bluuebunny/crossref_metadata_2025 huggingface dataset:
Control of lymphocyte recirculation in man. I. Differential regulation of the peripheral lymph node homing receptor L-selectin on T cells during the virgin to memory cell transition.
Authors: Picker L J , Treer J R , Ferguson-Darnell B , Collins P A , Buck D , Terstappen L W
Published: December 2022

Conventional virgin lymphocytes of a given class show relatively homogeneous recirculation through secondary lymphoid tissues, whereas memory/effector populations are composed of distinct subsets with differential, often tissue-selective migratory capability. In keeping with these observations, CD45RAhigh/ROlow "virgin" T cells in human peripheral blood uniformly express the peripheral lymph node homing receptor (HR) L-selectin, whereas among the CD45RAlow/ROhigh "memory/effector" subset, the expression of this HR is bimodal. To investigate the mechanisms responsible for the generation of memory/effector T cell subsets with differential homing potential, we developed a multiparameter flow cytometric technique that defines a common pathway of post-thymic T cell differentiation in secondary lymphoid tissues. Our analyses indicate that the virgin to memory T cell transition is characterized by a stepwise, unidirectional progression through distinct CD45RA+/RO+ intermediates that allow the in vivo discrimination of early, middle, and late transitional T cells. In normal peripheral blood, few T cells with a transitional phenotype are identified, but in secondary lymphoid tissues, 2 to 10% of T cells have this phenotype, including those CD45RA+ T cells that 1) are morphologically blasts, 2) are in S or G2/M phase of the cell cycle, or 3) express activation Ag. General adhesion molecules (LFA-1, LFA-3, ICAM-1) are uniformly up-regulated concordant with changes in T cell expression of CD45RA/RO in all tissues examined. Early in the transition, L-selectin is also uniformly up-regulated, but in subsequent stages, T cell expression of this HR is preferentially down-regulated in mucosal lymphoid tissues, and retained in peripheral lymph node. Differential regulation of L-selectin can also be demonstrated in vitro by the activation of virgin T cells in the presence of specific cytokines--IL-2 induces L-selectin down-regulation, whereas IL-6 and particularly TGF-beta 1 promote L-selectin up-regulation. Taken together, these findings support the hypothesis that local microenvironments within particular secondary lymphoid tissues influence HR expression at the time of CD45RA/RO conversion, and thereby contribute to the formation of CD45RAlow/ROhigh memory/effector T cell populations with tissue-selective homing behavior.
A monoclonal antibody to recombinant human IFN-alpha receptor inhibits biologic activity of several species of human IFN-alpha, IFN-beta, and IFN-omega. Detection of heterogeneity of the cellular type I IFN receptor.
Authors: Benoit P , Maguire D , Plavec I , Kocher H , Tovey M , Meyer F
Published: December 2022

A cDNA encoding a 63-kDa human IFN-alpha R (hIFN-alpha R) has recently been cloned. Mouse cells transfected with this cDNA failed to show any signal transmission for IFN-beta, suggesting the involvement of additional chains in the receptor complex. We have expressed in Escherichia coli and COS 7 cells a soluble recombinant protein (sIFN-alpha R) comprising the extracellular domain of the hIFN-alpha R fused at the carboxyl terminus to a sequence of five histidine residues. The sIFN-alpha R was affinity purified and used to generate mAb. One mAb, 64G12, which recognized the cellular receptor as well as the recombinant proteins was found to neutralize the biologic activity of IFN-alpha, -beta, and -omega and natural type I IFN, but not IFN-gamma. To our knowledge, this is the first report of a neutralizing mAb against the human IFN-alpha R. Interestingly, we found that the affinity of this antibody for the cellular receptor depended on the cell line used. These results provide strong evidence that the cloned hIFN-alpha R chain is required for binding and signal transmission of all subspecies of type I IFN and suggest a structural heterogeneity of the receptor at the cell surface.
Role of peripheral hemopoietic chimerism in achieving donor-specific tolerance in adult mice.
Authors: Maeda T , Eto M , Nishimura Y , Nomoto K , Kong Y Y , Nomoto K
Published: December 2022

The role of peripheral hemopoietic chimerism in the induction and maintenance of donor-specific tolerance was investigated by our tolerance-inducing method using cyclophosphamide (CP). As has been previously reported, CP injection at a dose of 200 mg/kg to C3H (Thy-1.2, Mls-1b) mice 2 days after priming with 10(8) viable AKR (Thy-1.1, Mls-1a) spleen cells (SC) resulted in both establishment of mixed chimerism and selective elimination of V beta 6+CD4+ T cells in the periphery. When, instead of viable SC, 1300 rad irradiated 10(8) AKR SC were used for priming to C3H mice, CP treatment 2 days after the priming also caused significant but, as compared with priming with nonirradiated viable cells, incomplete elimination of V beta 6+ T cells in the periphery. In these mice, no hemopoietic chimerism was found. In parallel with this incomplete elimination of peripheral V beta 6+ T cells, LN cells of these mice showed reduced but considerable response to AKR SC. However, once hemopoietic chimerism was introduced to these incompletely tolerant mice by an injection with donor-type viable [AKR x C3H]F1 SC 2 days after CP-treatment, LN cells from these newly established chimeras, irrespective of presence or absence of the thymus, became completely nonresponsive to AKR while preserving normal response to BALB/c (third party). This state of nonresponsiveness was accompanied by clonal elimination of the remaining V beta 6+ T cells in the periphery. These results indicate that peripheral chimerism promoted profound tolerance to donor-Mls Ag specifically. Furthermore, from experiments of skin grafting, we demonstrated that tolerance to minor histocompatibility Ag was also achieved in the presence of peripheral hemopoietic chimerism.
Characterization of responses of normal human T cells to Trypanosoma cruzi antigens.
Authors: Piuvezam M R , Russo D M , Burns J M , Skeiky Y A , Grabstein K H , Reed S G
Published: December 2022

