bluuebunny/crossref_metadata_2025 huggingface dataset:
Preferential involvement of a phospholipase A2-dependent pathway in CD69-mediated platelet activation.
Authors: Testi R , Pulcinelli F M , Cifone M G , Botti D , Del Grosso E , Riondino S , Frati L , Gazzaniga P P , Santoni A
Published: December 2022

CD69 is a signal transducing disulfide-linked homodimer functionally expressed on platelets, CD3bright thymocytes, and activated lymphocytes. In an attempt to investigate early molecular events in CD69-mediated cell activation we studied the relative contribution of phospholipase A2 (PLA2) and phosphatidylinositol-specific phospholipase C-dependent pathways during platelet activation induced by CD69 stimulation. Thromboxane A2 (TXA2) synthetase inhibitor and TXA2R inhibitor R68070 were able to inhibit platelet aggregation induced by CD69 stimulation, indicating that TXA2 was the main mediator of the response. CD69-induced arachidonic acid release and TXA2 production were essentially PLA2 dependent because they could be blocked by the PLA2 inhibitor quinacrine. Inositol 1,3,4-trisphosphate generation was clearly detectable after CD69 cross-linking, but it was completely abrogated by quinacrine and R68070 and therefore secondary to TXA2 release and TXA2R engagement. Finally, direct measurement of enzymatic activity in vitro using radiolabeled phospholipid vesicles showed that CD69 cross-linking resulted in PLA2-dependent arachidonic acid and lysophosphatidylcholine generation from phosphatidylcholine, which was sensitive to quinacrine but not to R68070. By contrast, CD69-induced 1,2-diacylglycerol release from phosphatidylinositol 4,5-bisphosphate was blocked by both inhibitors. These results indicate a preferential involvement of PLA2 in CD69-dependent signal transduction in platelets and provide evidence for the unique role of PLA2-mediated activation pathways in transmembrane receptor signaling.
CD4-CD8+ T lymphocytes mediate AKR/gross murine leukemia virus nonresponsiveness in moderately aged AKR.H-2b:Fv-1b mice.
Authors: Rich R F , Fujii T , Green W R
Published: December 2022

Previously we reported that as AKR.H-2b:Fv-1b mice become older than 9 wk of age they begin to specifically lose the ability to generate anti-AKR/Gross murine leukemia virus (MuLV) CTL responses after immunization and in vitro restimulation with cells expressing AKR/Gross MuLV-encoded Ag. Interestingly, the frequency of virus-specific precursor cytotoxic T lymphocytes (CTL) observed in moderately-aged AKR.H-2b:Fv-1b mice was not substantially decreased from that found in their young responder counterparts. To further investigate the mechanism(s) responsible for the inability of moderately-aged AKR.H-2b:Fv-1b mice to mount AKR/Gross MuLV-specific CTL responses, adoptive transfer experiments were performed in the present study. Transferring splenocytes from moderately-aged AKR.H-2b:Fv-1b donors into young AKR.H-2b:Fv-1b recipients resulted in inhibition of AKR/Gross MuLV-specific CTL responsiveness. Anti-Thy-1.1 plus complement depletion of T cells from the donor cell population before adoptive transfer resulted in a near complete restoration of AKR/Gross MuLV responsiveness of young recipient AKR.H-2b:Fv-1b mice suggesting that the inhibition observed in moderately aged mice was mediated by T lymphocytes. Additional experiments using depletion of T subsets before cell transfer demonstrated that inhibition of AKR/Gross MuLV-specific CTL responsiveness was mediated by a CD4-CD8+ T lymphocyte.
Expansion of murine T cells bearing a unique T cell receptor beta-chain in Friend virus-induced tumor in situ.
Authors: Suzuki M , Koseki H , Mizutani Y , Kuribayashi K , Kanno M , Taniguchi M
Published: December 2022

Heterogeneity of V alpha 1+ and V beta 10+ TCR alpha beta-chains, which are predominantly used in anti-FBL-3 CTL clones established in vitro, was investigated at a nucleotide level in FBL-3 tumor-infiltrating lymphocytes (TIL) in vivo. The majority (90%) of V beta 10+ beta-chains dominated in TIL used homogeneous V beta 10D beta 2.1 sequences identical to that used in the T cell clones with cytotoxic functions. The homogeneous TCR beta-chain expression was dominant and found to be about 10% of the total TCR beta-chains in the TIL population, which was a greater than 300-to 900-fold increase than in the regional lymph nodes. This is in good agreement with the in vitro data showing that about 11% CTL clones used the homogeneous V beta 10D beta 2.1+ beta-chain. However, the J beta segment does not seem to contribute greatly to the recognition and selection of this TCR because some of homogeneous VD+ beta-chains were associated with J beta segments other than J beta 2.7 of the CTL clones. The frequency of the V alpha 1J alpha 112-2+ alpha-chain expression of the CTL type was much less (3- to 80-fold increase compared to that of lymph node) and also varied in sample materials, indicating the lower contribution of the alpha-chain for the oligoclonality of the TCR. The results were also confirmed by quantitative PCR and RNase protection assays. This suggests that the dominant expression of the homogeneous TCR beta-chain is due to the expansion of the particular anti-FBL-3 CTL in the tumor in situ. Also, the TCR beta-chain, especially the V beta D beta region, rather than alpha-chain is more important for the recognition and selection of the anti-FBL-3 TIL with cytotoxic functions.
Formation of complexes between self-peptides and MHC class II molecules in cells defective for presentation of exogenous protein antigens.
Authors: Bikoff E K
Published: December 2022

