bluuebunny/crossref_metadata_2025 huggingface dataset:
Lack of target cell participation in cytotoxic T lymphocyte-mediated lysis.
Authors: Ratner A , Clark W R
Published: December 2022

Data on the subject of cell-mediated cytotoxicity suggest that no single mechanism is likely to provide a satisfactory explanation of this process. Lytic pathways have been proposed that involve both the effector cell and the target cell as active participants. In this report we describe a system in which the target cell is rendered unable to participate in its own demise. Using sheep E derivatized with CD3 antibodies, we show that metabolic inhibition of SRBC by depleting intracellular ATP with iodoacetamide, or even conversion of SRBC to "ghosts" by hypotonic lysis and resealing, has no effect on cytolysis. In the presence of EGTA or cholera toxin, both of which inhibit CTL degranulation, there is a strong suppression of both serine esterase release and cytolysis. These data show clearly that in some situations CTL are able to lyse target cells without any active participation by the target cells themselves.
Trichophyton tonsurans allergen. I. Characterization of a protein that causes immediate but not delayed hypersensitivity.
Authors: Deuell B , Arruda L K , Hayden M L , Chapman M D , Platts-Mills T A
Published: December 2022

Fungal infections of skin or nails are extremely common and often caused by dermatophyte fungi of the genus Trichophyton. These fungi are unusual in that they can give rise to delayed hypersensitivity (DH) or immediate hypersensitivity (IH) responses. Recently, IH to Trichophyton tonsurans has been demonstrated in patients by skin tests, serum IgE antibody test (RAST), and positive nasal and bronchial challenges. To further investigate the immunology of Trichophyton, a 30-kDa T. tonsurans allergen was isolated by gel filtration and hydrophobic interaction chromatography. This protein, Tri t I, gave a single band on SDS-PAGE, and the 30 amino-terminal amino acids have been determined. Among patients with positive IH skin tests, 34 of 48 (71%) had IgG antibody and 26 of 48 (54%) had IgE antibody to Tri t I. Among those who had positive responses to both skin tests and RAST, 22 of 30 (73%) had IgE antibodies to Tri t I; thus, this protein represents a major allergen. Twelve clones of murine IgG mAb antibodies were produced. Two clones, 2F2-F7 and 6B11-C2, were found to define separate epitopes on Tri t I and were used to develop an immunometric assay for the quantitation of Tri t I. Twenty-three of 38 volunteers with a history of athlete's foot were found to have either IH and/or DH to Trichophyton mix and underwent further testing with purified Tri t I. Of the nine found to have IH to the mix, eight were sensitive to Tri t I. Seven of these eight had IgG and IgE antibodies to Tri t I, by Ag-binding RIA, and all were RAST positive to the unpurified extract. An additional 14 had either DH alone (n = 7) or a wheal and flare response followed by DH at 48 h (n = 7). Of these 14 who had DH responses to Trichophyton mix, only one showed DH to an equivalent quantity of purified Tri t I; among this group, none showed IH or serum IgE antibodies and only one had detectable IgG antibody to Tri t I. The results suggest that the majority of subjects with DH to Trichophyton are responding to a protein other than Tri t I and that the wheal that precedes DH reactions is some patients is not associated with IgE antibodies.
Mutation of the disulfide loop in staphylococcal enterotoxin A. Consequences for T cell recognition.
Authors: Grossman D , Van M , Mollick J A , Highlander S K , Rich R R
Published: December 2022

The hallmark of T cell responses to staphylococcal enterotoxins (SE) and other super-Ag is a selective stimulation of cells expressing particular TCR-V beta segments. Our previous studies suggested that the disulfide loop in SE is critical for their interaction with the TCR. To investigate this concept in further detail we constructed disulfide loop mutants of staphylococcal enterotoxin A (SEA), and examined these altered toxins for mitogenicity, class II MHC binding, and V beta specificity. We found that substitutions of either Cys-96 or Cys-106 decreased mitogenicity by 100-fold without significantly affecting class II binding or resistance of the molecule to proteolysis. Several mutants lost the capacity to stimulate V beta 11+ cells, except a Cys-106----Gln mutant for which V beta 11-stimulatory activity was increased. By contrast, mutants containing Cys----Ala substitutions acquired the capacity to stimulate V beta 6+ cells. Despite these effects of V beta specificity, all mutants retained the predominant preference of SEA for V beta 3+ cells. Neither exchange of regions flanking the loop in SEA with corresponding residues in SEB, nor conversion of the entire loop region of SEA to that of SEE, were associated with transfers of V beta specificity. Our results suggest that the disulfide loop in SEA contributes to toxin avidity for the TCR, rather than specificity for particular V beta.
Development of a bone marrow culture for maintenance and growth of normal human B cell precursors.
Authors: Wolf M L , Buckley J A , Goldfarb A , Law C L , LeBien T W
Published: December 2022

