bluuebunny/crossref_metadata_2025 huggingface dataset:
Anergy and apoptosis in CD8+ T cells from HIV-infected persons.
Authors: Lewis D E , Tang D S , Adu-Oppong A , Schober W , Rodgers J R
Published: December 2022

CD8+T cells from HIV-infected persons increase early in infection, display increased levels of activation Ags, and abnormal MHC-restricted, HIV-specific and nonspecific cytotoxicity abilities. Paradoxically, these cells are also unresponsive to T cell signaling in vitro and have decreased in vitro cloning potential. HIV-specific CTL precursors also are lost late in infection. A quantitative Southern blotting technique showed that CD8+ T cells from asymptomatic, HIV-infected persons have increased DNA fragmentation after overnight incubation. DNA fragmentation was reduced by an endonuclease inhibitor but not by cycloheximide, suggesting that a pre-apoptotic state exists in vivo. Partial inhibition of DNA fragmentation also could be induced by IL-2 addition. No consistent difference in fragmentation was observed among CD8+ subpopulations from HIV-infected individuals, although only CD8+ T cells that did not express activation Ags (DR-, CD28+, CD57- phenotype) showed reduced fragmentation when incubated in IL-2. A dramatic increase in CD8+, CD28- cells was observed in asymptomatic HIV-infected people. A subset of CD8+, CD28- cells in both controls and HIV-infected people do not proliferate to T cell signals, and these cells from controls demonstrate increased DNA fragmentation in vitro after 3 days of incubation, regardless of stimulation conditions. This suggests that the cells are end-stage cells. Taken together, the data suggest an increase in anergic or apoptotic CD8+ T cells in HIV-infected persons. Eventual depletion of HIV-specific CD8+ T cells may occur through a process of proliferation, anergy induction, and apoptosis.
Arrested rearrangement of TCR V beta genes in thymocytes from children with X-linked severe combined immunodeficiency disease.
Authors: Sleasman J W , Harville T O , White G B , George J F , Barrett D J , Goodenow M M
Published: December 2022

Human X-linked severe combined immunodeficiency disease (SCID) is an immunodeficiency disorder in which T cell development is arrested in the thymic cortex. B lymphocytes in children with X-linked SCID seem to differentiate normally. X-linked SCID is associated with a mutation in the gene that encodes the IL-2R gamma-chain. Because TCR-beta gene recombination is a pivotal initial event in T lymphocyte ontogeny within the thymus, we hypothesized that a failure to express normal IL-2R gamma could lead to impaired TCR-beta gene recombination in early thymic development. PCR was used to determine the status of TCR-beta gene-segment rearrangements in thymic DNA that had been obtained from children with X-linked SCID. The initial step in TCR-beta gene rearrangement, that of D beta to J beta recombination, was readily detected in all thymus samples from children with X-linked SCID; in contrast, V beta to DJ beta gene rearrangements were undetectable in the same samples. Both D beta to J beta and V beta to DJ beta TCR genes were rearranged in the thymic tissues obtained from immunologically normal children. We conclude that TCR beta-chain gene rearrangement is arrested in children with X-linked SCID. Our results suggest a causative relationship between the failure of TCR beta-chain gene rearrangements to proceed beyond DJ beta rearrangements and the production of a nonfunctional IL-2R gamma-chain.
Activation of src family kinases in human pre-B cells by IL-7.
Authors: Seckinger P , Fougereau M
Published: December 2022

IL-7 was identified originally as a specific pre-B cell growth factor. We have investigated its signal transduction mechanism by using the human pre-B cell line Nalm-6, and have found that it stimulates tyrosine phosphorylation of various proteins: pp27, pp43, pp54, pp64, pp78, pp90, pp105, and pp120. Antiphosphotyrosine immunoprecipitates from IL-7-stimulated Nalm-6 showed two major proteins of M(r) = 60,000 and 55,000, capable of autophosphorylation. Autophosphorylation was maximal 10 min after the cells were challenged with the cytokine. Antiphosphotyrosine immunoprecipitates from IL-7-stimulated cells also increased tyrosine phosphorylation of the exogenously added substrate histone H2B. Furthermore, by using a polyclonal anti-IL-7 receptor (IL-7R) Ab in Western blotting analysis, we observed that antiphosphotyrosine immunoprecipitates were associated with the IL-7R in a transient manner. These data indicate that the IL-7R associates with tyrosine-phosphorylated proteins as its amino acid sequence is devoid of a putative site of tyrosine phosphorylation. These results were confirmed as several 32P-labeled proteins were visualized after immunoprecipitation by using anti-IL-7R Ab. Anti-IL-7R immunoprecipitates from IL-7-stimulated cells revealed a unique band of M(r) = 60,000 associated with the receptor able to autophosphorylate in the presence of ATP and Mn2+. Hence, we identified p59fyn and p53/56lyn to be stimulated by IL-7. In contrast to p53/56lyn, p59fyn was found to be associated constitutively with the cloned IL-7R. These data emphasize the role of the src family in hematopoiesis.
TGF-beta 1 is a potent inducer of human effector T cells.
Authors: Cerwenka A , Bevec D , Majdic O , Knapp W , Holter W
Published: December 2022

