bluuebunny/crossref_metadata_2025 huggingface dataset:
Glutathione depletion is an early and calcium elevation is a late event of thymocyte apoptosis.
Authors: Macho A , Hirsch T , Marzo I , Marchetti P , Dallaporta B , Susin S A , Zamzami N , Kroemer G
Published: December 2022

According to current understanding, several metabolic alterations form part of the common phase of the apoptosis process. Such alterations include a disruption of the mitochondrial transmembrane potential (delta psi(m)), a depletion of nonoxidized glutathione (GSH) levels, an increase in the production of reactive oxygen species (ROS), and an elevation in cytosolic free Ca2+ levels. Using a cytofluorometric approach, we have determined each of these parameters at the single cell level in thymocytes or T cell hybridoma cells undergoing apoptosis. Regardless of the apoptosis induction protocol (glucocorticoids, DNA damage, Fas cross-linking, or CD3epsilon cross-linking), cells manifest a near-to-simultaneous delta psi(m) dissipation and GSH depletion early during the apoptotic process. None of the protocols for apoptosis inhibition (antioxidants, delta psi(m) stabilization, Bcl-2 hyperexpression, or inhibition of IL-1-converting enzyme) allowed for the dissociation of delta psi(m) disruption and GSH depletion, indicating that both parameters are closely associated with each other. At a later stage of the apoptotic process, cells manifest a near-simultaneous increase in ROS production and intracellular Ca2+ levels. Whereas the thapsigargin- or ionophore-induced elevation of calcium levels has no immediate consequence on delta psi(m') cellular redox potentials, or ROS production, pro-oxidants and menadione, an inducer of mitochondrial superoxide anion generation, cause a rapid (15 min) Ca2+ elevation. Together, these data suggest a two-step model of the common phase of apoptosis. After an initial delta psi(m) dissipation linked to GSH depletion (step 1), cells hyperproduce ROS with an associated disruption of Ca2+ homeostasis (step 2).
CD19 expression levels regulate B lymphocyte development: human CD19 restores normal function in mice lacking endogenous CD19.
Authors: Sato S , Steeber D A , Jansen P J , Tedder T F
Published: December 2022

Establishing signal transduction thresholds that regulate B lymphocyte responses to foreign Ags and tolerance to self Ags is critical for humoral immune responses. The effects of altered signaling thresholds in B lymphocytes were examined in CD19-deficient mice and transgenic mice that expressed human CD19 at varying densities. Human CD19 restored normal B cell function and development to CD19-deficient mice when expressed at levels comparable to those of circulating human B cells. While CD19 expression levels were found to be developmentally regulated and tightly controlled in normal mice, two- or threefold changes in cell surface CD19 expression in transgenic mice dramatically affected B cell development, mitogen responses, serum Ig levels, humoral immune responses, and germinal center formation. B cells from mice that overexpressed CD19 also had decreased levels of surface IgM and a cell surface phenotype consistent with increased signaling in these cells. These results suggest that CD19 may serve similar functions in humans and mice and that CD19 defines signaling thresholds in vivo for the Ag receptor as well as other cell surface receptors that regulate B lymphocyte selection, activation, and differentiation.
CD40 is a functional activation antigen and B7-independent T cell costimulatory molecule on normal human lung fibroblasts.
Authors: Sempowski G D , Chess P R , Phipps R P
Published: December 2022

CD40 is an important signaling and activation Ag found on certain bone marrow-derived cells. Recently, CD40 also has been shown to be expressed by mesenchymal cells, including human fibroblasts. Little is known about the role of CD40 in fibroblasts. The current study investigates the hypothesis that CD40 expressed on lung fibroblasts is an activation structure and mechanism for interaction with hemopoietic cells. Communication between resident tissue fibroblasts and T cells is necessary for normal wound healing, and can be pathologic, resulting in tissue fibrosis. Signaling through CD40 with soluble CD40 ligand stimulated fibroblast activation, as evidenced by mobilization of nuclear factor-kappaB and by induction of the proinflammatory and chemoattractant cytokines IL-6 and IL-8. IFN-gamma-primed lung fibroblasts costimulate T lymphocyte proliferation utilizing CD40, but not the well-studied costimulatory molecules B7-1 and B7-2. Data reported herein support the hypothesis that cognate interactions between tissue fibroblasts and infiltrating T lymphocytes, via the CD40/CD40L pathway, augment inflammation and may promote fibrogenesis by activating both cell types.
CD16-expressing CD8alpha alpha+ T lymphocytes in the intestinal epithelium: possible precursors of Fc gammaR-CD8alpha alpha+ T cells.
Authors: She J , Simpson S J , Gupta A , Holländer G , Levelt C , Liu C P , Allen D , van Houten N , Wang B , Terhorst C
Published: December 2022

