bluuebunny/crossref_metadata_2025 huggingface dataset:
Limited usage of T cell receptor V beta genes by allopeptide-specific T cells.
Authors: Liu Z , Sun Y K , Xi Y P , Hong B , Harris P E , Reed E F , Suciu-Foca N
Published: December 2022

T cell reactivity to alloantigens results from direct and indirect recognition of allogeneic MHC molecules and/or peptides. Although direct recognition is not self-MHC restricted, indirect recognition, the result of alloantigen processing and presentation by host APC, is restricted by the self (responder) MHC molecule to which the allopeptide has bound. We have studied the MHC restriction and TCR usage in T cell alloreactivity to a synthetic peptide corresponding to amino acid residues 21-42 of the DR beta 1*0101 molecule. T cell lines were developed by in vitro immunization of T cells from three responders carrying the DR beta 1*1101 allele with this peptide. In all three responders, reactivity to peptide 21-42 was restricted by the DR11 molecule. A limited usage of V beta genes was found in these T cell lines, all of which shared the expression of V beta 13.2. Because indirect recognition of allopeptide may play an important role in chronic, antibody-mediated allograft rejection, study of the TCR gene usage may contribute to the development of specific immunosuppression therapy.
Invariant chain retains MHC class II molecules in the endocytic pathway.
Authors: Loss G E , Sant A J
Published: December 2022

During exocytosis of MHC class II, the class II alpha beta heterodimer associates with a third polypeptide termed invariant chain (Ii). Class II and Ii are coordinately processed and transported until proteolytic cleavage of Ii in an acidic compartment immediately before class II surface expression. Although the lysosomotropic agent chloroquine (CQ) prevents dissociation of class II/Ii within the cell, the ultimate fate of these complexes has not been determined. We considered two alternative possibilities. If Ii encodes an intracellular retention signal, then persistent association of Ii with class II could lead to intracellular accumulation of class II/Ii complexes. Alternatively, if Ii does not block further transport of class II, then CQ treatment should result in aberrant expression of class II/Ii complexes at the cell surface. Ltk- and EL4 cells transfected with I-A(d) alone or I-A(d) plus Ii were treated with CQ and examined for changes in surface class II and Ii expression. Anti-Ii mAb surface staining did not increase with prolonged CQ treatment, but a dramatic decrease in surface class II staining was observed. This decrease in class II was observed both with genomic Ii and p31 cDNA transfectants and was rapidly reversed upon drug removal. Accumulation of Ii and class II within treated cells was directly observed by intracellular staining. Similar effects on MHC surface expression were observed with the lysosomotropic agents primaquine and NH4Cl and the cysteine protease inhibitor leupeptin. Ii-negative cells treated in parallel displayed no effect of the lysosomotropic agents or leupeptin on class II surface staining. These results indicate that dissociation of Ii from newly synthesized class II is required for transport of the alpha beta dimer to the cell surface, and suggest that Ii serves to retain class II molecules in a post-Golgi endocytic compartment.
Acidification and disulfide reduction can be sufficient to allow intact proteins to bind class II MHC.
Authors: Jensen P E
Published: December 2022

The hypothesis that reduction of disulfide bonds and exposure to low pH may be sufficient to allow intact proteins to bind class II MHC was addressed in this study. Functional assays were used to determine minimal conditions sufficient to bypass the requirement for Ag processing. Fixed APC, pulsed with HEL at pH 5 in the presence of a reducing agent, were observed to stimulate I-Ed-restricted T cell hybridomas. Activity required both low pH and reducing agent. Fixed APC also stimulated cytochrome c-specific T cells after exposure to Ag at pH 5 but not pH 7. No reducing agent was required. Peptide was considerably more potent in these assays than intact cytochrome c. By contrast, highly purified cytochrome c was equal in potency with a peptide in competition binding assays using purified I-Ek. The protein did not bind I-Ad or I-Ak. This suggested that cytochrome c can efficiently bind I-Ek at low pH without the requirement for proteolytic cleavage. The capacity of HEL to bind purified I-Ed was strictly dependent on the presence of reducing agent. The reducing agent alone had no effect. A small panel of proteins was screened for class II binding at low pH in competition assays. Horse myoglobin and human hemoglobin were particularly potent in their capacity to inhibit binding of biotin-labeled peptide to purified class II. Unlabeled peptide inhibited binding of labeled peptide to I-Ad at pH 5 and pH 7. By contrast, intact horse myoglobin was active only at pH 5. This result suggested that low pH may enhance the binding of horse myoglobin to I-Ad through effects on the structure of both proteins. Our observations support the hypothesis that low pH and disulfide reduction can be sufficient to allow partially unfolded or structurally destabilized proteins to bind class II MHC.
Unexpected effects of the IL-2 receptor alpha subunit on high affinity IL-2 receptor assembly and function detected with a mutant IL-2 analog.
Authors: Kuziel W A , Ju G , Grdina T A , Greene W C
Published: December 2022