The absence of long term bone marrow cultures for studying the growth and differentiation of human B cell precursors (BCP) has placed restrictions on the ability to analyze the early stages of human B cell ontogeny. We now describe a bone marrow-derived adherent cell microenvironment that maintains human BCP for several weeks in vitro. The adherent cells are maintained in a serum-free tissue culture medium, and consist of a predominant population of CD10+ fibroblast-like cells and a minor population of CD10+/nonspecific esterase+ macrophages. Adherent cell cultures seeded with fresh or cryopreserved fetal bone marrow, or purified CD10+/surface IgM- cells, provide a supportive microenvironment for lymphoid cells with a predominant phenotype of CD10+/CD19+/HLA-DR+/surface IgM-. Supplementation of the adherent cell cultures with human IL-7 induces active growth of BCP during the first 14 to 21 days of culture. However, the expansion of these cells does not continue past 21 days, and the cultures undergo a steady decline in BCP. Analysis of adherent cell conditioned medium revealed the presence of an unidentified soluble factor (or factors) that acts in concert with IL-7 to promote the growth of CD10+/surface IgM- cells. This culture system will be useful in elucidating the patterns of gene expression and growth factor requirements that characterize normal human B cell ontogeny, and perturbations of normal B cell ontogeny that lead to immunodeficiency and leukemia.
Differentiation in mature T lymphoid leukemia cells is unstable and reversible to myeloid cells, without the involvement of a common stem cell.
Authors: Griesinger F , Jansen B , Kersey J H
Published: December 2022

Mechanisms for the unstable and reversible differentiation of a mature CD3+ CD7+TCR-alpha/beta+ T lymphoid leukemia into the myeloid lineage were investigated. Inasmuch as productive rearrangement of the TCR-alpha is a late determinant of T cell differentiation, the TCR-alpha rearrangement was sequenced to determine the state of differentiation of the leukemic multipotent cell. An identical productive rearrangement of J alpha C to a novel V alpha region was found in the myeloid and T lymphoid leukemic cells. Thus, a "terminally" differentiated T lymphoid leukemic cell after productively rearranging TCR-alpha and -beta continues to display potential for multilineage differentiation. Therefore, multilineage potential is due to an unstable and reversible differentiation in a mature T lymphocyte as opposed to differentiation of an uncommitted common T and myeloid precursor cell.
The IL-2 receptor alpha-chain alters the binding of IL-2 to the beta-chain.
Authors: Arima N , Kamio M , Okuma M , Ju G , Uchiyama T
Published: December 2022

The binding of IL-2 to its high affinity receptor results in the formation of the ternary complex consisting of IL-2, alpha-chain (p55, Tac) and beta-chain (p75). We studied the role of alpha-chain in IL-2 binding to the high affinity receptor using IL-2 analog Lys20 which was made by the substitution of Lys for Asp20 of wild-type rIL-2. Lys20 bound to MT-1 cells solely expressing alpha-chain at low affinity, but did not bind to YT-2C2 cells which solely expressed beta-chain. However, direct binding of radiolabeled Lys20 to ED515-D cells, an HTLV-I-infected and IL-2-dependent T cell line, revealed both high affinity and low affinity binding although the Kd value of high affinity binding was 50 to 100 times higher than that of the high affinity binding of wild-type rIL-2. High affinity binding of Lys20 was completely blocked by 2R-B mAb recognizing IL-2R beta-chain. Anti-Tac mAb recognizing IL-2R alpha-chain abolished all of the specific Lys20 bindings. In contrast to the replacement of cell bound 2R-B mAb with wild-type rIL-2 at 37 degrees C, the addition of an excess of Lys20 did not cause the detachment of cell-bound radiolabeled or FITC-labeled 2R-B mAb. Consistent with the results of binding studies, Lys20 induced the proliferation of ED515-D cells, but not large granular lymphocyte leukemic cells. The growth of ED-515D cells was completely suppressed by either anti-Tac mAb or 2R-B mAb. These results strongly suggest that coexpression of the IL-2R alpha- and beta-chains alters the binding affinity of Lys20 and that the interaction between IL-2 and the alpha-chain is a key event in the formation of the IL-2/IL-2R ternary complex.
Differential expression of Q4 proteins (Qb-1) in fibroblasts and lymphocytes.
Authors: Day P M , Frelinger J A
Published: December 2022

The Qb-1 protein is coded for by the Q4 gene. This gene appears to be widely transcribed in murine tissues. We have examined the subcellular localization and processing of Qb-1 in fibroblast cells. This protein has previously been reported to be secreted from activated lymphocytes. However, we report that high levels of Qb-1 are present on the surface of B10.P fibroblast cells. We also observed an unusual intracellular distribution for Qb-1. The protein appears to be highly concentrated in the endoplasmic reticulum, in addition to being on the surface. This distribution is not due to inefficient processing. The kinetics of Qb-1 processing and transport are not unusual for class I molecules. Complete resistance to endo-beta-N-acetylglucosaminidase H is acquired, indicating terminal processing. This unusual localization in fibroblast cells and the differential expression of this protein between cell types may reflect a specific role for Qb-1.
Protection of mice from fatal Toxoplasma gondii infection by immunization with p30 antigen in liposomes.
Authors: Bülow R , Boothroyd J C
Published: December 2022

Using liposomes as adjuvant, a purified membrane Ag from Toxoplasma gondii (p30) has been tested for its protective effect in mice. Immunization with p30 in liposomes resulted in only one in 15 mice dying from a challenge that killed 11 of 15 control mice (receiving only buffer or liposomes without p30). The p30 Ag alone gave intermediate levels of protection, with 5 of 15 animals dying. The source of the p30 Ag was the rapidly growing, laboratory-adapted strain of T. gondii, RH; challenge was with the recently isolated C strain which still has all the properties of a wild-type strain. To assess the validity of this combination, the amino acid sequence of p30 from these two strains (as predicted from the corresponding gene sequence) was compared and found to differ in only eight residues. This minimal variation argued that RH was a valid source of material for a subunit vaccine, as subsequently confirmed by the protection studies. These results indicate that p30 is an appropriate Ag for development into a subunit vaccine for immunization of humans and/or domestic livestock, which are a major source of human infection.
A purified parasite antigen (p30) mediates CD8+ T cell immunity against fatal Toxoplasma gondii infection in mice.
Authors: Khan I A , Ely K H , Kasper L H
Published: December 2022

