bluuebunny/crossref_metadata_2025 huggingface dataset:
Neutrophil activation detected by monoclonal antibodies.
Authors: Tetteroo P A , Bos M J , Visser F J , von dem Borne A E
Published: December 2022

Monoclonal antibodies have been produced against three neutrophil-associated membrane proteins (p 90, p 170, and p 70) expressed at different maturation stages of the cells. The reactivity of the antibodies against p 90 (B13.9) and p 170 (CLB-gran 10), as measured by quantitative flow cytofluorometry, increased after stimulation of the neutrophils by the calcium ionophore A23187, by phorbol myristate acetate, or by the chemoattractant formylmethionyl-leucyl-phenylalanine in combination with cytochalasin B. This increase is regulated independently of the simultaneously increased expression of the C3bi receptor, because neutrophils of a patient deficient for the C3bi receptor showed a normal increase in membrane expression of p 90 and p 170. Neutrophil cytoplasts were not inducible to increased membrane expression, suggesting that the cytoplasts lack the internal pool of these proteins. The reactivity of the antibody against p 70 (CLB-gram 5) was not affected by activation. The antibodies B13.9 and CLB-gran 10 may be useful to detect neutrophil activation.
Substrate specificity and partial characterization of the PAF-acylhydrolase in human serum that rapidly inactivates platelet-activating factor.
Authors: Wardlow M L , Cox C P , Meng K E , Greene D E , Farr R S
Published: December 2022

The structure of the potent inflammatory mediator, platelet-activating factor, is 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC, PAF-acether). Human sera contain an acid labile factor (ALF) that is a Ca+2-independent 2-acylhydrolase-specific for AGEPC and AGEPC-like molecules. The enzyme functions by catalytically removing the sn-2 acetyl moiety from AGEPC, producing the biologically inactive sn-2 hydroxy form or 2-lyso-GEPC. Incubation of ALF with sn-2 acyl PAF analogs indicated that the enzyme hydrolyzes the sn-2 fatty acid only if the chain length is five carbons or less, the sn-1 position fatty acid length is greater than 10 carbon units, and at least one methyl group is present on the terminal amine of the choline group. The enzyme was active with either an ether or ester linkage at the sn-1 position. ALF is inactivated by heating to 65 degrees C for 30 min. It is pronase and trypsin sensitive but resistant to papain and papain with dithiothreitol. Further characteristics of human ALF indicated a broad pH range of activity with an optimum of pH 6.2 and an isoelectric point of 6.2 to 6.7. The specificity and Ca+2 independence of human ALF sets it apart from phospholipase A2. It is proposed that human ALF be called human serum PAF-acylhydrolase to distinguish it from other hydrolases currently known to exist.
Mechanism of action of glucocorticoid-induced immunoglobulin production: role of lipoxygenase metabolites of arachidonic acid.
Authors: Goodwin J S , Atluru D
Published: December 2022

Glucocorticoids stimulate polyclonal immunoglobulin (Ig) production in cultures of human peripheral blood lymphocytes. The mechanism of action of glucocorticoids in this system, and indeed in any physiologic system, is unknown. Because glucocorticoids stimulate the production of phospholipase A2-inhibitory glycoproteins, we investigated whether glucocorticoids stimulate polyclonal Ig production by inhibition of arachidonic acid metabolism. Nonspecific lipoxygenase/cyclooxygenase inhibitors stimulate polyclonal Ig production in a manner similar to the effect of glucocorticoids, whereas specific cyclooxygenase inhibitors actually inhibit Ig production. Two specific 5-lipoxygenase inhibitors, with little or no activity against cyclooxygenase or other lipoxygenases, also stimulate Ig production. The dose-response effect of all of these drugs on Ig production was similar to the dose response of inhibition of 5-lipoxygenase. Leukotriene B4 (LTB4) added in low concentrations (10(-10)M) on days 1, 2, and 3 of a culture eliminated the stimulatory effect of glucocorticoids or 5-lipoxygenase inhibitors, whereas LTC4, LTD4, prostaglandin E, or 5-hydroxyeicosatetraenoic acid had no effect. These results suggest that the relevant action of glucocorticoids in stimulating Ig production might be in preventing endogenous arachidonic acid metabolism, perhaps the endogenous production of LTB4.
Adoptive immunotherapy of a syngeneic murine leukemia with a tumor-specific cytotoxic T cell clone and recombinant human interleukin 2: correlation with clonal IL 2 receptor expression.
Authors: Matis L A , Shu S , Groves E S , Zinn S , Chou T , Kruisbeek A M , Rosenstein M , Rosenberg S A
Published: December 2022

The successful adoptive immunotherapy of the syngeneic Friend virus-induced murine leukemia FBL-3 was mediated by a proliferative MHC-restricted, tumor-specific CTL clone in combination with recombinant human IL 2. This clone was previously shown to express the L3T4-, Lyt-1+, Lyt-2+ surface phenotype. Activation of the clone for 48 hr in vitro with irradiated tumor cells induced the expression of IL 2 receptors and markedly increased clonal proliferation in response to recombinant IL 2. Intravenous injection of 2 X 10(7) 48 hr in vitro-activated cloned cells, followed by 6 days of systemic (i.p.) administration of IL 2 resulted in the complete regression of tumors and the cure of 50% of the treated mice. IL 2 alone had no effect on tumor growth, whereas the injection of nonactivated (resting) clone plus IL 2 or activated clone without IL 2 had small but insignificant effects on tumor growth and survival. These results indicated that the in vivo effector functions of cloned T cells may be markedly enhanced by the concurrent systemic administration of recombinant IL 2 and by the induction of optimal IL 2 receptor expression on the cloned T cells at the time of cell administration.
Interdependence of the radioprotective effects of human recombinant interleukin 1 alpha, tumor necrosis factor alpha, granulocyte colony-stimulating factor, and murine recombinant granulocyte-macrophage colony-stimulating factor.
Authors: Neta R , Oppenheim J J , Douches S D
Published: December 2022

