Activation of HLA-restricted EBV-specific cytotoxic T cells does not require CD4+ (helper) T cells or exogenous cytokines

MHC-restricted, viral Ag-specific "memory" CTL are thought to play a decisive role in the defense against pathogenic viruses. However, the requirements for activating such CTL remain controversial. In particular, the role of CD4+ helper cells and their soluble products (e.g., IL-2) are uncertain. To approach these questions as they relate to EBV-specific CTL, highly purified CD8+ T cells from healthy EBV-seropositive individuals were cultured with autologous irradiated EBV-transformed B lymphoblastoid cell lines (LCL), in the presence or absence of autologous CD4+ cells or 1 to 10 U/ml purified rIL-2. The results indicate that the induction of CTL requires neither Th cells nor exogenous IL-2. The CTL generated from isolated CD8+ cells were HLA class I restricted as demonstrated by their ability to lyse targets sharing at least one HLA-A or -B Ag with the stimulating autologous LCL. Furthermore, a mAb (W6/32) to a common determinant on HLA class I Ag blocked both the generation and effector phases of killing, whereas an HLA class II directed mAb had no effect. Addition of an IL-2R-specific antibody (anti-Tac) to the culture medium blocked induction of CTL, suggesting that endogenously produced IL-2 plays an obligatory role in this system. Paraformaldehyde fixation of LCL abrogated their ability to function as stimulator cells; however, addition of 2 U/ml exogenous IL-2 to fixed LCL cultured with CD8+ cells allowed for the induction of highly specific CTL. These results indicate that EBV-specific memory CTL can be activated in the absence of CD4+ helper cells or their soluble products, but nonetheless require Ag and IL-2.     

Antigen-specific suppressor T lymphocytes in man make use of the same set of surface molecules as do cytolytic T lymphocytes: roles of Leu-2/T8, Leu-4/T3, Leu-5/T11, LFA-1 molecules

When cultured with autologous antigen-primed Leu-3+ lymphoblasts, Leu-2+ cells differentiate into suppressor T cells (Ts) that specifically inhibit the responses of fresh autologous Leu-3+ cells to the priming antigen. We have shown previously that the Leu-4/T3 (CD-3) molecular complex and HLA-A,B molecules on the surface of Leu-3+ inducer blasts are recognized by Leu-2+ Ts during their differentiation. This study examines the role of various cell surface molecules expressed by Leu-2+ Ts during the inductive and effector phases of suppression. Leu-2+ cells were treated in the absence of complement with a variety of monoclonal antibodies recognizing distinct human lymphoid antigens either before or after their activation with alloantigen-primed Leu-3+ blasts. Antibodies to Leu-2/T8 (CD-8) and lymphocyte function-associated antigen-1 (LFA-1) (CDw-18) molecules inhibited not only the generation but also the effector function of Leu-2+ Ts. Although antibodies to Leu-4/T3 (CD-3) and Leu-5/T11 (CD-2) molecules caused profound inhibition of the activation of Ts, these antibodies failed to inhibit the effector function of Ts. On the contrary, anti-Leu-4 antibody consistently augmented the suppressor effect of Ts. Antibodies directed against Leu-1/T1 (CD-5), Leu-3/T4 (CD-4), LFA-3, and class I (HLA-A,B,C) and class II (HLA-DR,DQ) major histocompatibility complex molecules had no effect on either the generation or the effector function of Ts. These results suggest the involvement of Leu-2/T8 (CD-8), Leu-4/T3 (CD-3), Leu-5/T11 (CD-2), and LFA-1 (CDw-18) molecules on the surfaces of Leu-2+ cells in the activation and effector functions of Ts.     

Functional characterization of human T lymphocyte subsets distinguished by monoclonal anti-leu-8

Previous studies have shown that monoclonal anti-Leu-8 antibody identifies functionally distinct subpopulations within both the Leu-2 (T8+) and Leu-3 (T4+) lineages of human T lymphocytes. We now report in detail on the tissue distribution of the Leu-8 antigen and on extensive functional studies of T cells subsets distinguished by their expression or lack of expression of this marker. Leu-8 is present on a wide variety of hematologic cells, including granulocytes, T and B lymphocytes, monocytes, and null or NK cells. Within lymph nodes and tonsils, Leu-8 is absent from both B and T cells within germinal centers but is present on nearly all paracortical lymphocytes. Leu-8 is present on most but not all EBV-transformed B cell lines, reflecting its presence on a subset of normal peripheral blood B cells. None of six malignant T cell lines tested were Leu-8+, whereas most circulating T cells are Leu-8+. Although standard immunoprecipitation techniques failed to demonstrate any specific bands on SDS polyacrylamide gels, the antigenic determinant recognized by anti-Leu-8 is protein or protein-associated, because brief treatment of target cells with pronase abrogated binding of anti-Leu-8. Both Leu-3+8+ and Leu-3+8- cells proliferated in response to several soluble antigens and to autologous and allogeneic non-T cells. Nonetheless, nearly all of the helper T cells for PWM- and AMLR-induced PFC were contained within the Leu3+8- subset. Optimal suppression of the PWM-induced PFC response required both Leu-2+8+ and Leu-2+8- cells, and irradiation of either subset with 3000 R abrogated the capacity of the recombined subsets to effect suppression. In contrast to help for B cell differentiation, both Leu-3+8+ and Leu-3+8- cells were capable of amplifying the development of allospecific T killer cells; precursor and effector T killer cells could be found within both Leu-2+8+ and Leu-2+8- subpopulations. The correlation between Leu-8 phenotype and selected immune functions of T cells (and B cells; see companion paper) indicates that anti-Leu-8 distinguishes important immunoregulatory T and B lymphocyte subsets in man.     