Binding of IL-2 to the high affinity IL-2R results in the formation of a stable complex consisting of IL-2 and at least three distinct, noncovalently associated receptor subunits: IL-2R alpha (p55), IL-2R beta (p70-75), and IL-2R gamma (p64). Ligand binding also stimulates the rapid endocytosis of this receptor complex. To gain further insight into the function of the various subunits of the multicomponent high affinity IL-2R complex, we have carried out binding, internalization, and proliferation assays using an IL-2 analog designated F42A that contains a single Phe for Ala amino acid substitution at position 42. This mutation markedly reduces the intrinsic affinity of the resultant IL-2 analog for the low affinity IL-2R alpha subunit although having little or no effect on binding to the IL-2R beta (or IL-2R beta/gamma) intermediate affinity receptor. We have confirmed that F42A does not bind to the IL-2R alpha chain when expressed alone on MT-1 cells or in the presence of the large or small excess of IL-2R beta chains present on either YT-1 cells or forskolin-induced YT-1 cells, respectively. However, although F42A does not interact with the large number of low affinity IL-2R alpha chains present on HUT 102B2 cells, this ligand does bind to the small number of IL-2R beta chains present on these cells with at least 10-fold higher than expected affinity. These findings indicate that excess IL-2R alpha chains may exert positive effects on binding perhaps by changing the conformation of IL-2R beta. In F42A-stimulated internalization assays on forskolin-induced YT-1 cells, the IL-2R alpha chain is consistently endocytosed together with the IL-2R beta subunit indicating that IL-2R alpha is stably associated with the F42A-IL-2R beta complex even though the alpha-subunit contributes little if any affinity to the F42A binding reaction. In proliferation assays on mouse IL-3-dependent pro-B BA/F3 cells stably coexpressing transfected human IL-2R alpha and IL-2R beta subunits (but a mouse IL-2R gamma subunit), F42A proved to be a more effective agonist of growth than wild-type IL-2. These results suggest that the interaction between wild-type IL-2 but not F42A and the IL-2R alpha subunit in this mixed species high affinity receptor complex may induce an unfavorable receptor conformation leading to diminished rather than enhanced growth signal transduction.
Amino acid changes in the peptide binding site have structural consequences at the surface of class I glycoproteins.
Authors: Pease L R , Horton R M , Pullen J K , Hunt H D , Yun T J , Rohren E M , Prescott J L , Jobe S M , Allen K S
Published: December 2022

Structural changes on the surface of the class I Ag binding domain resulting from point mutations localized inside the Ag binding cleft of the H-2Kb and Kf glycoproteins were revealed using mAb. Both the loss and gain of antibody binding sites found among naturally occurring K glycoproteins resulted from single amino acid substitutions at a variety of different positions buried within the Ag binding groove. Each of the amino acid replacements analyzed represented naturally occurring diversity known to exist among the functional class I Ag-presenting molecules of the mouse. The binding of the affected mAb was not significantly altered in Kb molecules expressed by transfected T2 cells. Because T2 cells have been shown to express Kb molecules that are either largely devoid of bound peptides or bind a vastly different set of low affinity peptides, it is unlikely that the detected structural changes were caused by alterations in the spectrum of peptides bound by the class I variant glycoproteins. Similarly, a class I point mutant, Kb-97R, that also has been shown previously to bind a very different set of peptides in comparison to the parental Kb molecule also displays normal antibody binding properties. We conclude from these studies that structural diversity within the Ag binding cleft indirectly influences the external surface of the Ag-presenting domain of the class I H chain. Significantly, this surface is the interface between the T cell receptor and MHC molecules and may make contributions to the fine specificity of allorecognition.
RANTES is a chemotactic and activating factor for human eosinophils.
Authors: Alam R , Stafford S , Forsythe P , Harrison R , Faubion D , Lett-Brown M A , Grant J A
Published: December 2022

RANTES is a member of the 8-kDa cytokine family that has been shown to possess chemotactic activity for monocytes and CD4 T cells. In this study, we investigated whether RANTES could affect eosinophil chemotaxis and function. Peripheral blood eosinophils from blood donors were isolated on Percoll gradients to > 98% purity and then used for chemotaxis, flow cytometry, eosinophil cationic protein release assay, and survival assay. We found that RANTES is chemotactic for eosinophils at 10(-9) to 10(-8) M concentrations. RANTES elicited 65% of the chemotactic response to 10(-7) M platelet-activating factor in all experiments. The mechanism of chemotaxis was investigated by studying the expression of adhesion molecules on eosinophils by flow cytometry. We found that RANTES up-regulated the expression of CD11b/CD18 on eosinophils in a dose-dependent manner. In another set of experiments, purified eosinophils incubated with various concentrations of RANTES released eosinophil cationic protein as measured by a RIA. We also investigated the effect of RANTES on eosinophil density. Leukocytes were incubated in the presence or absence of RANTES, and the distribution of eosinophils on discontinuous Percoll gradients was then examined. We found that eosinophils became hypodense (< 1.085) when incubated in RANTES. However, unlike IL-3, RANTES did not affect the survival of eosinophils in a 4-day culture system. Thus, we established that RANTES is a chemotactic and activating factor for eosinophils.
Transforming growth factor-beta mediates IL-1-dependent induction of IL-1 receptor antagonist.
Authors: Wahl S M , Costa G L , Corcoran M , Wahl L M , Berger A E
Published: December 2022

