Search

Objective: Immunoglobulin (Ig) G4-related aortic aneurysms (IgG4-AAs) are a special aortic aneurysm among IgG4-related diseases (IgG4-RDs), which are inflammatory and fibrous conditions characterized by tumorous swelling of affected organs and high serum IgG4 concentrations. Recently, IgG4-RD pathogenesis was shown to be associated with T-helper-2 (Th2) and regulatory T (Treg) dominant cytokine production, such as interleukin (IL)-4, IL-10, and IL-13. IL-6 is a key proinflammatory cytokine contributing to lymphocyte and plasmacyte maturation and to atherosclerosis and aneurysm development. We serologically and histopathologically evaluated the cytokine profile in IgG4-AA patients., Methods: Patients with IgG4-AAs (n = 10), non-IgG4-related inflammatory abdominal aortic aneurysms (non-IgG4-AAAs; n = 5), atherosclerotic AAAs (aAAAs; n = 10), and normal aortas without dilatation (n = 10) were examined for serum IL-10, IL-13, and IL-6 levels. Resected aortic tissues were evaluated for cluster of differentiation (CD) 34 (in the endothelial cells and mesenchymal cells) and CD163 (by macrophages) expression using immunohistochemistry and in situ hybridization., Results: Serum IL-10 levels were rather higher in IgG4-AA patients (median, 1.3 pg/mL) than in non-IgG4-AAA and aAAA patients and in patients with normal aortas. Elevated serum IL-13 levels relative to standard values were detected in two IgG4-AA patients but not in the other groups. Cells immunopositive for IL-10 and IL-13 were more frequent in IgG4-AAs and significantly correlated with serum IgG4 levels. Serum IL-6 levels (median, 78.5 pg/mL) were also significantly higher in IgG4-AA patients than in non-IgG4-AAA and aAAA patients and control patients with normal aortas (P = .01, P = .001, and P = .004, respectively). They positively correlated with serum IgG4 levels and adventitial thickness, but other cytokines did not. The number of IL-6-immunopositive cells in the adventitia was significantly higher in IgG4-AA patients (median, 17.8/high-power field) than in aAAA patients or patients with normal aortas (P =.001 and P = .002, respectively). In situ hybridization confirmed frequent IL-6 messenger (m)RNA expression in the endothelium, mesenchymal cells, and histiocytes in IgG4-AA adventitia. In the same cells of IgG4-AAs, coexpression of IL-6 and CD34 mRNA or CD163 mRNA was detected., Conclusions: The cytokine profiles of IgG4-AA patients had two characteristics: local IL-10 and IL-13 upregulation in IgG4-AAs was related to Th2 and Treg-predominant cytokine balance, similar to other IgG4-RDs, and IL-6 upregulation in the adventitia was characterized by activated immune reactions in IgG4-AA patients. IL-6 synthesis, through contributions of mesenchymal cells and macrophages in the adventitia, is strongly involved in IgG4-AA pathogenesis or progression, or both., (Copyright © 2017 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

The differential recognition of fungal cell wall polysaccharides that program innate and adaptive immunity to the human opportunistic fungal pathogen Aspergillus fumigatus has been a focus of considerable interest. In a mouse model of fungal conidia aspiration, decreased relative levels of cell wall core carbohydrates β-1,3-glucan to chitin in A. fumigatus isolates and mutant strains were correlated with increased airway eosinophil recruitment. In addition, an increase in fungal surface chitin exposure induced by the β-1,3-glucan synthesis-targeting drug caspofungin was associated with increased murine airway eosinophil recruitment after a single challenge of conidia. The response to increased A. fumigatus chitin was associated with increased transcription of IL-17A after a single aspiration, although this cytokine was not required for eosinophil recruitment. Rather, both RAG1 and γδ T cells were required, suggesting that this subset of innate-like lymphocytes may be an important regulator of potentially detrimental type 2 immune responses to fungal inhalation and infection., (Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Several CD4 + T cell subtypes contribute to immune homeostasis in malaria, but the markers that define the main suppressive T cell subsets induced by this infection remain largely unknown. Here we provide a detailed phenotypic characterization of immunoregulatory CD4 + T cell populations in uncomplicated human malaria. We found an increased proportion of CD4 + T cells expressing CTLA-4, OX40, GITR, TNFRII, and CD69 in acute-phase single-species infections with Plasmodium vivax, Plasmodium falciparum, or both. Such an increase was not proportional to parasite density in P. vivax infections, and did not persist after parasite clearance. Significantly, less than 10% of CD4 + T cells expressing these regulatory molecules had the classical T regulatory (Treg) phenotype (CD4 + CD25 + CD127 - FoxP3 + ). Two major Treg cell subpopulations, which together accounted for 19-23% of all Treg cells circulating in malaria patients, expressed surface receptors with opposing regulatory functions, either CTLA-4 or OX40. OX40 + Treg cells outnumbered their CTLA-4 + counterparts (1.8:1) during acute P. vivax infection, while a more balanced ratio (1.3:1) was observed following parasite clearance These data reveal new players in the complex CD4 + Treg cell network that maintains immune homeostasis in malaria and suggest potential targets for therapeutic interventions to improve parasite-specific effector immune responses., Competing Interests: The authors declare that no competing interests exist., (Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Although most autoimmune diseases are connected to major histocompatibility complex (MHC) class II alleles, a small number of these disorders exhibit a variable degree of association with selected MHC class I genes, like certain human HLA-A and HLA-B alleles. The basis for these associations, however, has so far remained elusive. An understanding might be obtained by comparing functional, biochemical, and biophysical properties of alleles that are minimally distinct from each other, but are nevertheless differentially associated to a given disease, like the HLA-B*27:05 and HLA-B*27:09 antigens, which differ only by a single amino acid residue (Asp116His) that is deeply buried within the binding groove. We have employed a number of approaches, including X-ray crystallography and isotope-edited infrared spectroscopy, to investigate biophysical characteristics of the two HLA-B27 subtypes complexed with up to ten different peptides. Our findings demonstrate that the binding of these peptides as well as the conformational flexibility of the subtypes is greatly influenced by interactions of the C-terminal peptide residue. In particular, a basic C-terminal peptide residue is favoured by the disease-associated subtype HLA-B*27:05, but not by HLA-B*27:09. This property appears also as the only common denominator of distinct HLA class I alleles, among them HLA-B*27:05, HLA-A*03:01 or HLA-A*11:01, that are associated with diseases suspected to have an autoimmune etiology. We postulate here that the products of these alleles, due to their unusual ability to bind with high affinity to a particular peptide set during positive T cell selection in the thymus, are involved in shaping an abnormal T cell repertoire which predisposes to the acquisition of autoimmune diseases., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Notch plays multiple roles in T cell development in the thymus and T cell differentiation in the periphery. In order to systematically examine the role of Notch in T cell biology, we determined the cell surface expression of all Notch receptors and ligands on various populations of T cells by using a panel of specific monoclonal antibodies we recently established. Notch1 and Notch3 were upregulated at double-negative (DN) 2-DN4 stages of immature thymocytes, then downregulated on mature single-positive thymocytes and peripheral T cells, but were rapidly upregulated again upon activation. Notch2 was consistently expressed on T cells while Notch4 was not. Jagged1 and Jagged2 were expressed at double-positive stage of immature T cells. Jagged2 was also inducible on mature T cells upon activation. In contrast, no Delta-like (Dll) 1 or Dll4 expression was observed on T cells. These comprehensive profiling of the expression of Notch receptors and ligands would be informative to fully understand the role of individual Notch receptors and ligands in T cell development and differentiation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In a proportion of patients with chronic myeloid leukemia (CML) being treated with dasatinib, we recently observed large granular lymphocyte (LGL) expansions carrying clonal T-cell receptor (TCR) gamma/delta gene rearrangements. To assess the prevalence and role of clonal lymphocytes in CML, we collected samples from patients (n = 34) at the time of diagnosis and during imatinib and dasatinib therapies and analyzed lymphocyte clonality with a sensitive polymerase chain reaction-based method of TCR gamma and delta genes. Surprisingly, at CML diagnosis, 15 of 18 patients (83%) had a sizeable clonal, BCR-ABL1 negative lymphocyte population, which was uncommon in healthy persons (1 of 12; 8%). The same clone persisted at low levels in most imatinib-treated patients. In contrast, in a distinct population of dasatinib-treated patients, the diagnostic phase clone markedly expanded, resulting in absolute lymphocytosis in blood. Most patients with LGL expansions (90%) had TCR delta rearrangements, which were uncommon in patients without an LGL expansion (10%). The TCR delta clones were confined to gammadelta(+) T- or natural killer-cell compartments and the TCR gamma clones to CD4(+)/CD8(+) alphabeta(+) fractions. The functional importance of clonal lymphocytes as a part of leukemia immune surveillance and the putative anergy-reversing role of dasatinib require further evaluation.