T lymphocytes normally express their Ag receptors in association with the CD3 proteins, which include CD3zeta. In CD3zeta eta(null) mice thymic and peripheral T lymphocytes do not express the TCR/CD3 complex on their surface due to retention in the endoplasmic reticulum of the remaining polypeptide chains. However, intestinal intraepithelial lymphocytes (iIEL) of CD3zeta eta(null) mice do express surface TCR, because the Fc epsilonRI gamma chain replaced the CD3zeta chain in the TCR/CD3 complex. Here we report that in a subset of CD8alpha alpha+ iIEL the presence of the Fc epsilonRI gamma chain could be accounted for by the surface expression of the Fc gammaRIII(CD16) complex. Because in wild-type (wt) mice only CD16+ iIEL coexpressed Fc epsilonRI gamma and CD3zeta, we concluded that the presence of Fc epsilonRI gamma was dictated by its required participation of CD16 complex. CD8alpha alpha+ iIEL bearing CD16 and B220 were also detected in the intestinal mucosa of RAG-2(null) mice from 12 days after birth onward. Two independent experimental settings were used in an attempt to demonstrate that CD16+ iIEL matured into CD16- T cells. First, in the RAG-2(null) mice, iIEL responded to in vivo administration of an anti-CD3epsilon mAb by progression to a more mature stage of development, characterized by a loss of CD16 and B220. Secondly, a conversion to CD16- iIEL occurred upon transfer of wt CD16+ iIEL into RAG-2(null) mice. We conclude from these experiments that in both RAG-2(null) and wt mice, a precursor/progeny relationship may exists between CD16+ B220+ CD8alpha alpha+ and CD16- B220- CD8alpha alpha+ iIEL.
Wortmannin, a phosphatidylinositol 3-kinase inhibitor, blocks antigen-mediated, but not CD3 monoclonal antibody-induced, activation of murine CD4+ T cells.
Authors: Shi J , Cinek T , Truitt K E , Imboden J B
Published: December 2022

Perturbation of several distinct T cell molecules, including the CD3/TCR complex, CD7, and CD28, activates phosphatidylinositol 3-kinase (PI3-K), but a clear consensus on the role of PI3-K in T cell activation has yet to emerge. We report here that CD3 mAb-induced IL-2 production by CD4+ T cells from DO11.10 TCR-alphabeta-transgenic mice is refractory to the potent PI3-K inhibitor, wortmannin, demonstrating that activation under these conditions is independent of PI3-K. In marked contrast, wortmannin substantially inhibits IL-2 production elicited by Ag (OVA(323-339) peptide) presented by appropriate APCs (syngeneic B7+ B cell blasts) and blocks Ag-induced differentiation of naive CD4+ DO11.10 T cells into IL-4-producing cells. Wortmannin inhibits Ag-induced conjugate formation between T cells and B7+ B cell blasts. Because T cell activation by Ag requires stable interactions with APCs, this inhibitory effect on conjugate formation may underlie the ability of wortmannin to block Ag-induced IL-2 production and differentiation.
CD28 engagement and proinflammatory cytokines contribute to T cell expansion and long-term survival in vivo.
Authors: Vella A T , Mitchell T , Groth B , Linsley P S , Green J M , Thompson C B , Kappler J W , Marrack P
Published: December 2022

To mount a productive response to Ag, CD4+ T cells in mice must divide, differentiate, and survive at least until the Ag has been eliminated. It has been suggested that to accomplish this, T cells must receive two signals, one through their TCRs and a second through CD28. The second signal through CD28 has been thought to fulfill two roles, to stimulate T cell proliferation and to promote T cell survival. In this paper we confirm that CD28 engagement can contribute to vigorous T cell expansion in mice injected with superantigens. However, CD28 engagement does not protect T cells produced during a superantigen-specific proliferative response from undergoing subsequent deletion. Even if CD28 is bound, 4 days after superantigen exposure, the majority of T cells produced in response to superantigen exposure are eliminated in vivo. In contrast, this loss of superantigen-stimulated T cells can be prevented by the inflammatory stimuli created by injection of bacterial LPS. This protection does not require engagement of CD28 by its ligands, B7-1 and B7-2. These data suggest that productive T cell responses in mice involve a number of signals, including those initiated through TCR and CD28, which are primarily involved in the activation and expansion of T cells, and others delivered by proinflammatory cytokines that protect an activated T cell from subsequent deletion.
CIITA-dependent and -independent class II MHC expression revealed by a dominant negative mutant.
Authors: Zhou H , Su H S , Zhang X , Douhan J , Glimcher L H
Published: December 2022

The MHC class II transactivator gene (CIITA) coordinately controls the expression of the three major human class II genes, HLA-DR, HLA-DQ, and HLA-DP. Indeed, patients with one form of MHC class II immunodeficiency disease, due to defective CIITA genes, lack expression of all three isotypes. Nevertheless, there is substantial evidence that human class II genes are not always coordinately regulated, raising the possibility that CIITA-independent, isotype-specific class II regulatory pathways exist. To address this issue, we have generated a dominant negative mutant of CIITA that lacks the acidic transcription-activating N terminus, but retains the proline/serine/threonine-rich domain. Three newly produced anti-CIITA mAbs revealed that this mutant protein lacked N-terminal epitopes. In this study, we report that this CIITA dominant negative mutant repressed the constitutive expression of all three class II isotypes in human EBV-B cell lines, as well as IFN-gamma-induced class II transcription in HeLa cells. However, in a CIITA-deficient, EBV-transformed B cell line, clone 13, the dominant negative mutant did not alter the endogenous expression of the HLA-DQ gene. Taken together, these data demonstrate the existence of both CIITA-dependent and -independent class II regulatory pathways. Furthermore, our data provide evidence that the latter pathways can be isotype specific.
Dyad symmetry within the mouse 3' IgH regulatory region includes two virtually identical enhancers (C alpha3'E and hs3).
Authors: Saleque S , Singh M , Little R D , Giannini S L , Michaelson J S , Birshtein B K
Published: December 2022

