bluuebunny/crossref_metadata_2025 huggingface dataset:
TCR usage and functional capabilities of human gamma delta T cells at birth.
Authors: Morita C T , Parker C M , Brenner M B , Band H
Published: December 2022

Little is known about the gamma delta T cells in human neonatal umbilical cord blood. To compare neonatal cord blood and adult blood gamma delta T cells, we studied the V gamma and V delta gene segment usage in these populations by flow cytometry, and we derived cord blood gamma delta T cell clones to determine their functional capabilities. Unlike adult blood gamma delta T cells that predominantly express V gamma 2V delta 2 TCRs, neonatal cord blood gamma delta T cells expressed diverse V gamma and V delta gene segments paired in a variety of combinations rarely observed in adults. gamma delta T cell clones derived from neonatal cord blood similarly expressed a diverse array of TCRs. These cord blood-derived gamma delta T cell clones had weak cytolytic activity when assayed for K562 tumor cell killing, lectin-mediated cytolysis, and redirected cytolysis. They also expressed lower levels of the CD2, LFA-1, and CD45RO cell surface receptors as compared with strongly cytolytic adult gamma delta T cell clones. These properties of the cord blood-derived gamma delta T cell clones, weak cytotoxic activity and low adhesion molecule expression, were similar to the properties of the CD4+ subset of adult gamma delta T cells. Thus, neonatal gamma delta T cells are functionally different from the majority of adult gamma delta T cells and display a distinct TCR repertoire and accessory molecule profile.
IL-12 stimulates the development of acute graft-versus-host disease in mice that normally would develop chronic, autoimmune graft-versus-host disease.
Authors: Via C S , Rus V , Gately M K , Finkelman F D
Published: December 2022

The injection of DBA/2 (D2) spleen cells into (C57BL/6 x DBA/2)F1 mice (BDF1) induces a chronic, autoimmune graft-vs-host disease (GVHD) that is characterized by: increased production of Th2-associated cytokines; increased levels of serum Ig, including IgE; increased production of IgG anti-DNA Abs; and no detectable antihost CTL activity. Experiments were performed to determine if treatment with the cytokine IL-12, which stimulates the production of Th1-associated cytokines and inhibits Th2-associated cytokine production, would inhibit humoral autoimmunity in this system. Treatment of mice with 100 ng IL-12 per day for 5 days, starting on the day of cell transfer, resulted in: 1) near complete suppression of autoantibody production; 2) decreased serum Ig levels; 3) detectable donor antihost CTL activity; and 4) greatly reduced numbers of host splenic B and T cells. Treatment of mice with a neutralizing anti-IFN-gamma mAb did not reverse these effects of IL-12. Thirty nanograms per day resulted in reduced numbers of host B cells and reduced serum anti-DNA levels, but no detectable antihost CTL activity. IL-12 treatment initiated 7 days after cell transfer had little effect on the development of autoimmune GVHD. These observations suggest the following: 1) IL-12 inhibits humoral autoimmunity in a murine parent-->F1 GVHD model by inducing the activation of host-reactive CTLs that reject the host immune system. 2) This effect is IFN-gamma-independent. 3) IL-12 needs to be present during the initial differentiation of T cells in this system to have this effect.
Topology of the loxoribine binding site. Studies with inactive loxoribine analogues.
Authors: Goodman M G , Goodman J H
Published: December 2022

We have previously described a class of immunostimulatory ribonucleoside, exemplified by loxoribine (7-allyl-8-oxoguanosine) and 8-bromoguanosine, that act on cycling B cells to promote nonspecific proliferation and differention, and that synergize with Ag to recruit quiescent Ag-reactive B cells to undergo differentiation and high level Ab production. In murine B cells, two distinct binding activities have been characterized that have dissociation constants that parallel their distinctive dose-response profiles. The SJL mouse strain, which is hypo-responsive to the B cell proliferative properties of these molecules, exhibits a dissociation constant of 10- to 20-fold lower affinity than that of normal murine strains; its Kd for the higher affinity-binding interaction, however, is essentially normal. These observations suggested the existence of two distinct binding activities. Recently, immunoselective subgroups of loxoribine have been described, with an ability to stimulate individual components of the immune response that correlates with specific changes in discrete domains of the nucleoside molecule. By using a panel of nonstimulatory loxoribine analogues as inhibitors, we now report further evidence for the existence of unique sites on loxoribine binding proteins that recognize distinct domains on nucleoside analogues; changes in these regions confer immunologic selectivity. These data confirm the conclusions of previous studies, extend our understanding of the topology of subsites that are otherwise silent, and provide insight into the binding site mediating adjuvanticity. These results allow more profound appreciation of the nature of structural changes that will permit a tighter fit between ligand and binding site, ultimately promoting the design of more potent immunoselective agents.
Murine VCAM-1. Molecular cloning, mapping, and analysis of a truncated form.
Authors: Kumar A G , Dai X Y , Kozak C A , Mims M P , Gotto A M , Ballantyne C M
Published: December 2022