Interleukin 1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF alpha), granulocyte-colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) are molecularly distinct cytokines acting on separate receptors. The release of these cytokines can be concomitantly induced by the same signal and from the same cellular source, suggesting that they may cooperate. Administered alone, human recombinant (hr)IL-1 alpha and hrTNF alpha protect lethally irradiated mice from death, whereas murine recombinant GM-CSF and hrG-CSF do not confer similar protection. On a dose basis, IL-1 alpha is a more efficient radioprotector than TNF alpha. At optimal doses, IL-1 alpha is a more radioprotective cytokine than TNF alpha in C57BL/6 and B6D2F1 mice and less effective than TNF alpha in C3H/HeN mice, suggesting that the relative effectiveness of TNF alpha and IL-1 alpha depends on the genetic makeup of the host. Administration of the two cytokines in combination results in additive radioprotection in all three strains. This suggests that the two cytokines act through different radioprotective pathways and argues against their apparent redundancy. Suboptimal, nonradioprotective doses of IL-1 alpha also synergize with GM-CSF or G-CSF to confer optimal radioprotection, suggesting that such an interaction may be necessary for radioprotection of hemopoietic progenitor cells.
Development of rat anti-mouse interleukin 3 monoclonal antibodies which neutralize bioactivity in vitro.
Authors: Abrams J S , Pearce M K
Published: December 2022

Several rat anti-mouse interleukin 3 (IL-3) monoclonal antibodies have been developed which inhibit the biologic activity of mouse IL-3. These antibodies were produced in rats immunized with preparations of purified, recombinant mouse IL-3, obtained from transiently transfected COS7 cell supernatant. Hybridomas secreting anti-IL-3 were selected initially either on the basis of their giving a positive signal in an indirect enzyme-linked immunosorbent assay, or for their ability to inhibit the proliferation of the IL-3-dependent mouse mast cell line, MC/9. Neutralizing rat monoclonal IgG1, IgG2a, and IgG2b antibodies have been identified; these also block IL-3-induced proliferation of the NFS-60 and IC2 cell lines. These antibodies also blocked the IL-3-induced proliferation of mouse bone marrow-derived colony-forming units-culture suggesting that the same epitopes on IL-3 influence receptor recognition for both the proliferation of factor-dependent cell lines as well as normal bone marrow cells. Fab fragments produced from certain of the IgG2a-neutralizing antibodies blocked as well as the parent IgG. Antibody cross-blocking studies identified one neutralizing antibody apparently recognizing an epitope that was spatially distinct from those recognized by the other blocking antibodies tested. The development of these neutralizing rat monoclonal antibodies to mouse IL-3 should facilitate further investigation on the role of this factor in hemopoietic regulation.
Human Ia alpha- and beta-chains are sulfated.
Authors: Sant A J , Zacheis M , Rumbarger T , Giacoletto K S , Schwartz B D
Published: December 2022

The human Ia antigens (DR, DQ, and DP), determined by genes with the HLA complex, are heterodimers consisting of a 34,000-Da alpha-chain glycoprotein and a 29,000-Da beta-chain glycoprotein. During the course of studies characterizing a recently described sulfated proteoglycan that is specifically associated with Ia, we discovered that there were also nonproteoglycan sulfated components present in the Ia immunoprecipitates. One-dimensional sodium dodecyl sulfate-gel analysis of these latter sulfated components derived from both DR and DQ immunoprecipitates indicated that these components have mobilities indistinguishable from conventional Ia alpha and beta glycoproteins. Two-dimensional gel analysis confirmed these findings and revealed that Ia-associated invariant proteins are sulfated as well. The sulfate moiety was not removed by endoglycosidase F treatment, suggesting that the protein portion of the molecule was sulfated. These results indicate that Ia alpha-, beta-, and invariant chains can be sulfated and raise the possibility that sulfation may play a role in the physiology of Ia molecules.
Immunoregulatory functions of paf-acether. I. Effect of paf-acether on CD4+ cell proliferation.
Authors: Dulioust A , Vivier E , Salem P , Benveniste J , Thomas Y
Published: December 2022

Paf-acether or platelet-activating factor (1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a phospholipid mediator of inflammation initially described as a potent platelet-aggregating compound. It is newly formed by a variety of cells including monocytes and is now recognized as a major mediator of cell-cell interactions. The present studies were undertaken to determine whether paf-acether could modulate T cell function. We found that addition of paf-acether to CD4+ cells cultured with phytohemagglutinin markedly inhibited the proliferative response in a dose-dependent manner. Maximal inhibition occurred when paf-acether was present during the first 24 hr of cell culture and the presence of paf-acether did not alter the kinetics of CD4+ cell proliferation. Importantly, the mechanism by which paf-acether inhibited the proliferative response was not related to inhibition of interleukin 2 (IL-2) secretion since the amount of IL-2 in cultures was not altered and addition of exogenous IL-2 failed to restore the CD4+ cell proliferative response. Further, as judged by indirect immunofluorescence, paf-acether did not inhibit IL-2 receptor expression. Taken together, these data indicate that paf-acether interferes with some processes leading to CD4+ cell proliferation. This new role for the chemically defined monokine paf-acether emphasizes the potential role of inflammatory lipid mediators in the regulation of T cell response.
Calcium-dependent intracellular acidification dominates the pH response to mitogen in human T cells.
Authors: Gelfand E W , Cheung R K , Grinstein S
Published: December 2022