The transcription of the murine Ig heavy chain locus is regulated not only by the intronic enhancer, E mu, but also by a 3' regulatory region located downstream of the C alpha membrane exon. Several DNase I-hypersensitive sites (hs1-4) and enhancer elements (e.g., C alpha3'E) have been identified in this 3' regulatory region, and some of these were suggested to comprise a locus control region. However, little is known about the coordinate regulation or function of these individual elements. Here we provide evidence that C alpha3'E and hs3 are virtually mirror images of each other and demarcate the edges of an approximately 25-kb region of quasi-dyad symmetry with 3'alphaE(hs1,2) at its center. Flanking 3'alphaE(hs1,2) are inverted repeats and families of repetitive sequences uniquely located in this region. We have observed that, like 3'alphaE(hs1,2) and hs3, C alpha3'E is DNase I hypersensitive in plasma cell lines, but not in a pre-B cell line. Additionally, we found that C alpha3'E and hs3 show significant transcriptional synergy in transfection assays only in a plasma cell line. The DNA topology of the 3' regulatory region coupled with new and existing data on the activity of its individual enhancers during B cell differentiation lead us to propose a biphasic model for the activity of this region. According to our model, one unit, consisting of the 3'-most enhancer, hs4, is active early and throughout B cell development. The second unit, which comprises C alpha3'E, 3'alphaE(hs1,2), and hs3, becomes active later in development, when it contributes to such processes as class switching and increased levels of Ig heavy chain gene transcription in plasma cells.
HLA-DMA and HLA-DMB gene expression functions through the conserved S-X-Y region.
Authors: Westerheide S D , Louis-Plence P , Ping D , He X F , Boss J M
Published: December 2022

The MHC class II homologous proteins HLA-DMA and HLA-DMB function in the loading of peptides onto class II molecules. Like the class II genes, the HLA-DM genes contain upstream regulatory sequences similar to the S-X-Y regulatory region as well as additional putative regulatory sites. To determine whether the DM genes are regulated in a similar manner as class II genes, a series of in vivo and in vitro analyses was performed. Deletion analysis showed that expression from the DM promoters is dependent on the conserved S-X-Y region. The class II-specific transcription factors RFX and CIITA are also required for expression, as cell lines deficient in these factors failed to allow transcription from the DM promoters. In addition, in vivo footprint analysis showed the putative X and Y boxes to be occupied by transcription factors in wild-type B cells, but not in RFX-deficient B cells. In astrocytes, IFN-gamma treatment induced increased occupancy of these sites. None of the other putative regulatory sites was occupied in vivo, indicating that they may not be functional. Finally, gel shift analysis showed synergistic complex formation between proteins that bind to the putative X boxes of the DM genes, as is found for the DRA gene. Therefore, the DM genes share a common mechanism of regulation with the class II genes.
Immunosuppressive activity of cloned natural killer (NK1.1+) T cells established from murine tumor-infiltrating lymphocytes.
Authors: Tamada K , Harada M , Abe K , Li T , Tada H , Onoe Y , Nomoto K
Published: December 2022

To elucidate the role of NK1.1+ T cells in the antitumor immune response, we established cloned NK1.1+ T cell lines from tumor-infiltrating lymphocytes (TIL) of B16 melanoma, and examined their mode of action in generating antitumor effector T cells both in vitro and in vivo. An NK1.1+ T cell clone (TM4.2) was phenotypically CD3+ TCR-alphabeta+ CD4- CD8- NK1.1+, and CD28+. The TM4.2 cells suppressed the in vitro generation of anti-B16 melanoma CTLs, but not the effector function of CTLs. The results using a transwell membrane suggested that their suppressive activity was mediated by both soluble factors and a direct cell to cell interaction. As for the soluble factors, the suppressive activity of the culture supernatant of TM4.2 cells was neutralized by anti-TGF-beta mAb, and the TM4.2 cells actually produced a considerable amount of TGF-beta. On the other hand, the TM4.2 cells showed a high level of cytolytic activity against B cell blasts and CD80-transfected P815, and such cytolytic activity was reduced by the addition of anti-CD80 mAb. In addition, NK1.1+ T cells in the freshly isolated TIL were revealed to express CD28. Furthermore, the TM4.2 cells suppressed the in vitro generation of anti-allo CTLs irrespective of the MHC haplotype. Finally, the TM4.2 cells suppressed the in vivo antitumor immune response. Collectively, these findings demonstrate that NK1.1+ T cells in TIL show immunosuppressive activity in the antitumor immune response through the production of TGF-beta and the preferential cytolysis of B7-expressing cells.
Early embryo loss is associated with the prior expression of macrophage activation markers in the decidua.
Authors: Haddad E K , Duclos A J , Lapp W S , Baines M G
Published: December 2022

In early embryo loss, the activation of maternal immune effector mechanisms play a critical role in determining the success or failure of a pregnancy. We have previously shown that increased nitric oxide production by decidual macrophages is involved in early embryo loss occurring at day 12 of gestation. In this study, using reverse transcription-PCR and Southern blotting, the expression of inducible nitric oxide synthases (iNOS) and TNF-alpha mRNA was determined to quantify macrophage activation in individual murine embryos in a model of spontaneous early embryo loss. At day 8 of gestation, 32 and 29% of embryos with no apparent pathology showed an increase in iNOS and TNF-alpha mRNA expression, respectively. This corresponds to the natural resorption rate seen in the mouse model. In addition, the percentage of embryos with increased iNOS and TNF-alpha mRNA expression was further augmented when pregnant mice were induced to abort at a higher rate. These results showed, for the first time, a correlation between increased iNOS and TNF-alpha expression and embryo resorption. The results provide evidence for the presence of activated macrophages at implantation sites before overt embryo damage occurs.
Inflammatory cytokines IFN-gamma plus TNF-alpha induce regulated expression of CD80 (B7-1) but not CD86 (B7-2) on murine fibroblasts.
Authors: Pechhold K , Patterson N B , Craighead N , Lee K P , June C H , Harlan D M
Published: December 2022