Vascular cell adhesion molecule-1 (VCAM-1) is a member of the Ig superfamily that shows increased expression in a number of pathologic conditions. The role of VCAM-1 in human disease remains undefined and murine models are being extensively studied to help define the importance of VCAM-1 in inflammatory disorders. We have cloned and characterized the murine Vcam1 gene including 3 kb of 5'-flanking sequences and mapped the gene to chromosome 3 near Amy1. cDNA clones isolated from a stimulated hepatic library were found to encode a truncated form of VCAM-1 (T-VCAM-1) which contains Ig domains 1 through 3 and has a unique alternative carboxyl terminus. This form arises by alternative splicing. High level expression of T-VCAM-1 in transfected L cells was sufficient to support adhesion of lymphocytes, and this adhesion was blocked by Abs to VCAM-1. Treatment of transfected COS cells with phospholipase C led to reduced levels of T-VCAM-1 on the cell surface consistent with glycosylphosphatidylinositol linkage. Northern blot analysis showed that mRNA for T-VCAM-1 is inducible in multiple tissues after stimulation with endotoxin. Both forms of VCAM-1 were expressed in cultured endothelial, fibroblast, and aortic smooth muscle cells, whereas neither form was observed in monocyte- and lymphocyte-derived lines. Differential regulation of both forms of VCAM-1 was observed in the three different cell types that are present in the vessel wall. Thus, expression of VCAM-1 is restricted and controlled at the level of transcription and by alternative splicing.
Elicitation of specific cytotoxic T cells by immunization with malaria soluble synthetic polypeptides.
Authors: Blum-Tirouvanziam U , Beghdadi-Rais C , Roggero M A , Valmori D , Bertholet S , Bron C , Fasel N , Corradin G
Published: December 2022

We have studied the immunogenicity of Plasmodium falciparum circumsporozoite (CS) protein-derived synthetic polypeptides in mice. These synthetic peptides correspond to the N- and the C-terminal domains 22-125 and 289-390, respectively of the P. falciparum 7G8 isolate CS protein expressed on the sporozoite surface. They comprise what is believed to be the mature protein, except for the central repetitive B cell domain. BALB/c (H-2d) mice were immunized s.c. with 50 micrograms soluble CS polypeptides emulsified in IFA. After a single immunization, CS-specific helper and cytotoxic T lymphocytes (CTLs) could be obtained. The resultant CTLs obtained by in vitro restimulation of primed lymph node (LN) cells recognized H-2Kd target cells in the presence of short synthetic peptides defined in the present study. These epitopes are contained within the N- and C-terminal regions of the CS protein, and correspond to sequences 39-47 and 333-342. In addition, these CTLs can specifically lyse H-2d target cells transfected with the CS gene. These results suggest that, by immunization of mice with large soluble CS synthetic polypeptides in IFA, it is possible to obtain MHC class I-restricted T cell responses specific for the CS protein. This approach might be advantageous in the formulation of efficient malaria subunit vaccines.
Mucosal exudation associated with a pulmonary delayed-type hypersensitivity reaction in the mouse. Role for the tachykinins.
Authors: Buckley T L , Nijkamp F P
Published: December 2022

In this study, the role for sensory neuropeptides in the induction of pulmonary inflammation associated with a delayed-type hypersensitivity (DTH) reaction in the mouse lung was investigated. Dinitrofluorobenzene (DNFB) was used as the sensitizing hapten and dinitrobenzene sulfonic acid was used as the intranasal challenge. Two hours after the challenge, no hapten-specific differences were observed between vehicle- and DNFB-sensitized mice. However, mucosal exudation and leukocyte accumulation (mononuclear leukocytes and neutrophils) were enhanced in the DNFB group 24 h after the challenge. In additional experiments we investigated whether the sensory nerves and specific sensory neuropeptides play a role in this pulmonary DTH reaction. The selective NK1 antagonist (RP 67580; 5.10(-9) mol/mouse), administered 5 min before and 1 h after the challenge, suppressed mucosal exudation and leukocyte accumulation in the airways 24 h after the challenge. In contrast, the selective calcitonin gene-related peptide (CGRP) antagonist, CGRP8-37 (5.10(-9) mol/mouse) had no effect on the pulmonary DTH reaction. Surprisingly, the depletion of all sensory neuropeptides resulted in an enhancement of mucosal exudation in vehicle- and DNFB-sensitized mice 24 h after the challenge. In conclusion, these results demonstrate that the tachykinins are important in mediating mucosal exudation and leukocyte accumulation associated with the DTH reaction in mouse airways. However, the findings in neuropeptide-depleted mice suggest that the sensory nerves may also play a protective role in mouse airways.
Alternatively processed human E-selectin transcripts linked to chronic expression of E-selectin in vivo.
Authors: Chu W , Presky D H , Swerlick R A , Burns D K
Published: December 2022

E-selectin, also known as endothelial leukocyte adhesion molecule-1 (ELAM-1), is transiently expressed on endothelial cells in response to inflammatory cytokines such as IL-1 and TNF-alpha and mediates adhesion of leukocytes. The genomic structure of E-selectin is highly conserved and includes multiple polyadenylation signals and a number of AUUUA transcript destabilizing elements within the 3'-untranslated region (UTR). Anchored-PCR analysis indicates that all three polyadenylation signals within the human E-selectin 3'-UTR are indeed functional, and three forms of E-selectin transcripts (Types I, II, and III) generated by differential usage of these polyadenylation signals were detected in both cytokine-stimulated primary human endothelial cells and human skin tissue cultures. Although the longest transcript (Type III) is the most abundant form found in cytokine-stimulated human endothelial cells and human skin tissue cultures in vitro, only the shortest transcript (Type I) is detected in human dermal biopsies expressing cell surface E-selectin. Interestingly, the Type I E-selectin transcript lacks six of the mRNA destabilizing elements that are thought to mediate rapid degradation of the corresponding mRNA. In agreement with the absence of these mRNA decay signals, we determined that the Type I E-selectin transcript is more stable than the full-length Type III E-selectin transcript in an in vitro chimeric mRNA stability assay. Therefore, the presence of only the Type I transcript in skin biopsies of chronic inflammatory lesions suggests that differential polyadenylation of E-selectin transcripts may provide the molecular basis for the observed chronic expression of E-selectin in human dermal disorders.
Functional regulation of platelet/endothelial cell adhesion molecule-1 by TGF-beta 1 in promonocytic U-937 cells.
Authors: Lastres P , Almendro N , Bellón T , López-Guerrero J A , Eritja R , Bernabéu C
Published: December 2022

