bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-2 enhances c-fms expression in human monocytes.
Authors: Espinoza-Delgado I , Longo D L , Gusella G L , Varesio L
Published: December 2022

We investigated the effects of IL-2 and IFN-gamma on the expression of the proto-oncogene c-fms mRNA, which encodes for the macrophage CSF receptor. Low constitutive expression of c-fms mRNA was detected in fresh human monocytes. Stimulation of monocytes with IL-2 induced a significant increase in c-fms mRNA relative to medium control that was observed as early as 6 h after IL-2 stimulation. Dose-response experiments showed that 100 U/ml of IL-2 were sufficient to enhance the expression of c-fms mRNA. In contrast, IFN-gamma did not modify the levels of c-fms transcript. Immunoprecipitation experiments demonstrated that IL-2 enhanced c-fms glycoprotein levels. Experiments in which monocytes were activated with IFN-gamma or IL-2 followed by macrophage CSF-1 and then tested for tumoricidal activity demonstrated that macrophage CSF-1 sustained the cytotoxic activity induced by IL-2 but not by IFN-gamma. These data demonstrate that IL-2 enhances c-fms mRNA and c-fms glycoprotein expression suggesting that IL-2, by augmenting expression of macrophage CSF-1 receptors, can lead to prolongation of monocyte-mediated tumoricidal activity obtained in the presence of exogenous macrophage CSF-1.
IL-4 inhibits the expression of high affinity IL-2 receptors on monoclonal human B cells.
Authors: Karray S , Dautry-Varsat A , Tsudo M , Merle-Beral H , Debre P , Galanaud P
Published: December 2022

In the presence of anti-mu antibodies (anti-microAb), monoclonal B lymphocytes from patients suffering from B type chronic lymphocytic leukemia (B-CLL) can respond to IL-2. In contrast to the effect it exerts on normal B cells, IL-4 does not promote DNA synthesis by B-CLL lymphocytes. Rather this interleukin inhibits the response to IL-2 in all patients' cells that responded to this interleukin. We thus examined whether IL-4 would modulate the number and/or the affinity of IL-2 receptors. A 3-day activation of cells by anti-microAb induced a few hundred high affinity IL-2 receptors (HA-IL-2R) on B-CLL cell surface, as determined by Scatchard analysis. Treatment of cells with IL-4 caused a marked decrease in the number of HA-IL-2R without interfering with the binding ability of IL-2. In contrast with this profound suppressive effect, IL-4 did not down-regulate the expression of each chain, alpha and beta (p55 and p75, respectively), of the HA-IL-2R heterodimer. In fact, the expression of alpha and beta induced by anti-microAb was enhanced by IL-4. Altogether, IL-4 exerts a critical influence on the function and the configuration of HA-IL-2R without inhibiting the expression of two subunits, alpha and beta.
IL-2 and IL-6 synergize to augment the pore-forming protein gene expression and cytotoxic potential of human peripheral blood T cells.
Authors: Smyth M J , Ortaldo J R , Bere W , Yagita H , Okumura K , Young H A
Published: December 2022

Our previous studies have demonstrated that high dose IL-2 (1000 U/ml) alone can induce human peripheral blood T cell pore-forming protein (PFP) mRNA expression and cytotoxic potential. We now report that the levels of IL-2 needed to induce these effects in T cells can be significantly reduced in the presence of IL-6. IL-6 and suboptimal doses of IL-2 (10 U/ml) were found to costimulate PFP mRNA expression and cytotoxic potential in resting human peripheral blood T cells, whereas IL-6 or low dose IL-2 alone had no effect. The induction of T cell PFP mRNA by IL-2/IL-6 was extremely rapid and increases in both PFP mRNA expression and cytotoxic potential were IL-6 dose dependent. The costimulatory effect of IL-6 did not appear to involve the IL-2/IL-2R pathway in as much as IL-6 did not induce IL-2 production or detectably increase IL-2R surface expression in T cells. These findings, in addition to the rapid induction of PFP mRNA by IL-2/IL-6, suggested that IL-6 can directly and independently provide an additional signal to augment the differentiation of CTL. In contrast to the results observed in T cells, IL-6 and IL-2 could enhance CD3- large granular lymphocyte (LGL) NK activity, but IL-6 either alone or in combination with IL-2 had no effect on constitutive PFP mRNA expression in resting LGL. These data further confirm that different mechanisms may be responsible for lymphokine activation of CTL and LGL in human peripheral blood. In particular it appears that IL-6 acts as a costimulatory signal with IL-2 in generating CTL and that IL-6 functions in part by acting in synergy with IL-2 to induce PFP, a major lytic protein involved in lymphocyte cytotoxicity.
Expression and characterization of a chimeric bifunctional antibody with therapeutic applications.
Authors: Phelps J L , Beidler D E , Jue R A , Unger B W , Johnson M J
Published: December 2022

A simple method is described for the generation of a biologically produced mouse/human chimeric hetero-bifunctional antibody that has dual specificity for human carcinoembryonic Ag and metal chelate haptens. Two large compound chimeric vectors each containing the genetic information to produce a single antibody specificity were sequentially electroporated into the murine nonsecreting hybridoma SP2/0. This led to the isolation of a clone expressing high levels of total IgG (up to 25 micrograms/ml/10(6) cells), 10 to 20% of which showed simultaneous reactivity with both Ag. Binding studies showed that the immunoreactivities and affinity constants for the individual arms of the bifunctional antibody were equivalent to those seen with the parental antibodies.
A specific defect of prosolin phosphorylation in T cell leukemic lymphoblasts is associated with impaired down-regulation of DNA synthesis.
Authors: Cooper H L , Fuldner R , McDuffie E , Braverman R
Published: December 2022

