bluuebunny/crossref_metadata_2025 huggingface dataset:
CD4-imitating human antibodies in HIV infection and anti-idiotypic vaccination.
Authors: Deckert P M , Ballmaier M , Lang S , Deicher H , Schedel I
Published: January 2023

Anti-idiotypic vaccination against HIV infection aims at inducing an anti-gp120 immune response through anti-CD4 Abs mimicking epitopes of the gp120 molecule. The mAb IOT4a induces anti-gp120 Abs in rabbits. This study investigates the presence of human serum Abs cross-reacting with anti-CD4 mAbs and gp120 during HIV infection and after parenteral vaccination with IOT4a. Ten HIV-infected volunteers without immunodeficiency were inoculated s.c. with 0.6, 1.2, or 2.4 mg IOT4a. Six booster injections followed until day 35. Sera from study patients, 80 HIV-positive, and 43 seronegative controls were examined by a panel of assays, including an ELISA using competition against biotinylated recombinant CD4, flow cytometry assaying inhibition of anti-CD4 mAbs and biotinylated gp120 binding to CD4-positive lymphocytes, and an IOT4a immunoblot. After vaccination, an increase in competitive binding activity, which was quantitative in ELISA and flow cytometry, was observed. In the ELISA, competition against biotin-CD4 was quantitatively quenched by preincubating sera with r-gp120 or anti-CD4 mAbs such as IOT4a and Leu3a. Naive sera or sera from blood donors had no such effect in the assays employed, while 5/80 HIV sera showed binding qualities similar to vaccinees. These results suggest that 1) CD4 internal-image Abs emerge in a small proportion of HIV-positive individuals, and 2) parenteral vaccination with mAb IOT4a can induce a gp120 cross-reacting immune response that inhibits gp120 binding to CD4.
Leukophysin: an RNA helicase A-related molecule identified in cytotoxic T cell granules and vesicles.
Authors: Abdelhaleem M M , Hameed S , Klassen D , Greenberg A H
Published: January 2023

Leukophysin (LKP) is a 28-kDa protein of CTL and U937 monocytic cells that is located in the membrane of high density granules as well as lighter cytoplasmic granules or vesicles. mAbs to KLP were used to clone a full length cDNA clone with an open reading frame coding for a 235-amino acid polypeptide with a molecular mass of 24.3 kDa and two potential transmembrane regions. The nucleotide sequence was highly homologous to the 3' end of human RNA helicase A. Expression of the LKP was confirmed as a reverse transcriptase-PCR product that may be an alternately spliced product of RNA helicase A. The cDNA contained a repetitive motif that was similar to synaptophysin 1, a protein that is important for synaptic vesicle exocytosis. A polyclonal Ab directed against the 17 carboxyl-terminal amino acids of LKP detected the same 28-kDa granule membrane protein as the D545, one of the mAbs used to clone the cDNA. In addition, the D545 mAb reacted strongly with the GST fusion protein of the bacterially expressed LKP cDNA. In confocal immunofluorescence studies, the anti-LKP peptide Ab reacted with granzyme A-negative granules and vesicles in CD8+ CTL lymphocytes from normal and Chediak-Higashi patients. Thus, based on the expression of the C-terminal LKP epitope, vesicular structures an granules have been detected in CTL that are distinct from classical granzyme-containing cytolytic granules.
Accelerated immune response in transgenic mice expressing rat CD44v4-v7 on T cells.
Authors: Moll J , Schmidt A , van der Putten H , Plug R , Ponta H , Herrlich P , Zöller M
Published: January 2023

Splice variants of the glycoprotein CD44 (CD44v) that confer metastatic behavior to noninvasively growing rat tumor cells are transiently expressed on lymphocytes during activation. A mAb directed against an epitope encoded by CD44 exon v6 blocks both metastasis formations and lymphocyte activation, implicating CD44v in normal immune function. To explore the nature of this function of CD44v, transgenic mice were generated that constitutively express rat CD44v4-v7 on thymocytes and peripheral T cells. The number of lymphocytes as well as the distribution of lymphocyte subpopulations were similar in nontransgenic and rat CD44v4-v7 transgenic mice. T cells of the transgenic mice, however, responded faster to activating stimuli, particularly during primary stimulations with T cell mitogens and T-dependent Ags in vivo and in vitro. This accelerated response depended on the expression of the transgene product, since a rat CD44v6-specific Ab reverted the response profiles of the transgenic mice to those of nontransgenic mice. Since the transgene gained in vivo and in vitro functional activity only upon antigenic stimulation, it is likely that CD44 variant isoforms are involved in the process of signal transduction during lymphocyte activation.
Chemokines and T lymphocyte activation: I. Beta chemokines costimulate human T lymphocyte activation in vitro.
Authors: Taub D D , Turcovski-Corrales S M , Key M L , Longo D L , Murphy W J
Published: January 2023

While chemokines primarily promote chemotaxis, it is apparent that these cytokines also modulate a number of other biologic activities, including adhesion, degranulation, cytotoxicity, and enzyme release. We demonstrate here that the beta chemokines, recombinant human macrophage inflammatory protein-1 alpha and -1 beta, RANTES (regulated upon activation, normally T cell expressed and secreted), and macrophage chemotactic peptide-1, are capable of directly costimulating purified human T cell and human T cell clone proliferation and IL-2 production in the presence of anti-CD3 mAb, but not phorbol esters, in vitro. This costimulatory activity was dose and donor dependent and required the presence of free extracellular calcium as well as endogenously produced IL-2. Chemokine treatment of human T cells in vitro increased the level of cell surface CD25 and soluble CD25. In addition, these chemokines enhanced both Ag- and alloantigen-specific T cell and T cell clone proliferation. This activity was further augmented in the presence of the CD28 ligand, B7-1. Neutralization analyses using chemokine-specific Abs revealed that endogenously produced chemokines are important costimulatory mediators in human T cell activation. Together, these results suggest that chemokines not only play an important role in lymphocyte recruitment to inflammatory sites, but also participate in T cell activation.
A combination of IL-10 and direct contact with plasma cell tumors decreases CD23 expression on splenic B cells.
Authors: Ruzek M C , Billadeau D , Mathur A
Published: January 2023