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a widely distributed cell adhesion molecule present on monocytes, macrophages, and monocytic cell lines. Treatment of the promonocytic cell line U-937 with TGF-beta 1 induces homotypic cellular aggregations, simultaneous with an increase in surface expression and specific transcripts of PECAM-1. The TGF-beta-induced cell adhesion phenomena are not dependent on LFA-1, intercellular adhesion molecule-1 (ICAM-1), very late Ag-4 (VLA-4), or very late Ag-5 (VLA-5). However, the phenomena seem to be directly mediated by PECAM-1 because 1) it is inhibited by the addition of Abs or an antisense oligonucleotide specific for PECAM-1; and 2) TGF-beta 1-treated U-937 cells bind to PECAM-1-expressing mouse transfectant fibroblasts, but not to mock transfectants. In addition, this aggregation phenomena are divalent cation-dependent and requires the integrity of the cytoskeleton. Analysis of the intracellular signaling pathways indicates that TGF-beta 1 induces protein kinase C activity, as well as PECAM-1 phosphorylation and association with cytoskeletal components. Furthermore, in this model, an autocrine mechanism for releasing the bioactive form of TGF-beta 1 operates, allowing PECAM-1 activation. These results provide evidence that TGF-beta 1 regulates PECAM-1 function by increasing the expression and activating the adhesion of PECAM-1 in monocytic cells. These two mechanisms seem to be necessary for adhesion because independent inhibition of either expression or activation of PECAM-1 leads to abrogation of cellular aggregation.
Thrombospondin promotes chemotaxis and haptotaxis of human peripheral blood monocytes.
Authors: Mansfield P J , Suchard S J
Published: December 2022

The presence of the extracellular matrix protein thrombospondin (TSP) at sites of tissue injury or inflammation may promote monocyte migration to these sites and play a central role in their eventual differentiation into tissue macrophages. Previously, we have shown that TSP promotes neutrophil adhesion and migration, and primes for oxidant generation. To examine the effect of TSP on monocyte motility, we conducted chemotaxis assays in modified Boyden chambers. TSP was chemotactic for monocytes, with a maximal response at 200 to 500 nM TSP. Checkerboard analysis confirmed that migration was directional. mAb C6.7, against the distal COOH terminus of TSP, inhibited chemotaxis, demonstrating specificity and indicating that the chemotactic activity resides in the COOH terminus. Consistent with the mAb data, the COOH-terminal 140-kDa proteolytic fragment of TSP was chemotactic for monocytes, whereas the NH2-terminal heparin-binding domain was inactive. A synthetic peptide containing the sequence CSVT, derived from the type I repeats of TSP, was also chemotactic. Thus, two different sites on the COOH terminus of TSP are capable of stimulating monocyte chemotaxis. Pertussis toxin, but not cholera toxin, completely inhibited TSP-mediated chemotaxis, suggesting the involvement of GTP-binding proteins. TSP bound to polycarbonate filters stimulated monocyte haptotaxis, with a maximal response at 4 pmol. The directional nature of this motility was confirmed by checkerboard analysis. Monocyte haptotaxis was inhibited by two different mAbs recognizing distinct sites on the COOH terminus. As with chemotaxis, the 140-kDa fragment, but not the heparin-binding domain, contained the haptotactic activity. The CSVT-containing synthetic peptide also promoted monocyte haptotaxis. But, in contrast to chemotaxis, neither pertussis toxin nor cholera toxin inhibited TSP-mediated haptotaxis, suggesting the involvement of a different signal transduction pathway. mAbs against GPIV, beta 1, beta 3, or alpha v integrins did not affect monocyte chemotaxis or haptotaxis, ruling out the involvement of these receptors. These results indicate that TSP is likely to play an important role in monocyte recruitment to an inflammatory or injury site.
IL-12 protects bone marrow from and sensitizes intestinal tract to ionizing radiation.
Authors: Neta R , Stiefel S M , Finkelman F , Herrmann S , Ali N
Published: December 2022

IL-12, a potent stimulator of hemopoietic progenitor cells, was evaluated as a potential protector against 60Co-gamma radiation-induced lethal hemopoietic syndrome in mice. Administration of IL-12 before lethal irradiation of genetically distinct strains of mice, B6D2F1 and C3H/HeJ, protected a significant fraction of both strains of mice from death. Radioprotection was associated with a fivefold increase in the number of bone marrow cells at 6 days after irradiation. Even at supralethal doses of radiation (1200 cGy), the number of c-kit+ bone marrow cells 3 days after irradiation was twofold greater in IL-12-treated mice than in saline-treated mice. However, mice that received IL-12 and 1200 cGy (B6D2F1) or 900 cGy (C3H/HeJ) died of the gastrointestinal syndrome, as was evident by gross necroscopy and histologic evaluation, within 4 to 6 days after irradiation. Induction of the gastrointestinal syndrome in mice not treated with IL-12 required radiation doses of 1500 cGy or greater in both strains. Thus, at doses of radiation at which IL-12 still protects c-kit+ hemopoietic cells, it sensitizes the intestinal tract to damage. Radioprotection with IL-12 was abrogated by anti-IL-1R or anti-stem cell factor Ab. Anti-IFN-gamma Ab did not affect IL-12-induced hemopoietic radioprotection, but abrogated sensitization of the intestinal tract by IL-12. The sensitizing effect of IL-12 may be related to its ability to prime mice to subsequent inflammatory challenge, as demonstrated by an almost 100-fold increase in circulating TNF and IL-6 levels in normal B6D2F1 mice challenged with IL-12 and LPS. This priming effect of IL-12 also was abrogated by anti-IFN-gamma Ab.
Heterogeneity of the circulating human CD4+ T cell population. Further evidence that the CD4+CD45RA-CD27- T cell subset contains specialized primed T cells.
Authors: Baars P A , Maurice M M , Rep M , Hooibrink B , van Lier R A
Published: December 2022