TGF-beta 1 is known to modulate lymphocyte activation affecting cell proliferation and the production of cytokines and Igs. Little is known about the characteristics of T cells grown in the presence of TGF-beta 1. We have stimulated human T cells with PHA in the presence of TGF-beta 1 under serum-free conditions for 7 days and characterized the resulting cell population. TGF-beta 1 (0.0032 to 10 ng/ml) affected neither [3H]thymidine incorporation (day 4) nor cell yield (day 7) in these cultures. However, cells activated in the presence of TGF-beta 1 proliferated vigorously in secondary cultures and produced highly elevated amounts of IL-2 (12 +/- 3-fold enhancement of IL-2 production in response to CD2 plus CD28 stimulation compared with control cells, mean +/- SEM; n = 10). The enhancing effects of TGF-beta 1 were demonstrable over a wide range of concentrations (0.4 to 10 ng/ml). The increased IL-2 protein production was paralleled by a dramatic up-regulation of IL-2 mRNA. In addition, cells precultured with TGF-beta 1 responded with enhanced cluster formation in the secondary cultures. With regard to their phenotype, we observed an increased expression of the alpha E beta 7-integrin human mucosal lymphocyte-1 and of the CD2-restricted epitope CD2R, whereas the expression of CD11a was slightly decreased. In contrast, TGF-beta 1 did not influence the constitutive or activation-induced expression of CD4, CD8, CD45RA, CD45RO, CD25, CD71, CD54, CD58, CD59, and B7. We conclude that TGF-beta 1 supports the generation of human effector cells with a strongly enhanced capacity to respond to subsequent restimulation.
Cloning and molecular characterization of mouse bcl-x in B and T lymphocytes.
Authors: Fang W , Rivard J J , Mueller D L , Behrens T W
Published: December 2022

We report the cloning and initial characterization of the mouse homologues of the bcl-2-related gene, bcl-x. We compare these to the two major isoforms of human bcl-x that were previously identified, bcl-xLong (bcl-xL) which has death repressor activity similar to bcl-2, and bcl-xShort (bcl-xS), an alternative mRNA splice product that deletes a highly conserved domain shared by bcl-2 family members and promotes cell death in transfection studies. In addition to murine (m) bcl-xL and mbcl-xS, we have cloned a novel cDNA isoform that we designate mbcl-x delta TM. This cDNA deletes, by means of alternative splicing, the carboxy terminal transmembrane domain of bcl-x and is predicted to be a soluble, rather than membrane-bound, protein. We found that mbcl-x mRNA is highly inducible in splenocytes stimulated with either anti-CD3 Abs or LPS/dextran sulfate, and that the major species of mbcl-x mRNA in a variety of cell lines and tissues was the xL isoform. Transfection of mbcl-xL or mbcl-xS into Hela cells resulted in targeting primarily to mitochondria, whereas mbcl-x delta TM localized diffusely throughout the cytosol. Overexpression of the novel delta TM isoform in an IL-3-dependent cell line delayed the onset of apoptosis induced by growth factor withdrawal. Thus, a naturally-occurring form of mbcl-x present in lymphocytes that does not localize to the mitochondrial membrane is functional in preventing apoptotic cell death. Moreover, these data suggest that bcl-x provides a life signal in activated lymphocytes.
Invariant-cognate peptide exchange restores class II dimer stability in HLA-DM mutants.
Authors: Monji T , McCormack A L , Yates J R , Pious D
Published: December 2022

Class II presentation mutants have mutations in the HLA-DMA or B genes and are defective in the presentation of whole exogenous Ags restricted by HLA-DR, -DQ, and -DP. The functional defect in Ag presentation is accompanied by an altered conformation of cell surface class II molecules and instability of extracted class II dimers in SDS-PAGE; the latter can be corrected by incubation of mutant cells in an acidic pH in the presence of cognate peptide. Here we investigated the basis for correction of class II dimer instability by acid/cognate peptide treatment and the extent to which this treatment corrects the class II conformational defect in DMB mutants. We found that an acidic pH generates peptide binding sites in class II molecules of DMB mutants by eluting invariant chain (li)-derived peptides from them. Cognate peptides can then bind to the empty binding sites of class II molecules in a pH-independent manner, which results in stabilization of class II dimers. Acid/peptide treatment also restores the DR polymorphic epitope recognized by mAb 7.3.19.1 but not the DR polymorphic epitope recognized by mAb 16.23; low pH gradually destroys the 16.23 epitope in nonmutant cells. Mutant 10.24.6, which has a mutation in the DRA coding region creating an extra glycosylation site, also has unstable DR dimers whose stability is restored by acid/peptide treatment. These results suggest that the primary phenotypic defect in both the DMB and 10.24.6 mutants is the abundance of li peptides and lack of cognate peptides bound to class II molecules.
CD8+ T lymphocyte-mediated lysis of Chlamydia-infected L cells using an endogenous antigen pathway.
Authors: Beatty P R , Stephens R S
Published: December 2022

Intracellular bacterial pathogens have evolved to either grow in the nutrient-rich cytoplasm or remain sequestered within a vacuole. One potentially important selective advantage for growth within a vacuole may be evasion of cell-mediated detection and cytolysis. To address this question we used the endosomally confined bacterium Chlamydia trachomatis, which naturally infects epithelial cells, to examine CTL-mediated lysis of nonphagocytic cells. CTL-mediated lysis of infected target cells was detected, although the increased expression of ICAM-1 by transfection was required. The elimination of CD8+ T cells or addition of brefeldin A or cycloheximide eliminated specific cytolysis, whereas conversely, treatment with chloroquine or ammonium chloride had only minor effects. These results implicate endogenous Ag processing for Chlamydia-specific cytolysis. This work demonstrates CTL-mediated lysis of cells infected with an intracellular bacterium that inhibits lysosomal fusion and is confined to an endosomal vacuole.
Toxoplasma gondii oral infection induces specific cytotoxic CD8 alpha/beta+ Thy-1+ gut intraepithelial lymphocytes, lytic for parasite-infected enterocytes.
Authors: Chardès T , Buzoni-Gatel D , Lepage A , Bernard F , Bout D
Published: December 2022