Prosolin is a major cytosolic phosphoprotein of proliferating normal PBL. Treatment of growing PBL with phorbol ester (12-O-tetradecanoylphorbol-13-acetate (TPA)) or calcium ionophore (A23187) for 1 h caused phosphorylation of prosolin with the production of up to four prominent phosphorylated forms differing in degree of phosphorylation and/or two-dimensional electrophoretic mobility (peptides B to E). Formation of these phosphopeptides coincided with rapid down-regulation of DNA synthesis. A23187 was particularly effective in inducing phosphorylation of the more highly phosphorylated peptides D and E, suggesting the existence of a (Ca2+)-activated mechanism in their phosphorylation. The T cell leukemia cell lines Jurkat, HuT-78, CCRF-CEM, and Molt-4 showed reduced to absent ability to phosphorylate prosolin peptides rapidly in response to A23187 and also showed diminished down-regulation of DNA synthesis. In leukemic cells treated with both TPA and A23187, peptides B and C were rapidly phosphorylated, but the phosphorylation of peptides D and E seen in normal PBL remained deficient. The T cell leukemic cells appear to have intact a TPA-activated mechanism for phosphorylating prosolin peptides B and C, but share an impairment of a specific Ca2(+)-activated mechanism, possibly a Ca2(+)-dependent protein kinase, required for phosphorylation of prosolin phosphopeptides D and E. The degree of rapid down-regulation of DNA synthesis was correlated with degree of phosphorylation of peptide E in PBL and in three of four T cell leukemic cell lines. Thus, rapid phosphorylation of prosolin may mediate responses to TPA and A23187 in normal proliferating PBL, including down-regulation of DNA synthesis. A deficiency of this pathway in leukemic T cells may impede their response to physiologic growth regulatory signals utilizing this pathway and contribute to unrestrained cell growth.
Role of CD4+ T cells and T-independent mechanisms in acquired resistance to Salmonella typhimurium infection.
Authors: Nauciel C
Published: December 2022

Two aspects of acquired resistance to Salmonella typhimurium infection in BALB/c mice, i.e., the ability to clear the primary inoculum from the spleen and resistance to a secondary challenge, were studied with the use of mAb against T cell subsets. The ability to clear a temperature-sensitive mutant of S. typhimurium from the spleen (assessed at day 21) was abrogated by in vivo treatment with anti-CD4 mAb. Accelerated bacterial clearance could be adoptively transferred into naive mice. In vitro depletion experiments also showed the role of CD4+ T cells in this phenomenon. Depletion of CD8+ T cells had only a marginal effect. Resistance to reinfection in the late phase of the primary infection (day 50) was markedly depressed by in vivo treatment with anti-CD4 mAb, whereas this was not the case during the early phase (day 14). Furthermore, during the early phase of infection athymic nude mice showed increased nonspecific resistance to reinfection. Taken together these results suggest that T-independent mechanisms play a major role in acquired resistance during the early phase of infection.
A T cell receptor V alpha region selectively expressed in CD4+ cells.
Authors: Jameson S C , Kaye J , Gascoigne N R
Published: December 2022

The peripheral TCR V beta repertoire is strongly influenced by the processes of negative selection (deletion) and positive selection in the thymus. In order to investigate whether such selection events influence the V alpha repertoire, we have produced an anti-V alpha 11 mAb. This antibody was made by immunization with a chimeric TCR:Ig protein containing V alpha 11 in place of the VH of an IgG2a, lambda Ig. This scheme optimizes the specificity of immunization and facilitates the screening procedure. The antibody recognizes a panel of V alpha 11-expressing T cell clones. Analysis of mouse strains indicates that the antibody recognizes V alpha 11 only in mice of the C57 background. The expression of the epitope on peripheral T cells is strongly biased to the CD4+ subset, suggesting positive selection of V alpha 11 on class II MHC molecules. In some strain comparisons, the percentage of V alpha 11-expressing T cells in the CD4+ subset was elevated in I-E+ relative to I-E- strains. These data suggest that V alpha 11 can differentially influence the selection of T cells into the CD4+/CD8+ subsets.
Angiotensin II (AII)-related idiotypic network. III. Comparative analysis of idiotopes and paratopes borne by monoclonal antibodies raised against AII (AB1) and its internal image (AB3).
Authors: Budisavljevic M , Ronco P M , Verroust P J
Published: December 2022

