Characterization of CD28CD4 T cells in living kidney transplant patients with long-term allograft acceptance

CD28⁻CD4⁺ T-cell subpopulation is expanded in kidney allograft patients with long graft survival. To seek for the roles of CD28⁻CD4⁺ T cells in the long-term acceptance of kidney allografts, we characterized this population by analyzing cell surface molecules, TCR V_β repertoire, mixed lymphocyte reaction (MLR), and cytokine production. The number of CD28⁻CD4⁺ T cells increased correlatively with time after transplantation in this group of patients. The CD28⁻CD4⁺ T cells did not express detectable levels of CD25, CD69, V24, or CTLA-4 but expressed heterogeneous amounts of CD45 RA on the surface. Freshly sorted CD28⁻CD4⁺ T cells revealed a restricted V_β repertoire, whereas the V_β usage of CD28⁺CD4⁺ T cells from the same patients was much diversified. Expression levels of TGF-β and IFNγ gene were significantly higher in the CD28⁻ CD4⁺ T cells than in the CD28⁺CD4⁺ T cells from the kidney allograft patients. These findings suggest that an oligoclonal CD28⁻ CD4⁺ T-cell population is continuously activated in patients with long allograft survival, which may be linked with the long-term acceptance.     

Analysis of the Conservation of T Cell Receptor Alpha and Beta Chain Variable Regions Gene in pp65 Peptide-Specific HLA-A＊0201-Restricted CD8^＋ T Cells

Many viral epitope specific T cell receptors （TCRs） in MHC-matched individuals have been demonstrated to involve conserved amino acid motifs in β chain complementarity-determining region 3 （CDR3）. However, it is not sure whether the conserved motifs can also be found in TCR β chain. In previous studies, we developed a modified method to enlarge the percentage of cytomegalovirus （CMV） pp65 peptide-specific CD8^＋ T cells in PBMC by continuous peptide stimulation in vitro, which provides sufficient number of specific T cells for detection. In this study, we further analyzed the restrictive usage of TCR Vα and Vβ gene families and investigated the CDR3 gene sequence of pp65 peptide-specific CD8β T cells. Analysis of CDR3 spectratypes suggested a restricted usage of TCR α chain AV8, AV12, AV21, AV31 families and TCR βchain BV3, BV14, BV21, BV23, BVll families in donor CD8^＋ T cells stimulated by pp65 peptide. The sequences of these T cells involved similar sequence （TX） G （X） A in CDR3 region of TCR α chain and L （XT） G （X） A in TCR β chain.     

Human extracellular proteins display a different pattern of local sequence similarity with the four classes of human T-cell receptor V-regions than foreign proteins and human intracellular proteins: a preliminary report

A pool of 110 randomly selected/generated amino acids sequences was used to perform specific local sequence similarity alignment analysis with the pool of 279 reported sequences of human T-cell receptor (TCR) V-regions. The 110 analyzed sequences were divided, according to their origin and nature, into six protein groups, as: human intracellular (hi), extracellular/transmembrane (he) and extracellular adhesive matrix (ha) proteins, ‘average' human proteins (hum), proteins of non-human origin (nhum) and randomly generated quasi-protein sequences (r). These sequences were decomposed into all their overlapping 11-mer segments, generating a total of 56 836 derived peptides (at least 8000 per group). Each derived peptide was aligned with the 279 human TCR V-regions and assigned to the category (-like, β-like, γ-like or δ-like) corresponding to the class (V, Vβ, Vγ or Vδ) of the V-region encompassing the most similar segment, as determined by the performed similarity-search. The six protein groups were found to differ significantly in their distribution of derived peptides among the four categories. According to the binomial tests results, human proteins from the extracellular compartment (he, ha) comprise a higher proportion of δ-like segments (P=2.3×10⁻² and P<10⁻⁸, respectively) than the ‘average' human proteins (hum). In addition, and in accordance with this finding, proteins that are normally not found in that topological compartment comprise a lower proportion of δ-like peptides (P=1.4×10⁻⁵ and P<10⁻⁸ for groups nhum and hi, respectively) than the ‘average' human proteins (hum). In contrast, these proteins comprise a higher proportion of γ-like segments (P=8.3×10⁻³, P=1.4×10⁻³ and P=1.7×10⁻⁴, for groups r, nhum and hi, respectively) than the ‘average' human proteins (hum). These findings indicate significant differences between proteins encountered in the extracellular compartment—that are normally immunologically tolerated—and those the presence of which is usually non-tolerated. The results suggest that the discrimination and the reaction of the human immune network to proteins found in the extracellular compartment correlate with the proteins' pattern of preferential local sequence similarity with the Vγ and Vδ classes of human TCR V-regions, implying a specific and an important role of γδ-cells in the maintenance of the immune homeostasis. Whether this implication represents a rule associated with self-tolerance, will be investigated by future analyses.     

