IL-4-producing CD8+ T cells with a CD62L++(bright) phenotype accumulate in a subgroup of older adults and are associated with the maintenance of intact humoral immunity in old age

An increased production of proinflammatory cytokines occurs in a high percentage of elderly persons and is associated with an impaired humoral immune response. However, high IL-4 production has also been observed in old age. We now demonstrate an IL-4-producing subpopulation of CD8+ T cells in a subgroup of healthy older adults. This T cell subset is substantial in size and has a characteristic phenotype expressing CD45RO, CD28, CD62L, and CD25. IL-4-producing CD8+ T cells produce large amounts of IL-2 but not IFN-gamma or perforin, and these cells do not have a regulatory suppressive effect on other T cells. In vivo IL-4-producing CD8+ T cells can be stably detected over a year. When put into culture they also have a stable cytokine production pattern but fail to produce perforin even in the presence of IL-12. This special T cell type does not occur in persons under the age of 40, but is present in 36% of the persons >60 years of age. In this age group, IL-4-producing CD8+ T cells are more frequent in persons who are still capable of raising a humoral immune response following immunization than in others who fail to produce protective Abs after vaccination. Our results suggest that CD8+ T cells with a CD62L++(bright) phenotype accumulate in a subgroup of older adults. Due to their phenotype that enables them to migrate into lymphoid tissues and to their capacity to produce IL-4, these cells may counterbalance the overproduction of proinflammatory cytokines in old age.     

Clonal analysis of the peripheral T cell compartment of the SCID-hu mouse.

Severe combined immunodeficiency (SCID) mice can be transplanted successfully with human fetal liver and thymus (SCID-hu mice). Precursor cells derived from the fetal liver differentiate in the thymus and migrate into the blood as mature T cells. In the present paper, the peripheral T cell compartment of such mice was studied. Peripheral WBC were activated by PHA and cultured in the presence of irradiated human feeder cells. The resultant cell population consisted exclusively of human CD1- CD2+ CD3+ CD7+ T lymphocytes; up to 4% of the T cells expressed the TCR gamma delta, whereas 95 to 100% were TCR alpha beta +. The CD4bright (42 to 66%) and CD8bright (30 to 54%) populations coexpressed variable but low levels of CD8 and CD4, respectively. The T cell cultures from the SCID-hu mice did not display reactivity towards the autologous human EBV-transformed B cell lines (B-LCL). On the other hand, these human T cells proliferated and were cytotoxic against allogeneic human B-LCL. T cell clones were established from cultured SCID-hu T cells. All T cell clones were TCR alpha beta + CD3+ CD2+; 61% of the clones were CD4+ CD8-, 27% were CD8+ CD4-, 11% were CD8+ CD4lo, and 2% were CD4+ CD8lo. None of these clones recognized the autologous B-LCL established from the fetal human donor. Fourteen of 100 T cell clones had specific alloreactivity, as tested on a panel of five B-LCL. Of these 14, two CD8+ CD4lo and two CD8+ CD4- clones were cytotoxic and did not proliferate in response to specific stimulator cells. Furthermore, two CD4+ CD8lo and eight CD4+ CD8- clones proliferated specifically in response to alloantigens. In conclusion, the peripheral human T cells of SCID-hu animals are functional and their TCR repertoire is polyclonal, alloreactive, and devoid of self-reactive cells. Therefore, the SCID-hu mouse can be a suitable model for the study of alloreactivity and allotolerance in vivo, as well as for the study of negative selection in the human thymus.     

