Blood Subpopulations Research Articles

Cortisol and epinephrine control opposing circadian rhythms in T cell subsets

AbstractPronounced circadian rhythms in numbers of circulating T cells reflect a systemic control of adaptive immunity whose mechanisms are obscure. Here, we show that circadian variations in T cell subpopulations in human blood are differentially regulated via release of cortisol and catecholamines. Within the CD4+ and CD8+ T cell subsets, naive cells show pronounced circadian rhythms with a daytime nadir, whereas (terminally differentiated) effector CD8+ T cell counts peak during daytime. Naive T cells were negatively correlated with cortisol rhythms, decreased after low-dose cortisol infusion, and showed highest expression of CXCR4, which was up-regulated by cortisol. Effector CD8+ T cells were positively correlated with epinephrine rhythms, increased after low-dose epinephrine infusion, and showed highest expression of β-adrenergic and fractalkine receptors (CX3CR1). Daytime increases in cortisol via CXCR4 probably act to redistribute naive T cells to bone marrow, whereas daytime increases in catecholamines via β-adrenoceptors and, possibly, a suppression of fractalkine signaling promote mobilization of effector CD8+ T cells from the marginal pool. Thus, activation of the major stress hormones during daytime favor immediate effector defense but diminish capabilities for initiating adaptive immune responses.

Extracorporeal Photopheresis Efficiently Impairs the Proinflammatory Capacity of Human 6-Sulfo LacNAc Dendritic Cells

Extracorporeal photopheresis (ECP) emerged as a promising treatment modality for steroid-refractory graft-versus-host disease (GVHD), which represents a major complication of allogeneic hematopoietic stem-cell transplantation. Dendritic cells (DCs) display an extraordinary capacity to induce T-cell responses and play a crucial role in the initiation and maintainance of GVHD. This study evaluated the direct impact of ECP on the proinflammatory capacity of 6-sulfo LacNAc (slan) DCs, representing a major subpopulation of human blood DCs. SlanDCs were isolated from ECP-treated or untreated blood of healthy donors or GVHD patients by immunomagnetic isolation. The maturation of slanDC was determined by flow cytometry. Cytokine production of slanDCs was measured by enzyme-linked immunosorbent assay. SlanDC-mediated T-cell proliferation was evaluated by H-thymidine incorporation. SlanDC-mediated T-cell programming was determined by flow cytometry. ECP efficiently impairs the spontaneous maturation and secretion of proinflammatory tumor necrosis factor-alpha, interleukin-1beta, and interleukin-12 by slanDCs. Furthermore, ECP markedly inhibits slanDC-induced proliferation of CD4 and CD8 T cells and polarization of naïve CD4 T lymphocytes into Th1 cells. These novel findings indicate that ECP efficiently impairs the proinflammatory capacity of slanDCs, which may represent an important mechanism for the therapeutic efficiency of ECP in GVHD.

Detection of JC Virus DNA and Proteins in the Bone Marrow of HIV‐Positive and HIV‐Negative Patients: Implications for Viral Latency and Neurotropic Transformation

We sought to determine the prevalence of JC virus (JCV) in bone marrow samples from human immunodeficiency virus (HIV)-positive and HIV-negative patients and to determine whether bone marrow is a site of latency and neurotropic transformation of JCV, the agent of progressive multifocal leukoencephalopathy (PML). We collected bone marrow aspirates, archival bone marrow samples, and blood and urine samples from 75 HIV-negative and 47 HIV-positive patients without PML as well as bone marrow and urine or kidney samples from 8 HIV-negative and 15 HIV-positive patients with PML. Samples were tested for JCV DNA by quantitative polymerase chain reaction and for JCV protein expression by immunohistochemical analysis. JCV regulatory regions (RRs) were characterized by sequencing. JCV DNA was detected in bone marrow samples from 10 (13%) of 75 and 22 (47%) of 47 of the HIV-negative and HIV-positive patients without PML, respectively, compared with 3 (38%) of 8 and 4 (27%) of 15 of the HIV-negative and HIV-positive patients with PML. JCV DNA (range, 2-1081 copies/microg of cellular DNA) was detected in multiple leukocyte subpopulations of blood and bone marrow samples. JCV large T antigen, but not VP1 capsid protein, was expressed in bone marrow plasma cells. Bone marrow JCV RR sequences were similar to those usually found in the brains of patients with PML. Bone marrow is an important reservoir and a possible site of neurotropic transformation for JCV.

