Reference values for T, B and NK human lymphocyte subpopulations in adults.

The data presented in this paper are reference ranges for frequencies of thirty-eight subpopulations of T, B and NK lymphocytes, established from a cohort of 253 healthy blood donors aged from 19 to 67. When relevant, the influence of age or sex was taken into account to calculate these reference values. This article is related to the research article entitled "Influence of age, sex and HCMV-serostatus on blood lymphocyte subpopulations in healthy adults" (Apoil et al., 2017) [1]. Immunophenotyping data obtained from each individual is made publicly available for extended analyses.

Influence of age, sex and HCMV-serostatus on blood lymphocyte subpopulations in healthy adults.

Using a standardized immunophenotyping procedure we studied thirty-eight distinct subpopulations of T, B and NK lymphocytes in 253 healthy blood donors aged from 19 to 67. We analysed the influence of age, sex and HCMV seropositivity on each lymphocyte subpopulations and established reference ranges. We observed that aging influences the largest number of lymphocyte subpopulations with a slow increase of CD8+ EMRA T lymphocytes and of the numbers of circulating Tregs. The proportion of HLA-DR+ cells among Tregs increased with age and was correlated to the proportion of HLA-DR+ cells among effector T CD4+ lymphocytes. Sex had a major impact on absolute counts of CD4+ T cells which were higher in females. HCMV-seropositivity was associated with higher frequencies of CD8+ EMRA memory T lymphocytes while a high frequency of terminally differentiated EMRA CD4+ T cells was observed in 80% of HCMV-positive individuals and in none of the HCMV seronegative individuals.

Methodological aspects of minimal residual disease assessment by flow cytometry in acute lymphoblastic leukemia: A French multicenter study.

Background: Minimal residual disease (MRD) assessment provides a powerful prognostic factor for therapeutic stratification in acute lymphoblastic leukemia (ALL). Multiparameter flow cytometry (MFC) has the potential for a rapid and sensitive identification of high risk patients. Our group has previously published that MRD levels analyzed by clone specific Ig/TcR-QPCR and MFC were concordant at a sensitivity of 10-4 . Here we report the MFC methodological aspects from this multi-center experience. Methods: MRD was assessed by MFC in 1030 follow-up samples from 265 pediatric and adult patients with de novo ALL treated in the FRALLE, EORTC or GRALL clinical trials. MRD assessment as applied by the eight participating MFC laboratories is described in detail regarding cell preparation, leukemia-associated immunophenotype (LAIP) markers and data analysis. Samples were obtained from bone marrow (BM) and peripheral blood (PB). Immunostaining was performed after erythrocyte lysis or Ficoll enrichment. Results: This study confirms the applicability of MFC-based MRD assessment in 97% of patients with ALL at the 10-4 cut-off. MRD values after Ficoll enrichment and erythrocyte lysis were found comparable. Higher MRD values were obtained in BM than in PB, especially for B-lineage ALL. Conclusions: Measurement of MRD by MFC at the 10-4 cut-off is applicable within a few hours for almost all patients and using a comparable analytical strategy allows for multicenter collaborative studies. The method can be introduced in a strategy aimed at defining the risk of failure of patients with childhood or adult ALL. © 2014 Clinical Cytometry Society.

Flow cytometry and IG/TCR quantitative PCR for minimal residual disease quantitation in acute lymphoblastic leukemia: a French multicenter prospective study on behalf of the FRALLE, EORTC and GRAALL.

