bluuebunny/crossref_metadata_2025 huggingface dataset:
Cells expressing an H chain Ig gene carrying a viral T cell epitope are lysed by specific cytolytic T cells.
Authors: Zaghouani H , Krystal M , Kuzu H , Moran T , Shah H , Kuzu Y , Schulman J , Bona C
Published: December 2022

The epitope corresponding to amino acid residues 147-161 of the nucleoprotein (NP) of influenza A virus is recognized by CTL in association with H-2Kd class I Ag. Herein, we engineered an Ig molecule carrying this CTL epitope by replacing the diversity gene segment of the H chain V region of an anti-arsonate antibody with an oligonucleotide that encodes the CTL epitope. The chimeric H chain gene was expressed either alone or together with the parental L chain in the nonsecreting BALB/c myeloma B cell line, SP2/0. The Ig produced by cells transfected with both the chimeric H chain and parental L chains genes expressed the NP epitope but lost the original arsonate binding activity. In addition, SP2/0 cells expressing the chimeric H chain either alone or together with the parental L chain were lysed by class I restricted NP-epitope specific CTL. By contrast, SP2/0 cells pulsed with soluble chimeric Ig molecules were not lysed by the specific CTL. These observations indicate that: 1) this particular CTL epitope can be expressed on Ig molecules without altering the H and L chain pairing; 2) this CTL epitope can be generated from this chimeric Ig in which it is surrounded by flanking regions distinct from those of the viral NP; and 3) the generation of this CTL epitope from the Ig molecule requires the endogenous pathway as do viral proteins.
Peripheral deletion of mature CD8+ antigen-specific T cells after in vivo exposure to male antigen.
Authors: Zhang L I , Martin D R , Fung-Leung W P , Teh H S , Miller R G
Published: December 2022

It has been well established that T cell tolerance to self Ag occurs primarily via clonal deletion of immature thymocytes in the thymus. Evidence also exists that there are additional mechanisms operative on mature T cells for establishing and maintaining tolerance in the periphery. To follow the fate of mature Ag-specific T cells in vivo, we used female transgenic mice, which contain a large population of male H-Y Ag-specific T cells that can be identified by immunostaining with mAb directed against CD8 and the transgenic TCR. H-Y Ag was introduced into these mice by injecting Ag-bearing male lymphocytes using conditions known to induce CTL precursor response reduction. The number of Ag-reactive CD8+ transgenic T cells in the periphery started to decrease after 2 days of in vivo exposure to male Ag. Decline was maximum (up to 80% of total) by 7 days, and stayed at this level for at least 6 wk. CD4+ cells and those CD8+ cells that did not carry the transgenic TCR were not affected. Most or all of the remaining Ag-reactive CD8+ cells in the periphery were fully responsive when stimulated by male Ag in vitro. Maturation of transgenic T cells in the thymus of injected mice remained the same as that of control animals. Our data provide direct evidence that mature Ag-reactive CD8+ cells are susceptible to clonal deletion in the periphery when exposed to the Ag in vivo. These findings suggest the presence of two types of APC in the periphery: stimulatory APC (e.g., macrophages and dendritic cells) required for initiating an active immune response; and functionally deleting APC (or veto cells) capable of deleting mature T lymphocytes that recognize Ag presented on their surface. Functionally deleting APC that present self Ag to peripheral T cells may provide a fail-safe mechanism against autoreactive cells that escaped deletion during differentiation in the thymus.
Alloantigenicity of human endothelial cells. 1. Frequency and phenotype of human T helper lymphocytes that can react to allogeneic endothelial cells.
Authors: Adams P W , Lee H S , Waldman W J , Sedmak D D , Morgan C J , Ward J S , Orosz C G
Published: December 2022

To determine the relative ability of allogeneic endothelial cells to stimulate helper T lymphocytes (HTL), human PBMC or purified T cells were incubated in conventional lymphocyte microcultures or in limiting dilution microcultures with allogeneic human umbilical vein endothelia (HUVE), with cytokine-treated allogeneic HUVE, or with allogeneic peripheral blood monocytes. These cultures were tested for IL-2 production as an index of HTL stimulation. Dose-response studies in conventional lymphocyte cultures indicated that allogeneic monocytes were better than allogeneic HUVE at stimulating IL-2 production. Limiting dilution analyses revealed that untreated HUVE and TNF-treated HUVE stimulated small numbers of HTL (approximately 1 HTL/30,000 PBMC), whereas 5 to 10 times more HTL were stimulated by IFN-gamma-treated HUVE and 10 to 20 times more HTL were stimulated by allogeneic monocytes. Serologic deletion studies revealed that most of the high frequency HTL responding to IFN-gamma-treated HUVE were CD4+, whereas most of the low frequency HTL responding to nontreated HUVE or to TNF-treated HUVE were CD8+. Interestingly, mAb to MHC class I and class II molecules, which significantly impaired HUVE-induced proliferation, caused little interference with HUVE-induced IL-2 production. Finally, polymerase chain reaction analysis demonstrated that untreated allogeneic HUVE cells could stimulate PBMC to produce mRNA for IFN-gamma, as well as for IL-2. These data demonstrate the following hierarchy of allogeneic stimulatory capacity for human HTL: monocytes greater than IFN-gamma-treated HUVE much greater than TNF-treated HUVE = nontreated HUVE. Further, these data suggest that non-activated allogeneic endothelial cells can initiate immune responses by inducing IL-2 and IFN-gamma. Because IFN-gamma can induce MHC class II expression by the endothelial cells, this could recruit large numbers of CD4+ T cells for IL-2 production.
A murine model of allergic bronchopulmonary aspergillosis with elevated eosinophils and IgE.
Authors: Kurup V P , Mauze S , Choi H , Seymour B W , Coffman R L
Published: December 2022

