bluuebunny/crossref_metadata_2025 huggingface dataset:
Natural killer (NK) cell stimulatory factor or IL-12 has differential effects on the proliferation of TCR-alpha beta+, TCR-gamma delta+ T lymphocytes, and NK cells.
Authors: Perussia B , Chan S H , D'Andrea A , Tsuji K , Santoli D , Pospisil M , Young D , Wolf S F , Trinchieri G
Published: December 2022

We have analyzed the effects of NK cell stimulatory factor/IL-12, on proliferation of PBL and their subsets. IL-12 synergizes with lectins and phorbol diesters to induce proliferation of CD4+ and CD8+ peripheral blood T lymphocytes. In the case of phorbol-diester-induced proliferation, the effect of IL-12 is in part mediated by induced IL-2 production, as suggested by the observation that IL-12 enhances IL-2 production in these cultures and that anti-IL-2 antibodies inhibit proliferation. IL-12 synergizes also with anti-CD3 antibodies and with allogeneic stimulation in MLC in inducing T cell proliferation. IL-12 alone is mitogenic for preactivated T and NK lymphoblasts. This mitogenic effect is observed with similar doses of IL-12 on NK lymphoblasts as well as on CD4+ and CD8+ TCR-alpha beta+ and on TCR-gamma delta+ lymphoblasts. On TCR-alpha beta+ T lymphocytes the effect of IL-12 is always additive to that of IL-2 over a wide dose range. The same effect is observed on highly activated, actively proliferating NK cells. However, on NK and TCR-gamma delta+ lymphoblasts reverting to a resting state after stimulation and on a TCR-gamma delta+ acute leukemia-derived T cell line, IL-12 inhibits significantly the proliferation induced by moderate to high doses (10 to 100 U/ml) of IL-2. This inhibitory effect is, at least in part, indirect, and depends on IL-12-induced production of TNF. Neutralizing anti-TNF antibodies, but not anti-IFN-gamma and anti-transforming growth factor antibodies, restore by more than 70% the inhibition of proliferation induced by IL-12 in these cultures. However, TNF alone cannot mimic the inhibitory effect of IL-12 on the IL-2-induced proliferation of NK and TCR-gamma delta+ lymphoblasts, suggesting the involvement of additional mechanisms. The relevance of these findings for the biology of lymphocyte subsets mediating MHC nonrestricted cytotoxicity is discussed.
Poly-(ADP-ribose) polymerase partially contributes to target cell death triggered by cytolytic T lymphocytes.
Authors: Redegeld F A , Chatterjee S , Berger N A , Sitkovsky M V
Published: December 2022

We hypothesized that CTL-induced target cell (TC) death is partially due to processes that follow the DNA damage in target cells and include the activation of poly-ADP-ribose transferase (PADPRT) by DNA strand breaks. According to this model, the activated PADPRT is expected to deplete NAD, ATP, and to contribute to the TC death. We used inhibitors of PADPRT and a PADPRT-deficient cell mutant, as well as other nucleated TC and SRBC to test the role of PADPRT in CTL-induced cytotoxicity. It is found that inhibitors of PADPRT (3-aminobenzamide, benzamide (aromatic amides)) and nicotinamide all inhibit the CTL-mediated lysis of both Ag-specific TC and of Ag-nonbearing TC. The effect of PADPRT inhibitors was not due to inhibition of the lethal hit delivery by CTL, because in parallel control experiments, the same inhibitors did not interfere with CTL-induced lysis of SRBC, cells that are devoid of nuclei and PADPRT. Moreover, the effect of inhibitors of PADPRT did not affect earlier stages of lethal hit delivery because 3-aminobenzamide and benzamide did not interfere with CTL-induced DNA fragmentation in TC at concentration which protected TC lysis. Importantly, a PADPRT-deficient cell line was also much more resistant to CTL-induced lysis as tested in retargeting (4 and 8 h) assays; this was expected if activation of PADPRT is indeed involved in TC death. Control experiments reveal that the relative resistance of the PADPRT-deficient cell mutant to CTL-induced lysis was not related to its impaired ability to form conjugates and to trigger CTL (as tested in granule exocytosis assay). In addition, PADPRT-deficient cells were as susceptible to CTL-induced DNA fragmentation as were the control cells; yet, they were resistant to CTL-induced 51Cr-release. Control cells and PADPRT-deficient mutant were equally susceptible to antibody+C'-mediated lysis. Our data support the view that the activation of PADPRT can contribute to the CTL-induced cytolysis of some TC, but is not involved in lysis of other TC, as evidenced by the ability of CTL to efficiently lyse SRBC. These data suggest that there could be multiple molecular pathways of TC death in CTL-mediated cytotoxicity and the relative contribution of PADPRT and/or other enzymes will reflect the individual make-up of a particular TC.
Negative selection of thymocytes. A novel polymerase chain reaction-based molecular analysis detects requirements for macromolecular synthesis.
Authors: D'Adamio L , Clayton L K , Awad K M , Reinherz E L
Published: December 2022

Self-tolerance is mainly established through clonal deletion of autoreactive T cells during thymic differentiation. The mechanisms by which deletion is achieved are poorly understood. Here we use a specific polymerase chain reaction-based system to characterize DNA fragmentation and show that after in vivo treatment of neonatal mice with staphylococcus enterotoxin B, selective apoptosis of V beta 8+ thymocytes occurs. This process precedes detectable deletion of V beta 8+ cells as determined by phenotypic analysis. Moreover, in vivo administration of cycloheximide and, to a lesser extent, actinomycin D, inhibits apoptosis of staphylococcus enterotoxin B specific thymocytes. Thus, macromolecular synthesis is a requirement for negative selection.
Granuloma T lymphocytes in murine schistosomiasis mansoni have somatostatin receptors and respond to somatostatin with decreased IFN-gamma secretion.
Authors: Blum A M , Metwali A , Mathew R C , Cook G , Elliott D , Weinstock J V
Published: December 2022