Effect of ACTH treatment on CSF and blood lymphocyte subpopulations in patients with multiple sclerosis.

Patients with active multiple sclerosis (MS) were examined for T lymphocytes (E-rosette-forming cells, E-RFC) and IgG concentrations in cerebrospinal fluid (CSF) and in blood, and for other lymphocyte subpopulations in blood, before and after treatment with adrenocorticotropical hormone (ACTH). Patients who responded to treatment had higher initial percentages of CSF T lymphocytes (93.1 +/- 3.0%) compared to patients who showed no clinical improvement (88.2 +/- 3.2%). After treatment, the responders had decreased CSF T lymphocyte levels (83.1 +/- 6.6%) while those of the nonresponders were unchanged (90.2 +/- 2.2%). In blood, T cell counts were unchanged, but "active" T cell levels showed a tendency to increase in responders and decreased significantly in non-responders. The percentage of Fc receptor-positive cells increased after treatment in both responders and nonresponders, and that of complement receptor-positive cells increased slightly in responders, but these changes were not statistically significant. Serum IgG concentrations decreased in eight of nine patients after treatment. CSF IgG concentrations were found to be increased, unchanged or decreased, after treatment with no significant differences between responders and nonresponders. The results suggests an involvement of the T lymphocyte in active MS.

Alterations of the immune system in acute pancreatitis and systematic inflammatory response syndrome

A considerable body of evidence indicates the contribution of the immune system to the mechanisms leading to acute pancreatitis [1]. Data focusing on the early course of events at the clinical ground of the process are lacking. The aim of the present study was to provide a complex evaluation of peripheral blood monocyte and subpopulations of lymphocytes in patients with acute pancreatitis and systemic inflammatory response syndrome.

Mononuclear cell subpopulations in CSF and blood of children with bacterial meningitis

To elucidate mononuclear cell subsets of the cerebrospinal fluid (CSF) in order to investigate the pathogenesis of bacterial meningitis (BM). Mononuclear cell and lymphocyte subsets in CSF and peripheral blood from 10 children with acute-stage BM before treatment on the same day were measured by flow cytometry. The control subjects for the subsets of peripheral blood were 15 healthy children. The percentages of CD14+ monocytes/macrophages (median: 56.5%), activated CD14+CD16+ monocytes/macrophages (20.9%), and CD14+CD16+ cells among total CD14+ cells (37.9%) in the CSF were significantly higher than those in the blood of children with BM (p<0.01, p<0.01, and p<0.05, respectively), which were significantly higher than those of the controls (p<0.001, p<0.001, p<0.05, respectively). The percentages of CD3+ (77.3%), CD4+ (45.2%), and CD8+ T cells (32.6%) in the CSF were significantly higher than those in the blood of affected children (p<0.01, p<0.01, and p<0.05, respectively). The percentages of CD3+, CD4+, and CD8+ T cells in the blood of children with BM were significantly lower than those of controls (all p<0.001). The percentages of CD20+ B cells (6.9%) in the CSF were significantly lower than those in the blood of affected children (p<0.01), which were significantly higher than those of controls (p<0.001). The percentages of monocytes/macrophages and T cells in CSF were higher than those in blood in children with BM.

A survey on comparison of Hemiscorpius lepturus and black scorpion effects on peripheral blood subpopulations of patients with scorpion bite under 15 years old referred to Ahwaz Abuzar Hospital

A survey on comparison of Hemiscorpius lepturus and black scorpion effects on peripheral blood subpopulations of patients with scorpion bite under 15 years old referred to Ahwaz Abuzar Hospital

Cytotoxic T lymphocyte activity and CD8 subpopulations in children at risk of HIV infection.