Transforming growth factor-beta (TGF-beta) is a potent immunomodulatory molecule that promotes inflammation through recruitment of monocytes and induction of IL-1 and other cytokines. These proinflammatory processes may be modulated by the ability of TGF-beta to induce de novo synthesis and secretion of an IL-1 receptor antagonist (IL-1ra) that binds to and blocks IL-1 receptors. In this study, we show that the addition of TGF-beta to human peripheral blood monocytes induced the sequential transcription of the 1.8-kb mRNA for IL-1 beta and for IL-1ra. The expression of detectable mRNA and synthesis of IL-1 beta peptide routinely preceded that for IL-1ra, suggesting possible dependency of IL-1ra generation on IL-1 beta. Antibody to IL-1 blocked TGF-beta induction of IL-1ra mRNA expression demonstrating an unique IL-1-dependent induction of its own antagonist. Confirmatory evidence that IL-1 participates in the generation of IL-1ra was obtained when exogenously added IL-1 induced and, IL-1 receptor antagonist blocked, IL-1ra transcription. Thus, these data suggest that TGF-beta, after release at a site of inflammation, induces synthesis of IL-1 that participates in the initial cytokine cascade central to an inflammatory response, and then triggers the generation of its own natural inhibitor by autocrine or paracrine pathways. This TGF-beta-induced IL-1-dependent induction of IL-1ra may provide a negative feedback loop, thereby promoting the resolution of an inflammatory response.
Comparison of HLA class II genes in Caucasoid, Chinese, and Japanese patients with primary Sjögren's syndrome.
Authors: Kang H I , Fei H M , Saito I , Sawada S , Chen S L , Yi D , Chan E , Peebles C , Bugawan T L , Erlich H A
Published: December 2022

To better define the genetic factors that predispose to primary Sjögren's syndrome (SS), we have used polymerase chain reaction in combination with oligonucleotide probe hybridization and DNA sequencing to analyze HLA-DRB1, -DQA1, -DQB1, and -DPB1 alleles in Caucasoid (California), Japanese (Tokyo), and Chinese (Shanghai and Beijing) SS patients. In comparison to local controls in each region, we found: 1) increased frequency of the predicted haplotype HLA-DRB1*0301-DRB3*0101-DQA1*0501-DQB1*0201 in Caucasoid patients (p < 0.001); 2) increased frequency of the predicted haplotype HLA-DRB1*0405-DRB4*0101-DQA1*0301-DQB1*0401 in Japanese patients (p < 0.05); 3) increased frequency of the predicted haplotype DRB1*0803-DQA1*0103-DQB1*0601 in Chinese patients (p < 0.05); and 4) no statistically significant association with DPB1 alleles in any group, although an increased number of Caucasoid and Japanese SS patients possessed DPB1*0301. Comparison of DNA sequences for the three disease-associated haplotypes in these ethnic groups revealed a shared region of predicted amino acids from positions 58 to 69 in the first domain of HLA-DQB1. These results extend previous studies by demonstrating that no single class II allele was associated with 1 degree SS in the different ethnic groups. However, a shared amino acid motif in the DQB1 first domain was present in each disease-associated haplotype.
An essential role for Langerhans cell-derived IL-1 beta in the initiation of primary immune responses in skin.
Authors: Enk A H , Angeloni V L , Udey M C , Katz S I
Published: December 2022

Langerhans cells (LC) are Ag-presenting cells required for induction of primary immune responses in skin. After activation by Ag, LC express increased levels of MHC class II Ag, exhibit increased accessory cell activity, and migrate to regional lymph nodes where they stimulate T cells. One of the earliest manifestations of LC activation is the accumulation of increased amounts of IL-1 beta mRNA in LC within 15 min after exposure to contact allergens in vivo. To determine if enhanced IL-1 beta production by LC could be causally linked to epicutaneous sensitization, we injected IL-1 beta intradermally into the ears of BALB/c mice and extracted total epidermal RNA 4 h later. A quantitative reverse transcriptase-polymerase chain reaction technique was used to compare changes in IL-1 alpha, IL-1 beta, macrophage inflammatory protein 2, IL-10, TNF-alpha, and 1-A alpha chain mRNA signals caused by intradermally-injected IL-1 beta to those caused by intradermal IL-1 alpha or TNF alpha, or by topical application of the contact allergen trinitrochlorobenzene (3% TNCB). Intradermal injection of 25 ng IL-1 beta resulted in 5-to 100-fold enhancement of mRNA signals for IL-1 alpha, IL-1 beta, MIP-2, IL-10, TNF alpha, and class II I-A alpha, mimicking the changes caused by allergen. In contrast, injection of equivalent amounts of IL-1 alpha or TNF alpha did not significantly alter the epidermal cytokine pattern. Simulating the effects of topically applied TNCB, intradermally-injected IL-1 beta (but not IL-1 alpha or TNF alpha) also caused enhancement of LC MHC class II expression. In addition, LC derived from IL-1 beta-injected skin were 2 to 3 times more potent accessory cells in an anti-CD3 proliferation assay than LC from IL-1 alpha or sham-injected skin. Finally, injection of hamster anti-mIL-1 beta mAb into the skin prior to TNCB treatment completely prevented sensitization to this allergen, although injections of similar amounts of hamster anti-mIL-1 alpha mAb or PBS were without effect. Taken together, our data indicate that dendritic cell-derived IL-1 beta may be a critical molecule required for initiation of primary immune responses in skin.
IFN induction and associated changes in splenic leukocyte distribution.
Authors: Ishikawa R , Biron C A
Published: December 2022