gamma-Glutamyl transpeptidase activity in Ascaris suum

Using histochemical techniques γ-glutamyl transpeptidase activity has been localized mainly in the cuticle-hypodermis in Ascaris suum. The specific activity of γ-glutamyl transpeptidase was 3.87 ± 0.49 μmol h⁻¹ (g tissue)⁻¹ in cuticle-hypodermis as compared to gastrointestinal tract, where it was 0.07 ± 0.02 μmol h⁻¹ (g tissue)⁻¹, and muscle and reproductive tract where it was <0.001 μmol h⁻¹ (g tissue)⁻¹. When cuticle sections were incubated with labelled glutamine a large portion of the glutamine nitrogen was recovered as ammonia which indicated glutamine uptake and utilization by the cuticle-hypodermis. Collectively these histochemical and biochemical data support the fact that γ-glutamyl transpeptidase activity is found in the cuticle-hypodermis of A. suum and this may be an indication that amino acid absorption could occur through the cuticle via the γ-glutamyl cycle.     

Organ-specific and differential requirement of TYK2 and IFNAR1 for LPS-induced iNOS expression in vivo

Lipopolysaccharide (LPS) is an integral structural component of the outer membrane of Gram-negative bacteria and the principal active agent in the pathogenesis of endotoxin shock. LPS is a potent inducer of a variety of cytokines and inflammatory agents that lead to a profound alteration of gene expression patterns in cells and organs. The gene coding for the inducible nitric oxide synthase (iNOS) is highly responsive to LPS in vitro and in vivo and accounts for the production of nitric oxide (NO). The Janus kinase (JAK) family member tyrosine kinase 2 (TYK2) is a constituent of the interferon (IFN) type I response pathway and an important effector in the progression of endotoxin shock. Macrophages deficient for IFNβ receptor chain 1 (IFNAR1) or TYK2 were shown to have an impaired LPS-induced iNOS expression. Here we determined the contribution of IFNAR1 and TYK2 to iNOS expression in vivo in a lethal LPS challenge model. TYK2 and IFNAR1 were found to be crucial for the LPS-induced iNOS mRNA and protein expression in spleen and lung that could be attributed to the Mac3-positive population. In liver LPS-induced iNOS mRNA expression was only partially impaired in TYK2-deficient mice and was unimpaired in IFNAR1-deficient mice, indicating organ specificity. TYK2^−/− and IFNAR1^−/− mice also differ with respect to IFNγ production upon LPS challenge in that TYK2^−/− mice show a defect while IFNAR1^−/− mice do not. Our data suggest that iNOS is induced through IFNAR1 and TYK2 in Mac3-positive cells which are the main source of iNOS in spleen and lung. The LPS-induced iNOS expression in liver is independent of IFNAR1 and partially dependent on TYK2, which is most likely due to the lack of IFNγ production in the absence of TYK2.     