HIV-specific cytotoxic T lymphocytes (CTL) are thought to play a major role in viral control in HIV-infected adults. Changes in the relative proportions of CD8 lymphocyte subpopulations are also thought to be associated with disease progression. Less is known about the relative effectiveness of CTL against different HIV targets, or about the relationship, if any, between CTL activity and CD8 subpopulations. We have measured CTL activity against four HIV gene products (gag, tat, pol and env) and expression of CD45RO, CD45RA, HLA-DR, CD29, S6F1, and CD57 surface markers on CD8 cells from nine HIV-infected and 11 HIV-uninfected children. Of nine HIV-infected children, six showed antigen-specific CTL activity on at least one occasion: 4/6 directed against tat, 6/6 against pol, 1/6 against env, and 1/6 against gag. However, the specificity of the CTL activity varied between children and within individual children with time. Furthermore, two uninfected children showed CTL activity, one to HIV-gag, -pol and -tat, and the other to HIV-pol. All the HIV-infected and two uninfected children had abnormal proportions of CD8 subpopulations in whole blood compared with age-matched controls. There was no correlation between CTL activity and CD8 subsets in whole blood. Five children changed from CTL-positive to CTL-negative (or vice versa) during the study. In these, the occasions when CTL activity was detected coincided with an increase in CD8 cells, an expansion of HLA-DR+ CD8 cells and a loss of CD45RA+ CD8 cells.

Leukocyte numbers and function in subjects eating n-3 enriched foods: selective depression of natural killer cell levels

IntroductionWhile consumption of omega-3 long-chain polyunsaturated fatty acids (n-3 LCPUFA) has been recommended for those at risk of inflammatory disease such as rheumatoid arthritis, the mechanism of their anti-inflammatory effect remains to be clearly defined, particularly in relation to the dose and type of n-3 LCPUFA. The objective of this study was to determine whether varying the levels of n-3 LCPUFA in erythrocyte membrane lipids, following dietary supplementation, is associated with altered numbers and function of circulating leukocytes conducive to protection against inflammation.MethodsIn a double-blind and placebo-controlled study, 44 healthy subjects aged 23 to 63 years consumed either standard or n-3 LCPUFA-enriched versions of typical processed foods, the latter allowing a target daily consumption of 1 gram n-3 LCPUFA. After six months, peripheral blood leukocyte and subpopulation proportions and numbers were assessed by flow cytometry. Leukocytes were also examined for lymphoproliferation and cytokine production, neutrophil chemotaxis, chemokinesis, bactericidal, adherence and iodination activity. Erythrocytes were analyzed for fatty-acid content.ResultsErythrocyte n-3 LCPUFA levels were higher and absolute leukocyte and lymphocyte numbers were lower in subjects consuming n-3 enriched foods than in controls. There were no changes in the number of neutrophils, monocytes, T cells (CD3+), T-cell subsets (CD4+, CD8+) and B cells (CD19+). However, natural killer (NK) (CD3-CD16+CD56+) cell numbers were lower in n-3 supplemented subjects than in controls and were inversely related to the amount of eicosapentaenoic acid or docosahexaenoic acid in erythrocytes. No significant correlations were found with respect to lymphocyte lymphoproliferation and production of IFN-γ and IL-2, but lymphotoxin production was higher with greater n-3 LCPUFA membrane content. Similarly, neutrophil chemotaxis, chemokinesis, bactericidal activity and adherence did not vary with changes in erythrocyte n-3 LCPUFA levels, but the iodination reaction was reduced with higher n-3 LCPUFA content.ConclusionThe data show that regular long-term consumption of n-3 enriched foods leads to lower numbers of NK cells and neutrophil iodination activity but higher lymphotoxin production by lymphocytes. These changes are consistent with decreased inflammatory reaction and tissue damage seen in patients with inflammatory disorders receiving n-3 LCPUFA supplementation.