The first line of defense of the host after primary infection by Toxoplasma gondii is the intestinal mucosal surface, which consists of epithelial cells, intraepithelial lymphocytes (IEL) (mainly of CD8 phenotype), mucus, and secretory Igs. T. gondii cysts were administered orally to CBA/J mice to determine whether parasite-specific cytotoxic IEL can be elicited within the intestine. We found that oral infection led to an increase in the relative percentages of the CD8 beta + and Thy-1+ IEL populations between day 9 and day 13 after infection. At these times, T. gondii-primed, but not control mice, generated parasite-specific cytotoxic effector IEL for toxoplasma-infected macrophages. This cytotoxic activity was genetically restricted. By using magnetically activated cell sorting, the effector IEL were shown to be CD8 alpha/beta + Thy-1+ T lymphocytes and FACScan analysis revealed that they mainly express TCR-alpha beta. A significant level of cytotoxicity was also observed against T. gondii-infected epithelial cells from the MODE-K cell line, suggesting that parasite-specific IEL may ensure epithelial integrity by rapidly killing infected enterocytes. Finally, toxoplasma stimulation in vitro led to IFN-gamma production by T. gondii-primed IEL. Taken together, these data suggest that antigen-specific IEL, bearing the CD8 alpha/beta + Thy-1+, TCR-alpha beta + phenotype, can act directly as effector cells through a specific CTL activity at the intestinal level and may indirectly activate other mucosal effector mechanisms through IFN-gamma secretion.
IL-2 can support growth of CD8+ T cells but not CD4+ T cells of human IL-2 receptor beta-chain transgenic mice.
Authors: Asano M , Ishida Y , Sabe H , Kondo M , Sugamura K , Honjo T
Published: December 2022

We have generated transgenic mice expressing the human (h) IL-2R beta-chain on lymphoid cells under the control of the mouse H-2Kd promoter. Spleen cells and thymocytes of the transgenic mice were cultured in the presence of 5 nM hIL-2. After a 10-day culture, the expanded populations were analyzed by flow cytometry and shown to be composed of CD8+ T cells and gamma delta T cells. Surprisingly, CD4+ T cells of the transgenic mice did not proliferate in response to hIL-2, although the CD4+ T cells expressed the transgenic hIL-2R beta-chain as well as the endogenous gamma-chain on their surface and bound 125I-labeled IL-2. When CD4+ T cells of the transgenic mice were stimulated with anti-CD3 mAb, the CD4+ T cells proliferated in response to hIL-2. These findings suggest that CD4+ T cells may require another triggering signal to respond to IL-2 even when IL-2Rs are expressed. By contrast, CD8+ T cells and gamma delta T cells respond to IL-2 as long as IL-2Rs are expressed.
Regulation of adhesion molecule expression by CD8 T cells in vivo. II. Expression of L-selectin (CD62L) by memory cytolytic T cells responding to minor histocompatibility antigens.
Authors: Mobley J L , Rigby S M , Dailey M O
Published: December 2022

Activation of murine T cells by Ag or mitogens results in changes in the expression of several cell-surface adhesion molecules that alter the way in which these cells migrate and localize in tissues in vivo. As naive CD8 precursor cells in lymph nodes (LN) differentiate into effector CTL in response to a skin allograft, they up-regulate their expression of Pgp-1 (CD44), VLA-4, and LFA-1 (CD11a/18), while down-regulating L-selectin (CD62L). All cytolytic activity is therefore present in this minor population of L-selectin- Pgp-1high LN T cells. We now report that, late after rejection of minor histocompatibility Ag-disparate skin grafts, the majority of memory CD8 T cells express both L-selectin and Pgp-1 and thus would be expected to migrate via the classical route of recirculation through LN. Furthermore, restimulation of these memory cells by Ag causes them to down-regulate L-selectin, so that memory-effector cells have the same L-selectin- Pgp-1high phenotype as do primary effector cells. These results are in contrast to recent reports that murine and ovine CD4 memory cells do not express L-selectin or recirculate through LN high endothelial venules. In addition, although virgin and naive CD4 cells may be divided based upon their expression of CD45RA or CD45RB, memory CD8 cells cannot be differentiated by their expression of CD45 isoforms.
An efficient Th2-type memory follows CD8+ lymphocyte-driven and eosinophil-mediated rejection of a spontaneous mouse mammary adenocarcinoma engineered to release IL-4.
Authors: Pericle F , Giovarelli M , Colombo M P , Ferrari G , Musiani P , Modesti A , Cavallo F , Di Pierro F , Novelli F , Forni G
Published: December 2022

A retroviral infection was used to introduce the cDNA coding for mouse IL-4 into the parental cells of a spontaneous adenocarcinoma of BALB/c mice (TS/A-pc). Four clones releasing between 5 to 40 U of IL-4 (10(5) cells) in 48 h culture were selected. The secretion of IL-4 does not affect their in vitro growth, whereas their ability to form tumor in vivo inversely correlates with the amount of IL-4 secreted. Although morphologic observation suggested that the rejection of clone D5.40 cells (releasing 40 U of IL-4) depends on eosinophil cytolysis, lymphocyte depletion experiments showed that this required CD8+ lymphocyte guidance. Mice that had rejected D5.40 cells were immune to a subsequent challenge with TS/A-pc. This memory rests on the interaction between noncytotoxic lymphocytes, eosinophils, and IgG1 and IgE anti-TS/A Abs. Comparison of these memory mechanisms with those elicited by IL-2 gene-transduced TS/A cells shows that the kind of cytokine released by the tumor cells determines the type of response. This Th2 memory seems to be more efficient in protecting against a subsequent challenge of TS/A-pc than the Th1-type memory elicited by IL-2 gene-transduced TS/A cells.
Monoclonal antibodies detect different distribution patterns of IL-8 receptor A and IL-8 receptor B on human peripheral blood leukocytes.
Authors: Chuntharapai A , Lee J , Hébert C A , Kim K J
Published: December 2022