The stimulation of normal human PBMC by Trypanosoma cruzi Ag was analyzed. PBMC showed significant in vitro proliferation in response to parasite lysate (Tct), with stimulation indices ranging from 10 to 400, peaking at 6 to 7 days. The cells stimulated with Tct produced significant levels of IL-2. To determine which cells proliferated in response to Tct, PBMC were separated into T- and B-enriched cell populations. Purified T cells, but not B cells, proliferated strongly to Tct. The T cell response required APC and was processing dependent. T cell lines generated against Tct proliferated in response to parasite lysate only in the presence of autologous APC and produced IL-2, IL-6, and IFN-gamma but not IL-4 in response to PMA plus ionomycin. Although there were a significant number of CD45Ra+ cells, the majority of the cells in these T cell lines were CD45Ro+. The V beta usage of Tct-responding T cells was heterogeneous, with most V beta genes represented among the responding cells. An immunodominant repeat Ag (TcD) and a ribosomal phosphoprotein (P0) of T. cruzi elicited strong proliferative responses in all subjects tested. These data indicate the presence of T cell-stimulatory Ag in Tct, characterized by nonpreferential usage of the V beta gene families. The strong stimulation of normal human PBMC by Tct may contribute to immunologic alterations seen in T. cruzi infection.
Mapping human T cell epitopes in leishmania gp63. Identification of cross-reactive and species-specific epitopes.
Authors: Russo D M , Jardim A , Carvalho E M , Sleath P R , Armitage R J , Olafson R W , Reed S G
Published: December 2022

Both a conserved surface metalloprotease of leishmania, gp63 as well as certain gp63-derived peptides, have been shown to have immunoprophylactic potential in mouse models of leishmaniasis. In addition, PBMC from individuals with cutaneous, mucosal, or cured visceral leishmaniasis respond in vitro to both native and rgp63. In this report, we mapped human T cell epitopes within gp63. T cells from leishmaniasis patients responded in vitro to certain peptides of gp63 by proliferation and IFN-gamma production. One peptide, (PT7), stimulated cells from all individuals tested (n = 7). Anti-PT7 T cell lines derived from PBMC of a mucosal leishmaniasis patient contained a heterogeneous population of cells which responded by proliferation and IFN-gamma production to in vitro stimulation with Leishmania promastigote lysate. Another peptide (PT1) derived from Leishmania chagasi gp63 stimulated PBMC from an L. chagasi patient although the corresponding Leishmania major-derived peptide did not. Both L. major PT7 and L. chagasi PT1 were able to induce anti-Leishmania-specific T cell lines from normal human PBMC. These T cell lines responded to in vitro stimulation with promastigote lysate indicating that both peptides were immunogenic for naive T cells in vitro. In conclusion, both antigenic and immunogenic gp63 peptide sequences have been defined, some appearing to be conserved among Leishmania species and at least one that appears to be species specific.
Cross-linking of CD14 molecules on monocytes results in a CD11/CD18- and ICAM-1-dependent adherence to cytokine-stimulated human endothelial cells.
Authors: Beekhuizen H , Blokland I , van Furth R
Published: December 2022

The myeloid differentiation protein CD14 that is expressed on the surface of mature monocytes contributes to the adherence of monocytes to cytokine-stimulated monolayers of human macrovascular endothelial cells (EC). It has also been observed that the initial adherence of monocytes to cultured cytokine-stimulated EC eventually results in an ICAM-1- and LFA-1 (CD11a/CD18)-dependent adherence, which coincides with stretching and lateral migration of the monocytes over the surface of EC. Recently, it was reported that CD14 mediates monocyte activation and can induce a change in the avidity of CD11a/CD18 for its ligand ICAM-1. The aim of the present study was to investigate whether activation of monocytes by CD14 elicits a CD11/CD18-dependent adhesion of monocytes to ICAM-1 on rIL-1 alpha-stimulated EC. Incubation of monocytes with murine anti-CD14 mAb alone did not mobilize intracellular calcium but the subsequent addition of F(ab')2 anti-mouse Ig, which caused cross-linking of CD14 on the surface of monocytes, induced a transient rise in cytosolic free calcium concentration and enhanced the percentage monocytes that adhered to monolayers of macrovascular venous EC stimulated with rIL-1 alpha for 24 h, but not to nonstimulated EC. The elevated adhesion was decreased with monocytes were preincubated with staurosporine, an inhibitor of intracellular protein kinase activity and was markedly inhibited by mAb against the common beta 2-subunit (CD18) of the CD11/CD18 molecules on monocytes and by mAb against ICAM-1 on 24-h rIL-1 alpha-stimulated venous EC. These studies provide evidence for the hypothesis that the binding of monocytes via CD14 to rIL-1 alpha-stimulated EC generates an intracellular response in monocytes and triggers an adhesion mechanism that allows CD11/CD18 molecules on monocytes to bind to ICAM-1 on EC.
Modulation of IL-1-induced neutrophil migration by dexamethasone and lipocortin 1.
Authors: Perretti M , Flower R J
Published: December 2022

