Different use of T cell receptor transducing modules in two populations of gut intraepithelial lymphocytes are related to distinct pathways of T cell differentiation.

Most gut intraepithelial cells (IEL) of the mouse are T cells that bear CD8 molecules, present either as alpha-beta chain heterodimers (CD8 beta+) or as alpha chain homodimers (CD8 beta-). All CD8 beta+ IEL bear alpha/beta T cell receptors (TCR); CD8 beta- IEL bear either alpha/beta or gamma/delta TCR and are considered to be a thymus-independent (TI) population, probably arising locally from a small fraction of CD3- IEL containing the recombinant activating gene RAG proteins. Here we report that TI CD8 beta- IEL, whether bearing alpha/beta or gamma/delta TCR, contain, in normal mice, mRNAs for both zeta and Fc epsilon RI gamma chains. These chains are present in their CD3-TCR complexes as homo- or heterodimers. In contrast, only zeta chain mRNA and homodimers are found in gut CD8 alpha/beta+ IEL and in peripheral T lymphocytes. Intestinal CD3- precursor cells contain only gamma chain, and CD3- IL-2R+ thymocyte precursors only zeta chain mRNAs. Only very primitive thymocyte precursors contain detectable gamma chain mRNA, and it thus appears that Fc epsilon RI gamma chain use is switched off at a very early stage during thymocyte differentiation. Thus, T cell differentiation in the gut epithelium differs from that occurring in the thymus, from which CD8 beta+ IEL appear to derive. Use of different TCR transducing modules and CD8 accessory molecules between the TI and the thymus-derived T cell populations provides an explanation for their difference in reactivity to antigenic stimulations and thus in selection of repertoires.

Identical T cell clones are located within the mouse gut epithelium and lamina propia and circulate in the thoracic duct lymph.

Murine gut intraepithelial (IEL) T cell receptor (TCR)-alpha/beta lymphocytes bearing CD8alpha/13 or CD8alpha/alpha coreceptors have been shown previously to express different oligoclonal TCR beta chain repertoires in the same mouse, in agreement with other evidence indicating that these two populations belong to different ontogenic lineages, with only CD8alpha/beta+ IELs being fully thymus dependent. CD8alpha/beta+, but not CD8alpha/alpha+, T lymphocytes are also present in the lamina propria. Here, we show that CD8alpha/beta+ lymphocytes from the lamina propria and the epithelium are both oligoclonal, and that they share the same TCR-beta clonotypes in the same mouse, as is also the case for CD4alpha T cells. Furthermore, identical T cell clones were detected among CD8alpha/beta IELs and CD8alpha/beta+ blasts circulating into the thoracic duct (TD) lymph of the same mouse, whereas TD small lymphocytes are polyclonal. These findings must be considered in light of previous observations showing that T blasts, but not small T lymphocytes, circulating in the TD lymph have the capacity of homing into the gut epithelium and lamina propria. These combined observations have interesting implications for our understanding of the recirculation of gut thymus-dependent lymphocytes and their precursors, and of the events leading up to the selection of their restricted TCR repertoire.

Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease.

Severe combined immunodeficiency-X1 (SCID-X1) is an X-linked inherited disorder characterized by an early block in T and natural killer (NK) lymphocyte differentiation. This block is caused by mutations of the gene encoding the gammac cytokine receptor subunit of interleukin-2, -4, -7, -9, and -15 receptors, which participates in the delivery of growth, survival, and differentiation signals to early lymphoid progenitors. After preclinical studies, a gene therapy trial for SCID-X1 was initiated, based on the use of complementary DNA containing a defective gammac Moloney retrovirus-derived vector and ex vivo infection of CD34+ cells. After a 10-month follow-up period, gammac transgene-expressing T and NK cells were detected in two patients. T, B, and NK cell counts and function, including antigen-specific responses, were comparable to those of age-matched controls. Thus, gene therapy was able to provide full correction of disease phenotype and, hence, clinical benefit.

Highly restricted human T cell repertoire in peripheral blood and tissue-infiltrating lymphocytes in Omenn's syndrome.

Omenn's syndrome is an inherited human combined immunodeficiency condition characterized by the presence of a large population of activated and tissue-infiltrating T cells. Analysis of the TCRB repertoire revealed a highly restricted TCRBV usage in three patients. More strikingly, T cell clones from the three patients expressed TCRB chains with VDJ junction similarities, suggesting a common antigenic specificity. Analysis of the TCRA repertoire in one patient also revealed a restricted TCRAV usage. Finally, analysis of the TCRBV repertoire of tissue-infiltrating T cells in one patient suggested nonrandom tissue migration. These results suggest that the oligoclonal expansion of T cells observed in Omenn's syndrome could be the consequence of autoimmune proliferation generated by a profound defect in lymphocyte development.