We have previously produced mAb against angiotensin II (AII), a phylogenetically conserved vasopressive octapeptide, and shown that they identify four distinct epitopes on the AII molecule. In addition we used internal image bearing polyclonal antiidiotypic antibodies raised against rabbit anti AII to produce mAb3. In this study we analyze the segregation of the idiotypic and paratopic repertoires of the mAb1 and mAb3. Analysis of mAb1 carried out with polyclonal Ab2 raised against the four distinct paratopes permitted classification of the mAb1 into four categories: (p+, id+) comprises antibodies with shared paratopic and idiotypic specificities: (p+, id-) is made up of antibodies that fail to express the Id defined by Ab2 raised against other antibodies pertaining to the same paratopic group; (p-, id+) includes antibodies that express cross-reactive Id on distinct paratopes; (p-, id-) refers to antibodies unrelated by their paratopes and Id mAb2 confirmed these results and showed expression of identical or closely related Id on clearly distinct paratopes. At the Ab3 level, using polyclonal Ab4, there was a higher degree of Id cross-reactivity between the two paratopes available. These data suggest that the parallel set concept may apply to the immune response to a natural peptidic Ag and its internal image. Comparison of idiotypic repertoires of mAb1 and mAb3 (using Ab2 and Ab4 antibodies) confirmed the lack of public Id and showed the predominance on mAb3 of "new" idiotypes absent from mAb1 molecules, as expected for internal image-induced antibodies. Cross-reactive idiotypes defined on mAb1 and conserved on mAb3 were expressed on the two paratopes defined at the Ab3 level. They were located on the H chain of the homologous paratope and required the association of H and L chains on the heterologous paratope. Our analysis suggests that, in the AII system, the idiotypic and paratopic repertoires segregate at least in part independently. The paratopic repertoire is limited to a small number of phylogenetically conserved specificities and may be encoded by germline genes. In contrast, the idiotypic repertoire is broader with respect to specificities, species, and localization on H and L chains. This extended diversity may be generated by somatic mutations or use of various combinations of H and L chains and/or V, D, J segments.
Phosphorylation-dephosphorylation of the CD6 glycoprotein renders two isoforms of 130 and 105 kilodaltons. Effect of serum and protein kinase C activators.
Authors: Cardenas L , Carrera A C , Yague E , Pulido R , Sánchez-Madrid F , de Landázuri M O
Published: December 2022

The molecular nature of the structural changes on the T cell-CD6 glycoprotein upon cell activation has been investigated. Cell surface 125I labeling and immunoprecipitation studies from PBMC revealed that after stimulation by different activators of protein kinase C, or after exposure to either human or FCS, the anti-CD6 mAb precipitated an additional protein of 130 kDa, together with the 105-kDa protein present in resting cells. Cell surface expression of this 130-kDa CD6 protein form could be detected as early as 15 min after PKC activation, without requiring de novo protein synthesis. Pulse and chase activation experiments of radioiodinated cells suggested that the 130-kDa molecule is the result of a posttranslational modification of the 105-kDa protein and that this conversion is a reversible process. Studies of 32P-cell labeling and immunoprecipitation by anti-CD6 mAb revealed that only the 130-kDa form was phosphorylated, whereas the 105-kDa protein was unphosphorylated both in resting and activated cells. Moreover, the removal of phosphate groups from the 130-kDa CD6-form by enzymatic treatment with alkaline phosphatase resulted in its conversion to the 105-kDa form. Taken together, these results demonstrate the existence of two CD6 molecular forms that are in a dynamic equilibrium and differ only at their degree of phosphorylation: a 105-kDa unphosphorylated form present in resting T cells that changes very rapidly to a 130-kDa phosphorylated form by exposure of cells either to serum or to activators of PKC.
Transmembrane signaling during natural killer cell-mediated cytotoxicity. Regulation by protein kinase C activation.
Authors: Leibson P J , Midthun D E , Windebank K P , Abraham R T
Published: December 2022

NK cells can mediate either FcR-dependent cytotoxicity against antibody-coated target cells or direct cytotoxicity against a variety of tumor cells. We used homogeneous, cloned populations of CD16+/CD3- human NK cells to characterize and compare the transmembrane signaling mechanisms used during these alternative forms of cytotoxicity. Cross-linkage of NK cell FcR with anti-FcR (anti-CD16) mAb or direct binding to NK-sensitive tumor targets resulted in a rapid release of inositol phosphates and increases in [Ca2+]i. The receptor-dependent [Ca2+]i increase (as monitored in indo-1 loaded NK cells by flow cytometry) consisted of an initial release of calcium from intracellular stores, followed by a sustained influx of calcium across the plasma membrane. To assess the potential regulatory feedback role of protein kinase C (PKC) activation in these proximal signaling events, NK cells were pretreated with either PKC-activating phorbol esters, nonactivating phorbol ester homologs, or synthetic diacylglycerols. Brief pretreatment with activating phorbol esters rapidly inhibited, in a concentration-dependent manner, both phosphoinositide hydrolysis and increases in [Ca2+]i induced by FcR ligation, whereas pretreatment with an inactive phorbol ester had no effect. This acute inhibitory effect was not explained by FcR down-regulation, which occurred with more prolonged exposure to phorbol esters. In contrast, the phosphoinositide turnover and [Ca2+]i increase in NK cells stimulated with NK-sensitive tumor targets were not affected by prior exposure to PKC-activating phorbol esters. This differential regulatory effect of phorbol ester on proximal signaling was paralleled by a corresponding effect on cytotoxicity, i.e., phorbol ester-induced activation of PKC inhibited FcR-dependent cytotoxicity, but did not alter direct cytotoxicity against NK-sensitive tumor cells. These results indicate that PKC activation can differentially regulate alternative forms of NK cell-mediated cytotoxicity by rapidly and specifically desensitizing the FcR.
Matrix-bound plasminogen activator inhibitor type 1 inhibits the invasion of human monocytes into interstitial tissue.
Authors: Kirchheimer J C , Binder B R , Remold H G
Published: December 2022