The Vent-Like Homeobox Gene VENTX2 Is Expressed in Human Hematopoietic Progenitor Cells and Promotes Human Myeloid Development In Vitro and In Vivo.

Recent data indicate that a variety of regulatory molecules active in early development may also play a role in the maintenance of hematopoietic stem cells with repopulating activity. Since it was shown, that the Xvent-2 homeobox gene is part of the BMP-4 signalling pathway in Xenopus, we sought to define the expression profile and function of the human homologue VENTX2 in hematopoietic development. When VENTX2 expression was assessed by TaqMan qRT-PCR in highly purified human cord blood subpopulations, expression of VENTX2 was detected in CD33 and CD15 positive myeloid cells as well as in B and T cells. In addition VENTX2 expression was observed in the primitive CD34+38- human subset as well as in 34+38+ compartment. Furthermore, VENTX2 expression was recurrently detected in bone marrow samples from AML patients at diagnosis as determined by TaqMan qRT-PCR (n=3). In an attempt to characterize the functional relevance of VENTX2 expression for hematopoietic development we retrovirally engineered murine hematopoietic progenitor cells to constitutively express the gene using a MSCV-based retroviral construct with an IRES-EGFP cassette. Successfully transduced cells were injected into lethally irradiated mice or used for in vitro experiments. At the level of the clonogenic progenitor VENTX2 induced myeloid and blocked erythroid differentiation with a significant 1.5 fold increase in the number of CFU-G (n=10; p<0.01) compared to the GFP control and a 5.4 fold decrease in the number of erythroid colonies (n=10; p<0.005) without a change of the total number of colonies produced indicating that VENTX2 promoted granulocytic differentiation in vitro. Re-plating assays showed an over 2 fold increase in the number of secondary CFU-G. In MS-5 murine assay VENTX2 transduced cord blood cells increase CD15+ cells 10.4 fold compared to the control (n=3)(p<0,01). When the effect of VENTX2 was determined on the frequency of LTC-IC by limiting dilution assay (n=2), no major differences were detected between the homeobox gene and the control arm. In NOD/SCID mice VENTX2 induced a 2.9 fold increase in the proportion of CD15+ mature myeloid cells as compared to the GFP control (VENTX2 mean 4 x 106 + 1 x 106 n=7; GFP mean 2x106 cells + 6 x 105 n=9; p<0.02). Using competitive repopulation unit assay (CRU) in NOD/SCID mice we could demonstrate that VENTX2 is able to increase the frequency of CRU cells by 1.8 over GFP control (n=4). These findings characterize VENTX2 as a novel regulatory protein of early human hematopoiesis and extend our knowledge on the regulatory role of non-clustered homeobox genes in hematopoietic development.

A sensitive assay for the quantification of integrin-mediated adhesiveness of human stem cells and leukocyte subpopulations in whole blood

Adhesion of leukocytes is an early step in the formation of adaptive or innate immunity. In chronic inflammatory pathologies like atherosclerosis, regulation of adhesiveness is pivotal for the accumulation of leukocytes within the vessel wall. Therefore, the quantification of adhesion is crucial for the understanding and monitoring of immune responses in patients. However, so far, functional analysis of leukocyte adhesion has been time consuming and required prior purification of cell populations from peripheral blood. This reduced the number of samples and cell populations that could be analysed from limited patient material. Here, we introduce a novel method involving rapid quantification of integrin-mediated leukocyte adhesion in human whole blood using flow cytometry. The quantification relies on soluble multivalent immunocomplexes and is thus called “ligand-complex-based adhesion assay” (LC-AA). LC-AA evaluates both integrin affinity and avidity in T-cells, NK-cells and monocytes from as little as 20 μl of whole blood. In marked contrast to T-cells and NK-cells, unstimulated monocytes show non-blockable background binding of the complexes. Therefore, for this subset only, the stimulation-induced integrin activation is measurable. With the LC-AA, for the first time, measurement of adhesiveness of extremely rare cell populations like CD34+ peripheral blood stem cells can be assessed in the absence of prior purification steps. Finally, the small blood volumes needed for adhesion analysis with the LC-AA allow the evaluation of multiple cell subpopulations in large sample collectives, e.g. required in clinical studies.