IL-1 is a pro-inflammatory cytokine which controls many features of the immune and inflammatory response. When injected into a mouse 6-day-old air-pouch, human rIL-1 (1 to 100 ng) induced in a dose-dependent fashion a migration of PMN that could be reliably assessed 4 h after injection. Both IL-1 alpha and IL-1 beta were active in this model. The effect of the cytokine was inhibited by local administration of actinomycin D (1 to 10 micrograms), alpha-melanocyte-stimulating hormone (200 micrograms), and a mAb recognizing IL-1R type I (10 micrograms). Indomethacin (1 mg/kg), an inhibitor of cyclo-oxygenase, and BW4AC (2 mg/kg), a selective lipoxygenase inhibitor, were without effect but moderate inhibition was seen with the platelet-activating factor antagonist WEB2086 (1 to 10 mg/kg). The glucocorticoid dexamethasone (0.015 to 1.5 mg/kg) potently inhibited the elicitation of neutrophils induced by IL-1 when given systemically 2 h before the cytokine. The steroid-induced anti-inflammatory protein lipocortin 1 (LC1) also produced a dose-dependent inhibition of PMN migration into the pouch with an ED50 of approximately 0.15 to 0.21 mg/kg. The denatured protein was without effect. Passive immunization of mice with a polyclonal sheep antiserum or a mAb raised against LC1 abolished the inhibitory action of dexamethasone whereas preimmune serum or control IgG were without significant effect. These findings provide further evidence that LC1 is involved in the anti-inflammatory action of glucocorticosteroids and suggest that this protein may act as an endogenous regulator of IL-1 action.
Inhibition of phorbol ester-induced T cell proliferation by bryostatin is associated with rapid degradation of protein kinase C.
Authors: Isakov N , Galron D , Mustelin T , Pettit G R , Altman A
Published: December 2022

The bryostatins (Bryo) represent a group of immunomodulators that counteract the tumor-promoting effects of PMA. In contrast to the mitogenic effect of PMA on human peripheral blood T lymphocytes, Bryo was nonmitogenic and, furthermore, it inhibited PMA-induced T cell proliferation in a dose-dependent manner when added up to 2 days after PMA. Because both Bryo and PMA bind to, and activate, protein kinase C (PKC), we compared their effects on PKC expression and activity in human PBL or leukemic T cells (Jurkat). After treatment for 5 to 60 min, both Bryo and PMA were found to: a) activate PKC in vitro with similar dose-response curves; b) induce a nearly complete cytosol-to-membrane translocation of enzymatically active, Ca(2+)-dependent PKC and of distinct immunoreactive PKC isoforms in intact PBL; and c) stimulate similar patterns of protein phosphorylation. After a longer, 20-h treatment with PMA (20 nM), a considerable portion of PKC was still membrane-associated, and the total amount of immunoreactive PKC was not reduced considerably. In contrast, Bryo induced a marked loss of cellular immunoreactive PKC, including PKC-alpha and -beta. These results were paralleled by measurements of total cytosol- or membrane-associated PKC enzymatic activity. Thus, substantial PKC activity was associated with the particulate fraction of PMA-, but not Bryo-stimulated PBL. Furthermore, inhibition of PMA-induced T cell proliferation by Bryo also correlated with a reduction in the amount of cytosolic and membrane-bound immunoreactive PKC and enzymatic activity, demonstrating the dominance of Bryo over PMA. We propose that Bryo inhibits PMA-induced T cell proliferation by causing rapid degradation of PKC, reflecting a requirement of persistent PKC stimulation (lasting approximately 48 h) for the activation of human T cells and progression through the cell cycle.
Expression and function of B7 on human epidermal Langerhans cells.
Authors: Symington F W , Brady W , Linsley P S
Published: December 2022

The B7 molecule is expressed by APC that can costimulate T cells by binding the T cell surface receptors CD28 and CTLA-4. The human epidermal Langerhans cell (LC) is one of the most potent APC, yet B7 expression by this cell type has not previously been assessed. We used a CTLA4-Ig fusion protein that binds B7 with high avidity to probe cell surface expression of B7 by cultured and noncultured LC. LC cultured for 1 or more days were specifically stained with biotinylated CTLA4-Ig and fluorescent streptavidin. In contrast, binding of CTLA4-Ig to freshly isolated LC was not detected. The cell surface distributions of B7 and of HLA-DR on cultured LC differed, as CTLA4-Ig binding was localized to discrete foci, whereas anti-DR mAb uniformly stained the LC plasma membrane. Analyses of epidermal cell (EC) mRNA indicated that the B7 gene is expressed by these cells. Thus, B7 gene probes specifically hybridized to polymerase chain reaction-amplified B7 mRNA isolated from cultured and noncultured EC. As LC are the only normal epidermal cell type that induces proliferation of allogeneic T cells, the role of B7 in this LC function was studied by coculturing highly purified resting CD4+ T cells and allogeneic EC in the presence of CTLA4-Ig, anti-CD54 (RR/1, anti-intercellular adhesion molecule-1) mAb, or both. CTLA4-Ig and RR/1 each inhibited CD4+ T cell responses to freshly isolated allogeneic EC, and cooperative inhibition of more than 90% was observed in cultures treated with both CTLA4-Ig and RR/1 at 5 micrograms/ml. CTLA4-Ig inhibited stimulation by either fresh EC or cultured EC, suggesting that the increased potency of cultured LC vs noncultured LC may reflect the time needed for noncultured LC to express cell surface B7 in vitro. These studies indicate that B7 is expressed on the cell surface of cultured LC, and that LC B7 costimulates the proliferation of resting allogeneic CD4+ T cells.
Increased function of P-glycoprotein in T lymphocyte subsets of aging mice.
Authors: Witkowski J M , Miller R A
Published: December 2022