Optimal T cell activation requires signals delivered via both the TCR and the costimulatory receptors. Considerable experimental data now suggest that this costimulatory signal is generated predominantly by CD28 when engaged by its ligands CD80 (B7-1) and/or CD86 (B7-2). Whether T cell activation is controlled in part by regulated CD80 and/or CD86 expression has been incompletely explored. Here, we report that CD80 can be expressed constitutively by murine fibroblasts and up-regulated after treatment with IFN-gamma plus TNF-alpha. CD80 expression and function was confirmed by 1) Northern analysis, 2) specific immunoprecipitation of a approximately 69-kDa surface protein that comigrated with CD80 precipitated from CD80-transfected CHO cells, and 3) two independent assays for costimulation of Ag-specific T cell activation. Taken together, these observations suggest that CD28/CTLA-4 ligands are expressed on a wider variety of tissues than previously suspected and that their expression is dynamically regulated. Consequently, these results might explain previous observations that inflammatory cytokines can result in autoimmune responses.
Adenosine metabolism during phorbol myristate acetate-mediated induction of HL-60 cell differentiation: changes in expression pattern of adenosine kinase, adenosine deaminase, and 5'-nucleotidase.
Authors: Spychala J , Mitchell B S , Barankiewicz J
Published: December 2022

Adenosine has potent immunosuppressive activity. Since the source of adenosine and the mechanism of its release in the immune system is largely unknown and may vary according to cell type, we have evaluated the relationship between adenosine metabolism and the enzymatic activities and mRNA levels of adenosine-metabolizing enzymes in myeloid and lymphoid cell lines. Induction of HL-60 cell differentiation along the macrophage lineage by PMA resulted in a reduction in the activities of adenosine deaminase (ADA), adenosine kinase (AK), and inosine monophosphate-specific cytosolic 5'-nucleotidase and an elevation of ecto-5'-nucleotidase (ecto-5'-NT). These changes were accompanied by an elevation of ecto-5'-NT mRNA and a decrease in ADA and AK mRNAs in a time-dependent fashion. Comparison of AK and ADA mRNA levels in several other leukemic cell lines revealed generally similar responses to PMA with much stronger suppression in immature T cells than in B cells. The metabolism of adenosine either through phosphorylation (AK) or deamination (ADA) was reduced in PMA-stimulated cells. Furthermore, the cumulative changes in enzyme expression resulted in a 2.5-fold increase in intracellular adenosine formation in PMA-stimulated cells. The inhibition of AK by 5'-iodotubercidin further increased adenosine formation by 6-fold over that in untreated cells. In accord with the increase in ecto-5'-NT activity, extracellular AMP dephosphorylation increased dramatically, but there was no increase in extracellular ATP degradation. These results indicate that a coordinated shift in adenosine-metabolizing enzyme levels during PMA-induced HL-60 cell differentiation is accompanied by a decrease in adenosine uptake and an increase in adenosine release.
Monocyte chemotactic protein-3 (MCP-3)/fibroblast-induced cytokine (FIC) in eosinophilic inflammation of the airways and the inhibitory effects of an anti-MCP-3/FIC antibody.
Authors: Stafford S , Li H , Forsythe P A , Ryan M , Bravo R , Alam R
Published: December 2022

Monocyte chemotactic protein-3 (MCP-3)/fibroblast-induced cytokine (FIC), a CC chemokine, is chemotactic for cells that typically infiltrate the late-phase allergic reaction. We developed a mouse model of airway inflammation to study the role of MCP-3/FIC. The immunization of mice with OVA resulted in Ag-specific IgE Ab production and the expression of mRNA for IL-4 in the lung tissue. Two weeks after immunization mice were challenged with the allergen by inhalation. Lungs were lavaged, and the tissue was examined at 2 or 24 h. Allergen challenge resulted in the increased recovery of leukocytes in the lavage fluid, but saline challenge did not. There was a significant increase in eosinophils (29 +/- 8% vs 1.2 +/- 0.2%) and lymphocytes (25 +/- 4% vs 5 +/- 2%) in the bronchoaveolar lavage fluid. Histologic examination of the lung demonstrated intense airway inflammation following OVA challenge. The expression of MCP-3/FIC and other CC chemokines (MCP-1, macrophage inflammatory protein-1alpha, and RANTES) was investigated by reverse transcription-PCR followed by densitometric analyses. The allergen challenge up-regulated the expression of mRNA for MCP-1, MCP-3/FIC, and macrophage inflammatory protein-1alpha at 2 and/or 24 h. Immunocytochemical staining for MCP-3/FIC showed that the allergen challenge induced the expression of MCP-3/FIC predominantly in the airway epithelium. Pretreatment of mice with an anti-MCP-3/FIC Ab significantly inhibited the OVA-induced airway inflammation and the bronchoalveolar lavage eosinophilia (8 +/- 2% vs 46 +/- 11% after control Ab, p < 0.03). We conclude that MCP-3/FIC plays a significant role in the allergen-induced eosinophilic inflammation of the airways.
Sezary lineage cells can be induced to proliferate via CD28-mediated costimulation.
Authors: McCusker M E , Garifallou M , Bogen S A
Published: December 2022