Induction of protective immunity against acute and chronic toxoplasmosis can be achieved using p30, the major membrane and excreted/secreted protein of Toxoplasma gondii. This protein, when administered to outbred mice in the presence of the saponin Quil A, is able to induce almost 100% protection against acute infection without evidence of intracerebral cyst development. Adoptive transfer of immune splenocytes from immunized inbred A/J mice conferred a significant level (p less than 0.001) of protection against subsequent challenge. Phenotypic analysis in outbred as well as two different strains of inbred mice (A/J and C57BL/6) demonstrated that CD8+ T cells are selectively stimulated by this immunization protocol. T cell depletion studies using specific mAb directed at either CD3+ or CD8+ T cell phenotype, followed by adoptive transfer, failed to confer protective immunity, whereas CD4+ depletion had no effect. These cytotoxic CD8+ T cells produced high titers of both IFN-gamma and IL-2. Moreover, these CD8+ T cells were directly parasiticidal against radiolabeled extracellular T. gondii, further supporting the critical immune function of these p30 Ag-specific CD8+ T cells in host immunity against T. gondii infection.
Vaccine-induced CD4+ T cells against the simian immunodeficiency virus gag protein. Epitope specificity and relevance to protective immunity.
Authors: Mills K H , Barnard A L , Williams M , Page M , Ling C , Stott E J , Silvera P , Taffs F , Kingsman A S , Adams S E
Published: December 2022

We have examined the induction and epitope specificity of T cells for the simian immunodeficiency virus (SIV) gag p27 protein in macaques immunized with either a recombinant SIV gag protein or an inactivated SIV vaccine. CD4+ MHC class II-restricted T cell lines and clones derived from five immunized macaques recognized a total of seven peptides in three immunodominant regions of p27. Two T cell clones generated from one of the lines, recognized a single 20 amino acid peptide that overlapped with a region previously shown to include a CTL epitope from SIV-infected macaques. Although this epitope is in a conserved region of the gag protein of SIV, its recognition by a CD4+ T cell clone was abrogated by sequence variation in the equivalent HIV protein. The specificity of the T cell lines for synthetic peptides demonstrated considerable overlap between T cells generated by immunization with the recombinant gag protein and inactivated SIV. However, in contrast to the protective efficacy of the whole virus vaccine in the syntex adjuvant formulation, immunization with the p27 protein with alum failed to generate a protective immune response. Furthermore, despite the consistent gag-specific T cell responses induced by the recombinant protein, there was no evidence of an enhanced antibody response to envelope (env) after live SIV challenge.
Epitope mapping of the Syrian hamster prion protein utilizing chimeric and mutant genes in a vaccinia virus expression system.
Authors: Rogers M , Serban D , Gyuris T , Scott M , Torchia T , Prusiner S B
Published: December 2022

The cellular prion protein (PrPc) is a host-encoded sialoglycoprotein bound to the external surface of the cell membrane by a glycosyl phosphatidylinositol anchor. A posttranslationally modified PrP isoform (PrPSc) is a component of the infectious particle causing scrapie and the other prion diseases. mAb have been raised against the protease-resistant core of Syrian hamster (SHa) PrPSc designated PrP 27-30. To map the epitopes within PrP reacting to these antibodies, we have expressed wild-type, chimeric mouse (Mo)/SHa and mutant MoPrP genes using recombinant vaccinia virus systems. The fidelity of the expression of recombinant PrPC was examined using vaccinia viruses expressing SHa-PrPC. It is full length, possesses Asn-linked carbohydrates and is attached to the external surface of the cell membrane by a glycosyl phosphatidylinositol anchor that is sensitive to cleavage by phosphatidylinositol-specific phospholipase C. We have tested 18 mAb for their ability to bind to chimeric prion proteins on immunoblots. Three distinct epitopes were identified that mapped to amino acid differences between SHa and MoPrP sequences. The first epitope, recognized by three of the antibodies tested, was defined by methionines at amino acids 108 and 111 in the mouse protein. The second epitope was dependent upon the presence of asparagines at positions 154 and 174 in MoPrP and was recognized by four of the antibodies tested. The third epitope mapped to a single amino acid substitution at residue 138 in MoPrP. mAb raised against SHaPrP 27-30 specific for this epitope are able to bind MoPrPC which has a single amino acid change (Ile to Met) at position 138. Eleven of the 18 antibodies tested mapped to this immunodominant epitope. It is located within a postulated amphipathic helix, a structure associated with immunodominant Ag. Inasmuch as PrPC, in its native form on the cell surface, is detected by the mAb 13A5 (a prototypic antibody of the immunodominant third epitope class), it is likely that this epitope is accessible in the native conformation of this protein.
Transforming growth factor-beta modulates T cell-mediated encephalitis caused by Borna disease virus. Pathogenic importance of CD8+ cells and suppression of antibody formation.
Authors: Stitz L , Planz O , Bilzer T , Frei K , Fontana A
Published: December 2022