mAbs previously reported to be specific for IL-8R type A (IL-8R-A) and mAbs specific for IL-8R type B (IL-8R-B), which are described in this paper, were used to investigate the expression of each receptor on various types of cells. We generated mAbs specific for IL-8R-B, 4D1, and 10H2 by immunizing mice with 293 cells that expressed IL-8R-B and by selecting hybridoma cell lines that secreted mAbs that bind to human neutrophils. Flow cytometry showed that mAbs 4D1 and 10H2 were specific for IL-8R-B, as determined by their exclusive binding to 293-27 cells that expressed IL-8R-B, but not to 293-71 cells that expressed IL-8R-A. Epitopes recognized by these IL-8R-B-specific mAbs were shown to be within the N-terminal residues 1-18 of the IL-8R-B on the basis of their binding to various N-terminal peptides, as measured by ELISA. These IL-8R-B-specific mAbs were able to inhibit up to 90 and 50% of the 125I-labeled IL-8 binding to 293-27 cells and human neutrophils, respectively. The combination of mAb 9H1 (anti-IL-8R-A) and mAb 10H2 (anti-IL-8R-B) inhibited approximately 70% of 125I-labeled IL-8 binding to human neutrophils. Flow cytometry showed a wide range of donor variation in the expression levels of IL-8R-A and IL-8R-B on various human peripheral blood leukocytes. All neutrophils, all monocytes, and 5 to 25% of total lymphocytes (CD8+ T cells and CD56+ NK cells) expressed IL-8R. Neutrophils expressed the highest level of both IL-8R-A and IL-8R-B, at an approximately equal ratio, whereas monocytes and IL-8R+ lymphocytes expressed higher levels of IL-8R-B than IL-8R-A. Double-color flow cytometric analysis showed that 7 to 42% of CD8+ T cells and 39 to 76% of CD56+ NK cells, but no CD 20+ B cells or CD4+ T cells, expressed IL-8R.
A transcription factor with AP3-like binding specificity mediates gene regulation after an allergic triggering with IgE and Ag in mouse mast cells.
Authors: Jarmin D I , Kulmburg P A , Huber N E , Baumann G , Prieschl-Strassmayr E E , Baumruker T
Published: December 2022

Mast cells are an important source of a number of lymphokines and chemokines primarily those released after challenge with the allergic trigger IgE and Ag. However, the mechanisms of lymphokine and chemokine gene activation in this cell type, as opposed to the mechanisms of activation in T cells, are poorly understood. As a model system, we addressed this issue in mast cells by using the recently cloned chemokine MARC gene, which belongs to the RANTES/sis gene family. After allergic stimulation, MARC induction is pronounced and mast cell specific. Northern blot analysis, in combination with two inhibitors, actinomycin D and cycloheximide, resulted in the formation of our initial hypothesis, which was that both transcriptional and post-transcriptional regulation are involved after stimulation through the Fc epsilon R. We performed a detailed promoter analysis of the cloned MARC gene by using transient assays of transfected reporter gene constructs. Thereby, two potential promoter regions were identified as being crucial for transcriptional stimulation. Additional fine mapping of the proximal element and subsequent electrophoretic mobility shift assays, combined with competitions of known transcription factor binding sites, identified one of the transcription factors in stimulated mast cells as an AP3 or AP3-like binding activity.
Regulation of intestinal epithelial barrier function by TGF-beta 1. Evidence for its role in abrogating the effect of a T cell cytokine.
Authors: Planchon S M , Martins C A , Guerrant R L , Roche J K
Published: December 2022

Maintenance of the integrity of the single-cell-thick intestinal epithelium as an in vivo barrier between environmental Ags and mucosal immunocytes is pivotal for health. The T cell cytokine IFN-gamma consistently disrupts this epithelial barrier in vitro, but the substances in mucosa that may be responsible for sustaining or enhancing barrier function have not been clearly identified. Therefore, we characterized the effect on the epithelial barrier of TGF-beta 1 and three prominent neuropeptides (VIP, substance P, somatostatin) by using a model system in which barrier function of a mature polar human colonic epithelial (T84) cell monolayer is reflected in 1) the electrical potential difference across the apical to basolateral surface of each cell, 2) the transmonolayer permeability to macromolecules such as horseradish peroxidase, and 3) lactate dehydrogenase release into the medium indicating epithelial cell cytolysis. Whereas T84 monolayers exposed to TGF-beta 1 alone demonstrated a modest increase in electrical resistance and barrier integrity, TGF-beta 1 showed a striking ability to reduce the capacity of IFN-gamma to disrupt epithelial barrier function. Characterization studies demonstrated that this TGF-beta 1 effect was prolonged (e.g., days) after a single exposure, progressive over the dose range 0.1 to 2.5 ng/ml, reversible with increased concentrations of IFN-gamma, and more pronounced when TGF-beta 1 exposure was to basolateral rather than to apical epithelial membranes. Macromolecular (horseradish peroxidase) penetration of epithelium was not simultaneously altered by TGF-beta 1 and epithelial cellular injury was minimal as gauged by lactate dehydrogenase release. Additional studies using a human pathogen demonstrated that TGF-beta 1 delayed and decreased the barrier disruption caused by exposure to Cryptosporidium parvum. TGF-beta 1 may be the first of a new class of cytokines that maintains and/or enhances barrier function of human enterocytes, in part by countering the effect of a T cell cytokine.
The repertoire of cytotoxic T lymphocytes in the recognition of mutant simian immunodeficiency virus variants.
Authors: Shen L , Chen Z W , Letvin N L
Published: December 2022