Invasion of tissue by monocytes in the course of cellular immune reactions is a multistep process that is thought to be based on the action of urokinase type plasminogen activator (u-PA), an ubiquitous serine protease able to convert the zymogen plasminogen into the active protease plasmin. Expression and occupation of urokinase-type plasminogen activator receptors (u-PA-R) are known to be up-regulated by IFN-gamma and TNF-alpha, and endogenously occupied u-PA-R were found to be instrumental in monocyte invasiveness. We used the amnion invasion assay to investigate whether monocyte invasiveness is affected by matrix-bound plasminogen activator inhibitors (PAI) and by fluid phase u-PA. We show in this study that preincubation of amnion membranes with 1.5 U/cm2 PAI-1 decreases invasion of IFN-gamma activated monocytes by 70% compared with controls. Anti-vitronectin antibodies, which block PAI-1 binding to the matrix, abrogate the inhibitory effect of PAI-1 on monocyte invasiveness, indicating that active PAI-1 is bound via matrix-associated vitronectin. In contrast, preincubation of the amnion membrane with PAI-2 which does not bind to the extracellular matrix has no effect on monocyte invasiveness. Finally, the inhibitory action of matrix-bound PAI-1 can be abrogated by addition of 5 IU/ml u-PA to the monocytes in the invasion chamber. These findings indicate that monocyte invasiveness might be regulated not only by expression and occupation of u-PA-R but also by matrix-bound PAI-1.
The in vivo generation of murine IgD-secreting cells is accompanied by deletion of the C mu gene and occasional deletion of the gene for the C delta 1 domain.
Authors: Mountz J D , Mushinski J F , Owens J D , Finkelman F D
Published: December 2022

Mature, resting rodent, and primate B lymphocytes express two membrane Ig isotypes, IgM and IgD. Although membrane IgD production by these cells is regulated at a transcriptional level, and does not require deletion of the C mu gene, C mu has been deleted in all of the IgD-secreting tumor cells that have been studied. These IgD-secreting tumors, which include two mineral oil-induced plasmacytomas and three IgD-switch variants of an IgM-secreting hybridoma, might not, however, be representative of the rare IgD-secreting cells generated in response to an immune stimulus. A recent study of mice injected with a goat antibody to mouse IgD has demonstrated the generation of a relatively large secretory IgD response in these animals. We have now produced hybridomas by fusing spleen cells from these mice with a non-Ig-secreting plasmacytoma. Two of these hybridomas, KWD-1 and KWD-2, secrete IgD and express cell membrane IgD. Both of these hybridomas were found to have deleted the C mu gene. KWD-2 produces a delta-chain mRNA and a delta-chain protein similar in size to those previously reported for normal secreted mouse IgD; however, KWD-1 synthesizes a secretory delta-chain mRNA that is approximately 0.25 kb smaller than the KWD-2 secretory delta-chain mRNA and secretes IgD with a delta-chain that is approximately 21 kDa smaller than the secretory delta-chain of KWD-2. ELISA studies with epitope-defined anti-delta mAb indicate that KWD-2 has both delta Fc (C delta 3) [corrected] and delta Fd (C delta 1) [corrected] determinants, whereas KWD-1 has delta Fc but not delta Fd. These studies also demonstrate that the Ag-binding site of KWD-1 is not deleted because KWD-1 specifically binds goat IgG. Northern blot analyses with exon-specific probes indicate that while both KWD-1 and KWD-2 synthesize kappa-chain mRNA and delta-chain mRNA that includes the VH, C delta hinge, and C delta 3 exons, the C delta 1 exon is present only on the KWD-2 delta-chain mRNA. Southern blot analysis confirms that the C delta 1 exon has been deleted in KWD-1, but not KWD-2. We have previously noted that a secretory delta-chain mRNA that is similar in size to that produced by KWD-1 accounts for approximately 25% of the splenic secretory delta-chain mRNA produced by goat anti-mouse IgD antibody-injected mice.(ABSTRACT TRUNCATED AT 400 WORDS)
BP-1/6C3 expression defines a differentiation stage of transformed pre-B cells and is not related to malignant potential.
Authors: Ramakrishnan L , Wu Q , Yue A , Cooper M D , Rosenberg N
Published: December 2022

BP-1 antibody recognizes a cell surface molecule related to the zinc-dependent metallopeptidases that is expressed during a narrow window early in B cell differentiation. Expression of the same molecule, as originally recognized by the mAb 6C3, is widely accepted to be associated with the complete malignant transformation of pre-B lymphoid cells. We have examined BP-1/6C3 expression in a panel of established Abelson virus-transformed cells that includes both cells analogous to pre-B cells and to less differentiated B lineage cells that have not yet completed Ig H chain gene rearrangement. This analysis reveals that many of the less differentiated transformants do not express BP-1/6C3 for an extended culture period. In contrast, virtually all transformants that are analogous to normal pre-B cells express the determinant early in their culture history. The BP-1/6C3 negative transformants are fully tumorigenic in syngeneic mice, demonstrating that BP-1/6C3 expression is not required for complete malignant transformation. Our data thus suggest that the pattern of BP-1/6C3 expression in Abelson virus-transformed cells mimics that observed in normal cells and is indicative of a differentiation event unrelated to the malignant potential of the cells.
Structurally interdependent and independent regions of allelic polymorphism in class II MHC molecules. Implications for Ia function and evolution.
Authors: Braunstein N S , Germain R N , Loney K , Berkowitz N
Published: December 2022