CD27, a member of the TNFR family, is expressed on most but not all peripheral blood CD4+ T cells. The small fraction of CD4+ T cells with a CD27- phenotype exclusively reside within the CD45RA-CD45RO+ subset. We previously provided evidence that CD27- cells are functionally differentiated cells that have lost CD27 expression as a result of persistent antigenic stimulation. We here show that compared with CD4+CD45RA-CD27+ cells, CD4+CD45RA-CD27- lymphocytes have a high expression of the beta 1 integrins VLA-4 and -5 and of the beta 2 integrin CD11b. Molecules implicated in homing of T cells to peripheral lymphnodes like CD31 and CD62L (L-selectin) are poorly expressed on CD27- cells, whereas receptors involved in organ-specific homing, e.g., cutaneous lymphocyte Ag and HML-1 (alpha E beta 7), are present on CD27- rather than CD27+ T lymphocytes. CD27+ and CD27- cells do not differ notably in the expression of activation molecules such as CD25, CD38, and CD70 (CD27 ligand) but CD7 is markedly absent on approximately half of the CD27- cells. Analysis of mutations in the HPRT gene, as measurement for the amount of cell divisions that have occurred in particular T cell populations in vivo, showed that CD45R0+ cells have a 2 to 5 times higher mutant frequency than CD45RA+ cells, whereas CD45R0+CD27- cells do not differ in this respect from CD45R0+CD27+ cells. In line with this latter finding, cells in G2M phase can only be found in the transitional, CD45RAbrightCD45R0bright subset but not in CD45R0+, CD45RA-, or CD27- cells. Our results imply that the CD27- population contains tissue-specific, specialized "primed" T cells that evolve in vivo independently from extensive cellular division.
Cell cycle control of apoptosis in human leukemic T cells.
Authors: Zhu L , Anasetti C
Published: December 2022

Eukaryotic cell cycle is regulated by feedback controls that can arrest the progress of the cell cycle at specific checkpoints. Failure of feedback controls may predispose cells to undergo apoptotic death in response to a variety of stimuli. Transformed cell lines often carry defects in one or more proteins that regulate cell cycle, and therefore can be used as a model to study how cell cycle controls cell death. The present study investigated whether TCR/CD3-induced apoptosis of human leukemic T cells is cell cycle dependent. Studies of Jurkat and Sup-T13, two human leukemic T cell lines, demonstrated that anti-CD3 Ab-driven activation results in a transient, reversible inhibition of DNA synthesis in Jurkat cells, but results in a persistent, irreversible inhibition of DNA synthesis in Sup-T13 cells. Anti-CD3 mAb blocked Jurkat cells at the G1/S interface without inducing cell death. In contrast, anti-CD3 mAb allowed Sup-T13 cells to enter S phase but inhibited progression into G2 phase and triggered cell death. Electron microscopy showed that anti-CD3 mAb-stimulated Sup-T13 cells acquired the characteristic morphology of apoptosis. The nuclear DNA appeared as oligonucleosome-sized fragments by gel electrophoresis, and as an apoptotic peak displaying lower fluorescence than the DNA content of cells in G1 phase by DNA flow microfluorometry. DNA fragmentation correlated with entry into S phase. The DNA synthesis inhibitor aphidicolin arrested Sup-T13 cells at the G1/S interface and prevented the apoptosis. Therefore, DNA synthesis is required for executing the apoptosis program initiated by TCR/CD3 stimulation of Sup-T13 cells. These data are consistent with the hypothesis that the suicide of activated cycling T cells upon encountering autoantigens may be one of the mechanisms for the immune system to prevent autoimmunity.
Enhanced cytotoxicity of IFN-gamma-producing CD4+ cytotoxic T lymphocytes specific for T. gondii-infected human melanoma cells.
Authors: Yang T H , Aosai F , Norose K , Ueda M , Yano A
Published: December 2022

CD4+ lines specific for Toxoplasma gondii-infected human melanoma P36 cells were established from PBL of a patient with chronic toxoplasmosis. CD4+ CTL lines were obtained by weekly in vitro stimulation with T. gondii-infected P36 cells that shared HLA-DR4 molecules with the patient. The lytic activity of CD4+ CTL lines against T. gondii-infected P36 or T. gondii-infected autologous EBV-transformed B lymphoma (EBV-Ya) was inhibited by anti-HLA-DR mAb, whereas anti-HLA-A, B, C mAb failed to block the lytic activity. Thus, the cytotoxicity of CD4+ CTL lines against T. gondii-infected P36 was restricted by HLA-DR molecules. In response to Ag-specific stimulation, CD4+ CTL lines produced significant levels of IFN-gamma. Exogenously added IFN-gamma up-regulated the surface expression of MHC class II, but not of class I in T. gondii-infected P36 cells. In addition, the CTL activity against T. gondii-infected P36 cells was augmented when target cells were co-cultured with IFN-gamma. These data indicate that CD4+ CTL-mediated cytotoxicity against T. gondii-infected melanocytes is enhanced by the autocrine production of IFN-gamma. Further, CD4+ CTL may play a role in the manifestation of toxoplasmic retinochoroiditis by killing T. gondii-infected melanocytes.
Experimental autoimmune encephalomyelitis-resistant mice have highly encephalitogenic myelin basic protein (MBP)-specific T cell clones that recognize a MBP peptide with high affinity for MHC class II.
Authors: Abromson-Leeman S , Alexander J , Bronson R , Carroll J , Southwood S , Dorf M
Published: December 2022