Sezary syndrome and mycosis fungoides are related chronic lymphoproliferative diseases caused by the malignant growth of CD4+ T lymphocytes that display hyperconvoluted nuclei and a predilection for skin homing. Despite the malignant nature of these cells, they paradoxically do not grow in vitro, either spontaneously or following exposure to mitogens. Partly because of this technical limitation, the cellular lineage and causes of abnormal growth resulting in a classical hyperconvoluted Sezary cell are poorly characterized. To better understand these aspects, we examined Sezary lineage cell growth in vitro. We found that, contrary to previous reports, Sezary lineage cells are capable of in vitro proliferation in response to either PHA or anti-CD3 mAb, if exogenous costimulation is provided. The CD28-B7 interaction provides at least one of the costimulatory signals capable of inducing Sezary lineage cell growth. Namely, Sezary lineage cells from three of six Sezary syndrome patients proliferated in response to PHA if an anti-CD28 mAb was also added to the in vitro culture. The remaining three patients' Sezary lineage cells were dependent upon CD28-B7-mediated costimulation, but in addition required other intercellular signals present on blood mononuclear cells. The relative lack of costimulation from the patients' own PBMC is not due to an intrinsic defect in the mycosis fungoides/Sezary syndrome patients' immune accessory cells. Rather, it appears primarily due to an inability of Sezary cells to significantly up-regulate CD40 ligand (gp39) following in vitro exposure to PHA.
Anti-IL-4 monoclonal antibody prevents antibiotics-induced active fatal anaphylaxis.
Authors: Park J S , Choi I H , Lee D G , Han S S , Ha T Y , Lee J H , Lee W H , Park Y M , Lee H K
Published: December 2022

We previously reported that anti-IL-4 mAb (11B11) failed to prevent protein-induced fatal murine anaphylaxis. To investigate the effect of anti-IL-4 on hapten-induced anaphylaxis, a model of murine anaphylaxis induced by antibiotics, penicillin V (Pen V) and cephalothin (CET), was developed, and the effect of anti-IL-4 on the anaphylaxis was observed. Pen V and CET induced 100 and 70 to 90% fatal reactions, respectively, when C57BL/6 mice were sensitized i.p. with 500 microg of antibiotic-OVA conjugate with 2 x 10(9) Bordetella pertussis and 1.0 mg of alum and challenged i.v. with 100 microg of antibiotic-BSA conjugate 14 days later. Serum taken from mice sensitized to Pen V passively sensitized normal mice to develop systemic anaphylaxis, and this ability of the serum was abrogated by heating at 56 degrees C for 2 h or depletion of IgE, but not IgG, Abs. Thus, the antibiotic-induced fatal reaction was an IgE-dependent anaphylactic reaction. Administration of anti-IL-4 at the beginning of sensitization completely prevented the fatal anaphylactic reactions to both Pen V and CET. This effect of anti-IL-4 was associated with its suppressive activity on antibiotic-specific serum IgE, but not IgG, levels. More importantly, anti-IL-4 therapy in previously sensitized mice was also effective in preventing the fatal reactions and rapidly reduced the established IgE levels. This study provides a new animal model of hapten-induced anaphylaxis and indicates that blocking of IL-4 activity may be beneficial in allergic diseases caused by a variety of haptens in which IgE Abs play a major role.
Somatic mutation leads to efficient affinity maturation when centrocytes recycle back to centroblasts.
Authors: Oprea M , Perelson A S
Published: December 2022

Although most mutations are deleterious, an interplay between somatic mutation and selection within germinal centers (GC) results in rapid generation of high affinity memory B cells. How high affinity B cells with large numbers of mutations are generated and preserved within GC containing at their peak only a few thousand cells has been puzzling. We have developed a model of somatic mutation and B cell expansion within a GC that resolves this puzzle. We show that the frequent recycling of Ag-selected centrocytes back into centroblasts can lead to efficient affinity maturation. Memory cells are generated in large numbers even when most of the selected centrocytes recycle back into centroblasts. Our model suggests that a germinal center reaction in which the output of cells is low up to the point of GC dissociation, followed by the release of centrocytes into the periphery, is advantageous for generating high affinity memory.
L-selectin is involved in lymphocyte migration to sites of inflammation in the skin: delayed rejection of allografts in L-selectin-deficient mice.
Authors: Tang M L , Hale L P , Steeber D A , Tedder T F
Published: December 2022

Adhesion of leukocytes to vascular endothelium is crucial for leukocyte migration into tissues. The contributions of L-selectin, P-selectin, and ICAM-1 to interactions between lymphocytes and endothelium was examined using allogeneic skin graft rejection as a model of cutaneous inflammation. L-selectin-deficient (L-selectin(-/-)) mice rejected both primary and secondary allogeneic (BALB/c) skin grafts significantly more slowly than L-selectin(+/+) littermates. Furthermore, skin graft rejection remained significantly delayed in L-selectin(-/-) mice, despite placement of grafts 7 days after i.p. immunization with allogeneic cells, when CTL responses in L-selectin(-/-) mice and L-selectin(+/+) littermates were confirmed to be equivalent. Indeed, specific CTL responses to BALB/c splenocytes were normal or elevated in L-selectin(-/-) mice following either skin grafts or immunization. However, the number of T lymphocytes within allogeneic grafts was lower in L-selectin(-/-) mice as compared with L-selectin(+/+) littermates. Therefore, delayed rejection of skin grafts by L-selectin(-/-) mice reflects impaired migration of effector cells into the graft rather than delayed or impaired generation of a CTL response. In contrast to L-selectin(-/-) mice, P-selectin-deficient and ICAM-1-deficient mice rejected allogeneic skin grafts normally. These findings delineate an important role for L-selectin in lymphocyte recruitment to cutaneous sites of inflammation.
Neutrophil production of IL-12 and IL-10 in candidiasis and efficacy of IL-12 therapy in neutropenic mice.
Authors: Romani L , Mencacci A , Cenci E , Spaccapelo R , Del Sero G , Nicoletti I , Trinchieri G , Bistoni F , Puccetti P
Published: December 2022