Possible interactions between regions of allelic polymorphism in the alpha- and beta-chains of class II MHC molecules were examined by measuring the efficiency of surface expression and the reactivity with mAb of wild-type and recombinant A alpha A beta-chain pairs from the b, d, and k haplotypes. These studies revealed regions of polymorphism within the alpha- and beta-chains that interact with complementary regions in the other chain. Unexpectedly, almost all the variable segments of both the class II MHC alpha- and beta-chains either directly contributed to or were near sites of interchain interactions. The exception was the beta HV3 (hypervariable (HV] segment (residues 61-71), which appeared to neither participate in nor be affected by interchain interactions. This division of the MHC molecule into interacting vs independent regions of allelic structural variation suggests that mutagenesis experiments involving the beta HV3 segment can be analyzed in a straightforward manner, as such mutations appear unlikely to alter the conformation of other molecular segments. Furthermore, functions attributed to the beta HV3 segment either experimentally or by population analysis should have a high probability of transfer by beta HV3 exchange (either experimentally or evolutionarily), because epitopes assigned to this region of the molecule are not affected by sequences outside this segment. This is of special importance because of the apparent involvement of this region in defining a potential site of interaction with antigenic peptides and TCR. In contrast, almost all other variable segments of the MHC molecule appear to have the capacity to contribute to interactions involving at least one other variable segment. This suggests not only that the experimental analysis of the contributions of these regions to various functions requires a consideration of inter- and intrachain interaction, but also that the transfer of function by genetic exchange of these structurally dependent regions is unpredictable. Selection must therefore operate on these interacting HV segments in the context of the complete alpha beta heterodimer. These results support our earlier arguments for cis-co-evolution of alpha- and beta-chain polymorphism and the absence of selection for F1 (hybrid) class II molecules. Finally, asymmetries observed in the contributions of particular pairs of HV segments to the efficient expression of Ia alpha beta heterodimers provide a basis for understanding mechanistically how cis-co-evolution may have occurred.
Differential sensitivity of specific and nonspecific antigen-presentation by B cells to a protein synthesis inhibitor.
Authors: Kakiuchi T , Watanabe M , Hozumi N , Nariuchi H
Published: December 2022

Specific and nonspecific Ag-presentation by B cells was examined for the sensitivity to the treatment with emetin, an irreversible protein synthesis inhibitor. For this aim, A20-HL B lymphoma cells expressing surface IgM receptors specific for TNP were used as APC. OVA and TNP-OVA were used as nonspecific and specific Ag, respectively. The treatment with emetin greatly impaired the ability of A20-HL cells to present specific Ag, but not nonspecific Ag, to 42-6A cloned T cells specific for OVA. The ability of the emetin-treated A20-HL cells to present nonspecific Ag indicates that the treated cells are able to process nonspecific Ag and to present processed Ag. Ag binding and the internalization by A20-HL cells through surface receptors were not affected by the emetin treatment. A20-HL cells took up specific Ag for stimulation of 42-6A cells in the presence of cycloheximide, a reversible protein synthesis inhibitor. These results suggest that the action of emetin is localized to the intracellular processing of specific Ag, not of nonspecific Ag. Thus, the processing pathway for specific Ag seems to be different from that for nonspecific Ag.
Inhibition of experimental autoimmune encephalomyelitis induction in SJL/J mice by using a peptide with high affinity for IAs molecules.
Authors: Lamont A G , Sette A , Fujinami R , Colón S M , Miles C , Grey H M
Published: December 2022

Blocking of the Ag presenting function of MHC by peptides capable of high affinity binding to this molecule has been proposed as a potential immunotherapeutic intervention in MHC-associated diseases. Recent studies have used this strategy to prevent the induction of experimental allergic encephalomyelitis (EAE) in mice. However, because of the close structural relationship between the inhibitor and encephalitogenic peptides, the results of these previous studies have been difficult to interpret with regard to whether MHC blockade was the mechanism by which the inhibitory peptides functioned. In our study, we have determined the capacity of unrelated peptides capable of binding with high affinity to IAs in inhibiting the induction of EAE in SJL/J mice after immunization with the autoantigenic peptide PLP 139-151. Prevention of the disease was accomplished by two methods: 1) when inhibitor was administered together with the encephalitogenic peptide at the time of immunization, as in previous studies, and 2) when inhibitor was administered at a separate site from the autoantigen 1 day before the immunization with that Ag. Inhibition was due to binding of the inhibitor to IAs, as evidenced by the fact that a control peptide incapable of binding to this MHC had no effect on the course of the disease. The finding that inhibitor could also be efficacious when administered at a separate site has implications for potential use of such a strategy to reverse ongoing autoimmune diseases. The inhibitor had to be present during the time of Ag stimulation, and had no long term inhibitory effects, in that a secondary immune response to the encephalitogenic peptide was not inhibited in animals given the inhibitory peptide before the induction of a primary response. This is compatible with the conclusion that MHC blockade was, in fact, the mechanism of the inhibition, rather than as a result of any long term suppressive effects on immunoreactive T cells. Finally, not only did administration of the inhibitory peptide lead to a prevention of the induction of EAE, but it could also be shown to decrease the T cell proliferative response in vitro to the autoantigen.
IL-7 supports the generation of cytotoxic T lymphocytes from thymocytes. Multiple lymphokines required for proliferation and cytotoxicity.
Authors: Bertagnolli M , Herrmann S
Published: December 2022

Current data suggest that the combination of IL-2 plus IL-6 or IL-4 supports the generation of a CTL response. In this report, we investigated whether IL-7 alone or in combination with other lymphokines supports the generation of active CTL from murine thymocytes. Added alone, IL-7 produced a modest CTL response, and this response was augmented (more than an additive response) by the addition of either IL-2, IL-6, or IL-4. After culture with IL-7, the removal of CD8+ cells correlated with a marked decrease in killing, whereas removal of the CD4+ population resulted in an enhanced CTL response. IL-7 generated a proliferative response to which the addition of other lymphokines resulted in an additive increase at best. To determine if the CTL response was found with IL-7 was dependent on the presence of endogenous lymphokines, we added blocking mAb and found that anti-IL-2 or anti-IL-6 blocked the level of CTL generated in the presence of IL-7. In contrast, anti-IL-4 was unable to inhibit CTL generation. The effect of the mAb on proliferation was somewhat paradoxical. The addition of anti-IL-2, anti-IL-6, or anti-IL-4 to cultures containing IL-7 all caused a significant decrease in proliferation that was overcome by the addition of the corresponding human lymphokines for IL-2 and IL-6. If thymocytes were fractionated and then cultured in the presence of IL-7, the double negative population gave the greatest proliferative response, the single positive thymocytes gave a response that was approximately 40% less, and the double positive thymocytes did not proliferate. These data indicate that IL-7 supports the induction of a cytotoxic response, however, this appears to require the presence of both IL-2 and IL-6.
Differential production of IL-2 and IL-4 mRNA in vivo after primary sensitization.
Authors: Mohler K M , Butler L D
Published: December 2022