BALB/c mice are resistant to disease induction when experimental protocols that induce experimental autoimmune encephalomyelitis (EAE) in susceptible strains of animals are used. We have previously described a panel of myelin basic protein (MBP)-specific CD4+ T cell clones from BALB/c mice, two of which induce moderate EAE when transferred to syngeneic recipients. These clones are I-E(d) restricted and recognize residues 151-160 of mouse MBP. Here, we describe a series of 17 MBP-reactive T cell clones, which were derived from two BALB/c mice. All are I-A(d) restricted and recognize nested epitopes in peptide 59-76 of mouse MBP. Four different TCR V beta chains are used by this panel of clones; these include V beta 8.2 (10/17), V beta 8.1 (2/17), V beta 7 (3/17), and V beta 14 (2/17). Twelve of fourteen clones tested adoptively transferred severe demyelinating EAE to syngeneic recipients. Studies of relative binding affinities of MBP peptides to class II molecules I-A(d) and I-E(d) show that peptide 59-76 binds with extremely high affinity to I-A(d), whereas three peptides that contains residues 151-160 bind poorly to I-E(d). These results are consistent with a growing number of reports that show that high affinity binding to class II is required for autoantigenic stimulation. Despite encephalitogenicity of 59-76-reactive T cells, active immunization of BALB/c mice with peptide 59-76 in adjuvant failed to induce either clinical or histologic signs of EAE. The implications of these findings for mechanisms of genetically determined EAE resistance are discussed.
Induction of peptide-specific CD8+ CTL clones in beta 2-microglobulin-deficient mice.
Authors: Cook J R , Solheim J C , Connolly J M , Hansen T H
Published: December 2022

We have examined the ability of beta 2-m- mice to produce CD4-8+ T cells by generating CD8+ CTLs to a defined ligand. We report here the first demonstration of peptide-specific, self-class I MHC-restricted CTLs from beta 2-m-deficient mice. We have used the KOD mouse, an H-2d beta 2-m- strain, to generate CTLs that recognize the class I MHC molecule Ld in association with one of two Ld-binding immunogenic peptides. Testing of these CTLs on a panel of Ld-binding peptides reveals a high degree of peptide specificity. Peptide-specific CTL bulk cultures from KOD mice differ from those generated in beta 2-m+ mice in that they possess altered affinities for their peptide ligands. In addition, we show that CTLs generated from beta 2-m- mice in the presence of beta 2-m+ stimulator cells and exogenous peptide are specific either for the exogenous peptide or for endogenous peptides that are present in association with Ld on the surface of beta 2-m+ cells, but are not present at detectable levels on beta 2-m- cells. These results demonstrate that positive selection of CD8+ CTLs can occur in vivo on the very low levels of class I MHC found in the KOD mouse. Furthermore, CTLs from the KOD mouse maintain a high degree of peptide specificity despite reduced levels of class I MHC.
Generation of T cells with differential responses to alloantigens in CD45 exon 6-deficient mice.
Authors: Kong Y Y , Kishihara K , Yoshida H , Mak T W , Nomoto K
Published: December 2022

We characterized the CD45+ population in the CD45 exon 6-deficient mice and addressed a question as to whether the T cells keep responsive properties to alloantigens. The peripheral CD45+ T cells from the untreated knockout mice showed a decreased expression level of CD45 but increased expression levels of LFA-1 alpha and CD44, indicating that the T cells are possibly in an activation state. The CD45 exon 6-deficient mice could readily reject fully allogeneic skin grafts but not non-MHC-disparate skin grafts. The skin allograft-rejected CD45 exon 6-deficient mice showed an increased number of peripheral CD8+ T cells expressing an isoform of CD45RB (CD45 zeta). In vitro assays including mixed lymphocyte reaction, CTL, and cytokine productions were consistent to the in vivo results. Furthermore, the effector cells shown in these assays were CD45+CD8+ T cells expressing a CD45RB isoform, CD45 zeta. Moreover, the CD45+ T cells had an abnormal regulation of CD45 expression level, as they could not increase their CD45 expression level after in vitro allogeneic stimulation. These results indicate that abnormal mature T cells in the periphery of CD45 exon 6-deficient mice cannot respond to nominal Ags presented by MHC but can react to allo-MHC. The reason that such functionally impaired T cells can be generated in the mice is still unknown but these results imply that there is differential requirement of CD45 function for alloreactive T cells in a thymic selection(s) and in effector functions.
Temporal loss of the activated L-selectin-low phenotype for virus-specific CD8+ memory T cells.
Authors: Tripp R A , Hou S , Doherty P C
Published: December 2022

Whether the L-selectin-low (L-sel-lo) phenotype of acutely stimulated CD8+ T cells is a permanent characteristic of long-term memory CTL precursors (p) is addressed for mice primed with an influenza A virus or the murine parainfluenza type 1 virus, Sendai virus. In both cases, many of the splenic CD8+ CTLp gradually lose the predominantly L-sel-lo profile associated with recently generated CTLp populations. The influenza-specific CTLp also tend to revert from the activated alpha 4-integrin-high to the resting alpha 4-integrin-low form. The kinetics of the switch back to the "naive" L-sel-hi phenotype differs for the influenza and Sendai virus models, perhaps reflecting events occurring during the acute phases of these responses. The return to being L-sel-hi is not due to irreversible lymphocyte senescence, because restimulation of this set with the inducing virus in vitro causes most of the cells to become L-sel-lo. Also, despite the time-related drift of these particular memory CTLp to the L-sel-hi state, the size of the total pool of L-sel-lo CD8+ T cells increases with age.
Sequence analysis of the gene for a novel superantigen produced by Yersinia pseudotuberculosis and expression of the recombinant protein.
Authors: Ito Y , Abe J , Yoshino K , Takeda T , Kohsaka T
Published: December 2022