The granulomas of mice infected with Schistosoma mansoni for 8 wk have macrophages that secrete somatostatin 1-14 (SOM). Within the granuloma, SOM has no known function. To uncover the possible significance of SOM produced within this granulomatous inflammation, we sought SOM receptors on distinct cellular components of the granuloma to identify cells targeted for SOM action. [125I]SOM 1-14 bound to dispersed granuloma inflammatory cells specifically and reversibly. Scatchard analysis suggested one receptor type (kDa 4.28 x 10(-9) M). Octreotide, a stable SOM derivative, displaced radioligand (kDa 1.01 x 10(-10) M), but SOM 1-28, substance P, and vasoactive intestinal peptide did not. The SOM receptor localized exclusively to a subset of granuloma CD4+ T lymphocytes. Using IL-5 and IFN-gamma ELISA, it was shown that granuloma T cells can secrete appreciable IL-5 and IFN-gamma when stimulated with Ag or mitogen. Both SOM and octreotide at concentrations as low as 10(-10) M substantially decreased IFN-gamma secretion from Ag or mitogen-stimulated T cells, but at concentrations as high as 10(-6) did not affect IL-5 production. Octreotide administered to animals in vivo decreased the intensity of the granulomatous response. Thus, some granuloma T cells have SOM 1-14 receptors. SOM 1-14, a product of granuloma macrophages, may participate in regulation of the granulomatous response by modulating the secretion of some lymphokines. Octreotide, a clinically useful SOM analog, mimics the action of SOM on the immune system.
The hamster immune response to tick-transmitted Borrelia burgdorferi differs from the response to needle-inoculated, cultured organisms.
Authors: Roehrig J T , Piesman J , Hunt A R , Keen M G , Happ C M , Johnson B J
Published: December 2022

The human immune response to natural infection with Borrelia burgdorferi appears to differ from that seen in small mammals infected by needle inoculation. In humans, antibody to outer surface proteins A and B (OspA and OspB) is not detectable until late in infection, but small mammals inoculated with B. burgdorferi produce early antibody to OspA and OspB. To investigate this disparity we compared the immune response in hamsters to B. burgdorferi after needle inoculation with cultured organisms or infected tick homogenates with the immune response after tick transmitted (natural) infection. We determined that the antibody response to OspA and OspB after natural infection of hamsters is similar to that seen in humans, and differs from the antibody response after hamster infection by needle inoculation. High titers of antibody to OspA and OspB were undetectable even 42 wk after bite by B. burgdorferi-infected ticks. The failure to produce antibody to OspA and OspB was not dependent on challenge dose, because animals inoculated by needle with low doses (1 x 10(5) to 1 x 10(6) cells) of B. burgdorferi produced antibody to OspA and OspB. A rapid but limited anti-41-kDa response was observed. One possible new Ag, 43 kDa (p43), was identified. The antibody response to p43 was independent of the route of inoculation. Our results suggest that the hamster immune response to tick-transmitted Borrelia burgdorferi differs from the response to needle inoculated, cultured organisms.
IL-3-dependent mast cells attach to plate-bound vitronectin. Demonstration of augmented proliferation in response to signals transduced via cell surface vitronectin receptors.
Authors: Bianchine P J , Burd P R , Metcalfe D D
Published: December 2022

The adhesive interactions of activated mast cells with the extracellular matrix play an important role in anchorage and cellular motility. In this report we demonstrate that IL-3-dependent bone marrow-derived mast cells adhere to plate-bound vitronectin with high affinity in a saturable and dose-dependent manner. This adhesion interaction is unique in that it does not require prior mast cell activation through Fc epsilon RI or after treatment with PMA. It is inhibited by divalent cation chelation and by competitive inhibition with a synthetic Arginine-Glycine-Aspartate-Serine tetrapeptide. Polyclonal antisera for alpha v beta 3, an integrin known to bind vitronectin, inhibits attachment to plate-bound vitronectin in a dose-dependent manner. Comparison of the adhesion interactions for vitronectin, fibronectin, and laminin indicate that adhesion to vitronectin is greater than that seen with either fibronectin or laminin, either in the presence or absence of PMA. FACS analysis using a monoclonal hamster anti-murine vitronectin receptor (alpha v) antibody followed by a fluorescein-conjugated rabbit anti-hamster IgG revealed no change in surface vitronectin receptor expression after Fc epsilon RI-mediated cell activation. Proliferation assays with correction for cell viability revealed a 25% increase in cell number above the maximal IL-3 response over a 24-h period of adhesion to a vitronectin-coated surface and a 41% increase over 96 h of adhesion to vitronectin. Binding to plate-bound vitronectin was not able to sustain cell viability in the absence of IL-3. Thus, IL-3-dependent bone marrow-derived mast cells adhere to vitronectin, an extracellular matrix protein present throughout connective tissues. This interaction generates a signal that results in the augmentation of the maximal IL-3-dependent mast cell proliferative response, thus demonstrating at least one way in which the interaction between mast cells and extracellular matrix alter the biologic responsiveness of the mast cell.
Priming of human eosinophils by platelet-activating factor enhances the number of cells able to bind and respond to opsonized particles.
Authors: Blom M , Tool A T , Roos D , Verhoeven A J
Published: December 2022