Cutaneous T-cell lymphoma (CTCL) encompasses leukemic variants (L-CTCL) such as Sézary syndrome (SS) and primarily cutaneous variants such as mycosis fungoides (MF). To clarify the relationship between these clinically disparate presentations, we studied the phenotype of T cells from L-CTCL and MF. Clonal malignant T cells from the blood of L-CTCL patients universally coexpressed the lymph node homing molecules CCR7 and L-selectin as well as the differentiation marker CD27, a phenotype consistent with central memory T cells. CCR4 was also universally expressed at high levels, and there was variable expression of other skin addressins (CCR6, CCR10, and CLA). In contrast, T cells isolated from MF skin lesions lacked CCR7/L-selectin and CD27 but strongly expressed CCR4 and CLA, a phenotype suggestive of skin resident effector memory T cells. Our results suggest that SS is a malignancy of central memory T cells and MF is a malignancy of skin resident effector memory T cells.

A DNA vaccine against infectious laryngotracheitis virus (ILTV) can induce specific humoral and cell-mediated immunity. However, compared to conventional vaccines, DNA vaccines usually induce poor antibody responses. To determine if co-expression of a cytokine can result in a more potent ILTV DNA vaccine, immunogenicity and protective efficacy of a monocistronic vector encoding the glycoprotein B (gB) of ILTV was compared to that of a bicistronic vector separately encoding the gB and chicken interleukin-18. Humoral and cellular responses induced by the DNA vaccines administered to the quadriceps muscle of chickens were evaluated. There were significant differences in antibody levels elicited by either monocistronic or bicistronic DNA vaccines as determined by ELISA. The percentages of CD3(+), CD3(+)CD8(+) and CD3(+)CD4(+) subgroups of peripheral blood T-lymphocytes in chickens immunized with the bicistronic DNA vaccine were higher than those in chickens immunized with monocistronic DNA vaccine. When chickens were challenged with a virulent CG strain of ILTV, the protective efficacy was enhanced significantly after immunization with the bicistronic DNA vaccine. These results demonstrated that co-expression of an adjuvant cytokine from a bicistronic DNA vaccine may be an effective approach to increasing ILTV DNA vaccine immunogenicity., (Copyright 2010 Elsevier B.V. All rights reserved.)

T-cell development follows a defined set of stage-specific differentiation steps. However, molecular and cellular events occurring at early stages of human T-cell development remain to be fully elucidated. To address this, human umbilical cord blood (UCB) hematopoietic stem cells (HSCs) were induced to differentiate to the T lineage in OP9-DL1 cocultures. A developmental program involving a sequential and temporally discrete expression of key differentiation markers was revealed. Quantitative clonal analyses demonstrated that CD34(+)CD38(-) and CD34(+)CD38(lo) subsets of UCB contain a similarly high T-lineage progenitor frequency, whereas the frequency in CD34(+)CD38(+/hi) cells was 5-fold lower. Delta-like/Notch-induced signals increased the T-cell progenitor frequency of CD34(+)CD38(-/lo) cells differentiated on OP9-DL1, and 2 distinct progenitor subsets, CD34(+)CD45RA(+)CD7(++)CD5(-)CD1a(-) (proT1) and CD34(+)CD45RA(+)CD7(++)CD5(+)CD1a(-) (proT2), were identified and their thymus engrafting capacity was examined, with proT2 cells showing a 3-fold enhanced reconstituting capacity compared with the proT1 subset. Furthermore, in vitro-generated CD34(+)CD7(++) progenitors effectively engrafted the thymus of immunodeficient mice, which was enhanced by the addition of an IL-7/IL-7 antibody complex. Taken together, the identification of T-progenitor subsets readily generated in vitro may offer important avenues to improve cellular-based immune-reconstitution approaches.

Sheep develop clinical diseases after 3-5 years after infection with Mycobacterium avium subsp. paratuberculosis (MAP). Clinical symptoms of paratuberculosis include persistent diarrhea and weight loss due to a chronic inflammation of the small intestine. Tissue alterations in the areas of the ileo-cecal junction are often observed. Here, we investigate the molecular processes underlying tissue damages in intestinal mucosa of 14 sheep showing either tuberculoid or lepromatous form of MAP enteritis. We found that E-cadherins, alpha-catenin and beta1-integrins were present at significant low levels in tissues of sheep affected by lepromatous form and that this pattern was associated with high expression of TGF-beta, IL-10, IL-1beta, and TNF-alpha and with a modest increase of CD4+ and CD25+ T cells. Tissues of sheep with the tuberculoid form showed high expression of IFNgamma, IL-12, and MCP-1 and a significant presence of CD4+ and CD25+ T cells. Finally, anti-transglutaminase (tTG) IgG1 antibodies were detected in sera of infected animal belonging to both groups, as already described for human inflammatory bowel diseases. Our results further stress the similarities in the clinical and histological features between ruminant paratuberculosis and human intestinal inflammatory diseases.