Elicitor-induced biosynthesis of psoralens in Ammi majus L. suspension cultures. Microsomal conversion of demethylsuberosin into (+)marmesin and psoralen

Suspension cultures of Ammi majus L. cells produce various linear furanocoumarins in response to treatment with elicitor preparations from either Alternaria carthami Chowdhury or Phytophthora megasperma f.sp. glycinea. Microsomes which were isolated from these cells 14 h after addition of the elicitor efficiently catalyzed the conversion of demethyl [3-14C]suberosin into labelled (+)marmesin in the presence of NADPH and oxygen. In contrast to the chemical cyclization of demethylsuberosin by m-chloroperoxybenzoic acid, the reaction catalyzed by the marmesin synthase proceeded rapidly and no intermediate demethylsuberosin epoxide could be recovered. Significant blue-light-reversible inhibition by carbon monoxide and inhibition by various chemicals known to inhibit reactions dependent on cytochrome P450 suggested that the marmesin synthase is a cytochrome-P450-dependent monooxygenase. Upon prolonged incubation, a subsequent major labelled product originated from (+)marmesin, which was identified as psoralen. The psoralen synthase was also characterized as a cytochrome-P450-dependent monooxygenase. Both the marmesin synthase and the psoralen synthase, as well as enzymes catalyzing the formation of demethylsuberosin and O-prenylumbelliferone from umbelliferone and dimethylallyl diphosphate, were associated with the endoplasmic reticulum in Ammi majus cells and their activities were concomitantly induced by elicitor treatment of the cells. We propose that in vivo these enzymes are active in the lumen of the endoplasmic reticulum from where the furanocoumarin phytoalexins are excreted into the cell culture fluid.     

Establishment and Characterization of PCL12, a Novel CD5+ Chronic Lymphocytic Leukaemia Cell Line

Immortalized cell lines representative of chronic lymphocytic leukemia (CLL) can assist in understanding disease pathogenesis and testing new therapeutic agents. At present, very few representative cell lines are available. We here describe the characterization of a new cell line (PCL12) that grew spontaneously from the peripheral blood (PB) of a CLL patient with progressive disease and EBV infection. The CLL cell origin of PCL12 was confirmed after the alignment of its IGH sequence against the “original” clonotypic sequence. The IGH gene rearrangement was truly unmutated and no CLL-related cytogenetic or genetic lesions were detected. PCL12 cells express CD19, CD20, CD5, CD23, low levels of IgM and IgD and the poor-outcome-associated prognostic markers CD38, ZAP70 and TCL1. In accordance with its aggressive phenotype the cell line is inactive in terms of LYN and HS1 phosphorylation. BcR signalling pathway is constitutively active and anergic in terms of p-ERK and Calcium flux response to α-IgM stimulation. PCL12 cells strongly migrate in vitro in response to SDF-1 and form clusters. Finally, they grow rapidly and localize in all lymphoid organs when xenotrasplanted in Rag2^-/-γc^-/- mice. PCL12 represents a suitable preclinical model for testing pharmacological agents.     

ACTION OF CYTOCHALASIN D ON CELLS OF ESTABLISHED LINES : I. Early Events

HeLa, Vero, L, HEp2, and MDBK cells respond immediately to 0.2–0.5 µg/ml cytochalasin D (CD) with sustained contraction (contracture), loss of microvilli, expression of endoplasmic contents (zeiosis), nuclear protrusion, and extension of cytoplasmic processes. The development of these changes is depicted, and the dose-response patterns in these cell lines are described. MDBK is generally most resistant and HeLa most sensitive to these effects of CD. Cells in G₁ are most sensitive to CD; responsiveness decreases progressively during early S and is least in mid S through G₂. CD inhibits transport of [¹⁴C]deoxyglucose in HeLa by about 45% but has no significant effect on hexose uptake in Vero and MDBK; sugar transport is thus apparently unrelated to any morphologic effect of CD. Although spreading and attachment are impeded, CD does not decrease and may even enhance the adhesiveness of established monolayers. Contraction appears to be a primary early effect of CD, upon which other visible changes follow. It is prevented by some inhibitors of energy metabolism (deoxyglucose and dinitrophenol) and does not occur in glycerinated models without ATP. The possible bases of the contractile response to CD are discussed. Although direct or indirect action of CD on some microfilaments may occur, a generalized structural disruption of contractile filaments by CD is considered unlikely.     

Transformation of T-lymphocyte subsets by Marek''s disease herpesvirus.