A model of allergic bronchopulmonary aspergillosis was developed by exposing BALB/c mice to Aspergillus fumigatus (AF) Ag. Animals immunized intranasally (i.n.) with soluble AF Ag produced low levels of serum IgE compared to animals given alum precipitated AF Ag i.p. The latter treatment also produced higher levels of serum IgG1 and AF-specific IgG1 than soluble AF given i.p. or i.n.. Blood and lung eosinophilia was detected in mice repeatedly exposed to AF by i.n. but not in the groups injected i.p. Particulate AF Ag-induced striking blood and lung eosinophilia and elevated levels of serum IgE in mice preexposed to AF Ag. The results indicate that route of inoculation and physical nature of Ag determine the immune response and can be manipulated to obtain enhanced IgE, eosinophils, or both in the animal model.
Local T cell responses induce widespread MHC expression. Evidence that IFN-gamma induces its own expression in remote sites.
Authors: Halloran P F , Autenried P , Ramassar V , Urmson J , Cockfield S
Published: December 2022

Local inflammation induces increased expression of MHC and other genes in the affected tissue because of the paracrine effects of cytokines such as IFN-gamma. We previously reported that one such process--local allograft rejection--was accompanied by increased expression of MHC in a remote tissue, namely kidney. To explore how local inflammation affects gene expression in remote tissues, we studied MHC, beta 2-microglobulin, and IFN-gamma expression in mice undergoing either of two T cell-dependent localized inflammatory processes: rejection of an ascites tumor allograft, and skin sensitization by oxazalone. As assessed by binding of radiolabeled mAb and by immunohistology, each stimulus increased MHC expression in many remote tissues, including liver, heart, pancreas, and kidney. This was associated with increases in steady state mRNA for class I, class II, and beta 2-microglobulin. MHC induction was inhibited by the in vivo administration of cyclosporine or anti-IFN-gamma mAb and did not occur in nude mice, confirming the key role of IFN-gamma released from T cells. When we examined tissues of mice with these localized inflammatory lesions for IFN-gamma mRNA levels by polymerase chain reaction, we found that IFN-gamma steady state mRNA levels were increased in the spleen and, more surprisingly, in the kidney, and in uninvolved skin. Moreover, anti-IFN-gamma inhibited the induction of IFN-gamma mRNA in the kidney, suggesting that IFN-gamma expression was induced by IFN-gamma in an autoregulatory fashion. Thus the systemic MHC induction accompanying local T cell-mediated inflammation reflects the release of IFN-gamma from the site of inflammation, but may be amplified by the ability of IFN-gamma to induce its own expression in remote tissues. This self-amplification of IFN-gamma may contribute to the ability of local inflammation to induce extensive systemic effects.
The lymphocyte-specific tyrosine protein kinase p56lck is endocytosed in Jurkat cells stimulated via CD2.
Authors: Marie-Cardine A , Maridonneau-Parini I , Ferrer M , Danielian S , Rothhut B , Fagard R , Dautry-Varsat A , Fischer S
Published: December 2022

Incubation of the human T cells, Jurkat, with two sets of activating anti-CD2 mAb (T11(2) + T11(3), D66 + T11(1)) induced delocalization of p56lck and CD2 receptors from the plasma membrane and increased the tyrosine kinase activity of p56lck. The anti-CD2 mAb combination (T11(2) + T11(3)) that produced the most rapid increase in p56lck kinase activity also induced the most rapid delocalization of the kinase. In stimulated cells, both p56lck and CD2 receptors are detected in cytoplasmic vesicles. The internalization of p56lck in endocytic vesicles was established by confocal microscopy. By double staining it was shown that only part of the p56lck colocalized with the internalized CD2 receptor suggesting distinct sorting processes. Internalization of p56lck appeared to be specific of CD2 stimulation as: 1) in Jurkat cells triggered with an anti-CD3 mAb, p56lck was not internalized whereas CD3 receptors were completely endocytosed; 2) when cells were stimulated via CD4, the kinase and CD4 receptors remained associated with the plasma membrane. In addition, internalization of p56lck upon stimulation of CD2 receptors was not modified in CD2+/CD3-Jurkat cells indicating that CD3 is not involved in this process. The identification of different subcellular localizations of p56lck in resting and stimulated T cells should represent an important step in the definition of its functional activity.
Adhesion of B lymphoid (MPC-11) cells to type I collagen is mediated by integral membrane proteoglycan, syndecan.
Authors: Sanderson R D , Sneed T B , Young L A , Sullivan G L , Lander A D
Published: December 2022