A vertebrate immune response is initiated by the presentation of foreign protein Ag to MHC class II-restricted T lymphocytes by specialized APC. Presentation of self-peptides in association with MHC class II molecules is also necessary for the induction of T cell tolerance. It is important to understand whether functionally divergent APC are responsible for delivering these distinct signals to class II-restricted T cells. Here we examine the ability of I-Ad surface molecules expressed in diverse cell types to stimulate I-Ad-restricted T cells. Recipients included J558L myeloma cells and EL4 lymphoma cells expressing barely detectable or undetectable levels of Ii chain mRNA. This allowed us to examine the influence of Ii expression on the presentation of intracellular Ag and thus test the hypothesis that Ii chain is necessary to prevent access of self-peptides to newly synthesized class II molecules. Ii chain expression did not restore the ability of transformants to process and present soluble protein Ag. A striking result was the finding that cells showing a defect in the exogenous class II presentation pathway were capable of functioning as stimulators when they expressed intracellular secreted but not signal-less V-CH3b Ag. Thus, so-called professional APC that can capture and process exogenous protein Ag may express a specialized set of proteins not required for the presentation of self-peptides.
IL-4 and IL-2 selectively rescue Th cell subsets from glucocorticoid-induced apoptosis.
Authors: Zubiaga A M , Munoz E , Huber B T
Published: December 2022

It is well established that T cell maturation and activation are negatively regulated by a mechanism termed apoptosis. We now present evidence that glucocorticoids, known to possess immunosuppressive properties, cause apoptosis in mature Th cells, similarly to what has been reported for thymocytes. Th cells treated with the synthetic glucocorticoid dexamethasone show genome fragmentation into oligonucleosomal fragments, and proliferation of growth factor stimulated Th cells is inhibited by glucocorticoids. We show that IL-4 specifically rescues Th2 cells from dexamethasone-mediated apoptosis, whereas IL-2 and IL-1 are ineffective in these cells. However, IL-2 is the relevant rescue-factor of glucocorticoid-treated Th1 cells. The rescue induced by IL-4 and IL-2 is thought to be mediated by protein kinases (possibly protein kinase C), as evidenced by the fact that the protein kinase inhibitor H7 blocks the action of IL-4 and IL-2 in glucocorticoid-treated cells. Our in vitro data show that mature T cells can be protected by their own growth factors from the deleterious effects of the synthetic glucocorticoid dexamethasone, and suggest that specific interactions occur between lymphokines and naturally produced glucocorticoids in vivo, which may play a role in the regulation of the immune response.
Granulocyte colony-stimulating factor (G-CSF) gene transduction in murine adenocarcinoma drives neutrophil-mediated tumor inhibition in vivo. Neutrophils discriminate between G-CSF-producing and G-CSF-nonproducing tumor cells.
Authors: Colombo M P , Lombardi L , Stoppacciaro A , Melani C , Parenza M , Bottazzi B , Parmiani G
Published: December 2022

We have previously demonstrated that the murine colon adenocarcinoma C-26 cell line transduced with the human gene for the granulocyte CSF (G-CSF) loses tumorigenic activity through a mechanism that involved massive targeting of neutrophils at the site of tumor injection. The suppression of tumorigenicity by G-CSF was limited to the G-CSF-producing cells and was not transferred to nonproducing C-26 cells in a mixed tumor transplantation assay. We present direct evidence that neutrophils are involved in this phenomenon. We firstly examined, by electron microscopy (EM), the morphology of tumor infiltrates obtained 2, 5, and 10 days after s.c. injection of a mixture of G-CSF-producing and -nonproducing C-26 cells into syngeneic BALB/c mice. The EM analysis showed at 5, but not at 2 or 10 days, the presence of neutrophils in intimate contact with tumor cells. We then investigated whether neutrophils discriminate between G-CSF-producing and -nonproducing C-26 cells. To this aim, C-26 cells were transduced, via retroviral vector, with the Escherichia coli LacZ gene and mixed tumor transplantation assays were performed by injecting a mixture of G-CSF-producing beta-gal- and G-CSF-nonproducing beta-gal+ C-26 cells at different ratios. Histologic and EM analysis of the tumors growing at the site of injection were carried out. Five days after injection, treatment with x-gal revealed, at the histochemical level, the presence of neutrophils around G-CSF producing beta-gal- cells; cell-cell contacts and fusion of cell membranes were detected by EM only between neutrophils and G-CSF-producing cells. In vitro experiments, performed in Boyden chambers, confirmed that the G-CSF produced by C-26 cells was a chemoattractant for neutrophils. In addition, a colorimetric, cytostatic assay revealed that neutrophils were able to inhibit the growth of G-CSF-producing but not of G-CSF-nonproducing C-26 cells. Thus the tumor take after injection of G-CSF-producing C-26 cells seems to be controlled in situ through two major mechanisms namely neutrophil chemotaxis and neutrophil-mediated tumor inhibition. The results indicate that neutrophils can discriminate between G-CSF-producing and -nonproducing tumor cells and that neutrophils infiltrate the tumor mixture as long as G-CSF-producing cells are present.
Antigen receptor-mediated protein tyrosine kinase activity is regulated by a pertussis toxin-sensitive G protein.
Authors: Melamed I , Wang G , Roifman C M
Published: December 2022