The age-associated decline in T lymphocyte function can be attributed in part to shifts between subsets (in particular the accumulation of memory T cells at the expense of naive T cells), and in part to alterations of function within the different T lymphocyte subsets. We show that the fluorochrome rhodamine-123 (R123) can distinguish further subdivisions within the naive and memory populations of both CD4 and CD8 T cells in the mouse. Aging leads to a progressive increase in the proportion of T cells in each subset that stain dimly with R123, and a corresponding decrease in the proportion of R123hi T cells, which are typical in young mice. We show that differential R123 staining between T cell subsets is due to variation in the activity of P-glycoprotein (P-Gp), the 170 kDa, ATP-dependent plasma membrane pump that mediates multiple-drug resistance. All T cells initially take up equivalent amounts of R123, but some T cells then can extrude the dye in a temperature-dependent process that is blocked by each of four pharmacologically disparate inhibitors of P-Gp function. Immunofluorescence experiments suggest that T cells from old and young mice have equal amounts of the P-Gp molecule itself, suggesting that differences in R123 extrusion may depend on posttranslational regulation of P-Gp activity.
Nonrandom T cell receptor usage in the allorecognition of HLA-DR1 microvariation.
Authors: Hurley C K , Steiner N , Wagner A , Geiger M J , Eckels D D , Rosen-Bronson S
Published: December 2022

Microvariation within the DR1 Ag family has created two DR molecules which differ only at beta-chain residues 85 (Val/Ala) and 86 (Gly/Val). TCR utilized by human alloproliferative T lymphocyte clones which can distinguish between these microvariants have been characterized by cDNA sequencing. The alpha- and beta-chain cDNA utilize a diverse set of variable (V) gene segments although the same V segment may be used by different individuals suggesting that V segment usage by the alloreactive T lymphocyte clones is nonrandom. There appears to be no difference in the repertoire of V segments utilized by T lymphocytes that preferentially recognize specific DR1 allelic products (DR(alpha,beta 1*0101) or DR(alpha,beta 1*0102)) and T lymphocytes that recognize both DR1 molecules. In contrast, the junctional regions of both alpha- and beta-chains are diverse in length and sequence although some common elements can be observed among TCR which share V gene segments. Two TCR which share V alpha and V beta gene segments differ in fine specificity for specific DR1 allelic products implicating the junctional regions of alpha- and beta-chains in the recognition of differentially bound peptides and/or in recognition of DR beta-chain residues 85 and 86. The stimulation of many diverse TCR by the limited allelic variation between DR(alpha,beta 1*0101) and DR(alpha,beta 1*0102) molecules suggests that the effect of DR microvariation on human immune responsiveness may be substantial.
Clonal analysis of a human antibody response. II. Sequences of the VH genes of human IgM, IgG, and IgA to rabies virus reveal preferential utilization of VHIII segments and somatic hypermutation.
Authors: Ikematsu H , Harindranath N , Ueki Y , Notkins A L , Casali P
Published: December 2022

The construction of mAb-producing cell lines has been instrumental in dissecting the fine specificities and genetic makeup of murine antibodies to exogenous and self Ag. The analysis of the genetic composition of human antibody responses has been hampered by the difficulty in generating human mAb of predetermined class and specificity. Using B lymphocytes from three healthy subjects vaccinated with inactivated rabies virus vaccine, we generated nine human mAb binding to rabies virus and analyzed the genes encoding their VH regions. Six mAb (five IgG1 and one IgA1) were monoreactive and displayed high affinities for rabies virus Ag. The remaining three mAb (IgM) were polyreactive and displayed lower affinities for rabies virus Ag. Seven mAb (3 IgG1, the IgA1, and the three IgM) utilized VH gene segments of the VHIII family. The remaining two IgG1 mAb utilized gene segments of the VHI and VHIV families. Of the seven expressed VHIII family genes, three were similar to the germline VH26c gene, two to the germline 22-2B gene, one to the germline H11 gene, and one to the germline 8-1B gene. The expressed VHI and VHIV genes displayed sequences similar to those of the germline hv1263 and V71-4 genes, respectively. The VH genes of all but one mAb (mAb55) resembled those that are predominantly expressed by C mu + clones in human fetal liver libraries. When compared with known germline sequences, the VH genes of the rabies virus-binding mAb displayed variable numbers of nucleotide differences. That such differences resulted from a process of somatic hypermutation was formally demonstrated (by analyzing DNA from polymorphonuclear neutrophil of the same subject whose B lymphocytes were used for the mAb generation) in the case of the VH gene of the high affinity (anti-rabies virus glycoprotein) IgG1 mAb57 that has been shown to efficiently neutralize the virus in vitro and in vivo. The distribution, mainly within the complementarity determining regions, and the high replacement-to-silent ratio of the mutations, were consistent with the hypothesis that the mAb57-producing cell clone underwent a process of Ag-driven affinity maturation through clonal selection. The D gene segments of the rabies virus-selected mAb were heterogeneous and, in most cases, flanked by significant N segment additions. The JH segment utilization was unbalanced and reminiscent of those of the adult and fetus. Four mAb utilized JH4, two JH6, two JH3, and one JH5; no mAb utilized JH1 or JH2 genes.(ABSTRACT TRUNCATED AT 400 WORDS)
Human cord blood antibody repertoire. Mixed population of VH gene segments and CDR3 distribution in the expressed C alpha and C gamma repertoires.
Authors: Mortari F , Wang J Y , Schroeder H W
Published: December 2022