Differentiating B lymphocytes undergo changes in cell-cell and cell-matrix adhesion that control their movement through a series of distinct microenvironments. The integral membrane proteoglycan, syndecan, is a candidate for mediating B lymphocyte-matrix interactions because it is expressed on B lymphocytes only at times when they associate with matrix, and because syndecan is known to behave as a matrix receptor on simple epithelia. However, syndecan from B lymphocytes is significantly smaller in molecular mass than syndecan from simple epithelia (85 vs 160 kDa) suggesting that syndecan may have distinct functions on these two cell types. Our study was undertaken to determine if syndecan mediates adhesion of B lineage cells to extracellular matrix. The murine myeloma cell line MPC-11 was used because syndecan is the only major heparan sulfate proteoglycan detected on these cells and because they express a form of syndecan almost identical to that found on normal B lymphocytes. Cell binding assays demonstrate that syndecan binds MPC-11 cells to type I collagen. Binding is inhibited by heparin, by pretreatment of cells with heparitinase or by growth of cells before the assay in chlorate, an inhibitor of sulfation. Solid phase assays show that syndecan purified from MPC-11 cells binds to type I collagen but not type IV collagen, laminin, or fibronectin. The interaction of MPC-11-derived syndecan with type I collagen is of relatively high affinity (Kd app = 143 nM) as measured by affinity coelectrophoresis. However, the 160-kDa form of syndecan isolated from epithelial cells has a greater than fourfold higher affinity for type I collagen (Kd app = 31 nM) than does the MPC-11 syndecan, suggesting that different molecular forms of syndecan have distinct ligand binding properties. These results demonstrate that syndecan can mediate B lymphocyte interactions with matrix and suggest that changes in syndecan expression during B cell differentiation are a mechanism for controlling B cell localization within specific microenvironments.
Identification and characterization of the effector region within human C5a responsible for stimulation of IL-6 synthesis.
Authors: Morgan E L , Sanderson S , Scholz W , Noonan D J , Weigle W O , Hugli T E
Published: December 2022

Human C fragment C5a is known to be a proinflammatory mediator and more recently shown to be a potent modulator of both humoral and cell-mediated immunity. We recently reported that natural and recombinant C5a induces the synthesis of IL-6-specific mRNA and secreted protein from human monocytes. Our studies using analogue peptides that are homologous to the carboxyl-terminal sequence of human C5a, indicate that the "effector" site for inducing IL-6 synthesis resides within the C-terminal region (C5a (70-74)) of the C5a molecule. C5a peptides containing the exact sequence of the natural factor were found to retain full agonist activity but exhibited low potency (0.01-0.1% of intact C5a). It was also shown that amino acid substitutions in the C5a peptides by aromatic/hydrophobic residues, outside the immediate effector site, resulted in analogue peptides with a substantial increase in potency relative to the most active natural peptide (C5a (56-74)). Moreover, these peptides approach the potency of natural C5a for induction of IL-6. Taken together, these results suggest that the inflammatory and immunoregulatory activities associated with C5a may, in part, be due to the synthesis of IL-6.
Effect of glucocorticoids on C3 gene expression by the A549 human pulmonary epithelial cell line.
Authors: Zach T L , Hill L D , Herrman V A , Leuschen M P , Hostetter M K
Published: December 2022

The third component of C, C3, is the key opsonin of the C cascade and is produced locally within the lung by pulmonary epithelial cells, macrophages, and fibroblasts. Because glucocorticoids regulate the maturation and expression of several physiologically important genes in pulmonary epithelial cells, we examined the effects of glucocorticoids on C3 mRNA expression and C3 synthesis by the human pulmonary epithelial cell line, A549. Treatment with dexamethasone enhanced C3 production in a time- and dose-dependent fashion such that concentrations of dexamethasone greater than or equal to 0.001 microM significantly increased C3 production on day 3 of culture. Natural glucocorticoids, corticosterone, cortisol, and 11-deoxycortisol also increased C3 concentrations in A549 supernatants. Both cycloheximide and the glucocorticoid receptor antagonist, RU486, individually inhibited the effect of dexamethasone on C3 production. Northern analysis demonstrated that the steady state 5.2-kb C3 message increased in A549 cells within 10 h of treatment with dexamethasone. RU486 inhibited the effect of dexamethasone on C3 mRNA expression. The integrity of the C3 thiolester bond, as measured by [3H]iodoacetic acid titration and hemolytic assay, was not disrupted by dexamethasone. We conclude that glucocorticoids such as dexamethasone enhance the expression of C3 mRNA and increase the production of functionally active C3 by A549 cells by a mechanism that is mediated by the intracellular glucocorticoid receptor.
Design and synthesis of a self-assembling peptide derived from the envelope proteins of HIV type 1. An approach to heterovalent immunogens.
Authors: Tripathy S P , Kumar A , Manivel V , Panda S K , Rao K V
Published: December 2022

A chimeric peptide that included sequences from gp120 and gp41 of HIV type 1 was synthesized. Cleavage from solid support yielded a composite of self-oligomerized products with molecular masses ranging from 5 to about 9 kDa. The oligomer but not its reduced, monomeric form was recognized by human anti-HIV sera and at least one of the two lysines in the sequence was involved in antibody binding. The oligomeric peptide was immunogenic, yielding a conformation-specific antibody response. Co-oligomerization of a hepatitis B surface Ag-derived peptide and the HIV type 1-derived peptide yielded a bivalent product in which conformational integrity of the individual components was maintained. Immunization with this hybrid peptide resulted in conformation-specific antibodies to both epitopes in all four murine strains tested. Lymphocyte proliferation assays revealed that the T epitopes resident in both peptide sequences remained active in the hybrid peptide. These results demonstrate the potential of this approach in generating multi- and heterovalent immunogens which may eventually find application as vaccines.
Inhibition of S-phase progression in macrophages is linked to G1/S-phase suppression of DNA synthesis genes.
Authors: Hamilton J A , Vairo G , Cocks B G
Published: December 2022

