn Jason R. , Maliszewski Charles , Kikutani Hitoshi , Noelle Randolph J. , Barth Richard J.
Published: December 2022

A critical role for CD40/CD154 interactions in the generation of protective cell-mediated tumor immunity has been demonstrated previously. Herein, we show that the failure to generate systemic tumor immunity in the absence of CD40/CD154 interactions correlates with an inhibition of Th1-type cytokine production following tumor vaccination. Furthermore, protective antitumor responses can be restored in CD40-deficient mice by the coadministration of CD40+/+ but not CD40−/− dendritic cells (DCs) with tumor Ag, suggesting that CD40 is critical for the maturation and function of DCs in vivo. Finally, we demonstrate that an IL-12-transduced but not a mock-transduced tumor vaccine induces systemic tumor immunity in anti-CD154-treated and CD154-deficient mice. These data suggest that impaired antitumor responses in the absence of CD40/CD154 interactions are the result of a lesion in APC function, namely IL-12 production, and that CD40 plays a critical role in the maturation of DCs in vivo.
β-Galactoside-Binding Protein (βGBP) Alters the Cell Cycle, Up-Regulates Expression of the α- and β-Chains of the IFN-γ Receptor, and Triggers IFN-γ-Mediated Apoptosis of Activated Human T Lymphocytes
Authors: Allione Alessandra , Wells Valerie , Forni Guido , Mallucci Livio , Novelli Francesco
Published: December 2022

In this paper, the effects of β-galactoside binding protein (βGBP), the LGALS1 gene product, on the cell cycle progression and expansion of activated human T lymphocytes were studied. βGBP drastically inhibits the IL-2 induced proliferation of PHA-activated T lymphocytes as well as the IL-2 independent proliferation of malignant T lymphocytes by arresting them in the S and G2/M phases of the cell cycle. In addition, βGBP up-regulates the expression of both the α- and the β-chains of the IFN-γR on activated T lymphocyte membrane. None of these effects depend on sugar binding: saturating amounts of lactose do not affect the cell cycle block nor IFN-γR up-modulation. The increased expression of both chains renders βGBP-treated T lymphoblasts sensitive to IFN-γ-induced apoptosis. Taken as a whole, these findings suggest that βGBP plays an important immunoregulatory role by switching off T lymphocyte effector functions. They also provide the first evidence of up-modulation of IFN-γR expression on T lymphocytes by a negative cell growth regulator.
Tumor-Infiltrating Lymphocytes Exhibiting High Ex Vivo Cytolytic Activity Fail to Prevent Murine Melanoma Tumor Growth In Vivo
Authors: Prévost-Blondel Armelle , Zimmermann Christine , Stemmer Christine , Kulmburg Peter , Rosenthal Felicia M. , Pircher Hanspeter
Published: December 2022

The identification of tumor-associated Ags recognized by CD8+ CTL and prevention of tumor outgrowth by adoptive transfer of these CTL demonstrates that CD8+ T cells play a major role in antitumor immunity. We have generated B16.F10 melanoma cells that express the glycoprotein epitope amino acid 33–41 (GP33) of the lymphocytic choriomeningitis virus (LCMV) to examine antitumor CD8+ T cell response in C57BL/6 mice immune to LCMV and in mice transgenic for the LCMV GP33-specific P14 TCR (P14 TCR mice). We find that B16.F10GP33 tumor cells grew in syngeneic C57BL/6 mice without inducing T cell tolerance. LCMV infection or adoptive transfer of LCMV-specific effector T cells delayed but did not prevent growth of preestablished tumors in these mice. However, B16.F10GP33 tumor cells were rejected in mice immune to LCMV and in mice treated with LCMV-specific effector T cells on the same day as the tumor. Surprisingly, B16.F10GP33 tumor cells grew in P14 TCR transgenic mice despite an abundance of tumor-associated Ag-specific CD8+ T cells. In these mice, freshly isolated tumor-infiltrating lymphocytes exhibited an activated phenotype and displayed high GP33-specific cytolytic activity when assessed ex vivo. Thus, B16.F10GP33 melanoma cells are able to initiate, but not to sustain, a GP33-specific CTL response sufficient to clear the tumor enduringly.
Transcription of a Minimal Promoter from the NF-IL6 Gene Is Regulated by CREB/ATF and SP1 Proteins in U937 Promonocytic Cells
Authors: Berrier Allison , Siu Gerald , Calame Kathryn
Published: December 2022