Addition of platelet-activating factor (PAF) to human eosinophils leads to the modulation of eosinophil responses. Earlier work from our laboratory has shown that the respiratory burst and homotypic aggregation response in these cells induced by opsonized particles (serum-treated zymosan, STZ), is strongly enhanced after pretreatment (priming) with PAF. In the present study we have investigated the effect of PAF on the binding of fluorescent STZ particles to human eosinophils. Addition of STZ to eosinophils isolated from the peripheral blood of normal donors results in an interaction of the STZ particles with only 30 to 40% of the cells. Treatment of the eosinophils with PAF (1 microM) for 2 min strongly enhanced the rate of particle binding and also doubled the percentage of eosinophils binding STZ. The effect of PAF priming is most likely mediated by a change in CR3, because it is reversed by mAb B2.12 blocking the iC3b binding site of CR3 and unaffected by mAb IV.3 blocking Fc gamma RII. This change is not an increase in cell surface expression of CR3, and it requires an active cellular metabolism to be maintained. The functional consequences of the effect of PAF on STZ binding were investigated in the nitro-blue tetrazolium dye slide test. PAF priming strongly enhanced the percentage of eosinophils producing oxygen radicals after STZ stimulation. Our findings indicate that the priming phenomenon observed in human eosinophils consists, at least in part, of a recruitment of cells able to interact with and to respond to opsonized particles.
Comparison of airway and blood eosinophil function after in vivo antigen challenge.
Authors: Sedgwick J B , Calhoun W J , Vrtis R F , Bates M E , McAllister P K , Busse W W
Published: December 2022

Eosinophils (EOS) are important effector cells in allergic diseases and asthma. However, functional characteristics of the EOS have been derived primarily from studies of blood cells, and it is unlikely that such assessments reflect events occurring in tissues or airways. To establish more precisely the function of airway EOS, segmental Ag challenge was used to elicit and isolate large numbers of these cells. Airway, as well as blood, EOS were isolated from allergic patients 48 h after segmental Ag challenge. Both blood and bronchoalveolar lavage (BAL) EOS were fractionated over Percoll density gradients; by using this protocol, three density-distinct populations of pure (>90%) EOS were obtained from BAL fluid (1.100, 1.095, and 1.090 g/ml) and one from blood (1.100 g/ml). The functions of these various populations were compared by measuring superoxide generation, adherence to collagen and endothelial cell monolayers, cell surface receptors, and in vitro survival. BAL EOS of all three densities had greater superoxide generation and adherence with FMLP activation than did corresponding blood EOS. In contrast, blood and airway EOS responded similarly to PMA. BAL EOS also had increased expression of CD11b/CD18 and HLA-DR. The intracellular calcium concentration ([Ca2+]i) was measured with the fluorescent marker indo-1/acetoxymethyl ester. FMLP caused a greater and more sustained increase in [Ca2+]i with BAL than blood EOS. EGTA blocked the sustained component of the [Ca2+]i response to FMLP. Our findings indicate that BAL EOS have an enhanced [Ca2+]i response to activation that may contribute to their functional up-regulation.
Intranasal immunization with liposomes containing IL-2 enhances bacterial polysaccharide antigen-specific pulmonary secretory antibody response.
Authors: Abraham E , Shah S
Published: December 2022

Secretory IgA (sIgA) present at mucosal surfaces such as the lungs and intestine plays an important role in resistance to infection occurring at these anatomic sites. Because IL-2 and IL-4 can augment B cell proliferation and Ig production, we investigated possible adjuvant effects of these cytokines on bacterial polysaccharide-specific pulmonary sIgA generation. As shown in previous studies, intranasal immunization with liposomes containing bacterial polysaccharide from Aerobacter levanicum and Pseudomonas aeruginosa resulted in increased numbers of bacterial polysaccharide-specific pulmonary plasma cells and sIgA titers, compared with those found in unimmunized mice. Inclusion of IL-2, but not IL-4, into the intranasally administered liposomes further increased titers of bacterial polysaccharide specific sIgA and pulmonary plasma cells. Intranasal vaccination with liposomes containing bacterial polysaccharide and 10 micrograms/kg IL-2 increased bacterial polysaccharide-specific pulmonary plasma cell numbers by more than 80-fold compared with the response in mice immunized with liposomes containing bacterial polysaccharide, but without IL-2. The percentage of pulmonary plasma cells producing antibody to polysaccharide from A. levanicum rose from 0.14% in mice intranasally immunized with liposomes containing only polysaccharide to 4.1% in animals vaccinated with liposomes containing polysaccharide and IL-2. Intranasal immunization with liposomes containing P. aeruginosa polysaccharide and IL-2 significantly reduced mortality from P. aeruginosa pneumonia. These results demonstrate that IL-2 has potent adjuvant effects on bacterial Ag-specific sIgA production in the lungs when included in intranasally administered liposomes.
Role of antigen-specific T cell help in the generation of in vivo antibody responses. I. Antigen-specific T cell help is required to generate a polyclonal IgG1 response in anti-IgD antibody-injected mice.
Authors: Morris S C , Lees A , Inman J , Finkelman F D
Published: December 2022