Minimal residual disease (MRD) quantification is widely used for therapeutic stratification in pediatric acute lymphoblastic leukemia (ALL). A robust, reproducible, sensitivity of at least 0.01% has been achieved for IG/TCR clonal rearrangements using allele-specific quantitative PCR (IG/TCR-QPCR) within the EuroMRD consortium. Whether multiparameter flow cytometry (MFC) can reach such inter-center performance in ALL MRD monitoring remains unclear. In a multicenter study, MRD was measured prospectively on 598 follow-up bone marrow samples from 102 high-risk children and 136 adult ALL patients, using IG/TCR-QPCR and 4/5 color MFC. At diagnosis, all 238 patients (100%) had at least one suitable MRD marker with 0.01% sensitivity, including 205/238 samples (86%) by using IG/TCR-QPCR and 223/238 samples (94%) by using MFC. QPCR and MFC were evaluable in 495/598 (83%) samples. Qualitative results (<0.01% or ≥0.01%) concurred in 96% of samples and overall positivity (including <0.01% and nonquantifiable positivity) was concurrent in 84%. MRD values ≥0.01% correlated highly (r(2)=0.87) and 69% clustered within half-a-log(10). QPCR and MFC can therefore be comparable if properly standardized, and are highly complementary. MFC strategies will benefit from a concerted approach, as does molecular MRD monitoring, and will contribute significantly to the achievement of 100% MRD informativity in adult and pediatric ALL.

Long-term evolution of lymphocytes subsets after induction therapy based on continuous versus discontinuous administration of anti-thymocyte globulins in renal-transplant patients.

UNLABELLED: The aim of our retrospective study was to assess the long-term evolution of lymphocyte subsets after two modes of administration of anti-thymocyte globulin (ATG) after renal transplantation. METHODS: Before 1993, patients (group I, n = 93) received fixed doses of RATG (1 mg/kg per day) for 8 consecutive days. Thereafter, RATG was either continued at the same dose for 15 days, in cases of delayed graft function, or was infused every other day at the same dose until the serum creatinine level became <150 micromol/L. After 1993, patients (group II, n = 66) received RATG at full dose (1 mg/kg per day) during the first 3 days and, thereafter, doses were adapted to target a CD2 T-cell count <50/mm3. RATG cumulative dose was significantly higher among group I than group II (9.7 +/- 4.5 versus 7.4 +/- 3.2 mg/kg, P = .0002). RESULTS: In both groups, total lymphocyte and T lymphocyte subset (CD4, CD8, CD2, CD3) counts decreased significantly during the first month after transplantation, increasing slowly between the first month and the third year posttransplantation. Thereafter it rose rapidly, which was greater in group II. At last follow up, total lymphocyte, T lymphocyte subsets and NK cell counts were similar to those observed before transplantation. At all monitoring times, T lymphocyte, B lymphocyte, and NK cell counts were similar in both group, except for the total lymphocyte count at 6 months and CD4 T lymphocyte count at 1 year, which were significantly higher in group II compared to group I. CONCLUSION: Induction therapy based on continuous or discontinuous administration of ATG is associated with profound depletion of T, B, and NK cells during the first 3 years, followed by a progressive reconstitution of the lymphocyte pool after 5 years.

Carcinocythemia as the single extension of breast cancer: report of a case and review of the literature.

Carcinocythemia (carcinoma cell leukemia) has been previously described as a rare, late and dramatic event occurring in widespread tumoral disease. We report a case of carcinocythemia occurring in a patient with a particularly indolent breast cancer. When a large amount of circulating tumor cells (CTC) appeared in the blood smears, neither visceral macrometastases nor massive bone marrow infiltration could be detected. We isolated CTC to perform cell cycle analysis and to study the expression of adhesion molecules. A fraction of the CTC was proliferating in the bloodstream but we detected no relevant anomaly of adhesion properties in the CTC. A post-mortem biopsy disclosed an exclusive sinusoidal infiltration of the liver. We propose that in this case, carcinocythemia resulted from the release of CTC from the visceral microcirculation where the proliferation occurred.

Inflammatory/haematopoietic cytokine production by human bone marrow adipocytes.

