Peripheral Lymphocyte Subsets Research Articles

Evaluation of the effect of selective serotonin-reuptake inhibitors on lymphocyte subsets in patients with a major depressive disorder

To date, only the effect of a short-term antidepressant treatment (<12 weeks) on neuroendocrinoimmune alterations in patients with a major depressive disorder has been evaluated. Our objective was to determine the effect of a 52-week long treatment with selective serotonin-reuptake inhibitors on lymphocyte subsets. The participants were thirty-one patients and twenty-two healthy volunteers. The final number of patients (10) resulted from selection and course, as detailed in the enrollment scheme. Methods used to psychiatrically analyze the participants included the Mini-International Neuropsychiatric Interview, Hamilton Depression Scale and Beck Depression Inventory. The peripheral lymphocyte subsets were measured in peripheral blood using flow cytometry. Before treatment, increased counts of natural killer (NK) cells in patients were statistically significant when compared with those of healthy volunteers (312+/-29 versus 158+/-30; cells/mL), but no differences in the populations of T and B cells were found. The patients showed remission of depressive episodes after 20 weeks of treatment along with an increase in NK cell and B cell populations, which remained increased until the end of the study. At the 52nd week of treatment, patients showed an increase in the counts of NK cells (396+/-101 cells/mL) and B cells (268+/-64 cells/mL) compared to healthy volunteers (NK, 159+/-30 cells/mL; B cells, 179+/-37 cells/mL). We conclude that long-term treatment with selective serotonin-reuptake inhibitors not only causes remission of depressive symptoms, but also affects lymphocyte subset populations. The physiopathological consequence of these changes remains to be determined.

Peripheral lymphocyte subset and serum cytokine profiles of patients with West syndrome

Objective: To clarify the immune pathophysiology of West syndrome (WS). Study design: We measured peripheral blood lymphocyte subset and serum cytokine profiles in 76 WS patients and 26 age-matched controls. Adrenocorticotropic hormone (ACTH) is one of the most effective therapy for WS and presumably immune-modulating; therefore, we compared the measured parameters between before ACTH (pre-ACTH) WS patients and controls, between cryptogenic and symptomatic WS patients before ACTH (pre-ACTH), and between before (pre-ACTH) and after (post-ACTH) ACTH WS patients. The post-ACTH group included those who received the last ACTH dose within 1 month of sampling. Results: CD3+ CD25+, CD19+, and CD19+ CD95+ cells were found to be significantly lower in the pre-ACTH group than in the controls. Interleukin (IL)-1 receptor antagonist (RA), 5, 6, and 15; eotaxin; basic fibroblast growth factor (bFGF); and interferon γ-inducible protein (IP)-10 levels were higher in pre-ACTH group than in the controls. No significant differences were found between the pre-ACTH cryptogenic and symptomatic groups. CD4+ cells, CD3+ cells, CD4+/8+ ratio, IL-1β, IL-12, and macrophage inflammatory protein (MIP)-1β were significantly higher in pre-ACTH group than in the post-ACTH group. Conclusions: Our study revealed immunological alterations in WS patients, and these responses were modified by ACTH therapy. Further study is needed to elucidate whether or how the immune system alteration is involved in the pathophysiology of WS.

A Phase I Study of the Anti-Natural Killer Inhibitory Receptor (KIR) Monoclonal Antibody (1-7F9, IPH2101) in Elderly Patients with Acute Myeloid Leukemia (AML): Clinical and Immunological Effects of a Single Dose Followed by Repeated Dosing.