Marek's disease herpesvirus (MDV)-transformed lymphoblastoid tumor cell lines were characterized for the presence of the surface markers. Monoclonal antibodies were used for CD3 (T-cell receptor [TCR] complex), TCR1, TCR2, and TCR3, CD4, CD8, and Ia antigen by indirect fluorescence staining followed by microscopic examination or flow cytometry. The lymphoblastoid cell lines were obtained from tumors from chickens infected with MDV (n = 44) or from local lesions induced by inoculation of allogeneic, MDV-infected chick kidney cells (n = 56). Lymphocytes were harvested from these lesions between 4 and 16 days postinoculation and cultured in vitro to establish cell lines. All cell lines expressed Ia antigen and CD3 and/or TCR and thus are activated T cells. Most of the cell lines developed from tumors were CD4+ CD8-; only one cell line was negative for both markers. Sixteen percent of the cell lines were TCR3+, while the remainder were TCR2+. The cell lines developed from local lesions were much more heterogeneous: 45% were CD4- CD8+, 34% were CD4- CD8-, and only 21% were CD4+ CD8-. The number of TCR3+ cell lines was larger than expected for the CD4- CD8+ and CD4- CD8- cell lines, as judged from the presence of these cells in the blood. These results indicate that several subsets of T lymphocytes can be transformed by MDV, depending on the pathogenesis of infection. Activation of T cells as a consequence of the normal pathogenesis or by allogeneic stimulation seem to be a first important step in the process of transformation.     

Leishmania: naive human T cells sensitized with promastigote antigen and IL-12 develop into potent Th1 and CD8(+) cytotoxic effectors.

Russo, D. M., Chakrabarti, P., and Higgins, A. Y. 1999. Leishmania: Naive human T cells sensitized with promastigote antigen and IL-12 develop into potent Th1 and CD8(+) cytotoxic effectors. Experimental Parasitology 93, 161-170. The differentiation of naive human T cells into Leishmania-specific Th1 or cytotoxic effector cells was examined by sensitizing T cells in vitro with dead Leishmania antigen in the presence or absence of IFN-gamma or IL-12. These Leishmania-specific T cell lines proliferated and produced cytokines in response to challenge with autologous Leishmania-infected macrophages. Sensitization in the presence of IL-12 or IFN-gamma induced Leishmania-specific human Th1 responses, with IL-12 inducing more potent Th1 responses. However, IL-12-induced Th1 responses were IFN-gamma dependent. T cell lines exhibited Th2 or Th0 phenotypes when primed in the absence of cytokines. Only T cell lines primed in the presence of IL-12 contained high percentages of CD8(+) cells. These cells lysed autologous Leishmania-infected but not uninfected macrophages in an MHC-dependent manner. Thus, this in vitro sensitization system can be used to delineate the conditions for optimally priming human Leishmania-specific effector cells.     

Modulation of in vitro autologous melanoma-specific cytotoxic T-cell responses by phorbol dibutyrate and ionomycin

Human melanoma-specific, HLA restricted, cytotoxic T-cell lines can be generated by in vitro stimulation and culturing of peripheral lymphocytes, or lymph node cells, with autologous or HLA-A region matched melanomas in the presence of a low concentration (5 U/ml) of IL-2. Stimulation is followed by a period of clonal expansion and differentiation into cytotoxic T-cells specific for melanoma. We investigated the effect of the PKC modulating drug phorbol dibutyrate combined with the calcium ionophore Ionomycin on growth and differentiation of the cell lines. The growth of the T-cell lines was substantially augmented in the presence of the drugs with increases of 10-fold or more in clonal expansion by 3 weeks of culture. The cell lines were IL-2 dependent for growth in the presence or absence of the drugs and the phenotypic distribution remained predominantly CD3+ T-cells of mixed CD4 and CD8 phenotypes. In spite of the increased rate of growth in the presence of the drugs, autologous melanoma-specific cytotoxicity was almost completely abrogated in those cultures. The cells were, however, nonspecifically lytic in the presence of concanavalin A. The melanoma-specific cytotoxic response was completely restored following culture with IL-2 alone. The results suggest that the human tumor-specific cytotoxic T-cell response can be induced and amplified in the presence of immune modulating drugs.     