The effects of a phorol ester and a mitogenic lectin on the intracellular pH (pHi) of human T lymphocytes was investigated. In contrast to the cytoplasmic alkalinization induced by 12-0-tetradecanoylphorbol-13-acetate, an acidification was recorded in cells treated with phytohemagglutinin. This decrease in pHi was magnified in Na+-free medium or in the presence of amiloride analogues, suggesting that activation of Na+/H+ exchange partially counteracts the phytohemagglutinin-induced acidification. The decrease in pHi was dependent on a sustained increase in cytosolic free Ca2+ and could be mimicked by addition of the divalent cation ionophore, ionomycin. The elevation of cytosolic free Ca2+ leads to metabolic H+ (equivalent) generation with consequent cytoplasmic acidification, which in human T cells predominates over the concurrent activation of the Na+/H+ antiport. These findings argue against the notion that activation of Na+/H+ exchange is a signal for the initiation of proliferation.
Patterns of immunoenhancement and suppression induced by Chlamydia trachomatis in vivo and in vitro.
Authors: Levitt D , Corlett R
Published: December 2022

We investigated the cycle of immune enhancement and suppression seen in mice infected with Chlamydia trachomatis by using in vivo and in vitro model systems. BALB/c mice injected intravenously with chlamydia reveal a three- to seven-fold increase in numbers of plaque-forming cells producing antibodies against sheep red blood cells (SRBC), when immunized with SRBC 0 to 5 days after chlamydia infection. When mice are injected with SRBC 10 to 15 days after initial chlamydia infection, the specific anti-SRBC plaque-forming cell response is suppressed two- to three-fold. In vitro, low numbers (2 to 5 X 10(6) bacteria/ml) of chlamydia stimulate potent proliferative responses by B lymphocytes while high numbers (25 X 10(6) bacteria/ml) of bacteria generate strong, general T suppressor activity. This model has important implications for regulation of immune responses that arise at different times during chlamydial infections, as well as for the potential effectiveness of chlamydial vaccines.
Genetics of resistance to the African trypanosomes. VI. Heredity of resistance and variable surface glycoprotein-specific immune responses.
Authors: De Gee A L , Levine R F , Mansfield J M
Published: December 2022

The question of genetic linkage of parasite-specific immune responses to resistance to infection in experimental African trypanosomiasis was addressed. For this purpose, major histocompatibility complex-compatible resistant and susceptible inbred mouse strains and their F1 hybrid, F2 hybrid, and backcross offspring were infected with Trypanosoma brucei rhodesiense LouTat 1. Immunologic control of the first peak of parasitemia and survival times were the parameters measured. As we have reported previously (R. F. Levine and J. M. Mansfield, J. Immunol. 133:1564, 1984), B10.BR/SgSnJ mice are relatively resistant and controlled the growth of the infecting variant antigenic type (VAT) by mounting an antibody response to exposed epitopes of the variable surface glycoprotein (VSG). Fluctuating parasitemias resulting from sequential growth of different variable antigenic types occurred subsequently, and these mice died with a median survival time of 48 days. C3HeB/FeJ mice, relatively susceptible, did not control the infecting VAT and did not exhibit VSG-specific antibodies. These mice died with a median survival time of 22 days. The (B10.BR X C3H)F1 hybrids derived from crosses between resistant and susceptible mice all exhibited VSG-specific antibody responses and controlled the infecting VAT population. However, the median survival time of the F1 hybrids (24 days) was not significantly different from the survival time of the susceptible C3H parent. These findings demonstrate for the first time that antibody-mediated control of parasitemia is inherited as a dominant trait; that overall resistance, as measured by survival time, is inherited as a recessive trait (e.g., susceptibility is dominant); and that the two events segregate independently of one another. Further analyses of the inheritance of immunity and resistance (survival time) were made in which the F2 hybrid and backcross studies revealed that there are multiple genes controlling the VSG-specific antibody response as well as determining susceptibility. An extension of the present studies to a similar but non-major histocompatibility complex-mouse model system of resistance and susceptibility (C57BL/6J and C3H/HeJ mice, F1 hybrids, and 11 recombinant inbred B X H strains derived from them) was made in order to link the strain distribution patterns of known genetic markers with control of VSG-specific antibody responses or with control of susceptibility. Results of this study showed that resistance varied independently of the ability to control parasitemia with VSG-specific B cell responses.(ABSTRACT TRUNCATED AT 400 WORDS)
Evidence for expression of two distinct T cell receptor beta-chain transcripts in a human diphtheria toxoid-specific T cell clone.
Authors: Triebel F , Breathnach R , Graziani M , Hercend T , Debre P
Published: December 2022

We have cloned and sequenced the human T cell receptor alpha- and beta-chains cDNA present in a lambda gt10 library derived from a human CD4+ diphtheria toxoid-specific T cell clone, PH28. Two 1.3-kb beta-transcripts within frame V, D, J joining were found. These two functional beta-transcripts use two members of different V beta-families, V beta 3 and V beta 4, one joined to D beta 1-1, J beta 1-1, C beta 1, the other joined to D beta 1-1, J beta 2-1, C beta 2. Moreover, we have cloned the two different V beta germ-line counterparts whose sequences confirm that these two V beta are not pseudogenes. Analysis of T cell receptor beta and alpha rearrangements in PH28 showed that a single rearrangement on each chromosome could be assigned either at the J beta or the J gamma loci. These results strongly suggest an absence of allelic exclusion for 1.3-kb mRNA beta-transcripts in a human antigen-specific T cell clone.
N-terminal and cDNA characterization of murine lymphocyte antigen Ly-6C.2.
Authors: Palfree R G , Sirlin S , Dumont F J , Hämmerling U
Published: December 2022