We and others have previously found that splenic B cells from plasma cell tumor-bearing mice exhibit decreased CD23 expression. In the present study we further examined the mechanism of CD23 down-regulation by plasma cell tumors. We show here that although direct contact is required between the tumor cells and B cells, it is not sufficient, since fixed tumor cells do not induce the same reduction in CD23 expression. We have identified IL-10, a cytokine produce by the tumors, as the sole soluble factor that contributes to decreased CD23 expression on B cells induced by plasma cell tumors because 1) Abs to IL-10 prevent the loss of CD23 induced by plasma cell tumors both in vitro and in vivo; 2) engineered IL-10 negative variants of these tumors are reduced in their ability to down-regulate CD23 expression; 3) rIL-10 alone induces partial, but significant, decreases in CD23 expression on normal splenic B cells; and 4) the addition of IL-10 and fixed tumor cells to cultures of normal splenocytes decreases CD23 expression to levels similar to those in cocultures with live tumor cells. Collectively, these results demonstrate that plasma cell tumors down-regulate CD23 expression on B cells by a coordinate mechanism of IL-10 plus contact-mediated events and reveal a novel role for IL-10 in the regulation of CD23 expression on B cells that is suggestive of host B cell activation in the presence of the tumor.
Regulation of B cell survival in xid mice by the proto-oncogene bcl-2.
Authors: Woodland R T , Schmidt M R , Korsmeyer S J , Gravel K A
Published: January 2023

CBA/N mice carry an X-linked immunodeficiency (xid) due to a point mutation in the Bruton's tyrosine kinase (btk) gene. xid mice have a smaller peripheral B cell pool than normal animals, lack CD5+ B cells (B1), and are hyporesponsive to mitogenic anti-Igs and thymus-independent type 2 Ags. The proto-oncogene bcl-2 affects B cell homeostasis by suppressing programmed cell death. We hypothesized that reduced bcl-2 expression could enhance programmed cell death in xid B cells, directly causing poor peripheral B cell survival and indirectly affecting Ag responsiveness. We measured and compared levels of endogenous Bcl-2 protein and spontaneous apoptosis in xid and normal B cells, and determined the effect of a human bcl-2/Ig minigene on B cell survival and Ag responsiveness in bcl-2 transgenics. The amount of endogenous Bcl-2 was reduced fivefold in freshly isolated xid B cells compared with that in normal cells, but was equal in xid and normal T cells. Attrition by spontaneous apoptosis was significantly higher in cultured xid B cells. Expression of the bcl-2 transgene suppressed apoptosis equally in normal and xid B cells, prolonged in vitro survival, and markedly expanded in vivo the follicular B cell population normally reduced in xid mice. However, most xid defects persisted; xid/bcl-2 mice remained deficient in B1 cells and hyporesponsive to anti-Igs, thymus-independent type 1 Ags, and thymus-independent type 2 Ags. The data suggest that signal transduction pathways using Btk independently regulate B cell survival and Ag responsiveness.
Defective interactions between TCR chains and CD3 heterodimers prevent membrane expression of TCR-alpha beta in human T cells.
Authors: Arnaud J , Chenu C , Huchenq A , Gouaillard C , Kuhlmann J , Rubin B
Published: January 2023

The human TCR complex is composed of two clonotypic polypeptide chains, TCR-alpha and TCR-beta (or TCR-gamma and TCR-delta) associated with CD3 gamma-, delta-, and epsilon-chains and zeta 2 homodimers. All six polypeptide chains are indispensable for TCR membrane expression and signaling function. In the present paper is described the analysis of a new TCR membrane-negative Jurkat T cell variant: E6.R3. The defect in this variant bears on the interaction between TCR and CD3 chains. E6.R3 cells have deleted three nucleotides in the TCR-alpha transmembrane (TM) region, which consequently lacks a leucine. This defect causes 1) lack of association between TCR alpha-chains and CD delta epsilon heterodimers; 2) lack of formation of disulphide-linked, fully glycosylated TCR-alpha beta heterodimers; and 3) lack of interaction between TCR-alpha beta/CD3 complexes and zeta-chains. Despite these defective interactions, TCR alpha-chains appear to become fully glycosylated, i.e., they are not retained in the endoplasmic reticulum but are further processed in the Golgi apparatus without such interactions. The defect may be due to the observation that in the E6.R3 TCR alpha- chains TM region, the two charged amino acids are situated on the same side of the alpha-helix; these two amino acids are exposed on opposite faces of the TM alpha-helix in normal TCR alpha-chains, possibly allowing TCR alpha-chains to interact with both CD3 delta- and CD3 epsilon-chains. Further possible consequences of the leucine deletion in the E6.R3 TCR-alpha TM region are discussed.
The in vivo mechanism of action of CTLA4Ig.
Authors: Judge T A , Tang A , Spain L M , Deans-Gratiot J , Sayegh M H , Turka L A
Published: January 2023