The outcome of classical Hodgkin lymphoma (cHL) patients may be related to the tumor microenvironment, which in turn may be influenced by Epstein-Barr virus (EBV) infection. To characterize the cHL microenvironment, a set of 63 cHL tissue samples was profiled using DNA microarrays. Their gene expression profile differed from that of histiocyte T cell-rich B-cell lymphoma (H/TCRBCL) samples that were used as controls, mainly due to high expression of PDCD1/PD-1 in H/TCRBCL. EBV(+) cHL tissues could be distinguished from EBV(-) samples by a gene signature characteristic of Th1 and antiviral responses. Samples from cHL patients with favorable outcome overexpressed genes specific for B cells and genes involved in apoptotic pathways. An independent set of 146 cHL samples was analyzed using immunohistochemistry. It showed a significant adverse value in case of high percentage of either TIA-1(+)-reactive cells or topoisomerase-2(+) tumor cells, whereas high numbers of BCL11A(+), FOXP3(+), or CD20(+) reactive cells had a favorable influence. Our results suggest an antitumoral role for B cells in the cHL microenvironment and a stronger stromal influence of the PD1 pathway in H/TCRBCL than cHL. The observation of Th1/ antiviral response in EBV(+) cHL tissues provides a basis for novel treatment strategies.

The tyrosine kinase JAK3 plays a well-established role during normal lymphocyte development and is constitutively phosphorylated in several lymphoid malignancies. However, its contribution to lymphomagenesis remains elusive. In this study, we used the newly identified activating JAK3A572V mutation to elucidate the effect of constitutive JAK3 signaling on murine lymphopoiesis. In a bone marrow transplantation model, JAK3A572V induces an aggressive, fatal, and transplantable lymphoproliferative disorder characterized by the expansion of CD8(+)TCRalphabeta(+)CD44(+)CD122(+)Ly-6C(+) T cells that closely resemble an effector/memory T-cell subtype. Compared with wild-type counterparts, these cells show increased proliferative capacities in response to polyclonal stimulation, enhanced survival rates with elevated expression of Bcl-2, and increased production of interferon-gamma (IFNgamma) and tumor necrosis factor-alpha (TNFalpha), correlating with enhanced cytotoxic abilities against allogeneic target cells. Of interest, the JAK3A572V disease is epidermotropic and produces intraepidermal microabscesses. Taken together, these clinical features are reminiscent of those observed in an uncommon but aggressive subset of CD8(+) human cutaneous T-cell lymphomas (CTCLs). However, we also observed a CD4(+) CTCL-like phenotype when cells are transplanted in an MHC-I-deficient background. These data demonstrate that constitutive JAK3 activation disrupts T-cell homeostasis and induces lymphoproliferative diseases in mice.

Recent reports have established an important role of CD4+CD25+ T cells in the immune regulation of infectious diseases, autoimmune disorders and cancer. In the present work, we investigated whether these cells had a regulatory role during Trypanosoma cruzi infection, using the Colombian strain. Inactivation of CD4+CD25+ cells in vivo conferred mice slightly more resistant to infection with the Colombian strain of T. cruzi, as evidenced by lower parasitemia and mortality rates. The augmented resistance to infection with Colombian strain did correlate with increased activation of effector CD4 cells. It was antibody-independent, since no difference in levels of IgM, IgG, IgG1 and IgG2a(b) recognizing T. cruzi antigens was observed throughout the infection of CD25-inactivated and control mice. Regarding pathogenesis, inflammatory infiltrate and frequency of CD4 and CD8 T cells or macrophages in the cardiac tissue was similar in both groups. Together, our data indicate that CD4+CD25+ cells have a limited role on host resistance during early T. cruzi infection. Despite exhaustive investigation, we did not observe any role for these regulatory cells in the pathogenesis of experimental chronic Chagas' disease.

Background: Abnormal cell proliferation, which results from deregulation of the cell cycle, is fundamental in tumorigenesis., Objectives: To investigate the expression of proliferation markers and cell cycle regulators in a range of T cell lymphoproliferative skin diseases., Methods: We studied skin specimens of 51 patients with parapsoriasis (PP), mycosis fungiodes (MF), or lymphomatoid papulosis (LyP). Immunohistochemistry was performed for Ki-67, proliferating cell nuclear antigen (PCNA), minichromosome maintenance protein 7 (MCM7), and p21., Results: MF with stage IIB-IV and LyP showed a significantly greater number of Ki-67-positive cells than PP (P=0.02 and 0.001) and MF I-IIA (P=0.019 and 0.003), respectively. MCM7 staining revealed significantly higher labeling indices for MF IIB-IV and LyP when compared to PP (P=0.002 and 0.04) and MF I-IIA (P=0.0005 and 0.01), respectively. Compared to PP and MF I-IIA, MF IIB-IV was associated with significantly higher labeling indices for PCNA (P=0.006 and 0.0004). p21 staining was significantly increased in MF IIB-IV and LyP when compared to PP (P=0.006 and 0.003) and MF I-IIA (P=0.003). However, p21 staining was all in all very weak., Conclusions: Ki-67 and PCNA seem to be useful immunohistological parameters for the correlation with the clinical stage of MF. In the differentiation and prognostication of T cell lymphoproliferative skin disorders, MCM7 may serve as a novel biomarker which is, in contrast to Ki-67 and PCNA, stable throughout the cell cycle.

T cells are thought to play a critical role in cerebral malaria pathogenesis. However, available evidences are restricted to rodent models in which V beta specific T cell expansion has been associated with neurological syndrome suggesting involvement of superantigens or dominant antigens. Using flow cytometry, we studied the peripheral V beta T cell repertoire of Ghanaian children with cerebral malaria, uncomplicated malaria and asymptomatic control children, to look for either expansion or deletion of specific V beta associated with cerebral malaria. At admission, the general pattern of the repertoire of the patients was very similar, with no major distortion compared to the control group a part a significant increase of the frequency of the V beta 21.3 subset correlating with disease severity and attributed to the CD4 subset. During convalescence very limited fluctuations were observed including a significant decrease of the V beta 21.3 subset and increase of the V beta 20 subset, a subset not detected at admission. The remarkable stability of the V beta repertoire observed in acute malaria either cerebral or uncomplicated argues against the idea that cerebral malaria would result from a T cell-mediated inflammatory shock syndrome driven by some dominant super-antigenic activity(ies). The significance of the reproducible increase of the CD4+V beta 21.3T cell subset deserves further investigations.

Natural killer (NK) cell-or T cell-mediated cytotoxicity traditionally is measured in 4-16 h (51)Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3(-)CD16(+)CD56(+)). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. The phenotype of effectors, viability of targets, the formation of tumor-effector cell conjugates and absolute numbers of all cells were measured based on light scatter (FSC/SSC), double discrimination of the fluorescence peak integral and height, and fluorescence intensity. Kinetic studies (0.5 and 1 to 4 h) at different effector to target (E:T) cell ratios (50, 25, 12, and 6) confirmed that the 3 h incubation was optimal. The FCC assay is more sensitive than the CRA, has a coefficient of variation (CV) 8-13% and reliably measures NK cell-or lymphokine-activated killer (LAK) cell-mediated killing of target cells in normal controls and subjects with cancer. The FCC assay can be used to study a range of phenotypic attributes, in addition to lytic activity of various subsets of effector cells, without radioactive tracers and thus, it is relatively inexpensive. The FCC assay has a potential for providing information about molecular interactions underlying target cell lysis and thus becoming a major tool for studies of disease pathogenesis as well as development of novel immune therapies.