The Ly-6C.2 molecule was purified from K36 tumor cells by affinity chromatography and gel filtration. The electrophoretically homogeneous preparation, with m.w. 15,000, was tested with a panel of antibodies that confirmed the presence of the LY-6C.2 epitope. An N-terminal sequence of 39 amino acids was obtained showing 59% homology with the corresponding portion of the Ly-6A.2 polypeptide. Based on the least homologous (29%) 14 amino acid segment, an oligonucleotide probe was constructed, and Ly-6C.2 cDNA was cloned from a BW5147 cDNA library. A 794-base pair cDNA containing the entire coding region had 82% homology with Ly-6A.2 cDNA. The encoded polypeptide sequence of 131 amino acids containing a perfect correlation with the N-terminal sequence data was 63% homologous with that of Ly-6A.2. The greatest homology was in the leader, first 16 N-terminal and last 39 C-terminal amino acids. The latter are likely to be important in determining the attachment of glycophosphatidylinositol. Despite results indicating fewer disulfide constraints in the Ly-6C molecule, the predicted sequence contains 10 cysteine residues nearly perfectly matched with those predicted in Ly-6A.
Cytotoxic T lymphocyte response to minor H-43a alloantigen in H-43b mice. Privileged H-2Kb restriction to the response is not due to immunodominance or epistatic effect but due to Ir gene function of H-2Kb itself.
Authors: Hino T , Ishikawa H , Saito K
Published: December 2022

Previous study demonstrated that anti-H-43a cytotoxic T lymphocyte (CTL) response of H-43b CWB (H-2b) stain carrying non-major histocompatability complex (MHC) genes of C3H and F1 strains raised by crossing CWB with various H-43b strains was restricted exclusively by self H-2Kb (Kb). In the present study, newly produced C3W strain (H-2k, H-43b), which is H-43-congenic to C3H/HeN (H-2k, H-43a), was used as H-43b mice, and possibility of immunodominance of Kb was examined. No anti-H-43a CTL response could be induced in C3W strain and F1 strains raised by crossing C3W with other H-43b strains not carrying Kb. Thus, the possibility of immunodominance of Kb over the other MHC class I alleles could not be supported. We also examined possibility of epistatic effect of I region genes and non-MHC genes on the Kb restriction. (C3W x C57BL/6)F1(I-Ak/b) and (C3W x B6.CH-2bm12)F1(I-Ak/bm12)mice showed equally anti-H-43a CTL response restricted exclusively by self Kb, and (C3W x B10.MBR)F1(Ik/k) mice also showed anti-H-43a CTL response restricted solely by self Kb. Cold target competition experiments demonstrated that H-43b C57BL/10 or A.BY mice, which do not have non-MHC genes of C3H mounted anti-H-43a CTL response restricted solely by self Kb. Thus, no relation of I region genes or non-MHC genes to the Kb restriction was shown. All the results indicate that H-43b mouse strains, including F1, can not achieve anti-H-43a CTL response unless they carry Kb allele. Notably, (C3W x C57BL/6)F1 mice mounted self Kb-restricted anti-H-43a CTL response, whereas (C3W x B6.CH-2bm1)F1 mice carrying mutated Kb could not mount anti-H-43a CTL response at all. These findings indicate strongly that Kb itself is classical Ir gene of anti-H-43a CTL response and directs self Kb restriction of the response.
cAMP-independent effects of cholera toxin on B cell activation. III. Cholera toxin A subunit-mediated ADP-ribosylation acts synergistically with ionomycin or IL-4 to induce B cell proliferation.
Authors: Francis M L , Okazaki I , Moss J , Kurosky A , Pecanha L M , Mond J J
Published: December 2022

To investigate whether ADP-ribosylation of proteins by cholera toxin could influence B cell activation, B cells were incubated with the A subunit of cholera toxin. Ionomycin acted synergistically to induce B cell proliferation with the A subunit of cholera toxin but not with cAMP-enhancing agents or with the B subunit of cholera toxin, suggesting that the synergistic effect of the A subunit was mediated via ADP-ribosylation and not via cAMP elevations or ganglioside GM1 binding. Indeed, inhibitors of ADP-ribosylation blocked the synergistic effect. Unlike anti-Ig, B cell proliferation stimulated by LPS or by the combination of the A subunit and ionomycin was observed in protein kinase C (PKC)-depleted B cells. However, neither the A subunit nor ionomycin enhanced B cell proliferation stimulated by low dose LPS, suggesting that the A subunit plus ionomycin stimulated an activation pathway distinct from the LPS-stimulated pathway. Additionally, unlike LPS, the A subunit plus ionomycin did not stimulate B cells in vitro to secrete Ig. IL-4 acted synergistically with the A subunit to induce B cell proliferation to the same extent as it did with anti-Ig; unlike the anti-Ig plus IL-4 synergy, however, the A subunit plus IL-4-mediated synergy persisted in PKC-depleted B cells. Taken together, our data suggest that cholera toxin A subunit-catalyzed ADP-ribosylation modifies a non-Gs protein involved in the activation of B cells, either through a novel pathway or at a point distal to the activation of PKC along the anti-Ig-stimulated pathway.
Nerve growth factor induces activation of MAP-kinase and p90rsk in human B lymphocytes.
Authors: Franklin R A , Brodie C , Melamed I , Terada N , Lucas J J , Gelfand E W
Published: December 2022

