bluuebunny/crossref_metadata_2025 huggingface dataset:
The age-associated increase in IFN-gamma synthesis by mouse CD8+ T cells correlates with shifts in the frequencies of cell subsets defined by membrane CD44, CD45RB, 3G11, and MEL-14 expression.
Authors: Ernst D N , Weigle W O , Noonan D J , McQuitty D N , Hobbs M V
Published: January 2023

The mouse model system was used to evaluate age-associated changes in the subset composition and function of the splenic CD8+ T cell pool. In response to stimulation with plate-bound anti-CD3 epsilon mAb, CD8+ cells from old C57BL/6NNia mice produced greater levels of IFN-gamma than cells from young-adult controls. This age-associated difference was apparent at the levels of both IFN-gamma mRNA accumulation and cytokine release, and was established within the first major cell cycle in culture. In addition, the capacity to produce IFN-gamma appeared to increase gradually with age as evidenced by studies on CD8+ cells from intermediate aged mice. In contrast to these findings, the peak S-phase responses by stimulated CD8+ cells from old mice were significantly reduced relative to young-adult controls. Immunophenotypic analyses of membrane CD44, CD45RB, 3G11, and MEL-14 expression by splenic CD8+ cells from young-adult, intermediate-aged, and old mice revealed an age-associated decrease in the frequencies of cells that expressed low levels of CD44 and high levels of CD45RB, 3G11, and MEL-14, whereas the reciprocal phenotypes increased with age. The correlated analysis of all four subset markers identified a composite phenotype (CD44loCD45RBhiMEL-14hi3G11lo/hi) which, based on past functional studies, is a candidate phenotype for naive cells. This "naive" phenotype dominated the CD8+ cell pool of young-adult mice but decreased in frequency with age. In contrast to the CD44lo cell group, the CD44hi cell fraction, which is associated with preactivated/memory CD8+ cells, was found to be uniformly 3G11lo but expressed heterogeneous levels of CD45RB and MEL-14, perhaps defining multiple subsets within the memory population. All of these latter subsets increased in frequency with age. Finally, we found that when CD8+ cells were fractionated based on CD44 expression the capacity to release measurable levels of IFN-gamma segregated entirely with the CD44hi fraction, irrespective of donor age. Together, these findings support the hypothesis that aging is accompanied by dramatic shifts in the subset compositions of splenic CD8+ cell pools, which contribute significantly to their increased capacity to produce IFN-gamma at the population level.
IL-4, but not tumor necrosis factor-alpha, increases endothelial cell adhesiveness for lymphocytes by activating a cAMP-dependent pathway.
Authors: Galéa P , Thibault G , Lacord M , Bardos P , Lebranchu Y
Published: January 2023

IL-4 and TNF-alpha increase endothelial cell adhesiveness for PBL by promoting the expression of adhesion molecules. We investigated the intracellular cAMP involvement in the increased endothelial cell adhesivity induced by IL-4 or TNF-alpha. We showed that both IL-4 and TNF-alpha increased intracellular cAMP in endothelial cells (EC). Furthermore, dibutyryl-cAMP and forskolin (which increased intracellular cAMP) increased basic EC adhesivity for PBL. The co-stimulation of EC with cAMP elevating agents and TNF-alpha, but not IL-4, resulted in an additive increase in EC adhesiveness. 2',5' dideoxyadenosine, an inhibitor of adenylate cyclase, decreased PBL adhesion to IL-4- but not TNF-alpha-treated EC. Similarly, HA1004, a protein kinase A inhibitor, totally reversed the IL-4 but not TNF-alpha effect on EC adhesiveness, whereas H7, a protein kinase C inhibitor, did not antagonise cytokine-enhanced EC adhesivity. These results indicate that IL-4, but not TNF-alpha, uses a cAMP-dependent pathway to increase PBL adhesion. Furthermore, we showed that cAMP elevation in EC did not induce vascular cell adhesion molecule 1, the only identified adhesion molecule induced by IL-4, indicating that a rise in cAMP in EC promotes an as yet unidentified adhesion pathway. Our results show that IL-4 increases EC adhesiveness for PBL through activation of protein kinase A by promoting an unidentified adhesion pathway.
Cognate peptide-induced destruction of CD8+ cytotoxic T lymphocytes is due to fratricide.
Authors: Su M W , Walden P R , Golan D B , Eisen H N
Published: January 2023

In the absence of other cells, cloned CTL in culture can undergo massive destruction upon the addition of a peptide that is recognized, in association with the CTL's class I MHC proteins, by the CTL's Ag-specific TCR. To determine whether the destruction is a result of the individual CTL's recognition via its own TCR of peptide-MHC-I complexes on its own surface ("suicide"), or to cytolytic attack by some CTL on others in the same culture ("fratricide"), we compared the rate of peptide-induced cell death in conventional cultures, where CTL are free to establish cell-cell contacts, with other cultures in which individual CTL were prevented from forming cell-cell contacts by encasing them individually in agarose gel microdrops. The differences were dramatic: in the presence of high concentrations of peptide (10 millionfold greater than is necessary to support 50% lysis of conventional target cells by these CTL) cell death was linear over 0 to 8 h in conventional cultures, at a rate of about 10% per hour, whereas in the presence of the same high concentration of peptide over the same time course, no death was detected among the cells encased in agarose gel microdroplets. The results demonstrate an absolute requirement for cell-cell contact in the destruction of cloned CTL in culture with their cognate peptides at high concentration. Using an increase of intracellular calcium ion concentration ([Ca2+]i) as a measure of T-cell activation, we also found that peptide-dependent activation of CTL likewise depends upon cell-cell contact.
A kinetic study on the deletion of thymic, peripheral, and gut-associated V beta 6+ T cells in an Mls-1b BALB/c colony infected with an exogenous mouse mammary tumor virus.
Authors: Tucek C L , Desaymard C , Papiernik M
Published: January 2023