Abstract 632Background: Non cytotoxic treatments such as immunotherapy represent promising approaches for the post-remission therapy of elderly patients with AML for which relapse free survival is short. The therapeutic potential of natural Killer (NK) cells has been revealed by the KIR mismatch allogeneic transplant model where the anti-leukemic effect of the graft is due to unleashed NK cells towards AML blasts, as suggested by enhanced in vitro NK lytic activity of KIR-HLA mismatched donor NK against recipient blasts (Miller et al. 2005; Ruggeri et al. 2002). To mimic this effect with a pharmaceutical agent, a fully human IgG4 anti-KIR mAb specific for KIR2DL1/2/3 (HLA-C specific KIRs), the 1-7F9/IPH2101, was generated (Romagne et al., Blood 2009). We present the results of the First in Human phase I trial of this agent in patients with AML in complete remission (CR). Methods: Patients aged 60-80 years with non promyelocytic AML in first CR following induction and 1-6 cycles of consolidation chemotherapy, normal renal and hepatic functions, KIR-expression on NK-cells and who signed informed consent were eligible for Protocol IPH2101-101. Dose escalation (IPH2101: 0.0003, 0.003, 0.015, 0.075, 0.3, 1, 3 mg/kg as iv infusion) was studied using a 3+3 scheme. Patients still in CR who had tolerated the single dose administration were eligible for an extension trial (Protocol IPH2101-102) investigating repeated doses of IPH2101 (1/ month x 6 months) using the dose the patient was allocated to in protocol IPH2101-101. Pharmacokinetic (PK) and circulating cytokines (IL-1b, IL-6, IFN-g, MIP-1β and TNF-a) were measured by ELISA. KIR occupancy and activation markers (CD69) were monitored by flow cytometry. Results: 23 patients, median age 71 years (range 61-79) in first CR of AML for a median of 20 weeks have been included in IPH2101-101. 3 patients (13%) had achieved CR after 2 induction courses, 5 patients (22%) had unfavorable cytogenetics, and 5 patients (22%) had high WBC at diagnosis. Planned dose escalation has been completed and no Dose Limiting Toxicity (DLT) has been recorded. 2 patients (in DL 4 and 5) have been replaced (one relapsed before 4 weeks of follow-up; the other had no detectable antibody after injection). Related Adverse Events seen in 14/23 patients (61%) were mild (grade ≤2) and transient. For doses above 0.075 mg/kg, ½ life ranged between 11-28 days. For doses above 0.3mg/kg, full saturation (>90% KIR occupancy) has been observed for 28 days to up to 6 weeks (at the 3 mg/kg DL). IPH2101 had no effect on the number and distribution of the peripheral lymphocyte subsets or the expression of NK receptors (KIRs, CD94/NKG2A, CD85j, NKp46, NKp30 or NKG2D). NK cell function tested by ex vivo cytotoxicity assay was not impaired after treatment. From dose 1 mg/kg (5/6 patients), IL-1b and IL-6 increased 6h post-dosing (2 to 8 fold increase and 2 to 15 fold increase compare to pre-dose, respectively) and decreased to pre-dose concentrations within 24h. Furthermore increase in TNF-a during the first hour was often correlated with CD69 up-regulation on NK cells. 8 patients received repeated doses of IPH2101 (0.0003 mg/kg: 2 patients, 0.003 mg/kg: 2 patients, 0.015 mg/kg: 1 patient, 1 mg/kg: 3 patients, 3 mg/kg pending) as part of the extension trial. No grade 3-4 related toxicities were observed. 2 patients completed the 6 courses, 5 patients were withdrawn for relapse and 1 patient is still ongoing at 1mg/kg. In accordance with the pre-clinical PK/PD model there is a clear relationship between exposure (Cmax) and KIR occupancy. Moreover the inter-patient variability is low (PK data) to moderate (KIR occupancy data). Median follow-up is 47 weeks. Among the 21 evaluable patients 11 patients are alive, 15 relapsed with 10 death, 6 are still in remission (1 patient in DL1, 2 patients in DL6 and 3 patients in DL7). Conclusions: Anti KIR treatment is safe and well tolerated and MTD has not been reached for doses producing full KIR saturation for 4 weeks, confirming the specificity of the immunological effects. Retreatment for prolonged period of time is feasible and safe. Signs of NK cell activation can be observed for doses above 0.3 mg/kg. IPH2101 deserves further investigation in patients with AML. Disclosures:Vey:INNATE PHARMA: Consultancy. d'Arnoux:INNATE PHARMA: Employment. Romagne:INNATE PHARMA: Employment. Andre:INNATE PHARMA: Employment. Tiollier:INNATE PHARMA: Employment. Squiban:INNATE PHARMA: Employment. Blaise:INNATE PHARMA: Consultancy. Olive:INNATE PHARMA: Research Funding.

Combined influence of adjuvant therapy and interval after surgery on peripheral CD4+ T lymphocytes in patients with esophageal squamous cell carcinoma

