Resistance and Susceptibility to Cyclosporin A as CD3− 4− 8− Human Thymocytes Differentiate In Vitro

Human T-cell development appears to be relatively resistant to cyclosporin A (CsA). Children exposed to CsA mw/eroaspart of kidney transplant maintenance have few abnormalities. The objective of the study described here was to analyse the effects of CsA on the development in vitro of human multinegative (MN) (CD3-4-8-) thymocytes as a model system for thymic progenitor development in vitro. MN thymocytes, prepared by depletion methods, differentiated in vitro to acquire CD3 and undergo transitions in CD45 isoform expression analogous to those postulated to occur in vivo. In this work MN thymocytes were cultured with IL-2 and on thymic epithelial cells (TEC) with or without lL-2, either in ihe presence or absence of CsA. For many thymocyte preparations, differentiation in the presence of CsA resulted in almost complete inhibition of the acquisition of CD3and ofthe low Mr isofomi CD45R0. Expression of CD45RA and of total CD45 were reduced but not eliminated and the density of CD29 was unaffected. For others, neither CD3 nor CD45 expression was affected., but selective inhibition of TCR5 expression occurred. At all doses of CsA (0.1–100 μg/ml), MN thymocytes continued to cycle indicating a CsA-resistant generative compartment. Treatment of peripheral blood T cells with CsA had no effect on surface expression of CD3 or CD45 isoforms but did reduce the amount of de novo-synthesized CD45R0 mRNA. Culture of MN thymocytes on TEC rendered them virtually resistant to the negative effects of CsA. CD3 acquisition was unhindered and total CD45 remained high, but the transition from CD45RA to CD45R0 appeared to be delayed. In the absence of TEC, expression of both TCRδ and of TCRδ was inhibited, but on TEC, TCRδ was actually up-regulated in some conditions. The effects of CsA on human thymocyte development appeared to be modulated by the physiological state of the donor and the growth conditions to which the ceils were subjected. Conditions which most closely approximated those manifest in vivo rendered thymocytes most resistant to the negative effects of CsA. The amount of CsA required to affect differentiation in vitro was significantly higher than could be attained in vivo suggesting that the immunomodulatory effects of CsA in the maintenance of organ transplants may from an as yet uncharacterized mechanism     

Glucagon activates Ca2+ and Cl− channels in rat hepatocytes

Glucagon is one of the major hormonal regulators of glucose metabolism, counteracting the hepatic effects of insulin when the concentration of glucose in the bloodstream falls below a certain level. Glucagon also regulates bile flow, hepatocellular volume and membrane potential of hepatocytes. It is clear that changes in cell volume and membrane potential cannot occur without significant ion fluxes across the plasma membrane. The effects of glucagon on membrane currents in hepatocytes, however, are not well understood. Here we show, by patch-clamping of rat hepatocytes, that glucagon activates two types of currents: a small inwardly rectifying Ca²⁺ current with characteristics similar to those of the store-operated Ca²⁺ current and a larger outwardly rectifying Cl⁻ current similar to that activated by cell swelling. We show that the mechanism of glucagon action on membrane conductance involves phospholipase C and adenylyl cyclase. Contribution of the adenylyl cyclase-dependent pathway to activation of the currents depended on Epac (exchange protein directly activated by cAMP), but not on protein kinase A. The activation of Ca²⁺ and Cl⁻ channels is likely to play a key role in the mechanisms by which glucagon regulates hepatocyte metabolism and volume.     

CD3−4−8− Thymocyte Precursors with Interleukin-2 Receptors Differentiate Phenotypically in Coculture with Thymic Stromal Cells

CD3⁻4⁻8⁻ interleukin-2 receptor positive (IL-2R⁺) thymocyte precursors from adult mice were cocultured with thymic stromal cells from syngeneic adult mice. The IL-2R⁺CD3⁻4⁻8⁻ thymocytes were obtained by positive panning of IL-2R⁺ cells followed by either sorting or negative panning of triple negative cells, and they were cocultured with primary or secondary cultures of heterogeneous thymic stromal cells. Phenotypic maturation of these precursor cells was extremely rapid. Within 2½ days significant numbers of CD4⁺8⁺ and CD3⁺4⁺8⁻ cell populations developed, the latter expressing the αβ T-cell receptor (αβ-TCR). Thus heterogeneous stromal cell cultures support the development of IL-2R⁺ precursors and with these methods it will now be possible to isolate the particular stromal cells involved at each stromal-dependent step.     