Hyaluronidase can modulate expression of CD44

CD44 is a family of transmembrane glycoproteins with multiple isoforms generated by alternative exon splicing of a single gene. CD44 and its variants are expressed on a wide variety of cells including cancer cells. The mechanisms by which splice variant exons are selected are unknown. The presence of hyaluronan in the environment of the cell appears to influence that selection process. The expression of particular splice variants of CD44 as well as the simultaneous presence of hyaluronan is important for motility, invasion, and the metastatic spread of some tumors. The influence of hyaluronidase digestion on the expression of CD44 in human cancer cell lines was examined. CD44 isoforms containing alternatively spliced exons were sensitive to hyaluronidase digestion in all lines examined, but differences between cell lines were observed. Expression of CD44s, the standard form, was resistant to digestion in two of three cell lines. A tentative model was formulated proposing that CD44 isoforms containing splice variants are unstable, requiring the continuous presence of ligand for expression. CD44s is relatively more stable, not requiring the continuous presence of hyaluronan. Additionally, a number of new CD44 variant isoforms, not previously observed, were identified.     

Interaction of CD44 with different forms of hyaluronic acid. Its role in adhesion and migration of tumor cells

Interaction between hyaluronic acid (HA) and CD44 has been considered a key event in tumor invasion and metastasis. HA is a linear, high molecular weight glycosaminoglycan in its native state, but fragmented low molecular forms are found at sites of neoplastic or inflammatory infiltrates. Both high and low molecular weights HA are involved in diverse biological functions. In this study, we used two clonal variants of a T cell murine lymphoma designated LBLa and LBLc. These cell lines were found to differ in their in vivo and in vitro growth rates. LBLa grew faster and exhibited an enhanced invasive capacity as compared to LBLc. In contrast, cell lines did not differ in the expression of surface markers (CD8, CD24, CD25, CD44, and CD18), or in their capacity to bind HA. However, LBLa cells exhibited higher capacity to migrate to low molecular weight HA than did LBLc. Migration was mediated by CD44 since it was abrogated by anti-CD44 monoclonal antibody as well as by hyaluronidase. We suggest that interaction between CD44 and low molecular weight HA may trigger migration mechanisms in LBLa cells, thus contributing to enhanced invasive cell capacity.     

Autocrine growth of CD4+ T cells. Differential effects of IL-1 on helper and inflammatory T cells

The growth factor requirements of cloned lines representing two major subsets of CD4+ T cells were examined. The helper subset, which produces IL-4 as its autocrine growth factor, proliferates in response to IL-2 or to IL-4 in the presence of IL-1. The inflammatory subset, which produces IL-2 as its autocrine growth factor, proliferates in response to IL-2 and, in the presence of limiting amounts of IL-2, shows increased proliferation in the presence of IL-4. The inflammatory subset does not proliferate in response to IL-1 plus IL-4. This ability to respond to the combination of IL-1 plus IL-4 correlates with the presence of IL-1R on the cloned lines tested. These data suggest that IL-1 may play a controlling role in the clonal expansion of CD4+ T cells of different functional types. This, in turn, suggests means by which the immune response could be directed into humoral or cell-mediated responses.     

Memory CD4 T cells that express CXCR5 provide accelerated help to B cells

CD4 T cell help for B cells is critical for effective Ab responses. Although many of the molecules involved in helper functions of naive CD4 T cells have been characterized, much less is known about the helper capabilities of memory CD4 T cells, an important consideration for the design of vaccines that aim to prime protective memory CD4 T cells. In this study, we demonstrate that memory CD4 T cells enable B cells to expand more rapidly and class switch earlier than do primary responding CD4 T cells. This accelerated response does not require large numbers of memory cells, and similar numbers of primary responding cells provide less effective help than do memory cells. However, only memory CD4 T cells that express the B cell follicle homing molecule, CXCR5, are able to accelerate the response, suggesting that the rapidity of the Ab response depends on the ability of CD4 memory T cells to migrate quickly toward B cells.     