A system in which injection of mice with an antibody to mouse IgD that they recognize as foreign stimulates a large, T cell-dependent IgG response was used to study whether Ag-specific T cell help is required to stimulate polyclonal (non-Ag-specific) IgG production in vivo. Igha x Ighb allotype heterozygous mice were injected with a conjugate of a foreign Ag coupled to a mAb specific for one of the two IgD allotypes expressed in these mice. This conjugate cross-links mIgD on B cells that express the recognized allotype. These cells process the conjugate and present the foreign Ag to Ag-specific T lymphocytes, which become activated. Thus, B cells of the recognized allotype can be stimulated by cross-linking of their mIgD, Ag-specific T cell help, non-Ag-specific cytokines, and non-Ag-specific contact with activated T cells. In contrast, B cells that express the Igh allotype not recognized by the Ag-anti-IgD antibody conjugate (bystander B cells) can be stimulated in this system only by non-Ag-specific cytokines and non-Ag-specific contact with activated T cells. Although both recognized and bystander B cells in conjugate-injected mice demonstrated substantial increases in size and Ia expression, only the recognized B cells were induced to synthesize DNA and to make a substantial polyclonal Ig response. Bystander B cells still failed to secrete IgG when mice were injected with an anti-IgD-Ag conjugate specific for the other Igh allotype as well as a mAb that cross-linked IgD of the bystander B cell allotype. These observations demonstrate that although non-Ag-specific cytokine and contact-mediated T cell help are sufficient to induce B cells to increase in size and Ia expression in anti-IgD antibody-injected mice, Ag-specific T cell help is required to stimulate the generation of an IgG response in these mice.
Systemic suppression of delayed-type hypersensitivity by supernatants from UV-irradiated keratinocytes. An essential role for keratinocyte-derived IL-10.
Authors: Rivas J M , Ullrich S E
Published: December 2022

Exposing murine keratinocyte cultures to UV radiation causes the release of a suppressive cytokine that mimics the immunosuppressive effects of total-body UV exposure. Injecting supernatants from UV-irradiated keratinocyte cultures into mice inhibits their ability to generate a delayed-type hypersensitivity reaction against allogeneic histocompatibility Ag, and spleen cells from mice injected with supernatant do not respond to alloantigen in the in vitro MLR. A unique feature of the immunosuppression induced by either total-body UV-exposure or injecting the suppressive cytokine from UV-irradiated keratinocytes is the selectivity of suppression. Although cellular immune reactions such as delayed-type hypersensitivity are suppressed antibody production is unaffected. Because the selective nature to the UV-induced immunosuppression is similar to the biologic activity of IL-10, we examined the hypothesis that UV exposure of keratinocytes causes the release of IL-10. Keratinocyte monolayers were exposed to UV radiation and at specific times after exposure mRNA was isolated or the culture supernatant from the cells was collected. IL-10 mRNA expression was enhanced in UV-irradiated keratinocytes. The secretion of IL-10 by the irradiated keratinocytes was determined by Western blot analysis. A band reactive with anti-IL-10 mAb was found in supernatants from the UV-irradiated but not the mock-irradiated cells. IL-10 biologic activity was determined by the ability of the supernatants from the UV-irradiated keratinocytes to suppress IFN-gamma production by Ag-activated Th 1 cell clones. Anti-IL-10 mAb neutralized the ability of supernatants from UV-irradiated keratinocytes to suppress the induction of delayed-type hypersensitivity in vivo. Furthermore, injecting UV-irradiated mice with antibodies against IL-10 partially inhibited in vivo immunosuppression. These data indicate that activated keratinocytes are capable of secreting IL-10 and suggest that the release of IL-10 by UV-irradiated keratinocytes plays an essential role in the induction of systemic immunosuppression after total-body UV exposure.
Increased avidity of mutant IgM antibodies caused by the absence of COOH-terminal glycosylation of the mu H chain.
Authors: Bazin R , Darveau A , Martel F , Pelletier A , Piché L , St-Laurent M , Thibault L , Demers A , Boyer L , Lemieux G
Published: December 2022

We have previously described the isolation of two hybridoma variants secreting higher avidity IgM (D5 and 7F5), starting from the E11 hybridoma cell line, which produces an antibody specific for the A Ag of the ABO blood group system. In order to explain at the molecular level this increased reactivity, cDNA encoding the H and L chains of the E11, D5, and 7F5 mAb were cloned and sequenced. Comparison of the nucleotide sequences showed a single point mutation in each of the two mAb produced by the hybridoma variants. The mutations were both located in the H chain C region and caused a Ser to Phe substitution at position 565 in the D5 mAb and a Asn to Tyr substitution at position 563 in the 7F5 mAb. Both substitutions modified the consensus glycosylation sequence (Asn-X-Ser/Thr) located in the tail piece of the secretory mu-chain. The absence of glycosylation at this site was confirmed by CNBr cleavage of the [14C]mannose-labeled mAb. The two single point mutations were solely responsible for the increased avidity of the antibodies, as confirmed by site-directed mutagenesis of the E11 mu-chain and serologic analysis of the mutated E11 antibodies. We conclude that the absence of glycosylation at Asn 563 is responsible for the increased avidity of the mutant, possibly by altering the quaternary structure of the IgM polymer. To our knowledge, this is the first report that point mutations in the H chain C region can influence the reactivity of IgM mAb.
L chain isotype regulation in horse. I. Characterization of Ig lambda genes.
Authors: Home W A , Ford J E , Gibson D M
Published: December 2022