The importance of the repertoire of a primed, AIDS virus-specific population of CTL in the recognition of emerging mutant viruses was assessed in simian immunodeficiency virus (SIV)-infected rhesus monkeys. These studies were done by using the well-characterized CTL recognition of the SIVmac Gag peptide 11C (p11C) epitope in rhesus monkeys expressing the MHC class I molecule Mamu-A*01. Lysis of peptide-pulsed targets by bulk PBL effector cells from SIVmac-infected, Mamu-A*01+ monkeys was significantly decreased by mutations at residues 2, 3, and 5 of the nine-amino-acid Gag p11C epitope. However, effector cell lysis of targets pulsed with p11C containing substitutions at residues 3 or 5 was substantially increased by in vitro incubation of the monkeys' PBL with these mutant peptides. This suggested that expandable populations of cells exist in the primed CTL of infected monkeys capable of recognizing mutant peptide sequences. In fact, without in vitro exposure of PBL to mutant peptides, SIVmac Gag-specific CTL could be cloned from PBL of infected monkeys that lysed targets pulsed with mutant peptides. These observations suggest that clones of effector cells capable of recognizing many viral mutants may be able to expand in vivo to control the spread of emerging variant AIDS viruses.
Phenotypic characterization of CD4-/alpha beta TCR+ and gamma delta TCR+ T cells with a transendothelial migratory capacity.
Authors: Galéa P , Brezinschek R , Lipsky P E , Oppenheimer-Marks N
Published: December 2022

During inflammation, both CD4+ and CD4- T lymphocytes extravasate into perivascular tissues by adhering to and migrating through the vascular endothelium. These studies were undertaken to characterize the phenotype of CD4- T cells that have a capacity to migrate through endothelium. Results show that CD4- T cells exhibit a greater capacity to migrate through endothelial cells (EC) than CD4+ T cells; and that TCR-gamma delta+ T cells exhibited the greatest migratory capacity. The migrating CD8+ T cell population was enriched in CD45RO+/L-selectin-/LFA-1bright/CD29bright/CD 44bright cells. TNF-alpha-activated EC did not support increased CD4- T cell transendothelial migration and changes in the phenotype of the migrating CD8+ T cells. The migrating CD4- T cell population was enriched in VLA-2+ T cells and expressed increased densities of VLA-4, VLA-5, and VLA-6. The migrating TCR-gamma delta+ T cell population contained both CD8dim and CD8- T cells. Moreover, the migrating gamma delta T cell population was not different from the initial or nonadherent population in that it contained CD45RO+, CD45RA+, and L-selectin+ cells. Finally, migrating TCR-gamma delta+ T cells contained cells expressing V delta 2 and V gamma 9 TCR chains, but these were not enriched compared with the initial population. These studies have characterized the CD4- T cells that are capable of transendothelial migration in vitro. The results are consistent with the conclusion that unique subpopulations of CD8+ alpha beta and CD8+ and CD8- gamma delta T cells gain access to inflammatory sites by virtue of their intrinsic ability to migrate across the endothelium.
The A-myb gene is preferentially expressed in tonsillar CD38+, CD39-, and sIgM- B lymphocytes and in Burkitt's lymphoma cell lines.
Authors: Golay J , Erba E , Bernasconi S , Peri G , Introna M
Published: December 2022

The A-myb gene is structurally related to the c-mby proto-oncogene, a transcription factor involved in the regulation of hemopoietic proliferation and differentiation. Recent evidence has shown that A-myb also functions as a transcriptional activator. We have previously demonstrated that A-myb RNA is not expressed in most mature human leukocytes at rest or after mitogenic or functional activation. We show here, by using cell sorting, PCR, and Western analyses that A-myb is most highly expressed in the subsets of human tonsillar B lymphocytes with the phenotypes CD38+, CD39-, and SIgM-. The preferential expression of A-myb in these populations was seen at both the RNA and protein levels. CD38 was consistently best at separating high from low A-myb-expressing cells, whereas other markers (CD10, 22, 23, 77, 11a, and 49d) did not correlate with A-myb expression. The CD38+ population expressing the highest levels of A-myb was shown to contain mostly cycling cells inasmuch as more than 95% were in the late G1, S, G2, and M phases of the cell cycle. In addition, A-myb expression always correlated with the percentage of cells in S/G2/M in the populations sorted with either CD38, CD39, or sIgM. Small resting tonsillar B lymphocytes induced to proliferate in vitro by several different polyclonal B cell activators did not, however, express detectable levels of A-myb, although these cells were demonstrated to express CD38 and enter the S/G2/M phases of the cell cycle. These data suggest that A-myb is a marker of in vivo-activated but not in vitro-activated B lymphocytes. Finally, A-myb was also found to be highly expressed in five of seven Burkitt's lymphoma lines and in none of three EBV lymphoblastoid cell lines. This finding is in agreement with the phenotype of the normal B cells that express high levels of A-myb in vivo and suggests that A-myb may be specifically induced within germinal center B cells.
ME1 epitope of HLA-B27 confers class I-mediated modulation of gram-negative bacterial invasion.
Authors: Kapasi K , Inman R D
Published: December 2022