Lack of stimulatory effect of dienogest on the expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 by endothelial cell as compared with other synthetic progestins

Objectives: Monocyte adhesion to endothelial cells is an important initial event at the onset of atherosclerosis. It is partially mediated by the expression of adhesion molecules on the endothelial cell surface. While estrogens inhibit the development of atherosclerosis, the effect of co-administered progestin remains controversial. We examined the effect of progestins on cytokine-stimulated human umbilical venous endothelial cell (HUVEC) expression of adhesion molecules. Methods: In HUVECs, mRNA expression of progesterone receptors (PRs) and androgen receptors (AR) was determined by RT-PCR. HUVECs were stimulated by interleukin-1β (IL-1β) for 24 h with or without various steroids, and then the cell-surface expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was semiquantified by ELISA. Results: In all preparations of HUVECs used in this study, RT-PCR confirmed mRNA expression of both isoforms of PR, PR-A and PR-B, as well as AR. Addition of progesterone (10⁻¹⁰–10⁻⁷ M) or dienogest (DNG) (10⁻¹⁰–10⁻⁸ M) did not affect IL-1β-stimulated ICAM-1 or VCAM-1 expression. In contrast, medroxyprogesterone acetate, norethindrone acetate and levonorgestrel (10⁻¹⁰–10⁻⁸ M) dose-dependently increased cell adhesion molecules. The progestin-induced increase was blocked by the concomitant addition of mifepristone, a PR antagonist, but not by hydroxyflutamide, an AR antagonist, indicating that the progestin stimulation was mediated predominantly via PR. Conclusions: These results suggest that DNG, unlike other synthetic progestins, lacks stimulation of cell adhesion molecules. For the prevention of atherosclerosis, estrogen in combination with DNG may be a suitable regimen in hormone replacement therapy in postmenopausal women.     

Cassette vectors directing expression of T cell receptor genes in transgenic mice

We describe a pair of cassette vectors that can be used to express rearranged T cell receptor genes in transgenic mice. Short DNA fragments containing rearranged V and Vβ segments are readily amplified from T cells and introduced between artificial cloning sites. Transgene-derived mRNAs are transcribed under the control of the natural TCR and -β promoter/enhancer elements. Using this vector, we have obtained transgenic mouse lines which display transgene-encoded TCR and β chains on a majority of T cells.     

Signalling initiated with CD4–TCR or TCR–TCR interactions: comparison of tyrosine phosphorylation patterns and CD45 effects

Antigen-triggered response in T cells is mediated by the T cell receptor (TCR)/CD3-complex. This signalling, however, is modulated by a number of other surface molecules. Among the most important of these is the CD4/CD8 molecule which associates with the TCR/CD3-complex and binds to the MHC complex. The molecular mechanisms involved in interactions between TCR–TCR and TCR–CD4 are not fully understood. We have earlier described an experimental model that allows us to dissect signals involving CD4–TCR interactions and those involving TCR–TCR interactions using a mouse CD4⁻CD8⁻ T cell hybridoma cell-line transfected either with the TCR from a mouse T-helper ₂ cell-line (D10) alone or with both the TCR and the CD4 molecule. To further characterize these two different modes of signalling in T lymphocytes we have studied the tyrosine phosphorylation patterns resulting from these interactions. In addition, we have studied the modulatory effect of the CD45 molecule on these interactions. In contrast to some earlier reports, we found that both the patterns of induced tyrosine phosphorylation and the effects of CD45 modulation were essentially similar in the CD4–TCR and the TCR–TCR signal transduction cascades. The results are consistent with a purely synergistically amplifying function for CD4 on the TCR-mediated signalling.     