186: Superior antitumor in vitro responses of allogeneic matched sibling compared to autologous patient CD8 + T cells

Allogeneic cell therapy as a means to break immunotolerance to solid tumors is increasingly used for cancer treatment. To investigate cellular alloimmune responses in a human tumor model, primary cultures were established from renal cell carcinoma (RCC) tissues of 56patients. In three patients with stable RCC line and human leukocyte antigen (HLA)-identical sibling donor available, allogeneic and autologous RCC reactivities were compared using mixed lymphocyte/ tumor cell cultures (MLTC). Responding lymphocytes were exclusively CD8 + T cells, whereas CD4 + T cells or natural killer cells were never observed. Sibling MLTC populations showed higher proliferative and cytolytic antitumor responses compared with their autologous counterparts. The allo-MLTC responders originated from the CD8 + CD62L(high) + peripheral blood subpopulation containing naive precursor and central memory T cells. Limiting dilution cloning failed to establish CTL clones from autologous MLTCs or tumor-infiltrating lymphocytes. In contrast, a broad panel of RCC-reactive CTL clones was expanded from each allogeneic MLTC. These sibling CTL clones either recognized exclusively the original RCC tumor line or cross-reacted with nonmalignant kidney cells of patient origin. A minority of CTL clones also recognized patient-derived hematopoietic cells or other allogeneic tumor targets. The MHC-restricting alleles for RCC-reactive sibling CTL clones included HLA-A2, HLA-A3, HLA-A11, HLA-A24, and HLA-B7. In one sibling donor-RCC pair, strongly proliferative CD3 + CD16 + CD57 + CTL clones with non-HLA-restricted antitumor reactivity were established. Our results show superior tumor-reactive CD8 responses of matched allogeneic compared with autologous T cells. These data encourage the generation of antitumor T-cell products from HLA-identical siblings and their potential use in adoptive immunotherapy of metastatic RCC patients. (Cancer Res 2006; 66(23): 11447-54)

Clusters Differentiation in HIV Patients According to Measurements of T Cells Subpopulations in Whole Blood After In Vitro Activation with Mitogen

Clusters Differentiation in HIV Patients According to Measurements of T Cells Subpopulations in Whole Blood After In Vitro Activation with Mitogen

Superior AntitumorIn vitroResponses of Allogeneic Matched Sibling Compared with Autologous Patient CD8+ T Cells

Allogeneic cell therapy as a means to break immunotolerance to solid tumors is increasingly used for cancer treatment. To investigate cellular alloimmune responses in a human tumor model, primary cultures were established from renal cell carcinoma (RCC) tissues of 56 patients. In three patients with stable RCC line and human leukocyte antigen (HLA)-identical sibling donor available, allogeneic and autologous RCC reactivities were compared using mixed lymphocyte/tumor cell cultures (MLTC). Responding lymphocytes were exclusively CD8(+) T cells, whereas CD4(+) T cells or natural killer cells were never observed. Sibling MLTC populations showed higher proliferative and cytolytic antitumor responses compared with their autologous counterparts. The allo-MLTC responders originated from the CD8(+) CD62L(high)(+) peripheral blood subpopulation containing naive precursor and central memory T cells. Limiting dilution cloning failed to establish CTL clones from autologous MLTCs or tumor-infiltrating lymphocytes. In contrast, a broad panel of RCC-reactive CTL clones was expanded from each allogeneic MLTC. These sibling CTL clones either recognized exclusively the original RCC tumor line or cross-reacted with nonmalignant kidney cells of patient origin. A minority of CTL clones also recognized patient-derived hematopoietic cells or other allogeneic tumor targets. The MHC-restricting alleles for RCC-reactive sibling CTL clones included HLA-A2, HLA-A3, HLA-A11, HLA-A24, and HLA-B7. In one sibling donor-RCC pair, strongly proliferative CD3(+)CD16(+)CD57(+) CTL clones with non-HLA-restricted antitumor reactivity were established. Our results show superior tumor-reactive CD8 responses of matched allogeneic compared with autologous T cells. These data encourage the generation of antitumor T-cell products from HLA-identical siblings and their potential use in adoptive immunotherapy of metastatic RCC patients.