Ligation of the Ag receptor on B cells is associated with a rapid increase in phosphorylation on tyrosine residues of multiple substrates. One of the substrates is the phosphoinositide-specific phospholipase C-gamma 1. Because activation of phospholipase C-gamma 1 seems to be dependent on tyrosine phosphorylation, it is assumed that the two signaling pathways, phosphatidylinositol turnover and tyrosine phosphorylation, might be linked. However, since the Ag receptor does not possess a kinase domain, it remains unclear how these signaling pathways are regulated by the Ag receptor. Previous studies have proposed the existence of a receptor-coupled G protein that regulates inositol phosphate production in B cells. We confirm that phosphoinositide turnover is regulated by a pertussis toxin (PT)-sensitive G protein, most probably by controlling phosphorylation of phospholipase C-gamma 1. We show that treatment of permeabilized B cells with a nonhydrolyzable GTP analogue guanosine 5'-[3-thio]triphosphate induced an increase in tyrosine phosphorylation of multiple substrates that are identical to the proteins phosphorylated after anti-IgM stimulation. Furthermore, binding of the inactive form of G proteins with guanosine 5'-[2-thio]-triphosphate blocked anti-IgM induced tyrosine phosphorylation in permeabilized B cells. The results indicate that an Ag receptor-coupled G protein controls protein tyrosine kinase activity. We show that this G protein is sensitive to PT because tyrosine phosphorylation mediated by the Ag receptor was inhibited by this toxin in a concentration-dependent manner. Similar concentrations of PT also blocked tyrosine phosphorylation on phospholipase C-gamma 1 and generation of inositol phosphates. Preincubation of intact B cells with PT resulted in inhibition of c-fos mRNA expression and DNA synthesis in anti-IgM stimulated B cells, indicating that post-transcriptional events are also controlled by the Ag-receptor coupled G protein. We conclude that Ag receptor-associated protein tyrosine kinase activity is regulated by a G protein. This PT-sensitive G protein also regulates phosphorylation and activation of phospholipase C-gamma 1 as well as later events in B cell activation such as c-fos mRNA expression and proliferation.
Simultaneous depletion of CD4+ and CD8+ T lymphocytes is required to reactivate chronic infection with Toxoplasma gondii.
Authors: Gazzinelli R , Xu Y , Hieny S , Cheever A , Sher A
Published: December 2022

C57BL/6 mice chronically infected with an avirulent strain (ME-49) of Toxoplasma gondii were used to study the mechanisms by which T lymphocytes and IFN-gamma prevent reactivation of latent infection. Infected animals were treated with mAb, either anti-CD8, anti-CD4, anti-CD4 plus anti-CD8, anti-IFN-gamma, or anti-CD4 plus anti-IFN-gamma and the mice followed for survival, histopathology, cyst numbers, and spleen cell cytokine responses. In agreement with previously published findings, treatment with anti-IFN-gamma antibodies fully reactivated the asymptomatic infection, inducing massive necrotic areas in the brain with the appearance of free tachyzoites and death of all animals within 2 wk. Mice treated with the combination of anti-CD4 plus anti-CD8 antibodies showed augmented pathology and mortality nearly identical to the anti-IFN-gamma- treated animals. In contrast, treatment with anti-CD4 or anti-CD8 mAb alone failed to result in significantly enhanced brain pathology or mortality. In additional experiments, full reactivation of infection was observed in mice treated with anti-CD4 plus anti-IFN-gamma indicating that CD4+ lymphocytes are not required for the pathology resulting from IFN-gamma neutralization. Cytokine measurements on parasite Ag-stimulated spleen cells from mAb-treated mice indicated that both CD4+ and CD8+ cells produce IFN-gamma whereas only CD4+ cells contribute to parasite Ag-induced IL-2 synthesis. Together, these results suggest that CD4+ and CD8+ lymphocytes act additively or synergistically to prevent reactivation of chronic T. gondii infection probably through the production of IFN-gamma.
Presentation of three different viral peptides, HTLV-1 Tax, HCMV gB, and influenza virus M1, is determined by common structural features of the HLA-A2.1 molecule.
Authors: Utz U , Koenig S , Coligan J E , Biddison W E
Published: December 2022

To determine whether similar or dissimilar molecular features of class I molecules are involved in the presentation of structurally distinct peptides, we have investigated the influence of different pockets of the HLA-A2.1 molecule on the presentation of three different viral peptides. HTLV-I Tax peptide 12-19, HCMV gB 619-628, and influenza M1 58-66 are minimal peptides that induce HLA-A2.1-restricted noncross-reactive CTL. A detailed analysis of the structural features of HLA-A2.1 that are involved in peptide presentation was undertaken using a panel of 11 HLA-A2 mutants with single amino acid substitutions within pockets present in the peptide binding site. Nine of the 11 mutants affected presentation of each of the three peptides, whereas the other two mutants had negative effects on presentation of only two of these viral peptides. These results indicate that common structural features in HLA-A2 determine the binding of different peptides, and help to provide a plausible explanation for how structurally diverse peptides bind to HLA-A2.
V beta gene repertoires in aging mice.
Authors: González-Quintial R , Theofilopoulos A N
Published: December 2022

To determine whether aging and thymic involution are associated with defects in intrathymic T cell selection or clonal instabilities, we compared transcript levels for 18 V beta genes in thymic and splenic T cells of young (2 month), adult (12 month), and old (20 month) mice of various Mls and MHC haplotypes (C57BL/6, BALB/c, and DBA/2). The results showed that the unselected thymic V beta repertoires remain stable throughout life, despite severe reduction in cellularity in the thymus of aged mice. Similarly, splenic CD4 and CD8 V beta repertoires showed no significant alterations, and no leakage to the periphery of endogenous superantigen-reactive V beta clones was observed with age, even in irradiated and bone marrow-reconstituted old mice. Responses in vitro to bacterial superantigens were undiminished with age but, significantly, some of these superantigens expanded V beta clones that are cross-reactive with endogenous superantigens and are normally partially (V beta 11 and -12) or severely (V beta 3.1) deleted in BALB/c mice. In the course of these studies, several previously unrecognized reactivities of V beta with staphylococcal toxins were also revealed.
Transcription of unrearranged Ig H chain genes in human B cell malignancies. Biased expression of genes encoded within the first duplication unit of the Ig H chain locus.
Authors: Sideras P , Nilsson L , Islam K B , Quintana I Z , Freihof L , Rosén A , Juliusson G , Hammarström L , Smith C I
Published: December 2022