A single dose of CTLA4Ig, an inhibitor of CD28-mediated T cell costimulation, given 2 days after transplantation induces specific unresponsiveness to alloantigens in vivo. However, the mechanisms responsible are unknown. Using pigeon cytochrome c as a model Ag, we monitored the effect of CTLA4Ig on the fate of Ag-reactive T cells in normal mice and on pigeon cytochrome c-specific TCR transgenic cells adoptively transferred into congenic mice. CTLA4Ig significantly inhibits immunization with pigeon cytochrome c. In particular, ELISA and ELISPOT assays indicate an 80 to 90% reduction in Th1 (i.e, IL-2 and IFN-gamma) cytokine production and in the numbers of cytokine-producing cells. Interestingly, despite this profound reduction in cytokine-producing cells, Ag-reactive T cells expand in CTLA4Ig-treated animals, although the degree of expansion is reduced by 50% compared with that in control Ig-treated animals. Thus, loss of Th1 cytokine production in CTLA4Ig-treated animals is not fully explained by the decreased expansion of Ag-specific T cells. These results suggest two mechanisms of action for CTLA4Ig in vivo: inhibition of expansion of Ag-reactive cells and induction of anergy in the residual population.
Identification and functional analysis of Ly-6A/E as a thymic and bone marrow stromal antigen.
Authors: Izon D J , Oritani K , Hamel M , Calvo C R , Boyd R L , Kincade P W , Kruisbeek A M
Published: January 2023

We recently described an mAb (MTS23) reactive with a membrane Ag expressed on a subset of thymic medullary stromal cells. This Ag is also constitutively expressed at high levels on peripheral B cells, macrophages, and thymic and splenic dendritic cells of C57BL/6 mice. A number of stromal cell lines derived from thymus and bone marrow also stain with MTS23, but thymocytes and peripheral T cells only weakly express the Ag detected by MTS23. Here we show that the molecule detected by MTS23 is a member of the Ly-6 family of phosphatidylinositol-anchored membrane proteins. Treatment of stromal cells with phosphatidylinositol-phospholipase C before staining completely abolished expression. Using transient expression of 293T cells and a cDNA library of a stromal cell line cloned into the pEF-BOS vector, a cDNA encoding the MTS23-target Ag was isolated. Partial sequencing and restriction enzyme mapping revealed that it represents the Ly-6A/E protein. While the physiologic significance of the presence of Ly-6 molecules on stromal cells is not clear, it has been known for some time that, at least in lymphocytes, cellular activation events can be induced upon Ly-6 engagement. We now demonstrate that Ly-6 also functions as a signal transduction molecule on stromal cells, in that granulocyte-macrophage CSF can be produced by a variety of stromal cell lines upon mAb-mediated cross-linking of Ly-6. Together with the dramatic up-regulation of Ly-6 expression on stromal cells upon IFN-gamma treatment, this is the first indication of a biologic function of an Ly-6 gene product on nonhemopoietic cells. The results suggest that Ly-6 may play a role in the cross-talk between lymphocyte precursors and stromal cells.
Gene-targeted mice lacking B cells are unable to eliminate a blood stage malaria infection.
Authors: von der Weid T , Honarvar N , Langhorne J
Published: January 2023

Mice deficient of mature B cells due to a targeted disruption of the transmembrane exon of the Ig mu-chain gene (mu-MT mice) can reduce a primary acute infection with the malaria parasite Plasmodium chabaudi chabaudi (AS strain) to low levels but are unable to eliminate parasites and instead develop chronic relapsing parasitemias. This model of B cell deficiency confirms previous findings using anti-mu-treated mice that B cells are required for final parasite clearance. Injection of B cells from immune donors into chronically infected mu-MT mice enabled them to clear their infection within 1 wk. When mu-MT mice that had been cured of their malaria infection by treatment with chloroquine were rechallenged with P. c. chabaudi (AS) they developed secondary infections of a magnitude similar to a primary infection, in contrast to wild-type mice in which a secondary challenge results only in a transient low patent parasitemia. These results suggest that B cell-dependent mechanisms play a crucial role in immunity to secondary infections. There is a pronounced expansion of gamma delta cells in the spleen of chronically infected mu-MT mice. After clearance of parasites in mu-MT mice either after adoptive transfer of immune B cells or by treatment with chloroquine, gamma delta T cells returned to levels observed in wild-type mice. This suggests that the expansion of gamma delta cells observed in mu-MT mice is due to the chronic persistence of parasites, rather than to the lack of B cells.
Pro-opiomelanocortin-derived peptides induce IL-10 production in human monocytes.
Authors: Bhardwaj R S , Schwarz A , Becher E , Mahnke K , Aragane Y , Schwarz T , Luger T A
Published: January 2023

There is strong evidence for the existence of a neuroimmune axis which is regulated by a network of interacting cytokines and neuropeptides. Accordingly, pro-opiomelanocortin-derived peptide hormones such as melanocyte-stimulating hormones (MSH), adrenocorticotropin, and beta-endorphin not only could be detected in many immunocompetent cells but also turned out to be potent immunomodulating and anti-inflammatory mediators, mainly through regulating cytokine production. Thus, it was investigated whether alpha-MSH, which is known to inhibit immune and inflammatory responses, would influence the production of the cytokine synthesis inhibitor IL-10 by human PBMC. Stimulation of PBMC with alpha-MSH resulted in a significantly enhanced release of, IL-10 protein. These data were confirmed by Northern blot analysis, which demonstrated increased IL-10 mRNA expression induced by alpha-MSH. This effect of alpha-MSH was dose-dependent; maximum IL-10 release and mRNA expression were obtained at a concentration of 10(-13) M. There is also clear evidence that only the C-terminal tripeptide of alpha-MSH was required to enhance IL-10 production. In addition, alpha-MSH and its tripeptide strongly induced IL-10 in purified monocytes. In contrast, neither unstimulated nor activated T lymphocytes produced increased amounts of IL-10 in response to alpha-MSH. These findings indicate that pro-opiomelanocortin peptides such as alpha-MSH are able to up-regulate the production of suppressor factors such as IL-10 in monocytes and thereby may contribute to immunosuppression.
Stress-induced enhancement of antigen-specific cell-mediated immunity.
Authors: Dhabhar F S , McEwen B S
Published: January 2023