Adiponectin Deficiency Protects Mice From Chemically Induced Colonic Inflammation

Adiponectin (APN) is an adipokine that regulates insulin sensitivity and is anti-inflammatory in atherosclerosis. The goal of this study was to investigate the role of APN in intestinal inflammation. APN knockout (KO) mice and their wild-type (WT) littermates received dextran sulfate sodium (DSS) or trinitrobenzene sulfonic acid (TNBS) to induce intestinal inflammation. Clinical and histologic scores and proliferation of epithelial cells were assessed. Cytokines and APN levels were measured. Expression of APN and heparin binding epidermal growth factor (HB-EGF) was analyzed by immunohistochemistry. Expression of APN and its receptors, HB-EGF, and basic fibroblast growth factor (bFGF) messenger RNA was assessed by reverse-transcription polymerase chain reaction. Association of serum APN with HB-EGF and bFGF was studied by coimmunoprecipitation. APN KO mice are protected from chemically induced colitis; administration of APN restores inflammation. APN is expressed in the colon, luminal APN associates with colonic epithelial cells. In vitro, APN increases production of proinflammatory cytokines from colonic tissue. Expression of colonic APN overlaps with that of bFGF and HB-EGF, which play a protective role in colitis. Circulating APN binds to bFGF and HB-EGF, likely inhibiting their protective activity. Inhibition of EGF receptor signaling, which is required for biologic activity of HB-EGF, restores inflammation in APN KO mice. APN deficiency is associated with protection from chemically induced colitis. APN exerts proinflammatory activities in the colon by inducing production of proinflammatory cytokines and inhibiting bioactivity of protective growth factors. Thus, in colitis, APN exerts an opposite role compared with atherosclerosis.

Rapid Expansion of Acute Myeloid Leukemia-Reactive Cytotoxic T Cells from CD8+CD62L+ Blood Lymphocytes of HLA-Matched Healthy Donors In Vitro

Allogeneic cytotoxic T-lymphocyte (CTL) therapy in acute myeloid leukemia (AML) is hampered by the poor efficiency of growing leukemia-reactive CTLs from healthy donors in vitro. We established an allogeneic mini-mixed lymphocyte-leukemia culture (MLLC) approach by stimulating comparably small numbers (104/well) of CD8+ T cells isolated from healthy donors against irradiated primary AML blasts in 96-well plates. Prior to use, CD8+ T cells were immunomagnetically separated into a CD62L(high)+ subset enriched for naive precursors and central memory cells as well as a CD62L(low)+/negative subset containing effector memory cells. The culture medium contained IL-7, IL-12, and IL-15. After 2 weeks, IL-12 was replaced by IL-2. Mini-MLLCs were performed in seven different healthy donor-AML pairs that were matched for HLA class I according to high-resolution molecular typing. Following 2 weekly re-stimulations with primary AML blasts, mini-MLLC responder populations were analyzed for reactivity on day 19 of culture using split-well IFN-gamma ELISPOT assays. AML-reactive CD8+ T-cell responders were obtained from all 7 donor-AML pairs. The majority of reactive cultures originated from the CD62L(high)+ subfractions. In 4 out of 7 pairs MLLC responder populations mainly recognized AML blasts, but not Epstein-Barr virus transformed B-lymphoblastoid cell lines of donor and patient origin. The AML-reactive CD8+ T cells were restricted by single HLA class I alleles as determined by blocking experiments using a panel of HLA allele-specific monoclonal antibodies. Representative mini-MLLC responders demonstrated strong cytotoxicity against primary AML blasts in 51Chromium-release assay. Cross-reactivity testing identified an HLA-A*0201-restricted CTL population that recognized AML blasts much stronger than non-malignant monocytes of the same patient. This CTL neither recognized recipient-derived primary fibroblasts nor other hematopoietic cells suggesting a leukemia-associated rather than a minor histocompatibility antigen as the target structure. Several MLLC-derived CTL populations expressed unique T cell receptor Vbeta chains consistent with clonal origin from AML-reactive precursors. Multiple CTL responders reached a cell yield exceeding 108 after 6 to 10 weekly re-stimulations with AML blasts. Our results suggest that in healthy individuals most AML-reactive CD8+ CTLs originate from the CD62L(high)+ peripheral blood subpopulation containing naive precursor and central memory T cells. This mini-MLLC approach allows the rapid expansion of AML-reactive CD8+ CTLs from HLA-matched healthy donors to cell numbers sufficient for antigen identification strategies or adoptive immunotherapy trials.