Plasma catecholamines after insulin hypoglycemia in Sheehan's syndrome.

The plasma catecholamine response to hypoglycemia was studied in a group of hypopituitary patients with Sheehan's syndrome before (group A) and after (group B) combined cortisol and thyroid hormone treatment as well as in a group of normal women (group C). The mean basal plasma norepinephrine (NE) level was significantly increased in group A compared to levels in groups B and C, in which values were similar. The mean basal plasma epinephrine (E) level was not significantly altered by hypopituitarism. The plasma NE response to hypoglycemia was similar in the three groups, while the plasma E response was blunted in groups A and B. However, the plasma E response was significantly decreased only in half of the patients. The basal E/NE ratio was similar in the three groups, but it was significantly decreased in groups A and B compared to that in group C at the peak. From these data we conclude that 1) hypopituitarism is characterized in the basal state by increased adrenergic tone, probably related to secondary hypothyroidism; and 2) during hypoglycemia adrenal stimulation is impaired only in some patients. The role of ACTH in the regulation of E secretion is minor. Impaired neurogenic regulation in some patients with Sheehan's syndrome could contribute to their illness.

Oral load of tyrosine or L-dopa and plasma levels of free and sulfoconjugated catecholamines in healthy men.

The levels of free and sulfoconjugated catecholamines were measured in the plasma of fasting, recumbent normal subjects before and after an oral load of the catecholamine precursors tyrosine or L-dopa. Basal values of sulfoconjugated catecholamines, measured in plasma samples diluted 1:100 were 7998 +/- 540 pg/ml for dopamine sulfate, 2938 +/- 281 pg/ml for norepinephrine sulfate, and 2958 +/- 288 pg/ml for epinephrine sulfate (n = 37 tests in 15 men); these basal values are higher than those reported previously. Neither free nor sulfoconjugated catecholamine concentrations were changed by a tyrosine load (100 mg/kg) that induced a doubling of the plasma tyrosine level or by a meal low in phenylalanine and tyrosine (but otherwise supplying constituents of normal nourishment) that induced a greater than 50% reduction in the plasma tyrosine concentration. After an oral load of L-dopa (125 mg) the following were observed. (1) An extremely large increase (greater than 100-fold) in dopamine sulfate levels was noted, an increase that was less marked in the same subjects given L-dopa (125 mg) plus the peripheral dopa-decarboxylase inhibitor carbidopa (12.5 mg); as expected, free dopamine concentration also was increased. (2) Neither free nor sulfoconjugated norepinephrine concentrations were altered. (3) Epinephrine sulfate but not free epinephrine concentration was increased (more than ten-fold) after L-dopa ingestion alone; this result was unexpected and has to be confirmed before considering its physiological meaning, if any.

Free and conjugated catecholamines in digestive tissues of rats.

Using a radioenzymatic technique, the highest concentrations of free catecholamines were found in the duodenum, and the lowest in the liver of untreated rats. When compared to the antrum, the concentration of free dopamine was higher, and that of norepinephrine lower in the fundus. As far as conjugated catecholamines are concerned, the tissue concentrations of both sulfo- and glucurono-conjugates were usually low, and often non detectable, with an exception: the concentration of glucurono-conjugated dopamine was very high in the duodenum, ileum, and liver of untreated rats.

Portal and arterial free and conjugated noradrenaline in two models of portal hypertension in rats.

Free and conjugated noradrenaline concentrations were measured in portal-venous and arterial plasma from sham-operated rats or rats with portal hypertension. Two types of portal hypertension in rats were evaluated: in portal vein stenosed rats, the liver was normal, whereas cirrhosis developed in bile duct ligated rats. In cirrhotic rats, arterial free noradrenaline level was higher than in both sham-operated and portal-stenosed rats, this indicating that enhanced sympathetic nervous activity depends on the development of cirrhosis. In all groups of rats, portal venous plasma free noradrenaline was higher than arterial plasma level, indicating a production of noradrenaline by splanchnic organs. Arterial noradrenaline level may be mainly dependent on this splanchnic production in case of portal hypertension. Sulfoconjugated and glucuronoconjugated noradrenaline plasma levels were similar in the three groups of rats. This shows that alteration in conjugation is not likely to be a major factor in the abnormal circulating levels of free noradrenaline observed in cirrhotic rats.