The studies described here demonstrate that the activation of the physiologic stress response systems of the body can enhance immune function in vivo. This enhancement is observed as a large and long lasting increase in allergic contact sensitivity or delayed-type hypersensitivity, an immune reaction which involves an Ag-specific, cell-mediated immune response. In contrast, acute stress has no effect on the course of irritant contact sensitivity, an immune reaction that does not involve an Ag-specific memory response. A comparison of infiltrating leukocyte numbers in sections of inflamed skin from unstressed and stressed animals shows that stress induces a significant and persistent increase in numbers of leukocytes at the site of the delayed-type hypersensitivity reaction. These results demonstrate that a relatively mild behavioral manipulation can enhance an important class of immune responses that mediate harmful (allergic dermatitis) as well as beneficial (resistance to certain viruses, bacteria, and tumors) aspects of immune function. The implications that these studies have for clinical, diagnostic, and experimental manipulations involving cell-mediated immune function are discussed.
Flanking residues alter antigenicity and immunogenicity of multi-unit CTL epitopes.
Authors: Bergmann C C , Yao Q , Ho C K , Buckwold S L
Published: January 2023

CD8+ CTL responses constitute a critical component for vaccines developed to eliminate intracellular pathogens. One approach to achieve broad CTL diversity is based on genetically linking immunogenic peptides from multiple proteins to form poly-epitope Ags. To address the influence of flanking residues on class I Ag presentation, H-2d-restricted HIV-1 and mouse hepatitis virus CTL epitopes were linked via various spacer residues. The resulting 20 to 31 amino acid peptides were expressed using recombinant vaccinia viruses to monitor both CTL recognition and induction. Our data indicate that recognition is profoundly influenced by the nature of intervening residues forming carboxyl-terminal flanks for one and amino-terminal flanks for the other epitope. Flanking amino acids with aromatic (tyrosine), basic (lysine), and small aliphatic side chains (alanine) supported efficient CTL recognition of both epitopes. By contrast, acidic and helix breaking residues (glycine, proline) specifically inhibited recognition of the adjacent amino-terminal epitope. Flanking residues inhibitory for recognition were also detrimental for CTL induction, suggesting similar processing mechanisms in vitro and in vivo. The ratios of peptide-specific CTL precursors primed by the tandem epitopes varied up to 50-fold depending on molecular context. These data demonstrate a substantial role of carboxyl-flanking residues in governing the efficiency of class I Ag presentation both in vitro and in vivo. The dramatic influence of flanking residues on the hierarchy of CTL responses indicates that CTL induction by poly-epitope Ags can be optimized by strategically linking epitopes via selection of appropriate spacer residues.
Cell surface ADP-ribosyltransferase regulates lymphocyte function-associated molecule-1 (LFA-1) function in T cells.
Authors: Nemoto E , Yu Y , Dennert G
Published: January 2023

Post-translational modifications are important in regulating the functions of signal proteins. This is well established for intracellular proteins, but little is known in the case of extracellular domains of cell surface molecules. We recently described a cell surface protein, mono-ADP-ribosyltransferase (ADPRT), on cytotoxic T cells and showed that it mediates attachment of ADP-ribose to cell surface proteins. Concomitantly, cytolytic activity and cell proliferation are inhibited. Here we report that one of the principal proteins modified by this enzyme is lymphocyte function-associated molecule-1 (LFA-1). While both chains are ADP-ribosylated on the extracellular domain of the molecule, persistence of the modification differs between the chains. Label is released from the beta-chain by 1 h, yet remains for at least 6 h on the alpha-chain. Loss of label is suppressed by phosphodiesterase inhibitors such as ADP-ribose and p-nitrophenylthymidine 5'-monophosphate, pointing to the involvement of this class of enzyme. Modification of LFA-1 requires expression of the cell surface ADPRT and causes the loss of epitopes recognized by alpha- and beta-chain-specific Abs. Concomitantly, the generation of inositol phosphates induced by Ab cross-linking of LFA-1 is significantly inhibited. Consistent with this effect, anti-LFA-1-induced homotypic cell adhesion is also inhibited. These effects are not seen in cells from which the ADPRT was removed by phospholipase C. Moreover, cells lacking the cell surface ADPRT are not inhibited by NAD in the cell adhesion assay, but gain this property upon transfection with the ADPRT gene. It is concluded that the cell surface protein mono-ADPRT regulates LFA-1 functions.
Control of endogenous mouse mammary tumor virus superantigen expression in SJL lymphomas by a promoter within the env region.
Authors: Zhang D J , Tsiagbe V K , Huang C , Thorbecke G J
Published: January 2023

SJL mouse lymphomas (reticulum cell sarcomas, or RCSs) of germinal center B cell origin express an endogenous mouse mammary tumor virus (mtv-29) superantigen (vSAg) that stimulates Vbeta16+ T cells to produce cytokines essential for RCS growth. Normal or LPS-activated SJL/J B cells contain two to three larger mRNAs for mouse mammary tumor virus-long terminal repeat (LTR) but not the 1.8-kb mRNA, which is prominent in RCS cells and encodes the vSAg-29. mRNAs from RCS and normal lymphoid cells were characterized by Northern hybridization using DNA probes from various regions of mtv-29, as well as by reverse transcription PCR, RNase protection, and primer extension. The larger mtv-29 transcripts, coding for envelope protein, are initiated in the 5' LTR, as expected. Surprisingly, the 1.8-kb mRNA, encoding the open reading frame of the LTR, is initiated in the middle of the env region and spliced in the 3' env. This is the first report of an mtv-vSAg transcript that is not controlled by promoter(s) located in the 5' LTR. The env initiation site appears identical to that of the mouse mammary tumor virus env transcriptional activator-directed PMA-induced defective LTR transcript in the C57BL6 T cell lymphoma, EL-4. The stimulus independence, B lymphoma specificity, and absence of deletions within either the 5' or 3' LTR regions of mtv-29 in RCS distinguish the situation in RCS cells from that in EL-4. These findings suggest that the novel mtv-29-vSAg transcript reflects an RCS-cell-specific regulation of transcription.
Key epitopes on the ESAT-6 antigen recognized in mice during the recall of protective immunity to Mycobacterium tuberculosis.
Authors: Brandt L , Oettinger T , Holm A , Andersen A B , Andersen P
Published: January 2023