The aim of this study was to investigate possible differences in cellular immunity between chemo- and/or radiotherapy groups during a long interval after surgery in esophageal squamous cell carcinoma (ESCC) patients. Cellular immunity was assessed as peripheral lymphocyte subsets in response to chemotherapy (CT), radiotherapy (RT) and CT+RT by flow cytometric analysis. There were 139 blood samples obtained at different time points relative to surgery from 73 patients with ESCC. The changes in the absolute and relative proportions of lymphocyte phenotypes were significant among the adjuvant therapy groups. There were significant differences in the absolute counts of CD4+ and CD8+ T cells among the interval groups, and a lower CD4/CD8 ratio was found in patients following a prolonged interval. RT alone had a profound effect on the absolute counts of CD3+, CD4+ and CD8+ T cells compared with the other groups. CD4+ T cells exhibited a decreasing trend during a long interval, leading to a prolonged T-cell imbalance after surgery. Univariate analysis revealed that the interaction of the type of adjuvant therapy and the interval after surgery was correlated only with the percentage of CD4+ T cells. The percentage of CD4+ T cells can be used as an indicator of the cellular immunity after surgery in ESCC patients. However, natural killer cells consistently remained suppressed in ESCC patients following adjuvant therapy after surgery. These findings confirm an interaction between adjuvant therapy and the interval after surgery on peripheral CD4+ T cells, and implies that adjuvant therapy may have selective influence on the cellular immunity of ESCC patients after surgery.

Assessing the immune state of dogs suffering from pituitary gland dependent hyperadrenocorticism by determining changes in peripheral lymphocyte subsets

In order to evaluate the immune state of dogs suffering from pituitary-dependent hyperadrenocorticism (PDH), peripheral lymphocyte subsets were examined. Twenty seven PDH dogs and eight healthy control dogs were used in the current study. Eight healthy dogs served as the control group. Twenty seven PDH dogs were categorized into 4 groups based on their post serum cortisol concentrations by ACTH stimulation test: 2-5, excellent control (n = 8); 5-20, fair control (n = 7); >20, poor control (n = 4); and untreated (n = 8). Cell counts were executed with white blood cells (WBC), lymphocytes, CD3(+) (T lymphocytes), CD4(+) (Helper T lymphocytes), CD8(+) (Cytotoxic T lymphocytes), CD21(+) (B lymphocytes) cells in addition to calculating CD4(+)/CD8(+) ratio. Results indicated a significant difference in lymphocyte numbers and lymphocyte subset populations (CD3(+), CD4(+), CD8(+), and CD21(+) cells) between PDH and control dogs. Moreover, comparison of the PDH groups (excellent control; fair control; poor control; untreated) demonstrated that all groups had a significant decrease in lymphocytes numbers (CD3(+), CD4(+) and CD21(+) cell counts) as compared to control group. Meanwhile, no significant differences were observed in WBC counts and CD4(+)/CD8(+) ratio between groups. Furthermore, lymphocyte subset distribution in excellent control PDH dogs without concurrent disease (n = 4) better resembled that of control dogs as compared to PDH dogs with concurrent disease (n = 4). PDH dogs may be suffering from an immuno-depressed state as evidenced by significant differences in lymphocyte subset populations. Furthermore, treatment of both PDH and concurrent disease might improve lymphocyte subset distribution.

Study on T cell subsets in HIV/AIDS patients

Analyze Naive and Mermory T cell subsets in HIV/AIDS patients and investigate their relationship with disease development. T cell subsets from 15 normal control subjects, 79 HIV/ AIDS patients were detected by FCM. With diesase progression, CD4+ Naive cell counts and ratio was both decreased obviously (P < 0.001); CD4+ Tcm cell counts was significantly decreased (P < 0.001), CD4+ Tcm cell ratio was obviously higher (P = 0.002); CD4 TEM cell ratio was obviously increased( P < 0.001); CD8+ T Naive cell counts and ratio was also decreased obviously (P < 0.05); CD8+ T(CM), T(EM), T(EMRA) are not significantly different. The peripheral lymphocyte subsets in HIV/AIDS patients changed obviously. The counts of naive T cell decreased, while the proportion of memory T cell increased significantly. It will help to understand pathogenesis of HIV.

Changes in Peripheral Lymphocyte Subsets in the Type 1 Diabetic Dogs Treated with Insulin Injections

Plasma metabolites and peripheral lymphocyte subsets were measured in ten diabetic and ten control dogs to investigate their significances as indicators to evaluate immune states in the diabetic dogs. Diabetic dogs were treated with insulin injections, however their plasma glucose and fructosamine concentrations were significantly higher than those of the controls. There were no significant differences in counts of total white blood cells (WBC) and lymphocyte CD8(+) cells (cytotoxic T cells) between the control and the diabetic dogs. In the diabetic dogs, the counts of CD3(+) (T cells), CD4(+) (Helper T cells) and CD21(+) (B cells) cells and the peripheral lymphocytes CD4/CD8 ratio were significantly lower than those in the control dogs. We confirmed abnormality of lymphocyte subsets in insulin treated diabetic dogs and it may relate to depression of immunocompetence and high susceptibility to common infectious diseases.