Our studies were undertaken to evaluate early events associated with IFN expression and IFN-induced cellular changes in the spleen. Polyinosinic-polycytidylic acid (poly(I:C)) was used to induce IFN in C57BL/6 mice. Biologically active IFN was present in spleens and sera of poly(I:C)-treated mice as early as 3 h and peaked at 6 to 12 h posttreatment. Neutralization assays and Northern blot analyses demonstrated that IFN-beta was the form of IFN preferentially induced in the spleens. Immunohistochemical staining and in situ hybridization studies of splenic tissue sections demonstrated that, although many cells were induced to express low levels of IFN-beta, low frequency cells were particularly potent expressers of IFN-beta. Perivascular cells expressed high levels of IFN-beta. Other positive cells were localized in red pulp at early times and in white pulp at later times posttreatment. Histologic examination of splenic sections showed that the IFN production was accompanied by dramatic architectural changes. There were significant 1.4-fold increases in the proportion of white pulp area and > 50% decreases in red pulp leukocyte number. These architectural changes appeared at 6 h and lasted through 36 h after poly(I:C) treatment. They were not due to increased cell proliferation as splenic weights and cell yields were not elevated. The changes in splenic leukocyte distribution were shown to be a result of IFN induction as: 1) treatment with anti-IFN antibodies inhibited poly(I:C)-induced depletion of red pulp leukocytes, and 2) administration of purified IFN-beta induced both an increase in white pulp area and a decrease in red pulp leukocytes. Splenic leukocytes were labeled with the lipophilic fluorescent dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate, to evaluate cell traffic after IFN induction. Poly(I:C) enhanced accumulation of cells in white pulp regions. In contrast to the 76 +/- 2 labeled cells that accumulated in 0.5 mm2 of white pulp area in control PBS-treated mice, 131 +/- 4 labeled cells accumulated in 0.5 mm2 of white pulp area in poly(I:C)-treated mice. The poly(I:C)-enhanced leukocyte accumulation in short term cell migration to white pulp regions was dependent on preexposure of both splenic leukocytes and recipient environments to the IFN inducer. These data demonstrate that poly(I:C) is a potent and rapid inducer of IFN-beta production by splenic cells and that IFN induces cellular redistribution in the spleen. The results suggest that margination of leukocytes into splenic white pulp may be another important immunoregulatory function of IFN.
Cross-linking of B cell receptor-related MB-1 molecule induces protein tyrosine phosphorylation in early B lineage cells.
Authors: Matsuo T , Nomura J , Kuwahara K , Igarashi H , Inui S , Hamaguchi M , Kimoto M , Sakaguchi N
Published: December 2022

Cross-linking of surface B cell Ag receptor (BCR) induces tyrosine phosphorylation of BCR-associated components through a receptor-mediated signal transmission pathway. B cell-specific mb-1 and B29 genes encode the alpha/beta components of the BCR-associated complex in mature sIgM+ B cells. Here, we studied the involvement of the mb-1 gene product, MB-1, in the BCR-related structure of immature B cells. Affinity-purified anti-MB-1 antibody coprecipitated mu chain/20-kDa/15-kDa proteins together with monomer MB-1 and Ig-alpha/Ig-beta heterodimer components from digitonin lysates of the pre-B cell line 18.81. The monomer MB-1 and Ig-alpha in the pre-B cell line were shown to migrate with identical patterns in nonequilibrium pH gradient gel electrophoresis/SDS-PAGE. Western blot analysis showed that MB-1 protein is coprecipitated with mu chain from the pre-B cell line. We studied the tyrosine phosphorylation response of bone marrow B lineage cells as well as spleen B cells after cross-linking of BCR-related components with anti-mu, anti-kappa, and anti-MB-1 antibodies. We identified the activation of tyrosine kinase by direct cross-linking of MB-1 expressed on the surface of early B lineage cells. Anti-mu antibody stimulation induced the activation of tyrosine kinase in early (5- to 10-min) and late (30- to 120-min) responses in bone marrow early B lineage cells. Anti-MB-1 mAb (11-18-5) induced the late response exclusively but anti-kappa antibody induced only the early response. These results clearly indicate that MB-1 acts in the BCR-mediated signal transmission in early B lineage cells. To explore the molecular mechanism of protein tyrosine phosphorylation in bone marrow B lineage cells, we studied associated components of the BCR complex by using an in vitro kinase reaction and observed the phosphorylation of a 60-kDa protein in pre-B cell lines. The 60-kDa phosphoprotein coprecipitated with MB-1 and the BCR-related complex is very similar to the Src-type Fyn tyrosine kinase or a Fyn-related protein.
Ig repertoire expression of BALB/c primary and secondary B cell precursors specific for phosphorylcholine.
Authors: Wu P , Ward R E
Published: December 2022

To better understand the regulation of Ig repertoire expression and the generation of memory B cells, we have used the splenic focus assay to analyze primary and in vitro generated secondary phosphorylcholine (PC)-specific BALB/c B cell precursors. A quantitative analysis of the kinetics of appearance of these precursors over the time of the culture period was performed. The precursors were analyzed for TEPC15 Id expression, H chain isotype, relative affinity, and epitope specificity (PC vs p-nitrophenyl PC). In addition, we tested the hypothesis that primary and secondary precursors could be distinguished on the basis of the expression of the cell-surface Ag defined by the mAb JIId. We found that secondary in vitro PC-hemocyanin stimulation resulted in an increase in the anti-PC precursor frequency, relative to primary stimulation, of 37%. This increase was caused almost entirely by the secondary stimulation of non-TEPC15 expressing precursors. Relative to the primary precursors, the secondary precursors also: 1) expressed a lower percentage of IgM and a higher percentage of IgG, 2) had a higher relative affinity, and 3) expressed a lower level of JIId-defined Ag. Very little difference was observed in the epitope specificity of the primary vs secondary precursor repertoire. Based on an analysis of the kinetics of the response, together with JIId cell sorting and transfer experiments, our results support a model whereby a portion of the secondary anti-PC response is derived from secondary precursors arising from primary precursors, and the other portion is derived from distinct secondary precursors expressing a relatively low amount of the JIId Ag.
Differential expression of IgE and IgG4 specific antibody responses in asymptomatic and chronic human filariasis.
Authors: Kurniawan A , Yazdanbakhsh M , van Ree R , Aalberse R , Selkirk M E , Partono F , Maizels R M
Published: December 2022