The recall of long-lived immunity in a mouse model of tuberculosis (TB) is defined as an accelerated accumulation of reactive T cells in the target organs. We have recently identified Ag 85B and a 6-kilodalton early secretory antigenic target, designated ESAT-6, as key antigenic targets recognized by these cells. In the present study, preferential recognition of the ESAT-6 Ag during the recall of immunity was found to be shared by five of six genetically different strains of mice. Overlapping peptides spanning the sequence of ESAT-6 were used to map two T cell epitopes on this molecule. One epitope recognized in the context of H-2b,d was located in the N-terminal part of the molecule, whereas an epitope recognized in the context of H-2a,k covered amino acids 51 to 60. Shorter versions of the N-terminal epitope allowed the precise definition of a 13-amino acid core sequence recognized in the context of H-2b. The peptide covering the N-terminal epitope was immunogenic, and a T cell response with the same fine specificity as that induced during TB infection was generated by immunization with the peptide in IFA. In the C57BL/6j strain, this single epitope was recognized by an exceedingly high frequency of splenic T cells (approximately 1:1000), representing 25 to 35% of the total culture filtrate-reactive T cells recruited to the site of infection during the first phase of the recall response. These findings emphasize the relevance of this Ag in the immune response to TB and suggest that immunologic recognition in the first phase of infection is a highly restricted event dominated by a limited number of T cell clones.
Synthesis of the CC-chemokines MIP-1alpha, MIP-1beta, and RANTES is associated with a type 1 immune response.
Authors: Schrum S , Probst P , Fleischer B , Zipfel P F
Published: January 2023

Lymphocytes regulate the immune response by secreting cytokines that control the activity and function of effector cells. Chemokine subsets are ideal candidates for recruitment of specific effector cells to inflammatory sites or to other lesions because of their selective chemoattractant activities. Given the Th1-Th2 model of immune regulation and the particular role of leukocyte recruitment for the outcome of the response, we analyzed whether a subset of human chemokines is associated with a specific type of immune response. Therefore, we have analyzed the human T cell response to Ags prepared from Yersinia enterocolitica and Ascaris suum with respect to cytokine mRNA-synthesis and secretion. For the Gram-negative bacterium Y. enterocolitica, induction of a type 1 response is indicated by IL-2 and IFN-gamma production, and for the nematode A. suum, a type 2 response is based on IL-4 and IL-5 production. Interestingly, expression of three CC-chemokines (i.e., MIP-1alpha, MIP-1beta, and RANTES) correlated with the type 1 response induced by Y. enterocolitica Ag. Chemokine secretion is not restricted to T lymphocytes; therefore, synthesis of MIP-1alpha, MIP-1beta, and RANTES was also characterized in human T cell clones that display a cytokine pattern indicative of the Th2, Th0, or Th1 phenotype. Again CC-chemokine secretion correlated with the Th1-like phenotype. In six analyzed IL-2- and IFN-gamma- secreting Th1 clones and in two Th0 clones, MIP-1alpha, MIP-1beta, and RANTES were detected, while none or only minimal secretion of these CC-chemokines was observed in three IL-4- and IL-5-producing Th2 cell clones.
IgE autoantibodies directed against the major bullous pemphigoid antigen in patients with a severe form of pemphigoid.
Authors: Delaporte E , Dubost-Brama A , Ghohestani R , Nicolas J F , Neyrinck J L , Bergoend H , Janin A , Capron M
Published: January 2023

Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease characterized in part by circulating and tissue-bound IgG autoantibodies directed against the basement membrane zone. In addition, most of the patients with BP have increased serum IgE levels which seem to be correlated with the disease activity, whereas the presence of circulating anti-basement membrane zone IgE Abs has been reported in some patients. To elucidate whether IgE-dependent mechanisms play a role in the physiopathology of BP, we looked for the presence of IgE Abs specifically directed against the major BP Ag (BPAg1) in sera of BP patients at the onset and after remission of the disease. A radioimmunoassay and a 55-kDa recombinant protein (rBP55) obtained from a cDNA sequence, encoding the C-terminal region of the BPAg1 and containing the BPAg1 immunodominant epitopes, were used. Anti-rBP55 IgE Abs were found in 12 of the 19 sera tested. When the patients were divided into two groups according to the disease severity, anti-rBP55 IgE Abs were found only in patients with a severe form of the disease. Cytophilic IgE was detected on approximately 20% of peripheral blood eosinophils purified from BP patients. Immunohistochemistry studies suggested that some of the IgE-bearing cells in the lesional skin of BP patients are eosinophils. Immunostaining experiments revealed the existence of FcepsilonRI on both peripheral blood and tissue eosinophils. Taken together, these results suggest that IgE-dependent mechanisms could participate in the constitution of the lesions in BP.
Molecular bases of combined subtotal deficiencies of C6 and C7: their effects in combination with other C6 and C7 deficiencies.
Authors: Fernie B A , Würzner R , Orren A , Morgan B P , Potter P C , Platonov A E , Vershinina I V , Shipulin G A , Lachmann P J , Hobart M J
Published: January 2023