Some of the important controlling events regulating eukaryotic S-phase progression are considered to occur late in the G1 stage of the cell cycle. We show here that stimulation of DNA synthesis in bone marrow-derived macrophages (BMM) by macrophage CSF-1 is preceded by G1 expression of three genes which encode proteins associated with the DNA synthesis machinery--the M1 and M2 subunits of ribonucleotide reductase and proliferating cell nuclear Ag (PCNA). Increased expression for these genes correlated well with the mitogenic response and sustained expression required de novo RNA and protein synthesis and also the presence of CSF-1 for at least most of G1. Inhibitors of BMM proliferation (LPS, TNF-alpha, IFN-gamma, and cAMP elevating agents) suppressed CSF-1-induced expression of M1, M2, and PCNA mRNA measured at 22 h. This suppression occurred even when added up to 12 h after the CSF-1, a period coinciding with the G1/S-phase boundary. The delayed kinetics of this effect parallels the ability of these agents to maximally inhibit CSF-1-induced BMM DNA synthesis when added at similar times. Decreased expression of M1, M2, and PCNA was not merely a consequence of DNA synthesis inhibition because the S-phase inhibitor, hydroxyurea, did not suppress CSF-1-induced gene expression. These results suggest that inhibition of DNA synthesis by antiproliferative agents involves inhibition of expression of several genes associated with the DNA synthesis machinery.
Unique palindromic sequences in synthetic oligonucleotides are required to induce IFN [correction of INF] and augment IFN-mediated [correction of INF] natural killer activity.
Authors: Yamamoto S , Yamamoto T , Kataoka T , Kuramoto E , Yano O , Tokunaga T
Published: December 2022

Thirty-mer single-stranded oligonucleotides, with a sequence chosen from the known cDNA encoding the 64-kDa protein named Ag A or the MPB-70 protein of Mycobacterium bovis BCG and the human cellular proteins such as complement component 1 inhibitor and Ig rearranged lambda-chain, were used to dissect the capability to induce IFN and to augment NK cell activity of mouse spleen cells by coincubation in vitro. Three with the hexamer palindromic sequence as GACGTC were active, whereas two kinds of oligonucleotides with no palindrome were inactive. The oligonucleotides containing at least one of the different palindromic sequences showed no activity. When a portion of the sequence of the inactive oligonucleotides was substituted with either palindromic sequence of GACGTC, AGCGCT, or AACGTT, the oligonucleotide acquired the ability to augment NK activity. In contrast, the oligonucleotides substituted with another palindromic sequence such as ACCGGT was without effect. Furthermore, exchange of two neighboring mononucleotides within, but not outside, the active palindromic sequence destroyed the ability of the oligonucleotides to augment NK cell activity. Stimulation of spleen cells with the substituted oligonucleotide, A4a-AAC, induced production of significant amounts of IFN-alpha/beta and small amounts of IFN-gamma. Augmentation of NK activity of the cells by the oligonucleotide was ascribed to IFN-alpha/beta production. These results strongly suggest that the presence of the unique palindromic sequences, such as GACGTC, AGCGCT, and AACGTT, but not ACCGGT, is essential for the immunostimulatory activity of oligonucleotides.
In vivo T cell activation, in vitro defective IL-2 secretion, and response to influenza vaccination in elderly women.
Authors: Huang Y P , Pechere J C , Michel M , Gauthey L , Loreto M , Curran J A , Michel J P
Published: December 2022

IL-2 secretion in response to mitogenic stimulation, assayed in vitro, is significantly reduced in circulating T lymphocytes isolated from healthy old people, but the significance of this abnormality and how it relates to in vivo IL-2 secretion remain unclear. We found that IL-2 secretion in response to PHA plus PMA by peripheral blood T cells isolated from 10 out of 32 (31%) healthy old individuals (mean age 86 yr, range 74-97) was significantly decreased compared with results obtained in 23 younger individuals (mean age 34 yr, range 23-46). This IL-2 secretion defect in vitro was reversible after a 3-day incubation in the absence of activators. The 10 healthy old individuals who had defective IL-2 secretion in vitro also showed increased levels of serum IL-2. T cells from 22 healthy old and 22 young individuals, who had normal IL-2 secretion (geometric mean +/- log of 1 SD: 139 +/- 0.3 U/ml and 212 +/- 0.31 U/ml, respectively) in vitro, showed a remarkable transient T cell defect in IL-2 secretion (15 +/- 0.47 U/ml for the old, 54 +/- 0.28 U/ml for the young) 15 days after influenza vaccination. IL-2 secretion became normal again 30 days after vaccination. The T cell-IL-2 activity, expressed as a T cell-IL-2 activity score (calculated as the logarithm of the serum IL-2 U/ml divided by the logarithm of the IL-2 secretion U/ml, in vitro) was significantly increased in elderly non-responders after influenza vaccination (mean +/- 1 SD: 1.4 +/- 0.51) compared with elderly (0.44 +/- 0.13) and younger responders (0.3 +/- 0.2). Our data suggest that in vitro defective IL-2 secretion is a consequence of T cell activation which seems to occur in a significant proportion of healthy elderly individuals and may be clinically relevant inasmuch as it appears to prevent the normal vaccine-induced antibody response.
T cell activation-inducing epitopes of the house dust mite allergen Der p I. Proliferation and lymphokine production patterns by Der p I-specific CD4+ T cell clones.
Authors: Yssel H , Johnson K E , Schneider P V , Wideman J , Terr A , Kastelein R , De Vries J E
Published: December 2022