A population of 164 adult individuals resident in an area endemic for Brugia malayi lymphatic filariasis has been studied for humoral immune responses to filarial parasites. Antibody levels to Ag extracted from adult worms were determined for each of the IgG subclasses, for IgM and for IgE. The dominant isotype of antifilarial antibody was IgG4, which represented 88% of total IgG in asymptomatic microfilaremics, most of whom possessed 100 to 1000 micrograms/ml of specific antibody of this subclass (geometric mean 762 micrograms/ml). Patients with chronic disease (elephantiasis), who were generally amicrofilaremic, had substantially higher levels of IgG1, IgG2, and IgG3, but a 3.4-fold lower geometric mean level of specific IgG4 (222 micrograms/ml) than asymptomatics with or without microfilaremia. In contrast, specific IgE antibody levels in cases of elephantiasis were on average 4.5 times higher than those found in the asymptomatic carrier state. The majority of microfilaremics were therefore typified by extremely high specific IgG4 concentrations and relatively low IgE reactivities, whereas clinical cases tended to show the reverse relationship. The possible roles of these isotypes and the implications of changing IgG4/IgE balances in disease are discussed.
Ity influences the production of IFN-gamma by murine splenocytes stimulated in vitro with Salmonella typhimurium.
Authors: Ramarathinam L , Niesel D W , Klimpel G R
Published: December 2022

The Ity-Lsh-Bcg genetic locus in the mouse has been documented to confer innate resistance to at least three intracellular pathogens: Salmonella typhimurium, Leishmania donovani, and Mycobacterium. Expression of the resistance gene(s) results in a slower net growth of these pathogens in the reticuloendothelial system early postinfection. Although it is clear that the resident macrophages in resistant mice are functionally superior with regard to antimicrobial activity, the exact mechanism(s) underlying the control exerted by this gene is not understood. Using S. typhimurium infection as a model, we have examined the influence of this resistance gene(s) on the production of IFN-gamma, a cytokine known to play an important role in host-defense against several intracellular pathogens. We compared IFN-gamma production by splenocytes from resistant (Ity(r)) and sensitive (Ity(s)) inbred mouse strains after stimulation in vitro with S. typhimurium. Spleen cells from Ity(r) mouse strains produced significantly higher levels of IFN-gamma when compared to spleen cells obtained from Ity(s) mouse strains. Enhanced IFN-gamma production was not a generalized response to bacteria. Listeria monocytogenes induced comparable levels of IFN-gamma production from both Ity(r) (CBA/J) and Ity(s) (C57BL/6) mice. Splenocytes from Ity congenic mouse strains displayed similar differences in the level of IFN-gamma produced after S. typhimurium stimulation, with spleen cells from the Ity(r) strain producing significantly higher levels of IFN-gamma when compared to spleen cells from the Ity(s) strain. A requirement for adherent cells and/or adherent cell-derived factors has been documented for IFN-gamma production by S. typhimurium-stimulated splenocytes. Interestingly, supernatant from adherent cells obtained from Ity(r) mouse strains was found to induce the production of significantly higher levels of IFN-gamma when compared to adherent cell supernatant from Ity(s) strains. Nylon wool nonadherent cells from Ity(s) mouse strains produced high levels of IFN-gamma when exposed to supernatants obtained from adherent cells of Ity(r) mouse strains. In contrast, nylon wool nonadherent cells from Ity(r) mouse strains produced reduced levels of IFN-gamma when exposed to supernatant obtained from adherent cells of Ity(s) mouse strains. Thus, modulation of IFN-gamma production appears to be a function of the Ity(r) gene(s). This study documents for the first time that the Ity locus may play a role in controlling resistance to Salmonella infection by regulating IFN-gamma production by NK cells.
Salmonella typhimurium induces IFN-gamma production in murine splenocytes. Role of natural killer cells and macrophages.
Authors: Ramarathinam L , Niesel D W , Klimpel G R
Published: December 2022

IFN-gamma is a cytokine known to play an important role in host defense against Salmonella typhimurium. The lymphoid cells required for in vitro production of IFN-gamma after S. typhimurium stimulation of mouse spleen cells was investigated. Spleen cells depleted of cells bearing NK1.1, asialo GM1, Thy 1.2, or CD5 resulted in a significant reduction in IFN-gamma production after stimulation with S. typhimurium. In contrast, Con A-induced IFN-gamma production was only slightly reduced after depletion of NK1.1- or asialo GM1-bearing cells. Spleen cells from SCID mice produced elevated levels of IFN-gamma after stimulation with S. typhimurium. IFN-gamma production by SCID spleen cells was dependent upon asialo GM1+ T cells, suggesting that NK cells were the cells producing IFN-gamma in response to S. typhimurium. Splenic adherent cells were required for optimal IFN-gamma production. However, direct contact between the adherent and nylon wool nonadherent (NWNA) cell populations was not essential. IFN-gamma production was observed when the adherent and NWNA cell populations were physically separated or when supernatant from S. typhimurium-stimulated adherent cells was added to NWNA cells. Optimal IFN-gamma production was dependent on the presence of TNF-alpha, inasmuch as addition of antibody to TNF-alpha to spleen cell or NWNA cell cultures significantly reduced IFN-gamma production. However, addition of rTNF-alpha did not induce IFN-gamma production by NWNA cells. These findings document the existence of a T-independent mechanism for early IFN-gamma production in response to S. typhimurium, and show that TNF-alpha is necessary but not sufficient for the production of IFN-gamma.
Toxoplasma gondii induces a T-independent IFN-gamma response in natural killer cells that requires both adherent accessory cells and tumor necrosis factor-alpha.
Authors: Sher A , Oswald I P , Hieny S , Gazzinelli R T
Published: December 2022