Neutrophil defects predispose to severe fungal infections, yet the immunomodulatory role of these cells is poorly defined. The contribution of neutrophils to the early cytokine balance governing Th1 and Th2 cell development was examined in mice with candidiasis. Neutrophils secreted IL-12 and IL-10, correlating with the respective development of self-limiting (Th1-associated) and progressive (Th2-associated) disease. Exogenous IL-12 was effective in protecting neutropenic hosts susceptible to infection. These results suggest that 1) neutrophils, via their ability to release cytokines, play an active role in determining the qualitative development of the T cell response, and 2) their early role in anticandidal immunity can be replaced by exogenous IL-12.
Ultraviolet light suppresses IFN-gamma-induced IL-7 gene expression in murine keratinocytes by interfering with IFN regulatory factors.
Authors: Aragane Y , Schwarz A , Luger T A , Ariizumi K , Takashima A , Schwarz T
Published: December 2022

UV light is a potent stimulus for keratinocytes to release several cytokines. Recently, UV light was shown to inhibit keratinocyte release of IL-7, a growth factor for dendritic epidermal T cells. Since to date IL-7 is the only keratinocyte-derived cytokine down-regulated by UV light, we addressed the molecular mechanisms involved. IFN-gamma treatment of the murine keratinocyte cell line Pam 212 resulted in an up-regulation of IL-7 mRNA, while IL-7 transcripts were suppressed in cells exposed to UV before IFN-gamma. Because IFN-gamma induces IL-7 via activation of an IFN-stimulated response element (ISRE) located in the 5' upstream region of the IL-7 gene, bandshift assays were performed using the ISRE sequence from the IL-7 gene. Nuclear extracts from untreated cells revealed two bands, a slower migrating band identified by supershift analysis as IFN regulatory factor-2 (IRF-2), a transcriptional repressor, and a more rapidly migrating band identified as IRF-1, a transcriptional activator. IFN-gamma significantly induced IRF-1 binding, whereas UV treatment plus IFN-gamma decreased IRF-1 binding, suggesting that UV light suppresses IFN-gamma-induced expression of IL-7 by interfering with IRF-1. Chloramphenicol transferase assay confirmed functional relevance, showing that the minimal promoter sequence for the ISRE explicitly responded to IFN-gamma, which was suppressed by UV irradiation. Northern blot analysis using an IRF-1 cDNA probe revealed that UV light reduced IFN-gamma-induced IRF-1 mRNA. This study demonstrates that UV light can inhibit cytokine activities by interference with transcriptional activators. This newly described ability of UV light may contribute to its immunosuppressive properties.
A TCR V alpha CDR3-specific motif associated with Lewis rat autoimmune encephalomyelitis and basic protein-specific T cell clones.
Authors: Buenafe A C , Tsu R C , Bebo B , Bakke A C , Vandenbark A A , Offner H
Published: December 2022

To investigate TCR V alpha gene expression in the Lewis rat model of experimental autoimmune encephalomyelitis, we obtained V alpha chain sequences from two V beta8.2+-encephalitogenic, BP72-89-specific T cell clones. Two different V alpha genes, a V alpha2 gene and a V alpha23 gene, are utilized, but both were found to contain an asparagine repeat (Asn3+) sequence present in the V alpha CDR3 region. This Asn3+ motif is also present in the previously reported sequence of a BP68-88-specific hybridoma, 510, which utilizes a different V alpha2 gene family member. In further experiments, spinal cord T cells were isolated at the onset of basic protein (BP)-induced disease and sorted for the OX-40 activation marker, which we have previously used to enrich for specifically activated T cells. Analysis of V alpha expression in the OX-40+ population revealed the biased use of three V alpha genes, V alpha1, V alpha2, and V alpha23. The Asn3+ motif was present in the V alpha CDR3 region of V alpha1, V alpha2, and V alpha23 cDNA derived from OX-40+ spinal cord T cells but found to be generally absent in the OX-40- spinal cord population. Since these Asn3+ motif-bearing V alpha chain sequences are nearly identical to those utilized by the BP-specific encephalitogenic clones described, it is likely that these V alpha sequences are derived from disease-associated T cells in the spinal cord. Thus, we demonstrate that the Asn3+ V alpha CDR3 motif is strongly associated with experimental autoimmune encephalomyelitis in the Lewis rat and propose that it plays a role in TCR recognition of a specific BP peptide/MHC complex.
Concomitant killing in vitro of both gp120-coated CD4+ peripheral T lymphocytes and natural killer cells in the antibody-dependent cellular cytotoxicity (ADCC) system.
Authors: Jewett A , Cavalcanti M , Giorgi J , Bonavida B
Published: December 2022

NK cells play an important immunoregulatory role in first line defense mechanisms against infection. As disease progresses, HIV-1 infected patients show loss of NK cytotoxic function, down-modulation and/or loss of expression of both CD16 and CD56 surface Ags on NK cells and a gradual loss of both CD4+ T cells and NK cell numbers. A potential mechanism by which these manifestations may occur in vivo was investigated. We hypothesized that NK-mediated Ab-dependent cellular cytotoxicity (ADCC), using gp120-coated CD4+ peripheral T lymphocytes as targets and anti-HIV serum, will result in the concomitant killing of the CD4+ T lymphocyte targets and the NK lymphocytes. This hypothesis was examined in an in vitro model system. The findings demonstrate that gp120-coated peripheral T lymphocytes can serve as targets and are killed in ADCC. Further, the NK cells that recover from the ADCC reaction show a loss of cytotoxic function, acquire the CD16(dim/-) CD56(dim/-) phenotype and a significant fraction is killed by activation-induced cell death or apoptosis. These findings are reminiscent of the properties of circulating NK cells in HIV-infected patients. The implication of these findings in the pathogenesis of AIDS is discussed.
Corticosteroids inhibit IL-12 production in human monocytes and enhance their capacity to induce IL-4 synthesis in CD4+ lymphocytes.
Authors: Blotta M H , DeKruyff R H , Umetsu D T
Published: December 2022