The study of lymphokines has been almost entirely conducted by utilizing in vitro assay systems, long term cell lines, and clones. Thus, little information is available concerning the production of lymphokines/cytokines in vivo after specific antigenic stimulation. In order to address this limitation, we have modified the mRNA phenotyping system to allow for the quantitation of lymphokine mRNA after antigenic stimulation in vivo. We report here the production of both IL-2 and IL-4 mRNA in vivo after primary sensitization with picryl chloride. However, the time course of IL-2 and IL-4 mRNA production is discordant. The majority of IL-2 mRNA expression occurs from 1 to 3 days after antigenic exposure, whereas IL-4 mRNA expression occurs mainly from day 3 through day 5. Thus, the production in vivo of these two lymphokine mRNA after sensitization with picryl chloride appears to occur as a "cascade." These results 1) demonstrate that IL-4 mRNA is induced during a primary immune response in vivo and 2) raise the possibility that the generation of an immune response in vivo may involve a specific sequential production of certain lymphokines.
Serotonin-activated signal transduction via serotonin receptors on Jurkat cells.
Authors: Aune T M , Kelley K A , Ranges G E , Bombara M P
Published: December 2022

Serotonin (5-hydroxytryptamine) (5HT) a neurotransmitter and vasoactive amine, is a major storage product of platelets that are released at sites of inflammation. Several different subtypes of serotonin receptors have been defined. 5HT receptors have been divided into three major families based on molecular, biochemical, and pharmacologic properties. Binding of serotonin to the 5HT1 family results in inhibition of adenylate cyclase whereas binding to the 5HT2 family results in stimulation of phosphatidylinositol turnover and mobilization of intracellular Ca2+. 5HT has been shown to have effects on lymphoid cells. The question of whether human T lymphocytes express receptors for 5HT and transduce signals through 5HT receptors has not been adequately addressed. As a model system, Jurkat cells (a transformed human T lymphocyte line) were examined to determine if they expressed 5HT receptors and whether 5HT stimulated an increase in inositol phosphates or affected adenylate cyclase activity. The results show that Jurkat cells bind 5HT with an average dissociation constant of 90 nM and that 5HT stimulates an increase in inositol phosphate and intracellular Ca2+ levels. These results link the 5HT receptor on Jurkat cells to the 5HT2 family; however, studies with 5HT receptor agonists and antagonists failed to clearly classify the 5HT receptor on Jurkat cells as a known member of the 5HT2 family.
Compartmentalization of low molecular mass GTP-binding proteins among neutrophil secretory granules.
Authors: Dexter D , Rubins J B , Manning E C , Khachatrian L , Dickey B F
Published: December 2022

Neutrophils contain several distinct classes of secretory granules that may sequentially fuse with the phagosome after the ingestion of particulates, or that may be differentially exocytosed after cellular activation with soluble stimuli. The exocytosis of neutrophil secretory granules has been shown to be GTP-dependent at a step distal to activation of the transductional G proteins. Inasmuch as ras-related low molecular mass GTP-binding proteins have been shown to play regulatory roles in vesicle sorting in the secretory pathway in yeast, the differential mobilization of neutrophil granules might be regulated by distinct GTP-binding proteins. We therefore explored the distribution and identity of low molecular mass GTP-binding proteins in neutrophil secretory granules and other subcellular fractions. After lysis by nitrogen cavitation, four highly resolved fractions were harvested from discontinuous Percoll gradients: a microsomal fraction enriched for plasma membranes, specific granules, primary granules, and cytosol. At least seven bands of distinct Mr were detected by probing protein blots with [32P]GTP. Microsomes contained a prominent GTP-binding band at 26 kDa and weaker ones at 24 and 22.5 kDa; specific granules contained bands at 26, 24, 22, and 20 kDa; primary granules showed bands at 24 and 23 kDa; cytosol showed strong bands at 23.5 and 19 kDa and a weak band at 26 kDa. Antiserum against ADP-ribosylation factor reacted strongly with the 19-kDa band in cytosol but with none of the membrane fractions. None of these proteins was recognized by antibodies against ras or against Sec4p. Botulinum exoenzyme C3 labeled bands of molecular mass 20 and 21 kDa in cytosol and microsomes that have distinct mobilities from all the blotted [32P]GTP-binding proteins. The highly compartmentalized subcellular distribution of the blotted [32P]GTP-binding proteins in neutrophils is consistent with a regulatory role in the differential mobilization of granule compartments during cellular activation.
MHC and non-MHC-restricted recognition of filarial surface antigens in mice transplanted with adult Brugia malayi parasites.
Authors: Kwan-Lim G E , Maizels R M
Published: December 2022