Spleen cells from scid mice produce high levels of IFN-gamma when exposed to either live tachyzoites of Toxoplasma gondii or a soluble parasite extract. Small numbers of parasites are sufficient to stimulate this response, which is also induced by cell-free supernatants of cultured tachyzoites. The parasite molecules responsible for triggering IFN-gamma production are heat-labile but resistant to freezing and thawing. Depletion of NK cells or adherent cells from the splenocyte population abolishes the response. Moreover, cultured bone marrow-derived NK cells are stimulated by Toxoplasma to produce IFN-gamma, but only when supplemented with adherent peritoneal washout or thioglycollate-induced exudate cells. Supernatants of macrophages preincubated with T. gondii extract also induce IFN-gamma synthesis by cultured NK cells. Addition of neutralizing mAb against TNF-alpha abolishes the IFN-gamma response of scid spleen cells exposed to the parasite or of NK cells incubated with supernatants of adherent cells stimulated with T. gondii extract. Moreover, splenic adherent cells produce low levels of TNF-alpha in response to the parasite. Nevertheless, TNF-alpha alone is not sufficient to trigger IFN-gamma production from purified NK cell populations. These findings provide the first example of the stimulation of T-independent IFN-gamma production by a protozoan. The ability of T. gondii to trigger this pathway may underlie its induction of strong IFN-gamma-dependent nonspecific and specific cell-mediated immunity.
Increased expression of CD11b and functional changes in eosinophils after migration across endothelial cell monolayers.
Authors: Walker C , Rihs S , Braun R K , Betz S , Bruijnzeel P L
Published: December 2022

Eosinophils from sputum, nasal polyps, and bronchoalveolar lavages of asthmatics demonstrated a considerably increased CD11b expression, compared with blood eosinophils. Furthermore, the tissue eosinophils expressed ICAM-1 (CD54) and HLA-DR, whereas peripheral blood eosinophils did not. In vitro migration of peripheral blood eosinophils across IL-1-activated human umbilical vein endothelial cell monolayers caused a considerable up-regulation of CD11b and CD35 expression, no induction of ICAM-1 or HLA-DR, and a small but significant decrease in CD11a, CD29, and CD32 expression. These changes were only partially inducible with supernatants from nonactivated or IL-1-activated endothelial cells, platelet-activating factor, or a variety of recombinant cytokines. Thus, cell-cell interactions mediated by receptor-ligand binding or endothelial cell membrane-bound mediators, rather than soluble factors, are responsible for the altered eosinophil surface marker expression. Indeed, preparations of membrane fragments from IL-1-stimulated endothelial cells were able to induce up-regulation of CD11b, which was not inhibitable with the platelet-activating factor antagonist WEB 2086 or antibodies against ELAM-1, VCAM-1, or ICAM-1. Investigation of the functional significance of the increased CD11b expression on eosinophils revealed only minimal changes in the adherence or transmigration capacity. Nevertheless, increased CD11b expression was related to an increased capacity to generate superoxide after stimulation with opsonized zymosan. Thus, cell-cell interactions between eosinophils and endothelial cells induce a considerable up-regulation of CD11b and CD35 on eosinophils and an increased capacity to generate an oxidative burst.
Functional and molecular characterization of tumor-infiltrating lymphocytes transduced with tumor necrosis factor-alpha cDNA for the gene therapy of cancer in humans.
Authors: Hwu P , Yannelli J , Kriegler M , Anderson W F , Perez C , Chiang Y , Schwarz S , Cowherd R , Delgado C , Mulé J
Published: December 2022

TNF is effective in causing the regression of selected murine tumors when administered at high concentrations. Therapeutic levels in humans cannot be obtained systemically, however, because of dose-limiting toxicity. The development of immunotherapy with IL-2 and tumor-infiltrating lymphocytes (TIL), which can accumulate at tumor sites in some patients, and of efficient retroviral techniques for gene transfer into eukaryotic cells has allowed new therapeutic approaches using TNF. We have retrovirally transduced human TIL with the gene for TNF in an attempt to deliver high concentrations of TNF to the tumor site without dose-limiting systemic toxicity. Successful gene insertion was confirmed by Southern hybridization in 16 of 16 transduced and selected TIL cultures from 15 different patients, with an estimated 28 to 93% transduced cells within each culture. Transduced selected TIL cultures produced greater amounts of TNF, compared with nontransduced controls, in 11 of 16 cultures evaluated. However, overall production of TNF was > 30-fold lower, compared with a transduced and highly selected tumor cell line control (MEL-TNF). In addition, steady state levels of vector-derived transcript in nine of 10 transduced selected TIL cultures were < 14% of the amount seen in the MEL-TNF control line. In an attempt to increase TNF production, TIL were transduced with a mutated form of TNF containing the IFN-gamma signal peptide in place of the transmembranous region, to enhance secretion into the endoplasmic reticulum. By using this vector, TNF production increased by an average of fivefold. These studies demonstrate that TIL can be genetically modified to express and secrete a protein for use in targeted cancer therapy but that partial expression blockades exist that prevent maximal cytokine production by introduced genes in TIL.
Selective and nonselective stages in homing of T lymphocytes to the central nervous system during experimental allergic encephalomyelitis.
Authors: Karin N , Szafer F , Mitchell D , Gold D P , Steinman L
Published: December 2022