A previous report from this laboratory demonstrated that human B lymphocytes expressed nerve growth factor (NGF) receptors on their surface. On the basis of NGF enhancement of B cell proliferation these receptors are presumed to be functional. We have now characterized one of the signaling pathways that NGF may utilize in the functional activation of B lymphocytes. Stimulation of three different human B-lymphoblastoid cell lines with NGF induced the tyrosine phosphorylation and activation of the p42erk-2 isoform of MAP-kinase (MAPK). In addition, NGF induced shifts in the mobility of p90 ribosomal S6 kinase (p90rsk) on immunoblots and increased p90rsk kinase activity in immunoprecipitates. NGF-induced shifts in p90rsk mobility displayed similar dose and time kinetics as NGF-induced MAPK activation. Activation of both MAPK and p90rsk occurred with doses of NGF as low as 400 pg/ml. Preincubation of NGF with anti-NGF Ab inhibited NGF-induced activation of MAPK and p90rsk. These results demonstrate that the interaction of NGF with its receptor on human B cells results in the stimulation of major components of the signaling pathway also initiated by NGF-receptor ligation in cells of neuronal origin.
IL-9 induces expression of granzymes and high-affinity IgE receptor in murine T helper clones.
Authors: Louahed J , Kermouni A , Van Snick J , Renauld J C
Published: December 2022

Interleukin 9 (IL-9) is a TH2 cytokine that has been shown to promote the antigen-independent growth of some mouse T helper clones. To characterize the specificity of IL-9-mediated T cell activation, we used a murine T cell clone that could grow with either IL-9 or IL-2. After differential hybridization of a cDNA library, we isolated three genes that were expressed preferentially in the presence of IL-9. Two of them correspond respectively to granzyme A and granzyme B, two proteases expressed by activated T cells. By Northern blot hybridization and functional assays, we found that IL-9 induced the expression of granzyme B in several T cell clones as well as in mast cell lines. In addition, other proteases such as the mouse mast cell proteases were also found to be expressed by IL-9-activated T cell clones. The third IL-9-induced cDNA corresponds to the alpha-chain of the high-affinity receptor for IgE. Several T cell clones expressed this IgE receptor mRNA and were able to bind IgE with high affinity. Taken together, our results indicate that IL-9 induces a mast cell-like phenotype in T cell clones.
Adoptive cell transfer of contact sensitivity-initiation mediated by nonimmune cells sensitized with monoclonal IgE antibodies. Dependence on host skin mast cells.
Authors: Matsuda H , Ushio H , Paliwal V , Ptak W , Askenase P W
Published: December 2022

A role for mast cell release of serotonin (5-HT), via Ag-specific factors derived from Thy-1+ B220+ lymphoid cells in the initiation of murine contact sensitivity (CS) has been suggested. However, because CS in mast cell-deficient mice was intact, a role for mast cells in CS initiation was unclear. Therefore, we examined whether CS could be initiated by i.v. injection of nonimmune mixed lymphoid cells that were sensitized in vitro with IgE. When naive mice received IgE-sensitized nonimmune spleen or lymph node cells, or IgE-sensitized purified mast cells, together with immune CS-effector B220- T cells, which therefore were depleted of CS-initiating, Thy-1+, B220+ cells, which could not transfer CS, then reconstitution of CS occurred. Mast cell-deficient W/Wv mice could not elicit this IgE-dependent CS ear swelling, but when mast cell deficiency was reversed by ear injection of normal bone marrow-derived cultured mast cells, then CS was restored. In vitro pretreatment with irrelevant monoclonal anti-OVA IgE prevented CS initiation mediated by Ag-specific, IgE mAb-sensitized cells, presumably by blocking sensitization with IgE. Thus Fc epsilon R on the normal lymphoid cells were involved. When ketanserin, a 5-HT2 receptor antagonist, was injected i.v. before cell transfer, CS initiation via IgE-sensitized cells and CS were no longer elicited. Thus, in this system, IgE Abs bound to circulating IgE Fc epsilon R bearing lymphoid cells sensitized in vitro (most likely basophils), probably mediated early activation of these circulating basophils to release mediators, causing 5-HT release from cutaneous mast cells, to mediate CS initiation.
Synergistic effects of IL-7 and IL-12 on human T cell activation.
Authors: Mehrotra P T , Grant A J , Siegel J P
Published: December 2022

We have previously demonstrated that human rIL-12 alone can augment the development of cytotoxic activity in stimulated CD8+ T cells. The present study was undertaken to examine the interactions of rIL-7 and rIL-12 on human peripheral blood T cell activation and CTL differentiation. Purified T lymphocytes were pulsed overnight with immobilized alpha-CD3 and then cultured for 3 additional days with IL-7 and/or IL-12. The combination of IL-7 and IL-12 synergistically enhanced the proliferation of either fresh CD3+ T cells or an IL-2-dependent CD4+ T cell line, Kit-225-K6. This synergy was seen on both subsets of T cells; however, CD8+ T cells were usually more responsive to IL-7 and IL-12 at lower concentrations than were CD4+ T cells. Furthermore, these cytokines additively/synergistically augmented the cytotoxic activity of CD8+ T cells. Abs to IL-2 and IL-2R alpha blocked the synergistic effect on proliferation of CD4+ T cells, but had a minimal effect on the synergistic response of the proliferative and cytotoxic activity of CD8+ T cells. Examination of the effects of IL-7 and IL-12 on the expression of IL-12 receptor on T cells revealed an increase in the subunit of IL-12R by IL-7 as determined by flow cytometric analysis. We analyzed the effects on IFN-gamma production by CD8+ T cells and found that IL-7 alone did not induce detectable levels of IFN-gamma production but together with IL-12 it synergistically enhanced the production of IFN-gamma. We also found that IFN-gamma was probably not required for enhanced CTL activity of CD8+ T cells, because Ab to human IFN-gamma did not block additive/synergistic effects of either cytokine. The synergistic stimulatory activity of IL-7 and IL-12 may be of significance in vivo and may provide an alternative mechanism of stimulating T cells for use in immunotherapy.
Cellular distribution and costimulatory function of rat CD28. Regulated expression during thymocyte maturation and induction of cyclosporin A sensitivity of costimulated T cell responses by phorbol ester.
Authors: Tacke M , Clark G J , Dallman M J , Hünig T
Published: December 2022