We have previously demonstrated that the presence of the MHC class I molecule, HLA-B27, on the surface of transfected fibroblasts differentially alters Gram-negative bacterial invasion as compared with class I alleles that are not implicated in the seronegative spondyloarthropathies. We have now extended this analysis to show that fibroblasts transfected with HLA-B7, a cross-reactive allele with HLA-B27, also demonstrate a similar altered bacterial invasion phenotype. The decrease in the ability of the bacteria to penetrate the HLA-B27 and HLA-B7 transfectants is an invasion-mediated event, as demonstrated by differential invasion events using Escherichia coli transfected with the inv gene of Yersinia enterocolitica. The lysine at position 70, although unique to the HLA-B27 subtypes, is shown to be not involved in mediating the decrease in invasion. However, the ME1 epitope is the critical factor in determining allele-specific alteration in invasion on the basis of the following: 1) ME1 mAb preincubation reverses the decrease; 2) ME1-binding alleles act like HLA-B27; 3) a class I allele that is intermediate in ME1 binding (HLA-B14) also demonstrates a relative decrease in invasion; and 4) mutation at residue 67 (C-->Y) in HLA-B27, which eliminates the ME1 epitope, normalizes the decreased invasion seen in the native HLA-B27-transfected cells. Thus, the ME1 epitope relates to the disease susceptibility for reactive arthritis that is conferred by both HLA-B27 and cross-reactive group Ags.
Antigen-dependent selection of T cells that are able to efficiently regulate free cytoplasmic Ca2+ levels.
Authors: Rajasekar R , Augustin A
Published: December 2022

In T helper cells, the process of memory acquisition is reflected in the expression of phenotypic markers. However, little is known regarding the functional changes that occurred in T helper cells selected after a primary Ag-specific response. We now present data that indicate that such T helper cells acquire the ability to down-regulate high concentrations of free cytoplasmic Ca2+. This property renders them resistant to intense inductive stimuli, such as high concentrations of ionomycin. The accumulation of cells that display this ability parallels the progressive proliferative enrichment of Ag-specific T cells and correlates with the surface expression of the CD45RB(low) isoform. As previously shown, persistent high levels of cytoplasmic Ca2+ are responsible for death via apoptosis in virgin T cells. In contrast, the ability to regulate cytosolic Ca2+ allows survival and clonal expansion. We suggest that memory T cells behave differently from virgin T cells when interacting with Ag-presenting B cells (as reported by others) because they have acquired this new physiologic property. Ag-presenting B cells deliver an intense stimulatory signal to T cells because of a high multiplicity of cognate interactions. Thus, Ag-driven T cell proliferation results in the selection of "resistant" T helper cells that can be successfully stimulated by memory B cells. In contrast, naive T cells, which cannot modulate high levels of cytosolic Ca2+, are deleted as a consequence of Ag presentation by B cells.
Role of intrathymic clonal deletion and peripheral anergy in transplantation tolerance induced by bone marrow transplantation in mice conditioned with a nonmyeloablative regimen.
Authors: Tomita Y , Khan A , Sykes M
Published: December 2022

We have investigated the mechanism of tolerance induced by allogeneic bone marrow transplantation (BMT) in mice conditioned with a nonmyeloablative regimen. Permanent mixed chimerism and skin allograft tolerance were achieved when recipient B10 (H-2b) mice were pre-treated with anti-CD4 and anti-CD8 mAbs, thymic irradiation, and 3 Gy whole body irradiation, followed by injection of B10.A (H-2a) bone marrow cells. V beta 11+ T cells are normally deleted in I-E+ B10.A mice, but not in I-E- B10 mice. In spleens of mixed B10.A-->B10 chimeras, V beta 11+ B10 T cells were reduced but detectable in the first 6 wk after BMT, and later became undetectable. Splenic V beta 11+ T cells were unresponsive to receptor cross-linking with V beta 11-specific mAb, demonstrating anergy. B10 V beta 11+ T cells were also detected in spleens of mice that were thymectomized before BMT. In PBL of chimeras, V beta 11+ T cells were undetectable for at least 1.5 yr. Thymocyte chimerism and extensive clonal deletion of mature V beta 11+ B10 thymocytes were observed as early as 10 days post-BMT and at all subsequent time points in thymi of mixed chimeras. Rare I-E+ donor cells were detected in day 10 thymi, and these increased progressively in frequency at later times. In chimeras prepared in the reciprocal strain combination, B10-->B10.A, B10 donor V beta 11+ T cells were undetectable in PBL. Together, our results implicate both central clonal deletion and peripheral clonal anergy in tolerance induced with our regimen. A very low number of intrathymic donor cells or low density of Ag expression is sufficient to mediate extensive clonal deletion of thymocytes that recognize donor Ags.
A V lambda x-bearing monoclonal antibody with similar specificity and sequence to encephalitogenic T cell receptors.
Authors: Maier C C , Galin F S , Jarpe M A , Jackson P , Krishna N R , Gautam A M , Zhou S R , Whitaker J N , Blalock J E
Published: December 2022

The fine specificity of mAb F28C4 to myelin basic protein (MBP), acetyl residues 1-9, has been compared with the previously described specificity of an encephalitogenic T cell clone, PJR-25. F28C4 has been found to express a cross-reactive idiotope (CRI) that is shared with MBP acetyl peptide 1-9-specific TCR. The CRI seems to be located at or near the Ag-combining site of F28C4 and the TCR and, thus, might possibly result from overlapping epitope specificity. We tested the fine epitope specificity of F28C4 by using alanine-substituted peptide analogues and found that residues critical for TCR recognition, Cln3 and Pro6, are also necessary for F28C4 recognition. By using nuclear magnetic resonance, we found that the MBP acetyl peptide 1-9 binds F28C4 in an extended conformation and that the central residues are more tightly bound than the terminal residues, much like the MBP-TCR interaction. Furthermore, sequence homology (75% overall) was found between the regions that contained CDR3 of F28C4 VL and VH and the VDJ junction of the TCR V beta. This homology is not shared by other Ig CDR3 regions and arises, in part, because F28C4 uses an unusual V lambda light chain, V lambda x. Thus, F28C4 shares a CRI with the TCRs, possibly as a result of having similar fine epitope specificity and sequence homology. The anti-CRI mAb can down-modulate experimental allergic encephalomyelitis; thus, it is possible that Abs that are similar to F28C4 may play an important immunoregulatory role in experimental allergic encephalomyelitis in vivo.
Limited T cell response to donor MHC peptides during allograft rejection. Implications for selective immune therapy in transplantation.
Authors: Benichou G , Fedoseyeva E , Lehmann P V , Olson C A , Geysen H M , McMillan M , Sercarz E E
Published: December 2022