[Plasma catecholamines, free and conjugated, in the hemodialyzed chronic renal failure patient]. / Catécholamines plasmatiques, libres et conjuguées, chez l'insuffisant rénal chronique hémodialysé.

Free, sulfo and glucuro-conjugated catecholamines (dopamine, noradrenaline and adrenaline) were measured to study their metabolism in 35 non-selected patients with chronic renal failure, and under hemodialysis for various periods of time. Our data demonstrate a statistically significant increase of free dopamine, and free noradrenaline concentration in these patients, while that of free adrenaline was not different from controls. However a careful scrutiny of 35 individual data suggests that sub-groups of patients with either high normal or low plasma free noradrenaline concentration could exist; this likely heterogeneity could be a good explanation for conflicting conclusions provided by previous reports. Suspecting that conjugated catecholamines might be altered in these patients, plasma sulfo and glucuro-conjugated amines were measured. We have found a predictable and highly significant increase of sulfo-conjugated catecholamines; glucuroconjugated dopamine and noradrenaline were unchanged, while glucuroconjugated adrenaline was significantly increased. The physiological meaning, if any, of these new observations on conjugated catecholamines cannot be assessed at the moment.

[Urinary excretion of catecholamines in the dog: physiopathologic hypotheses in man]. / Excrétion urinaire des catécholamines chez le chien: hypothèses physiopathologiques chez l'homme.

Renal handling of free catecholamines (dopamine, norepinephrine and epinephrine) was studied in 36 hydropenic anesthetized mongrel dogs in accordance with the clearance technique. There was no statistically significant correlation between plasma concentrations and either systemic (mean blood pressure, or cardiac output) or renal (clearance of PAH or glomerular filtration rate) hemodynamics. Net tubular transport (NTT) was calculated as the difference between filtered load and urinary excretion for any catecholamine. The mean NTTs of free catecholamines were as follows: --2,72 ng/min for dopamine, --3,18 for norepinephrine, and --1,36 for epinephrine, showing that they are mostly reabsorbed. However the use of averages is misleading inasmuch as tubular transport of free catecholamines is a heterogeneous phenomenon: a secretion appears to predominate when plasma concentrations are low and a reabsorption predominates when they are high. Whether such a heterogeneity is due to either a genetic heterogeneity in mongrel dogs, or an age-related difference is discussed.

Comparative analysis of CD4-mediated down-regulation of T cell adhesion to B cells by flow cytometry and fluorescence microscopy.

We have previously shown by means of fluorescence microscopy that antigen-independent adhesion of resting CD4 T cells to EBV-transformed B cells can be down-regulated by ligand interaction with CD4. In this study we used flow cytometry analysis of conjugate formation to confirm these findings. No conjugates between resting CD4 + T cells and B cells were initially detected in the latter method, because flow velocity in the flow chamber induces hydrodynamic elongation forces which disrupt low-affinity conjugates. After forcing cell conjugation by low-speed centrifugation of T and B cells, conjugates became detectable although in smaller numbers than in fluorescence microscopy. "Forced" cell conjugates had similar characteristics to their unforced counterparts, i.e., 37 degrees C temperature dependency, mediation by LFA-1/ICAM-1 and CD2/LFA-3 pathways, and transiency. The latter characteristic was at least partly mediated by CD4/HLA class II interaction, since adhesion of CD4 + T cells to HLA class II-B cells was more stable. In addition, adhesion was inhibited by anti-CD4 antibodies but not by an HLA DR-derived peptide known to inhibit unforced CD4 + T cell adhesion to B cells. This blocking effect was partially reproduced by reducing the centrifugation time prior to the adhesion assay. These results show that a) CD4-mediated down-regulation of T cell adhesion can be observed by means of two different techniques, and b) analysis of cell-cell adhesion after increasing centrifugation times (and possibly speeds) is a simple way of measuring adhesion forces semiquantitatively.

The activation of protein kinase C is not necessary for the monoclonal antibody-induced modulation of CD3 and CD4 antigens.

We studied the effect of staurosporine, a potent inhibitor of protein kinase C (PKC) activity, on the phorbol ester- or monoclonal antibody (mAb)-induced modulation of CD3 and CD4 surface antigens. Staurosporine (10(-5) M) completely inhibited phorbol ester-induced modulation but had no effect on that induced by mAb. These results indicate that the down-regulation of CD3 and CD4 observed after activation of the cells by the corresponding mAb is independent from PKC-mediated phosphorylations, and thus that the activation of PKC is sufficient but not necessary to induce the modulation of CD3 and CD4 antigens.