CD28 has been identified in man and mouse as a potent costimulatory receptor on T cells. We have generated a mAb, called JJ319, to rat CD28 and show that it is expressed on virtually all peripheral rat alpha beta and on most gamma delta T cells, and on about half of NK cells. In contrast to the mouse but as in humans, most immature CD4+8+TCRlow thymocytes express little or no CD28, whereas CD28 expression is high on TCRintermediate and TCRhigh cells. mAb JJ319 very effectively costimulates T cell proliferation and IL-2 secretion by resting rat T cells. In contrast to results obtained in mice and humans, phorbol ester did not synergize in T cell activation with CD28-specific mAb but even induced sensitivity to cyclosporin A in T cell cultures that were optimally costimulated by mAbs to the TCR and to CD28. This result points to a novel effect of protein kinase activation by phorbol ester on signal transduction by TCR plus CD28 costimulation which only becomes apparent if, as in the rat, the TCR-mediated signal cannot be replaced by phorbol ester.
T cell development in TCR-alpha beta transgenic mice. Analysis using V(D)J recombination substrates.
Authors: Capone M , Curnow J , Bouvier G , Ferrier P , Horvat B
Published: December 2022

The major pathway of intrathymic T cell differentiation leads CD4-8- (DN) T lineage-committed precursors to TCR-alpha beta+ CD4+8- or CD4-84+ (SP) T lymphocytes. The expression of functionally rearranged TCR-alpha beta transgenes (Tg-TCR) may influence thymocyte development by affecting the various selection events that control T cell differentiation. To gain insights into these processes, we have produced double transgenic animals carrying V(D)J recombination substrates in addition to the MHC class I (H-2Kb) allospecific KB5C20 Tg-TCR. We have analyzed substrate rearrangements in purified populations of Tg-TCR+ thymocytes in the situation of positive or negative selection. The profile of rearrangements found in SP thymocytes, positively selected for the Tg-TCR, suggests that expression of the KB5C20 Tg-TCR has only a minimal influence on substrate V(D)J recombination in cells differentiating along the major alpha beta T cell developmental pathway. In contrast, Tg-TCR+ DN thymocytes, in both positively and negatively selecting haplotypes, presented a profile that implies premature cessation of substrate rearrangements. This profile was maintained in peripheral Tg-TCR+ DN cells and was distinct from the one found in CD25+, alpha beta+, or gamma delta+ DN cells purified from mice transgenic for the recombination substrates only. These results are discussed with respect to the possible origin and differentiation pathway of Tg-TCR+ DN and SP cells.
Structure of the human C7 gene and comparison with the C6, C8A, C8B, and C9 genes.
Authors: Hobart M J , Fernie B A , DiScipio R G
Published: December 2022

The seventh component of complement is a single chain plasma glycoprotein that is involved in the cytolytic phase of complement activation. We have determined the structure of the C7 gene, which is encoded by 18 exons whose sizes vary from 56 to 244 bp. For the most part, the exons do not correspond to the protein homology units. However, two intron/exon boundaries occur at junctions between different functional parts of the protein. The first is at a site between the end of the C9 homology unit and the carboxyl-terminal extension which is also a feature of C6. The second of these boundaries occurs between the regions encoding two pairs of cysteine-rich modules (the short consensus repeats and the factor I modules) located in the carboxyl-terminal part of C7. In contrast to the exons, the introns range considerably in size from 0.5 to 8.5 kbp. The complete analysis indicates that the gene encoding C7 is approximately 80 kbp in length. We show here that the C7 gene is highly homologous to that for C6, and also to C8A, C8B, and C9, confirming and extending the published data. With the exception of exon 1, all intron/exon boundaries are preserved with respect to phase when compared with C6.
Myelin basic protein peptide complexes with the class II MHC molecules I-Au and I-Ak form and dissociate rapidly at neutral pH.
Authors: Mason K , Denney D W , McConnell H M
Published: December 2022

The acetylated N-terminal peptide of myelin basic protein (MBP) is the immunodominant T cell epitope in the induction of experimental autoimmune encephalomyelitis in the I-Au- and I-Ak-expressing mouse strains. We used a direct binding assay to examine the kinetics of binding and dissociation of a series of MBP peptide analogues with the affinity-purified class II MHC molecules I-Au and I-Ak. We observe much faster in vitro rates of binding and dissociation than has been reported previously for other immunogenic peptides at neutral pH. The kinetics also reveal inactivation of the peptide-free class II MHC molecules. These results are consistent with previously proposed mechanisms for tolerance escape and autoimmune disease.
DNA sequencing of the gene encoding a bacterial superantigen, Yersinia pseudotuberculosis-derived mitogen (YPM), and characterization of the gene product, cloned YPM.
Authors: Miyoshi-Akiyama T , Abe A , Kato H , Kawahara K , Narimatsu H , Uchiyama T
Published: December 2022