Cancer results from a tumor cell intrinsic dysregulation of oncogenes, tumor suppressor and stability genes as well as from the avoidance of immunosurveillance. A complex network of cellular interactions allows one to mount cognate anti-tumor immune responses. Recently, discoveries have been made regarding the links between innate and cognate antitumor immunity eliciting protective T-cell responses. The intricate differentiation pathway, whereby dendritic cells can efficiently mature in the tumor microenvironment, appears crucial for the priming of T cells. Transformed cells might deliver danger signals directly to the dendritic cell. Alternatively, other cell types belonging to the innate immune system can sense transformed cells through a specific set of receptors and then interact with dendritic cells to modulate their activation state. A novel subset of innate effector cells called interferon-producing killer dendritic cells are multitasking chimeras that can recognize and kill transformed cells, and undergo a maturation state of antigen presentation. Also, evidence has been produced suggesting that cell death promoted by conventional chemotherapy or radiotherapy might elicit interactions between the innate and the cognate immune system that result in anti-tumor immune responses.

In HLA-incompatible hematopoietic stem cell transplantation, alloreactive donor T cells recognizing recipient mismatch HLA cause severe graft-versus-host disease (GVHD). Strategies allowing the selective depletion of alloreactive T cells as well as the enhancement of graft-versus-malignancy immunity would be beneficial. We generated donor CD8 T-cell lines in vitro using allogeneic recipient cells mismatched at a single HLA class I allele or haplotype as stimulators. Recipient cells were obtained from acute myeloid leukemias, renal-cell carcinomas, and CD40L-induced B lymphoblasts. Resulting alloreactive T cells were activated by incubating day 21 T-cell cultures with HLA-mismatch transfected K562 cells or recipient-derived fibroblasts. Selective allodepletion (SAD) was subsequently performed by a newly developed immunomagnetic depletion approach targeting the tumor necrosis factor receptor molecule CD137 (4-1BB). Compared with other activation-induced antigens, CD137 showed a superior performance based on a consistently low baseline expression and a rapid up-regulation following alloantigen stimulation. In 15 different SAD experiments, the frequency of alloreactive CD8 T cells was reduced to a median of 9.5% compared with undepleted control populations. The allodepleted T-cell subsets maintained significant antitumor and antiviral CD8 responses. In vitro expansion of tumor-reactive T cells followed by CD137-mediated SAD might enhance the antitumor efficacy of T-cell allografts with lower risk of inducing GVHD.

In compliance with federal regulations, blood banks routinely use leukocyte depletion filters to eliminate contaminating leukocytes from blood products such as red blood cell and platelet concentrates. We developed and optimized conditions to elute leukocytes adsorbed to these filters; resulting in leukocyte suspensions which we termed Filter Buffy Coats (FBCs). These Filter Buffy Coats can replace standard buffy coats for various research applications. After optimizing both the filter elution medium as well as elution protocols, we compared commonly used leukocyte depletion filters from four different manufacturers. Relative fractions as well as total recoveries of leukocyte subsets, such as lymphocytes, monocytes and granulocytes, found in Filter Buffy Coats were identified and compared among the filters as well as to standard buffy coats and whole blood. Flow cytometric analysis of Filter Buffy Coats confirmed the presence of T- and B-lymphocytes, NK cells and monocytes. Furthermore, a significant quantity of CD34(+) hematopoietic stem or progenitor cells (HSC/HPC) was detected in Filter Buffy Coats prepared from different filters, thus making FBCs a valuable source for research on HSC/HPC. Colony assays revealed that most of these CD34(+) cells are functional. Using immunomagnetic cell sorting (MACS), we isolated a variety of leukocyte populations from FBC mononuclear cells (Filter-PBMCs) including T lymphocytes (CD4(+), CD8(+), CD3(+)), B lymphocytes (CD19(+)), NK cells (CD56(+)), HSC/HPC (CD34(+), CD133(+)) or dendritic cells (BDCA-4(+)). Functional properties of Filter-PBMCs, as well as of some of these isolated leukocyte populations, were confirmed using standard assays. In summary, Filter Buffy Coats are a valuable and convenient source of different peripheral leukocyte populations and can replace standard buffy coat preparations for research applications.

Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells use the perforin/granzyme pathway as a major mechanism to kill pathogen-containing cells and tumor cells.(1,2) Dysregulation of this pathway results in several human diseases, such as hemophagocytic lymphohistiocytosis. Here we characterize the single-cell expression pattern of granzymes A and B in human lymphocytes using a flow cytometry-based assay. We demonstrate that most circulating CD56(+)8(-) NK cells, and approximately half of circulating CD8(+) T lymphocytes, coexpressed both granzymes A and B. In contrast, few circulating CD4(+) T lymphocytes expressed granzymes A or B. Activation of CD8(+) T lymphocytes with concanavalin A (ConA)/interleukin-2 (IL-2), and activation of CD4(+) T lymphocytes with antibodies to CD3/CD28 or CD3/CD46 (to generate T regulatory [Tr1] cells), induced substantial expression of granzyme B, but not granzyme A. Naive CD4(+)CD45RA(+) cells stimulated with antibodies to CD3/CD46 strongly expressed granzyme B, while CD3/CD28 stimulation was ineffective. Finally, we show that granzyme B-expressing CD4(+) Tr1 cells are capable of killing target cells in a perforin-dependent, but major histocompatibility complex (MHC)/T-cell receptor (TCR)-independent, manner. Our results demonstrate discordant expression of granzymes A and B in human lymphocyte subsets and T regulatory cells, which suggests that different granzymes may play unique roles in immune system responses and regulation.

We report on the characteristics of 21 patients with hepatosplenic gammadelta T-cell lymphoma (HSgammadeltaTCL), an entity recognized since 1994 in the Revised European American Lymphoma (REAL) classification. Median age was 34 years. Patients had splenomegaly (n = 21), hepatomegaly (n = 15), and thrombocytopenia (n = 20). Histopathologic findings were homogeneous and showed the presence of medium-sized lymphoma cells within the sinusoids of splenic red pulp, liver, and bone marrow. Marrow involvement was usually mild but could be demonstrated by phenotyping in all patients. Cells were CD3+CD5-, expressed the gammadelta T-cell receptor, and had a nonactivated cytotoxic cell phenotype (TIA-1+, granzyme B-). Most patients were CD4-/CD8- (16 of 18); CD56+ (15 of 18), expressed the Vdelta1epitope (Vd1+/Vd2-/Vd3-) (9 of 12); and were negative for Epstein-Barr virus (EBV) (18 of 20). Isochromosome arm 7q was documented in 9 of 13 patients. Eight patients had previously undergone kidney transplantation or had a history of systemic lupus, Hodgkin disease, or malaria. Prognosis was poor; median survival time was 16 months, and all but 2 patients ultimately died despite consolidative or salvage high-dose therapy. In conclusion, HSgammadeltaTCL is a disease with distinctive clinical, histopathologic, and phenotypic characteristics. Bone marrow biopsy with combined phenotyping is sufficient for diagnosis, and splenectomy is therefore unwarranted. Current treatment modalities appear to be ineffective in most patients.