Developmental regulation of the diversity of the antibody repertoire is associated with the controlled stepwise acquisition of Ag responsiveness during ontogeny. The development of IgG and IgA serum levels in humans lags far behind the maturation of the rest of the immune response. We cloned cord blood mononuclear cell C gamma and C alpha transcripts and compared them with a large database of > 250 published and unpublished sequences, including C mu transcripts from this same cord blood cDNA library, to determine whether restrictions in the expressed antibody repertoire contributed to the limitations of the neonatal IgG and IgA response. We found a diverse antibody repertoire containing a high frequency of the same VH gene segments first identified in fetal liver, as well as VH elements homologous to those in the cord blood C mu transcripts. Although VH, DH, and JH utilization differed, the distribution of CDR3 lengths in the C gamma transcripts was similar to that found in fetal liver C mu transcripts, whereas the distribution of CDR3 lengths in the C alpha transcripts was similar to that found in cord blood C mu. This comprehensive analysis of the C mu, C gamma, and C alpha V domain repertoires provides new insights into our understanding of the timing of sequential B cell waves that seed peripheral lymphoid compartments during development.
HLA-unrestricted killing of HSV-1-infected mononuclear cells. Involvement of either gamma/delta+ or alpha/beta+ human cytotoxic T lymphocytes.
Authors: Maccario R , Revello M G , Comoli P , Montagna D , Locatelli F , Gerna G
Published: December 2022

The characterization of HSV-specific human CTL, obtained in short term cultures by stimulating PBMC of healthy HSV-immune donors with autologous, PHA-activated, HSV-1-infected mononuclear cells, is described. CTL induced by using this technique are able to mediate a strong lytic activity against both HSV-1- and HSV-2-infected targets, whereas they do not kill autologous EBV-lymphoblastoid cell lines unless they are superinfected with HSV-1. TCR-gamma/delta+ cells are mainly responsible for HSV-specific cytotoxic activity in some donors, whereas TCR-alpha/beta+ CTL are primarily involved in other subjects. The large majority of HSV-specific CTL bearing either TCR-gamma/delta or TCR-alpha/beta also express CD8 and/or CD56 molecules. Virus-specific CTL, here described, require the expression of HLA class I Ag on the surface of target cells to mediate lytic activity. Nevertheless, the response is apparently HLA-unrestricted in that HSV-1-induced CTL are also able to lyse target cells mismatched for A, B, C, DR, and DQ loci. Our data suggest that both TCR-gamma/delta+ and TCR-alpha/beta+ CTL may play a role in the immune response to HSV in humans.
Effector functions of circumsporozoite peptide-primed CD4+ T cell clones against Plasmodium yoelii liver stages.
Authors: Rénia L , Grillot D , Marussig M , Corradin G , Miltgen F , Lambert P H , Mazier D , Del Giudice G
Published: December 2022

Previously, CD4+ T cell lines and clones were isolated after immunization of BALB/c and C57BL/6 mice with the Py1 peptide, a 21-mer synthetic peptide corresponding to a N-terminal segment of the circumsporozoite protein of Plasmodium yoelii. The clones were separated into the Th1 and Th2 subsets on the basis of lymphokine production. It was observed that immunization with the Py1 peptide induced preferentially Th1 cells in BALB/c and Th2 cells in C57BL/6 mice. These clones were then tested for their cytolytic ability in vitro. Some of the clones from BALB/c and C57BL/6 mice eliminated liver stage parasites from cultured hepatocytes in a MHC restricted manner. Nevertheless, none of these clones was able to lyse Py1 peptide-pulsed target cells. It was also found that two clones could protect BALB/c mice against a sporozoite challenge. These results provide evidence that CD4+ T cells, induced after priming with a defined peptide, could participate in the effector mechanisms against malaria liver stages.
Induction of leukemia inhibitory factor in human synovial fibroblasts by IL-1 and tumor necrosis factor-alpha.
Authors: Hamilton J A , Waring P M , Filonzi E L
Published: December 2022

The cytokines, IL-1 alpha and TNF-alpha, induced a dose-dependent production of leukemia inhibitory factor (LIF) in cultured human synovial fibroblast-like cells, as measured by a radioreceptor competition assay. Significant levels of LIF were first detected in cell supernatants between 2 and 4 h after cytokine addition. Increases were also observed in LIF mRNA steady-state expression. Evidence is presented for down-regulation of the IL-1-induced LIF activity by an endogenous cyclo-oxygenase product(s); the glucocorticoid, dexamethasone, lowered the IL-1-induced LIF activity and mRNA expression. A synergistic effect was noted between the actions of IL-1 alpha and TNF-alpha, and between IL-1 and transforming growth factor-beta. IFN-gamma could not induce LIF formation in the synovial cells but inhibited the stimulatory effect of IL-1. These results suggest that cytokine-stimulated synovial fibroblasts may be a major source of intraarticular LIF production in the joints of patients with inflammatory arthritis. Synoviocyte-derived LIF may activate monocyte/macrophages in the lesions and may contribute to the bone changes and certain systemic manifestations in patients with inflammatory joint disease.
Receptor-activated calcium influx in human monocytes exposed to monocyte chemotactic protein-1 and related cytokines.
Authors: Sozzani S , Molino M , Locati M , Luini W , Cerletti C , Vecchi A , Mantovani A
Published: December 2022