Previously, we have demonstrated that during allograft rejection, MHC molecules of the donor are processed and presented to alloreactive CD4+ T lymphocytes in the form of peptides associated with the MHC class II molecules of the recipient. There is an increasing body of evidence that this indirect pathway of allorecognition may play a major role in allograft rejection. Herein, we have used a series of overlapping MHC peptides progressing along the sequence of the donor MHC molecule in single residue steps. We have mapped all potential MHC Ag determinants to which T cell responses could be generated after s.c. injection of allogeneic splenocytes. We have shown that splenic T cell proliferative responses to the beta 1 domains of donor Ak, Ad, and A(s) mouse MHC class II molecules were directed toward a single immunodominant determinant in each of three donor/recipient combinations. Interestingly, after allogeneic spleen cell transplantation, an additional determinant on donor MHC could be detected in the draining lymph nodes. This result shows that the fine specificity of T cell response to donor transplantation Ags can differ between lymphoid organs. Then, we investigated whether limitation of T cell response to donor MHC peptides applies to the clinical situation of a graft. We have shown that after an allogeneic skin graft, self-restricted alloreactive T cells proliferated to the same determinant on the donor MHC molecule. These results indicate that immune intervention, such as tolerance induction to the dominant T cell determinant on donor MHC molecules, may be developed for the prevention of allograft rejection.
Peptides corresponding to the CD8 and CD4 binding domains of HLA molecules block T lymphocyte immune responses in vitro.
Authors: Clayberger C , Lyu S C , DeKruyff R , Parham P , Krensky A M
Published: December 2022

CD4 and CD8 are cell surface glycoproteins that serve as co-receptors for Ag with the TCR. Recent studies have shown that both CD4 and CD8 interact with conserved regions of MHC class II and class I, respectively. To investigate further the roles of CD4 and CD8 in the immune response, we prepared synthetic peptides corresponding to the HLA sequences with which CD4 and CD8 are thought to interact. The peptide corresponding to residues 222 to 235 of the HLA class I heavy chain blocked the differentiation of human CTL precursors into active effect cells but affected neither the ability of PBLs to proliferate in response to mitogen nor the cytotoxic activity of established CTLs. In contrast, the peptide corresponding to residues 134 to 152 of the HLA-DR beta-chain inhibited the differentiation of CTL precursors, the proliferative response of freshly isolated PBL, and the proliferation of an established alloreactive CD4+ T cell clone to Ag. The inhibitory effect of the DR.134-152 peptide on CTL differentiation could be overcome by addition of exogenous IL-2 to the limiting dilution cultures, whereas the effect of the HLA-1.222-235 peptide was unaffected by exogenous IL-2. These results directly demonstrate a functional role for these regions of MHC molecules and underscore the central role of both CD4 and CD8 in the effective initiation of a CTL response.
Human T cells express specific binding sites for C1q. Role in T cell activation and proliferation.
Authors: Chen A , Gaddipati S , Hong Y , Volkman D J , Peerschke E I , Ghebrehiwet B
Published: December 2022

Although the receptor that binds to the collagen-like domain of human C1q (C1qR) is expressed on a wide variety of cell types, the presence or absence of this receptor on human T lymphocytes has been debatable. The current studies were undertaken to re-examine whether human T cells possess specific binding sites for C1q by using a combination of techniques, including radioligand binding studies, flow cytometric analysis, and epifluorescence imaging techniques. Radioligand binding studies indicate that both peripheral T cells and the cultured T cell line, MOLT4, bind 125I-labeled C1q in a specific and apparently saturable manner, reaching equilibrium within 30 min at 37 degrees C under conditions of subphysiologic (90 mM NaCl) ionic strength. Western blot analysis with anti-C1qR of membrane proteins derived from Raji and MOLT4 cells showed an apparent single band of approximately 60 kDa under nonreducing conditions. Furthermore, when peripheral blood T cells were stimulated with 12,-o-tetradecanoyl phorbol-13-ester acetate for 5 days at 37 degrees C and assessed by FACS for their ability to bind anti-C1qR, the mitogen-induced cells were found to bind 40 to 50% more than their unstimulated counterparts. In addition, both CD4+ and CD8+ T cells were found to bind anti-C1qR. When the cells were mitogen induced with either 12,-o-tetradecanoyl phorbol-13-ester acetate, Con A, or PWM for 48 h in the presence or absence of 50 micrograms/ml C1q then pulsed with 1 microCi [3H]thymidine for 16 h at 37 degrees C, proliferation was significantly inhibited (40 to 80%, n = 7) as assessed by reduced [3H]thymidine incorporation. Taken together, the data suggest that: 1) Human T cells express C1qR in which immunoblots reveal a 60-kDa single chain protein. 2) C1qR expression is up-regulated by mitogens that induce T cell proliferation. 3) The primary ligand, C1q, induces an antiproliferative signal, which suggests that the C1qR plays a role in T cell activation and proliferation. In addition, the data contribute to the characterization of C1qRs on cells in peripheral blood and indicate that all cells, with the exception of erythrocytes, bear functional C1q receptors.
Engagement of the common leukocyte antigen CD45 induces homotypic adhesion of activated human T cells.
Authors: Spertini F , Wang A V , Chatila T , Geha R S
Published: December 2022