Increased occurrence of IgE+ and FcεRI+ cells in adenoids from atopic children

BACKGROUND: To examine the influence of atopy on the different cell populations in adenoids, we investigated the presence of IgE+ cells, cells expressing the high-affinity receptor for IgE (Fc(epsilon)RI), and various other cell populations in adenoid tissue, in atopic and nonatopic children with otitis media with effusion (OME) or adenoid hyperplasia (AH). METHODS: Cryostat sections of adenoids from 14 atopic and 16 nonatopic children suffering from long-lasting OME (n=15) or obstructive AH (n=15) were investigated with immunohistochemical markers for T-cell subsets, mast cells, eosinophils, plasma cells, CD25, CD1a, IgE, and Fc(epsilon)RI. RESULTS: Sensitization to allergens was correlated to an increase of IgE+ cells in the epithelium (P<0.01), the extrafollicular area (P<0.0001), and the follicles (P<0.001) of the adenoids and an increase of Fc(epsilon)RI+ cells in the extrafollicular area (P<0.01). A minority of the IgE+ cells were plasma cells. No significant differences in cells stained for IgE, Fc(epsilon)RI, or the other markers were observed between patients with OME and AH. CONCLUSIONS: Atopy is associated with increased numbers of IgE+ and Fc(epsilon)RI+ cells in adenoids irrespective of whether the child has OME or AH.     

CD8+ T Cells Induce Medullary Thymic Epithelium and CD4+CD8+CD25+ TCRβ− Thymocytes in SCID Mice

During T-cell development the transition in the thymus of CD4-CD8- double negative (DN) progenitor T cells into CD4+CD8+ double positive (DP) cells is dependent on the expression of a T-cell receptor (TCR)-beta-chain protein. In this study purified peripheral CD4+ and CD8+ T lymphocytes from the C.B-17 strain of mice were adoptively transferred into syngeneic, neonatal SCID mice, where donor cells resided at constant numbers in thymus from 2 weeks until 10 weeks post cell transfer. In the recipient thymus the CD8+ donor cells outnumbered the CD4+ cells by a factor of three to five and both subsets contained a large fraction of activated cells. During the late phase of treatment, CD8+ T cells induced high numbers of DP thymocytes in the SCID mice, a process accompanied by the maturation of medullary epithelial cells. Such thymic development in the SCID mouse was inhibited by coresiding CD4+ donor T cells. These results indicate a regulatory role by mature peripheral T cells on medullary epithelial growth and thymocyte development in the treated SCID mice.     

CD4+, CD8+, and CD4− CD8− T Cells in CSF and Blood of Patients with Multiple Sclerosis and Tension Headache

Two-colour flow cytometric analysis was performed on paired samples of peripheral blood (PB) and cerebrospinal fluid (CSF) of patients with untreated multiple sclerosis (MS) and, for reference, subjects with muscular tension headache (TH) using anti-CD3, anti-CD4, anti-CD8, and anti-HLA-DR monoclonal antibodies in different combinations. CD4+/CD8+ T-cell ratio was increased in CSF compared to PB in both MS patients and TH subjects to a similar extent. This was mainly due to higher CD4+ T-cell levels in the CSF compartment. The proportion of HLA-DR+ T cells was higher in CSF than PB in both MS and TH; this increase of DR+ T cells in CSF was more prominent in MS. The level of CD4+ CD8+ T cells, which represent a subset of activated T cells, was not different between CSF and PB, either in MS or in TH. The proportion of CD4- CD8- T cells, which were found generally not to be blast cells, was lower in CSF compared to PB in both patient groups. However, their CSF level was higher and their PB level lower in MS compared to TH. Results point to an accumulation of activated T-helper cells in the CSF of both MS patients and healthy subjects. Fetal-type CD4- CD8- T cells bearing the unusual T-cell receptor gamma/delta seem to be selectively recruited to the CSF of MS patients.     