We examined the effects of corticosteroids on IL-12 production by human monocytes and on cytokine synthesis in T cells. To distinguish the effects of corticosteroids on the APC used to activate the T cell from direct effects of corticosteroids on the T cell, experiments were performed by exposing the APC and not the T cell to corticosteroids. We found that corticosteroids significantly inhibited the production in monocytes of IL-12, a cytokine that is extremely potent in enhancing IFN-gamma and inhibiting IL-4 synthesis in T cells. We demonstrated that reduced production of IL-12 in corticosteroid-treated monocytes resulted in a decreased capacity of the monocytes to induce IFN-gamma and an increased ability to induce IL-4 in T cells. These results suggest that although corticosteroids may be beneficial for the treatment of asthma or allergic disease due to direct inhibitory effects of corticosteroids on cytokine synthesis in T cells, chronic corticosteroid therapy may indirectly exacerbate the long-term course of allergic disease. This deleterious effect of corticosteroids would result from a limitation in IL-12 production in tissue monocytes and macrophages, which would enhance production of Th2 cytokines (which augment allergic disease), and would reduce production of Th1 cytokines (which attenuate allergic disease) in T cells that subsequently infiltrate the tissues.
Streptococcal preparation OK-432 is a potent inducer of IL-12 and a T helper cell 1 dominant state.
Authors: Fujimoto T , Duda R B , Szilvasi A , Chen X , Mai M , O'Donnell M A
Published: December 2022

Streptococcal preparation OK-432 is a bacterial immunopotentiator extensively used in Japan for adjuvant cancer therapy. Using a C57BL/6 mouse model, OK-432 was found to induce multiple cytokines including the Th1 polarizing cytokine IL-12. Expression of IL-12 protein by murine splenocytes was restricted to macrophages and B cells and led to high levels of IFN-gamma production from both CD4+ and CD8+ T cells. Of the Th2 cytokines IL-4 and IL-10, only IL-10 protein was detected and originated primarily from the adherent cell population. Its expression was delayed relative to IL-12. A similar pattern of cytokine induction was observed from human PBMCs. OK-432-driven IFN-gamma production was inhibited by anti-IL-12 Ab, anti-IL-2 Ab, anti-TNF-alpha Ab, and anti-IL-2R alpha Ab, suggesting that IFN-gamma production from Th1 cells is induced by the cooperation action of these cytokines through the IL-2R alpha pathway. When compared with another widely used immunopotentiator bacillus Calmette-Guérin (BCG), OK-432 was a stronger IL-12 and IFN-gamma inducer. Furthermore, the mechanism of IFN-gamma induction by OK-432 differed from BCG in that coincident granulocyte-macrophage CSF and IL-1 expression played little to no role. These results suggest that OK-432 is a potent multicytokine inducer, specifically a strong inducer of IL-12, and that OK-432 may exert its antitumor effect by promoting a Th1-dominant state.
Evidence for self and nonself peptide partial agonists that prolong clonal survival of mature T cells in vitro.
Authors: Matsushita S , Kohsaka H , Nishimura Y
Published: December 2022

When examining the effects of peptide analogues without proliferation-inducing activity on three human CD4+ T cell clones with distinct TCRbeta recognizing a nonself mycobacterial bacillus Calmette-Guerin a (BCGa) peptide fragment (EEYLILSARDVLAVVSK)/HLA-DR14 complex, we found that 1) stimulation of T cells with a one-residue-substituted analogue or a minimally homologous self peptide fragment derived from human connexin 26 (IMILVVAAKEVWGDEQA) can prolong the in vitro survival of T cells, in a clone specific-manner; 2) this prolongation is associated with the up-regulation of Bcl-xL without proliferation; and 3) these peptide-clone combinations are capable of inducing lymphokine secretion. Thus, peptide partial agonism may play a role in the survival of not only thymocytes but also mature T cells, in the absence of wild-type ligands.
IL-6 rescues resting mouse T cells from apoptosis.
Authors: Teague T K , Marrack P , Kappler J W , Vella A T
Published: December 2022

It has previously been demonstrated that mature mouse T cells live for many weeks in vivo. In contrast, explanted lymph node or splenic T cells undergo spontaneous death within days, suggesting that survival factors supplied in vivo are not present in normal tissue culture medium. We discovered that IL-6 can rescue resting T cells from apoptosis in vitro. We show that recombinant mouse IL-6 as well as IL-6 in endothelial cell supernatants are sufficient to rescue T cells from death in the absence of additional cytokines. We show that CD4+ T cells express Bcl-2 immediately following isolation from the mouse, but after 24 h in culture Bcl-2 is undetectable. If during this time period the T cells are incubated with rIL-6, Bcl-2 expression is not down-regulated. It is, therefore, possible that IL-6 rescue from death is mediated by maintenance or induction of Bcl-2 expression. Addition of rIL-6 does not by itself induce blastogenesis or proliferation, and therefore, this cytokine appears to be a true survival factor rather than a mitogenic factor for resting T cells. Together, these results support a potential role for IL-6 as one of the factors important for prolonging resting T cell survival in vivo.
A naturally processed peptide presented by HLA-A*0201 is expressed at low abundance and recognized by an alloreactive CD8+ cytotoxic T cell with apparent high affinity.
Authors: Wang W , Gulden P H , Pierce R A , Shabanowitz J , Man S T , Hunt D F , Engelhard V H , Shabanowitz J A
Published: December 2022