Abrogation of the Allelic Exclusion in a T Cell Receptor β Chain Gene Transgenic Mouse Strain

The expression of endogenous T cell receptor (TcR) β chains in a TcR β chain gene transgenic mouse (TGM) strain was examined. Unlike many other TGM strains reported, a considerable proportion of T cells from the thymus and spleen as well as organ cultured fetal thymus from our TGM express endogenous TCR β chains on their surface. Compatible with this was the elucidation of VDJ rearrangement of endogenous β chain genes by PCR. Three color flowcytometric analysis of thymus cell subpopulations revealed that the expression levels of both endogenous and transgenic TcR β genes are regulated in a maturational stage specific manner. Splenic T cells contained a several fold higher percentage of endogenous TcR β positive cells than thymus cells, suggesting a role of TcR on T cell peripherization. Vβ6 positive cells were deleted in the TGM carrying minor lymphocyte stimulating (Mls)-l^a antigen, indicating that the endogenous TcR β is functional in terms of transmiting a signal for clonal deletion.     

Heterogeneity in terms of interleukin 4-dependent regulation of Fce receptor/CD23 expression on chronic B-lymphocytic leukemia cells

To discriminate the stages of maturation arrest of leukemic B cells, we have investigated the cell surface expression of FcεR_1l (H107 antigen) on leukemic B cells from 6 patients with chronic type B-lymphocytic leukemia(B-CLL) by a double staining method combined with cytoflorometry, and their production of soluble FceR_ll ⁺ by an ELISA technique. FceR_ll was expressed onμ⁺/δ⁻ cells of case 5 as well as on μ⁺/δ⁺cells of cases 1,2 and 4, but not on μ⁺/δ⁺cells in cases 3 and 6. The cultivation of leukemic cells with IL-4 not only increased the percentage of FceR_ll⁺cells but also enhanced the production of soluble Fcer_llin most cases. However, IL-4 had no effects on μ⁺/δ⁻/Fcεr_ll⁺ cells of cases 5, which appeared to correspond to a rather late     

BACE1 gene deletion: impact on behavioral function in a model of Alzheimer's disease

Accumulation of cerebral amyloid-β (Aβ) has been implicated as a putative causal factor in the development of Alzheimer's disease (AD). Transgenic mice like the PDAPP line overexpress human mutant Amyloid Precursor Protein (hAPP) and recapitulate many features of AD, including amyloid neuropathology and cognitive deficits. Inhibition of the β-site aspartyl cleaving enzyme (BACE1) enzyme responsible for the first proteolytic cleavage that ultimately generates Aβ has been proposed as a strategy for AD therapy. To assess the theoretical repercussions of β-secretase activity reduction in an in vivo model of AD, BACE1^−/− mice bred to the PDAPP line were examined in a series of behavioral tasks. Although BACE1 gene ablation abolished hAβ accumulation, BACE1^−/− mice had unexpected sensorimotor impairments, spatial memory deficits, and displayed seizures, phenotypes which were severe on the PDAPP background. These results suggest that while excess Aβ is functionally pathological, BACE1-mediated processing of APP and other substrates play a role in “normal” learning, memory and sensorimotor processes.     

Human suppressor T cells: Induction, differentiation, and regulatory functions

T-cell-mediated suppression of human immune responses involves a complex interaction between distinct lymphocyte subsets with suppressor-inducer and suppressor-effector functions. Recent studies with subset-specific monoclonal antibodies have defined a characteristic phenotype of suppressor-inducer cells (CD4⁺ Leu8⁺ 2H4⁺ 4B4⁻) that can be distinguished from that of helper cells for antibody synthesis (CD4⁺ Leu8⁻ 2H4⁻ 4B4⁺). Similarly, suppressor-effector cells (CD8⁺ CD11⁺ Tp44⁻ can typically be defined as a subset separable from cytotoxic T cells (CD8⁺ CD11⁻ Tp44⁺). Both antigen-specific and nonspecific interactions are important in suppressor T-cell activation and function. Soluble signals required for differentiation of CD8⁺ suppressor cells include an indomethacin-sensitive monocyte product and interferon gamma. In contrast, proliferation of the CD8⁺ suppressor cell subset depends on stimulations first by a product of CD4⁺ Leu8⁺ cells, T suppressor cell growth factor, and second by interleukin 2. Although the molecular basis of antigen-specific interactions between CD4⁺ and CD8⁺ cells in suppressor cell generation has not been defined, it may involve both conventional, presumably MHC-restricted, interactions between antigen and antigen receptors, as well as anti-idiotypic interactions of suppressor-effectors with determinants on suppressor-inducer receptors. Progress in elucidating requirements for activation, growth, and differentiation of suppressor cells should facilitate long-term culture of such cells and lead to clearer understanding of mechanism of suppressor-cell mediated immunoregulation.     