Adipocytes are now considered as secretory and endocrine cells. White and brown adipocytes synthesize and secrete a variety of cytokines, among a number of peptide and non-peptide products. Some of these cytokines, particularly IL-6 and TNF-alpha, appear multifunctional since they may be involved in the control of adipose mass, inflammatory response and haematopoiesis. Bone marrow adipocytes are another abundant type of adipocytes, but their precise role in humans is poorly understood. In the present study, we demonstrate that, in contrast to non-medullary adipocytes, human bone marrow adipocytes in primary culture secrete only trace amounts of IL-1 beta and TNF-alpha, but, they secrete significant and regulated levels of IL-6. These results reinforce the concept of functional heterogeneity of adipose tissues according to their anatomical localization, and indicate that bone marrow adipocytes may contribute to the complex network of cytokines involved in the control of haematopoiesis.

[Management of intractable cancer pain: from intrathecal morphine to cell allograft]. / Traitement des douleurs chroniques rebelles d'origine cancéreuse: de la morphinothérapie intrathécale à la thérapie cellulaire.

The durable effectiveness of intrathecal morphine administration is well established for the management of intractable cancer pain, after failure of systemic opioids, secondary to the persistence of non-reversible undesirable side effects. Many patients are referred to late in the disease course. This conservative method to control pain of malignant origin must not be reserved for last resort treatment for terminal patients. Intra-cerebro-ventricular morphine administration is a very effective and generally safe method for controlling intractable cancer pain. Because of the chronic implantation of an intra-ventricular catheter this method is somewhat invasive. Its indications remain a simple and effective alternative when the topography of nociceptive pain is diffuse or cephalic. In clinical practice, intrathecal and/or intra-cerebro-ventricular administration of opioids is limited by cost, the need for specialized maintenance and mechanical malfunctions if implantable drug delivery systems, or by the risk of bacterial contamination and ambulatory constraints when repeated daily injections via an intrathecal access port are used. To answer these limitations, cell therapy using intrathecal chromaffin cell allograft is a promising approach for the management of cancer pain refractory to traditional drug therapy and pain lesion surgery. The basic rationale and preclinical studies on experimental pain models have enabled starting prospective clinical trials. Prior to transplantation, handling and preparation of the chromaffin tissue is critical for allograft viability. The initial results of clinical trials with human chromaffin cell grafts from intractable cancer pain have reported long-lasting pain relief, in correlation with met-enkephalin release into the CSF. Convincing evidence will require controlled studies. The limitations of this innovative cell therapy and especially the lack of human adrenal gland availability point to the need for new sources of cells. Perspectives include xenogenic or engineered cell lines.

Relationship between the fatty acid composition of rat lymphocytes and immune functions.

The effects of dietary lipids on the fatty acid composition, activation and proliferation of lymphocytes were investigated. Weanling male Wistar rats were fed for 8 weeks on one of two low-fat diets which contained 50 g lipid/kg, or one of two high-fat diets containing 200 g lipid/kg, from either coconut oil or soyabean oil. The fatty acid composition of phospholipids from splenocyte membranes was affected by dietary lipid manipulation, and these differences influenced lymphocyte functions. Increased levels of linoleic acid in spleen lymphocytes correlated negatively with interleukin-2 receptor alpha-chain expression determined either by measuring the mean fluorescence or by the proportion of cells staining positive for CD25, and with the cell proliferation index. However, we found a positive correlation between interleukin-2 receptor alpha-chain expression determined by measuring the mean fluorescence and the cell proliferation index with the oleic acid concentration of spleen lymphocytes. Since phospholipid hydrolysis occurs early in lymphocyte activation, immunosuppressive effects induced by polyunsaturated fatty acids, described in the literature, could be due to an increase of linoleic acid or a decrease of oleic acid affecting many components of plasma-membrane-associated events involved in lymphocyte activation.

Adrenal medullary explants as an efficient tool for pain control: adhesive biomolecular components are involved in graft function ex vivo.

