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.

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.

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.

A subset of HLA-DR9 molecules is detected by a polymorphic monoclonal antibody on lymphoblastoid cell lines but not on peripheral blood lymphocytes.

Some mAbs recognizing polymorphic epitopes of HLA-DR molecules exhibit striking differences of reactivity with the same HLA-DR molecules expressed by different cell types. In this study, we investigated the basis for the differential reactivity of the polymorphic anti-DR mAb OHA TM901 with HLA-DR9 molecules expressed by human PBLs or LCLs. By immunoprecipitation experiments we showed that OHA TM901 recognizes a subset of HLA-DR9 molecules from LCLs. This subset corresponds to HLA-DR9 molecules containing immature-type oligosaccharides. The absence of OHA TM901 reactivity with HLA-DR9 PBLs, as revealed by cytofluorometry analysis, suggests that this subset is either not expressed or expressed at a very low level on PBLs. These results indicate that overexpression of HLA-DR molecules in immortalized LCLs could lead to cell-surface expression of underglycosylated forms which are generally not found on the cell surface of PBLs.

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.

One-year chromaffin cell allograft survival in cancer patients with chronic pain: morphological and functional evidence.

The control of chronic pain through transplantation of chromaffin cells has been reported over the past few years. Analgesic effects are principally due to the production of opioid peptides and catecholamines by chromaffin cells. Clinical trials have been reported with allografts consisting of whole-tissue fragments implanted into the subarachnoid space of the lumbar spinal cord (14,19,36). In the present study, allogeneic grafts were successfully used to control chronic pain in two patients over a period of 1 yr based on patient reported pain scores, morphine intake, and CSF levels of Met-enkephalin. Macroscopic examination at autopsy located the transplanted tissue fragments in the form of multilobulated nodules at the level of the spinal axis and cauda equina. Immunocytochemical microscopy showed neuroendocrine cells are positive for chromagranin A (CGA), and enzymes tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DbetaH). The results suggest that there is a relationship between analgesic effect, Met-enkephalin levels in CSF, and the presence of chromaffin cells surviving in spinal subarachnoid space.

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.

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.

In vitro interleukin-1 and 2 production and interleukin 2 receptor expression in the rhesus monkey.

Anti-human monoclonal antibodies were used to detect and quantify interleukins-1 and 2 and interleukin-2 receptor expression in peripheral blood mononuclear cells from a rhesus monkey. Interleukin-1 production could be induced by phorbol esters (PMA) and was potentiated by phytohemagglutinin (PHA). Interleukin-2 secretion could also be induced by the combination of PHA and PMA, but only weakly with PHA alone. Interleukin-2 receptor expression was present in a subpopulation of unstimulated lymphocytes and could be enhanced by PHA or PMA. These data show once again that the rhesus monkey immune system is cross-reactive with the human one and that rhesus macaque could be a good model to study interleukin therapy.

Transplantation of human chromaffin cells for control of intractable cancer pain.

Adrenal medullary chromaffin cells produce high levels of endogenous opioid peptides. Recent data suggest that transplantation injected locally into the spinal subarachnoid space reduced intractable malignant pain. In order to determine the feasibility, the efficacy and the risks of using adrenal medullary tissue for control of irreducible pain, we have developed a transplantation protocol on cancer pain patients selected when they required chronic intrathecal injection of morphine and progressively increasing doses to maintain the level of analgesic effects. At the present time, our clinical trial involves 8 patients. We report here our initial results (mean follow-up: 5 months). The various data collected before and after the intrathecal administration of chromaffin cells included: 1) Pain evaluation over time, with concomitant narcotic intake, 2) CSF sampling through an implanted access port to determine the following biological parameters: biochemical assay for opioid peptides, cell count and phenotyping of lymphocytes, 3) peripheral blood samples for lymphocyte typing. The results confirm the efficacy of adrenal medullary transplantation into spinal CSF for controlling irreducible cancer pain. Complementary intrathecal and oral morphine were totally stopped in 2 cases and stabilized in 5 others. It seems essential to have an important volume of grafted tissue to achieve analgesia with high levels of metenkephalin in CSF. A progressive decrease in metenkephalin release was observed from 2 to 4 months after the transplantation. Two patients with a long-term follow-up (8 and 12 months) needed another intrathecal chromaffin cell graft.

[Analysis of the cerebrospinal fluid by isoelectrofocusing on agarose in multiple sclerosis]. / Analyse du LCR par isoélectrofocalisation en agarose dans la sclérose en plaques.

Intrathecal synthesis of immunoglobulins can be proved by means of two methods: quantitatively by immunoglobulins titration in CSF, the results expressed with several ratios; qualitatively by demonstration of oligoclonal distribution of gammaglobulins. IEF is the most sensitive of the qualitative methods. From a technical point of view Agarose Isoelectrofocusing seems to be a better method than polyacrylamide isoelectrofocusing and permits, when the interpretation is difficult, immunofixation into the gel. The authors report the results of a comparative study between the evaluation of the IgG Index and agarose isoelectrofocusing of 281 CSF divided into 113 CSF from patients with Multiple Sclerosis (MS) and 168 CSF from patients with other neurological diseases (OND). Sensitivity of IEF was higher than IgG index to prove intrathecal IgG synthesis: in the group of patients with MS, 91 p. 100 of CSF were abnormal instead of 72 p. 100 of IgG Index. In the group of patients with OND, abnormalities in IEF were low (5 p. 100) but the number of inflammatory diseases was poor. These results were similar with the findings of many authors using the same methods. In our opinion, IEF is the best technique which a specialized laboratory can use in routine to prove an immunoglobulin intrathecal synthesis.

[Study of CD45 isoforms on T CD8 lymphocytes, in the peripheral blood and the graft in patients after kidney transplantation]. / Etude chez le transplanté rénal des isoformes du CD45 sur les lymphocytes T CD8, dans le sang circulant et dans le greffon.

We report characterization of CD45 isoforms expressed by CD8+ lymphocytes in peripheral blood and in the graft of 40 kidney transplanted patients who underwent kidney biopsy on the basis of clinical signs suggesting rejection. Standard histological examination of the biopsy fragments and three-color cytofluorimetric analysis of lymphocytes extracted from the same fragments by mechanical and enzymatic treatment were performed simultaneously and compared to the peripheral blood lymphocytes. In 14/40 biopsies where lymphocyte extraction succeeded, the predominant subset was CD8 (CD4/CD8 mean ratio was 0.53). Almost all CD8+ cells were activated: among these CD8+ cells, 55 percent were HLA-DR+, and 68 percent CD45RO+, i.e. of a memory cell type with cytotoxic activity. This situation resembles the in vitro observation made during mitogenic stimulation of lymphocytes by phytohemagglutinin, OKT3 or CML ("culture mixte lymphocytaire"). Beside their evident interest for the diagnosis, these data could be useful for our understanding of the physiopathology of the rejection crisis.

[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.

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.