Mls-1a expression results in the deletion of T cells bearing V beta 6 chains of the TCR. However, V beta 6+ T cells are also deleted in Mls-1b BALB/c mice that have been infected with an exogenous mouse mammary tumor virus (swiss mice) via maternal milk intake, and whose open reading frame region is markedly similar to that of the provirus Mtv-7. In this report we describe the kinetics of V beta 6+ T cell deletion in the thymus, spleen, lymph nodes, and gut-associated lymphoid populations of these BALB/c mice from the early weeks of life to 6 mo of age. Deletion of V beta 6+ T cells within the CD4+ T cell population was more obvious in the thymus than in the spleen at 8 wk of age. However, the earliest incidence of deletion was observed in the gut intraepithelial lymphocyte population at 5 wk of age. Furthermore Mtv-7 (SW) transcripts were only found in the gut in the first wk of life, whereafter they could be detected in the thymus, spleen, and lymph nodes. This report suggests that after entering the intestinal tract of host mice, mouse mammary tumor virus (swiss mice) is subsequently transferred to the thymus and peripheral lymphoid organs resulting in the deletion of CD4+V beta 6+ T cells in that order.
V gene analysis of anticardiolipin antibodies from MRL-lpr/lpr mice.
Authors: Kita Y , Sumida T , Ichikawa K , Maeda T , Yonaha F , Iwamoto I , Yoshida S , Koike T
Published: January 2023

We analyzed the VH and V kappa genes of 14 hybridomas producing anticardiolipin antibodies (aCL) from MRL-lpr/lpr mice, in an attempt to address the question of whether aCL are generated by Ag-driven stimulation or by polyclonal B cell activation. Five of six aCL from one mouse and both aCL from the other mouse carried 98 to 100% homologous VH and V kappa genes, respectively, thereby indicating that aCL were derived from the oligoclonal B cell precursor in that individual mouse. Of nine clones, six (67%) used the VH gene of the J558 family, and three (43%) of seven clones used the V kappa gene of the V kappa 23 group. Three to five somatic mutations were detected in all VH and V kappa genes of the examined aCL. These results suggest the possibility that usage of VH/V kappa genes in aCL is not random and that aCL consist of somatically mutated Ig genes.
Inability of truncated recombinant Osp A proteins to elicit protective immunity to Borrelia burgdorferi in mice.
Authors: Bockenstedt L K , Fikrig E , Barthold S W , Kantor F S , Flavell R A
Published: January 2023

The murine immune response to Borrelia burgdorferi (Bb), the etiologic agent of Lyme disease, is characterized by the development of antibodies reactive with the outer surface protein (Osp) A. It has been demonstrated that passive immunization of mice with at least some Osp A antibodies, including an Osp A mAb (CIII.78) that binds to a conformational epitope in the carboxyl-terminus of Osp A, provides protection against Bb challenge. Active immunization of mice with Osp A also confers protection, making Osp A a candidate for a vaccine Ag. To determine the regions of the Osp A protein that can elicit protective immunity, we immunized boosted mice with overlapping recombinant truncated fragments of Osp A, then challenged them with Bb. All groups of mice developed IgG Osp A antibodies detectable by immunoblotting with sera diluted at least 5000-fold. As expected, vaccination with full-length recombinant Osp A protected mice from challenge infection. In contrast, none of the mice vaccinated with the truncated Osp A proteins demonstrated immunity, even those immunized with an Osp A fragment binding the neutralizing mAb CIII.78. Osp A antibodies contained in the truncated Osp A antisera also failed to immunoprecipitate in vitro translated full-length Osp A and did not bind, as demonstrated by indirect immunofluorescence, to live or acetone-fixed Bb. Taken together, these results suggest that neutralizing Osp A antibodies are induced by vaccination with the full-length recombinant Osp A protein but not by vaccination with recombinant fragments.
Effect of in vivo inhibition of nitric oxide production in murine leishmaniasis.
Authors: Evans T G , Thai L , Granger D L , Hibbs J B
Published: January 2023

In vitro experiments suggest that cytokine induced nitric oxide (NO) synthesis from L-arginine is a major effector mechanism against prokaryotic and eukaryotic intracellular pathogens. N omega monomethyl L-arginine (MLA), an active site inhibitor of the cytokine induced NO synthase, inhibits cytokine induced resistance of mammalian cells to intracellular microbes in vitro. In our experiments, we show that Leishmania infection markedly increases NO synthesis in the genetically resistant C3H/HeN mouse strain. In addition, administration of 50 mM MLA in the drinking water inhibits endogenous NO synthesis as well as natural resistance to footpad Leishmania infections in both susceptible BALB/c and resistant C3H/HeN mice. Leishmania parasites continued to proliferate in MLA-treated C3H/HeN mice after footpad inoculation. Similarly C3H/HeN mice treated for 3 wk with MLA had an increased parasite load and sloughing of the footpad. Footpad size of C3H/HeN mice not treated with MLA returned to base-line diameter and the regional nodes contained few amastigotes. These in vivo effects were paralleled by endogenous NO synthesis (high when Leishmania was controlled and low when Leishmania was not controlled). In addition, inhibition of NO synthesis in Leishmania-infected mice leads to a cachectic state not caused by infection alone or inhibition of NO synthesis alone. The cachexia appeared to be due to decreased food intake that occurs when NO synthesis is inhibited in Leishmania-infected mice. Our results show that cytokine induced NO has a major effector role in resistance of murine hosts to Leishmania infection.
IFN-gamma involvement in the severity of gram-negative infections in mice.
Authors: Kohler J , Heumann D , Garotta G , LeRoy D , Bailat S , Barras C , Baumgartner J D , Glauser M P
Published: January 2023