PSTAT1, pSTAT3, and T‐bet expression in peripheral blood mononuclear cells from relapsing‐remitting multiple sclerosis patients correlates with disease activity

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system, and it is considered to be a T helper 1 (Th1) cell-mediated autoimmune disease. T-bet has been identified as a key transcription factor for the development of Th1 cells and the induction of interferon (IFN)-gamma production. T-bet is induced during T-cell activation by the IFN-gamma signal transducer and activator of transcription (STAT)-1 signalling pathway. In this study we found an up-regulation of T-bet and pSTAT1 in peripheral blood CD4+ and CD8+ T cells and monocytes from relapsing-remitting MS patients in relapse compared with patients in remission and with healthy subjects. The increased expression of pSTAT1 strongly correlated with T-bet expression in CD4+ and CD8+ cells and monocytes from patients in relapse and was associated with an increased production of IFN-gamma by peripheral blood mononuclear cells (PBMCs). pSTAT3 was also up-regulated in CD4+ and CD8+ cells and monocytes from patients in relapse and was associated with an increased production of interleukin (IL)-10 but not of IL-6. pSTAT1, pSTAT3, and T-bet expression strongly correlated with Gd-DTPA-enhanced lesions on brain and spinal cord magnetic resonance imaging. Our data show for the first time that there is an up-regulation of type 1 immunity-correlated transcription factors such as STAT1 and T-bet in peripheral blood subpopulations of MS patients in the active phase of disease. The evaluation of T-bet and pSTAT1 expression in peripheral blood CD4+, CD8+ T cells and monocytes could be used as a marker of disease activity in relapsing-remitting MS.

Characterization of WC1 co-receptors on functionally distinct subpopulations of ruminant γδ T cells

WC1 molecules are implicated in augmenting cellular activation as well as inducing cell cycle arrest of γδ T cells. Since WC1 is a large multigene family differences in outcome could result from modulation of different WC1 molecules. To further investigate this family of molecules, peripheral blood WC1 + γδ T cell subpopulations were evaluated by 2-D Western blotting and RT-PCR. We found 13 cDNA intracytoplasmic tail sequences with differences in signaling motifs among them and at least 20 biochemically distinguishable WC1 spots associated with cell membranes, with some in lipid rafts. An understanding of the diversity of 2-D spots could not be resolved by evaluating T cell clones, removing sialyated carbohydrates or blotting with anti-WC1.1 or anti-WC1.2-specific antibodies. Nevertheless, while the major γδ T cell subpopulations in blood (WC1.1 +/WC1.2 − and WC1.2 +/WC1.1 −) both had complex 2-D patterns, virtually all spots associated with WC1.2 +/WC1.1 − cells bore the WC1.2 epitope, distinguishing them from the WC1.1 + cells.