Combined subtotal deficiency of C6 and C7, in which both proteins are expressed at very low levels, has been observed in homozygous form in two families. A defect at the 5' splice donor site of intron 15 of the C6 gene explains the low molecular weight of the C6 protein and is probably responsible for its low expressed concentration. The C7 defect is more enigmatic: the protein is of normal molecular weight, low circulating concentration, and altered isoelectric point. An Arg > Ser codon substitution in exon 11 is the only molecular alteration within the mature C7 protein. These defects are associated with a characteristic set of polymorphic DNA markers in the C6/C7 region, forming a distinct haplotype. The haplotype has been found in combination with a number of other haplotypes containing defective genes that lead either to C6 or C7 deficiency, but with different consequences. Where it is combined with a C6-deficient gene, the serum C7 levels can be surprisingly high, possibly because there is no C6 generating C56 to consume the C7. In contrast, where the C7 genes are both defective (but still partially functional), there may be a profound deficit of circulating C7 because there is ample C6 to produce C56 and consume the already small amount of C7. Each molecular defect has also been found in isolation and has the expected effect.
RIP-beta 2-microglobulin transgene expression restores insulitis, but not diabetes, in beta 2-microglobulin null nonobese diabetic mice.
Authors: Kay T W , Parker J L , Stephens L A , Thomas H E , Allison J
Published: January 2023

Beta2m-deficient nonobese diabetic (NODbeta2mnull) do not develop insulitis or diabetes. Expression of a beta2m transgene controlled by the rat insulin promoter (RIP-beta2m) in NODbeta2mnull mice resulted in reconstitution of IFN-gamma-inducible cell surface MHC class I protein on pancreatic beta-cells. These mice developed insulitis, but did not develop diabetes. Transfer of T cells from diabetic NOD mice to NODbeta2mnull recipients resulted in insulitis, which took several months to progress to diabetes. In contrast, transgenic RIP-beta2m/NODbeta2mnull mice with islet MHC class I reconstitution developed diabetes rapidly after transfer of diabetic NOD spleen cells. Administration of cyclophosphamide, which accelerates diabetes in NOD mice, resulted in 43% of RIPbeta2m/NODbeta2mnull mice becoming diabetic compared with 75% of wild-type mice and 0% of NODbeta2mnull mice. Acceleration of diabetes by cyclophosphamide was prevented by anti-CD8 mAb treatment. FACS analysis of peripheral blood and lymphoid organs from transgene-bearing animals did not show an increase in the number of CD8+ T cells compared with that in NODbeta2mnull mice. In summary, beta-cell expression of beta2m in NODbeta2mnull mice resulted in a return of insulitis, but not spontaneous diabetes. These studies demonstrate that beta2m and cell surface MHC class I expression on beta-cells are essential for the initiation of diabetes in the NOD mouse and further confirm that efficient progression to diabetes requires both CD4+ and CD8+ T cells.
Nonrheumatoid IgM in human hepatitis C virus-associated type II cryoglobulinemia recognize mimotopes of the CD4-like LAG-3 protein.
Authors: Mecchia M , Casato M , Tafi R , Filocamo G , Bonomo L , Fiorilli M , Cortese R , Migliaccio G , Nicosia A
Published: January 2023

Type II mixed cryoglobulinemia (CryoII) is an autoimmune disorder frequently associated to human hepatitis C virus (HCV) infection and characterized by the presence of cold-insoluble immunocomplexes containing IgM with rheumatoid activity. To identify disease-related epitopes, we screened a phage-displayed random peptide library using purified IgM from patients with HCV-associated CryoII(CryoII/HCV). A dominant population of phage isolates bearing the HPLAP pentapeptide consensus motif was identified and shown to be recognized by a nonrheumatoid IgM species strongly associated to CryoII/HCV. The phage-borne mimotopes (phagotopes) displayed a strong homology with an exposed extra-loop region of human lymphocyte activation 3 gene (LAG-3) product. Consistently, rabbit sera raised against a synthetic LAG-3 peptide efficiently recognized the selected phagotopes. Furthermore, one such phagotope was revealed to be a good immunogenic mimic of LAG-3 when injected into rabbits. IgM purified from CryoII/HCV patients' sera specifically reacted with the LAG-3 peptide in ELISA, and this binding was inhibited by the selected phagotopes. These results provide experimental support for a general strategy to identify novel autoantigens.
Site-directed mutagenesis of recombinant human beta 2-glycoprotein I identifies a cluster of lysine residues that are critical for phospholipid binding and anti-cardiolipin antibody activity.
Authors: Sheng Y , Sali A , Herzog H , Lahnstein J , Krilis S A
Published: January 2023

beta2-Glycoprotein I (beta2GPI) is a phospholipid-binding serum protein with anticoagulant properties. It plays a vital role in the binding of anti-cardiolipin Abs purified from patients with autoimmune disease when assayed in a cardiolipin (CL) ELISA. Based on a three-dimensional model of beta2GPI, electrostatic calculations, and earlier peptide studies, a highly positively charged amino acid sequence, Lys282-Asn-Lys-Glu-Lys-Lys287, located in the fifth domain of beta2GPI, has been predicted to be the phospholipid binding site. We tested this hypothesis by site-directed mutagenesis of residues in the predicted phospholipid binding site and by assessing the mutants for phospholipid binding and anti-beta2GPI activity. A single amino acid change from Lys286 to Glu significantly decreased the binding of beta2GPI to CL. Double and triple mutants 2k (from Lys286, 287 to Glu286, 287), 2ka (from Lys284, 287 to Glu284, 287), and 3k (from Lys284, 286, 287 to Glu284, 286, 287) possessed no binding of Ab to beta2GPI in a CL ELISA, as well as no inhibitory activity on the binding of iodinated native beta2GPI to CL. These results indicate that the residues Lys284, Lys286, and Lys287 in the fifth domain of beta2GPI are critical for its binding to anionic phospholipids and its subsequent capture for binding of anti-beta2GPI Abs.
CD30 induction and cytokine profiles in hepatitis C virus core-specific peripheral blood T lymphocytes.
Authors: Woitas R P , Lechmann M , Jung G , Kaiser R , Sauerbruch T , Spengler U
Published: January 2023