Contrary to our abundant knowledge about the sensitization phase of human contact hypersensitivity, little is known about the cell types orchestrating the effector phase. In order to address this issue, we phenotypically analyzed biopsies from 72 h epicutaneous patch test reactions (n=10) and normal human skin (n=5) for the presence of various leukocyte differentiation antigens. The inflammatory infiltrate was dominated by CD3+/CD4+ T cells with approximately 30% of the cells coexpressing CD25 and CTLA-4, a phenotype consistent with either activated effector or regulatory T cells. In our search for professional antigen-presenting cells, we were surprised to find not only sizeable numbers of CD1a+ dendritic cells and CD1c+ dendritic cells, but also of CD123+, CD45RA+, BDCA-2+, CLA+, and CD62L+ plasmacytoid dendritic cells. Although virtually absent in normal human skin, these cells were detectable already 6 h after hapten challenge and were often found in close proximity to CD56+ natural killer cells, indicative of a functional interaction between these cell types. The detailed knowledge of the cellular composition of the inflammatory infiltrate in allergic contact dermatitis and its kinetics should form the basis for the investigation of the immunologic and molecular events operative in the perpetuation and resolution of the eczematous response.

The importance of alpha beta versus gamma delta T-cell subset antigen expression in the classification of peripheral T-cell lymphomas is still unclear. The objective of this study was to investigate the prognostic value of T-cell receptor-delta 1 (TCR delta 1) expression in primary cutaneous T-cell lymphomas. TCR delta 1 cellular expression was assessed in skin biopsy specimens of 104 individuals with cutaneous T-cell lymphoma by immunohistochemistry. Both univariate (Kaplan-Meier) and multivariate (Cox regression) analyses were conducted to determine which variables (T-cell subtype, hemophagocytosis, histologic profile, age, sex, and adenopathy) were significantly associated with survival. Univariate analysis indicated that there was a statistically significant difference in survival between the patients with alpha beta cutaneous T-cell lymphoma and patients with gamma delta cutaneous T-cell lymphoma (P <.0001). There was also a statistically significant decrease in survival among patients who had subcutaneous involvement compared with patients who had epidermotropic and/or dermal involvement (P <.0001). Cox model analysis indicated that TCR delta 1 expression was the factor that was most closely associated with decreased survival (P <.0001). Among those patients with cutaneous gamma delta T-cell lymphoma (n = 33), there was a trend for decreased survival for patients who had histologic evidence of subcutaneous fat involvement in comparison with patients who had epidermotropic or dermal patterns of infiltration (P =.067). No other prognostic factors were identified as having a notable association with outcome in this subgroup. TCR delta 1 expression in primary cutaneous lymphomas is an independent prognostic factor associated with decreased survival.

We have analyzed the phenotype, cytokine profile, and mitotic history (telomere length) of monoclonal T-cell expansions in 5 CD3(+) T-cell large granular lymphocyte (TLGL) leukemia patients by fluorescence activated cell sorting (FACS) and single-cell polymerase chain reaction (PCR). We confirm that the common phenotype of TLGL leukemia is CD3(+)CD8(+)CD45RA(+)CD27(-)CD94(+)(CD57(+)). Interestingly, the C-type lectin-like type killer cell receptor CD94 was invariably associated with the activating form of its signal-transducing molecule NKG2. Furthermore, when judged by criteria such as interferon gamma (IFN-gamma)/tumor necrosis factor (TNF) production, expression of granzyme, FasL, and NKG2D, the TLGL cells had all the features of a cytotoxic effector T cell. Telomere shortening in TLGL cells was in the normal range for CD8(+) T cells, indicating that they had not divided significantly more than chronically stimulated CD8(+) T cells in healthy individuals. In 25 of 27 controls, cells with a TLGL phenotype occurred at low (1%-3%) frequencies. However, in the other 2 individuals (ages 28-36 years), large stable (> 3 years) monoclonal expansions of CD3(+)CD8(+)CD45RA(+)CD27(-)CD57(+)CD94(+) NKG2C(+) were found which rendered these controls phenotypically indistinguishable from TLGL leukemia patients. We believe that the TLGL clonopathy, rather than being of a neoplastic nature, is more likely an extreme manifestation of the large and stable clonal size characteristic of CD8(+) effector cells. Such a TLGL clone consisting of cells without any particular pathologic trait might exist in a considerable number of individuals. Clinical symptoms may occur in individuals in whom the TLGL clone encounters antigen and is triggered to produce large amounts of effector molecules that dysregulate the immune system, which could manifest itself as autoimmunity or as a FasL-mediated neutropenia.

Whereas most peripheral CD8(+) alphabeta T cells highly express CD8alphabeta heterodimer in healthy individuals, there is an increase of CD8alpha(+)beta(low) or CD8alphaalpha alphabeta T cells in HIV infection or Wiskott-Aldrich syndrome and after bone marrow transplantation. The significance of these uncommon cell populations is not well understood. There has been some question as to whether these subsets and CD8alpha(+)beta(high) cells belong to different ontogenic lineages or whether a fraction of CD8alpha(+)beta(high) cells have down-regulated CD8beta chain. Here we assessed clonality of CD8alphaalpha and CD8alpha(+)beta(low) alphabeta T cells as well as their phenotypic and functional characteristics. Deduced from surface antigens, cytotoxic granule constituents, and cytokine production, CD8alpha(+)beta(low) cells are exclusively composed of effector memory cells. CD8alphaalpha cells comprise effector memory cells and terminally differentiated CD45RO(-)CCR7(-) memory cells. T-cell receptor (TCR) Vbeta complementarity-determining region 3 (CDR3) spectratyping analysis and subsequent sequencing of CDR3 cDNA clones revealed polyclonality of CD8alpha(+)beta(high) cells and oligoclonality of CD8alpha(+)beta(low) and CD8alphaalpha cells. Importantly, some expanded clones within CD8alphaalpha cells were also identified within CD8alpha(+)beta(high) and CD8alpha(+)beta(low) subpopulations. Furthermore, signal-joint TCR rearrangement excision circles concentration was reduced with the loss of CD8beta expression. These results indicated that some specific CD8alpha(+)beta(high) alphabeta T cells expand clonally, differentiate, and simultaneously down-regulate CD8beta chain possibly by an antigen-driven mechanism. Provided that antigenic stimulation directly influences the emergence of CD8alphaalpha alphabeta T cells, these cells, which have been previously regarded as of extrathymic origin, may present new insights into the mechanisms of autoimmune diseases and immunodeficiencies, and also serve as a useful biomarker to evaluate the disease activities.

T cell proliferative responses decrease with age, but the mechanisms responsible are unknown. We examined the impact of age on memory and naive CD4(+) T cell entry and progression through the cell cycle using acridine orange to identify cell cycle stage. For both subsets, fewer stimulated cells from old donors were able to enter and progress through the first cell cycle, with an increased number of cells arrested in G(0) and fewer cells in post G(0) phases. The number of dead cells as assessed by sub-G(0) DNA was also significantly greater in the old group. CD4(+) T cells from old mice also exhibited a significant reduction in clonal history as assessed by CFSE staining. This was associated with a significant decline in cyclin D2 mRNA and protein. We propose that decreases in cyclin D2 are at least partially responsible for the proliferative decline found in aged CD4(+) T cells.