Stimulation of human monocytes with monocyte chemotactic protein-1 (MCP-1) resulted in an increase of [Ca2+]i. The [Ca2+]i rise was dependent on external Ca2+, could be reconstituted by the addition of external Ca2+ and was blocked by Ni2+. Agonist-stimulated Ca2+ influx was demonstrated directly by the use of Mn2+: in the presence of extracellular Mn2+, MCP-1 and FMLP stimulated a dose-dependent quench in fluorescence of Fura-2-loaded monocytes. This quench was the result of stimulation of Mn2+ influx and was blocked by Ni2+ and by the Ca2+ channel inhibitor SC38249. Pretreatment of monocytes with verapamil and nifedipine or high depolarizing [K+] had no effect. MCP-1 did not induce production of inositol triphosphate nor turnover of phosphatidylinositol 4,5-biphosphate. Collectively, these results show that MCP-1 does not induce discharge of intracellular stores. This chemokine stimulates divalent cation entry (Ca2+ or Mn2+) by a mechanism independent of changes in [Ca2+]i, unrelated to voltage-dependent Ca2+ channels and presumably involving receptor-activated channels. In addition to MCP-1, also two other members of the chemokine Cys-Cys family, RANTES and MIP-1 alpha, stimulated [Ca2+]i increase. Exposure of human monocytes to either RANTES or MIP-1 alpha, had no effect on a subsequent stimulation by MCP-1. On the contrary, MCP-1 cross-desensitized monocytes for a subsequent stimulation with RANTES and MIP-1 alpha. These results suggest a certain level of receptor sharing among members of the Cys-Cys chemokine family.
Overlap of the anti-Sm and anti-DNA responses of MRL/Mp-lpr/lpr mice.
Authors: Bloom D D , Davignon J L , Cohen P L , Eisenberg R A , Clarke S H
Published: December 2022

Autoantibodies specific for the Sm ribonucleoprotein are spontaneously produced in patients with SLE and in mice of the MRL mouse strains. We have previously reported the characterization of the clonality and V region gene use of 41 MRL/Mp-lpr/lpr (MRL/lpr)-derived B cell hybridomas selected for Sm binding. In this report, we show that many of the expressed V genes of these hybridomas are also expressed by anti-DNA hybridomas of MRL/lpr mice. Moreover, the anti-Sm hybridomas from nine clonal groups produce antibodies that bind ssDNA, and those of five clones produce antibodies that also bind dsDNA. Sm/DNA-specific hybridomas, but not Sm-only-specific hybridomas, have a higher than expected content of arginine residues in CDR3 of the H chain, similar to MRL/lpr hybridomas selected on the basis of DNA binding. One clone displays intraclonal differences in DNA binding, inasmuch as the most extensively mutated members produce antibodies that are able to bind dsDNA and have a higher affinity for ssDNA than the least mutated members of this clone. Thus, DNA appears to be a selecting Ag in this response. These data indicate an overlap in the anti-Sm and anti-DNA autoimmune responses in MRL mice that may have implications for the activation of anti-Sm B cells, and for defining the spectrum of Ag targeted in SLE.
B cell tolerance induction by cross-linking of membrane IgM, but not IgD, and synergy by cross-linking of both isotypes.
Authors: Gaur A , Yao X R , Scott D W
Published: December 2022

Previous studies in our laboratory demonstrated that overnight exposure of adult splenic B cells to anti-Ig resulted in an unresponsive state characterized by decreased antibody synthesis but normal mitogen-driven proliferation (i.e., energy). Because both anti-F(ab')2 and anti-mu were equally effective at inducing tolerance, it was important to determine whether cross-linking of IgD together with or separately from IgM influenced the induction of unresponsiveness. Although anti-mu induced significant unresponsiveness, treatment of adult splenic B cells with anti-delta alone generally failed to reduce the subsequent response to either LPS or fluoresceinated Brucella abortus. Interestingly, anti-delta synergized with suboptimal concentrations of anti-mu to induce tolerance. Synergy could be observed in this system when anti-delta was added either simultaneously with or before (but not after) anti-mu; moreover, anti-delta was effective in a pretreatment (wash-out) protocol. To investigate the role of protein tyrosine kinase (PTK) activity in tolerance induction, splenic B cells were treated with tyrphostin before treatment with either anti-mu or anti-delta. We found that pretreatment with tyrphostin for 2 h before the addition of anti-mu prevented the induction of unresponsiveness with this antibody, whereas this PTK inhibitor facilitated tolerance when used with anti-delta treatment only. We propose that cross-linking of surface IgM directly or indirectly invokes a tyrphostin-sensitive, PTK-dependent pathway leading to the early events in tolerance induction, which can be augmented under limiting conditions by anti-IgD. Because cross-linking of either receptor initiates several common pathways, simultaneous cross-linking can lead to synergy and a dominance of the IgM signal. In contrast, IgD alone may fail to elicit tolerance because this isotype may also be associated with different PTK that cause positive signaling.
Effect of prostaglandin E2 (PGE2) on IL-3/granulocyte-macrophage colony-stimulating factor production by T helper cells. Mode of stimulation and presence of costimulation can determine response to PGE2.
Authors: Li T K , Fox B S
Published: December 2022

PGE2 is an immunomodulator that selectively inhibits the production of lymphokines associated with Th1 cells (IL-2 and IFN-gamma) but not Th2 cells (IL-4 and IL-5). We examined the effect of PGE2 on the production of IL-3 and granulocyte-macrophage (GM)-CSF from murine Th1 and Th2 clones. When the T cells were stimulated with Ag and APC, PGE2 inhibited IL-3/GM-CSF production from 3 Th1 clones and 1 Th2 clone, but enhanced production from 3 Th2 clones. A more specific bioassay demonstrated that IL-3 production was differentially affected by PGE2 in the Th clones. These data suggested that the effect of PGE2 on IL-3 production is dependent, not on a property of the lymphokine, but on a property of the T cell. The responsiveness to PGE2 did not consistently differ between Th1 and Th2 cells, and the observed heterogeneity in the response of Th2 clones did not correlate with the ability to induce increases in intracellular [Ca2+]. However, we postulated that signaling differences between the clones might explain the varied responsiveness to PGE2. If so, then the mode of stimulation might be expected to activate different pathways and thus affect the PGE2-responsiveness. Stimulation with ionomycin induced variable levels of IL-3/GM-CSF from the T cell clones. APC-derived costimulation dramatically enhanced IL-3/GM-CSF; the cells which produced high levels in response to ionomycin alone were not detectably costimulating each other. Interestingly, PGE2 enhanced IL-3/GM-CSF (and IL-3 alone in at least some cases) from cells stimulated with ionomycin alone, demonstrating that the mode of stimulation affects the PGE2-responsiveness. Addition of APC not only enhanced lymphokine production, but also altered the PGE2-responsiveness of the Th1 cells. In these cells, PGE2 either inhibited IL-3 and GM-CSF production or had no effect, in no case was the lymphokine production enhanced by PGE2 as it had been with ionomycin alone. These data indicate that the presence of APC-derived costimulatory signals can alter the effect of PGE2 on Th cell lymphokine production.
Costimulatory signals are required for optimal proliferation of human natural killer cells.
Authors: Robertson M J , Manley T J , Donahue C , Levine H , Ritz J
Published: December 2022