Analysis of the H chain class switch recombination events on the productive and nonproductive alleles of several human B cell lines has demonstrated that, in the majority of cases, the two loci had rearranged different CH genes. The absence of a strict correlation between the two rearrangement events has been interpreted in favor of a stochastic switching mechanism in human B cells. Based on the accessibility model for the H chain class switch, we have considered an alternative explanation, namely, that transcriptional activation of more than one CH gene could render more than one isotype accessible for recombination. In order to test this possibility, we have stimulated a number of IgM-bearing B cell tumors and analyzed the transcriptional response of their unrearranged C gamma and C alpha H chain genes at the Ig class level by Northern hybridization and at the subclass level by RNA-RNA solution hybridization. Our data show that human clonal B cell populations can simultaneously express germ-line transcripts of both gamma and alpha genes. Our data also demonstrate a bias in the expression of the different isotypes in the B cell chronic lymphocytic leukemias analyzed, with the genes present with the first duplication unit of the H chain locus (gamma 3, gamma 1, and alpha 1) being expressed almost exclusively. These findings indicate that transcriptional activation of large chromosomal regions containing more than one CH gene could render all the genes available for recombination and, therefore, provide an explanation for the asymmetry in the rearrangement events that have previously been described in human B cell tumors. The possible relevance of our findings to the molecular mechanism of the H chain class switch is discussed.
Augmentation of in vivo cytotoxic T lymphocyte activity and reduction of tumor growth by large multivalent immunogen.
Authors: Rogers J , Mescher M F
Published: December 2022

Class I alloantigen incorporated into cell-size supported membranes provides an effective stimulus for in vitro stimulation of CTL responses. When alloantigen-bearing cell-size (5 microns) microspheres, termed large multivalent immunogen (LMI), were administered in vivo, no primary cytotoxic response to the Ag could be detected. However, coadministration of LMI and allogeneic tumor stimulator cells resulted in substantial augmentation of the resulting CTL response, compared with that obtained from mice that received just stimulator cells. Responses were augmented only when the same alloantigen was present on the LMI and on the stimulator cells, and the effector cells remained specific for the cognate alloantigen-bearing targets. The physical form of the alloantigen was critical for augmentation; alloantigen in liposomes had no effect on response levels. Tumor cell Ag in the form of purified plasma membrane vesicles can also be incorporated onto the surface of cell-size microspheres. As with allogeneic responses, tumor Ag on LMI specifically augmented the in vivo CTL activity generated in response to irradiated tumor cells in syngeneic mice. Administration of Ag-bearing LMI to mice inoculated i.p. with live P815, EL4, or RDM4 tumor cells resulted in a significant reduction in growth of the tumors in their syngeneic hosts. Similarly, LMI treatment significantly reduced growth of P815 as a solid s.c. tumor. LMI-mediated growth reduction occurred only when plasma membrane Ag from the cognate tumor was used to prepare the LMI, and Ag in the form of free plasma membrane vesicles was not effective. Although Ag has been used to manipulate in vivo humoral and Th responses, this has proven to be much more difficult for CTL responses. The ability of Ag-bearing LMI to affect significantly the in vivo levels of cytolytic response and to reduce syngeneic tumor growth has potential for application to tumor immunotherapy and, possibly, treatment of other diseases in which CTL can provide a protective effect.
Evidence for clonal expansion of T cell receptor V gamma II+ T cells in the synovial fluid of patients with arthritis.
Authors: Doherty P J , Yang S X , Laxer R M , Silverman E D , Inman R , Pan S
Published: December 2022

We have demonstrated among synovial fluid T cells a unique profile of V gamma II sequences likely arising from clonally expanded T cells. We have determined the junctional diversity associated with each expressed V gamma family by resolving amplified fragments of cDNA into component parts on large denaturing gels. Among synovial fluid T cells we frequently find dominant fragments of a unique size clearly smaller than the dominant band observed with peripheral blood T lymphocytes. In some cases the dominant bands are 12 or 15 nucleotides smaller than the corresponding most abundant band from peripheral blood T lymphocytes. Patterns of lower m.w. species not typical of a polyclonal population argues that clones of T cells expressing the V gamma II family are expanding in the joint and that a high proportion of these cells do not express the V gamma IIJP sequence typical of peripheral blood but rather express V gamma II in combination with a shorter J fragment, JP1, JP2, J1, or J2. In addition by examining joint effusions from the left and right knees from the same individual we have shown that the profiles of V gamma II sequences derived from the fluids are identical to each other but clearly distinct from that of peripheral blood. We have, in addition, quantitated with a series of synthetic internal standards the relative usage of each V gamma family expressed by T cells in the synovial fluid and peripheral blood of seven patients with arthritis including six patients who were either children or adolescents and one adult patient. All patients showed a reduction in the relative expression of V gamma II in synovial T cells relative to peripheral blood T lymphocytes and a corresponding increase in the expression of V gamma I or V gamma III or both. We did not detect expression of V gamma IV in either lymphocyte population.
Immunogenetics of collagen-induced arthritis in rats. Both MHC and non-MHC gene products determine the epitope specificity of immune response to bovine and chick type II collagens.
Authors: Griffiths M M , Cremer M A , Harper D S , McCall S , Cannon G W
Published: December 2022