T-lymphocyte subsets and thymic size in malnourished infants in Egypt: a hospital-based study

Thymus size was assessed ultrasonographically and correlated to the percentage of CD4 and CD8 T-lymphocytes in peripheral blood in 32 infants with protein-energy malnutrition (PEM) and compared with 14 healthy control infants. The study revealed thymus atrophy in patients with PEM, especially the oedematous type, accompanied by changes in the peripheral lymphocyte subsets. These changes were reversible after nutritional rehabilitation. However, they may affect the immune status of PEM patients and may require a longer duration of nutrition rehabilitation than required for recovery of anthropometric measures. We recommend proper assessment of the immune functions of PEM patients during nutritional rehabilitation until full recovery.

Absence of CD4CD25 regulatory T cell expansion in renal transplanted patients treated in vivo with Belatacept mediated CD28–CD80/86 blockade

Aims Belatacept is a new recombinant molecule (CTLA4-Ig) that interferes with the second activation signal of T lymphocytes. CTLA4-Ig induced T cell allograft tolerance in rodents but not in primates. We examined the changes in peripheral lymphocyte subsets, including regulatory T cells, in renal transplant patients treated with Belatacept. Methods A cross-sectional immunological study was carried out 6 months after transplantation in 28 patients enrolled in the Belatacept phase II study. Eighteen patients received Belatacept, mycophenolate mofetil and steroids (Belatacept group), while the control group of 10 patients received cyclosporine, mycophenolate mofetil and steroids (CsA group). Lymphocyte subsets were examined by flow cytometry. Foxp3 mRNA expression was measured by quantitative PCR. Results The number of T lymphocytes and the percentage of CD3+ T cells were similar in both groups. However, the percentage of CD3+ CD4+ T cells was lower in the Belatacept group than in the control CsA group ( B = 42.5% ± 13.7 vs CsA = 52.9% ± 9, p < 0.005), and the percentage of CD3+ CD8+ cells was higher in the Belatacept group than in the control ( B = 32.9% ± 6.7 vs CsA = 19.5% ± 8.2, p < 0.0002). The percentage of CD19+ cells was similar in both groups. Among CD56+cells, only the percentage of CD16+ cells was significantly higher in the Belatacept group than in the control ( B = 82% ± 12 vs CsA = 59.7% ± 25, p = 0.01). Among CD4 and CD8 T cells the percentage of activated lymphocytes expressing CTLA4, HLA-DR or CD40L was similar in both groups. The percentage of CD4+CD25+ T cells was higher in the CsA group. The percentage of regulatory CD4+CD25+ cells with bright CD25 staining was similar in both groups ( B = 3.6 ± 2.3% vs CsA = 4.7 ± 1.9%, ns) as was the expression of FoxP3. Conclusion Our results indicated that Belatacept did not induce regulatory T cell expansion in vivo. We suggest that Belatacept treatment should be maintained after transplantation to allow graft acceptance.

Changes in peripheral lymphocyte subsets in patients after partial microwave ablation of the spleen for secondary splenomegaly and hypersplenism: A preliminary study

Purpose: Microwave ablation therapy for secondary splenomegaly and hypersplenism has been shown to be effective from pre-clinical animal models and clinical investigations. This study was performed to determine its effects on the status of peripheral lymphocyte subsets in patients receiving microwave ablation of the spleen.Materials and methods: Ten patients with secondary splenomegaly and hypersplenism received microwave ablation of the spleen during laparoscopy or percutaneously under ultrasound guidance. The percentage peripheral blood T cells, B lymphocytes and NK cells were measured using flow cytometry before and on days 1, 3 and 7 after therapy, as well as 1 and 3 months afterwards.Results: Percentages of CD3+ and CD4+ cells increased rapidly 1 month after therapy. There was no significant change in CD8+, CD4+/CD8+ or NK cells of the pre- and post-therapy levels and B lymphocytes increased significantly after therapy. In patients with an ablation volume (AV) less than 20% (group A), T cells increased 1 month after ablation but decreased 3 months after ablation. B lymphocytes increased significantly after surgery. Levels of NK cells were lower than that before therapy on each testing. In patients with 20–40% AV (group B), levels of T cells, B lymphocytes and NK cells showed an increase. Levels of CD4+ cells were significantly higher in group B than in group A, 3 months after therapy.Conclusions: Microwave ablation therapy for splenomegaly and hypersplenism appears to have a favourable effect on peripheral lymphocyte subsets. A relationship may exist between the ablation volume and the level of peripheral lymphocyte subsets.