Sphingosine kinase 1 promotes cerebral ischemia-reperfusion injury through inducing ER stress and activating the NF-κB signaling pathway

Endoplasm reticulum stress and inflammation response have been found to be linked to cerebral ischemia-reperfusion (IR) injury. Sphingosine kinase 1 (SPHK1) has been reported to be a novel endoplasm reticulum regulator. The aim of our study is to figure out the role of SPHK1 in cerebral IR injury and verify whether it has an ability to regulate inflammation and endoplasm reticulum stress. Hydrogen peroxide was used to induce cerebral IR injury. Enzyme-linked immunosorbent assay, quantitative polymerase chain reaction, western blots, and immunofluorescence were used to measure the alterations of cell viability, inflammation response, and endoplasm reticulum stress. The results demonstrated that after exposure to hydrogen peroxide, cell viability was reduced whereas SPHK1 expression was significantly elevated. Knockdown of SPHK1 attenuated hydrogen peroxide-mediated cell death and reversed cell viability. Our data also demonstrated that SPHK1 deletion reduced endoplasm reticulum stress and alleviated inflammation response in hydrogen peroxide-treated cells. In addition, we also found that SHPK1 modulated endoplasm reticulum stress and inflammation response to through the NF-κB signaling pathway. Inhibition of NF-κB signaling pathway has similar results when compared with the cells with SPHK1 deletion. Altogether, our results demonstrated that SPHK1 upregulation, induced by hydrogen peroxide, is responsible for cerebral IR injury through inducing endoplasm reticulum stress and inflammation response in a manner working through the NF-κB signaling pathway. This finding provides new insight into the molecular mechanism to explain the neuron death induced by cerebral IR injury.     

Effect of CD4 gene expression on adenovirus replication.

The gene encoding the CD4 receptor was introduced into KB cells to establish the KBT4 cell line, a cell line susceptible to infection with human immunodeficiency virus type 1. Adenovirus replication was found to be significantly less in these cells than in the parental KB cells. Similar decreased adenovirus type 5 (Ad5) replication occurred in HeLaT4 cells compared with the original HeLa cells. The presence of CD4 did not alter the cell surface population of KB cell adenovirus receptors, since viral adsorption was similar in the two cell lines. Moreover, addition of soluble CD4 did not reduce viral replication in either KB or KBT4 infected cells. Uncoating of viral DNA was also unchanged in KBT4 cells compared with the parental KB cells. In contrast, migration to or entrance of viral DNA into nuclei and synthesis of early viral RNAs was delayed and reduced in KBT4 cells. These effects were more pronounced for Ad7 than for Ad5. The yields of infectious viruses were the same in both cell lines, however, after transfection of naked viral DNAs to initiate infection. These results imply that the expression of the CD4 gene in KBT4 cells interfered with passage of uncoated virus across endosomal vesicles and/or transfer of uncoated core viral DNA into the nucleus.     

Alpha(1,3)-fucosyltransferase VII and alpha(2,3)-sialyltransferase IV are up-regulated in activated CD4 T cells and maintained after their differentiation into Th1 and migration into inflammatory sites.

Activated Th1 CD4 T cells bind to P-selectin and migrate into inflamed tissue, whereas Th2 cells do not. We show that alpha(1, 3)-fucosyltransferase VII (FucT-VII) and alpha(2, 3)-sialyltransferase IV (ST3GalIV), which are crucial for the biosynthesis of functional P-selectin ligands, are absent in naive CD4 T cells, but are rapidly up-regulated upon activation. Th1 or Th2 differentiation in the presence of polarizing cytokines leads to down-regulation of FucT-VII mRNA selectively in Th2 but not in Th1 cells. Influencing the differentiation by varying the priming dose of antigenic peptide results in similar FucT-VII down-regulation only in Ag-specific Th2 cells. ST3GalIV levels remain elevated. FucT-VII and ST3GalIV mRNAs are also up-regulated by Th1 cells primed in vivo and recruited into the lymph nodes draining delayed-type hypersensitivity sites. We identify FucT-VII gene expression as a principal difference between Th1 and Th2 cells, and underscore the importance of FucT-VII and ST3GalIV expression for the biosynthesis of functional selectin ligands.     