Seven inbred, RT1-congenic rat strains were immunized with native bovine (BII), porcine (PII), or chick (CII) type II collagen and observed for onset, incidence, and severity of arthritis. Clinical results were compared with IgG reactive with native rat type II collagen (RII) and the purified, renatured cyanogen-bromide peptides of BII, CII, or RII. Immunodominant responses to CB11, CB9,7, and CB12 of RII were identified. Secondary responses to CB8 and CB10 also occurred. Reproducible patterns of peptide reactivity were defined in each strain and reflected both RT1 and non-RT1 genotypes plus the species of immunizing collagen. BN non-RT1 gene products moderated clinical arthritis but increased the levels of reactivity to CB11 in three strains carrying RT1l,n,av1 haplotypes. WF (RT1u) rats were susceptible to collagen-induced arthritis (CIA) and developed very high levels of autoantibodies with dominant responses to rat CB11 after CII injections and to rat CB11 and CB9,7 after BII injections. DA (RT1av1) rats developed the most severe arthritis but had only moderate (total) levels of anti-RII IgG: a broad response to CB11, CB10, and CB9,7 after CII injections but predominantly to CB12 and CB9,7 after BII injections. Three RT1n strains--DA.1N(BN), WF.1N(MAXX), and BN--were resistant to BII-induced CIA but developed mild arthritis after immunization with CII. After BII: BN IgG reacted with CB9-7, CB11, and CB12; DA.1N and WF.1N IgG reacted with CB9,7 and CB12. After CII: BN IgG reacted broadly with CB11, CB9-7, CB12, and CB8; WF.1N IgG reacted to CB9-7, CB11, CB8, and CB12; DA.1N IgG reacted with CB8, CB11, and CB9-7. Thus, selective induction of CIA in BN, WF.1N, and DA.1N rats by CII correlated with serum IgG reactivity to rat CB11, but overall strain results identified no single cyanogen-bromide peptide as expressing the sole "arthritogenic" epitope in CIA.
Enhancement of human neutrophil functions by a monoclonal antibody directed against a 19-kDa antigen.
Authors: Mollinedo F , Burgaleta C , Velasco G , Arroyo A G , Acevedo A , Barasoain I
Published: December 2022

A mouse IgG mAb termed P1C3 was raised against A23187-treated human peripheral blood neutrophils and has been shown to recognize an Ag with an apparent molecular mass of 19 kDa, herein named p19. This p19 Ag was weakly expressed at the cell surface of resting human peripheral blood neutrophils and monocytes, but its cell surface expression was dramatically increased upon activation of these cell types with different secretagogues, including FMLP, PMA, and the calcium ionophores A23187 and ionomycin. A large latent pool of p19 molecules became accessible by immunofluorescence flow cytometry after cell permeabilization of resting neutrophils. A practically total translocation of the intracellular pool of this p19 molecule to the plasma membrane was achieved under appropriate cell stimulation, which induced an almost total degranulation of neutrophil secretory granules. The p19 Ag was absent from platelets, PBL, as well as from the human promyelocytic cell line HL-60, the human promonocytic cell line U937, and the human lymphoid cell lines Daudi and Jurkat. The p19 Ag was also expressed by circulating and/or interstitial neutrophils and monocytes in distinct tissues examined. The mAb P1C3 was found to enhance several neutrophil responses, such as chemotaxis, cell adhesion, phagocytosis, and respiratory burst. These data indicate that the mAb P1C3 recognizes an intracellular Ag in human resting mature neutrophils and monocytes, which upon cell activation is translocated to the cell surface and is able to affect cell functionality.
In vitro induction of CD8 expression on thymic pre-T cells. II. Characterization of CD3-CD4-CD8 alpha + cells generated in vitro by culturing CD25+CD3-CD4-CD8- thymocytes with T cell growth factor-beta and tumor necrosis factor-alpha.
Authors: Suda T , Zlotnik A
Published: December 2022

We previously reported that CD25 (IL-2R p55)-positive CD3-CD4-CD8- murine thymocytes can be induced to express CD8 alpha (Lyt-2) by transforming growth factor-beta plus TNF-alpha in the presence of IL-7 (which is necessary to maintain the viability and differentiation capacity of CD25+CD3-CD4-CD8- thymocytes in vitro). The majority of cells recovered after 2 to 3 days from these cultures expressed CD8 alpha (but not CD3 or CD4). In this study, we have characterized these in vitro generated CD3-CD4-CD8 alpha + thymocytes and compared them with normal CD3-CD4-CD8+ thymocytes. Unlike normal CD3-CD4-CD8+ thymocytes that express CD8 alpha and CD8 beta (Lyt-3-chain) simultaneously, only a fraction of in vitro generated CD3-CD4-CD8 alpha + cells expressed CD8 beta. However, along with the induction of CD8 alpha and CD8 beta expression, the expression of other T cell differentiation markers (including CD2, CD25, and CD44) also changed in a manner corresponding to physiologic differentiation. Cell-surface phenotyping suggests that CD8 alpha + beta - cells are less mature than CD8 alpha + beta + cells. These in vitro generated CD3-CD4-CD8 alpha + thymocytes expanded and differentiated into the CD4+CD8+ stage as well as mature (CD3+) single positive (CD4+CD8-) and CD4-CD8+) stages in fetal thymus organ culture that had been depleted of lymphoid cells by treatment with 2-deoxyguanosine. The latter observation indicates that these in vitro generated CD3-CD4-CD8 alpha + thymocytes are responsive to other differentiation-inducing signals (including those that induce CD4) that exist in fetal thymus organ culture. These results suggest that in vitro generated CD3-CD4-CD8 alpha + thymocytes represent intermediate differentiation stages between CD25+CD3-CD4-CD8- and CD3-CD4-CD8+ cells found in normal thymus.
High efficiency presentation of TNP-conjugated islet antigen to islet-specific T cells by a hybrid B cell line expressing TNP-specific surface Ig.
Authors: Wegmann D , Norbury-Glaser M , Bergman B , Haskins K
Published: December 2022