Adolescents and young adults successfully restore lymphocyte homeostasis after intensive T‐cell depleting therapy for cancer

Long-term impairment of T-cell regeneration has been widely recognized in humans rendered lymphopenic by disease or therapy (Hakim & Gress, 2005). Successful restoration of T-cell homeostasis following lymphodepletion requires residual thymic function, and age-associated declines in thymic function prevent full immune reconstitution in a substantial percentage of adolescents and adults following the induction of lymphopenia. These observations raise the possibility that only young children are capable of full restoration of T-cell homeostasis following lymphocyte depletion. However, most studies demonstrating incomplete restoration of lymphocyte homeostasis are either limited by relatively short follow-up periods (<2 years) (Hakim et al, 2005) or are confounded by ongoing thymotoxic diseases, such as graft-versus-host disease (Weinberg et al, 2001) or human immunodeficiency virus infection (Douek et al, 2000). In one study, persistent depletion of naïve subsets was observed as long as 30 years following treatment for Hodgkin lymphoma (Watanabe et al, 1997), but an important effect of thymic irradiation in this population could not be ruled out. In order to address whether populations rendered lymphopenic during late childhood, adolescence and young adulthood are capable of full immune reconstitution, we analysed peripheral lymphocyte subsets as part of a cross-sectional study examining late effects of treatment for paediatric sarcomas. Thirty-two patients, described previously (Mansky et al, 2006), treated for the Ewing sarcoma family of tumours (ESFT), rhabdomyosarcoma (RMS) and non-rhabdomyosarcoma soft tissue sarcomas (NR-STS) at the Pediatric Oncology Branch of National Cancer Institute (1965–1998), were studied. All patients received multimodality therapy known to induce profound lymphopenia, which included high dose ciclophosphamide (mean 16·7 gm/m2, range 5·8–18·4 gm/m2). Peripheral blood CD4, naïve CD4 (CD4+CD62L+CD45RA+), total CD8, naïve CD8 (CD8+CD62L+CD45RA+), B (CD20), and natural killer (NK; CD16+CD56+CD3+) subsets were enumerated using standard techniques and compared with 53 healthy controls studied in the same laboratory. Comparisons were made using two-tailed t-tests, with P ≤ 0·01 considered significant. Median age at treatment was 15·4 years (range 7·1–34·2 years), median age at time of analysis was 37·4 years (range 17·5–55·4 years) and median time elapsed from completion of therapy to analysis was 17·3 years (2·9–32·6 years). Median age of controls was 38·6 years (range 25·2–62·8 years). Sarcoma survivors demonstrated normal levels of total circulating CD4 and CD8 T cells, and NK cells and a modest increase in total B cells (Fig 1). Furthermore, they all demonstrated recovery of normal naïve CD4 subsets and there was a statistically significant increase in naïve CD8+ cells, indicative of thymic-dependent immune reconstitution. No patient presented clinical evidence for immune deficiency or autoimmunity following completion of therapy. Thus, in the absence of intercurrent disease and high-dose mediastinal irradiation, humans that were rendered lymphopenic during adolescence and early adulthood successfully restored lymphocyte homeostasis when followed for prolonged periods. Whether populations of older individuals also reliably restore lymphocyte homeostasis with prolonged follow-up will require long-term studies. Adolescents and young adults restore normal T-cell homeostasis following lymphodepleting chemotherapy. Patients, treated with intensive chemotherapy a median of 17 years earlier for sarcoma, showed no evidence for depletion of any lymphocyte subset. Treated patients demonstrated slightly higher median levels of CD8+ naïve T cells and B cells compared with controls.

Identification of N-arachidonylglycine as the endogenous ligand for orphan G-protein-coupled receptor GPR18

An orphan G-protein-coupled receptor, GPR18, was cloned on the basis of degenerate-oligonucleotide PCR analysis of HUT 102 cells using primers designed from the conservative regions of the human chemokine receptor. GPR18 was expressed significantly in lymphoid cell lines, but not in non-lymphoid hematopoietic cell lines. Moreover, the expression of the GPR18 gene was higher in peripheral lymphocyte subsets (CD4+, CD4+CD45RA+, CD4+CD45RO+, CD8+, and CD19+) than in monocytes and lymphoid cell lines, and was increased after stimulation with phytohemagglutinin. By screening using a lipid library, N-arachidonylglycine (NAGly) induced an increase in intracellular Ca2+ concentration in GPR18-transfected cells, which was significantly greater than that in mock-transfected cells. NAGly also inhibited forskolin-induced cAMP production in a pertussis toxin-sensitive manner in the GPR18-transfected CHO cells. This is the first study to demonstrate that NAGly is a natural ligand for GPR18.