The transmembrane tyrosine phosphatase CD45 plays an important role in TCR/CD3-mediated signaling. We demonstrate in this study that ligand binding to the CD45 molecule induces homotypic cell adhesion of activated, but not resting, T lymphocytes. mAbs to CD45 (4B2 and 10G10) and to CD45RO (UCHL1), but not to CD45RA (IOL2), caused sustained adhesion of alloreactive T cell lines. In contrast, none of the anti-CD45 mAbs induced aggregation of resting peripheral T cells. CD45-mediated adhesion of activated T cells involved both CD11a/18-dependent as well as CD11a/18-independent mechanisms. mAb 4B2 induced a strictly CD11a/18-dependent adhesion that was completely inhibited by both the protein kinase C (PKC) inhibitor sphingosine and the protein tyrosine kinase (PTK) inhibitors genestein and herbimycin A. In contrast, mAb 10G10, which recognized an epitope on CD45 distinct from the one recognized by mAb 4B2, induced CD11a/18-independent adhesion that was inhibited by sphingosine, but not by genestein or herbimycin A. Biochemical studies revealed direct evidence for activation of protein kinase C and protein tyrosine kinase after engagement of CD45 on activated T cells by mAb 4B2. These results indicate that in addition to its role in TCR/CD3-mediated activation, engagement of CD45 transduces signals that result in enhanced adhesiveness of activated T cells.
Ligand motifs of HLA-DRB5*0101 and DRB1*1501 molecules delineated from self-peptides.
Authors: Vogt A B , Kropshofer H , Kalbacher H , Kalbus M , Rammensee H G , Coligan J E , Martin R
Published: December 2022

Antigenic peptides are presented to CD4+ T cells by MHC class II molecules via a highly polymorphic peptide-binding groove. The two HLA-DR alleles isotypically expressed on HLA-DR15Dw2-positive cells, DRB1*1501 (DR2b) and DRB5*0101 (DR2a) molecules, show a number of differences in polymorphic residues of the beta-chain, including the Gly-Val-dimorphism at position beta 86. Therefore, different requirements for interaction of peptides with these alleles must be expected. In this study, naturally processed self-peptides were eluted from purified HLA-DR15Dw2 molecules and related to DRB1*1501 or DRB5*0101 molecules by binding assays. An alignment of self-peptides and foreign peptides allowed the delineation of putative anchor motifs. N- and C-terminally truncated and alanine-substituted derivatives of the DR15Dw2 restricted myelin basic protein epitope MBP(85-105) confirmed their validity. Thus, DRB5*0101 requires a bulky hydrophobic residue (F or Y) at position i as a primary anchor, and Q or an aliphatic residue, such as V, I, or M, at position i + 3; positively charged residues at positions i + 7 and i + 8 are secondary anchors. For DRB1*1501, a nonaromatic, hydrophobic anchor (L, V, or I) at position i is supplemented by a bulky hydrophobic residue (F or Y) at position i + 3 as primary anchor; an additional hydrophobic side chain represented by M, I, V, or F occurs at position i + 6. Therefore, MBP(85-105) seems to contain two MHC interaction sites for DRB1*1501 and DRB5*0101, respectively, that may contribute to its immunodominance. Because HLA-DR15 Dw2 is associated with susceptibility to develop multiple sclerosis, the delineation of ligand motifs of the two DR2 alleles may help to study the interaction between potential autoantigenic peptides and these molecules in the future.
A low molecular weight phagocytosis-inhibitory factor obtained from human erythrocyte membranes specifically down-regulates Mac-1 activity on tetradecanoyl phorbol acetate-stimulated monocytic cell lines in a Ca(2+)-dependent manner.
Authors: Baranji K , Baranyi L , Yoshida T , Okada N , Okada H
Published: December 2022

A low molecular mass (< 3 kDa) phagocytosis-inhibitory factor (PIF), was partially purified from human erythrocyte membranes. PIF inhibits latex phagocytosis and C, as well as FcR-mediated phagocytosis, by macrophage-like cells in a Ca(2+)-dependent manner. This phagocytosis-inhibitory activity is reversible because removal of PIF restores phagocytic capability of cells. After treatment with PIF, Mac-1 Ag (CR3 or CD11b) becomes almost undetectable on the cell surface by immunofluorescence staining using the mAbs D-12 and BEAR-1, whereas staining with the LM2/1 anti-Mac-1 mAb proved that Mac-1 is still present on the cell surface, thus, indicating a possible conformational change in Mac-1. PIF has no significant inhibitory effect on staining of CR1 (CD35), CR2, (CD21), 20-kDa homologous restriction factor (CD59), decay-accelerating factor (CD55), LFA-1 (CD11a), or p150.95 (CD11c). Although binding of Mac-1-bearing U-937 cells to C3bi-opsonized beads is completely blocked, binding via Con A and FcRs remains unaffected by PIF treatment. In addition to the suppressive effect on phagocytosis, inhibition of cell adhesion was observed as well. The inhibitory effect of PIF on cell adhesion is not monocyte specific, because after exposure to PIF the TGW neuroblastoma cell line lost its ability to attach to the tissue culture plate, but retained its ability for homotypic aggregation. The possibility that PIF is a natural regulator of erythrophagocytosis is suggested.