Characteristics of antigen-independent and antigen-dependent interaction of dendritic cells with CD4+ T cells.

Dendritic cells (DC) are the main antigen-presenting cells for the initiation of primary T cell-mediated immune responses. In the first stage of activation, T cells bind to DC in an antigen-independent manner. We studied the adhesion characteristics of human CD4+ T cells to DC generated from CD34+ hematopoietic progenitors following 12 to 13 days of culture in the presence of granulocyte/macrophage colony-stimulating factor and tumor necrosis factor-alpha. A majority of these cells had the morphology, phenotype and functions of DC. CD4+ T/DC adhesion was measured by means of fluorescence microscopy and flow cytometry. Four independent receptor/ligand pathways, LFA-1/ICAM, ICAM/LFA-1, CD2/LFA-3 and CD28/CD80, were involved in the transient adhesion of DC to CD4+ T cells in antigen-independent and specific alloantigen-dependent situations, as shown by blocking experiments using monoclonal antibodies. The antibodies also blocked a primary mixed lymphocyte reaction (MLR) in which DC were used as stimulatory cells. Adhesion of alloreactive CD4+ T cells to antigen-presenting DC was stronger than that of resting CD4+ T cells, while peak adhesion occurred after 5 and 20 min, respectively. The LFA-1 ligands involved in adhesion of resting CD4 T cells to DC and alloreactive CD4+ T cells to specific DC differed in part, since ICAM-3 on resting T cells and ICAM-1 on alloreactive T lymphocytes preferentially bound LFA-1. Studies of interactions between DC and phorbol ester-activated T cells expressing the CD40 ligand revealed a fifth independent adhesion pathway, CD40/CD40 ligand. CD4-mediated regulation of CD4+ T/DC adhesion was suggested by the observation that preincubation of CD4+ T cells and DC individually with anti-CD4 antibodies inhibited adhesion. In addition, antibodies specific for HLA class II molecules inhibited adhesion when used to pretreat DC but not alloactivated CD4+ T cells.

Is dopamine a physiological natriuretic hormone in the dog?

Both plasma and urinary dopamine and noradrenaline were measured as free and sulphate conjugates, by a radioenzymatic method, before and during extracellular volume expansion (ECVE) with hypo-, iso- or hyper-tonic fluid (usually sodium chloride solution) in dogs. During ECVE there was a decrease in plasma catecholamine concentration. For all cases except noradrenaline, this is probably due to a dilution phenomenon since when results were expressed as pg/mg of protein, ECVE had no effect. This change in noradrenaline accounted for the increase in the dopamine/noradrenaline ratio. As expected, there was an increase in the urinary excretion of dopamine during ECVE with both iso- and hyper-tonic fluid. This increase was not observed in the group of dogs given hypotonic fluid, although the increase of fractional excretion of sodium was of a similar order of magnitude. The increase in the urinary excretion of dopamine was apparently not affected by an increase in plasma sodium concentration and/or osmolality. The demonstrated dissociation between sodium and dopamine in urine does not support a physiological role for dopamine in renal handling of sodium during ECVE, and raises the question of its specificity.

Opposing effects of protein tyrosine kinase inhibitors on the monoclonal antibody induced internalization of CD3 and CD4 antigens.

We have previously demonstrated that the monoclonal antibody (mAb)-induced modulation of CD3 and CD4 antigens from the surface of human peripheral blood lymphocytes is not dependent from protein kinase C activity (Thuillier et al., Eur. J. Immunol. 1990. 20:1197). In the present report we study the effect of genistein and of herbimycin A, two potent inhibitors of protein tyrosine kinases (PTK), on the mAb-induced modulation of CD3 and CD4 surface antigens. Both genistein and herbimycin inhibited the mAb-induced internalization of CD3 and, in contrast, facilitated that of CD4 antigen. These results indicate that the mAb-induced modulation of CD3 is essentially dependent on the PTK pathway, whereas PTK appear to negatively regulate the mAb-induced modulation of CD4.

Germ-line transcription and methylation status of the TCR-J alpha locus in its accessible configuration.