Previously, we found a novel bacterial superantigen from Yersinia pseudotuberculosis, designated Y. pseudotuberculosis-derived mitogen (YPM). In the present study, we analyzed the DNA sequence of the gene encoding YPM. The YPM gene was cloned into a plasmid vector pMW119 and expressed in Escherichia coli DH10B. Like the native YPM, the cloned YPM required the expression of MHC class II molecules on accessory cells in the induction of IL-2 production by human T cells. TCR-V beta repertoire of human T cells reactive with the cloned YPM was V beta 3, V beta 9, V beta 13.1, and V beta 13.2. This repertoire is the same as that of T cells reactive with the native YPM. These results indicate that the cloned YPM expressed in E. coli is identical to the native YPM. Sequencing of the YPM gene revealed that the gene contained an open reading frame of 456 base pairs encoding a precursor form of 151 amino acid residues with m.w. 16,679 that is processed into a mature form of 131 amino acid residues with m.w. 14,529. Homology analysis revealed that the homology of amino acid sequence is quite low among YPM and other well known bacterial superantigens. We designated the gene encoding YPM as ypm.
The interferon-stimulated response element and a kappa B site mediate synergistic induction of murine IP-10 gene transcription by IFN-gamma and TNF-alpha.
Authors: Ohmori Y , Hamilton T A
Published: December 2022

The present study investigates mechanisms involved in cooperation between IFN-gamma and TNF-alpha to promote transcription from the IP-10 gene in NIH 3T3 cells. IFN-gamma synergistically enhanced TNF-alpha-induced levels of IP-10 mRNA, whereas levels of JE (MCP-1) or KC (GRO/MGSA) mRNA induced by TNF-alpha were unaffected by IFN-gamma. The cooperation between IFN-gamma and TNF-alpha for induction of IP-10 mRNA was independent of de novo protein synthesis and mediated at least in part by increased transcription. Transient transfection analysis with a 243-bp fragment flanking the transcription start site of the murine IP-10 gene indicated that synergy between the two stimuli was dependent upon occupancy of at least two of three critical regulatory sequence elements: an IFN-stimulated response element (ISRE) and one of two kappa B sites. IFN-gamma and TNF-alpha independently activated nuclear factors capable of specific interaction with the ISRE and kappa B sites, respectively. IFN-gamma induced two ISRE binding complexes, one of which was protein synthesis independent, appeared within 15 min of stimulation, and contained p91 or signal transducer and activator of transcription (STAT) 1. TNF-alpha induced only one ISRE binding activity, which was dependent upon protein synthesis. TNF-alpha also induced kappa B binding activity that was composed of NF-kappa B1 (p50) and RelA (p65) whereas IFN-gamma had no detectable effect on kappa B binding activity. Together these results indicate that the highly synergistic transcriptional activation of the IP-10 gene by IFN-gamma and TNF-alpha involves the cooperation between factors that are independently activated by the two stimuli and that bind to independent sites.
Effect of amino acid substitutions within the region 62-76 of I-A beta b on binding with and antigen presentation of Torpedo acetylcholine receptor alpha-chain peptide 146-162.
Authors: Oshima M , Atassi M Z
Published: December 2022

Previous study has shown that reduced T cell response to peptide alpha 146-162 of Torpedo californica acetylcholine receptor (tAChR) in B6.C-H-2bm12 (bm12) mice, a mutant of C57BL/6 (B6) mice, correlated with its nonsusceptiblity to experimental autoimmune myasthenia gravis. There are three amino acid differences between the I-A beta b of the two strains (positions 67, 70, and 71). We synthesized peptides I-A beta b62-76 (peptide b6), I-A beta bm1262-76 (peptide bm), and three additional peptides, b6(67F), b6(70Q), and b6(71K), and determined their ability to bind peptide alpha 146-162 and the dissociation constants (Kd) of the binding. Peptide alpha 146-162 bound with a significantly higher affinity to peptide b6 than to peptides bm or b6(71K), suggesting that the lower affinity of peptide alpha 146-162 to I-Abm12 is a factor in the reduced response to this peptide by bm12 T cells. This was confirmed by measurement of the Kd values of the binding of peptide alpha 146-162 to the I-A molecules of B6 and bm12. Furthermore, APC of bm12 presented the peptide, or tAChR, poorly to peptide-specific or to tAChR-specific B6 T cells. The major effect is caused by the change of Thr-71 in I-A beta b to lysine in I-A beta bm12. However, APC of B6 also presented peptide alpha 146-162 much less efficiently to peptide-specific T cells of bm12. This demonstrated that these three amino acid changes also influence the T cell receptor recognition of peptide-MHC complex and that both B6 and bm12 T cells recognizing peptide alpha 146-162 or tAChR are under a high H-2 restriction.
Assembly of IgM. Role of disulfide bonding and noncovalent interactions.
Authors: Wiersma E J , Shulman M J
Published: December 2022