Adrenal medullary (AM) tissue transplantation into the central nervous system has been reported as a potential source of opioid peptides and catecholamines, which have analgesic effects useful in the control of chronic pain. Clinical trials, involving allogeneic graft of whole tissue explants into the subarachnoid space of the lumbar spinal cord, have already been reported. The aim of the present study was to determine whether adhesion and function of AM explants were related in some extent and how this relationship could account for improvement of AM tissue in terms of analgesic activity before grafting. Our experiments demonstrated a significant correlation between the adherent state of AM organoids during culture and a sustained secretion of Met-enkephalin and catecholamines by chromaffin cells (CC). These findings suggest that optimal culture condition for AM organoid adhesion can be defined for maintenance of tissue, prior to transplantation. Using immunocytochemistry, flow cytometry, and ELISA assays we showed that different cell adhesion molecules (CAMs) and extracellular matrix ECM proteins were expressed and released by AM cells during culture. Adherent AM organoids expressed increased levels of specific neural CAMs (NCAM and HNK-1 epitope) and integrin chains (beta1, alpha1, alpha2, alpha4, alpha5) and deposited markedly higher levels of fibronectin, but also laminin and collagen IV. Those molecules and probably adhesion processes they control might be involved in the maintenance of the CC-secreting neuroendocrine phenotype through cellular signaling pathways.

[Clinical perspectives of xenografts: encapsulated chromaffin cells and pain]. / Perspectives cliniques des x¿enogreffes: cellules chromaffines encapsul¿ees et douleur.

Intrathecal allograft of chromaffin cells can be effectively used in replacement of more conventional therapies for treating intractable chronic pain, such as in cancer. The efficacy of this technique depends on the ability of those cells to produce analgesic opioids and on the immuno-privileged property of the central nervous system, in which rejection risks are limited. However, there are some limitations to the generalization of this new therapy, mainly due to the low number of available grafts. Thus other sources than humans have to be considered. Here we discuss the pros and cons of the xenogeneic chromaffin cells of bovine or porcine origin. Graft immuno-isolation, for example, by using cell encapsulation, seems to be unavoidable in spite of the graft site.

Intrathecal grafting of unencapsulated adrenal medullary tissue can bring CD4 T lymphocytes into CSF: a potentially deleterious event for the graft.

Adrenal medullary tissue including chromaffin cells was grafted intrathecally in cancer patients to relieve intractable pain. The central nervous system (CNS) is considered an immune privileged site. Therefore, non-HLA-matched and unencapsulated tissue was grafted in 15 patients and 1 sham control in a series of at least 20 grafts. We observed an increase in CSF lymphocyte counts in 15/20 allografts (75%). In contrast to peripheral blood, CD4 T cells predominated in the CSF, but failed to exhibit an activated phenotype (CD25+ CD45RO+ HLA-DR+). The positive effect of graft on pain, the high met-enkephalin levels, the absence of any increase in CSF cytokine levels particularly for IFN-gamma or IL-2 (but not IL-10 and IL-6), indirectly indicated that the graft was tolerated despite the presence of CSF lymphocytes. The single treatment failure and three of four cases of partial efficacy occurred in grafts where CSF lymphocytes were present. Moreover, when assayed (n = 7), the CD4+ CSF lymphocytes still retained the capacity to exhibit ex vivo a normal or enhanced frequency of T CD4 cells producing IFN-gamma and IL-2. Taken together, our observations indicate that impairment of the local immunosuppressive balance can lead to activation of those CSF CD4 T cells and drive a rejection process. This study suggests further work on the purification and/or the immunoisolation of tissues grafted in the CNS will be necessary, particularly when the possibility of long-term and repeated grafting is considered.

In vivo effects of olive oil-based lipid emulsion on lymphocyte activation in rats.