The role of IFN-gamma in the regulation of inflammation leading to gram-negative septic shock is still poorly understood. IFN-gamma blockade has been shown to improve the survival of animals challenged with i.v. bolus injections of LPS and gram-negative bacteria. We have investigated a model of focal Escherichia coli infection leading to peritonitis and septic shock. Mice were challenged i.p. with an inoculum near the LD50. The addition of rIFN-gamma together with bacteria increased the mortality and the level of blood TNF-alpha and IL-6. Conversely blockade of IFN-gamma with a neutralizing mAb significantly improved the survival of the mice. This beneficial effect was not associated with a stringent decrease in blood bacterial counts and TNF-alpha and IL-6 levels in survivors. In this model, the protective effect of anti-IFN-gamma mAb contrasted with the ineffectiveness of a neutralizing anti-TNF-alpha mAb. These findings suggest that overproduction of IFN-gamma might have a more detrimental role than overproduction of TNF-alpha during focal gram-negative infections.
Binding, internalization, and deacylation of bacterial lipopolysaccharide by human neutrophils.
Authors: Luchi M , Munford R S
Published: January 2023

Bacterial LPS is a potent agonist for priming and stimulating neutrophils (PMN). Although much has recently been learned about the binding receptors for LPS on these and other cells, little is known about the subsequent fate of LPS that has bound to the cell surface. In these studies, we evaluated three events in the interaction of Escherichia coli [3H]LPS with human PMN: 1) binding to the plasma membrane; 2) translocation to an intracellular compartment; and 3) enzymatic deacylation. Our results suggest that PMN bind LPS by at least two mechanisms: when serum is present, LPS binds almost entirely to CD14, whereas in the absence of serum, other binding mechanisms predominate. Serum thus augments CD14-mediated LPS binding, although the total amount of cell-associated LPS increases only by a factor of two, on average, when serum is added. Binding outpaces intracellular movement of the LPS, yet at least 1%/min of the cell-associated LPS is translocated to an intracellular compartment. In the absence of serum, LPS internalization occurs in the presence of a mAb that blocks LPS-CD14 binding, suggesting that an interaction with CD14 is not essential for LPS to traffic beyond the plasma membrane. LPS deacylation, which occurs over several hours, is inhibited by agents that reduce lysosomal (endosomal) acidification. This finding is consistent with a deacylating role for acyloxyacyl hydrolase, which has an acid pH optimum, and suggests that LPS moves at least transiently into an acidic intracellular compartment. These experiments provide a new temporal framework for evaluating LPS-neutrophil interactions.
Cytoplasmic tails of human complement receptor type 3 (CR3, CD11b/CD18) regulate ligand avidity and the internalization of occupied receptors.
Authors: Rabb H , Michishita M , Sharma C P , Brown D , Arnaout M A
Published: January 2023

To evaluate the role of the cytoplasmic tails of CD11b and CD18 in ligand binding and internalization of the human complement receptor type 3 (CR3, CD11b/CD18), wild type, and truncation mutants of CD11b and CD18 cDNA were expressed in COS fibroblastoid cells and assayed for surface membrane expression, ligand binding, and internalization. Truncated CD11b (alpha 1119T) and CD18 (beta 728T) subunits retaining, respectively, two and five residues of the putative cytoplasmic tails were generated by in vitro mutagenesis. Binding of alpha 1119T beta and alpha beta 728T heterodimeric receptors to iC3b was increased by 330% and 210%, respectively, relative to wild type CR3, suggesting that both cytoplasmic tails of CR3 negatively control receptor avidity. Internalization of antibody cross-linked wild type CR3 occurred predominantly through coated pits. Truncation of the cytoplasmic tail of CD18 but not of CD11b markedly reduced coated pit internalization of CR3, an effect also produced by a single F754A mutation involving one of two putative NPXF internalization signals in CD18.
CD48 is a low affinity ligand for human CD2.
Authors: Sandrin M S , Mouhtouris E , Vaughan H A , Warren H S , Parish C R
Published: January 2023

COS cells transiently transfected with human CD48 were found to bind human PBL, whereas mock or CD7-transfected COS cells failed to bind human lymphocytes. Binding of PBL to CD48 transfectants was almost totally inhibited by either CD48 mAb pretreatment of COS cells or CD2 mAb pretreatment of PBL, implying an interaction between CD2 and CD48. This conclusion was confirmed by the demonstration that a highly fluorescent, multimeric form of rCD2 bound to CD48 transfected COS cells in a CD48-dependent manner. Additional mAb blocking studies revealed that CD48 interacts with the T11(1) region of CD2, the same region of CD2 that binds LFA-3 (CD58). Thus, CD48 and CD58 represent alternative and possibly competing ligands for CD2, although based on blocking studies with soluble CD2, CD48 interacts with CD2 with approximately a 100-fold lower affinity that CD58.
Characterization of a cell surface glycoprotein IPO-3, expressed on activated human B and T lymphocytes.
Authors: Sidorenko S P , Clark E A
Published: January 2023