Longitudinal analysis of immune cell phenotypes in early stage multiple sclerosis: distinctive patterns characterize MRI-active patients

To investigate whether peripheral immune abnormalities are associated with brain inflammation in multiple sclerosis, and whether differences in MRI activity are paralleled by changes in leukocyte composition, we conducted a prospective longitudinal study in patients at their clinical onset. Twenty patients presenting a first inflammatory event in the central nervous system suggestive of multiple sclerosis underwent, every 45 days for one year, immunophenotyping of 98 blood cell subsets together with brain MRI and clinical evaluation. Six patients showed intense MRI activity, six patients did not display MRI activity, while the remaining 8 patients had low (i.e. intermediate) MRI activity during the follow-up. Our results show that MRI-active and MRI-inactive patients display significant differences in ten lymphocyte subsets. Among these, there are both effector (CCR7-CD45RA-CD4+ alphabeta T cells, CCR5+ gammadelta T cells) and regulatory (DN CD28+ alphabeta T cells and CD25+CD8+ alphabeta T cells) lymphocytes pertaining to the innate and the acquired arms of the immune system. Moreover, these differences were, upon employment of a class prediction procedure based on "support vector machines" algorithm utilizing leave-one-out cross validation procedures, able to correctly assign patients to their respective MRI activity group. All 6 MRI-active and 6 MRI-inactive patients were correctly classified, and, upon application of a class prediction model in an unsupervised manner to the 8 patients with intermediate MRI activity, 6 were predicted as MRI-active and 2 as MRI-inactive patients. Also, when the mean values of the first three time points (T0, T1 and T2) were used for the prediction of all patients, the selected lymphocyte subsets correctly classified 90% of patients. Sensitivity was 91.7% and specificity was 87.5%. These results provide evidence showing that brain inflammation in multiple sclerosis is associated with distinct changes in peripheral lymphocyte subsets, and raise the possibility that the identified subsets may, after adequate validation, assist in the prediction of MRI activity in the early stages of multiple sclerosis.

Dual Effect of Methylprednsolone Pulses on Apoptosis of Peripheral Leukocytes in Patients with Renal Diseases

It is well known that change in apoptosis may modulate the natural story of illness, and that many drugs may act through modulation of apoptosis, but the role of steroids in acting through apoptosis in different settings, including renal diseases, has still to be elucidated. We studied the in vivo effects of steroids by oral assumption (10 to 25 mg/deltacortene) or by intravenous pulses (300 to 1000 mg/dose) on apoptosis and cellular subsets of peripheral lymphocytes, by evaluating DNA-fragmentation and lymphocyte subsets in 79 subjects: 22 controls and 57 patients with various renal diseases (25 Lupus-GN, 19 membranous-GN (MGN), 6 rapidly progressive-GN (RPGN), 2 acute interstitial nephritis (AIN), 5 on chronic dialysis. Baseline apoptosis was present in 1/22 (4.5%) of controls, 3/25 (12%) SLE, 2/6 (33.3%) RPGN and 10/19 (52.6%) MGN. A significant decrease in CD3+CD8+ cell count and a significant increase of the CD3+CD4/CD3+CD8+ ratio were found in apoptosis-positive subjects. DNA fragmentation did not change after oral steroids, paralleling a 22 to 32% decrease in total lymphocytes. Following intravenous methylprednisolone pulses, a deeper drop of all lymphocyte subsets was observed, while DNA fragmentation turned from present to absent in 2 MGN, but not in 2 RPGN, and from absent to present in 1 ARF and 1 SLE, independently of the dosage. We demonstrated that the presence of apoptosis in renal diseases is associated with decreased CD3+CD8+ cell count. Furthermore, steroid intravenous pulses, besides inducing a profound decrease in lymphocyte subsets, do exert a dual effect on baseline leukocyte apoptosis, eventually leading to a reversal of baseline patterns, either turning from negative to positive or from positive to negative. Oral steroid therapy did not influence baseline apoptosis.