We previously reported that the Gram-negative bacterium Yersinia pseudotuberculosis produces a superantigen (YPM, Y. pseudotuberculosis-derived mitogen) that expands T cells bearing V betas 3, 9, 13.1, and 13.2 in an MHC class II-dependent manner. Based on the previously determined N-terminal 23 amino acids of YPM (T-D-Y-D-N-T-L-N-S-I-P-S-L-R-I-P-N-I-A-T-Y-T-G- (one-letter code)), we cloned the ypm gene and analyzed the nucleotide sequence. The gene encodes a 151-amino acid protein with a 20-amino acid signal peptide at its N terminus. The recombinant YPM expressed by the cloned gene exerted a mitogenic activity on human PBMC at a concentration of approximately 1 pg/ml. T cells bearing V beta 13.3 were preferentially expanded as well as T cells bearing the same V beta repertoires stimulated by native YPM. T cells were stimulated by the recombinant YPM in the presence of either fixed or unfixed HLA class II-transfected mouse fibroblasts. Furthermore, sequence diversity in the junctional region of the TCR beta-chain containing the V beta 3 element could be observed after stimulation by the recombinant YPM. These results indicate that YPM belongs to the category of superantigens and should be included as a novel member. The amino acid sequence of the mature protein showed no significant homology to other superantigens derived from Gram-positive bacteria such as Staphylococcus aureus and Streptococcus pyogenes. This observation, together with the substantially smaller m.w. suggest that ypm must have evolved from a different ancestral gene.
Self and foreign 60-kilodalton heat shock protein T cell epitope peptides serve as immunogenic carriers for a T cell-independent sugar antigen.
Authors: Könen-Waisman S , Fridkin M , Cohen I R
Published: December 2022

Healthy individuals manifest natural T cell reactivity to epitopes of the 60-kDa heat shock protein (hsp60) of both self and bacterial origin. The present studies were done to learn whether defined peptides of hsp60 could function as T cell carrier epitopes for a poorly immunogenic T-independent capsular polysaccharide, the Vi Ag of Salmonella typhi. Homologous peptides were synthesized from the mouse self-hsp60 molecule (CP1m), from the closely related human hsp60 molecule (CP1h), and from the more distant Escherichia coli (CP1ec) and mycobacterial (CP1mt) hsp60 molecules. The peptides were conjugated to Vi and tested for their immunogenicity in BALB/c (H-2d) and H-2 congenic mice (H-2k and H-2b). We now report that the self-CP1m and cross-reactive CP1h peptides were as immunogenic as was the non-cross-reactive foreign CP1ec peptide. Small amounts of the CP1 peptide, even in PBS, sufficed to induce anti-Vi Abs of the IgG1 (T-dependent) isotype in naive mice. The carrier effect was associated with the ability of the peptides to bind to APC and to induce T cell proliferation. H-2d and H-2k mice, but not H-2b mice responded to CP1m/h and CP1ec. None of the mice responded to CP1mt. No signs of inflammation or autoimmune disease were detected. Thus, natural T cell autoimmunity exists and can be harnessed to provide T cell help for Ab production to a foreign bacterial molecule in a synthetic vaccine.
Infection of IL-4-deficient mice with the parasitic nematode Brugia malayi demonstrates that host resistance is not dependent on a T helper 2-dominated immune response.
Authors: Lawrence R A , Allen J E , Gregory W F , Kopf M , Maizels R M
Published: December 2022

Resistance of intact mice to infection with the filarial nematode, Brugia malayi is dependent on the presence of T cells. To investigate the role of Th2 cells in this protection, mice with a targeted disruption of the IL-4 gene were infected with different developmental stages of B. malayi. We examined the phenotypic changes in the immune response and the survival of each stage in these mice. In wild-type mice, adult female worms induce Th2 responses, characterized by antigen-specific IgG1 production, elevated IgE, and marked IL-4 secretion by splenocytes stimulated in vitro with Brugia extract. However, first stage larvae (microfilariae), induce Th1 responses with the appearance of antigen-specific IgG2a, IgG2b, and IgG3 and IFN-gamma secretion by splenocytes. Infection of IL-4-deficient mice revealed a dramatic change in the response to adult worms, with a severe reduction in IgG1 production and a corresponding increase in the production of IgG2a, IgG2b, IgG3, and IFN-gamma release. The switch to Th1-type responses was particularly marked in IL-4-deficient recipients of female worms, which continually release live microfilariae. In the absence of IL-4, down-regulation of the microfilarial-induced Th1 response does not occur. Despite these profound alterations to the immune response in IL-4-deficient mice, survival of infective larvae, adult worms, or microfilariae in the peritoneal cavity was unaffected. In mice, therefore, the prominent Th2-type response elicited by filarial parasites may not be an essential component of the host protective immune response.
HIV-1 infection of human macrophages impairs phagocytosis and killing of Toxoplasma gondii.
Authors: Biggs B A , Hewish M , Kent S , Hayes K , Crowe S M
Published: December 2022