HLA-DRB1 molecules and antigenic experience shape the repertoire of CD4 T cells

Forces influencing the composition of the mature TCR repertoire have been well studied in the mouse. In particular, the contribution of MHC molecules in negative and positive selection events of T lymphocytes has been established. To understand whether the allelic polymorphism of HLA-DRB1 molecules can shape the human TCR repertoire, we compared the usage of TCR Vβ segments in two cohorts of unrelated individuals who were selected for the expression of HLA-DRB1 alleles. To investigate the potential role of antigenic experience in shaping the TCR repertoire, we compared the usage of Vβ gene elements in CD45RO⁻ CD4⁺ (naive) T cells versus CD45RO⁺ CD4⁺ (memory) T cells. A correlation between Vβ gene segment usage and HLA-DRB1 alleles could be demonstrated for the repertoire of the naive CD4⁺ T cells, suggesting a shaping force of the HLDRB1 allele on the peripheral TCR repertoire. While the HLA-DRB1 imposed profile in Vβ distribution was maintained in CD45RO⁺ CD4⁺ T cells, it was less pronounced, indicating that antigenic experience modulates the functional TCR repertoire.     

Description of a mouse monoclonal anti-HLA-B27 antibody HLA-ABC-m3

The production and characterization of a new anti-HLA-B27 monoclonal antibody HLA-ABC-m3 is described. This cytotoxic IgG2a antibody binds protein A and is able to precipitate cell surface molecules of 43,000 and 12,000 daltons corresponding to the HLA heavy chain and β2-microglobulin. Population testing revealed that the HLA-ABC-m3 antibody reacted with the peripheral blood lymphocytes of 47/47 individuals conventionally typed as HLA-B27⁺ and with 5/105 HLA-B27⁻ individuals. These five extra reactions were with individuals expressing the cross-reactive HLA-B7 alloantigen, although the affinity of the monoclonal antibody for B27 heterozygous individuals (approx 10⁹ M⁻¹) was tenfold greater than with B7 individuals (approx 10⁸ M⁻¹). In addition, HLA-ABC-m3 reactivity segregated with HLA-B27 in two families. This monoclonal antibody should be of value in the investigation of the role of HLA-B27 in disease.     

Rearrangements of t-cell receptor β-chain genes in human leukaemias

Rearrangements of the T-cell receptor (TCR), β-chain genes and immunoglobulin (Ig) heavy chain genes in several T-cell leukaemias (T-ALL and ATL), and some B-cell and myelogenous leukaemias were investigated. Two out of 15 cases of T-cell leukaemia tested failed to show a rearrangement pattern of TCR β genes although both expressed mRNA for this gene. The remaining 13 cases showed diverse patterns of rearrangements involving either Cβ₁, Cβ₂ or both. Cβ₁, but not Cβ₂ was deleted in s the T-cell leukaemias. Polyclonal T cells from four normal individuals showed the germ line pattern and an additional two bands in Hind III digested DNA. Except for one, all cases of C-ALL (B-cell leukaemia) showed a rearranged J_H locus which was not evident in any of T-cell leukaemias studied. One case of B-cell leukaemia showed a rearrangement of both TCR β genes and J_H genes. The results of these studies suggest that rearrangement of TCR and Ig genes occurs at a very early stage of differentiation of stem cells and does not appear to play a direct role in leukaemogenesis per se.     