CD56dim NK cells, which comprise approximately 90% of human peripheral blood NK cells, respond to IL-2 with cytokine production, up-regulation of functionally relevant surface molecules, and augmented cytolytic activity. Nevertheless, CD56dim NK cells proliferate poorly in response to IL-2 alone. We found that other NK cell mitogens, including IL-4, IL-7, and IL-12, also induced little proliferation of CD56dim NK cells. Indeed, IL-2 stimulated at least 10-fold more NK cell proliferation than did IL-4, IL-7, or IL-12. In contrast, leukocyte-conditioned medium (LCM) induced two- to threefold greater proliferation of CD56dim NK cells than did optimal concentrations of IL-2. Although the calcium ionophore ionomycin did not stimulate proliferation by itself, it markedly augmented LCM-induced proliferation of CD56dim NK cells. Proliferation in response to either LCM alone or LCM together with ionomycin was almost completely abrogated by anti-IL-2R antibodies. Thus, IL-2 appears to be necessary but not sufficient for optimal proliferation of CD56dim NK cells. LCM-induced proliferation of ionomycin-activated CD56dim NK cells was inhibited 24% by anti-IL-1 heteroantisera and 57% by anti-TNF antisera; a combination of both antisera inhibited proliferation by 73%. Furthermore, although rIL-1 and TNF did not induce proliferation by themselves, both cytokines could augment IL-2-induced proliferation of resting or ionomycin-activated NK cells. Hence IL-1 and TNF do not appear to be primary NK cell mitogens, but rather accessory factors that can enhance IL-2-dependent NK cell proliferation. Stimulation through CD2 or CD16 Ag did not enhance LCM-induced NK cell proliferation. However, stimulation with NK-sensitive K562 cells strongly augmented CD56dim NK cell proliferation to LCM or to IL-2, IL-1, and TNF in combination. NK-resistant Daudi cells did not promote the proliferation of highly purified NK cells. Thus, NK cell proliferation may be enhanced by triggering through putative receptors for natural killing, and ionomycin may mimic such triggering. Although IL-2 by itself can induce NK cell proliferation, most NK cells resemble T and B lymphocytes in that they require multiple signals for optimal proliferation.
DNA regions flanking the mouse Ig 3' alpha enhancer are differentially methylated and DNAase I hypersensitive during B cell differentiation.
Authors: Giannini S L , Singh M , Calvo C F , Ding G , Birshtein B K
Published: December 2022

Two B cell-specific enhancer elements are associated with the IgH gene cluster. One enhancer is located within the J-C mu intron (E mu), whereas a second enhancer (3' alpha E) is approximately 12.5 kb 3' of the C alpha membrane exon. In an attempt to understand the function of 3' alpha E, we have characterized its surrounding structural milieu during various stages of B cell differentiation through analysis of methylation patterns and the identification of DNAse I-hypersensitive sites. We observed a correlation between the chromatin structure of this region and the differentiation state of the cell. Compared to liver and brain, the region 3' of alpha was hypermethylated in pre-B and T cell lines and became progressively demethylated as B cell differentiation continued. A DNAse I-hypersensitive site was present in pre-B cell lines about 17 kb 3' of 3' alpha E. In fully differentiated myeloma cell lines, a second cluster of DNAse I-hypersensitive sites was present immediately 5' of 3' alpha E. Our data indicate that the 3' alpha enhancer is relatively sequestered during early stages of B cell differentiation and becomes increasingly accessible at later stages.
Verification of a model of a F(ab) complex with phenylarsonate by oligonucleotide-directed mutagenesis.
Authors: Sompuram S R , Sharon J
Published: December 2022

A model of the combining site of a mouse antibody specific for p-azophenylarsonate was tested by oligonucleotide-directed mutagenesis of the proposed hapten-contacting residues, Arg96 in the L chain, and Asn35, Trp47, Tyr50, Ser95, and Tyr100b in the H chain. The affinity and relative affinity for p-azophenylarsonate-N-acetyl-L-tyrosine of mutant antibodies expressed in transfectomas were determined by fluorescence quenching and by inhibition ELISA, respectively. The results show that alteration of the proposed contacting residues has drastic effects on hapten binding, and that the hydroxyl groups of Tyr50 and Tyr100b appear to orient the phenyl rings for optimal aromatic-aromatic interactions with the phenyl ring of the hapten. They further indicate a tight packing of the contacting residues around the hapten, which cannot accommodate changes in the positions of the functional groups of Asn35 and Ser95.
Characterization of an unusual cell type (CD4+ CD3-) expanded by helminth infection and related to the parasite stress response.
Authors: Estes D M , Turaga P S , Sievers K M , Teale J M
Published: December 2022