Numerous studies suggest that immune function may be compromised by lipid emulsions rich in polyunsaturated fatty acids, especially linoleic acid. In our study, we compared the effect of a new olive oil-based lipid emulsion (ClinOleic(R)) containing 18% linoleic acid, and an emulsion based on soybean oil (Ivelip(R); 52% linoleic acid) on lymphocyte functions. Weaning Wistar rats (n= 24) were fed for 4 weeks on an oral diet that contained 12% of total energy as lipids from soybean oil. Then they received, during 6 days, a total parenteral nutrition (260 kcal/kg/d) in which 12% of total energy was brought by one of the two lipid emulsions. The fatty acid profile of spleen lymphocyte phospholipids reflected lipid intakes, with a higher content of oleic acid in ClinOleic(R) group and linoleic acid in Ivelip(R) group. A greater proportion of cells expressed the interleukin-2 receptor a-chain (CD25) after administration of ClinOleic(R) when compared to Ivelip(R) (55.43 +/- 3.47 vs 45.48 +/- 3.26%, P<< 0.05). Moreover, the CD25 expression was positively correlated with oleic acid content of spleen lymphocyte phospholipids (r= 0.500, P<< 0.018). These results show that ClinOleic(R) is able to induce, in vivo, a greater proportion of cells expressing CD25, and suggest that oleic acid could have a role in the observed effects.

Ex vivo flow cytometry determination of intracytoplasmic expression of IL-2, IL-6, IFN-gamma, and TNF-alpha in monocytes and T lymphocytes, in chronic hemodialysis patients.

AIMS: To determine the intracytoplasmic expression of TNF-alpha, IL-2, IL-6 and IFN-gamma, ex vivo and in vitro, in both monocytes and T lymphocytes by flow cytometry after appropriate stimulation using phorbol myristate acetate (PMA)/ionomycin or lipopolysaccharides (LPS) in the presence of monensin, in order to assess the bio(in)compatibility of different dialysis membranes. METHODS: We examined monocytes and T lymphocytes taken from chronic hemodialysis patients (using either cuprophane (CUP), n = 6; polyacrylonitrile (AN 69), n = 6; or polysulfone (PS), n = 6 membranes), before and after a dialysis session. We compared the results with those obtained from end-stage chronic renal failure patients (n = 3) and healthy volunteers (n = 11). RESULTS: Before any stimulation there was a statistically significant difference in the percentages of TNF-alpha, IL-6, and IFN-gamma- expressing monocytes with respect to the dialysis membrane used. The highest percentages were observed for CUP and AN69 patients with figures of around 30% for each cytokine; the lowest percentages were found in PS patients and healthy volunteers. One hour after LPS stimulation the patterns remained unchanged for TNF-alpha and IFN-gamma, whereas the percentages of IL-6-expressing cells in PS patients and in healthy volunteers reached the figures obtained in the other groups. When we examined the percentage of IFN-gamma-, TNF-alpha- and IL-6-expressing monocytes in patients before and after a dialysis session, before any stimulation, we found that the results were significantly different for the three membranes (p = 0.01). Thus, a dialysis session with polysulfone membranes had no significant effect on the precentages of IFN-gamma-, TNF-alpha-, and IL-6-expressing monocytes, whereas percentages were significantly lower after the dialysis session when using cuprophane or AN69 membranes, suggesting a release of these cytokines by the monocytes during dialysis. A significant number of IFN-gamma- and IL-2-expressing T lymphocytes were only detected after 18 hours of PMA/ionomycin stimulation. The percentages of IFN-gamma-expressing T cells recorded for the different membranes were not statistically different from those recorded for healthy subjects or pre-dialysis patients, i.e., they were between 11.5 and 20%. However, the percentages of IL-2-expressing T lymphocytes were significantly different between the 5 groups, i.e., 31.3, 30.5, 18.6, 13.9 and 7. 6%, respectively, for CUP patients, pre-dialysis patients, healthy volunteers, PS and AN69 patients. This suggests that pre-dialysis and CUP patients have, at baseline, a stimulation of their T lymphocytes. Finally, a 4-hour dialysis session had no impact on the percentages of IL-2-expressing T lymphocytes, whereas it was associated with a significant decrease in the percentage of IFN-gamma-expressing cells, but only when cuprophane membranes were used. CONCLUSION: Cytokine flow cytometry enables one to study, ex vivo, i.e., without any stimulation of the cells, and in vitro after appropriate stimulation, the bio(in)compatibility of dialysis membranes assessed by the intracytoplasmic cytokine profiles of TNF-alpha, IFN-gamma, IL-6 and IL-2, evaluated at the single cell level.