Effect of Wuye Decoction on lymphocyte phenotype in patients with non-small cell lung cancer

To investigate the effect of Chinese recipe, Wuye Decoction (WYD), on immune function in patients with non-small cell lung cancer (NSCLC). Eighty-two patients of NSCLC with pathologically confirmed diagnosis, who had received operative treatment and completed the post-operational chemotherapy, were randomly assigned into 2 groups. Group A (42 cases) received WYD and Group B (40 cases) received no specific medicine. Positive rate of various peripheral lymphocyte subsets, including CD3, CD4, CD8, CD16, CD19 and CD25, in both groups was observed immediately after chemotherapy (T(0)) and 3 months later (T(1)), the same indexes of 20 healthy volunteers allocated in Group C were also determined at T(0) for control. The positive rates of CD4, CD4/CD8, CD16, CD19 and CD25 were significantly lower (P < 0.05) while that of CD8 was significantly higher (P < 0.05) in Group A and B at T(0) than those in Group C; at T(1), these indexes, except CD25, got significantly restored in Group A with the level approaching normal range (P > 0.05), and showed significant difference from those in Group B (P < 0.05), since these indexes in that group remained unchanged at the corresponding period. As for CD25, it was insignificantly changed in Group A after WYD treatment, and thus, at T(1), it was still lower than that in Group C (P < 0.05) and showed insignificant difference as compared with that in Group B (P > 0.05). Comparison of CD3 among the 3 groups showed no significant difference (P > 0.05). WYD could activate the immune function of NSCLC patients, and so it is recommended to be used in the treatment of NSCLC in clinical practice.

Rapid helper T-cell recovery above 200 × 106/l at 3 months correlates to successful transplant outcomes after allogeneic stem cell transplantation

The current study evaluates the role of quantitative measurement of peripheral lymphocyte subsets, especially CD4+ helper T-cell recovery, in predicting transplant outcomes including overall survival (OS) and non-relapse mortality (NRM) after allogeneic stem cell transplantation. A total of 69 allogeneic recipients were included with following diagnoses: acute myeloid leukemia 42, acute lymphoblastic leukemia 5, chronic myeloid leukemia 15, non-Hodgkin's lymphoma 5 and high-risk myelodysplastic syndrome 2. The peripheral lymphocyte subset counts (CD3+ T cells, CD3+4+ helper T cells, CD3+8+ cytotoxic T cells, CD19+ B cells, and CD56+ natural killer cells) were measured at 3, 6 and 12 months. The CD4+ helper T-cell reconstitution at 3 months was strongly correlated with OS (P<0.0001), NRM (P=0.0007), and opportunistic infections (P=0.0108) at the cutoff value of 200 x 10(6)/l CD4(+) helper T cells. Rapid CD4+ helper T-cell recovery was also associated with a higher CD4+ helper T-cell transplant dose (P=0.006) and donor type (P<0.001). An early CD4+ helper T-cell recovery at 3 months correlated with a subsequent faster helper T-cell recovery until 12 months, yet not with B-cell recovery. In a multivariate analysis, rapid recovery of CD4+ helper T cells at 3 months was a favorable prognostic factor together with higher CD34+ cell transplant dose in terms of OS (P=0.001) and NRM (P=0.005).

Modulation of Natural Killer (NK) Receptors on NK (CD3 −/CD56 +), T (CD3 +/CD56 −) and NKT-like (CD3 +/CD56 +) Cells after Heart Transplantation

After undergoing heart transplantation and the subsequent compulsive immunosuppressive treatments, patients are at risk of rejection episodes, infectious complications or cancer development. Thus, it is probable that the various subsets of peripheral cytotoxic lymphocytes are modulated in such patients. This area of study can now be investigated by examining the numerous recently described natural killer (NK)-cell-related surface receptors. A prospective cohort of 60 heart transplant recipients and 60 controls was studied. The partitioning of lymphocyte subsets, especially NK (CD3-/CD56+), T (CD3+/CD56-) and NKT-like (CD3+/CD56+) cells, was compared in both groups using multi-parametric flow cytometry. Moreover, expression of a series of seven NK-related receptors was compared on the three subsets defined by CD56 expression. A significant increase in NK-cell levels was observed in transplanted patients, as compared with controls, whereas T and NKT-like cells were in similar proportions in both groups. Two NK-related receptors showed significantly different levels of expression in heart transplant recipients: the cytotoxic effector, CD244, which was in a significantly increased proportion on T and NKT-like cells; and the activating receptor, CD161, which was expressed significantly less on NK and NKT-like cells, but more on T cells. These findings indicate that cytotoxic NK-related cells, increased in proportion, also display increased levels of activity-associated markers in heart transplant recipients. Viral infection or the immunosuppressive regimen could be responsible for the modulation of regulatory receptors on NK and NKT-like cells in heart transplant recipients.