An alternative approach to the assessment of γδ T-cell clonality in celiac disease intestinal lesions through cDNA heteroduplex analysis of T-cell receptor VJ junctions

We have investigated the clonality of the γδ T lymphocytes infiltrating the intestinal mucosa of CD patients and control subjects by means of a simple and powerful method based on the heteroduplex analysis of the TCR VJ junctions. Each V-specific TCR chain, amplified either from fresh biopsy material or intestinal T-cell-line cDNA, is denatured and renatured to allow the random reshuffling of the various strands carrying different junctional sequences, coamplified in the same reaction. The mismatched chains (heteroduplexes) are separated from the matched ones (homoduplexes) through polyacrylamide gel electrophoresis, and whenever one or more T-cell clones are emerging over the polyclonal background, discrete bands are visible by ethidium-bromide staining. Through this method, we have estimated the diversity of the Vδ1–3 chains and a newly described V gene (Vδ8) whose homologue in mice is abundantly expressed in γδ iLs. We demonstrate that the well-documented expansion of Vγ1⁺ γδ lymphocytes in the jejunum of CD patients is polyclonal. Overall, the heteroduplex analysis on fresh intestinal and peripheral blood lymphocytes from both healthy and affected subjects shows a polyclonal pattern of all the Vδ⁺ subsets. In contrast, most intestinal T-cell lines produce oligoclonal patterns, suggesting a dramatic in vitro selection effect. The cell expansion in culture is generally not required for the TCR heteroduplex analysis, which can therefore be applied to rapidly monitor the T-cell response in a variety of physiologic and autoimmune reactions, substituting the standard approach of TCR cloning and multiple VJ sequencing. Human Immunology 40, 303–311 (1994)     

A quantitative analysis of rat adrenal chromaffin tissue: Morphometric analysis at tissue and cellular level correlated with catecholamine content

A morphometric analysis of normal Wistar rat adrenal medulla following perfusion fixation and Araldite embedding, was correlated with catecholamine levels on fresh tissue, measured by high-performance liquid chromatography. The mean volume of whole adrenal is 13.2 mm³ and the mean medullary volume 1.3mm³. Volume density estimates showed that the medulla is composed of 63% chromaffin tissue with an adrenaline to noradrenaline storing cell ratio of 4.4:1. The vasculature occupies 20%, neuronal tissue 5% and interstitial tissues 12% of the medulla. A comparison was made of cell volumes, cell numbers and volume and surface density estimates of cytoplasmic organdies in adrenaline and noradrenaline storing cells. The mean cell volume of adrenaline storing cells at 1300 μm³ is larger than that of noradrenaline storing cells at 980 μm³. A single adrenal medulla contains4.4−5.7 × 10⁵ adrenaline cells and1.5−1.9 × 10⁵ noradrenaline cells. Chromaffin granules account for approximately 30% of the volume of the cytoplasm; the numerical density of granules at different sites in the cell was calculated for adrenaline cells. The volume density of mitochondria (4%) and the surface density of mitochondrial membranes (the ratio of outer to inner membrane being approximately 1:2.3) were similar in both cell types. Rough endoplasmic reticulum was the only organelle to show a significant difference in volume and surface density between the two cell types. Adrenaline storing cells have stacks of rough endoplasmic reticulum which have two to three times the surface and volume densities of that found diffusely scattered throughout noradrenaline cells. The adrenaline content of an adrenaline storing cell is0.14 × 10⁻⁶ μM and that of a granule 3.0 × 10⁻¹² or3.8 × 10⁻¹² μ moles depending on the method of calculation. The noradrenaline content of noradrenaline storing cells can only be calculated on the assumption that all noradrenaline is stored in this cell type though it is likely that some is contained within adrenaline cells. Based on this assumption the noradrenaline content is0.17 × 10⁻⁶μ moles per cell and5 × 10⁻¹² μ moles per granule. The present study provides baseline morphometric data on the rat adrenal medulla at tissue and cellular level correlated with amine levels in adrenaline and noradrenaline storing cells and granules.