Head-down tilt bed rest and immune responses.

Head-down tilt bed rest (HDT) is used as a model for studying the physiological changes occurring in weightlessness during spaceflight. In the present study, eight volunteers were subjected to a strict HDT of -6 degrees for 42 days. Blood samples were obtained 37 and 13 days before, at days 13, 34, and 41 during, and 12, 33, and 47 days after HDT. FACScan analysis was used to determine cell subpopulations. Plasma was used to quantify various circulating hormone levels. Whole blood and reconstituted blood were stimulated with various activators such as phytohaemagglutinin-P (PHA), PHA combined with phorbol-12-myristate 13-acetate (PMA), anti-CD2, anti-CD3, and lipopolysaccharide. Supernatants were collected and analysed for the interleukins IL-1beta, IL-2, IL-6, and IL-10, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha). The total number of T lymphocytes and monocytes did not change significantly, whereas the number of polymorphonuclear cells increased during HDT. The percentage of CD2+ and CD3+ cells was increased at day 35 of HDT. The percentage and total number of natural killer cells (CD2+/CD3-/CD56+) was increased 12 days before and 14 days after HDT. TNF-alpha secretion did not change significantly during HDT. IL-2, IL-10 and IFN-gamma were increased at day 34 of HDT. IL-1beta levels were increased before and during HDT compared to post-HDT measurements. No significant changes were observed in plasma immunoglobulin, complement factors and other factors of the inflammatory system. Prolactin levels increased slightly but significantly at day 35 of HDT, thyreotropin and growth hormone levels remained virtually unchanged. Cortisol decreased slightly but significantly over the entire duration of the study. The changes observed during HDT do not indicate that the immune system is blunted, and these changes do not seem to correlate with the duration of HDT. Taken together these results show that a HDT does not reproduce the changes in immune responses observed after spaceflight.

Predicting factors of long-term results of OKT3 therapy for steroid resistant acute rejection following cadaveric renal transplantation.

In this retrospective study, we evaluated the histological and biological predictors of long-term response of renal transplant (RT) patients treated with orthoclone OKT3 for steroid resistant acute rejection (AR). Seventy-three patients, aged 37 +/- 12 years, were included in this study between March 1987 and December 1996. All the patients but one had received sequential quadruple immunosuppression (polyclonal antilymphocyte globulins; steroids; azathioprine, and cyclosporin A). OKT3 (5 mg/day for 10 days) was administered for biopsy-proven steroid resistant AR i.e., after 3 consecutive pulses of methylprednisolone (10 mg/kg each). This was the first AR in 46 cases, the second AR in 22 cases and the third AR in 4 cases. Renal histology (Banff) showed borderline (BL) changes in 18 patients, grade I AR in 28 patients; grade II AR in 22 patients, and grade III AR in 5 patients. When treatment with OKT3 commenced (107 +/- 18 days post-transplantation) the mean serum creatinine (SCr) level was 325 +/- 195 micromol/l; this had decreased to 191 +/- 106 micromol/l by the end of OKT3 therapy. The immediate response to OKT3 therapy i. e., within the first month, was not dependent on the histological score. Twenty-six patients (35%) subsequently experienced at least one more AR episode of whom 4 were retreated with OKT3. The overall patient's survival was 94.5% at last follow-up. The overall cumulative graft survival was 64.5% at 2 years, 52.5% at 5 years, and 40.5% at 8 years. The graft survival (5 years) tended to depend on the initial histological score, i.e. BL 30%; grade I 66%; grades II and III 55.5% (p = 0.08). In a multiple logistic regression analysis we tried to identify independent factors that would predict that a graft would still be functioning at least 2 years after OKT3 therapy. We therefore analyzed the following parameters: donor and recipient's age; gender; cold ischemia time; HLA matching; panel reactive antibodies (PRA) prior to grafting; previous transplantation(s); total number of AR episodes; the time of onset of the AR treated by OKT3 compared to the other AR; the time of onset of the AR treated by OKT3; SCr levels at days 0, 10 and 30 after OKT3 therapy; histological score (Banff) i.e., the magnitude of AR and the presence or absence of chronic lesions. The only independent factors which would predict that a graft was still functioning 2 years after OKT3 therapy were: PRA <25% (Odds ratio (OR) 7.68 (1.15-51.3); p = 0.035); a grade I AR (OR 10.52 (1.18-93. 5); p = 0.035); SCr level 1 month after OKT3 therapy (OR 0.935 (0. 87-1.002); p = 0.05). HLA matching and the presence of histological chronic lesions were nearly significant (p = 0.06 and 0.09 respectively). In conclusion, this retrospective study shows that independent predictors of the long-term response to OKT3 therapy for AR in RT patients are the magnitude of pre-transplant PRA, the histological score, and the SCr level one month after OKT3 therapy.