Cloned human CD4+ T cell lines specific for the house dust mite Dermatophagoides pteronyssinus were used to map minimal T cell activation-inducing epitopes on the Group I allergen in D. pteronyssinus extracts (Der p I) molecule. Most of these Der p I-specific T cell clones expressed different TCR V alpha and V beta gene products. Using recombinant deletion proteins, three T cell epitopes were identified on the Der p I molecule; p45-67 and p117-143 were recognized by HLA-DR7-restricted T cells, whereas p94-104 was recognized in the context of HLA-DR2, DRw11 (DR5), and -DR8 molecules. This degenerate class II MHC restriction appears to be due to shared Phe and Asp residues at positions 67 and 70, respectively, in the third variable domain of the HLA-DR beta chain. All three T cell epitopes induced Th2-like cytokine production profiles by the Der p I-specific T cell clones, which were characterized by the production of very high levels of IL-4 and IL-5, as compared with those secreted by tetanus toxin-specific T cell clones derived from the same patients, but no or low amounts of IL-2 and IFN-gamma. This Th2-like production profile was, however, not an intrinsic property of the Der p I-specific T cells, but was dependent upon their mode of activation. Stimulation with Con A also induced very low or no measurable levels of IL-2 and IFN-gamma, whereas activation with TPA and the calcium ionophore A23187 resulted in the production of high levels of IL-4, IL-5, IL-2, and IFN-gamma. These results indicate that Der p I-specific T cell clones are not defective in their capacity to produce high levels of Th1 cytokines.
Failure of IL-1 receptor antagonist and monoclonal anti-IL-1 receptor antibody to inhibit antigen-specific immune responses in vivo.
Authors: Faherty D A , Claudy V , Plocinski J M , Kaffka K , Kilian P , Thompson R C , Benjamin W R
Published: December 2022

rIL-1R antagonist (rIL-1ra) and 35F5, a neutralizing mAb, have been shown to inhibit the ability of IL-1 alpha and IL-1 beta to bind to type I but not type II murine receptors. Additionally, IL-1ra and 35F5 inhibit a variety of inflammatory responses in vitro and in vivo. In the present report we have evaluated the activity of human IL-1ra and 35F5 in murine Ag-specific cell-mediated and humoral immune response models. Administration of IL-1ra, either twice daily or as a continuous infusion, did not inhibit the cytolytic T lymphocyte response to allogeneic splenocytes. The CTL response was also not inhibited by daily administration of 35F5. The delayed type hypersensitivity response to oxazolone was similarly refractory to administration of IL-1ra and 35F5. In the humoral immune response models, neither the splenic plaque response to SRBC nor the IgG or IgM response to TNP-keyhole limpet hemocyanin was inhibited by treatment with IL-1ra or 35F5. These data suggest that signaling through the type I IL-1R is not required for these Ag-specific immune responses.
IL-10, T lymphocyte inhibitor of human blood cell production of IL-1 and tumor necrosis factor.
Authors: Ralph P , Nakoinz I , Sampson-Johannes A , Fong S , Lowe D , Min H Y , Lin L
Published: December 2022

We have identified and purified a factor that inhibits the production of IL-1 beta and TNF by stimulated human mononuclear cells. The activity is produced by the T cell lines Hut-78 and Mo constitutively under serum-free conditions. Crude conditioned media have titers of up to 100 U/ml (one unit defined as the reciprocal of the dilution producing 50% inhibition). The activity resides mainly in a single size peak of 30 to 35 kDa and an isoelectric point around 8. Other cytokines in this size range that have been reported to be inhibitory for IL-1 and TNF production include TGF-beta, IL-4, and IL-6; these factors were excluded by lack of detection, neutralizing antibody, and low activity compared with our factor. Another factor with these size and charge properties is IL-10, which inhibits T cell cytokine production. By polymerase chain reaction analysis, Mo and HuT-78 lines contain IL-10 transcripts whereas JURKAT is negative; this correlates with inhibitor bioactivity from the three lines. Use of mAb specifically showed the inhibitor to be IL-10.
Promiscuous malaria peptide epitope stimulates CD45Ra T cells from peripheral blood of nonexposed donors.
Authors: Fern J , Good M F
Published: December 2022

PBL from individuals with no history of malaria exposure, as well as cord blood lymphocytes, were tested for proliferation to T cell epitopes from the malaria circumsporozoite proteins of Plasmodium falciparum and Plasmodium vivax. Cells from many individuals proliferated in response to these peptides, but for two peptides (P. vivax317-336 and P. falciparum CS331-350) the response rate ranged from 64 to 93%, with the specific stimulation indices reaching as high as 38. The phenotype of the cells responding to PfCS331-350 was predominantly CD4+,CD8-,CD45Ra+,CD45Ro-, which was the inverse of the phenotype of the cells responding to tetanus toxoid with respect to CD45 isoforms. T cell clones from different individuals specific for PfCS331-350 were restricted by at least four different HLA-DR molecules and there was no evidence that the peptide was a "superantigen." Overlapping peptides were used to demonstrate that clones had different fine specificities although the peptide specificities of the DR4-restricted and DR11-restricted clones were similar. Although the individuals tested here have had no history of malaria exposure, these data demonstrate that they have T cells specific for malaria sequences present in high frequency that proliferate as intensely as some memory responses. Although one clone from an individual with a history of BCG vaccination did react strongly with PPD, the phenotype of these cells suggests that they are not classical memory cells for a cross-reactive recall Ag. Such cells may affect the induction or expression of malaria immunity.
Constitutive versus cell cycle regulation of c-myb mRNA expression correlates with developmental stages in murine B lymphoid tumors.
Authors: Catron K M , Purkerson J M , Isakson P C , Bender T P
Published: December 2022