Polymeric IgM is usually envisaged as an array of mu 2L2 monomers in which the mu heavy chains are held together by disulfide bonds involving cysteines at positions 337, 414, and 575. We have studied the importance of inter-mu-chain disulfide bonds for formation of IgM polymers and monomers by analyzing the effects of eliminating one or more of these disulfide bondings. Ablation of all inter-chain bonds by either chemical reduction and alkylation or by mutagenesis resulted in the exclusive production of halfmers (muL) molecules. IgM composed of mu-chains bearing each of the other six possible combinations of cysteine to serine replacements was produced as different mixtures of polymers, monomers, and halfmers. Cysteine 575 was both necessary and sufficient for efficient assembly of IgM polymers and sufficient but not necessary for efficient assembly of monomers. Cysteine 337 was sufficient but not necessary for efficient assembly of monomers, and was neither sufficient nor necessary for formation of polymers. Cysteine 414 was neither necessary nor sufficient for efficient formation of either monomers or polymers. Data also suggest that noncovalent interactions between C mu 2 domains are stronger than the interactions between C mu 4/tail domains.
IL-2 enhances the function of recombinant poxvirus-based vaccines in the treatment of established pulmonary metastases.
Authors: Bronte V , Tsung K , Rao J B , Chen P W , Wang M , Rosenberg S A , Restifo N P
Published: December 2022

Neoplastic cells are generally poor immunogens. Transfection of the murine tumor CT-26 with beta-galactosidase (beta-gal), a protein from Escherichia coli, did not alter its growth rate in vivo, or its lethality, and did not elicit a measurable anti-beta-gal immune response. Immunization with beta-gal-expressing recombinant vaccinia viruses (rVV) elicited specific anti-beta-gal cytolytic T lymphocytes, but rVV-beta-gal was only marginally therapeutic when given to tumor-bearing mice. With the aim of expanding the immune response against beta-gal, used here as a model tumor Ag, we gave mice exogenous IL-2 starting 12 h after the poxvirus. The therapeutic effectiveness of the combination of poxvirus and IL-2 was far greater than either of these treatments alone. When the cDNA for IL-2 was inserted into the viral genome of the rVV construct to make a double recombinant (drVV), antitumor activity was further augmented. One mechanism of action may be the enhanced activation or expansion of cytotoxic T cells, because a marked increase in primary cytotoxic responses against vaccinia determinants was observed. Interestingly, other cytokines (mGM-CSF, mTNF-alpha, and mIFN-gamma) inserted into the rVV genome did not modify the efficacy of the rVV constructs. The increase in specific CTL responses against beta-gal by drVV expressing the tumor-associated Ags (TAA) and IL-2 was more pronounced in mice bearing the lacZ-transduced tumor than in those bearing the parental cell line, suggesting that the TAA presented by growing tumor cells can either pre-activate or otherwise amplify the immune response induced by the rVV. Unfortunately, in several long-term surviving mice, tumor recurred that no longer expressed beta-gal. These results indicate that treatment of disseminated tumors by using recombinant viruses expressing TAA can be enhanced by IL-2 provided exogenously, or encoded within the recombinant virus.
Lymph node (but not spleen) invasion by murine lymphoma is both CD44- and hyaluronate-dependent.
Authors: Zahalka M A , Okon E , Gosslar U , Holzmann B , Naor D
Published: December 2022

Similar to activated T cells, LB T cell lymphoma expresses the CD44 cell surface Ag. In addition, the vast majority of LB cells also express the beta 2 (CD18) and alpha L (CD11a) chains of LFA-1 integrin. In view of the finding that anti-CD18 mAb blocked spleen, but not lymph node invasion by LB cells inoculated s.c. into BALB/c mice, we tested the ability of anti-CD44 mAb (IM 7.8.1) to block the infiltration of LB cells into the lymph nodes. We found that, as opposed to anti-CD18 mAb, anti-CD44 mAb, as well as its F(ab')2 or Fab fragment, prevented lymph node infiltration but had no effect on spleen invasion. This conclusion was based on histologic examination and [3H]thymidine incorporation into proliferating LB cells invading the lymphoid organs. Histologic analysis further demonstrated that LB cells invade the lymph node via the afferent lymphatics. The surface expression of CD44 molecules on LB cells was enhanced after PMA activation. PMA activation also enabled in vitro binding of the lymphoma to hyaluronic acid (HA), a known ligand of CD44. Because anti-CD44 mAb, its F(ab')2 or Fab fragment, and hyaluronidase blocked this binding, we also tested the ability of the enzyme to inhibit lymph node invasion by LB cells. We established through histologic examination and [3H]thymidine incorporation that hyaluronidase protected the lymph node, but not the spleen, from invasion by the lymphoma.
Biologic activities of the beta-chemokine TCA3 on neutrophils and macrophages.
Authors: Devi S , Laning J , Luo Y , Dorf M E
Published: December 2022

Previous in vivo and in vitro studies demonstrated that the murine beta-chemokine TCA3 is a chemoattractant for monocytes/macrophages and neutrophils. The ability of TCA3 to activate these cell populations is now evaluated. Treatment with 10 to 20 nM rTCA3 induced a respiratory burst with the production of superoxide and hydrogen peroxide in both casein-elicited and unstimulated neutrophil and macrophage populations. In addition, TCA3 treatment induced the production of reactive nitrogen intermediates, whereas stimulation with higher concentrations (100 nM) of TCA3 induced the exocytosis of lysozyme and elastase in the presence of cytochalasin B (7 micrograms/ml). Subnanomolar concentrations (100 pM) of TCA3 also caused integrin-mediated increases of adhesiveness to fibrinogen by neutrophils and macrophages. Increased adhesiveness is the most sensitive assay for TCA3 bioactivity. TCA3 treatment appears to involve signaling through a G-protein-linked receptor as Pertussis toxin abolished the TCA3-mediated increase of adhesiveness and the production of reactive nitrogen intermediates. The dose dependence of the TCA3-mediated activities indicate a coordinated inflammatory response mediated by varying concentrations of TCA3.