Analysis of 10 cDNA encoding lambda L chains of horse Ig indicated that this species may employ a relatively small number of variable region (V lambda) genes in the splenic B cell population. The V lambda sequences of all of the cDNA analyzed were closely related (> 88% identity at the nucleotide level) and were characterized by a deletion of the amino acid residue at position 3 compared with V lambda sequences so far described in other species. The 10 V lambda sequences could be grouped into three groups, V lambda 1 to V lambda 3, on the basis of a number of linked substitutions. Sequences within the groups showed the greatest divergence in the third cdr regions and at the V-J junctions. The junctional variation included amino acid substitutions on both sides of the V-J junction as well as the insertion or deletion of two to four amino acid residues. Four C lambda genes were identified in genomic blots of horse DNA, and three of these were found expressed in splenic cDNA. The fourth C lambda gene may represent a pseudogene, inasmuch as the associated J region possessed a defective heptamer joining sequence. Six of the nine possible V lambda-C lambda combinations were found in the cDNA analyzed, suggesting that genes belonging to groups V lambda 1 through V lambda 3 may rearrange to any one of three J lambda-C lambda genes. One V lambda germline gene was characterized and found to represent a distinct V lambda group (V lambda 4), not represented in the cDNA sequences analyzed. The number of germline V lambda genes was estimated to be 20 to 30, based on analysis of restriction fragments hybridizing with V lambda probes. On the basis of these data, we propose that the V lambda repertoire in horse may consist of relatively limited number of genes, of which only a few may be used at high frequency in the splenic B cell population. The results indicate that predominance of lambda-chains in horse Ig may not simply be due to the presence of a large germline V lambda gene repertoire.
V delta 1+ subset of human gamma delta T cells responds to ligands expressed by EBV-infected Burkitt lymphoma cells and transformed B lymphocytes.
Authors: Häcker G , Kromer S , Falk M , Heeg K , Wagner H , Pfeffer K
Published: December 2022

Human gamma delta T cells of peripheral blood can be divided in two groups in terms of their TCR as well as their behavior upon in vitro stimulation. The major subset expresses the TCR V-segments V gamma 9 and V delta 2 and proliferates in response to ligands revealed by various microorganisms, and the cell line Daudi in addition. The minor group is less homogenous on the gamma-chain but is almost completely identified by mAb against the V delta 1 segment; there is no ligand known to promote growth of these cells. Here we demonstrate that gamma delta T cells out of this subgroup are strongly stimulated in vitro by cells from several Burkitt's lymphoma cell lines. EBV infection of the Burkitt's lymphoma cell lines enhanced the stimulatory ability towards the T cells. Although EBV infection influenced the expression of a variety of cell surface molecules including ICAM-1 and LFA-3, no correlation to the gamma delta T cell-stimulating capacity became apparent. We conclude that Burkitt's lymphoma cells and transformed B cells express ligands of cellular origin for a hitherto poorly characterized subgroup of human gamma delta T cells.
Highly diverse T cell recognition of a single Plasmodium berghei peptide presented by a series of mutant H-2Kd molecules.
Authors: Jaulin C , Casanova J L , Romero P , Luescher I , Cordey A S , Maryanski J L , Kourilsky P
Published: December 2022

We have tested 21 independent CTL clones for recognition of a single peptide derived from the Plasmodium berghei circumsporozoite protein in the context of 13 mutants of the murine MHC class I molecule H-2Kd. In this series of Kd mutants, amino acid residues located on the upper surface of the alpha-helices were individually substituted by alanine. Remarkably, most clones displayed individual recognition patterns on the Kd mutants. We had previously found that this series of CTL clones was likewise highly diverse in terms of both TCR primary structure and peptide fine specificity. Our data thus reinforce the concept that multiple T cell epitopes are available on the surface of a single peptide-MHC class I complex for recognition by specific TCR.
Transfection of mouse cytotoxic T lymphocyte with an antisense granzyme A vector reduces lytic activity.
Authors: Talento A , Nguyen M , Law S , Wu J K , Poe M , Blake J T , Patel M , Wu T J , Manyak C L , Silberklang M
Published: December 2022

Murine CTL have seven serine proteases, known as granzymes, in their lytic granules. Despite considerable effort, convincing evidence that these enzymes play an obligatory role in the lytic process has not been presented. To investigate the function of one of these proteases, granzyme A (GA), we utilized an antisense expression vector to lower the level of the enzyme in the cells. An expression vector containing antisense cDNA for GA and the gene for hygromycin B resistance was constructed and electroporated into the murine CTL line, AR1. Transfectants were selected based on resistance to hygromycin B, and a number of stable lines were developed. One of the antisense lines had greatly reduced levels of GA mRNA, when compared to the parental cells or to control lines transfected with the vector lacking the antisense DNA. The message levels for two other CTL granule proteins, granzyme B and perforin, were unaffected by the antisense vector. The amount of GA, as measured by enzymatic activity, was 3- to 10-fold lower in the transfectant. Most significantly, this line also consistently showed 50 to 70% lower ability to lyse nucleated target cells and to degrade their DNA. Furthermore, it exhibited 90 to 95% lower lytic activity to anti-CD3-coated SRBC. Conjugate formation with target cells, however, was normal. These data provide strong evidence that GA plays an important role in the cytolytic cycle, and that the quantity of enzyme is a limiting factor in these cytolytic cells.
Infection of human natural killer (NK) cells with replication-defective human T cell leukemia virus type I provirus. Increased proliferative capacity and prolonged survival of functionally competent NK cells.
Authors: Lo K M , Vivier E , Rochet N , Dehni G , Levine H , Haseltine W A , Anderson P
Published: December 2022