The diversity of Ag-specific receptors on T cells homing to an inflammatory infiltrate in the central nervous system has been analyzed. Experimental autoimmune encephalomyelitis, a T cell-mediated inflammatory disease of the central nervous system, was induced in Lewis rats with a CD4+, CD8- T cell line specific for peptide 68-86 of myelin basic protein. Within the line a wide array of TCR V beta genes was transcribed including the V beta 8, V beta 10, V beta 15, V beta 16, and V beta 19 families. Accumulation of T cells at the site of inflammation was determined by using RNA-polymerase chain reaction amplification of rearranged TCR V beta transcripts derived from brain. By 8 to 10 h after i.p. infusion of the pathogenic T cell line, TCR V beta transcripts, including mainly V beta families that were predominantly rearranged by the line, could be identified in brains. Restricted TCR V gene transcripts with predominance of the V beta 8 family were identified in brain 48 h after injection, before onset of disease. Paralysis was apparent by 4 to 5 days after injection. At this time diverse V beta gene transcripts were detected in brain, reaching a maximum by day 9, when paralyzed rats have recovered. By day 14 a second stage of limited heterogeneity in the T cell infiltrate could be identified with predominant expression of V beta 8, V beta 9, V beta 10, and V beta 19. Interestingly, three out of these four V beta families were predominantly expressed within the encephalitogenic line. Thus, T cell migration to brain in experimental autoimmune encephalomyelitis is characterized by a rapid penetration of T cells followed by a selective trapping of T cells before the clinical manifestations of disease. When clinical disease was present the T cell infiltrate was diverse, whereas in the post-acute phase of disease the T cells in the central nervous system had limited heterogeneity with selective accumulation of T cells transcribing the same V regions that were detected in the line that incited disease.
Functional effect of IL-7-enhanced CD19 expression on human B cell precursors.
Authors: Wolf M L , Weng W K , Stieglbauer K T , Shah N , LeBien T W
Published: December 2022

We have previously demonstrated that IL-7 can sustain the growth of normal human B cell precursors (BCP) for several weeks on bone marrow-derived stromal cells. Flow cytometric analysis of BCP recovered from IL-7 supplemented cultures revealed two- to threefold higher levels of cell surface CD19, compared with BCP maintained without IL-7. Short term culture of BCP showed that IL-7 enhancement of CD19 was dose-dependent, with increases detected by day 1 and plateauing by days 3 to 4. IL-7 increased cell-surface CD19 on small lymphoid cells, and to a greater degree on lymphoblasts, whereas cell-surface CD10 was unchanged. The CD34+/CD19+ pro-B cell population showed a greater increase in cell-surface CD19 compared with pre-B and immature B cells. IL-1, IL-3, IL-4, IL-6, and stem-cell factor had no effect on CD19. The potential functional significance of IL-7-enhanced cell-surface CD19 was examined using a F(ab')2 fragment of anti-CD19. This reagent had no effect on [3H]TdR incorporation in BCP cultured in the absence or presence of IL-7 for 5 days, but homotypic adhesion of BCP was induced at a concentration as low as 1.0 ng/ml F(ab')2 anti-CD19. IL-7 enhanced the F(ab')2 anti-CD19 induced homotypic adhesion of BCP in a dose-dependent manner. Blocking antibody studies indicated that members of the beta 1 or beta 2 integrin families did not mediate anti-CD19-induced homotypic adhesion, even though the adhesion was completely ablated by 10 mM EDTA. The pre-B and immature leukemic B cell lines NALM-6 and 1E8 expressed comparable levels of cell-surface CD19, and exhibited comparable increases after IL-7 stimulation. However, their homotypic adhesion responses to anti-CD19 were different. NALM-6 cells exhibited a strong homotypic adhesion response to anti-CD19 that was EDTA-resistant, and beta 1/beta 2 integrin independent. 1E8 cells only responded to anti-CD19 after IL-7 stimulation; this response was EDTA-sensitive and beta 1/beta 2 integrin independent. These collective results indicate that IL-7 not only acts as a growth factor for human BCP, but also regulates signal transduction through cell-surface CD19.
Structure of the human C2 gene.
Authors: Ishii Y , Zhu Z B , Macon K J , Volanakis J E
Published: December 2022

A human genomic cosmid clone, S22A, was used to characterize the structure of the human C2 gene. Eighteen exons spanning 18 kb of DNA were mapped by nucleotide sequencing of exon-containing subclones and by Southern blotting. Introns vary in length between 83 bp and 4.4 kb and all intron/exon boundaries follow the AG/GT consensus rule for splicing. Exon 1 encodes 270 bp of the 5'-untranslated region of the C2 cDNA C2HL5-3 and 15 amino acids of the leader peptide. The three short consensus repeats of C2b are encoded by the single exons 2, 3, and 4, respectively. The von Willebrand factor type A-like domain of C2a is encoded by exons 6-10 and the serine protease domain by exons 11-18. Exons 5 and 15 appear to be unique to the C2 and factor B genes.
Monoclonal antibody that distinguishes between a phosphorylated, beta 2-microglobulin-associated, and a free, nonphosphorylated, chain of MHC class I.
Authors: Thor G , Sepulveda H , Chada S , Dutton R W
Published: December 2022