There is compelling evidence from animal models that type I diabetes is a consequence of T cell-mediated destruction of islet beta-cells. The recent isolation of islet-specific T cell clones from nonobese diabetic mice provides a means of identification of the Ag on islet cells that are responsible for stimulation of autoreactive T cells. We describe an APC line constructed by fusion of spleen B cells obtained from nonobese diabetic mice to a B lymphoma that was transfected with the H and L chains of an IgM specific to the hapten TNP. Using this hybrid APC we have observed a dramatic increase in the efficiency of presentation of TNP-conjugated islet cell protein preparations compared to that seen with conventional APC. Our results illustrate the potential use of this APC line for isolation and characterization of islet Ag relevant to the T cell response.
Murine AIDS superantigen reactivity of the T cells bearing V beta 5 T cell antigen receptor.
Authors: Kanagawa O , Nussrallah B A , Wiebenga M E , Murphy K M , Morse H C , Carbone F R
Published: December 2022

A B cell line, B6-1710, that expresses the defective virus known to induce murine AIDS stimulates a large fraction of nonprimed splenic T cells. Analysis of the T cell population responding to the B6-1710 for TCR V beta-chain usage revealed that, in addition to the previously reported V beta 5-chain-positive T cells, T cells bearing V beta 11 and V beta 12 are also specifically enriched. We have established V beta 5+ T cell lines, clones, and hybridomas expressing identical TCR with different CD4/CD8 phenotypes and demonstrated that T cell reactivity to B6-1710 is, although not absolute, dependent on the presence of CD4 molecules. Further analysis of T cell hybridomas with known J beta-chain usage revealed that D beta- and J beta-chain usage do not play crucial roles in T cell reactivity to B6-1710 B cells. However, T cell hybridomas derived from TCR-V beta gene transgenic mice were found to be heterogeneous for their reactivity to B6-1710, suggesting that the V alpha-chains associating with the transgenic V beta-chain determine T cell responsiveness to B6-1710. These data clearly demonstrate that T cell reactivity to a murine AIDS virus expressing B cell line resembles that previously reported for Mls-like superantigens.
Up-regulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors in murine peritoneal exudate macrophages by both GM-CSF and IL-3.
Authors: Fan K , Ruan Q , Sensenbrenner L , Chen B D
Published: December 2022

Murine peritoneal exudate macrophages (PEM) display multiple CSF receptors. In this study, the expression of granulocyte-macrophage (GM)-CSF receptors in PEM was studied. PEM displayed over 5000 single type, high affinity GM-CSF receptors/cell with a Kd = 38 to 42 pM and an apparent molecular mass of 86,000 Da. Treatment of PEM with low, but not high, concentrations of recombinant murine (rMu) GM-CSF continuously for 24 h resulted in a marked up-regulation of GM-CSF receptors in PEM. A similar up-regulation of GM-CSF receptors also was detected in PEM cultures treated with rMuIL-3 (1-100 ng/ml) for 24 h or longer, regardless the doses of rMuIL-3 added in this case. Scatchard analysis of equilibrium binding showed that the enhanced binding activities in both cases were due to an increase in total number of GM-CSF receptors rather than changes in receptor affinity. Contrariwise, treatment with recombinant human macrophage-CSF (greater than 100-1000 ng/ml) partially inhibited the expression of GM-CSF receptors in PEM. Removal of rMuGM-CSF from culture medium 24 h after treatment led to a further up-regulation of GM-CSF receptors over a 4 to 24-h period, depending on the doses of initial treatment. On the other hand, removal of rMuIL-3 from culture medium after prolonged treatment did not result in further increase in GM-CSF receptors. The protein synthesis inhibitor cycloheximide abrogated GM-CSF receptor up-regulation induced by both rMuIL-3 and rMuGM-CSF, whereas actinomycin D inhibited only the second (8-24 h) phase of GM-CSF receptor up-regulation induced by exposure to high concentrations rMuGM-CSF (10 ng/ml). These findings suggest that rMuGM-CSF and rMuIL-3 up-regulate GM-CSF receptors in PEM in part through similar or identical metabolic pathways and provide further evidence of a close linkage between IL-3 and GM-CSF receptors.
Basement membrane and its components on lymphocyte adhesion, migration, and proliferation.
Authors: Li Y Y , Cheung H T
Published: December 2022

During inflammation and recirculation, lymphocytes migrate into tissues by traversing the capillary endothelium, a process known as extravasation. After crossing the endothelial cells, lymphocytes come into contact with the basement membrane, which is a specialized layer of extracellular matrix containing predominantly laminin, collagen type IV, entactin, and heparan sulfate proteoglycans. In tissue invasion by inflammatory cells and metastatic tumor cells, the basement membrane serves as a substratum for cell adhesion and migration. However, the role of basement membrane in lymphocyte extravasation remains unclear. In this study, we investigated the effect of basement membrane on lymphocyte adhesion, migration, and proliferation, using matrigel as a model for basement membrane. We observed that matrigel promotes both lymphocyte adhesion and migration, with entactin primarily responsible for promoting adhesion and laminin for promoting migration. In addition, activation of lymphocytes by anti-CD3 enhances their adhesion and migration on matrigel-coated substratum. We also observed that matrigel inhibits the proliferation of lymphocytes stimulated by Con A. Furthermore, we demonstrated that laminin is the matrigel component responsible for inhibiting lymphocyte proliferation. However, matrigel has no effect on the proliferation of lymphocytes stimulated by LPS. These results suggest that matrigel has different effects on lymphocyte subpopulations. In agreement with the results on proliferation, matrigel also inhibits the production of IL-2 by Con A-stimulated lymphocytes.
Tumor necrosis factor-alpha-dependent production of reactive nitrogen intermediates mediates IFN-gamma plus IL-2-induced murine macrophage tumoricidal activity.
Authors: Cox G W , Melillo G , Chattopadhyay U , Mullet D , Fertel R H , Varesio L
Published: December 2022