We purified a 70 kDa O-glycoprotein that binds to the GalNAc specific lectin from Amaranthus leucocarpus (ALLr) and determined its expression pattern on T lymphocytes from different murine lymphoid organs. High level of ALLr expression was demonstrated in 95-98% of both CD4(+)8(+) and CD4(-)8(+) thymocytes, and in 80-95% of CD8(+) T cells from peripheral blood, lymph nodes, and spleen, whereas a minor fraction of CD4(+)8(-) thymocytes (46-67%) and peripheral CD4(+) T cells (9-40%) showed low ALLr expression. Peripheral CD19(+) B cells were ALLr negative and most of the peripheral ALL(+) T cells showed a CD62L(hi)CD45RB(hi)CD44(lo/-) phenotype, indicating features of naive cells. Mitogenic activation of peripheral T cells increased 3-fold the number of ALL(+)CD4(+) T cells 24 h after stimulation, as opposed to a >80% decrease in CD8(+) T cells 72 h after stimulation. Our results suggest that ALL detects a non-described surface O-glycoprotein selectively expressed by naive CD8(+) T cells and by early activated CD4(+) T cells.

The "conventional" NK1.1(-) T cells from mouse blood and marrow were compared with regard to surface receptors, cytokine secretion, and function. Most blood NK1.1(-) CD4(+) and CD8(+) T cells expressed the naive CD44(int/lo)CD62L(hi)CD45RB(hi) T-cell phenotype typical of those in the peripheral lymphoid tissues. In contrast, most marrow NK1.1(-) CD4(+) and CD8(+) T cells expressed an unusual CD44(hi)CD62L(hi)CD45RB(hi) phenotype. The blood NK1.1(-) CD4(+) T cells had a naive T-helper cytokine profile and a potent capacity to induce lethal graft versus host (GVH) disease in a C57BL/6 donor to a BALB/c host bone marrow transplantation model. In contrast, the marrow NK1.1(-) CD4(+) T cells had a Th0 cytokine profile and failed to induce lethal GVH disease, even at 20-fold higher numbers than those from the blood. NK1.1(-) CD8(+) T cells from the blood but not the marrow induced lethal GVH disease. Nevertheless, the marrow NK1.1(-) CD8(+) T cells induced potent antitumor activity that was augmented by marrow NK1.1(-) CD4(+) T cells and facilitated hematopoietic progenitor engraftment. The inability of marrow CD4(+) and CD8(+) T cells to induce GVH disease was associated with their inability to expand in the blood and gut of allogeneic recipients. Because neither the purified marrow CD4(+) or CD8(+) T cells induced GVH disease, their unique features are desirable for inclusion in allogeneic bone marrow or hematopoietic progenitor transplants.

Strains of human immunodeficiency virus (HIV) transmitted between individuals use the CCR5 coreceptor, but no preferential depletion of particular Th-lymphocyte subpopulations has been reported during primary HIV infection (PHI). In contrast, gut-associated Th lymphocytes are preferentially depleted in macaques recently infected by simian immunodeficiency virus. The expression of CCR5 and the intestinal homing receptor integrin alpha4beta7 on subpopulations of Th lymphocytes was studied in 12 patients with PHI. There was a profound decrease of circulating alpha4beta7+ Th lymphocytes and CCR5+ memory Th lymphocytes with nonlymphoid homing potential (CD62L-CD45RO+). Unlike other Th lymphocytes, this cell population remained depleted despite early control of viral replication under antiretroviral treatment. Therefore, HIV preferentially targets a specific CCR5+ subpopulation of Th lymphocytes early during infection, inducing its persistent depletion despite treatment. Protective immunity in vivo depends on Th lymphocytes carrying homing capacity to nonlymphoid tissue, and therefore these data may explain the persistent abnormalities of immune functions in patients infected with HIV.

There is no reliable laboratory indicator of the onset of chronic graft-versus-host disease (cGVHD). This study looks at whether the expression of OX40, a member of the tumor necrosis factor receptor family, is related to the development of cGVHD in patients who underwent allogeneic hematopoietic stem cell transplantation. Peripheral blood mononuclear cells from 22 patients after day 100 were subjected to multicolor flow cytometry. The percentages of both OX40+CD4+ and OX40+CD8+ T cells were significantly higher in patients with cGVHD than those without (P <.0001 and P =.001, respectively). Serial analyses showed that OX40+CD4+ T cells elevated before the onset of cGVHD and closely correlated with the therapeutic response. The expression of CD25, CD69, and HLA-DR was partially detectable on OX40+ T cells. These results indicate that serial measurement of OX40+ T cells is useful for predicting the onset as well as the therapeutic response of cGVHD and raise a possibility that the OX40/gp34 system is involved in the pathogenesis of cGVHD.

Despite thymic deletion of cells with specificity for self-antigens, autoreactive T cells are readily detectable in the normal T-cell repertoire. In recent years, a population of CD4(+) T cells that constitutively express the interleukin-2 receptor-alpha chain, CD25, has been shown to play a pivotal role in the maintenance of self-tolerance in rodent models. This study investigated whether such a regulatory population exists in humans. A population of CD4(+)CD25(+) T cells, taken from the peripheral blood of healthy individuals and phenotypically distinct from recently activated CD4(+) T cells, was characterized. These cells were hyporesponsive to conventional T-cell stimuli and capable of suppressing the responses of CD4(+)CD25(-) T cells in vitro. Addition of exogenous interleukin-2 abrogated the hyporesponsiveness and suppressive effects of CD4(+)CD25(+) cells. Suppression required cell-to-cell contact but did not appear to be via the inhibition of antigen-presenting cells. In addition, there were marked changes in the expression of Notch pathway molecules and their downstream signaling products at the transcriptional level, specifically in CD4(+)CD25(+) cells, suggesting that this family of molecules plays a role in the regulatory function of CD4(+)CD25(+) cells. Cells with similar phenotype and function were detected in umbilical venous blood from healthy newborn infants. These results suggest that CD4(+)CD25(+) cells represent a population of regulatory T cells that arise during fetal life. Comparison with rodent CD4(+)CD25(+) cells suggests that this population may play a key role in the prevention of autoimmune diseases in humans.

Depending on the microenvironment, murine gamma delta T cells differentiate into either Th1 (IFN-gamma-producing) or Th2 (IL-4-producing) cells. It is unclear, however, whether circulating human peripheral blood gamma delta T cells can be driven into Th1 or Th2 cells by modulation of the priming cytokine milieu. In this study, peripheral blood gamma delta T cells were stimulated by phosphoantigen (isopentenyl pyrophosphate) or Daudi lymphoma cells in the presence of Th1-priming (rIL-12, anti-IL-4 Ab) or Th2-priming (rIL-4, anti-IL-12 Ab) conditions. Single-cell analysis of cytokine secretion (IFN-gamma and IL-4) was performed by flow cytometry after 18 h and after restimulation of expanded gamma delta T cells. The early activation of resting gamma delta T cells was characterized by the induction of IFN-gamma. Priming under Th1 conditions induced a Th1 profile characterized by increased secretion of IFN-gamma and TNF-alpha, while Th2 conditions caused increased production of IL-4 (Th2 profile) by the gamma delta T cells. These results indicate that the major subset of human gamma delta T cells can be polarized into either Th1 or Th2 cytokine pattern depending on the cytokine milieu in which contact with antigen occurs.