Mice infected with the parasite Mesocestoides corti develop hypergammaglobulinemia, hepatomegaly, and splenomegaly. The immune response to M. corti infection is directed, in part, at molecules secreted by the organism. Two of these molecules have been shown to be hsp70 and hsp60 homologues. In this study it was found that incubation of splenocytes from infected animals with M. corti-secreted molecules or the isolated M. corti hsp70 results in the expansion of an unusual cell type with the morphology of large granular lymphocytes. The cell lines express Thy-1, CD4 (low), and CD45RB but lack TCR alpha beta, TCR gamma delta, CD3, CD8, and slg. The lack of a TCR suggested NK cells, but no cytolytic activity could be detected. In addition, the cell lines constitutively produce IL-6 and can be induced to express IL-2, but not IL-4, IL-5, or IFN-gamma. Given the phenotype of these cells, it is possible that they represent T lineage precursors or some type of effector cells. Notably, CD3- CD4+ cells appear to be expanded in the spleens and livers of M. corti-infected animals, suggesting an important role in infection. Moreover, the selective growth of this cell type with M. corti hsp70 suggests that the outgrowth and in vivo expansion of these cells may be related to the stress response of the parasite.
Leishmania major interferes with antigen presentation by infected macrophages.
Authors: Fruth U , Solioz N , Louis J A
Published: December 2022

Impairment of the Ag-presenting capacity of macrophages harboring intracellular Leishmania might represent one of the several mechanisms by which these parasites can evade host-protective T cell responses. Thus, the present study was designed to investigate the ability of macrophages, parasitized with Leishmania major, to present Ag to relevant T cell hybridomas. Results show that bone marrow-derived macrophages from BALB/c mice, after infection with L. major, have a greatly reduced capacity to present OVA, beta-galactosidase, and L. major-derived Ag to specific T cell hybrids derived from mice immunized with those Ag. In contrast, after pulsing with relevant peptide, macrophages containing L. major have a normal Ag-presenting capacity. The inhibition of presentation of native Ag did not appear to result from decreased endocytosis or catabolism. Inasmuch as the inhibition of presentation could not be attributed to an impaired processing of the Ag or an unavailability of MHC class II molecules on the surface of infected cells, these results could indicate that the presence of L. major interferes with the intracellular loading of MHC class II molecules with antigenic peptides.
Vaccination with recombinant filarial paramyosin induces partial immunity to Brugia malayi infection in jirds.
Authors: Li B W , Chandrashekar R , Weil G J
Published: December 2022

Vaccination with irradiated infective larvae induces partial protective immunity to infection with the filarial nematode Brugia malayi in jirds. Prior studies have shown that such immunization stimulates a much stronger antibody response to recombinant and native filarial paramyosin than that seen after normal infection. To determine whether vaccination with recombinant paramyosin could induce protective immunity to larval challenge, jirds were immunized with either B. malayi paramyosin and maltose binding protein (BM5-MBP) (fusion protein of B. malayi paramyosin and maltose-binding protein), MBP alone, or recombinant Dirofilaria immitis paramyosin. Animals were challenged with 100 infective larvae s.c. 8 wk after the second immunization. Necropsies were performed 16 wk after challenge. Vaccination with BM5-MBP induced significant protective immunity; adult worm recoveries, worm lengths, and blood microfilaria counts were reduced in the BM5-MBP group relative to the MBP control group. The reductions in adult worm recoveries (43%) and female worm lengths (10%) were statistically significant (p < 0.05). Interestingly, protective immunity was not induced by immunization with D. immitis paramyosin. Additional studies are needed to identify mechanisms involved in protective immunity induced by BM5-MBP and to understand the differential activity of the two closely related recombinant paramyosin proteins in this model.
Activation of tumor necrosis factor-alpha production from human neutrophils by IL-2 via IL-2-R beta.
Authors: Wei S , Blanchard D K , Liu J H , Leonard W J , Djeu J Y
Published: December 2022

In addition to T cells, NK cells, B cells, and monocytes, we provide new evidence that human polymorphonuclear neutrophils (PMN) can be functionally activated by IL-2 via binding to IL-2R beta expressed on the cell surface. Brief exposure of normal PMN to human rIL-2 enhanced both transcriptional and translational expression of TNF-alpha. The release of TNF-alpha protein by IL-2-treated PMN was inhibitable by a specific mAb against human IL-2-R beta. The response to IL-2 was dose and time dependent with the increase in TNF-alpha mRNA detected maximally 3 h after IL-2 exposure, followed by a continuous maintenance of high mRNA levels up to 18 h. The TNF-alpha mRNA was significantly increased above the medium control level, with as little as 10 U/ml of IL-2. Maximal transcription was obtained with 1000 U/ml of IL-2, which achieved the level observed with known neutrophil activating factors such as granulocyte-macrophage-CSF, IL-8, and Candida albicans. Using actinomycin D, it was found that new and continuous synthesis of a labile TNF-alpha mRNA was responsible for the observed high levels of transcripts. Of significance was the observation that cycloheximide could selectively modulate TNF-alpha mRNA transcription in neutrophils, depending on the cytokine used. Cycloheximide did not affect or alter TNF-alpha mRNA induction in IL-2-treated neutrophils but abrogated it in granulocyte-macrophage-CSF-treated neutrophils and superinduced transcription in C. albicans-treated neutrophils. Thus various control elements must be involved in the transcription of the TNF-alpha genes that are responsive to different cytokines and activating factors. The induction of TNF-alpha and functional activation of neutrophils by IL-2 is therefore an important immunomodulatory property of IL-2 that has not heretofore been recognized.