We have previously established that IFN-gamma plus IL-2 induces murine macrophage tumoricidal activity. The purpose of this study was to identify the effector molecules that account for the IFN-gamma plus IL-2-induced macrophage cytotoxicity against P815 mastocytoma cells. ANA-1 macrophages and normal thioglycollate-elicited mouse peritoneal macrophages produced little or no detectable nitrite (NO2-) after incubation with IFN-gamma alone or IL-2 alone; however, IL-2 synergized with IFN-gamma for the production of NO2-. IFN-gamma plus IL-2 did not induce NO2- production or tumoricidal activity in ANA-1 macrophages that were cultured in medium devoid of L-arginine or in ANA-1 macrophages that were incubated with NG-monomethyl-L-arginine. As observed previously with ANA-1 macrophage tumoricidal activity, IL-4 inhibited IFN-gamma plus IL-2-induced, but not IFN-gamma plus LPS-induced, NO2- production. IL-4 also selectively decreased the ability of IFN-gamma and/or IL-2 to augment TNF-alpha mRNA expression in ANA-1 macrophages. Lastly, incubation of ANA-1 macrophages with anti-TNF mAb selectively inhibited the ability of IFN-gamma plus IL-2 to induce NO2- production and tumoricidal activity. These results indicate that IFN-gamma plus IL-2-induced tumoricidal activity is dependent upon the metabolism of L-arginine to reactive nitrogen intermediates, and they establish a role for TNF-alpha as a required intermediate for IL-2-dependent NO2- production and tumoricidal activity.
Role of IL-6, IL-2, and IL-4 in the in vitro induction of cytotoxic T cells.
Authors: Quentmeier H , Klaucke J , Mühlradt P F , Drexler H G
Published: December 2022

The mode of IL-6-induced differentiation of Con A-stimulated CD4-CD8+ CTL-P was examined. Through application of neutralizing anti-IL-6, anti-IL-2, and anti-IL-4 mAb it was shown that IL-6 is an "early acting" factor for development of accessory cell-depleted thymocytes. IL-2 and IL-4 are obligatory "late acting" factors for this process. In accordance IL-4, but not IL-6, induced active CTL from CD4-CD8+ CTL-P. The increase of CD4-CD8+ CTL originates at least partially from CD4+CD8+ cells differentiating to active CTL in vitro. CTL development was paralleled by an increase in CD4-CD8+ cells and by a distinct increase in [3H]TdR uptake on day 2 of cultivation. Our data suggest that IL-6 induces Th cells to produce IL-2 and IL-4, the mediators for final differentiation of CD4-CD8+ cells.
Role of P2z purinergic receptors in ATP-mediated killing of tumor necrosis factor (TNF)-sensitive and TNF-resistant L929 fibroblasts.
Authors: Pizzo P , Murgia M , Zambon A , Zanovello P , Bronte V , Pietrobon D , Di Virgilio F
Published: December 2022

Two closely related cell lines were characterized in their responses to extracellular ATP (ATPo): the fibroblast cell line L929 and a TNF-resistant variant L929/R. Both lines showed ATPo-activated increases in intracellular Ca2+, inward current, and sustained depolarization of the plasma membrane, cell responses compatible with activation of purinergic receptors of the P2y, P2x, or P2z subtype; however, only the L929/R variant was susceptible to ATPo-dependent early permeabilization of the plasma membrane to hydrophilic solutes of M(r) below 900, a response uniquely caused by the activation of P2z receptors. Both cell types were susceptible to the cytotoxic effect of ATPo, but killing of the L929/R variant required much shorter incubations in the presence of this nucleotide. Morphologic examination of ATPo-challenged L929 and L929/R cells showed that cell death occurred by two alternative mechanisms: colloido-osmotic lysis or apoptosis. Occurrence of apoptosis was confirmed by agarose gel analysis of cellular DNA. Although ATPo caused a fast mobilization of intracellular Ca2+, neither colloido-osmotic lysis nor apoptosis were Ca2+ dependent. Our results show that the L929/R variant, but not the L929 parental fibroblast cell line, expresses functional purinergic receptors of the P2z subtype. The presence of P2z receptors confers to L929/R cells enhanced susceptibility to ATPo-mediated cytotoxicity.
Immunization of mice with lipopeptides bypasses the prerequisite for adjuvant. Immune response of BALB/c mice to human immunodeficiency virus envelope glycoprotein.
Authors: Martinon F , Gras-Masse H , Boutillon C , Chirat F , Deprez B , Guillet J G , Gomard E , Tartar A , Levy J P
Published: December 2022

In vivo priming of CTL requires the association with MHC class I molecules of peptides derived from the processing of endogenously produced proteins. Immunization with exogenous proteins or peptides rarely induces MHC class I-restricted CTL unless they are associated with lipidic compounds. The capacity to induce CTL was compared in synthetic peptides and simple lipopeptides containing the Immunodominant MHC class I H-2Dd-restricted T-cell epitope of HIV-1 gp160. In contrast with free peptides in saline, lipopeptides induced strong primary CTL responses in vivo. These CTL were able to lyse cells infected with a recombinant vaccinia virus expressing the HIV-1 env gene. Priming of CTL was also successful when using 16-amino acid lipopeptides as 34-amino acid lipopeptides, suggesting that several epitopes might be included in a single construct. In vivo priming of CTL also requires CD4+ T cell help. We therefore searched for Th cell activation after priming with lipopeptides. Our results show that, as with CTL induction, Th cell activation with lipopeptides did not require mixing with adjuvant. In addition, lipopeptides were also efficient at stimulating antibody-mediated responses. Our results show that a single lipopeptidic construct can induce a total immune response, which is of importance in vaccine development.
Characterization of the Mlsf system. I. A novel "polymorphism" of endogenous superantigens.
Authors: Abe R , Foo-Phillips M , Granger L G , Kanagawa O
Published: December 2022