Borna disease is a virus-induced, immune-mediated encephalomyelitis based on a delayed-type hypersensitivity reaction. The severity of clinical symptoms after intracerebral infection of rats with Borna disease virus was reduced after treatment with transforming growth factor (TGF-beta 2). Intraperitoneal injection of the recombinant molecule, rTGF-beta 2, started on the day of infection at a dose of either 1 micrograms given every day or every other day for 8 consecutive days or 2 micrograms every third day, was found to result in the absence of typical Borna disease symptoms at 14 days after infection in most of the TGF-beta-treated rats, a time point at which all infected control animals not treated with rTGF-beta 2 showed distinct signs of Borna disease. The inhibition of the disease was paralleled by a significant reduction of the inflammatory reaction in the brain. However, the efficacy of treatment with rTGF-beta 2 was transient, because after day 21 only a slight or no reduction of the inflammatory reaction and, consequently, symptoms of Borna disease could be observed. Immunohistologic investigations revealed reduced CD4+ T cell numbers and no changes in macrophage counts in encephalitic lesions of rTG-beta treated rats. However, CD8+ cells were markedly decreased in the encephalitic lesions. Furthermore, the expression of MHC class II Ag was significantly reduced in the brain of rTGF-beta 2 treated Borna disease virus-infected rats, whereas MHC class I Ag expression was not. Most treated animals showed a reduction of Borna disease virus-specific serum antibodies, the result of an inhibition of the IgG response. The results presented here suggest a distinct influence of rTGF-beta 2 on T cell-mediated immune functions during the early phase of Borna disease virus-induced encephalomyelitis.
Specific pathogen-free BALB/cAn mice are refractory to plasmacytoma induction by pristane.
Authors: Byrd L G , McDonald A H , Gold L G , Potter M
Published: December 2022

Forty to sixty percent of conventionally (CON) raised BALB/cAnPt (BALB/c) mice develop plasmacytomas (PCT) when injected with three 0.5 ml i.p. injections of pristane. When CON-BALB/c mice were converted to specific pathogen free (SPF) status by foster nursing caesarean delivered term mice on C3H/HeN SPF mothers and maintained under strict SPF conditions, less than 5% of the mice developed pristane-induced PCT. FACS analysis of the cellular composition of oil granulomatous tissue revealed a dramatic influx of CD4+ cells in CON mice that was significantly reduced in SPF mice. Moreover, while both CON and SPF mice had similar patterns of gut flora colonization, only CON-BALB/c mice had occasional circulating antibodies to mouse hepatitis virus and Sendai viruses. Maintenance in strict SPF conditions, therefore, results in a prolonged state of relative Ag deprivation and a failure to continuously activate new T and B cell populations. The results suggest that PCT formation depends on exogenous antigenic stimulation and that the presence of minimal gut flora is insufficient to render these mice susceptible to PCT induction.
Identification and characterization of a T cell-inducing epitope of bovine ribonuclease that can be restricted by multiple class II molecules.
Authors: Chen J S , Lorenz R G , Goldberg J , Allen P M
Published: December 2022

An immunodominant epitope of bovine RNase restricted by I-Ek molecules was identified using a T cell hybridoma recognizing RNase. This epitope was localized to the peptide RNase(90-105). Single conservative amino acid substitutions were made at each of the positions 94 through 105. It was found that only at one position, Asn-103, were conservative substitutions not allowed. This residue was shown to be the critical residue in determining T cell specificity. The ability of RNase(90-105) and the well-defined T cell epitope, HEL(46-61) to stimulate mouse strains expressing different independent H-2 haplotypes was examined using a T cell proliferation assay. The response to HEL(46-61) was completely restricted to mice expressing an I-Ak molecule. In striking contrast, 6 of 10 different mouse strains, H-2b,f,k,q,s,u, mounted vigorous T cell responses to RNase(90-105). The response was restricted to both I-A and I-E molecules, including I-Ab, I-Af, I-Ek, I-Aq, and I-As. H-2d mice were nonresponders to RNase(90-105), which was shown to be due to the failure of RNase(90-105) to bind to I-Ad molecules. A variant RNase(90-105) peptide was generated, containing an I-Ad binding motif, that could bind to I-Ad molecules. Despite its ability to bind, this variant peptide was not able to stimulate a response in H-2d mice. This result demonstrates that the ability of a peptide to bind to an Ia molecule is necessary but not always sufficient for a response to occur. Thus, in contrast to the highly restricted HEL(46-61) determinant, the RNase(90-105) determinant is permissive in its binding to Ia molecules. These results show that in the universe of T cell inducing epitopes contains both highly restricted and broadly restricted epitopes are found.
IgE and IgG binding of peptides expressed from fragments of cDNA encoding the major house dust mite allergen Der p I.
Authors: Greene W K , Cyster J G , Chua K Y , O'Brien R M , Thomas W R
Published: December 2022