We characterize the expression, biochemical structure, and function of a novel glycoprotein, IPO-3, up-regulated on activated human lymphocytes. IPO-3 is found on activated B cells, B cell lines, and hairy cell leukemias but is not expressed on T cell or nonlymphoid cell lines. IPO-3 is not B cell-specific as it is detected at low levels on CD45RO+ CD45RA- peripheral blood T cells and CD4+CD8+CD45RO+ CD45RA- thymocytes. The IPO-3 Ag is a single-chain heavily N-glycosylated phosphoglycoprotein approximately 75 to 95 kDa in size with a 42-kDa protein core. In vitro kinase assays revealed that IPO-3 has a protein kinase activity associated with it that is maintained even in Nonidet P-40 lysates. IPO-3 is up-regulated on resting B cells within 16 h after activation with different signals including anti-IgM, IL-4, or mAb to CD40, CD20, or Bgp95. It could also be induced on T cells via CD3-cross-linking, but the kinetics of IPO-3 induction was slower on T cells than on B cells. Cross-linking IPO-3 on B cells with mAb did not induce proliferation alone but did augment proliferation promoted by IL-4 and anti-CD40 and did trigger increases in [Ca2+]i in resting B cells. Binding of IPO-3 could not be inhibited by a variety of mAb to previously identified activation markers. Thus, the IPO-3 glycoprotein appears to be a novel marker of activated B and T lymphocytes, which may play a role in the regulation of lymphocyte activation.
Unexpected lack of reactivity of allogeneic anti-Ia monoclonal antibodies with MHC class II molecules expressed by mouse intestinal epithelial cells.
Authors: Vidal K , Samarut C , Magaud J P , Revillard J P , Kaiserlian D
Published: January 2023

We have examined MHC class II molecules expression by murine gut epithelial cells using a large panel of anti-Ia antibodies. In contrast to conventional APC (i.e., B cells and macrophages), only two anti-Ia antibodies reacted with enterocytes: a xenogeneic rat anti-Ia mAb (CD311) directed against a monomorphic class II determinant, and a polyclonal antiserum directed against both I-A and I-E heterodimers. In contrast, allogeneic anti-Ia mAb were either unreactive (17 of 20) or reacted weakly (3 of 20) with enterocytes, even after in vivo treatment with IFN-gamma. This pattern of Ia reactivity of epithelial cells was tissue specific (restricted to gut mucosa) and cell specific (restricted to gut epithelial cells). Biochemical and molecular studies confirmed that enterocytes expressed I-A and I-E isotypes on their cell surface and contained mRNA of both subregion loci. Interestingly, enterocytes appeared deficient in expression of the MHC class II-associated invariant chain, and are not able to stimulate allogeneic T cells. These data suggest that gut epithelial cells express a conformation of class II molecules, antigenically distinct from that expressed on conventional APC.
Structural analysis of the CD3 zeta/eta locus of the rat. Expression of zeta but not eta transcripts by rat T cells.
Authors: Itoh Y , Matsuura A , Kinebuchi M , Honda R , Takayama S , Ichimiya S , Kon S , Kikuchi K
Published: January 2023

We analyzed the structure and pattern of expression of rat TCR zeta- and eta-chains to investigate if these components function in activation and development of rat T cells. The rat zeta cDNA contained the complete open reading frame coding for a polypeptide of 164 amino acids and the 5' and 3' noncoding sequences. Comparison of the amino acid sequence to those of mouse and human counterparts revealed a high degree of similarity, more than 85% homology among all three species except for the signal peptide, which was especially high in the cytoplasmic domain including the nucleotide binding site and the possible tyrosine phosphorylation sites. Furthermore, we determined the nucleotide sequences of a rat genomic eta-like sequence located in the 3' region of the rat zeta-gene. Although it showed a high level of nucleotide similarity to mouse and human counterparts, 90.4 and 78.9%, respectively, the deduced polypeptide was very short (only 28 residues) and markedly divergent from the mouse and human eta-specific polypeptides due to frameshift mutations. Transcription of rat zeta was shown to be highly restricted to T cells; abundantly in thymocytes and scarcely in peripheral T cells. Surprisingly, the rat eta transcript could not be detected in any rat tissues so far tested by Northern blot analysis and even by the sensitive reverse transcription-polymerase chain reaction method, whereas it was readily detected in mouse thymus. These findings suggest that the zeta-chain has conserved roles in the TCR assembly and TCR-mediated signaling. However, the eta-chain seems not to be indispensable because of its structural diversity among these three species characterized to date and the apparent lack of eta expression in the rat.
Switch recombination breakpoints occur at nonrandom positions in the S gamma tandem repeat.
Authors: Kenter A L , Wuerffel R , Sen R , Jamieson C E , Merkulov G V
Published: January 2023

Ig switch (S) recombination is clearly focused on S regions. It is possible that S-specific DNA binding proteins facilitate the alignment of these regions before recombination. The S gamma 3-specific DNA binding proteins, SNAP and SNIP/NF-kappa B, interact with two discrete regions of the S gamma 3 tandem repeat, the A and B sites. Recombination breakpoints in the S gamma 3 region were found to significantly correlate with the binding sites of the S gamma 3 binding proteins. We now report the conservation of the SNIP and SNAP binding sites in S gamma 2b and S gamma 1 DNA. SNIP/NF-kappa B interacts with its cognate sites in S gamma 2b and S gamma 1 DNA as determined by mobility shift assays, competition binding studies, and supershift analysis using an antiserum specific for the p50 component. SNAP binds specifically to S gamma 2b and S gamma 1 as measured by mobility shift assays and competition binding studies. SNAP is composed of two closely traveling mobilities that do not separate on partial purification. SNIP and SNAP are expressed in nuclear extracts derived from murine splenic B cell cultures stimulated with mitogen or mitogen and IL-4. No new DNA binding proteins specific for S gamma 1 tandem repeats are detectable in nuclear extracts from B cells stimulated with mitogen and IL-4. The sites at which recombination occurs in the S gamma 2b and S gamma 1 regions have been analyzed statistically and found to correlate with the SNIP/NF-kappa B and SNAP binding sites. Distinctions have been found regarding the use of DNA substrates within the tandem repeat between primary (mu-->gamma) and successive (gamma-->x) S recombination.
Self-peptides from four HLA-DR alleles share hydrophobic anchor residues near the NH2-terminal including proline as a stop signal for trimming.
Authors: Kropshofer H , Max H , Halder T , Kalbus M , Muller C A , Kalbacher H
Published: January 2023