Age-associated thymic atrophy has been proposed to be due to changes in both the thymic microenvironment and in the intrinsic properties of the early T cell progenitors, the CD44(+)CD25(-)CD3(-)CD4(-)CD8(-) cells. We have purified these cells from the thymus of both old and young mice and demonstrate no age-associated defect in their ability to differentiate into their progeny in vitro when used to reconstitute fetal thymic organ cultures. We also demonstrate that in the presence of anti-IL-7, CD44(+)CD25(-)CD3(-)CD4(-)CD8(-) cells from young mice show reduced thymocyte development in fetal thymic organ cultures compared with controls. Finally we have shown that old mice treated with IL-7 show improved thymopoiesis compared with control groups. The increased thymopoiesis seen in the old animals occurs in the sequential manner which would be anticipated for an agent working directly on the early stages, including the CD44(+)CD25(-)CD3(-)CD4(-)CD8(-) cells.

The aim of this study was to investigate the distribution of leucocyte subsets in the small intestine of healthy adult cats (n=16). Immunohistochemical methods were used to identify leucocyte subsets within the mucosa of the duodenum, jejunum and ileum. Computer-aided morphometry was used to enumerate cells within the epithelial compartment, villous lamina propria and lamina propria adjacent to upper and lower crypt. Throughout the small intestine, IgA+ and IgM+ plasma cells were more prominent in the lamina propria adjacent to the lower crypt than in the villus, whereas IgG+ plasma cells were present in equal numbers in the crypt and villous regions. Overall, IgA+ plasma cells predominated and IgM+ plasma cells were higher in number than IgG+ plasma cells at each of the three anatomical locations. By contrast, T cells (CD3+) and T-cell subsets (CD4+ and CD8+) were present in greater numbers in the villous lamina propria than in the lamina adjacent to the crypts. Intraepithelial lymphocytes (IELs) were also characterized phenotypically, the majority being CD8+ T lymphocytes. Lamina propria macrophages and dendritic cells were characterized by expression of L1 and major histocompatibility complex (MHC) class II, and MHC class II expression by enterocytes overlying Peyer's patches, although rare, was also shown. The qualitative and quantitative data from this study provide a basis for comparison with cats with inflammatory enteropathies., (Copyright Harcourt Publishers Ltd.)

We have investigated lymphocyte subpopulations and blood mononuclear cell (MNC) adhesion to activated endothelial monolayers in patients with human T lymphotropic virus type I (HTLV-I) associated myelopathy (HAM), in HTLV-I asymptomatic carriers (carriers), and in seronegative controls. HAM patients and carriers had higher levels of CD4(+)CD29(+) "memory cells" than controls (P < 0.05). The percentage of CD3(+)CD27(-) "primed T cell" was elevated in patients with HAM (P < 0.05), but not in carriers. HAM patients had higher levels of CD8(+)CD57(+) "cytotoxic cells" (P < 0.05) than controls and carriers. The percentages of CD4(+) cells coexpressing activation markers HLA-DR and CD25, and of CD8(+) cells expressing HLA-DR, were significantly higher in HAM patients and carriers than in controls. Functional experiments indicated that MNC from HAM patients adhered more to activated endothelial monolayers than MNC from carriers or controls. Blocking studies demonstrated that the adhesion molecules VLA-4 and ICAM-1 and also L-selectin all contributed to increased binding. Analysis of expression of molecules involved in adhesion indicated that in HAM patients, L-selectin (CD62L) expression on CD4(+) and CD8(+) subsets was lower than in controls. Interestingly, HAM patients had a lower percentage of CD4(+) subsets expressing L-selectin than carriers (P < 0.05). In contrast, the percentage of CD4(+) and CD8(+) cells expressing VLA-4 (CD49d) was found to be higher in both HAM patients and carriers compared with controls. After 2 days in culture without mitogen, the percentage of T cells expressing ICAM-1 (CD54) increased in culture in carriers and more profoundly in HAM, but not in controls (P < 0. 05). After culture, T cells expressing the early activation antigen CD69 were also increased in HAM and carriers (P < 0.05) but not in controls. Interestingly, the levels of CD8(+) cells coexpressing activation antigen HLA-DR and CD38 were higher in HAM patients compared with both carriers and controls (P < 0.05) after culture. These findings are consistent with the observations that HTLV-I produces chronic lymphocyte activation with increased adhesion. This may be sufficient to initiate events leading to central nervous system inflammation and ultimately to HAM., (Copyright 1999 Academic Press.)

Detailed characterisation of the T cell receptor (TCR) repertoire expressed by peripheral blood lymphocytes has been used to study specific T cell responses in disease conditions. The methods have mostly involved molecular biology analysis of transcribed gene products isolated from T cell subsets or individual clones. Extensive characterisation of the TCR Vbeta chain repertoire by flow cytometry is now possible due to the recently increased availability of specific monoclonal antibodies. However, there are major logistical problems inherent in this analysis relating to the number of cells required to obtain accurate results and the vast amounts of data generated. To reduce these factors to a practical level, we have performed a detailed study to define the limits of precision of cell subset analysis by flow cytometry. Maximal achievable precision was obtained by analysing 10(4) lymphocytes; no significant improvement was obtained by analysing greater numbers of cells up to 10(5) cells, even for cell subsets present at frequencies as low as 0.5%. Careful application of these precision profiles will also permit more effective use of clinical research samples for flow cytometry when the availability of cells is limited.

Cytokine production by human lymphocytes was assessed by a flow cytometric procedure involving staining of intracellular cytokines by the paraformaldehyde-saponin procedure. The production of interleukin 2 (IL2), interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) was determined in T-helper (Th) and cytotoxic T-cells (CTL) as well as in naive and memory cells after stimulation with phorbol-12-myristate-13-acetate (PMA) and ionomycin under the influence of monensin. Kinetic studies on IL2, IFNgamma and TNFalpha in lymphocyte subpopulations showed that TNFalpha was the first cytokine produced by T-cells. Production of this cytokine peaked at 2 h and declined rapidly thereafter. The peak of IL2 and IFNgamma production was at 8 h, and production of IL2 exceeded that of IFNgamma in T-cells at all times. IL2 production declined markedly after 8 h, while IFNgamma remained relatively stable for 24 h. IL2 and TNFalpha were mainly produced by Th cells while CTL primarily expressed IFNgamma. At all times a higher percentage of memory cells stained cytokine positive compared to naive cells and production of cytokines increased more rapidly in the memory cells. Naive cells produced primarily IL2, while memory cells expressed all the studied cytokines in substantial amounts. Kinetic studies between 1 and 24 h showed that 5 h was the optimal time point for evaluating the cytokines studied; hence normal values obtained from 50 healthy blood donors were evaluated after 5 h continuous PMA and ionomycin stimulation.