Large peptides expressed from cDNA fragments of a clone encoding the mite allergen Der p I were able to bind IgE and IgG in sera from allergic individuals. The binding was found for peptides from sequences throughout the molecule, with at least five regions, comprising residues 1-56, 53-99, 98-140, 166-194, and 188-222. The only limitation was that more than 30 amino acid residues were required for consistent binding. Each of seven sera examined showed a different profile of antibody binding to the peptides. For the most part the pattern of IgE and IgG binding to the peptides for each serum was similar, demonstrating a concordant repertoire. In 5/7 sera, however, IgG bound to some peptides which had little or no IgE binding activity, thus showing more diverse specificities. It is suggested that some divergence of repertoire can develop during the maturation of the B cell response.
Tumor necrosis factor-alpha regulates expression of receptors for formyl-methionyl-leucyl-phenylalanine, leukotriene B4, and platelet-activating factor. Dissociation from priming in human polymorphonuclear neutrophils.
Authors: O'Flaherty J T , Rossi A G , Redman J F , Jacobson D P
Published: December 2022

TNF-alpha enhances polymorphonuclear responses to many stimuli, including chemotactic peptide FMLP. It also promotes expression of FMLP receptors and thus may prime polymorphonuclear neutrophils to this and other agonists by up-regulating signal recognition molecules. However, we find that the cytokine's actions on FMLP receptors lagged priming of FMLP-induced degranulation. Moreover, TNF-alpha enhanced degranulation responses to leukotriene B4 and platelet-activating factor but paradoxically down-regulated leukotriene B4 receptors and only transiently up-regulated platelet-activating factor receptors. Hence, TNF-alpha has pleiotropic effects on receptor expression; these effects diverge from priming; and a large part of the primed state must reflect enhancement of post-receptor events.
Rapid increases in the membrane expression of neutral endopeptidase (CD10), aminopeptidase N (CD13), tyrosine phosphatase (CD45), and Fc gamma-RIII (CD16) upon stimulation of human peripheral leukocytes with human C5a.
Authors: Werfel T , Sonntag G , Weber M H , Götze O
Published: December 2022

Rapid increases in the membrane expression of C3 receptors on granulocytes and monocytes in response to the anaphylatoxin C5a have previously been described. In this study we demonstrate increases in the membrane expression of neutral endopeptidase (NEP, CD10, CALLA), aminopeptidase N (APN, CD13), tyrosine phosphatase (CD45/CD45Ro) and the Fc R Fc gamma-RIII (CD16) on granulocytes within minutes of treatment with human C5a. Monocytes responded to C5a with increases in CD13 and CD45/CD45Ro. These membrane modulations could be prevented by preincubating the C5a preparations with anti-C5a mAb C17/5 but not by pretreating the cells with cycloheximide. Increases of CD10, CD13, and CD11b but not CD11a (LFA-1) were also observed in leukocytes from patients undergoing hemodialysis with cuprophan membranes. The increase of CD16 on granulocytes was dependent on the presence of plasma during in vitro activation with C5a indicating that plasma contains inhibitors which prevent the previously described loss of Fc gamma-RIII upon stimulation of the cells.
Treatment of mice bearing BCL1 lymphoma with bispecific antibodies.
Authors: Brissinck J , Demanet C , Moser M , Leo O , Thielemans K
Published: December 2022

Bispecific antibodies with specificity for the CD3/TCR complex of CTL and a target cell Ag can bridge both cell types and trigger cellular cytoxicity. We have produced bispecific antibodies, directed against the surface-expressed Id of the mouse BCL1 lymphoma and the mouse CD3 complex, by hybrid-hybridoma fusion. Two recombination Ig were purified to homogeneity: B1 X 7D6F, which is univalent for Id and CD3 binding and B1 X 7D6M, which is univalent for Id binding but has lost the CD3 binding because of association of the anti-CD3 H chain with the inappropriate L chain. In vitro studies indicate that bridging the TCR/CD3 complex of resting T cells with tumor IgM Id and the appropriate bispecific antibody induced proliferation and secretion of IL-2. Furthermore, in cytotoxicity assays using 51Cr-labeled tumor cells, preactivated T cells could be targeted with the bispecific antibody to give complete lysis of the Ag+ tumor. Finally, the activity of the bispecific antibody was confirmed in vivo. Animals treated i.v. with 5 micrograms of bispecific antibody 9 days after receiving BCL1 cells were cured. Furthermore, when these animals were checked at 150 days for dormant or variant tumors, as have been reported after other forms of immunotherapy in this model, none could be found. Immunotherapy experiments comparing a mixture of control antibodies with the bispecific antibody demonstrate that tumor cell-T cell bridging is established in vivo and is required for therapeutic success. These results indicate the importance of bispecific antibodies as a novel form of treatment for cancer.
Prolonged expression of high molecular mass CD45RA isoform during the differentiation of human progenitor thymocytes to CD3+ cells in vitro.
Authors: Deans J P , Wilkins J A , Caixia S , Pruski E , Pilarski L M
Published: December 2022