Since an efficient control of virus infections may depend on the appropriate lymphokine profile, we studied cytokine responses and CD30 induction, a recently proposed surrogate marker of type 2 cells, in 10 healthy anti-hepatitis C virus (HCV)-seropositive blood donors without viremia (group A) and in 15 patients with hepatitis C (group B). Intracytoplasmic IFN-gamma, IL-2, IL-4, and IL-10 were determined by triple-color flow cytometry in the CD3+ and CD3+/CD30+ lymphocyte subsets after stimulation of PBMC with rHCV core protein and five core-derived peptides corresponding to the four immunodominant Th epitopes C.T1 to C.T4. In group A, more type 1 cytokines were induced by the rHCV core protein and all immunodominant core peptides (p < 0.05), whereas IL-10-producing T cells were found more frequently in group B. Induction of CD30+ T cells was found almost exclusively in group B (p < 0.01). The difference in cytokine responses was due to the CD3+/CD30- T cell subset and not the CD3+/CD30+ subset, which predominantly produced both IL-10 and IFN-gamma, but only small amounts of IL-2 and IL-4. We conclude that immunodominant HCV core peptides induce preferentially type 1 cytokines in healthy anti-HCV-positive blood donors and CD30 expression in patients with chronic hepatitis C. However, in both groups, CD30+ T lymphocytes produce an intermediate Th0-like cytokine profile. Thus, chronicity in HCV infection may reflect a lack of type 1 cytokine production.
Cytokine-deficient CD8+ Tc1 cells induced by IL-4: retained inflammation and perforin and Fas cytotoxicity but compromised long term killing of tumor cells.
Authors: Sad S , Li L , Mosmann T R
Published: January 2023

After antigenic stimulation, naive CD8+ T cells differentiate into cytotoxic Tc1 cells secreting the cytokines IL-2, IFN-gamma, and TNF, which aid their proliferation and effector functions. We have previously shown that IL-4 acts directly on differentiated Tc1 cells to impair subsequent Con A-induced IL-2 production. As IL-4 may be produced in the vicinity of Tc1 cells during normal immune responses, we have further analyzed the short and long term functions of IL-4-treated Tc1 cells. We now show that these cells also have a defect in the synthesis of IFN-gamma, TNF, and IL-10 in response to antigenic stimulation. IL-2 synthesis was the most sensitive, as stimulation of IL-4-treated Tc1 cells with higher numbers of APCs partially restored IFN-gamma, TNF, and IL-10, but not IL-2, synthesis. Injection of allo-specific Tc1 cells into mice expressing the target Ag revealed reduced cytokine synthesis in vivo by IL-4-treated Tc1 cells. Loss of cytokine synthesis did not impair the short term effector functions of Tc1 cells, as they induced adoptively transferred delayed type hypersensitivity in recipient mice and retained both perforin- and Fas-dependent cytolytic mechanisms in vitro. Long term coculture of tumor targets and tumor-specific Tc1 cells indicated that normal Tc1 cells proliferated and killed tumor cells, whereas IL-4-treated Tc1 cells failed to proliferate and hence were unable to curtail the proliferation of tumor cells. These results suggest that IL-4 synthesis in vivo would not affect immediate effector functions of differentiated Tc1 cells, but would compromise immunity by reducing their long term functional capability.
Different modes of peptide interaction enable HLA-DQ and HLA-DR molecules to bind diverse peptide repertoires.
Authors: Raddrizzani L , Sturniolo T , Guenot J , Bono E , Gallazzi F , Nagy Z A , Sinigaglia F , Hammer J
Published: January 2023

The role of HLA-DQ molecules in Ag presentation has, thus far, remained elusive. Here we report that two DQ allotypes, DQ7 (DQA1*0501/B1*0301) and DQ9 (DQA1*0201/B1*0303), are capable of binding peptide repertoires in complementarity with DR molecules. The results reflect fundamental differences in the binding modes of these two HLA class II isotypes, in that DQ7 and DQ9 but not DR molecules appear to have the capacity to bind peptide structures without type 1-like anchor residues. Consistent with this is our observation that none of the amino acid side chains of the class II-associated invariant chain peptides (CLIP) are required for association with DQ7 and DQ9, even though many of them are essential for CLIP-DR interaction. Together, these data reveal a functional complementarity of HLA-DR and -DQ molecules in Ag presentation.
Interleukin-10 prevents dendritic cell accumulation and vaccination with granulocyte-macrophage colony-stimulating factor gene-modified tumor cells.
Authors: Qin Z , Noffz G , Mohaupt M , Blankenstein T
Published: January 2023

A wide variety of human tumors express IL-10 for reasons poorly understood. We have analyzed the effect of spontaneous IL-10 expression by a mouse tumor (J558L) on its immunoparalyzing effect. Because "cross-priming" of T cells by host Ag-presenting cells for MHC class I-restricted tumor Ags is a major pathway for induction of tumor immunity and that is enhanced by granulocyte-macrophage (GM) CSF, we expressed this cytokine in J558L cells. GM-CSF-secreting cells were not effective when used for immunization against challenge with the parental tumor. Inhibition of IL-10 expression through an IL-10 antisense retrovirus restored the vaccine efficacy of GM-CSF-producing J558L cells, demonstrating a direct role of IL-10 in paralyzing the GM-CSF-induced antitumor immune response. Since the tumor used for challenge produced IL-10, we conclude that IL-10 interfered primarily with the initiation but not the effector phase of the immune response. Immunohistochemical analysis of the vaccine site showed a GM-CSF-induced accumulation of dendritic cells (DC) (MHC class II+ and DEC-205+) in the absence of IL-10. In the presence of IL-10, DC accumulation was completely inhibited. Together, our results demonstrate an antagonistic effect of IL-10 with respect to GM-CSF-induced DC accumulation and tumor immunity and suggest a new mechanism by which tumors escape immune recognition: namely by preventing APC from obtaining access to tumor Ags.
IL-2 and IL-12 act in synergy to overcome antigen-specific T cell unresponsiveness in mycobacterial disease.
Authors: de Jong R , Janson A A , Faber W R , Naafs B , Ottenhoff T H
Published: January 2023