This study was performed in order to investigate the type of T cells recruited to the blood in response to an acute bout of exercise with regard to mean lengths of telomeric terminal restriction fragments (TRF) and surface activation markers and with special emphasis on age-associated differences. Ten elderly and ten young humans performed maximal bicycle exercise. There was no difference in the number of recruited CD4+ and CD8+ cells between the young and elderly group. In both age groups the immediate increases could be ascribed to recruitment of CD28- cells (CD8+ and CD4+ cells) and memory cells (only CD8+ cells). Furthermore, after exercise mean TRF lengths were significantly reduced in blood mononuclear cells and in CD8+ cells from young subjects and in CD4+ cells from elderly subjects compared with lengths pre-exercise. These findings suggest that the mobilization of T lymphocytes during acute exercise is mainly a redistribution of previously activated cells with an increased replicative story than cells isolated from the blood at rest. Furthermore, elderly humans fulfilling the Senieur protocol have a preserved ability to recruit T lymphocytes in response to acute physical stress.

The helper T cell precursor (HTLp) assay is of value for predicting graft-versus-host disease after allogeneic bone marrow transplantation. The assay requires reliable detection of the amount of interleukin 2 (IL-2) produced by one cell. To optimize the IL-2 sensitivity of our HTLp assay we tested an IL-2 dependent cell line, CTLL-2, with two different measurement methods: a colorimetric assay with tetrazolium (MTT) and an isotope incorporation assay with 3H-thymidine (3H-TdR). The test conditions examined encompassed: time without IL-2, preincubation time in IL-2, CTLL-2 cell concentration and different human sera. Due to the different measurement procedures, the volumes of the IL-2 dilutions were 75 microl in assays with MTT and 150 microl in assays with 3H-TdR. We found that it was the amount of IL-2, not the concentration, that limited the growth of CTLL-2 cells. In the most optimal setting the MTT assay could detect 0.6 pg IL-2/well, corresponding to 8 pg/ml. For the 3H-TdR assay the sensitivity was 0.6 pg/well, corresponding to 4 pg/ml. Because of the possibility of IL-2 detection in the whole culture volume (150 microl), we found that the 3H-TdR assay was superior to the MTT assay with a 10-fold better sensitivity in different human sera.

Estrogen was administered to B6 (NK1.1+ strain), BALB/c (Mls-1b2a, V beta 3+ cells being forbidden clone), or (B6 x BALB/c) F1 mice (1 mg/mouse). On days 3 and 10, the number of cells yielded by the liver doubled, whereas that yielded by the thymus decreased prominently. The numbers of cells in the spleen, bone marrow, and blood were unchanged. c-kit+ stem cells, which give rise to multilineage cells, were present in the liver and bone marrow. The proportion of such c-kit+ cells in the liver increased while that in the bone marrow decreased on day 3. Therefore, the absolute number of c-kit+ stem cells increased severalfold in the liver and clusters of lymphoid cells became visible in the parenchymal space. At that time, the expression of recombination activating gene-1 and -2 mRNAs became prominent. Reflecting these phenomena, the number and proportion of IL-2R beta+ CD3int cells (i.e., primordial T cells) increased in the liver on days 3 and 10. An increase in the number of proportion of such CD3int cells was seen even in the thymus and uterus. In parallel with the increase of CD3int cells, the proportion of granulocytes also increased in various organs on day 3. Forbidden clones were present in either the NK1.1+ or the NK1.1- subset of CD3int cells in (B6 x BALB/c) F1 mice treated with estrogen and liver mononuclear cells in such mice acquired potent cytotoxicity against syngeneic thymocytes. These results reveal that estrogen has the ability to potentiate the generation of self-reactive T cells and granulocytes in the liver and other organs.

Analysis of the V beta-repertoire of antigen-reactive T cell populations can be approached using either flow-cytometry or PCR-based techniques. While the former method requires a complete set of V beta-specific monoclonal antibodies (mAbs) and large cell numbers for analysis, the latter is both time-consuming and labour-intensive. To circumvent the drawbacks of both these methods we have employed the recently developed technique of TaqManR PCR to analyse the V beta-usage of human T cell populations. TaqManR PCR is based on the 5'-->3' nuclease activity of Taq polymerase. During PCR amplification an internal oligonucleotide probe, that is labelled with a fluorescent reporter and a quencher dye, is cleaved by Taq polymerase. After cleavage, quenching of the reporter dye is lost and reporter fluorescence can be detected with a fluorescence plate reader. Using one C beta-specific fluorogenic probe and a panel of V beta-specific primers, we show that fluorescence-detected amplification of TCR beta cDNA is V beta-specific and linear within a 2-3-log range of template concentration. The sensitivity of TaqManR PCR is comparable to conventional detection of PCR-products by agarose gel staining, while processing time is reduced. Furthermore, superantigen-induced skewing of the V beta-repertoire of human T cells is readily detected with this method. Thus TaqManR PCR is a reliable and fast method for semiquantitative analysis of the V beta-repertoire of human T cell populations.

Infection with human immunodeficiency virus 1 causes profound changes in the lymph nodes of infected patients. In particular, large numbers of CD8+CD45RO+ T cells infiltrate both the paracortex and the germinal centers. These cells contained the cytotoxic granule-associated protein TIA-1 but showed no detectable levels of perforin and shared the same characteristics of the expanded, activated, short-lived CD8+ population found during acute viral infections. These cells expressed low levels of Bcl-2 and are likely to be short-lived in vivo as evidenced by the direct observation of CD8+ apoptotic cells in the paracortical areas of the infected nodes. Changes in the paracortical nonlymphoid populations were also seen. There were reactive changes in the blood vessels, and the macrophage population was expanded and activated. Furthermore, apoptotic bodies were seen in the cytoplasm of the activated CD68+RFD-7+RFD-1+ macrophages pointing to the phagocytic capacity of these cells and their role in the clearance of the apoptotic cells from the tissues. These observations suggest that the persistance of CD8+ population in human immunodeficiency virus 1 infection is not a result of the presence of an abnormal CD8+ population but rather a result of an inappropriate over-stimulation of the CD8+ cells.

Lymphocytes expressing alpha beta or gamma delta T-cell receptors (TCR) represent distinct T-cell populations. Because TCR delta genes lie within the TCR alpha locus, the rearrangement processes, transcription, and translation of TCR delta or TCR alpha variable domain exons require tight regulation. Human precursor B-cell leukemias (eg, the REH cell line) constitute an interesting model to study TCR delta/alpha recombination because they rearrange TCR delta/alpha loci along a hierarchically ordered pathway in which V delta 2D delta 3 segments are joined to the J alpha cluster. We now show for REH cells that chimeric TCR delta/alpha variable domain exons are posttranscriptionally modified by alternative splicing resulting in truncated V delta 2C alpha transcripts. This process also takes place during thymic differentiation. CD7+/CD3- T-cell precursors exhibit V delta 2D delta 3 rearrangements. Further differentiation into CD7+/CD3+ thymocytes is associated with the expression of a truncated V delta 2C alpha RNA species. In contrast, chimeric TCR delta/alpha rearrangements containing a V delta 1 segment (but no D delta sequences) are predominantly expressed as full-length V delta 1J alpha C alpha transcripts. These data suggest that alternative splicing constitutes a mechanism that restricts the production of distinct chimeric TCR alpha chains.