CD45, the leukocyte common Ag, has been shown to characterize T cell development both within the thymus and among peripheral T cells. The work reported here demonstrates that human multinegative (MN) thymocytes, depleted of cells bearing CD3, CD4, CD8, and CD19, express predominantly the high molecular mass CD45RA isoform, and lack low molecular mass CD45RB isoforms and CD45R0 as detected by immunofluorescence. By immunoprecipitation of surface-labeled CD45 molecules from MN thymocytes, a proportion of the CD45 is in fact of low molecular mass but does not include epitopes recognized by CD45R0, nor by CD45RB mAb specific for the p190. This suggests either glycosylation variants of CD45RB/CD45R0 undetectable by our mAb, or underglycosylated CD45RA. MN thymocytes lack TCR-alpha beta mRNA confirming their early developmental stage. Upon culture with IL-2 or with mitogenic combinations of anti-CD2/CD28 mAb, MN thymocytes differentiate to acquire CD3, TCR-alpha beta, and in some cases CD4 and/or CD8. We have predicted that maintenance of CD45RA and lack of CD45R0 expression is fundamental to generative thymic development. If correct, this demands that unlike peripheral T cells, differentiation of MN thymocytes should be accompanied by prolonged expression of high molecular mass CD45 isoforms. Analysis of CD45 isoform expression during MN thymocyte development confirms this prediction and indicates that expression of CD45RA is maintained, at increasing density, for at least 8 to 12 days of culture. Unlike peripheral blood T cells, this is accompanied by the gradual acquisition of firstly the p190 isoforms of CD45RB and later by CD45R0, resulting in a population of CD3+TCR-alpha beta cells coexpressing CD45RA/RBp190/R0. Dot blot analysis of mRNA from differentiating MN thymocytes indicates prolonged expression of mRNA encoding CD45 exons a, b, and c, again in contrast to peripheral T cells which lose all mRNA for alternatively spliced CD45 exons within the first 24 h poststimulation. This is discussed in the context of negative selection during thymic development and interconversion of T cell subsets.
In vivo induction of cytotoxic T cell response by a free synthetic peptide requires CD4+ T cell help.
Authors: Fayolle C , Deriaud E , Leclerc C
Published: December 2022

Most attempts to induce CTL responses by in vivo priming with free synthetic peptides have been unsuccessful so far. However, two separate studies have recently succeeded in inducing antiviral CTL responses by immunizing mice with unmodified free synthetic peptides derived from nucleoproteins from either lymphocytic choriomeningitis virus or Sendai virus. In the present study, we have analyzed the cellular mechanisms by which the lymphocytic choriomeningitis virus synthetic peptide induced CTL responses. We demonstrated that this peptide, which was previously shown to be recognized by CD8+ T cells, also contains a helper CD4+ T cell epitope. It stimulates in vivo both CD4+ T cell-mediated CTL response. The in vivo elimination of CD4+ T cells by treatment with a mAb was shown to strongly reduce the antipeptide CTL response. This study therefore demonstrates that to be able to induce CTL responses, a peptide has to stimulate both CD4+ and CD8+ T cell subset.
A recombinant C-terminal fragment of staphylococcal enterotoxin A binds to human MHC class II products but does not activate T cells.
Authors: Hedlund G , Dohlsten M , Herrmann T , Buell G , Lando P A , Segrén S , Schrimsher J , MacDonald H R , Sjögren H O , Kalland T
Published: December 2022

Binding of staphylococcal enterotoxin A (SEA) to MHC class II encoded proteins is a prerequisite for its subsequent activation of a large fraction of T lymphocytes through interaction with variable segments of the TCR-beta chain. We cloned SEA in Escherichia coli and produced four recombinant fragments covering both the N- and C-terminal regions. These fragments were used to analyze the interaction between SEA and the human MHC class II products. A C-terminal fragment of SEA, representing amino acids 107-233 bound to HLA-DR and HLA-DP but did not activate T cells. The three other fragments (amino acids 1-125, 1-179 and 126-233) neither bound to MHC class II Ag nor activated T cells. SEA apparently bind to HLA-DR and HLA-DP through its C-terminal part, whereas T cell activation is dependent on additional parts of the protein.
Chemically modified antigen-presenting cells induce T lymphocyte allospecific hyporesponsiveness.
Authors: Ibrahim M A , Coode W K , Takei F , Ellis J S , Chain B M , Katz D R
Published: December 2022

We have investigated the interaction between murine T lymphocytes and allogeneic APC in an in vitro proliferative mixed leukocyte reaction. Our results demonstrate that freshly isolated potentially alloreactive murine splenic T lymphocytes, in primary culture, can be induced to develop a state of allospecific proliferative hyporesponsiveness in vitro by exposure to 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide-modified allogeneic APC, a method similar to that previously used to induce nonresponsiveness in murine Ag-specific self-MHC-restricted T lymphocyte clones. This hyporesponsiveness was: specific for the allohaplotype of inducing APC, maintained for 96 h in vitro, not due to cellular inhibitory mechanisms, and associated with reduced ability to secrete IL-2 but not IL-3. Induction of this hyporesponsiveness was not due to altered expression of class II MHC gene products on the APC but was associated with markedly reduced T lymphocyte-APC adhesive interactions despite the lack of a detectable immunophenotypic change in lymphocyte function-associated Ag 1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1) expression on the modified APC. Therefore, we propose that TCR occupancy in the absence of normal T lymphocyte-APC adhesive clustering may induce T lymphocyte tolerance.
Induction of in vivo hyporesponsiveness to contact allergens by hapten-modified Ia+ keratinocytes.
Authors: Gaspari A A , Katz S I
Published: December 2022