Ultrastructural development of the saccus vasculosus in rainbow trout (Salmo gairdneri)

Summary The morphology of crown cells and supporting cells of the saccus vasculosus has been described by numerous investigators. A third type of cell has been mentioned by several authors and referred to variously as undifferentiated crown cells, pseudo-coronet cells, pear-shaped cells and, most recently, as liquor-contact neurons. A developmental study of the organ was undertaken as a possible means of characterizing this third cell type and determining its origin.The epithelium of the saccus vasculosus and the ependyma of the third ventricle are different and distinguishable at the time of hatching in rainbow trout. Initially, apical protrusions from crown cells extend slightly into the lumen and a few end knobs or motile cilia project from them. Basal bodies with cross-striated rootlets occur frequently. In swim-up fry, end knobs are more numerous and heavily vacuolated, although cross-striated rootlets are less apparent.Evidence is presented that is consistent with a hypothesis of secretory activity in the crown cells. Further, portions of end knobs containing this material appear to be pinched off from the remainder of the crown cell. The possible presence of bipolar neurons is also discussed.Supported by Research Grant 5 R01 NS0627 from the National Institute of Neurological Diseases and Stroke.     

Anatomical and cellular requirements for the activation and migration of virus-specific CD8+ T cells to the brain during Theiler's virus infection

Theiler's murine encephalomyelitis virus (TMEV) infection of the brain induces a virus-specific CD8(+) T-cell response in genetically resistant mice. The peak of the immune response to the virus occurs 7 days after infection, with an immunodominant CD8(+) T-cell response against a VP2-derived capsid peptide in the context of the D(b) molecule. The process of activation of antigen-specific T cells that migrate to the brain in the TMEV model has not been defined. The site of antigenic challenge in the TMEV model is directly into the brain parenchyma, a site that is considered immune privileged. We investigated the hypothesis that antiviral CD8(+) T-cell responses are initiated in situ upon intracranial inoculation with TMEV. To determine whether a brain parenchymal antigen-presenting cell is responsible for the activation of virus-specific CD8(+) T cells, we evaluated the CD8(+) T-cell response to the VP2 peptide in bone marrow chimeras and mutant mice lacking peripheral lymphoid organs. The generation of the anti-TMEV CD8(+) T-cell response in the brain requires priming by a bone marrow-derived antigen-presenting cell and the presence of peripheral lymphoid organs. Although our results show that activation of TMEV-specific CD8(+) T cells occurs in the peripheral lymphoid compartment, they do not exclude the possibility that the immune response to TMEV is initiated by a brain-resident, bone marrow-derived, antigen-presenting cell.     

CD163 expression confers susceptibility to porcine reproductive and respiratory syndrome viruses

Direct functional screening of a cDNA expression library derived from primary porcine alveolar macrophages (PAM) revealed that CD163 is capable of conferring a porcine reproductive and respiratory syndrome virus (PRRSV)-permissive phenotype when introduced into nonpermissive cells. Transient-transfection experiments showed that full-length CD163 cDNAs from PAM, human U937 cells (histiocytic lymphoma), African green monkey kidney cells (MARC-145 and Vero), primary mouse peritoneal macrophages, and canine DH82 (histocytosis) cells encode functional virus receptors. In contrast, CD163 splice variants without the C-terminal transmembrane anchor domain do not provide PRRSV receptor function. We established several stable cell lines expressing CD163 cDNAs from pig, human, and monkey, using porcine kidney (PK 032495), feline kidney (NLFK), or baby hamster kidney (BHK-21) as the parental cell lines. These stable cell lines were susceptible to PRRSV infection and yielded high titers of progeny virus. Cell lines were phenotypically stable over 80 cell passages, and PRRSV could be serially passed at least 60 times, yielding in excess of 10(5) 50% tissue culture infective doses/ml.