Naturally processed MHC class II-associated peptides proved to be heterogeneous in size, varying from 13 to 25 amino acids. Truncation variants suggested sequence motifs that afford the amino termini to be shifted for obtaining an alignment: a 9- to 11-residue core region that is bordered by primary anchor residues is surrounded by extra sequences of variable lengths and hitherto unknown functions. Herein we present bulk sequencing analyses of self-peptides from four HLA-DR alleles and HLA-DQw7 clearly showing that the length of most of the NH2-terminal preanchor sequence is limited to 1 to 3 residues. Most strikingly, proline is the dominant residue reappearing at positions 2 and 3 in any allele. Proline revealed to function as a stop signal for NH2-terminal trimming as well as a secondary anchor: crude cytosolic and endosomal peptide fractions could be processed by aminopeptidases in vitro, whereupon DR1 binding peptides with increased affinity were generated. In addition, aminopeptidase treatment of DR1: self-peptide complexes implied that proline together with sterical constraints of the MHC molecule do protect the peptides' NH2-termini from further processing, whereas their COOH-termini were accessible to cathepsin B processing. Finally, bulk sequencing profiles contained signals from further putative anchor residues clustering in the NH2-terminal region:tyrosine, phenylalanine, leucine, isoleucine, and valine are enriched at positions 2 to 4 in DR1, DR5, and DR6, however, at positions 4 to 6 in DR3. Isotype-specificity is demonstrated by DQw7 displaying glutamine and asparagine at position 2. Obviously, the degenerate occurrence of aromatic or aliphatic side chains close to the NH2-terminal guarantees for essential interactions with a hydrophobic pocket of the investigated DR molecules. Most probably, this pocket is located in the nonpolymorphic DR alpha-chain rationalizing previous findings of promiscuous peptide binding to different DR alleles.
Biochemical identity of the mouse Ly-19.2 and Ly-32.2 alloantigens with the B cell differentiation antigen Lyb-2/CD72.
Authors: Robinson W H , Landolfi M M , Schäfer H , Parnes J R
Published: January 2023

Lyb-2/CD72 is a 45-kDa mouse B cell surface protein that binds CD5 (Ly-1) and has been shown to induce B cell proliferation upon mAb binding. The serologically defined Ly-19.2 and Ly-32.2 lymphocyte alloantigens have mouse strain distribution patterns similar to that of the Lyb-2/CD72 alleles and map to the same region on chromosome 4 as Lyb-2/CD72. Our recent isolation of the Lyb-2a, -2b, and -2c cDNA has enabled us in this report to examine the relationship between Ly-19, Ly-32, and Lyb-2/CD72. A rat T cell line transfected with a mouse Lyb-2a cDNA is recognized by Ly-19.2-specific mAb, whereas transfectants expressing the Lyb-2b cDNA are recognized by both Ly-19.2 and Ly-32.2-specific mAb. Cell surface iodination immunoprecipitation analysis from Lyb-2a cDNA transfectants using Lyb-2a- and Ly-19.2-specific mAb as well as from Lyb-2b cDNA transfectants using Lyb-2b-, Ly-19.2-, and Ly-32.2-specific Ab, produced immunoprecipitates containing comigrating 45-kDa polypeptides. Preclearing studies with these transfectants indicate that the immunoprecipitated proteins represent the same polypeptide chain. These results demonstrate that the mouse Ly-19.2 and Ly-32.2 alloantigens are in fact the B cell differentiation Ag Lyb-2/CD72.
cDNA cloning of a major allergen from timothy grass (Phleum pratense) pollen; characterization of the recombinant Phl pV allergen.
Authors: Vrtala S , Sperr W R , Reimitzer I , van Ree R , Laffer S , Müller W D , Valent P , Lechner K , Rumpold H , Kraft D
Published: January 2023

We isolated a cDNA encoding a major grass pollen allergen from a timothy grass (Phleum pratense) pollen expression cDNA library using allergic patients' IgE. The complete cDNA encoded an allergen that binds IgE from about 80% of grass pollen-allergic patients. Significant sequence homology was found to other major grass pollen allergens from Kentucky bluegrass (Poa pratense) as well as from rye grass (Lolium perenne) which originally were believed to form different identities. Using different monoclonal and polyclonal antibodies raised against group V allergens we identified the recombinant protein as a group V allergen from timothy grass, Phl p V. In IgE-binding studies it is demonstrated that the rPhl p V allergen can be used to block binding of patients' IgE to natural group V isoallergens on two-dimensional immunoblots. IgE inhibition experiments show that up to 60% of grass pollenspecific IgE can be preadsorbed with the rPhl p V allergen from patients sera. The purified rPhl p V induced specific histamine release of blood basophils from grass pollen-allergic patients. This emphasizes the usefulness of the rPhl p V for diagnostic and therapeutic purposes and corroborates the view that specific diagnosis and therapy of type l allergy could be performed with a limited panel of relevant recombinant allergens.
Expression of a protective intestinal immune response can be inhibited at three distinct sites by treatment with anti-alpha 4 integrin.
Authors: Bell R G , Issekutz T
Published: January 2023