Human T-cell leukemia virus type I (HTLV-I) can infect a variety of human cell types, but only T lymphocytes are efficiently immortalized after HTLV-I infection. This study reports an attempt to infect and to immortalize NK cells with HTLV-I. Co-cultivation of freshly isolated NK cells with a HTLV-I-producing T cell line did not result in NK cell infection. However, NK cells activated with an anti-CD16 mAb and co-cultivated with a HTLV-I-producing T cell line were reproducibly infected by HTLV-I. HTLV-I infection was documented in NK cell lines and clones by the detection of defective integrated provirus by both Southern blot and polymerase chain reaction analysis. Although HTLV-I-infected NK cells produced viral proteins, they did not produce infectious viral particles. HTLV-I-infected NK cells were phenotypically indistinguishable from their uninfected counterparts (CD16+, CD2+, CD56+, CD3-). They also retained the ability to mediate both natural and antibody-dependent cell cytotoxicity. The IL-2-dependent proliferation of HTLV-I-infected NK cells was significantly greater than that of uninfected NK cells. The doubling time of this infected population was reduced from 9 days to 3 days, and the overall survival of the culture in the absence of restimulation was extended from 5 wk to 18 wk. Unlike T lymphocytes, HTLV-I-infected NK cells were not immortal, implying a fundamental difference between these two lymphocyte populations.
Transcription and recombination of the murine RS element.
Authors: Daitch L E , Moore M W , Persiani D M , Durdik J M , Selsing E
Published: December 2022

The deletion of C kappa is a frequent event in lambda-producing B cells in both mice and humans. Deletions of the murine C kappa gene are mediated by recombination events that involve the RS (recombining segment) element located downstream of the C kappa gene. RS recombinations appear to be mediated by the same mechanisms involved in Ig and TCR gene rearrangement. It has been suggested that RS recombinations might activate a factor that is involved in the initiation of lambda gene rearrangement in maturing pre-B cells. We have identified a unique RNA transcript derived from the recombined RS element present in some pre-B cell lines. However, gene transfer studies indicate that this RS transcript is not sufficient to induce lambda gene recombination in pre-B cell lines. We also find that recombination of the RS element in pre-B cell lines is closely correlated with changes in chromatin structure and transcriptional activation. Thus, recombination of the RS element in pre-B cells appears to be regulated in a manner similar to the regulation of antibody gene VDJ joining.
Human leukocyte activation antigen M6, a member of the Ig superfamily, is the species homologue of rat OX-47, mouse basigin, and chicken HT7 molecule.
Authors: Kasinrerk W , Fiebiger E , Stefanová I , Baumruker T , Knapp W , Stockinger H
Published: December 2022

Peripheral granulocytes from rheumatoid arthritis and reactive arthritis patients were recently found to express higher levels of a newly defined Ag, termed M6, in comparison to granulocytes from healthy subjects. We present here the molecular characterization of M6 Ag and show that it is a novel human leukocyte activation-associated cell surface glycoprotein. Peripheral lymphocytes do not significantly express M6 Ag, however, it appears upon 3-day PHA-activated T blasts. On monocytes, which constitutively express M6 Ag, it is down-regulated on day 1 but re-induced on day 3 of granulocyte-macrophage CSF stimulation. SDS-PAGE analysis of M6 immunoprecipitates shows a single band of 54 kDa under nonreducing conditions that shifts to 65 kDa under reducing conditions. Endoglycosidase F treatment of M6 immunoprecipitate reveals that 50% of the M6 molecule is composed of N-linked carbohydrates. By modifying the COS cell cloning strategy, we have isolated cDNA clones encoding M6 Ag. M6 cDNA hybridizes with a single mRNA transcript of approximately 1.7 kb in Northern blotting. Comparison analysis of the M6 sequence indicates that M6 Ag is a member of the Ig superfamily and the species homologue of rat OX-47 Ag, mouse basigin (gp42), and chicken HT7 molecule. The highly conserved remarkable transmembrane domain suggests that the M6 Ag may be a component of a multichain complex in the plasma membrane.
Granulocyte colony-stimulating factor treatment protects rodents against lipopolysaccharide-induced toxicity via suppression of systemic tumor necrosis factor-alpha.
Authors: Görgen I , Hartung T , Leist M , Niehörster M , Tiegs G , Uhlig S , Weitzel F , Wendel A
Published: December 2022

Pretreatment with recombinant human granulocyte CSF (G-CSF) protected mice in two different models of septic shock. Intravenous injection of 250 micrograms/kg G-CSF to mice prevented lethality induced by 5 mg/kg LPS. Injection of 50 micrograms/kg G-CSF protected galactosamine-sensitized mice against LPS-induced hepatitis. In either case, this protection was accompanied by a suppression of LPS-induced serum TNF activity. In contrast, when galactosamine-sensitized mice were pretreated with 50 micrograms/kg murine recombinant granulocyte/macrophage CSF instead of G-CSF and subsequently challenged with LPS, serum TNF activity was significantly enhanced and mortality was increased. The suppressive effect of G-CSF on LPS-induced TNF production was also demonstrated in rats. In vivo, no TNF was detectable in the blood of LPS-treated rats, which had been pretreated with G-CSF. Ex vivo, alveolar macrophages, bone marrow macrophages, Kupffer cells, or peritoneal macrophages prepared from G-CSF-treated rats produced significantly less TNF upon stimulation with LPS than corresponding populations from control rats. However, when these macrophage populations were incubated with G-CSF in vitro, LPS-induced TNF production was unaffected. These data suggest that the G-CSF-mediated suppression of TNF production is not a direct effect of G-CSF on macrophages. To examine whether, independent of the protection against LPS, G-CSF treatment still activated neutrophils, it was demonstrated that granulocytes from G-CSF-treated rats were primed for PMA-induced oxidative burst and for ionophore/arachidonic acid-stimulated lipoxygenase product formation. The experiments of this study support the notion that G-CSF is a negative feedback signal for macrophage-derived TNF-alpha production during Gram-negative sepsis.
A kinetic study of immune mediators in the lungs of mice infected with influenza A virus.
Authors: Hennet T , Ziltener H J , Frei K , Peterhans E
Published: December 2022