NF-IL6 is an important transcriptional regulator of genes induced in activated monocytes/macrophages, and NF-IL6 is the only CCAAT/enhancer-binding protein (C/EBP) family member whose steady-state mRNA levels increase upon activation of monocytes (1). We show that increased transcription of the NF-IL6 gene is responsible, at least in part, for induction of NF-IL6 mRNA following activation of U937 promonocytic cells. We have identified a 104-bp minimal promoter region of the NF-IL6 gene that is sufficient for basal and activation-dependent induction of transcription in U937 cells. This region contains binding sites for the cAMP response element-binding protein/activation transcription factor (CREB/ATF) and Sp1 families of transcription factors. Each site is functionally important and contributes independently to transcription of the NF-IL6 gene in U937 cells.
Rheumatoid Factor Specificity of a VH3-Encoded Antibody Is Dependent on the Heavy Chain CDR3 Region and Is Independent of Protein A Binding
Authors: Zhang Meilin , Majid Adrian , Bardwell Philip , Vee Chris , Davidson Anne
Published: December 2022

Rheumatoid factors (RF) recognize conformational determinants located within the Fc portion of IgG. By analyzing a panel of monoclonal rheumatoid arthritis (RA)-derived RFs, we previously demonstrated that the somatically generated light chain complementarity-determining region 3 (CDR3) contributes to RF specificity. We have now generated a panel of heavy chain mutants of the B′20 Ab, a high affinity RA-derived IgM RF. B′20 also binds avidly to protein A and weakly to ssDNA and tetanus toxoid. B9601, a RF negative Ab that is highly homologous to B′20 but does not bind any of the Ags tested, and RC1, a low affinity polyreactive RF, were used to generate heavy chain mutants with framework (FR) and CDR switches. The mutated heavy chains were cotransfected into a myeloma cell line with the germline counterpart of the B′20 light chain, and the expressed Ig tested for antigenic specificity. We show that both RF specificity and polyreactivity of B′20 is dependent on its unique heavy chain CDR3 region. Replacement with a B9601 CDR3 shortened to the same length as the B′20 CDR3, and with only 5 amino acid differences, did not restore Fc binding. Conversely, absence of protein A binding of B9601 is due to the presence of a serine residue at position 82a in the B9601 heavy chain FR3 region. Together, our data suggest that Ig gene recombination events can generate B cells with autoantibody specificities in the preimmune repertoire. Abnormal release, activation, expansion, or mutation of such cells might all contribute to the generation of a high titer RF response in patients with RA.
Asymmetric Contribution to Ig Repertoire Diversity by Vκ Exons: Differences in the Utilization of Vκ10 Exons
Authors: Fitzsimmons Sean P. , Rotz Benjamin T. , Shapiro Marjorie A.
Published: December 2022

The mouse has approximately 140 germline Vκ genes, and functional Vκ exons are expressed at roughly equivalent levels in the preimmune repertoire. We have examined the expression of individual members of the Vκ10 family. Vκ10A and Vκ10B genes have been utilized in numerous hybridomas and myelomas, while Vκ10C has not. In this study, we have cloned the Vκ10C gene and shown that it is structurally functional, has the expected promoter elements and recombination signal sequences, and that it is capable of recombination. Vκ10C mRNA, however, is present at levels at least 1000-fold lower than Vκ10A and Vκ10B in adult spleens. While there are no sequence differences in the octamer or TATA box between Vκ10C and Vκ10A, there are three nucleotide changes in the promoter region. These promoters equally drive the expression of a reporter gene in B cells or plasma cells, but the Vκ10A promoter is able to drive expression in pre-B cell lines significantly better than the Vκ10C promoter (p < 0.05). Vκ10C rearrangements can be detected in bone marrow and splenic DNA. Therefore, the lack of Vκ10C expression may reflect the inability of Vκ10C-rearranged cells to undergo positive or negative selection. Our results suggest that the available Ab repertoire is shaped not only by the number of structurally functional genes, but also by the ability of assembled genes to be expressed at critical points during B cell maturation.
The Assembly and Stability of MHC Class II-(αβ)2 Superdimers
Authors: Schafer Peter H. , Malapati Sunil , Hanfelt Kimberly K. , Pierce Susan K.
Published: December 2022