In contrast to T cells that respond to peptides presented by self MHC molecules, alloreactive T cells recognize determinants expressed on nonself MHC molecules. Because current positive selection models suggest that T cell affinity toward a nonself MHC molecule would be lower than that toward a self MHC molecule, we previously proposed that vigorous alloreactive responses would be generated preferentially toward those antigenic peptide complexes presented at the highest density on the cell surface. The high abundance of two class I MHC-associated peptides that have been identified as allo- or xenoantigens is consistent with this hypothesis. We report here the identification of a naturally processed peptide YLDPAQQNL that is presented by HLA-A*0201 and recognized by an alloreactive T cell clone. This peptide appears to originate from an unknown member of the zinc finger proteins. Quantitation by mass spectrometry indicates that this peptide is present on the surface at 85 to 125 copies per cell, comparable with the density of several other epitopes presented by HLA-A*0201 to self MHC-restricted T cells. In addition, based on the affinity of the peptide for HLA-A*0201 and the half-maximal peptide concentration required for T cell sensitization, this alloreactive T cell appears to have an affinity similar to or higher than that of many self MHC-restricted T cells. These data suggest that allogeneic responses can be directed against antigenic determinants of low abundance and that recognition of alloreactive peptides is not limited by a lower affinity of T cells for nonself MHC molecules.
Glycosylphosphatidylinositol-anchored mucin-like glycoproteins isolated from Trypanosoma cruzi trypomastigotes initiate the synthesis of proinflammatory cytokines by macrophages.
Authors: Camargo M M , Almeida I C , Pereira M E , Ferguson M A , Travassos L R , Gazzinelli R T
Published: December 2022

Components of Trypanosoma cruzi able to induce the production of IL-12 and other proinflammatory cytokines by macrophages were identified. Murine inflammatory macrophages were cultured with live parasites or with cellular components from different developmental forms of T. cruzi (i.e., trypomastigotes, amastigotes, metacyclic trypomastigotes, and epimastigotes), and the cytokine levels were measured after 24 and 48 h. Our results indicate that live trypomastigotes or live amastigotes (but not live epimastigotes or live metacyclic trypomastigotes) as well as trypomastigote extracts (but not extracts derived from epimastigotes) induce IL-12 and TNF-alpha synthesis by macrophages. Such biological activity is enhanced in membrane preparations from trypomastigotes. Further enrichment of the trypomastigote-derived monokine-inducing factor was obtained by solvent extraction and hydrophobic-interaction chromatography. The resultant purified molecules are a family of closely related glycoconjugates with predominant species at 70 to 80 and 120 to 200 kDa. These molecules are composed of carbohydrate chains O-linked to a polypeptide backbone that is anchored to the trypomastigote membrane via a glycosylphosphatidylinositol structure. The trypomastigote-derived glycoconjugates are active in inducing cytokine synthesis by macrophages at concentrations of 100 ng/ml. These effects are highly potentiated by IFN-gamma. Mapping of the glycoconjugate molecules to characterize the structural requirements for macrophage activation suggested that nonsaturated acyl fatty acid chains and periodate-sensitive units from the glycosylphosphatidylinositol anchor are important elements for the infective trypomastigote form to initiate cytokine synthesis by macrophages.
The differential ability of IL-8 and neutrophil-activating peptide-2 to induce attenuation of chemotaxis is mediated by their divergent capabilities to phosphorylate CXCR2 (IL-8 receptor B).
Authors: Ben-Baruch A , Grimm M , Bengali K , Evans G A , Chertov O , Wang J M , Howard O M , Mukaida N , Matsushima K , Oppenheim J J
Published: December 2022

IL-8 and neutrophil-activating peptide-2 (NAP-2) are two closely related C-X-C chemokines that differ in their abilities to induce chemotaxis of human polymorphonuclear leukocytes (PMN). Although two IL-8R types are expressed by PMN, only CXCR2 binds NAP-2 and IL-8 with equally high affinity. By using enriched CXCR2-transfected 293 cells, we show that high doses of IL-8 induce attenuation of chemotaxis, while equivalent doses of NAP-2 do not. Phosphorylation analysis shows that IL-8 induces higher levels of phosphorylation of the carboxyl terminus of CXCR2 than does NAP-2, suggesting that the level of phosphorylation contributes to the ability of the chemokines to attenuate the chemotactic response. To directly evaluate this difference, we analyzed the ability of receptors mutated to delete regions that highly express potential phosphorylation sites to be phosphorylated and to mediate chemotactic attenuation. We found that a carboxyl terminus-truncated mutant of CXCR2 was not phosphorylated by high doses of IL-8, as determined by in vivo phosphorylation assays and by analysis of the electrophoretic mobility of the receptors on SDS-PAGE gels. This mutated receptor had a significantly lower ability to attenuate IL-8-induced chemotaxis, indicating that the attenuation of chemotaxis is mediated by chemokine-induced receptor phosphorylation. In conclusion, the data show that the greater ability of IL-8 to induce receptor phosphorylation contributes to its more potent attenuation of chemotaxis as compared with NAP-2. This differential phosphorylation by IL-8 and NAP-2 of CXCR2 provides a basis for the divergent outcome of PMN-induced inflammation in response to these two closely related C-X-C chemokines.