The expression of c-myb mRNA is differentially regulated in murine B lymphoid tumors such that B cell lymphomas and plasmacytomas contain significantly less c-myb mRNA than pre-B cell lymphomas. To examine the low level of c-myb mRNA expression in the murine B cell lymphoma cell line BCL1, nonessential amino acid starvation was used to block these cells in a G1 state. When BCL1 cells were released from this block, a 7- to 10-fold increase in c-myb mRNA was detected in late G1 and S phase cells relative to that detected in exponentially growing BCL1 cells. This increase was not inhibited by aphidicolin. To determine whether cell cycle regulation of c-myb mRNA expression occurred during exponential growth in both murine pre-B cell lymphoma and B cell lymphoma cell lines, elutriation was used to separate exponentially growing cell populations. An increase in c-myb mRNA expression was seen in late G1 and S phase fractions from B cell lymphoma cell lines. In contrast, c-myb mRNA levels remained constant in elutriation fractions isolated from pre-B cell lymphoma cell lines. Expression of c-myb mRNA was not detected in exponentially growing or in Go serum-stimulated murine fibroblasts. These results indicate that constitutive vs cell cycle regulation of c-myb mRNA expression is related to the state of differentiation in murine B lymphoid tumors and suggest that a switch in regulation may occur during normal B cell development.
IL-7 receptor mediates tyrosine phosphorylation but does not activate the phosphatidylinositol-phospholipase C-gamma 1 pathway.
Authors: Roifman C M , Wang G X , Freedman M , Pan Z Q
Published: December 2022

IL-7 is a glycoprotein involved in the regulation of lymphocyte precursor growth. In addition, it has a comitogenic effect on mature T cells but not on mature B cells. The exact mechanism whereby IL-7R mediates these cell growth properties remains unknown. Because many growth factor receptor systems on various cell types transduce signals by activating a tyrosine kinase, we have studied here the effect of IL-7R ligation on protein tyrosine phosphorylation. We found that human rIL-7 consistently induced tyrosine phosphorylation of five major proteins, of 175, 155, 135, 110, and 85 kDa, and five minor proteins. The effect of human rIL-7 on tyrosine phosphorylation of these substrates was concentration and time dependent. One of the known substrates that is phosphorylated on tyrosine residues after binding of growth factors to their receptors is the phosphoinositide-specific phospholipase C. Several phospholipase C isozymes have been recently recognized; one isozyme, phospholipase C-gamma 1, was demonstrated to be phosphorylated rapidly after ligand binding to the platelet-derived growth factor receptor and the T cell Ag receptor. We show here that, in contrast to Ag receptor ligation, activation of IL-7R does not induce tyrosine phosphorylation on phospholipase C-gamma 1. Consistent with these results, human rIL-7 failed to increase phosphatidylinositol turnover and did not induce a rise in cytosolic free Ca2+ in the thymocytes, mature T cells, or pre-pre-B cells. The results indicate that the IL-7R mediates the activation of the tyrosine phosphorylation pathway but does not induce the phosphatidylinositol-phospholipase C pathway.
HLA-Bw22: a family of molecules with identity to HLA-B7 in the alpha 1-helix.
Authors: Hildebrand W H , Madrigal J A , Little A M , Parham P
Published: December 2022

Various HLA-B molecules exhibit serologic cross-reactions with HLA-B7, including HLA-B27, B40, Bw42, and Bw22. Of this group, primary structures for the three serologic subdivisions of HLA-Bw22, HLA-Bw54, Bw55, and Bw56, have yet to be determined. Here, we describe the nucleotide sequences of five distinctive HLA-Bw22 alleles isolated from cells of different ethnic origins typed either for Bw54, Bw55, or Bw56. Heterogeneity in molecules typed as Bw55 and Bw56 was defined. The five HLA-Bw22 alleles form a closely related family that appears to have evolved by a series of simple gene conversion events, all of which alter the antigen recognition site of the encoded proteins. Of note, HLA-Bw54 is the product of a gene conversion between HLA-B and C alleles. All the Bw22 alleles encode an alpha 1-helix identical in amino acid sequence to that of HLA-B7 and Bw42, a feature almost certainly responsible for the serologic cross-reactivity of these molecules. Shared substitutions in the alpha 1-helix can also explain the cross-reactivity of Bw22 and B7 with B27. Patterns of amino acid substitution in the alpha 2-domain of Bw22 heavy chains correlate with certain antibody and T cell cross-reactivities, thereby implicating particular amino acids in their target epitopes.
Expression of human IL-1 beta in Salmonella typhimurium. A model system for the delivery of recombinant therapeutic proteins in vivo.
Authors: Carrier M J , Chatfield S N , Dougan G , Nowicka U T , O'Callaghan D , Beesley J E , Milano S , Cillari E , Liew F Y
Published: December 2022

The feasibility of using Salmonella typhimurium aroA mutant (SL3261) to deliver protein therapeutic agents was investigated in a murine model system. We have constructed an Escherichia coli expression plasmid designed to express the human protein IL-1 beta. This plasmid expresses IL-1 beta to high levels (greater than 30% total cell protein) in E. coli. In Salmonella the IL-1 beta is expressed constitutively to about 10% total cell protein, as verified by Western blotting analysis using polyclonal rabbit anti-IL-1 beta antibody. The protein is produced in a soluble and biologically active form. BALB/c mice administered orally or i.v. with S. typhimurium aroA mutants carrying the plasmid produced highly significant antibody responses against human IL-1 beta as determined by a solid-phase RIA. Furthermore, mice injected with the construct were significantly protected against lethal gamma-irradiation (850 rad). This study therefore demonstrates that the vaccine strain of Salmonella mutants can also be used effectively to deliver therapeutic proteins in vivo.
IL-4 induces a Th2 response in Leishmania major-infected mice.
Authors: Chatelain R , Varkila K , Coffman R L
Published: December 2022