X-ray crystallography of several MHC class II molecules revealed a structure described as a dimer of heterodimers, or a superdimer. This discovery led to the hypothesis that MHC class II molecules may interact with the TCR and CD4 as an (αβ)2 superdimer, potentially providing more stable and stimulatory interactions than can be provided by the simple αβ heterodimer alone. In this study, using chemical cross-linking, we provide evidence for the existence of the superdimers on the surface of B cells. We further characterize the superdimers and demonstrate that in lysates of B cells, I-Ek dimers and superdimers are derived from the same population of I-Ek molecules. Purified, I-Ek molecules in solution also exist as a mixture of 60-kDa dimers and 120-kDa superdimers, indicating that I-Ek has an intrinsic ability to form 120-kDa complexes in the absence of other cellular components. Peptide mapping showed that the αβ and (αβ)2 complexes are closely related and that the superdimers do not contain additional polypeptides not present in the dimers. The (αβ)2 complex displays thermal and pH stability similar to that of the αβ complex, both being denatured by SDS at temperatures above 50°C and at a pH below 5. These data support the model that MHC class II has an intrinsic ability to assume the (αβ)2 superdimeric conformation, which may be important for interactions with the TCR and CD4 coreceptor.
B and T Cells Are Required for Mouse Mammary Tumor Virus Spread Within the Mammary Gland
Authors: Golovkina Tatyana V. , Dudley Jaquelin P. , Ross Susan R.
Published: December 2022

Mouse mammary tumor virus (MMTV) is an infectious retrovirus transmitted through milk from mother to newborns. MMTV encodes a superantigen (SAg) whose activity is indispensable for the virus life cycle, since a genetically engineered virus with a mutation in the sag gene neither amplified in cells of the immune system of suckling pups nor infected their mammary glands. When wild-type MMTV was injected directly into the mammary glands of uninfected pubescent mice, their lymphoid as well as mammary gland cells became virus infected. To test whether this infection of lymphoid cells was dependent on SAg activity and required for virus spread within the mammary gland, we performed mammary gland injections of wild-type MMTV(C3H) into two strains of transgenic mice that lacked SAg-cognate, Vβ14+ T cells. Neither the MTV-ORF or LEL strains showed infection of their mammary glands. Moreover, no MMTV infection of their peripheral lymphocytes was detected. Similar experiments with mice lacking B cells (μ-chain knockouts) showed no detectable virus spread in the mammary glands or lymphoid tissues. These data suggest that SAg activity and MMTV-infected lymphocytes are required, not only for initial steps of viral infection, but also for virus spread within the mammary gland. Virus spread at late times in infection determines whether MMTV induces mammary tumors.
Migration Inhibitory Factor Induces Killing ofLeishmania majorby Macrophages: Dependence on Reactive Nitrogen Intermediates and Endogenous TNF-α
Authors: Jüttner Stefan , Bernhagen Jürgen , Metz Christine N. , Röllinghoff Martin , Bucala Richard , Gessner André
Published: December 2022

Macrophage migration inhibitory factor (MIF) is a product of activated T cells, anterior pituitary cells, and macrophages. MIF plays an important role in LPS-induced shock and delayed-type hypersensitivity. Furthermore, MIF exhibits a proinflammatory spectrum of action, promoting TNF-α production by macrophages, and counter-regulates glucocorticoid suppression of cytokine production. Here, we report that purified recombinant MIF activates murine macrophages to kill Leishmania major, with maximal effects at concentrations above 1 μg/ml. This MIF-mediated activation is specific, since it can be blocked completely by anti-MIF mAb. The MIF-mediated activation is dependent on TNF-α produced endogenously by macrophages, because the administration of anti-TNF-α antiserum markedly reduced the MIF effect. No MIF-mediated activation was observed in macrophages derived from TNF receptor p55 knockout mice, thus demonstrating the requirement of the smaller TNF receptor molecule for autocrine TNF-α signaling. A highly specific inhibitor of the inducible nitric oxide synthase (iNOS), l-N6-(1-iminoethyl)lysine, dihydrochloride, also inhibited the action of MIF, suggesting an important role for iNOS in the antiparasitic properties of MIF. In line with this, no MIF-mediated activation was detected analyzing macrophages derived from iNOS-deficient mice. The effect of MIF was blocked completely by the macrophage-deactivating cytokines IL-10, IL-13, and TGF-β. Finally, the expression of MIF mRNA and protein was up-regulated in lymph nodes of mice during the first week after infection with L. major. MIF therefore represents a cytokine involved not only in the recruitment of proinflammatory cells during infection but also in the complex regulation of the antimicrobial activity of these cells.