Characterization of T-Cell Receptor Vβ Repertoire in Ovarian Tumour-Reacting CD3+ CD8+ CD4− CTL Lines

T cells from tumour infiltrating lymphocytes (TIL) cultured in media containing IL-2 were shown to mediate in vitro and in vivo antitumour responses. To characterize the T-cell antigen receptor (TCR) Vβ expression in autologous cytotoxic effectors we isolated CD3⁺ CD8⁺ CD4⁻ cells from cultures of TIL and tumour-associated lymphocytes (TAL) and analysed the TCR Vβ repertoire of CD3⁺ CD8⁺ CD4⁻ lines of known HLA-A, -B and -C phenotype, using polymerase chain reaction (PCR). These lines showed preferential lysis of autologous tumours and lysed, to a much lesser extent, NK and LAK cellsensitive targets. Tumour lysis was inhibited by antibodies to CD3 and MHC class I antigens indicating that they are cytotoxic T lymphocytes (CTL). These CD8⁺ CTL lines expressed a broad distribution of TCR Vβ repertoire which was dominated by particular groups of Vβ families in each CTL line. However, no predominant expression of one or the same Vβ segment in all CTL lines was observed although statistical correlations between Vβ family usage and magnitude of the antitumour cytolytic response were found. These results suggest that certain TCR Vβ families may be selected by antigen in ovarian tumour-reactive T cells and this selection may be affected by Ag expression, and/or host factors. To our knowledge, this is the first documentation of TCR Vβ repertoire of human ovarian tumour-reactive CD3⁺ CD8⁺ CD4⁻ CTL from different individuals of known HLA types.     

Leu 7+(HNK-l+) Cells

In the present study, combined methods (indirect immunofluorescence with monoclonal antibodies, Percoll density fractionation, FACS analysis, and the cytotoxicity test) were used for further characterization of peripheral blood Leu 7+ cells (human NK and K cells). The Leu 7+ cell content was found to be relatively higher in the low-density cell fraction in which cells of large granular lymphocyte morphology predominated. However, Leu 7+ cells were also present in intermediate and high-density fractions. Low-density Leu 7+ cells were characterized by both Leu 2 (T suppressor/cytotoxic) and OKM1 (myelomonocytic) markers, whereas among high-density Leu 7+ cells the Leu 2 phenotype strictly predominated. Depletion of OKT3+ cells from the non-adherent cell population caused a decrease of cells with T helper and T suppressor phenotypes but did not have this effect on Leu 7+ and OKM1+ cells. After depletion of Leu 7+ cells from the OKT3- population the content of both T suppressor and OKM1+ cells decreased. Both the present results and previous reports enable us to conclude that two main Leu 7+ cell subpopulations are present in blood, namely Leu 7+Leu 2+/Leu 4+ and Leu 7+/OKM1+ cells. The presence of small and large Leu 7+ cells was also shown by FACS analysis.     

Leu 7+(HNK-l+) Cells

In the present immunohistochemical studies, Leu 7+ (HNK-1+, human natural killer and killer) cells were found to occupy preferentially germinal centres of follicles in lymph nodes and tonsils. Leu 7+ cells were also present in germinal-like zones of spleen follicles and in mantle zones of hyperplastic thymus follicles and varied in localization in lymph nodes involved in different types of follicular centre cell-derived malignant lymphomas. Most of the Leu 7+ cells in the follicles expressed the Leu 3 (helper/inducer) marker. Double staining studies of tonsil sheep erythrocyte-rosetting and peripheral blood mononuclear cell suspensions showed that two main, mutually complementary, subpopulations of Leu 7+ cells could be distinguished in both cases, namely Leu 7+/Leu 4+ (subdivided into Leu 2+ (suppressor/cytotoxic) and Leu 3+) and Leu 7+/Leu 4-, including mostly cells with OKM 1 (myelomonocytic) characteristics. Thus, in the tonsil cell suspension the cells with Leu 7+ Leu 3+/OKM 1- immunophenotype strongly predominated, whereas among peripheral blood mononuclear cells Leu 7+Leu 2+/OKM 1- and Leu 7+/OKM 1+ immunophenotypes were mostly observed.