Because our previous in vitro studies of hapten-modified Ia+ keratinocytes (KC) indicated that these cells induced anergy in Ag-specific Th1 cells, we assayed such cells for their ability to induce unresponsiveness in an in vivo animal model system of delayed type hypersensitivity (allergic contact dermatitis). Naive animals that were treated with i.p. injections of FITC-modified Ia+ cultured Langerhans cells (cLC) developed allergic contact dermatitis to subsequent hapten challenge; whereas, animals treated with similar doses of FITC-Ia+ KC failed to become sensitized to epicutaneous application of FITC, as evidenced by absent ear swelling responses to a FITC challenge. Those animals that were first treated with intraperitoneal injections of hapten modified Ia+ KC could not be sensitized when they were subsequently exposed to sensitizing doses of FITC; whereas a similar first exposure to FITC-cultured Langerhans cells did not interfere with epicutaneous sensitization. This hyporesponsiveness to sensitization was hapten specific, as FITC-Ia+ KC-treated animals were hyporesponsive only to FITC but not to the irrelevant hapten, TNCB. Additionally, Ia- KC failed to induce unresponsiveness. Additional studies indicate that the hyporesponsiveness was not passively transferrable with splenocytes and was not related to the I-J MHC locus. In contrast to our in vitro studies, the unresponsiveness induced by hapten-modified Ia+ KC in vivo was transient in nature. These data indicate that hapten-modified Ia+ KC function in vivo as nonstimulatory accessory cells, by generating down-regulatory signals that can interfere with the induction of contact hypersensitivity.
T cell receptor gamma and delta gene rearrangements in scid thymocytes. Similarity to those in normal thymocytes.
Authors: Kienker L J , Kuziel W A , Garni-Wagner B A , Kumar V , Tucker P W
Published: December 2022

The nature of TCR gamma and delta gene rearrangements in 4- to 6-week-old scid thymocytes was examined by using the polymerase chain reaction technique, cloning, and DNA sequencing. Analysis of 78 sequences indicates that TCR gamma and delta gene rearrangements in scid mice generally resemble those in thymocytes from normal young adult mice. V gamma 1, V gamma 2, and V gamma 5 rearrangements are heterogeneous, with extensive N region addition and nucleotide excision from the recombining coding segments. In addition, homogeneous and fetal-like V gamma 3, V gamma 4, and V delta 1 rearrangements are observed. These rearrangements are currently difficult to interpret but may be significant with respect to whether certain homogeneous joints in normal mice are due to cellular selection or to the rearrangement process. scid TCR gamma and delta gene nucleotide sequences also reveal direct V-J delta joining, inter-(V-J-C gamma) cluster joining, and the possibility of inversional rearrangement at the gamma locus. Short sequence homologies may contribute to V(D)J recombination and to the rescue of blocked coding joints.
Expression of protein kinase C isoenzymes in thymocyte subpopulations and their differential regulation.
Authors: Freire-Moar J , Cherwinski H , Hwang F , Ransom J , Webb D
Published: December 2022

The expression of the different protein kinase C (PKC) isozymes in mouse thymocytes was studied to determine if there is a correlation between isozyme expression and thymocyte phenotype. Expression of PKC isozymes in thymocyte subsets (distinguished by the CD4 or CD8 Ag) was determined by message amplification phenotyping. The expression of mRNA for PKC-alpha, -beta, -epsilon, and -zeta, but not -gamma or -delta isozymes, was detected in all of the unstimulated thymocyte subpopulations analyzed. Thus no differences in the pattern of PKC isozyme expression were found that could be correlated with thymocyte phenotype. However, it was noted that the levels of PKC mRNA expression were affected by different stimuli in unfractionated thymocytes. Whereas mRNA levels of PKC-alpha and -beta were down-regulated by PMA and ionomycin treatment, no significant changes were seen in the levels of PKC-epsilon mRNA with these agents. PKC-epsilon mRNA decreased in thymocytes exposed to Con A similar to what has been reported for PKC-epsilon protein. PKC-zeta mRNA was also down-regulated by PMA or ionomycin, and the combination of both compounds caused a more rapid and drastic effect. Finally, PKC-delta mRNA expression was induced transiently in thymocytes only after exposure to PMA or Con A, and this induction was inhibited by ionomycin treatment. These results indicate that message levels of specific isoforms of PKC are uniquely regulated and suggest an additional level of control of PKC activity in activated lymphocytes.
Role of intrastructural/intermolecular help in immunization with peptide-phospholipid complexes.
Authors: Goodman-Snitkoff G , Good M F , Berzofsky J A , Mannino R J
Published: December 2022

The design of effective subunit vaccines requires the inclusion of both B and T cell epitopes. The best mechanism for including both types of epitopes within an Ag is dependent upon how the Ag is processed by the APC for presentation to a responsive Th cell. If it is more efficient to process a single molecule for both helper and primary epitopes, than covalent linkage of B cells and T cell epitopes for intramolecular presentation of help would be recommended. If however, separate peptides containing either B or Th cell epitopes could be included within a single complex for the elicitation of intermolecular/intrastructural help, more antigenically diverse structures could be designed. This paper reports that it is possible to generate intermolecular/intrastructural help within an antigenic peptide-phospholipid (PL) complex. These peptide-PL complexes use well defined epitopes from Plasmodium falciparum as Ag. In addition to generating intrastructural help, we have shown that the Ir to these peptide-PL complexes is controlled by Ir genes and is similar to the Ir to the circumsporozoite protein of this pathogen.
In vitro unresponsiveness to autologous sequences of the immunopathogenic autoantigen, S-antigen.
Authors: Fling S P , Donoso L A , Gregerson D S
Published: December 2022