The alpha 4 integrins mediate lymphocyte adhesion to Peyer's patch high endothelial vessels and homing to Peyer's patch, as well as to mesenteric lymph nodes. In rats, immunity to infection with the nematode Trichinella spiralis is known to be mediated by CD4+ OX22- (CD45RC-) cells that home to the intestine in large numbers. These experiments were conducted to determine whether the alpha 4 integrins or LFA-1 were involved in the expression of intestinal immunity to T. spiralis. Injection of the anti-alpha 4 integrin, mAb TA-2, but not anti-LFA-1, mAb TA-3, impaired the expression of immunity. An effect of TA-2 was measured at three distinct sites along the activation pathway leading to the migration of protective CD4+ OX22- cells to the intestine. Injection of TA-2 on the same day as infection prevented normal rejection of the parasite and abrogated the characteristic appearance of blast cells in draining lymph 3 days after infection. A similar effect on the migration of blast cells at day 3 was seen when TA-2 was injected 1 day after infection, and injection 2 days after infection still reduced the number of protective cells entering TD lymph on day 3. The effect of TA-2 and TA-3 on homing of dividing cells to the gut was examined by injecting dividing cells i.v. at the same time as antibody. Under these conditions migration of dividing cells to the gut was reduced by 90 to 95% and their capacity to adoptively transfer worm rejection blocked. Furthermore, TA-2 treatment also inhibited protection when it was injected 12, 18, or 24 h after the transfer of protective cells, when these cells had already entered the gut, but not when TA-2 injection was delayed for 36 h. These results indicate the involvement of alpha 4 integrins at the following points in the generation, dissemination, and function of CD4+ OX22- effectors: 1) initial activation during the first 48 h of infection; 2) migration of protective cells to and extravasation in the gut; 3) a function after entry into gut tissues. The results suggest that entry of dividing cells into the gut is critical for the adoptive transfer of protection and that alpha 4 integrin has multiple roles in the manifestation of intestinal immunity.
Profiles of cytokine production in relation with susceptibility to cerebral malaria.
Authors: de Kossodo S , Grau G E
Published: January 2023

Infection with Plasmodium berghei ANKA (PbA) leads, in susceptible strains of mice, to the development of cerebral malaria (CM), a lethal syndrome that reproduces some features of human CM. To study a possible relationship between genetic susceptibility to CM and the cytokine expression pattern, we quantitatively evaluated gene expression on RNA extracted from various organs of malaria-infected mice, using strains that are susceptible and resistant to CM. Northern blot analysis and semi-quantitative PCR showed that CM is associated with an increased TNF-alpha mRNA accumulation in the brain of mice developing the neurologic complications of CM. An increased IFN-gamma mRNA accumulation and a decreased expression of IL-4 and TGF-beta genes were also observed in mice susceptible to CM. In vitro restimulation studies using crude malarial Ag showed that lymphoid cell proliferation was higher in CM-susceptible than in CM-resistant infected mice. Moreover, susceptible mice produced large amounts of IFN-gamma, in a dose-dependent manner, in response to PbA Ag, whereas cells from resistant mice failed to produce significant amounts of this cytokine. Conversely, IL-2 and IL-4 production was significantly higher in infected CM-resistant mouse cells. No difference was seen in the production of IL-3 and IL-5 between resistant and susceptible PbA-infected mice. Upon stimulation with various malarial Ag, comparable amounts of TNF-alpha were produced by macrophages of either strain of mice. Taken together, these findings indicate that susceptibility to CM resides at the level of T cells rather than macrophages. Furthermore, the cytokine production profile is consistent with a predominant Th1-like response in mice developing cerebral complications of malaria.
Resistance of mice to experimental leishmaniasis is associated with more rapid appearance of mature macrophages in vitro and in vivo.
Authors: Sunderkötter C , Kunz M , Steinbrink K , Meinardus-Hager G , Goebeler M , Bildau H , Sorg C
Published: January 2023

Resistance to murine leishmaniasis has been related to the propagation of specific Th cell subsets (Th1 and Th2). This study shows that there are differences between resistant and susceptible mice in the initial myelomonocytic infiltrate, which precede the specific T cell response. After subcutaneous injection of 2 x 10(7) Leishmania major into footpads of resistant C57Bl/6 and susceptible BALB/c mice we performed immunohistochemical studies on the infiltrate. Two days after infection the percentage of more mature, F4/80-positive macrophages in the lesion increased faster in C57Bl/6 mice (63%) than in BALB/c mice (29%). The same strain-specific differences were observed after infection of corresponding strains of athymic mice (57.2% in C57Bl/6 nu/nu; 33.6% in BALB/c nu/nu), thus excluding a T cell-controlled phenomenon. After 1 wk the infiltrate in susceptible mice began to reveal significantly more cells containing MRP14, which is expressed by granulocytes and less mature monocytes but not by mature macrophages. No corresponding differences were found between athymic strains, suggesting that at this point organization of the infiltrate falls under control of protective T cells. In bone marrow cultures of BALB/c and C57Bl/6 mice, the percentage of F4/80-positive macrophages was also increasing faster in C57Bl/6 mice than in BALB/c mice. Increased expression of the F4/80 Ag was associated with higher leishmanicidal activity of C57Bl/6 macrophages. MRP14-positive bone marrow cells on the other hand were rarely infected by parasites. We suggest 1) that the earlier appearance of leishmanicidal macrophages in lesions of C57Bl/6 mice could influence propagation of either Th1 or Th2 cells by reduction of parasite load or by differential secretion of decisive cytokines and 2) that the diffuse accumulation of granulocytes and inflammatory monocytes in susceptible mice facilitates spread of disease.
IL-4 inhibits growth factor-stimulated rheumatoid synoviocyte proliferation by blocking the early phases of the cell cycle.
Authors: Dechanet J , Briolay J , Rissoan M C , Chomarat P , Galizzi J P , Banchereau J , Miossec P
Published: January 2023