The susceptibility of patients with AIDS to certain opportunistic infections is due to defective cell-mediated immunity. The contribution of direct infection of macrophages with HIV-1 to this defect is unknown. To address this issue, we infected normal human monocyte-derived macrophages with a monocytotropic strain of HIV-1 and examined their ability to phagocytose and kill the opportunistic pathogen, Toxoplasma gondii. Phagocytosis of heat-killed T. gondii was reduced in HIV-infected macrophages compared with mock-infected controls. Opsonization of heat-killed T. gondii increased phagocytosis by both mock- and HIV-infected macrophages, but phagocytosis in HIV-infected cultures remained lower than in controls. Internalization of live T. gondii by macrophages was unaffected by HIV infection. Intracellular replication of live T. gondii was enhanced by HIV infection, as shown in four experiments, each using monocyte-derived macrophages from a different donor. Treatment of HIV-infected macrophages with IFN-gamma decreased parasite replication but not to control levels. These findings suggest that infection of macrophages by HIV may be a contributing factor to the reactivation of T. gondii infection in patients with AIDS.
High prevalence of serum IgA HIV-1 infection-enhancing antibodies in HIV-infected persons. Masking by IgG.
Authors: Kozlowski P A , Black K P , Shen L , Jackson S
Published: December 2022

IgA and IgG purified from sera of 20 HIV-infected persons were separately examined for ability to mediate Ab-dependent enhancement (ADE) of HIV-1 infection of U937 cells. Both isotypes were capable of enhancing infection of these cells. However, IgA from twice as many persons (14/20) displayed infection enhancement when compared with IgG. This activity was predominantly observed with IgA from asymptomatic HIV-seropositive subjects (9/9). Enhanced HIV infection by IgA was observed most often at concentrations equivalent to serum dilutions in the range 10(-3) to 10(-5) and could be inhibited by preincubation of U937 cells with a mAb specific for the Fc alpha receptor. Concentrations of IgG mediating ADE were generally present in sera at dilutions from 10(-4) to 10(-6). When IgG was adjusted to physiologic concentration and combined with an enhancing concentration of IgA, enhancement was not observed unless IgG was also present at a concentration which exhibited this activity. These results suggest that, in comparison with IgG, HIV-infected individuals more often produce IgA Abs reacting with enhancing determinants of HIV. IgA-mediated ADE of HIV infection may not play a significant role in facilitating systemic dissemination of HIV because of the presence of higher concentrations of IgG. However, the production of IgA HIV-enhancing Abs at mucosal sites, where fewer IgG plasma cells are present, could contribute to the pathogenesis of HIV infection and interfere with development of vaccines designed to induce HIV IgA Abs at mucosal surfaces.
Apparent split tolerance of CD8+ T cells from beta 2-microglobulin-deficient (beta 2m-/-) mice to syngeneic beta 2m+/+ cells.
Authors: Jhaver K G , Rao T D , Frey A B , Vukmanović S
Published: December 2022

beta 2-microglobulin-deficient (beta 2m-/-) mice express reduced levels of MHC class I molecules and, consequently, have impaired positive selection of CD8+ T lymphocytes in the thymus. However, small numbers of CD8+ CTLs can be found in beta 2m-/- mice after immunization with allogeneic as well as syngeneic beta 2m+/+ tumor or spleen cells. It has been proposed, therefore, that because of the low ligand density in beta 2m-/- mice, negative selection does not remove cells capable of recognizing syngeneic MHC class I expressed at normal levels. We report here that beta 2m-/- CD8+ T cells are partially tolerant to syngeneic beta 2m+/+ cells. Despite the ability of beta 2m-/- mice to raise CD8+ CTLs against syngeneic beta 2m+/+ cells, these CD8+ cells do not proliferate and do not secrete IFN-gamma or IL-3/granulocyte-macrophage-CSF upon in vitro stimulation with syngeneic beta 2m+/+ cells. In contrast, all of these cellular responses are displayed by the beta 2m-/- CD8+ cells upon recognition of the allogeneic MHC class I. These in vitro findings of partial responsiveness to syngeneic and of full responsiveness to allogeneic MHC class I correlate well with the ability of beta 2m-/- mice to reject allogeneic, but not syngeneic, tumors in vivo. It appears, thus, that the significantly reduced levels of MHC class I molecules found in beta 2m-/- mice, although not capable of inducing deletion of all reactive clones, can induce deletion of high affinity clones and, therefore, maintain tolerance to self-MHC class I, even when expressed at much higher (beta 2m+/+) levels.
Regulation of synthesis and activity of the PLSTIRE protein (cyclin-dependent kinase 6 (cdk6)), a major cyclin D-associated cdk4 homologue in normal human T lymphocytes.
Authors: Lucas J J , Szepesi A , Modiano J F , Domenico J , Gelfand E W
Published: December 2022

The PLSTIRE protein (cyclin-dependent kinase 6 (cdk6)), which shares extensive sequence homology (approximately 70%) with cdk4, was identified as the earliest inducible member of the cdk family of proteins in human T lymphocytes induced to proliferate in vitro by stimulation either with phorbol 12,13-dibutyrate and ionomycin (PDB/I) or PHA. The p40cdk6 protein was present in resting cells and increased amounts were detected 6 h after stimulation. It increased in amount throughout the first cell cycle but was present in reduced amounts at later times. Activity of the kinase, determined by in vitro phosphorylation of recombinant truncated retinoblastoma tumor suppressor gene (Rb) protein (p60Rb), paralleled p40cdk6 protein amounts. Cyclins D2 and D3 were the major cyclins associated with p40cdk6, with D2 predominating in early G1 phase. Both PDB and ionomycin were required for maximal accumulation of p40cdk6, but either agent alone stimulated some increase in amount and activity of the protein. p40cdk6 also increased in amount in cells activated in the presence of cyclosporin A or FK506, drugs that inhibit production of IL-2 and cell proliferation, suggesting that initial induction occurred independently of IL-2-mediated cell cycle progression. Furthermore, increased accumulation of p40cdk6 protein and activity occurred in cells rendered "competent" (responsive to IL-2) by a brief treatment with PDB/I. Thus, increased accumulation of the protein and its activity begin before IL-2/IL-2 receptor interaction, suggesting that the cdk6-cyclin D2 complex might be involved in acquisition of the competent state in human T lymphocytes.
Analysis of direct and inverted DJH rearrangements in a human Ig heavy chain transgenic minilocus.
Authors: Tuaillon N , Miller A B , Tucker P W , Capra J D
Published: December 2022