Previous analyses of T cell recognition sites on immunopathogenic neural autoantigens have demonstrated, using LEW rats, the functional dissociation of in vitro proliferative responses and the ability to actively induce autoimmune diseases. In experimental autoimmune uveoretinitis, immunization of LEW rats with bovine retinal S-Ag reveals the presence of three immunodominant T cell recognition sites located in regions containing sequence differences between bovine and rat S-Ag. Immune responses of LEW rats to self (rat) and nonself (bovine and human) peptide homologues representing these three sites were compared. The immunodominant sequences of heterologous S-Ag were found to predict new pathogenic T cell recognition sites in the corresponding autologous rat sequence. Furthermore, in vitro proliferative responses to the pathogenic autologous sequences are dramatically diminished relative to the responses of lymphocytes raised to the non-self homologues. A pathogenic T cell line, R858, efficiently transferred disease, but was unresponsive to the autologous S-Ag peptide in proliferation assays. However, responses to autologous peptides were readily detected using nonirradiated splenic APC. Detection of responses to non-self peptides was independent of this radiosensitive Ag-presenting activity. The lack of in vitro proliferative responses to pathogenic autologous sequences by T cells bearing self-specific receptors, contrasted with the strong proliferation induced by non-self peptide homologues, suggests a mechanism of unresponsiveness to self.
Defective immune adherence and elimination of hepatitis B surface antigen/antibody complexes in patients with mixed essential cryoglobulinemia type II.
Authors: Madi N , Steiger G , Estreicher J , Schifferli J A
Published: December 2022

Mixed essential cryoglobulinemia type II (monoclonal Ig/polyclonal IgG) is characterized by systemic vasculitis caused by the deposition of circulating immune reactants that include the monoclonal component. Such reactants may include immune complexes (IC) formed from exogenous Ag. IC binding to E C receptor type 1 appears to play a role in transport and buffering of such IC (immune adherence: IA). To define the mechanisms responsible for immune deposition, 7 patients with cryoglobulinemia type II (IgM kappa/polyclonal IgG) and 14 normal volunteers were injected i.v. with hepatitis B surface Ag/antibody complexes. Two minutes after injection, only 19.4% (mean) of the circulating complexes were bound to E in patients as compared with 63.1% in normal subjects. This IA correlated directly with C4 and inversely with the IgM rheumatoid factor (RF) titer. Disappearance of IC was faster in patients (mean elimination rate: 15.7%/min) than in normal subjects (9.3%). In vitro experiments demonstrated that C depletion, interference with IC opsonization by monoclonal IgM RF, and decreased binding of opsonized IC in the presence of monoclonal RF are each associated with decreased IA. These observations suggest that, in patients with cryoglobulinemia type II, monoclonal IgM RF and low C contribute to reducing IA of circulating IC that might be rapidly trapped in tissues, resulting in injury.
Stable expression of a secreted form of the mouse IFN-gamma receptor by rat cells.
Authors: Fernando L P , LeClaire R D , Obici S , Zavodny P J , Russell S W , Pace J L
Published: December 2022

The biologic effects of IFN-gamma are mediated through a receptor that is expressed in relatively low abundance on normal mammalian cells. As a consequence, investigations of the physicochemical and ligand-binding properties of the purified receptor have been limited. The work reported here characterizes a secreted form of the receptor for mouse IFN-gamma, made by deletion of the nucleotides that code for the anchoring domain from a cDNA that encodes the receptor binding protein and its related signal peptide. When transfected into rat XC cells, this construct produced up to approximately 1 mg/liter of a secreted protein that had the characteristics of the native receptor. Both the secreted protein and its mRNA were of sizes that were consistent with loss of the transmembrane region. The protein was detectable by a mAb that is specific for an epitope that is found in the ligand binding site of the receptor for mouse IFN-gamma, as well as by a goat polyclonal IgG that is monospecific for the mouse IFN-gamma R. Supernates that contained the secreted protein blocked binding of IFN-gamma to mouse IFN-gamma R and inhibited in a dose-dependent manner the IFN-gamma-mediated priming of mouse bone marrow culture-derived macrophages for tumor cell killing. Availability of relatively large amounts of a secreted protein that retains ligand-binding activity should facilitate purification and basic studies of the receptor binding protein and could provide new approaches to the treatment/prevention of diseases that arise due to inappropriate response of cells to IFN-gamma. In addition, because this secreted receptor, unlike others, consists of both the extracellular and intracellular domains, it is likely that it will be useful in determining how the cytoplasmic portion of the receptor is involved in receptor function.
Protein kinase C mobilization in B lymphocytes. Differential isoenzyme translocation upon activation.
Authors: Márquez C , Martínez C , Boscá L
Published: December 2022

The subcellular distribution of protein kinase C has been analyzed in murine B lymphocytes exposed to LPS, anti-IgM antibodies and phorbol dibutyrate. An accurate determination of the enzyme mobilized from the soluble to the particulate fractions by these activators, has been made possible by the use of B cells in which the major part of the activity was present in the cytosol. Upon stimulation, we have analyzed the isoenzymatic forms translocated to the B cell membrane, showing a differential pattern of isoenzyme mobilization between LPS and anti-IgM antibodies. These data, together with the different Ca2+ requirements for the activation of the translocated protein kinase C isoenzymes, might help to unravel the mechanism responsible for the clonal expansion and differentiation of B lymphocytes, induced by the two ligands.