IL-12 secretion by APC is critical for the development of protective Th1-type responses in mycobacterial (Mycobacterium avium and Mycobacterium tuberculosis) infections in mice. We have studied the role of IL-12 and IL-2 in the generation of Mycobacterium leprae-specific T cell responses in humans. Leprosy patients were defined as low/nonresponders or high responders based on the level of T cell proliferation in M. leprae-stimulated PBMC. In high responders, M. leprae-induced proliferation was markedly suppressed by neutralizing anti-IL-12 mAb (inhibition 55 +/- 6%). Neutralization of IL-2 activity resulted in an inhibition of 77 +/- 4%. Given the importance of endogenous IL-2 and IL-12 in M. leprae-induced responses, we investigated the ability of rIL-2 and rIL-12 to reverse T cell unresponsiveness in low/nonresponder patients. Interestingly, rIL-12 and rIL-2 strongly synergized in restoring both M. leprae-specific T cell proliferation and IFN-gamma secretion almost completely to the level of responder patients. A similar synergy between rIL-2 and rIL-12 was also observed in high responders when suboptimal M. leprae concentrations were used for T cell stimulation. Our data demonstrate a crucial role for endogenous IL-12 and IL-2 in M. leprae-induced T cell activation. Most importantly, we show that rIL-2 and rIL-12 act in synergy to overcome Ag-specific Th1 cell unresponsiveness. These findings may be applicable to the design of antimicrobial and antitumor vaccines.
The CDK inhibitor, p27Kip1, is required for IL-4 regulation of astrocyte proliferation.
Authors: Liu J , Flanagan W M , Drazba J A , Estes M L , Barnett G H , Haqqi T , Kondo S , Barna B P
Published: January 2023

IL-4 is a pleiotrophic cytokine that has been shown to affect cells of the central nervous system. We have demonstrated that IL-4 inhibits DNA synthesis and proliferation in human astroglia expressing IL-4 receptors. In this study, we sought to identify mechanisms that could account for the antimitogenic effects of IL-4. Epidermal growth factor (EGF)-stimulated human astroglia were arrested in G1 phase by IL-4, even though IL-4 stimulated levels of the G1 cyclins, D1 and E. Histone H1 kinase activity of cdk2 immunoprecipitates, however, was sharply reduced by IL-4; impairment of kinase activity was also evident in cyclin E immunoprecipitates, which contained evidence of hypophosphorylated (inactive) cdk2 product. Reduced cyclin E-associated cdk2 activity was not due to impaired cyclin-dependent kinase-activating kinase (CAK) activity, which was unaffected by IL-4. Inactive cyclin E/cdk2 complexes from IL-4 + EGF-treated cells contained, however, strikingly elevated p27Kip1 cdk inhibitor. Elevated p27 was also detectable in whole cell lysates after 24 and 48 h of IL-4 treatment; by 72 h, p27 was no longer elevated. Pretreatment with antisense but not mismatch p27 oligonucleotides attenuated the inhibitory effects of IL-4 on DNA synthesis and histone kinase activity of cyclin E/cdk2 complexes. Antisense p27 also abrogated IL-4-mediated elevation of p27 in whole cell lysates and cyclin E/cdk2 complexes. These findings demonstrate that IL-4 regulates the cell cycle machinery of astroglial cells via a p27Kip1 braking mechanism.
IL-4 and IL-10 modulation of CD40-mediated signaling of monocyte IL-1beta synthesis and rescue from apoptosis.
Authors: Poe J C , Wagner D H , Miller R W , Stout R D , Suttles J
Published: January 2023

Previous studies have demonstrated that the interaction of CD40 on monocytes with CD40 ligand, present on activated CD4+ T cells, induces monocyte inflammatory cytokine synthesis and rescues monocytes from apoptosis. These findings suggest a role for CD40 signaling of monocyte activation in the maintenance and/or exacerbation of nonseptic (e.g., autoimmune) inflammatory responses. In the present study the effects of the modulatory cytokines IL-4 and IL-10 on CD40-mediated signaling of monocyte IL-1beta synthesis and rescue from apoptosis were examined. Both IL-4 and IL-10 decreased CD40-dependent IL-1beta synthesis in a dose-dependent manner individually and synergized in this effect when used concurrently, with minimal effect on CD40 surface expression. CD40 signaling of IL-1beta synthesis was shown to be dependent on the induction of protein tyrosine kinase (PTK) activity, and both IL-4 and IL-10 diminished CD40-mediated tyrosine phosphorylation of monocyte cellular proteins. However, IL-4, but not IL-10, blocked CD40-mediated rescue from apoptosis, an event that we have demonstrated previously to be dependent on PTK activity as well. Together these results suggest that in monocytes 1) both IL-4 and IL-10 target CD40-induced PTK activity in the down-regulation of IL-1beta synthesis; and 2) IL-4 and IL-10 have divergent effects on the CD40 signaling pathway, in that these cytokines are synergistic with respect to their abilities to inhibit CD40-mediated IL-1beta synthesis and differ in their abilities to block CD40-mediated rescue from apoptosis.