Differences in Type 1 and Type 2 intracytoplasmic cytokines, detected by flow cytometry, according to immunosuppression (cyclosporine A vs. tacrolimus) in stable renal allograft recipients.

Recent multicenter, randomized clinical trials have shown that in renal transplant patients tacrolimus (FK506) was more efficient than cyclosporine A (CsA) at preventing acute rejection. In order to try and evaluate whether this difference was related to a different in vivo T-cell suppression we assessed, in a prospective study, the frequencies of interleukin (IL)-2-, IL-4-, IL-5-, IL-6-, IL-10-, interferon-gamma (IFN-gamma)- and double-positive IL-2/IFN-gamma-producing whole T cells, CD4 + and CD8 + T-cell subsets by means of cytokine flow cytometry. This was performed after in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with phorbol myristate acetate (PMA) and ionomycin, in the presence of monensin, in 14 healthy volunteers (controls) and in 14 renal transplant patients. The immunosuppression of the latter was based either on CsA (n = 7) or on FK506 (n = 7). Cytokine-expressing T-cell frequencies were assessed immediately pretransplantation (DO), and subsequently 3 months (M3) and 6 months (M6) afterwards in fasting patients prior to the morning intake of the immunosuppressive drug. We found that at DO the frequencies of IL-2-(22 +/- 2% vs. 22.2 +/- 2%), IFN-gamma-(26 +/- 3% vs. 29 + 3.4%) and IL-4-(0.8 +/- 0.2% vs. 1.4 +/- 0.2%)-expressing T lymphocytes were not significantly different between the controls and the patients, respectively. Conversely, the frequency of IL-2/IFN-gamma double positive cells was higher in the latter (9.3 +/- 1.6%) than in the controls (5.6 +/- 0.8); p = 0.06. Finally, on D0 the frequencies of IL-5-, IL-6-, and IL-10-producing T lymphocytes were lower than 1%, in both groups, as well as after grafting, i.e. on M3 and M6. As compared to baseline (DO): (a) chronic immunosuppression significantly decreased the frequencies of IL-2-, IL-4- and IL-2/IFN-gamma-expressing T cells, whereas those of IFN-gamma, IL-5, IL-6, and IL-10 were not significantly affected; (b) the frequencies of cytokine-expressing T cells were not statistically different between M3 and M6; (c) the decrease in the frequencies of IL-2- and IL-2/IFN-gamma-expressing T cells affected CD4 + and CD8 + cells equally; (d) there was a marginal decrease in the frequency of IFN-gamma-expressing cells only in the CD4 + subset but not in the CD8 population; and (e) for CsA, but not for FK506, the frequency of the IL-2-expressing T cells was negatively correlated with the whole blood trough levels. When we compared the frequencies of cytokine-expressing cells in FK506- and CsA-treated patients, we found that the frequency of IL-2-expressing T cells was significantly lower with FK506 (10.9+/-1.61%) than with CsA (16.3 +/- 1.8%; p = 0.03), whereas the frequencies of the other cytokine-expressing cells were not statistically different between the two groups. In conclusion, our study clearly demonstrated that studied ex vivo, FK506 and CsA decrease the frequencies of cells expressing IL-2, IL-4 and IL-2/IFN-gamma in vivo but do not affect those expressing IFN-gamma. Meanwhile, the frequency of IL-2-producing T cells was more affected with FK506 than with CsA and was negatively correlated with the CsA trough level. Finally, our results regarding IL-2 might explain to some extent the higher efficiency of FK506 in vivo than CsA.