We have generated two in vivo mouse models to study the regulation of DNA accessibility to the V(D)J recombinase machinery in the T cell receptor (TCR)-J alpha locus. In recombination activating gene (RAG)-deficient mice, both injection of a TCR-beta chain transgene (RTB mice) or anti-CD3-epsilon treatment in vivo (RT3 mice) lead to the same phenotype with homogeneous thymocyte populations blocked at the CD4+ CD8+ double positive (DP) stage. At this developmental stage, the TCR-alpha rearrangements are about to start, and the TCR-J alpha locus is frozen in an accessible but yet unrearranged configuration in these mice. We show high level of TCR-alpha germ-line transcription in thymocytes from RTB and RT3 mice. Transcripts are skewed towards the 5' end of the TCR-J alpha locus, and the T early alpha (TEA) sterile transcript is predominant and therefore provides a useful marker for the TCR-J alpha locus opening. Analysis of the DNA methylation status reveals a global surmethylation of the TCR-J alpha locus in the thymus in comparison with non-lymphoid cells in these mice. We propose that hypermethylation of the locus could precede a progressive demethylation, providing a specific protective and regulatory role in the rearrangement events.

Subsets of CD3+ (T cell receptor alpha/beta or gamma/delta) and CD3- lymphocytes isolated from normal human gut epithelium display phenotypical features different from their counterparts in peripheral blood.

Intestinal intraepithelial lymphocytes (IEL) were studied, after isolation in humans, for their surface antigens with a large variety of monoclonal antibodies. They show peculiar characteristics when compared with peripheral blood lymphocytes and intestinal lamina propria lymphocytes. Although a majority of human intraepithelial lymphocytes (IEL) express an alpha/beta type of T cell receptor (TcR), 13% express a gamma/delta TcR, a percentage which was significantly higher than that found in blood and in lamina propria. In contrast to observations in mice, there was no evidence that normal human TcR gamma/delta+ intestinal IEL might use preferential variable segments of gamma genes. About 10% of human intestinal IEL expressed the alpha chain but not the beta chain of CD8, thus resembling a subset of CD8 alpha+beta- IEL, which was recently described in mice and found to be of thymoindependent origin. In addition, 10% of human IEL had a unique phenotype of immature T cells, as they bore only CD7, but no other T cell or natural killer cell markers. Finally, even the major population of IEL which expressed the usual markers of the T cell lineage (CD3, TcR alpha/beta, CD2, CD4 or CD8 alpha/beta) differed from peripheral blood T lymphocytes by their peculiar expression of surface antigens associated with activation. Indeed, 80% of IEL were CD45R0+, CD45A-, but co-expression of CD11a, CD29 and LFA-3 was inconstant. In addition, 90% of IEL expressed HML-1.

Free and conjugated catecholamines in patients with cirrhosis.

A defect of conjugation may play a role in the elevated plasma free norepinephrine observed in patients with cirrhosis. Plasma free, sulfoconjugated, and glucuronoconjugated catecholamine concentrations were assessed in 15 patients with cirrhosis and in 15 age-matched control subjects. Plasma free norepinephrine and epinephrine levels were significantly higher in patients with cirrhosis (481 +/- 75 and 96 +/- 16 pg/ml, respectively) than in those of the control group (307 +/- 33 and 42 +/- 10 pg/ml, p less than 0.05 and p less than 0.01, respectively). Plasma free dopamine levels were similar in both groups. Sulfoconjugated catecholamines were the predominant form in plasma from both cirrhotic patients and control subjects. The ratio of conjugated to total catecholamines was similar in the two groups. Therefore, it is unlikely that a defect in conjugation of catecholamines is contributing to the excessive plasma free norepinephrine and epinephrine concentrations found in patients with cirrhosis. Moreover, in patients with cirrhosis, no significant relation was found between plasma conjugated catecholamines and the severity of liver disease. This study shows that cirrhosis does not induce alteration in conjugation of catecholamines and that hepatocellular function is not essential for conjugation of circulating catecholamines.

Complexity of the mouse gut T cell immune system: identification of two distinct natural killer T cell intraepithelial lineages.

Gut thymo-dependent (CD8 alpha + beta + or CD4+) or -independent (CD8 alpha + beta -) intraepithelial lymphocytes (IEL) mediate cytotoxicity following T cell receptor (TCR)-CD3 signaling, but only TCR gamma delta + and alpha beta + thymo-independent IEL show cytotoxicity of natural killer (NK) and antibody-dependent cell-mediated cytotoxicity types. Moreover, TCR alpha beta + and gamma delta + thymo-independent IEL express NK receptors, and may therefore be referred to as NK-TIEL. NK-TIEL cytotoxicity is mediated through perforin, Fas, or both pathways. In contrast to that of other NK cells, this cytotoxicity is not negatively regulated by signals delivered through the recognition of major histocompatibility complex class I molecules. Thus, gut IEL include T cell subsets with unique specificities and functions, ontogenically distinct from other T cell lineages, which may increase the antigenic repertoire diversity of the immune system participating in the defense of the epithelial barrier.