We describe here the genetic control of humoral responses to filarial nematode Ag elicited by live adult Brugia malayi parasites in mice. Inbred and congenic mice of two different MHC haplotypes, H-2k and H-2d, were examined. Serologic analysis showed that the humoral responses to the major surface 29-kDa glycoprotein of adult parasites and a 40-kDa Ag from the surface of the microfilarial stage were restricted to mice with H-2k alleles (B10.BR, CBA/Ca, and CBA/N), whereas mice of the H-2d haplotype (B10.D2/n and BALB/c) were nonresponsive to these Ag. Conversely, internal adult Ag of molecular mass of 24 and 66 kDa were recognized only by animals with the H-2d haplotype. Apart from MHC-restricted recognition, the level of responses to phosphorylcholine and to a 15-kDa adult surface molecule were found to be influenced by non-MHC genes. A sharp restriction was also observed to an adult surface Ag complex of 17 to 200 kDa, which was recognized only by BALB/c mice. Thus, multiple examples of both H-2 and background genetic effects on the immune response to distinct filarial Ag can be found.
Individual VH genes detected with oligonucleotide probes from the complementarity-determining regions.
Authors: Guillaume T , Rubinstein D B , Young F , Tucker L , Logtenberg T , Schwartz R S , Barrett K J
Published: December 2022

The germ-line and expressed Ig repertoire was examined with three oligonucleotide probes from the CDR regions of VH18/2, a VH gene from the largest human VH gene family, VHIII. Each oligonucleotide probe detected small numbers of germ-line bands (1-5) under conditions in which single base differences can be detected; more than half of these bands were polymorphic. The combined results from pairs of oligonucleotides from CDR1 and CDR2 identified a single band on Southern blots, as did a probe from the 5' end of CDR2. This band contains the 18/2 germ-line gene. The nucleotide sequence of expressed VH genes that hybridized to both CDR probes or to the 5' CDR2 probe were greater than or equal to 97% homologous to 18/2 in both the framework and CDR regions. This group of closely related VH genes, the 18/2 CDR family, appears to be overexpressed. The role of polymorphisms and differential expression of individual V genes in multigenic autoimmune diseases, as well as the organization and expression of individual V genes, can be examined with pairs of oligonucleotides from CDR1 and the 3' end of CDR2, or with probes from the 5' end of CDR2.
Class II genes of miniature swine. IV. Characterization and expression of two allelic class II DQB cDNA clones.
Authors: Gustafsson K , LeGuern C , Hirsch F , Germana S , Pratt K , Sachs D H
Published: December 2022

Two cDNA clones coding for allelic miniature swine MHC class II Ag DQB chains have been isolated, characterized, and shown to be expressed after transfection into mouse fibroblasts. The two alleles differ at the nucleotide level by an overwhelming proportion of replacement substitutions, suggesting the influence of selection for polymorphism. Most of the resulting predicted amino acid replacements are in regions commonly polymorphic in mouse Ab and human DQB sequences, corresponding to the predicted Ag recognition site. Nucleotide and amino acid sequence comparisons to homologous mouse and human sequences show more similarity between swine and man than between either swine and mouse or man and mouse. This tendency is most pronounced when comparing the 3' untranslated regions. However, an examination of unique cross-species sharing of amino acid residues suggests a closer relationship between both man and miniature swine and man and mouse than between miniature swine and mouse. The simplest explanation we can envision for these findings is that the mouse DQB gene homologue (Ab) has been subject to a higher substitution rate than either swine or human DQB genes. An additional cytoplasmic exon expressed in mouse Ab gene products and in putative human DQB2 gene products is lacking in both swine and human DQB cDNA clones. Its absence suggests either that the expression of this exon in mouse Ab genes was activated after mammalian speciation or that the expression of this exon was independently inactivated in swine DQB and human DQB1 genes. Alternatively, the mouse Ab gene may be derived from the same primordial gene as human DQB2, whereas the pig DQB gene may be derived from the same primordial gene as the human DQB1 gene.
Expression cloning of a cDNA encoding M1/69-J11d heat-stable antigens.
Authors: Kay R , Takei F , Humphries R K
Published: December 2022

The differentiation Ag identified by the mAb M1/69 and J11d (commonly referred to as heat-stable Ag) are found in structurally heterogeneous forms on the surfaces of many types of murine hemopoietic cells. The extinction of expression of these antigens is associated with thymocyte maturation and Ig class switching in B cells, as well as terminal differentiation of macrophages. A cDNA encoding the M1/69-J11d peptide was cloned from a hemopoietic progenitor cell line by immunoselection of COS cells transfected with expression libraries. The cloned cDNA is a copy of a gene that is transcribed in M1/69-J11d+ lymphoid, myeloid, and erythroid cells. This gene could be responsible for the expression of all forms of the M1/69-J11d Ag, although there are homologous genes that may encode some forms of the Ag that are specifically expressed in bone marrow. The cloned cDNA encodes a surprisingly small peptide, predicted to contain only 30 amino acids after removal of a signal sequence and displacement of the C-terminal region by the glycosyl-phosphatidylinositol group that anchors the peptide to the cell surface. Almost all of the mass of the M1/69-J11d Ag accumulates through extensive N- and O-linked glycosylation at multiple sites in the short peptide. These carbohydrates are likely to execute the functions of M1/69-J11d Ag, which could be specialized to each cell type as a consequence of differential glycosylation.
Effects of stress on lysability of tumor targets by cytotoxic T cells and tumor necrosis factor.
Authors: Sugawara S , Nowicki M , Xie S , Song H J , Dennert G
Published: December 2022