Enumeration of peripheral lymphocyte subsets using 6 vs. 4 color staining: A clinical evaluation of a new flowcytometer

Technological advances in instruments allow the evaluation of many lymphocyte subsets in one step. The aim of this study was to evaluate the new FACSCanto flowcytometer in routine conditions, using a 6 color combination, single platform, whole blood, lysis, no wash protocol. Two systems were simultaneously compared on 67 blood samples and external quality controls, using CD3,CD4, CD8, CD19, CD16/56, and CD45 in one tube TRUCOUNT beads (BD Biosciences) or two tubes (TetraChrome and Flowcount, Beckman-Coulter and DakoCytomation). The day-to-day instrument detection but automatic compensations were stable. Manual compensation settings were satisfactory using available facilities. Commercial and UK NEQAS quality control results were acceptable. The intra-experiment reproducibility was good (coefficient of variation (CV)<3%) but highly operator-dependent (CD4+ T cell count CVs from 1.2 to 9.7, six operators). Storage of samples was acceptable, but storage of stained samples altered absolute count reliability. Serial dilutions show a good count accuracy. The FACScanto T subsets and B cell data were highly correlated with our reference values (r2>0.87) and absolutes values were very close (slopes>0.89). The gating strategy, fluorochrome choice, and compensation setting are discussed. A few improvements are expected (sample loader, data management, auto-gating, acquisition parameters, sample mixing, absolute values calculation, etc). In conclusions, despite its complexity, 6 color staining is a reliable, stable, and highly informative technique for lymphocyte subset monitoring but remains to be optimized.

Rapid Helper T-Cell Recovery at 3 Months Correlates to Successful Transplant Outcomes after Allogeneic Stem Cell Transplantation.

Background: The current study attempted to evaluate the role of a simple quantitative measurement of peripheral lymphocyte subsets, especially CD4+ helper T-cell recovery, in predicting transplant outcomes, including overall survival (OS), non-relapse mortality (NRM), and opportunistic infections, after allogeneic stem cell transplantation (SCT).Methods: A total of 69 patients receiving an allogeneic SCT were included. The disease entities were as follows: AML 42, ALL 5, CML 15, NHL 5, and high-risk MDS 2. The peripheral lymphocyte subset counts, such as CD3+ T-cells, CD3+4+ helper T-cells, CD3+8+ cytotoxic T-cells, CD19+ B-cells, and CD56+ natural killer (NK) cells, were measured 3, 6, and 12 months post-transplant.Results: The CD19+ B-cell reconstitution was slow, while a rapid CD56+ NK cell recovery was noted. The CD4+ helper T-cell reconstitution at 3 months was strongly correlated with OS (p<0.0001), NRM (p=0.0007), and opportunistic infections (p=0.0108) when stratifying patients with cut-off value of 200×106/L CD4+ helper T-cells. A rapid CD4+ helper T-cell recovery was also independently associated with a higher CD4+ helper T-cell transplant dose (p=0.006) and donor type (p<0.001) in a regression analysis. An early CD4+ helper T-cell recovery at 3 months was associated with a subsequent faster helper T-cell recovery until 12 months, yet not with B-cell recovery. In a multivariate survival analysis, a combination of a higher CD34+ cell dose and rapid recovery of CD4+ helper T-cells at 3 months was found to a have favorable prognosis in terms of OS (p=0.001, hazard ratio [HR] 3.653) and NRM (p=0.005, HR 4.836), yet not relapse.Conclusion: A rapid recovery of the CD4+ helper T-cell count above 200×106/L at 3 months seemed to correlate with a faster immune reconstitution and predict a successful transplant outcome. [Display omitted]

T-Cell Subsets and Interleukin-6 Response in Rasmussen’s Encephalitis

To evaluate the immunopathogenesis in Rasmussen's encephalitis, peripheral lymphocyte subsets and interleukin-6 analysis were performed in three patients. Magnetic resonance spectroscopy and diffusion-weighted magnetic resonance imaging were performed to assess neuronal injury in the affected hemisphere. Before initiation of immune therapy, percentage of cytotoxic T cells was found to be increased in peripheral blood obtained from patients compared with a group of age-matched normal control subjects. During follow-up, percentage of cytotoxic T cells returned to the normal ranges only in one patient who had an early functional hemispherectomy. All three patients had significantly increased interleukin-6 concentration in cerebrospinal fluid and serum compared with the mean values of patients with acute viral encephalitis. The magnitude of interleukin-6 response in the patients correlated with the neuronal loss and atrophy on magnetic resonance spectroscopy and diffusion-weighted magnetic resonance imaging studies. The patient, who had a fulminant course and an early hemispherectomy, had higher interleukin-6 concentration in cerebrospinal fluid and serum than those of the other two. Detection of an increased percentage of cytotoxic T cells in peripheral blood supports the presence of a T cell-mediated inflammatory pathogenesis in Rasmussen's encephalitis. However, elevated interleukin-6 response might reflect the magnitude of the inflammatory process in the affected hemisphere.