Expression of Runx1 Isoforms in Sub-Populations of Human Blood and Bone Marrow Cells.

Runx1 is a pivotal regulator of hematopoiesis. Deficiency of Runx1 leads to complete block of definitive hematopoiesis in mouse embryos. Runx1 protein forms a heterodimer with CBFβ and binds to the specific DNA sequence TGT/cGGT to regulate the expression of a number of hematopoietic genes. Runx 1 has been recognized to have three different isoforms (a, b, and c) due to differential splicing and promoter utilization. The distal (Runx1c) promoter region was shown to have binding sites mediating repression of Runx1 transcription. Data on the pattern of Runx1 isoforms expression in different hematopoietic lineages are scant. The functional impact of different Runx1 isoforms on commitment and differentiation of blood cells is also essentially unknown. To further explore this, we studied Runx1 isoform expression in different subpopulations of human hematopoietic cells. Total RNA was extracted from peripheral blood and unstimulated bone marrow. Also, Lin-CD34+CD38+, and Lin-CD34+CD38− cell populations were sorted from G-CSF-stimulated bone marrow and total RNA extracted. Expression of Runx1 isoforms was assessed by semiquantitative RT-PCR analysis, using GAPDH as a control. All 3 isoforms were present in all studied cell populations. Runx1a was uniformly a minor form with lowest expression. Runx1c isoform was a dominant one in peripheral blood as opposed to Runx1b in bone marrow. Most interestingly, the ratio of Runx1b to other isoforms was significantly increased in Lin-CD34+CD38- subpopulation compared to Lin-CD34+CD38+ cells. The prevalence of Runx1c isoform in peripheral blood as compaired to bone marrow is most likely related to a different cellular composition of these tissues and suggests that Runx 1c isoform may be predominantly expressed in some of the mature cell lineages. Since Lin-CD34+CD38− cells represent most primitive progenitor cell population with multi-lineage repopulating ability, the observed overexpression of Runx1b may be related to transcriptional accessibility of these cells. Upregulation of Runx 1b with G-CSF in this subpopulation can not be ruled out as well. These preliminary data suggest that the expression of Runx1 alternative splicing isoforms is both lineage and differentiation stage-specific in human hematopoietic cells. We will also test whether Runx1 isoform expression has a consistent pattern in different types of leukemia and if it is resembles such of mature blood counterparts or primitive progenitors. These studies, as well as the studies of differential expression of Runx1 isoforms in different mature blood lineages and progenitor cell sub-populations are underway and results will be presented. These data may further shed a light on functional differences in Runx1 isoforms and mechanisms of hematopoiesis.

The effect of anti-lymphocyte serum on subpopulations of blood and tissue leucocytes: possible supplementary mechanisms for suppression of rejection and the development of opportunistic infections

Xenogeneic anti-lymphocyte serum (ALS) remains a major reagent for immunosuppression in clinical practice, but mechanisms of action and risks of opportunistic infection have not been considered in the context of innate immunity and its role in immune responsiveness. Rabbit anti rat ALS was administered intraperitoneally. Blood was taken for flow cytometry to establish absolute counts of leucocyte subsets. Tissues were harvested for immunohistology to evaluate interstitial dendritic cells and tissue macrophages. At day 2 of ALS therapy, T cells are completely depleted, as anticipated. B cells are undiminished and form approximately 90% of blood leucocytes. Monocytes and natural killer (NK) cells are substantially (approximately 80%), but not completely, depleted, and there is a trend for diminished numbers of putative dendritic cells. Neither interstitial dendritic cells nor tissue macrophages in heart are affected. The results at day 7 were very similar to day 2. Substantial depletion of blood monocytes and NK cells might attenuate the innate immune system, and represent a possible supplementary mechanism (in addition to T cell depletion) for suppression of rejection. It might be of particular importance in reducing defences against infections. Monitoring these parameters could be of clinical value.