A major feature of rheumatoid arthritis is an uncontrolled proliferation of synoviocytes. This is consistent with the active production of factors such as platelet-derived growth factor (PDGF) and IL-1 by the synovitis, which act in vivo as well as in vitro as potent synoviocyte growth factors. We have previously shown that IL-4 is able to inhibit growth factor production in an ex vivo model of synovitis. Herein, we show that IL-4 strongly inhibited PDGF and IL-1 beta stimulated rheumatoid arthritis synoviocyte proliferation in a dose-dependent manner and through its 130 kDa receptor. This antiproliferative effect of IL-4 directly correlated with a blockade of the synoviocyte cell cycle at the G0 + G1 phases. We also observed that IL-4 induced striking morphologic changes in IL-1 beta or PDGF-stimulated synoviocytes, including increased volume and granulosity. These changes led to major perturbations of the cell monolayer, associated with a marked decrease of synoviocyte viability. Taken together, these data indicate that IL-4 inhibits growth factor-induced proliferation of synoviocytes by interfering with the cell cycle, and by decreasing cell survival.
GM-CSF regulates human eosinophil responses to F-Met peptide and platelet activating factor.
Authors: Tomioka K , MacGlashan D W , Lichtenstein L M , Bochner B S , Schleimer R P
Published: January 2023

Evidence is accumulating that suggests that granulocyte macrophage CSF (GM-CSF) may have inflammatory actions in addition to its effects as a hematopoietic growth factor. In these studies, the effects of culture of purified human eosinophils with GM-CSF on cellular responses, including intracellular calcium responses, the expression of adherence molecules, and adhesion, were examined. In freshly isolated eosinophils expression of the adhesion molecule CD11b was only weakly increased by a 10-min incubation with platelet-activating factor (PAF) (118 +/- 3, 121 +/- 4, 117 +/- 2, n = 8) and FMLP (100 +/- 1, 106 +/- 6, and 105 +/- 4% of control for 10(-8), 10(-7), and 10(-6) M, respectively, n = 4). Lyso PAF never increased CD11b expression. Exposure of eosinophils to GM-CSF (0.5 ng/ml for 2 days) induced an increase in CD11b responses to PAF (132 +/- 3, 144 +/- 4, and 140 +/- 7) and FMLP (123 +/- 8, 145 +/- 10, and 137 +/- 4% of control for 10(-8), 10(-7), and 10(-6) M concentrations, respectively, n = 4). In 3-day culture studies, responses were greatest on days 2 and 3. The half-maximal GM-CSF concentration for the increase in the response to FMLP (10(-7) M, day 2) was approximately 100 pg/ml. GM-CSF (0.5 ng/ml) induced an increase in basal CD11b expression by day 1, which declined toward the initial baseline on days 2 and 3. Dexamethasone (10(-7) M) hastened the decline in baseline CD11b expression but did not inhibit the increase in the response to either PAF or FMLP. After 3 days of culture with GM-CSF (0.08 ng/ml), FMLP (10(-6) M)-induced elevations of intracellular calcium were increased (277 +/- 27% of day 0 control, n = 4, p < 0.01), whereas the response induced by PAF was not (70 +/- 36% of day 0 control, p = NS). A 30-min exposure to GM-CSF caused a potentiation of both PAF- and FMLP-induced adherence of eosinophils to gelatin-coated plastic. Longer incubation with GM-CSF (3 days) potentiated only FMLP-induced adherence. We conclude that expression of adherence molecules, transmembrane signaling, and adherence responses of eosinophils are enhanced by exposure to GM-CSF. These effects of GM-CSF could potentiate the adherence to endothelium and subsequent migration of eosinophils to inflammatory sites in vivo.
Degenerate recognition of a dissimilar antigenic peptide by myelin basic protein-reactive T cells. Implications for thymic education and autoimmunity.
Authors: Bhardwaj V , Kumar V , Geysen H M , Sercarz E E
Published: January 2023

The key event in the induction of an immune response is the recognition by a T lymphocyte of an antigenic peptide bound to a MHC molecule. In the absence of structural coordinates of the TCR and class II MHC molecules, various models of T cell recognition have been proposed, with the emerging dogma that T cell recognition is exquisitely specific. We show here that T cell clones specific for the N-terminal fragment of myelin basic protein, acetylated Ac1-11, recognize a set of unrelated peptides in the context of the same I-Au molecule. Moreover, immunization with the peptide mimic is sufficiently cross-reactive with Ac1-9 to either induce the Ac1-9-reactive clones or to induce tolerance as well as protection against Ac1-9-induced EAE. This observed degeneracy in T cell recognition has important implications for thymic selection and induction of autoimmune disease states by "molecular mimicry."
Elevated IFN-gamma and decreased IL-2 gene expression are associated with HIV infection.
Authors: Fan J , Bass H Z , Fahey J L
Published: January 2023