In addition to Mlsa (Mls-1a) and Mlsc (Mls-2a, Mls-3a), we and others have recently described a third set of stimulatory minor lymphocyte stimulating (Mls) determinants, which are ligands for "I-E related" V beta, V beta 5, V beta 11, and V beta 12. Although all V beta associated with the recognition of the conventional Mls determinants are, in general, uniformly deleted in those animals expressing relevant Mls, expression of Mlsf-related V beta reveals various deletion patterns among different strains. Here we describe extensive genetic studies to evaluate the relationship among the self-Ag responsible for clonal deletion of T cells bearing Mlsf-related V beta by using antibodies specific for TCR V beta chain. In addition, a panel of T cell clones specific for the Mlsf determinant were generated and employed to analyze the determinant specificity, which is recognized by Mlsf-reactive T cells in vitro as well as the role of class II molecules in T cell recognition of the Mlsf determinants. The results of these two independent approaches provide evidence that the Mlsf system is composed of a set of gene products that reveal a unique polymorphism in the induction of clonal deletion in vivo and in T cell activation in vitro. One of these gene products causes almost complete deletion of the self-Mlsf reactive T cell repertoire in vivo and elicits a strong proliferative response to Mlsf-specific T cell clones. Expression of the other gene products results in the clonal deletion of only part of the Mlsf-reactive T cell repertoire. Furthermore, the response pattern of Mlsf-specific clones to intra-MHC recombinant inbred strains and the inhibition pattern of these clones by anti-class II antibody suggested that although expression of the I-E molecule is essential for T cell recognition of Mlsf determinants, the A beta gene may also contribute to the efficient presentation of Mlsf determinants by forming unique class II E alpha A beta molecules.
Characterization of the Mlsf system. II. Identification of mouse mammary tumor virus proviruses involved in the clonal deletion of self-Mlsf-reactive T cells.
Authors: Foo-Phillips M , Kozak C A , Principato M A , Abe R
Published: December 2022

The genetic linkage of loci encoding stimulatory Mlsa and Mlsc determinants with proviruses of mouse mammary tumour viruses (MMTV) has been shown. We previously have reported that the ligand(s) for V beta 5, V beta 11, and V beta 12 behaves as a novel minor lymphocyte-stimulating (Mls) determinant(s), Mlsf, to induce the strong proliferation of unprimed T cells, and that this ligand(s) also functions as a self-Ag for the clonal deletion of self-reactive T cells. In the accompanying paper (Part I), a unique polymorphism characteristic of the Mlsf gene product is presented. In order to determine the genetic basis for this novel Mls system, we examined the progeny of multiple genetic crosses to identify the MMTV proviral loci involved in the clonal deletion of self-Mlsf-reactive T cells. Results from these investigations indicated that at least three known MMTV proviruses, Mtv-8, Mtv-9, and Mtv-11 are involved in the expression of Mlsf gene products. Presence of Mtv-9 results in the complete deletion of V beta 5, V beta 11, and V beta 12; Mtv-8 is associated with the complete deletion of V beta 12, but only a partial deletion of V beta 11 (primarily CD4-positive T cell subset) with little or no deletion of V beta 5; and Mtv-11 induces the complete deletion of V beta 11 and V beta 12, but no deletion of V beta 5. Given the significant sequence homology in the C-terminal portion of the open reading frame (ORF) region among these three MMTV and the almost equivalent effect of these three MMTV provirus upon the V beta 12 repertoire, their apparent hierarchic effect upon the V beta 5 and V beta 11 repertoires suggests that affinity differences in recognition of the same determinant by different TCR V beta may play a significant role in the clonal deletion of self-reactive T cells.
Enhanced antigen presentation using human Fc gamma receptor (monocyte/macrophage)-specific immunogens.
Authors: Gosselin E J , Wardwell K , Gosselin D R , Alter N , Fisher J L , Guyre P M
Published: December 2022

A major new challenge for vaccine development is to target APC such as monocytes and macrophages for efficient Ag processing and presentation. It has been shown that Fc gamma R-mediated uptake of Ag-antibody complexes can enhance Ag presentation by myeloid cells at least 100-fold, and directing Ag to Fc gamma R in mice brings about a substantial increase in the effectiveness of immunization while eliminating the requirement for adjuvant. It has not been determined which of the three subclasses of human Fc gamma R on myeloid cells (Fc gamma RI, Fc gamma RII, or Fc gamma RIII) function to enhance Ag presentation. We have targeted our Ag (TT) to each of the three subclasses of human Fc gamma R on monocytes using Fc gamma R subclass-specific mAb-TT conjugates, and have measured TT presentation by monitoring T cell proliferation in response to TT. In addition, we have examined enhanced Ag presentation mediated by a human IgG1 (HIgG1) anti-TT mAb. All anti-Fc gamma R-TT conjugates enhanced Ag presentation. HIgG1 anti-TT, in monomeric form, enhanced Ag presentation through Fc gamma RI only. Anti-Fc gamma RI-Ag conjugates appear to be optimal for application as vaccines. They are monocyte/macrophage-specific, are very efficiently processed and presented, and enhance Ag presentation despite occupation of Fc gamma RI with HIgG.