D to JH rearrangement can occur either by a deletional or an inversional mechanism. In this study, we have analyzed deletional and inversional D to JH recombination in a human Ig heavy chain transgenic minilocus. The analysis of these events in a transgenic minilocus rather than in vivo in the human is simplified by the presence of a limited number of well defined VH, D, and JH gene segments in the minilocus. We show that in the transgenic minilocus, all D gene segments can be rearranged by deletion and virtually all can be rearranged by inversion. We also show that depending upon the D gene segment, rearrangement by deletion occurs approximately 1 to 1000 times more frequently than rearrangement by inversion. Our data suggest that in vivo, signal and coding end sequences are the major influences on the rearrangement frequency of a particular gene segment in a given orientation rather than its position within the transgenic locus. Additionally, our data indicate that intronic and recombination signal sequences are involved in the bias for deletion over inversion rather than the recombinase machinery itself.
Platelet-activating factor directly stimulates in vitro migration of endothelial cells and promotes in vivo angiogenesis by a heparin-dependent mechanism.
Authors: Camussi G , Montrucchio G , Lupia E , De Martino A , Perona L , Arese M , Vercellone A , Toniolo A , Bussolino F
Published: December 2022

The aim of the present study was to investigate the angiogenic properties of platelet-activating factor (PAF). In vitro PAF was shown to induce a dose-dependent migration of human endothelial cells (EC) across the polycarbonate filters in Boyden's chambers. In contrast, D-PAF, the biologically inactive enantiomer, and Lyso-PAF did not stimulate a significant migration of EC. This effect of PAF was not associated with a proliferative response of EC to this mediator. Moreover, the ability of PAF to stimulate the migration of EC was independent of the presence of heparin in the medium. WEB 2170, a specific PAF receptor antagonist, prevented the migration of EC induced by PAF, thus suggesting a receptor-dependent stimulation. The expression of PAF receptor gene by EC was confirmed by reverse transcriptase-PCR and Southern blot analysis. The in vivo angiogenic effect of PAF was studied in mice using a model in which Matrigel was used for the delivery of mediators. PAF induced a dose-dependent angiogenic response, which at pharmacologic concentrations (1-5 microM) did not require heparin, but at physiologic concentrations (5-50 nM) required the presence of heparin at doses that were not angiogenic per se. The angiogenesis induced by 50 nM PAF was, indeed, inhibited both by protamine and by the PAF receptor antagonist WEB 2170. The angiogenic effect of D-PAF and Lyso-PAF was not significant. Neutralizing Abs to basic fibroblast growth factor induced a slight but not statistically significant reduction of the angiogenesis induced by PAF.
Phosphorylation of complement component C3 and C3 fragments by a human platelet protein kinase. Inhibition of factor I-mediated cleavage of C3b.
Authors: Ekdahl K N , Nilsson B
Published: December 2022

Phosphorylation of C3 in vitro has been shown previously to lead to significantly altered function of the protein. Platelets are known to contain and release considerable amounts of protein kinases and ATP, which are prerequisites for protein phosphorylation. The aim of the present study was to investigate whether C3 is phosphorylated extracellularly by human platelets. Platelet-rich plasma was stimulated by human aggregated gamma-globulin or ADP. The remaining cells were removed by centrifugation, and the plasma was incubated with [gamma-32P]ATP. After precipitation with Sepharose-bound Abs to C3c followed by SDS-PAGE, it was shown that C3 was phosphorylated in the alpha-chain by a protein kinase dependent on Mn2+, Ca2+, or Mg2+ ions. The supernatant from washed, activated platelets was incubated with purified C3 or soluble or activated thiol Sepharose-bound C3b, together with [gamma-32P]ATP. Phosphorylation was seen in the alpha-chain of C3, and to the same extent in the alpha'-chain of both C3b preparations. The analysis of acid hydrolysate demonstrated that C3 contained 32P-labeled Thr and 32P-labeled Ser. After extensive proteolysis with trypsin, the major phosphorylation site was located to a peptide of 3 to 4 kDa that was bound to the activated thiol Sepharose via the free sulphydryl group in the C3d fragment. Incubation of phosphorylated C3b with factors I and H showed that phosphorylation inhibited the cleavage of the alpha'-chain of C3b. The results in this study suggest that phosphorylation is a regulator of C3 during platelet activation induced, for example, by immune complexes.
Recombinant soluble CD14 prevents mortality in mice treated with endotoxin (lipopolysaccharide).
Authors: Haziot A , Rong G W , Lin X Y , Silver J , Goyert S M
Published: December 2022

Endotoxic shock is a life-threatening condition mediated by cytokines released after exposure to bacterial LPS/endotoxin. Activation of monocytes and neutrophils by the binding of LPS to the membrane receptor, CD14, plays a key role in this response. Furthermore, a soluble form of the CD14 receptor enhances the endothelial cell response to LPS. We show here that despite the agonist effects of soluble CD14 on the endothelial cell response to LPS, recombinant soluble CD14 is able to protect mice from LPS-induced lethality. This protection appears to be associated with the inhibition of TNF-alpha release. These results suggest that the soluble CD14 receptor may represent a new form of therapy for endotoxic shock in humans.