The infection of mice with Leishmania major can cause either a fatal disseminated disease or a localized healing disease, depending on the genetic background of the mice. A strong correlation has been shown between disease outcome and the nature of the T cell response, with healer strains developing a Th1-like response and nonhealer strains a Th2-like response. The treatment of nonhealer BALB/c mice with a single dose of an anti-IL-4 antibody, given at the time of infection with L. major, allowed these mice to develop healing Th1-like responses, suggesting that IL-4 is required in BALB/c mice for the differentiation of Th cells into Th2 cells. Anti-IL-4 had to be present during the first 2 wk of infection to have this effect. Anti-IL-4 caused a marked shift from a Th2 to a Th1 pattern of cytokine expression within 4 days, in vivo, and injections of IL-4 had the opposite effect on the early response in healer C3H/HeN mice. These findings demonstrate that IL-4 can induce the development of Th2 response to L. major infection in vivo.
Microanatomic clonality of gamma delta T cells in human leishmaniasis lesions.
Authors: Uyemura K , Klotz J , Pirmez C , Ohmen J , Wang X H , Ho C , Hoffman W L , Modlin R L
Published: December 2022

T cells bearing gamma delta Ag receptors accumulate in the lesions of patients with localized American cutaneous leishmaniasis (LCL), and are thought to be involved in immunity to the parasite. To obtain clues as to the nature of the Ag recognized by these cells, we analyzed the diversity of the TCR delta-chain in LCL lesions. Using mAb against variable (V) encoded determinants with immunoperoxidase, both V delta 1 and V delta 2 subpopulations were identified in the dermal granulomas. However, within the epidermis of LCL lesions, the majority of the gamma delta T cells were V delta 1 positive. PCR analysis of lesion-derived DNA using oligonucleotide primers for V and junctional (J) gene segments revealed preferential usage of J delta 1 in lesions compared with the peripheral blood of these patients. Nucleotide sequence analysis of the V-J junction indicated limited diversity of gamma delta T cells within specific microanatomic regions. In addition, use of a single diversity (D) gene segment, D delta 3, in V delta 2 cells in lesions was observed, as opposed to multiple D delta gene segment usage in the blood of the same individuals. The distribution, gene segment usage and clonality of gamma delta T cells in lesions of leishmaniasis was remarkably similar to that observed in leprosy. Therefore, gamma delta T cells responding to infection may recognize a limited set of nominal Ag, perhaps common to distinct pathogens and/or those expressed by the host. Our findings are most consistent with a model in which specific gamma delta T cells are clonally selected by these Ag in lesions and undergo oligoclonal expansion within a microanatomic region.
Differential expression of related HLA class II DQ beta genes caused by nucleotide variation in transcriptional regulatory elements.
Authors: Shewey L M , Beaty J S , Andersen L C , Nepom G T
Published: December 2022

HLA class II genes comprise a large multigene family with intra- and interlocus variation in structure and expression. Within this family of related genes, the HLA-DX alpha and beta loci (HLA DQA2 and DQB2) are highly homologous to functional HLA-DQ loci (HLA DQA1 and DQB1) but are frequently termed pseudogenes because DX gene transcription has not been observed, even in cells expressing HLA-DQ. Analysis of upstream transcriptional regulatory elements for the DX beta and DQ beta genes identified a high degree of nucleotide homology, consistent with their derivation from a common ancestral class II gene. However, transient expression assays with plasmids utilizing promoter elements from the DX beta gene had no activity in transfected human B cells, in contrast to homologous DQ beta sequences. Reciprocal exchange of specific sequences from the DQ beta gene with those of the DX beta gene restored expression to wild-type DQ beta levels, as did mutagenesis of only three DX-specific nucleotides in the upstream regulatory region. These three nucleotides mark two binding sites for distinct nuclear DNA-binding proteins that differentially recognize DQ beta and DX beta sequences. Transcription of these genes is critically dependent on interactions between these two upstream regulatory region sites which distinguish DX beta from its closely related homologue, DQ beta.
The cDNA structure, expression, and chromosomal assignment of the mouse Fas antigen.
Authors: Watanabe-Fukunaga R , Brannan C I , Itoh N , Yonehara S , Copeland N G , Jenkins N A , Nagata S
Published: December 2022

The cell surface Fas antigen is a membrane-associated polypeptide which can mediate apoptosis. cDNA clones encoding the Fas antigen were isolated from a cDNA library constructed with mRNA from the mouse macrophage cell line BAM3. The nucleotide sequence and the deduced amino acid sequence of the mouse Fas antigen were 58.5 and 49.3% identical, respectively, to the corresponding sequences of human Fas antigen cDNA. The mouse Fas antigen consists of 306 amino acids with a calculated Mr of 34,971 and contains a single transmembrane domain which divides the molecule into extracellular and cytoplasmic domains. A 2.1-kb mRNA coding for the Fas antigen was detected in the mouse thymus, heart, liver, and ovary but not in brain and spleen. The expression of the Fas antigen gene in mouse fibroblast L929 and macrophage BAM3 cell lines was significantly induced by treatment with IFN-gamma but not by IFN-alpha/beta. Interspecific backcross analysis indicated that the gene coding for the Fas antigen is in the distal region of mouse chromosome 19.
Peptide-presenting similarities among functionally distant HLA-B27 subtypes revealed by alloreactive T lymphocytes of unusual specificity.
Authors: López D , Rojo S , Calvo V , López de Castro J A
Published: December 2022