We investigated a broad spectrum of immunoactive mediators in a mouse model of influenza. ICR mice (4-5 wk old) that were infected with a 10 LD50 dose of influenza A/PR8/34 virus died after 6 days without evidence of bacterial superinfection. Maximal virus titers were reached by day 2 postinfection, whereas the multifocal pneumonia with mononuclear cell infiltration reached its maximum at the end of infection. We measured the cytokines IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-6, IFN-gamma, TNF-alpha, granulocyte (G)/macrophage (M)-CSF, G-CSF, M-CSF, and the lipid mediators leukotriene B4 and platelet-activating factor in the cellfree bronchoalveolar lavage fluid of mice during infection. We found an early increase of IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, GM-CSF, IFN-gamma, and leukotriene B4. Levels of these factors peaked between 36 h and day 3 postinfection, with the exception of IL-6 that remained at elevated levels throughout infection. G-CSF and M-CSF increased slowly and reached a maximum by day 5 postinfection. We were unable to detect IL-2, IL-3, or IL-4. PAF remained at the same level throughout infection. Our results suggest that lung-resident cells, and possibly the alveolar macrophages, participate actively in the onset of the inflammatory response against the invading virus. The inability to detect the T cell products IL-2, IL-3, and IL-4 was unexpected considering the role of T cells in the elimination of the virus in infected mice. Our observation confirms thus earlier findings about the inability of specific T cell clones to elicit an unspecific antiviral effect.
Identification of new epitopes recognized by human monoclonal antibodies with neutralizing and antibody-dependent cellular cytotoxicity activities specific for human T cell leukemia virus type 1.
Authors: Kuroki M , Nakamura M , Itoyama Y , Tanaka Y , Shiraki H , Baba E , Esaki T , Tatsumoto T , Nagafuchi S , Nakano S
Published: December 2022

We have generated a number of EBV-transformed B cell lines producing human mAb against human T cell leukemia virus type 1 (HTLV-1) from the peripheral blood B lymphocytes obtained from patients with HTLV-1-associated myelopathy/tropical spastic paraparesis. Various synthetic peptides corresponding to antigenic regions of HTLV-1 gag and env proteins were used for the screening of antibodies in ELISA. In our study, four IgG mAb to the gag p19 amino acids 100 to 130, and 5 IgG mAb to the env p46 amino acids 175 to 199 were characterized. An immunofluorescence assay showed that all of these mAb specifically bound to the surface of HTLV-1-bearing cell lines. Among these mAb, one anti-gp46 mAb, designated KE36-11, neutralized the infectivity of HTLV-1 as determined by both the inhibition of HTLV-1-induced syncytium formation and transformation assays in vitro. An antibody-binding assay using overlapping oligopeptides revealed that KE36-11 recognized a new epitope locating between the gp46 amino acid sequence 187-193 (Ala-Pro-Pro-Leu-Leu-Pro-His). Another anti-gp46 mAb, designated KE36-7, showed antibody-dependent cellular cytotoxicity against HTLV-1-bearing cell line. KE36-7 bound strongly to the 10-mer peptide-gp46 187-196, and weakly to peptides containing the gp46 amino acid sequence 191-196 (Leu-Pro-His-Ser-Asn-Leu). These two epitopes, which are associated with HTLV-1 neutralization and antibody-dependent cellular cytotoxicity, are thus the first epitopes identified in human HTLV-1 infection. It is possible that passive immunization of humans with these two human mAb are effective on the protection of HTLV-1 infection in vivo.
Cognate interaction between T helper cells and B cells. VII. Role of contact and lymphokines in the expression of germ-line and mature gamma 1 transcripts.
Authors: Noelle R J , Shepherd D M , Fell H P
Published: December 2022

Complete reconstitution of Th cell function for B cell growth and differentiation was provided by plasma membranes (PM) derived from activated Th (PMAct) in combination with IL-4 and IL-5 (IL-4/IL-5). IL-5 has been shown to be essential for the secretion of all Ig isotypes by resting, conventional B cells activated by PMAct and IL-4. It was shown that in the presence of PMAct/IL-4/IL-5, a high frequency of resting B cells differentiated to express IgG1. IL-4 alone transiently induced the expression of germ-line gamma 1 transcripts. PMAct alone were ineffective at inducing germ-line gamma 1 transcript expression by resting B cells suggesting that Th-B cell contact was an insufficient signal to cause a detectable increase in the steady-state levels of gamma 1 germ-line transcripts. PMAct alone or PMAct/IL-4 did not induce the appearance of transcripts for secreted mu or mature gamma 1. IL-5, in combination with PMAct/IL-4, provided the necessary signal(s) required for the expression of secreted mu and mature gamma 1 transcripts. Therefore, IL-5 appeared to be an important if not essential differentiation factor for conventional B cells that have been activated by cognate help. It appeared that IL-5 promoted the secretion of Ig by inducing the synthesis of mature Ig transcripts.
CD11b (Mac-1): a marker for CD8+ cytotoxic T cell activation and memory in virus infection.
Authors: McFarland H I , Nahill S R , Maciaszek J W , Welsh R M
Published: December 2022