We have shown that a mAb, 7.2.14, recognizes a conserved sequence in exon 7 of a number of murine MHC class I molecules. 7.2.14 binding is abolished when the molecule is phosphorylated, presumably at a serine residue in exon 7, whereas treatment of material in cell lysates with alkaline phosphatase increases the intensity of the binding. A genomic construct containing Dd was transfected into human fibroblasts and a clonal cell line expressing high levels of surface MHC was selected. Cell lysates were prepared from surface-iodinated cells and analyzed by using a panel of antibodies. An apparent size heterogeneity was detected in the MHC class I gene product precipitated by different anti-class I MHC antibodies, suggesting that more than one conformational species of Dd was present. This was further investigated regarding the molecules precipitated by antibodies 34-2-12, M1/42, and 7.2.14. After preclearing of surface-iodinated cell lysates by using one antibody, challenge with the others still precipitated a Dd molecule, confirming that there were three independent conformations of the Dd gene product. A similar complexity could be observed in the lysates of surface-labeled spleen cells from C57BL/6 mice. A major polypeptide at approximately 48 to 50 kDa, representing the MHC H chain, was seen, and one or two as yet unidentified but strongly associated polypeptides at 41 kDa and 56 kDa were also visible. Sequential clearing of surface-iodinated material with one antibody followed by precipitation with the other confirmed that the 7.2.14-reactive material was distinct from that which reacted with M1/42. We propose that the 7.2.14-reactive 50-kDa band is the nonphosphorylated form of class I MHC, which exists in a conformation different from that of the conventional 48-kDa, phosphorylated, beta 2-microglobulin-associated entity.
Substance P modulates antigen-induced, IFN-gamma production in murine Schistosomiasis mansoni.
Authors: Blum A M , Metwali A , Cook G , Mathew R C , Elliott D , Weinstock J V
Published: December 2022

In murine Schistosomiasis mansoni, granuloma eosinophils make SP. We investigated whether SP affects lymphokine secretion in murine schistosomiasis. SP at > or = 10(-10) M, and other tachykinins at much higher concentrations, substantially increased IFN-gamma secretion from spleen or granuloma inflammatory cells primed in vitro by suboptimal stimulatory concentrations of egg Ag or mitogen. Cells receiving maximal antigenic or mitogenic stimulation were affected marginally. Also, tachykinins induced no IFN-gamma from resting cells receiving no Ag or mitogen stimulation. There are three distinct tachykinin receptors, called NK-1, NK-2 and NK-3. SP binds the NK-1 receptor with highest affinity. Specific NK-1 receptor antagonists blocked all tachykinin-induced, IFN-gamma secretion. An NK-2 receptor inhibitor had no effect. Thus, SP and other tachykinins were acting through an NK-1 receptor. Inflammatory cells from 4-day-old granulomas cultured in vitro secrete IFN-gamma. Yet, there was no measurable IFN-gamma when SP receptor antagonists were added to the cultures. Moreover, animals treated in vivo with the NK-1 receptor antagonist CP-96,345 produced smaller granulomas. This suggested that endogenous SP may be necessary for normal induction of granuloma IFN-gamma secretion and a normal granulomatous response. Granuloma macrophages make somatostatin (SOM) that can decrease IFN-gamma secretion. Yet, IFN-gamma secretion was unaffected when both SP and SOM were in the cell cultures. In conclusion, SP modulates Ag-driven IFN-gamma secretion through a NK-1 receptor. Also, SP and SOM may be components of a natural circuit within inflammation that regulates IFN-gamma production.
Synergistic cooperation between T cell lymphokines for induction of the nitric oxide synthase gene in murine peritoneal macrophages.
Authors: Deng W , Thiel B , Tannenbaum C S , Hamilton T A , Stuehr D J
Published: December 2022

The ability of T cell-derived cytokines to induce the expression of the nitric oxide synthase (NOS) gene in murine peritoneal macrophages was examined. IL-2 or TNF-alpha alone had no effect either on gene expression or enzyme activity, whereas IFN-gamma had only modest activity. When IL-2 or TNF-alpha were used in combination with IFN-gamma, there was a marked cooperative induction of both mRNA and enzyme activity. The cooperative effects were truly synergistic, as the consequences of combined cytokine treatment were many times greater than was seen with any of the agents acting independently. The expression of NOS mRNA and enzyme activity in response to combined lymphokine treatments was a continuous process reaching optimal levels between 24 and 48 h after stimulation. Concentration dependency for both IL-2 and TNF-alpha suggested that their effects were mediated through interaction with the corresponding defined cell surface receptors. Human rTNF-alpha was as effective a stimulus as murine TNF-alpha; because human TNF-alpha is recognized only by the p55 Type II TNF receptor, this structure appears to mediate the response to TNF-alpha. When IL-2 and TNF-alpha were added at saturating doses in the presence of IFN-gamma, there was an additive effect on NOS mRNA expression suggesting that IL-2 and TNF-alpha cooperate with IFN-gamma through at least partially distinct intracellular signaling pathways. Expression of NOS mRNA in response to IFN-gamma/IL-2 or IFN-gamma/TNF-alpha treatment required protein synthesis, suggesting that cooperative cytokine induction of NOS involves the intermediate expression of new gene products. Such molecular controls for regulation of inducible macrophage gene expression can be contrasted with regulatory control of other inflammatory genes such as IP-10 and TNF-alpha.