Functional dissection of HLA-B27 subtypes using alloreactive or B27-restricted CTL has shown that the structurally related B*2704 and B*2706 are the most distant subtypes relative to the prototype B*2705. In particular, previous studies have failed to find anti-B*2705 CTL cross-reacting with B*2704 or B*2706. Such failure can be accounted for by the drastic effect on T cell recognition of the change at residue 152 in both subtypes relative to B*2705, as established with site-directed mutants. B*2704 and B*2706 are also related in ethnic distribution, as they are restricted to Orientals, jointly being the predominant HLA-B27 subtypes in this population. As far as it is known, there are no differences relative to B*2705 in their linkage to ankylosing spondylitis. In our study, 5 of 13 examined anti-B*2705 limiting dilution CTL lines from a particular HLA-B27- individual were shown to crossreact with B*2704, B*2706 or both. The monoclonal nature of this cross-reaction was established by cold target competition analysis. This result demonstrates that the apparent differences in T cell antigenicity among anti-B27 subtypes are strongly influenced by the responder individual, as the spectrum of clonal specificities in anti-B27 responses may show significant differences among unrelated responders. Fine specificity differences among the cross-reactive CTL allowed unambiguous functional distinction between B*2704 and B*2706. The molecular basis of such cross-reactivity was examined by correlating CTL reaction patterns with the structure of both subtypes, which differ only by two residues located in the beta-pleated sheet bottom of the peptide binding site, and with site-directed mutants mimicking HLA-B27 subtype polymorphism. The results suggest that: 1) distinct peptides are involved in the allospecific epitopes recognized by the various crossreactive CTL, and 2) B*2704, B*2706, and B*2705 differ in their peptide-presenting specificity, but can present some identical or structurally similar peptides.
Immunologic disparity in the hypopituitary dwarf mouse.
Authors: Cross R J , Bryson J S , Roszman T L
Published: December 2022

The Snell-Bagg hypopituitary dwarf mouse has been shown to be deficient in growth hormone, thyroxine, and prolactin. There are reports indicating that in addition to these neuroendocrine abnormalities, development of immune competence is also severely impaired in these animals. However, other studies indicate that the immunologic potential of these mice does not differ from their heterozygous littermate controls. Our data show that dwarf mice weaned at 21 days of age and killed at that time, or 7 days later, have reduced numbers of cells in both the spleen and thymus and the mitogen responsiveness of these cells is impaired. However, if mice weaned on day 21 are analyzed at 32 days of age or the mice are weaned at day 30 and analyzed 7 days later the ability to respond to mitogenic stimulation does not differ from controls. Further experiments show that dwarf mice weaned at 30 days of age have a normal complement of V-beta TCR as evidenced by immunofluorescence analysis as well as a primary antibody response to SRBC equivalent to that observed in normal littermates. Immunofluorescence analysis of CD4 and CD8 expression on thymocytes obtained from dwarf mice shows a distinct pattern dependent on the time of weaning and time of analysis. Initial analysis of thymocytes from dwarf mice weaned and killed at 21 days of age do not differ from controls. However, cells from dwarf mice weaned on day 21 and killed on day 28 are markedly different with a loss of immature CD4+/CD8+ cells and a corresponding increase in CD4+ and CD8+ mature thymocytes. In contrast, the phenotype of thymocytes obtained from dwarf mice weaned at 30 days of age and killed on day 37 did not differ from normal littermates. Collectively these studies indicate that hypopituitary dwarf mice lag behind their heterozygous littermates with respect to development of immunocompetence but normal immune responsiveness does develop by 32 days of age when the mice are weaned on day 21.
Diversity in fine specificity and T cell receptor usage of the human CD4+ cytotoxic T cell response specific for the immunodominant myelin basic protein peptide 87-106.
Authors: Martin R , Utz U , Coligan J E , Richert J R , Flerlage M , Robinson E , Stone R , Biddison W E , McFarlin D E , McFarland H F
Published: December 2022

Multiple sclerosis (MS), a human demyelinating disease, is thought to be caused by an autoimmunologic process, and myelin basic protein (MBP) is considered a likely autoantigen. Studies of T cell lines (TCL) responding to different parts of the MBP molecule have indicated that amino acids 87 through 106 contain an immunodominant epitope of MBP. We have demonstrated previously that amino acids 89 through 99 represent the core of this 87-106 peptide epitope. Importantly, this epitope is not only encephalitogenic in SJL/J mice and Lewis rats but also has been shown to be recognized by human cytotoxic TCL in the context of four HLA-DR molecules that are associated with MS in different geographic areas. If the immune response to MBP peptide 87-106 was homogeneous with respect to epitope specificity and TCR usage, specific immunotherapies targeting the interaction of peptide, MHC, and TCR might be possible. In this study, the fine specificity of 29 CD4+ cytotoxic, long term, and limiting dilution TCL that had been generated against whole MBP and were derived from four MS patients and two healthy relatives was dissected using truncated and alanine-substituted peptides for the 87-106 peptide. In addition, the TCR alpha and beta chain usage of 15 CD4+ TCL was determined. Using truncated peptides, the presence of several nested immunogenic epitopes within amino acids 87 to 106 was demonstrated. TCL with identical restriction elements and similar responses to truncated peptides could be differentiated further using alanine-substituted peptides. Finally, heterogeneity of TCR usage was shown not only for those lines that differed in their peptide specificity but also for some that showed identical responses and were restricted by the same HLA-DR antigen. In conclusion, the CD4+ cytotoxic T cell response to the immunodominant MBP peptide 87-106 demonstrates a high degree of heterogeneity at the level of fine specificity and TCR usage. These findings indicate that specific immunotherapies aimed at TCR in MS will probably be more complicated than previously anticipated.