We have found that CD11b, a cell surface integrin of macrophages, granulocytes, and NK cells, is expressed by a subset of CD8+ T cells that include both the active virus-specific CTL and the virus-specific memory CTL populations. CD8+CD11b+ cells comprise less than 3% of naive mouse splenocytes, but after lymphocytic choriomeningitis virus (LCMV) infection increase by 9- to 12-fold by the peak (day 8) of the virus-specific CTL response. Depletion of day-8 splenocytes with anti-Mac-1 and C' or enrichment by sorting for CD11b+ or CD8+CD11b+ spleen cells demonstrated that LCMV-specific CTL are CD11b+. The CD11b+ subpopulation also contained the bulk of the IL-2-responsive CD8+ cells. MEL-14, a homing marker down-regulated on activated T cells, was down-regulated on the majority of CD8+ cells that became CD11b+. Less than 1% of LCMV-immune splenic lymphocytes expressed CD11b. Antibody and C' depletion of this population severely impaired the ability of immune splenocytes to respond to in vitro secondary stimulation with LCMV-infected peritoneal macrophages, but did not affect the generation of a primary allospecific CTL response in MLC. Mixing of CD8-depleted and CD11b-depleted LCMV-immune splenocytes failed to restore the ability of these cells to mount a virus-specific memory CTL response, indicating that a cell coexpressing CD8 and CD11b is essential for this response. As determined by limiting dilution analysis, the precursors for the LCMV-specific memory CTL response were enriched in the CD11b+ population of LCMV-immune splenocytes. CD11b stained far fewer CD8+ splenocytes from naive mice than did CD44 (Pgp-1), and among immune splenocytes it identified a small subpopulation of CD44hi cells, indicating that CD11b may be the best single marker available for discriminating between naive and memory CD8+ T cells.
Inhibition of phospholipase A2 activity in guinea pig eosinophils by human recombinant IL-1 beta.
Authors: Debbaghi A , Hidi R , Vargaftig B B , Touqui L
Published: December 2022

The effect of human rIL-1 beta on the release of arachidonic acid (AA) and on the phospholipase A2 (PLA2) activity in guinea pig eosinophils was investigated. Stimulation of [3H]AA-labeled eosinophils with the ionophore A23187 resulted in a time and concentration-dependent release of AA in parallel to hydrolysis of endogenous phosphatidylcholine (PC). Both events were abrogated by the chelation of intracellular free calcium, but not by its depletion from the medium, suggesting that the ionophore-induced AA release involves a PLA2 activity dependent on the mobilization of intracellular calcium. Addition of human rIL-1 beta (0.01 to 100 ng/ml) to eosinophils for 15 min had no effect on the release of AA induced by the ionophore. However, prolonged incubation with human rIL-1 beta (30 to 180 min) inhibited in a concentration- and time-dependent manner the release of AA and the hydrolysis of phosphatidylcholine in ionophore-stimulated eosinophils. Our results also showed that eosinophil homogenates contain a calcium-dependent PLA2 whose activity was markedly reduced when eosinophils were pretreated with human rIL-1 beta. The inhibition was time and concentration dependent and was observed in the presence of calcium and phospholipid excess. Finally, studies with Fura-2-loaded eosinophils showed that the ionophore A23187 stimulated an increase in intracellular calcium concentration that was not altered by pretreating the eosinophils with human rIL-1 beta. These results suggest that human rIL-1 beta inhibits the release of AA by eosinophils via the inhibition of a PLA2 activity and through a calcium-independent mechanism. Inhibition by human rIL-1 beta required a prolonged incubation (30 to 180 min) and was observed after its removal from the medium, suggesting that human rIL-1 beta did not interact directly with the PLA2 itself, but with a metabolic process involved with the regulation of its activity in eosinophils.
Ontogeny and distribution of the murine B cell Fc gamma RII.
Authors: Foy T M , Lynch R G , Waldschmidt T J
Published: December 2022

The distribution and expression of the IgG FcRII (Fc gamma RII) on normal murine B cells was examined. Using multicolor flow cytometry, spleens from neonatal mice of increasing age and adult bone marrow were analyzed for expression of the Fc gamma RII. In addition, B cells from peripheral lymphoid organs, as well as panel of B cell tumors, were tested. The results demonstrate that the Fc gamma RII is expressed on all pre-B cells and immature B cells in the neonatal spleen and adult bone marrow, on all mature B cells in peripheral lymphoid organs, and on switched B cells in Peyer's patches. Furthermore, the Fc gamma RII was found to be present on B cell tumors representative of all stages of B cell maturation and differentiation. Taken together, the results indicate that Fc gamma RII is expressed during the entire lifetime of the B cell. In addition, examination of spleen cells from neonatal mice revealed a large number of pre-B cells, phenotypically defined as B220+, IgM-. These pre-B cells were present at birth, peaked in number between 2 and 3 wk of age, and became a minor population by day 30. Further phenotypic analysis of these cells demonstrated the expression of the BLA-1 and BP-1 Ag, and the lack of T cell and NK cell markers, thus confirming their assignment to the B cell lineage. Finally, the Fc gamma RII present on these pre-B cells was shown to be functional, by virtue of its ability to bind aggregated IgG.
The CD28 ligand, B7, enhances IL-2 production by providing a costimulatory signal to T cells.
Authors: Norton S D , Zuckerman L , Urdahl K B , Shefner R , Miller J , Jenkins M K
Published: December 2022

Previous studies demonstrated that a human pre-B acute lymphoblastic leukemia cell line, NALM-6, failed to stimulate a primary MLR, despite expression of class II MHC and adhesion molecules. Here we demonstrate that this is the result of the fact that NALM-6 cells do not express the ligand for CD28, namely B7. NALM-6 transfectants that expressed high levels of B7 gained the capacity to stimulate IL-2 production by class II MHC molecule-specific alloreactive T cells and to costimulate a polyclonal population of purified T cells cultured with immobilized anti-CD3 mAb. In the presence of PMA, NALM-6 cells transfected with B7 polyclonally stimulated T cells in a cyclosporine A-resistant fashion, a property previously attributed only to agonistic anti-CD28 mAb. The gain of these functions could not be explained solely by an increased capacity of the transfectants to form conjugates with T cells, suggesting that the CD28/B7 interaction transduces a costimulatory signal in T cells.