The effects of stress on four tumor cell lines are analyzed in view of the possibility that stress protects tumor cells against immune attack. We show that stress causes resistance to CTL and TNF in two cell lines. Induction of resistance is time dependent and reversible and not due to failure of killer cells to interact with stressed targets. It is shown that stress induces stress proteins concomitant with induction of resistance to killer cells and TNF. Moreover experiments are presented suggesting that resistance to either immune effector is due to independent mechanisms. The conclusion that stress can induce mechanisms in targets that interfere with the action of TNF as well as with target lysis following a lethal hit by CTL is discussed.
DNP-specific/class I MHC-restricted suppressor molecules bear determinants of the T cell receptor alpha- and beta-chains. The V beta 8+ chain dictates restriction to either K or D.
Authors: Fairchild R L , Kubo R T , Moorhead J W
Published: December 2022

To examine in greater detail the relationship between DNP-specific/class I MHC-restricted suppressor molecules (SSF) that inhibit contact sensitivity to 2,4-dinitrofluorobenzene and the receptors on the T cells that produce them, we have generated two T cell hybridomas that can be induced to produce and secrete these molecules. In order to become activated to produce SSF, the Ts 15.15 and 15.31 cells required recognition of complexes of DNP/Dd on presenting cells. The suppressor molecules produced by each of the Ts hybrids had the same specificity, recognizing DNP/Dd on cells in the immune lymph node cell target population. The activation of the Ts hybrids was blocked when the cells were treated with the anti-V beta 8 antibody F23.1 before coculture with the DNP-presenting cells. Reduction of the 15.15 and 15.31 SSF followed by affinity chromatography on DNP-bovine-gamma-globulin-Sepharose beads indicated that these molecules are dimers and that one of the chains (Ag-binding(AgB] binds to cellfree DNP and one (non-Ag-binding (NAgB) chain) does not. The AgB chain was found to express an epitope bound by a mAb specific for a TCR alpha-chain-constant region determinant. Alternatively, the NAgB chain expressed an epitope bound by the anti-V beta 8 mAb F23.1. Active hybrid suppressor molecules were generated by combining the NAgB chain from a DNP-specific/H-2Kd-restricted SSF (produced by Ts hybridoma 3-10) with the AgB chain from Ts 15.31 and by combining the NAgB chain from Ts cell 15.15 with the 3-10 AgB chain. In each case, the class I MHC element (i.e., Kd or Dd) restricting the activity of these hybrid SSF correlated with the source of the V beta 8+, NAgB chain. Thus, these secreted immunoregulatory molecules have the Ag/MHC specificity of the T cells producing them and are structurally and serologically related to the TCR-alpha/beta. The results further suggest that for some hapten-specific/class I MHC-restricted TCR, the alpha-chain may have avidity for the hapten and the beta-chain may dictate the MHC restriction element (K or D) recognized by the receptor.
Regulatory function for murine intraepithelial lymphocytes. Two subsets of CD3+, T cell receptor-1+ intraepithelial lymphocyte T cells abrogate oral tolerance.
Authors: Fujihashi K , Taguchi T , McGhee J R , Eldridge J H , Bruce M G , Green D R , Singh B , Kiyono H
Published: December 2022

The murine intraepithelial lymphocyte (IEL) population is enriched in T cells that express the gamma delta-TCR, however, the biologic function served by these T cells remains obscure. IEL are considered to be major effector cells in mucosal immunity, and we have investigated whether IEL subsets could reverse orally induced systemic unresponsiveness (oral tolerance; OT) and support secondary type responses when adoptively transferred to mice orally tolerized with SRBC. When purified CD3+ IEL from mice orally primed with SRBC were transferred to adoptive hosts and challenged with SRBC, splenic IgM, IgG1, IgG2b, and IgA anti-SRBC plaque-forming cell responses were observed. However, CD3+ IEL from HRBC orally primed mice did not abrogate SRBC induced OT. Further, HRBC-primed CD3+, IEL converted HRBC-specific OT but not SRBC-specific OT. CD3+ IEL could be separated into four subsets based on expression of CD4 and CD8. CD3+, CD4-, 8+ T cells were the major subset (74.5%), with smaller numbers of CD4- and CD8- (double negatives, DN) (7.8%), CD4+, 8- (7.6%) and CD4+, CD8+ (double positives) (10.1%) T cells. Interestingly, both the CD3+, CD8+, and the CD3+, DN IEL subsets abrogated OT, resulting in significant IgM, IgG1, IgG2b, and IgA anti-SRBC plaque-forming cell responses when adoptively transferred to mice with OT. However, neither CD3+, CD4+, CD8-, nor double positive T cells affected OT when studied in this system. The CD3+, CD8+ IEL subset could be further separated into Thy-1+ (16.6%) and Thy-1- (83.4%) cells; adoptive transfer of Thy-1- cells abrogated oral tolerance whereas the Thy-1+ subset was without effect. When the expression of TCR on IEL with this biologic function was determined by use of monoclonal anti-alpha beta TCR (H57.597), TCR2-, CD3+ IEL possessed immunoregulatory function whereas the alpha beta-TCR+ (TCR2+) fraction did not abrogate OT. Immunoprecipitation of membrane fractions obtained from purified CD3+, CD4-, CD8+, Thy-1- IEL with polyclonal anti-delta peptide (Tyr-Ala-Asn-Ser-Phe-Asn-Asn-Glu-Lys-Leu) antibody revealed bands of 45 and 35 kDa, corresponding to the delta- and gamma-chains, respectively. These results suggest that gamma delta-TCR+ IEL possess a regulatory function, namely the restoration of immune responses in a state of oral tolerance. Further, both CD3+, CD4-, CD8+, Thy-1-, and CD3+, DN IEL T cells exhibit this effector contrasuppressor function.