Because cytokines have a central role in the regulation and function of the human immune system, expression of several key cytokine genes in HIV infection was compared by quantitative polymerase chain reaction studies in lymphocytes from HIV-seronegative and -seropositive subjects. Elevated levels of IFN-gamma mRNA and lowered IL-2 mRNA were found in the PBMC of eight seropositive men with CD4 T cells over 500/mm3 (mean, 647/mm3), whereas IL-4 and IL-10 mRNA were not changed significantly. PBMC obtained 2 yr later from four of these patients with stable disease status (unchanged CD4 T cell number) showed median mRNA levels that were nearer normal for IFN-gamma and for IL-2. Four other men whose CD4 levels fell more than 200/mm3 in the following 2 yr, however, showed increased IFN-gamma and lowered IL-2. Purified CD4 and CD8 T cells from 10 HIV-seropositive and 10 -seronegative homosexual men were compared. Cytokine gene expression was found to be markedly different in CD4 and CD8 T cells from HIV-seropositive men. In CD8 T cells on a per-cell basis, the levels of cytokine mRNA were substantially lower than in CD4 T cells and were not markedly changed in HIV infection. In the CD4 T cells, on a per-cell basis, the mean mRNA levels of IFN-gamma, IL-10, and TNF-alpha were increased substantially (p < 0.001) in HIV infection. IL-2 gene expression was not increased significantly. Thus, the low IL-2 mRNA expression seen in PBMC is primarily due to the reduced CD4 T cell numbers. Increased expression of IFN-gamma genes in CD4 T cells, however, indicates that these cells may be responsible for substantial amounts of circulating IFN-gamma that occur in HIV infection. The striking difference in the effect of HIV infection on the expression of IFN-gamma and IL-2 genes indicates that these cytokines are under separate control. IL-4 mRNA levels were not changed. IL-10 gene expression, however, was increased more in early HIV infection, with less of an increase later. Expression of all cytokines in CD4 T cells appeared to subside late in HIV infection. However, the balance of cytokine expression was altered in all stages of HIV infection.
Induction of peri-insulitis but not diabetes in islet transplants expressing a single foreign antigen. A multi-stage model of disease.
Authors: Holers V M , Hollis G F , Schwartz B D , Evans R J , Strauss-Schoenberger J , Carel J C , Barney D L , Li B , Stafford-Hollis J , Lacy P E
Published: January 2023

We have created a new transgenic model in which the human Epstein-Barr virus receptor, CR2 (CD21), has been expressed in pancreatic beta-cells. Mice derived from three transgenic founders bred into H-2b (C57BL/6) or H-2k (CBA) genetic backgrounds did not become spontaneously diabetic. After transplantation of CR2-expressing islets under the kidney capsule of genetically matched recipients, a histologic picture of peri-insulitis was found. However, animals did not manifest cellular invasion of the islets, destruction of the islets, or diabetes for at least 230 days. Significant numbers of both T and B lymphocytes were found in the cell population surrounding the islets. A pronounced serologic response to CR2 was also present and appeared to precede the onset of peri-insulitis. Thus, in this model, we have separated the process of diabetes induction into at least two phases. One is associated with peri-islet infiltration and an antibody response. However, at least one second signal is likely necessary for the process of islet destruction to follow.
Inhibition of inflammatory liver injury by a monoclonal antibody against lymphocyte function-associated antigen-1.
Authors: Tanaka Y , Kobayashi K , Takahashi A , Arai I , Higuchi S , Otomo S , Habu S , Nishimura T
Published: January 2023

When mice were given an i.v. injection of LPS 7 days after an i.v. injection of Propionibacterium acnes, liver injury and a rapid increase of serum alanine aminotransferase and asparagine acid aminotransferase occurred. The in vivo administration of mAb against LFA-1 on days 1, 2, and 3 after the i.v. injection of P. acnes resulted in a potent inhibition of all these dysfunctions. Using P. acnes and the LPS model, we found that anti-LFA-1 mAb protected the mice from P. acnes and LPS-induced lethal shock. During the course of P. acnes and LPS-induced liver injury, inflammatory cells infiltrated the liver and caused a massive hepatic cell necrosis. Flow cytometry revealed that the liver-infiltrating cells were mainly leukocytes expressing a higher level of LFA-1 antigen than that seen in the normal liver. These results suggested that the LFA-1 molecule on liver-infiltrating leukocytes may play an important role in the induction of inflammatory liver injury.
Polyclonal B cell activation induced by herpesvirus saimiri-transformed human CD4+ T cell clones. Role for membrane TNF-alpha/TNF-alpha receptors and CD2/CD58 interactions.
Authors: Del Prete G , De Carli M , D'Elios M M , Fleckenstein I M , Fickenscher H , Fleckenstein B , Almerigogna F , Romagnani S
Published: January 2023

We have shown that in vitro infection herpesvirus saimiri (HVS) can transform human CD4+ T cell clones with defined Th1 or Th2 cytokine profiles to continuous growth. We report here that transformation with HVS enabled both Th1 and Th2 clones to stimulate proliferation and Ig production by autologous or allogeneic B cells in the absence of stimulants. The polyclonal B cell-activating property of HVS-transformed clones was not related to free virus or soluble cytokines, but rather was dependent on an Ag-nonspecific, MHC-unrestricted, contact-dependent mechanism. T blasts from unstimulated HVS-transformed clones did not express CD40 ligand (CD40L) mRNA or CD40L protein, whereas a proportion of them constitutively expressed membrane TNF (mTNF)-alpha. Both CD40L and mTNF-alpha were detectable on either uninfected or HVS-transformed clones upon mitogen stimulation. The activation of high-density B cells by unstimulated HVS-transformed clones was not inhibited by soluble CD40-Ig fusion protein, but was strongly reduced by either anti-TNF-alpha or anti-TNF-alpha receptor (TNF-alpha R) mAbs. Addition of anti-CD2 and/or anti-CD58 mAbs was also inhibitory, but no additive effect with anti-TNF-alpha and/or anti-TNF-alpha R mAbs was observed. Neither anti-IL-2 nor CD40-Ig inhibited the proliferation of naive IgD+ B cells cocultured with fixed unstimulated HVS-transformed clones, whereas a combination of anti-TNF-alpha and anti-TNF-alpha R mAbs was inhibitory. In addition, fixed unstimulated HVS-transformed clones induced Ig synthesis in IgD+ naive B cells even in the absence of exogenous IL-2. Data suggest that both the mTNF-alpha/TNF-alpha R and the CD2/CD58 pathways, but not the CD40L-CD40 interaction plus secreted cytokines, are involved in the unusual mode of B cell activation exerted by CD4+ HVS-transformed clones.