Decreased peripheral blood T cell cytokine gene expression in rheumatoid arthritis.

Currently, few informations are available about spontaneous production of T cell cytokines in rheumatoid arthritis (RA) peripheral blood (PB), because these cytokines are generally under the detection threshold of ELISAs. Because the Th1/Th2 balance could help to determine the outcome of RA, we used a sensitive and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method to mesure spontaneous T cell production of IL-2, IL-4, IL-10, and IFN-gamma mRNAs using unstimulated PBMC from 25 active RA patients, not taking any DMARDs for at least 6 weeks, and 19 healthy controls. Spontaneous IL-2 and IL-4 mRNA expressions are significantly lower in RA patients compared to healthy controls. Levels of IL-10 and IFN-gamma are similar in the two groups. No correlation was found between cytokine mRNA levels and clinical parameters. Spontaneous IL-4 and IL-10 mRNA levels are respectively correlated to the number of CD4+ T cells and to the number of monocytes in PB. After in vitro stimulation, IFN-gamma mRNA production by RA PBMC is significantly decreased. Most of the patients cannot be classified as having a T cell cytokine type 1 or type 2 secretion pattern in PB. IL-2 and IL-4 mRNAs in PB of active RA are produced at a low spontaneous level and the response to in vitro activation by mitogen is weak.

The CBF.78 monoclonal antibody to human sialophorin has distinct properties giving new insights into the CD43 marker and its activation pathway.

We confirm here the CD43 specificity of the CBF.78 monoclonal antibody (mAb) and compare its phenotypic and functional capacities to classical group-A mAbs (DFT1, MEM-59) and to 2 other CD43 mAbs (RDP/AD9, 161-46). It reacts with stable human CD43 transfectants in a sialic acid independent way and blocks completely cell binding of RDP/AD9 or 161-46 more or less but not DFT1 and MEM-59. Its distribution differs from all other CD43. B lymphocytes, but surprisingly the majority of granulocytes or monocytes are CBF.78 negative. CBF.78 is expressed on all T lymphocytes, but the number of CBF.78 molecules/cell is low and equally represented on resting T CD4 and CD8 cells. In comparison to naive T lymphocytes, CD45RO cells increase their CBF.78 epitopes much more than other CD43 epitopes. At a single cell level, confocal microscopy shows that CBF.78 can exist independently of other epitopes. CBF.78 is able to induce homotypic adhesion in different cell lines but not in peripheral blood lymphocytes and is unable to relocalise the targeted molecules. U937 cell line that is not agglutinated by CBF.78 (or RDP/AD9) undergoes a stronger adhesion with PMA, when this reagent is combined with this mAb. By itself CBF.78 is unable to activate T lymphocytes and to costimulate CD3 mAbs but partially blocks PMA. The phosphorylation of the tyrosine kinase p59fyn and p56lck, driven by CBF.78, is weak and almost blocked by PMA. Altogether these data support the hypothesis that there are at least 3 modes of interaction between PKC and CD43 pathways: each pathway is inhibitory towards the other but the CD43 one can also be synergistic.