Blocking of human T lymphocyte functions by anti-Leu-2 and anti-Leu-3 antibodies: differential inhibition of proliferation and suppression

We have shown previously that monoclonal antibodies to the Leu-2 and Leu-3 T cell antigens block the response of their respective subsets in allogeneic MLR. The present study was an effort to explore the mechanism of inhibition and to determine if anti-Leu-2 and anti-Leu-3 antibodies affect the responses to stimuli in addition to alloantigens. Our results indicate that antibodies to Leu-2 and Leu-3 have profound inhibitory effects on proliferation by their respective T cell subsets responding to a variety of stimuli, including specific soluble antigens and alloantigen. This effect was characterized by the following features: a) For optimal inhibition of proliferation, antibody must be present at the onset of antigenic stimulation. b) Inhibition is augmented by increasing the concentration of antibody or decreasing the concentration of antigen. c) Fab fragments of both anti-Leu-2a and anti-Leu-3a antibodies also block proliferation. In addition to their effects on T cell proliferation, anti-Leu-3 antibody blocked T cell-dependent lg synthesis induced in MLR, and anti-Leu-2 antibody prevented the induction, in vitro, of Leu-2+3- suppressor cells of lg synthesis. Taken together, these results suggest that antibodies to antigenic determinants on the Leu-2 and Leu-3 molecules competitively block segments of these structures that bind to alloantigen or nominal antigen. On the other hand, anti-Leu-2a antibody failed to block suppression of the MLR by in vivo activated, antigen-specific Leu-2+3- suppressor cells, which suggests that the Leu-2a epitope does not transmit antigen-specific signals from these differentiated suppressor T cells.     

Immunochemical variants of HLA-B27

Detailed study of HLA-B27 was prompted by the extremely strong associations between this antigen and spondyloarthropathies. Despite the relative homogeneity of this antigen when defined by alloantisera, B27 reactivity with the monoclonal antibody B27M2 suggests previously unrecognized heterogeneity. To define and confirm this heterogeneity on a molecular level, detergent extracts were prepared from B cell lines derived from individuals reactive (+) or unreactive (-) with the B27M2 antibody. Extracts were immunoprecipitated by specific allogeneic or monoclonal antibodies and analyzed by two-dimensional polyacrylamide gel electrophoresis. By this method the B27M2+ and B27M2- variants of HLA-B27 had different isoelectric points (pl) and could be distinguished from each other and from a different (Bw44) control alloantigen. Blockade of glycosylation by pretreatment of cells with tunicamycin did not alter pl but did reduce HLA antigens by approximately 3000 daltons. These data demonstrate that B27 antigens can be subdivided into subsets with different molecular composition. The effects of this heterogeneity upon the associations of B27 and disease are not yet known.     

Differences in surface phenotype and mechanism of action between alloantigen-specific CD8+ cytotoxic and suppressor T cell clones

We have previously demonstrated that fresh CD8+ T cells proliferate in response to autologous, alloantigen-primed CD4+ T cells, and differentiate into Ts cells, which inhibit the response of fresh T cells to the primary allogeneic stimulator cell but not irrelevant stimulators. Although such Ts do not have discernible cytolytic activity, like classical cytotoxic T cells (Tc) they express CD3 and CD8 on their surface and function in a class I MHC-restricted manner. Our study was an attempt to compare the surface phenotype and mechanism of action of Ts and Tc clones derived from the same individual. Ts clones were generated from donor JK by repeated stimulation of CD8+ T cells with an autologous CD4+ T inducer line specific for an allogeneic lymphoblastoid cell line (LCL). These clones were noncytolytic for either the inducer line or the allogeneic stimulator LCL. Tc clones, generated by direct stimulation of JK CD8+ T cells with the same allogeneic LCL, mediated potent, alloantigen-specific cytolysis. All Tc clones were alpha, beta TCR+, CD3+, CD4-, CD8+, CD11b-, and CD28+. Ts clones were also alpha, beta TCR+, CD3+, and CD8+, but in contrast to Tc clones, Ts clones were CD11b+ and CD28-. When added to MLR both Ts and Tc clones inhibited the response of fresh JK CD4+ T cells to the original but not irrelevant allogeneic LCL. However, Ts inhibited the response of only those CD4+ T cells that shared class I)MHC determinants with the Ts donor, whereas Tc inhibited the response of CD4+ T cells from all responders, regardless of HLA type. Pretreatment of Ts clones with mAb to CD2, CD3, or CD8 blocked suppression, whereas similar pretreatment of Tc clones blocked cytotoxicity in 4-h 51Cr release assays but had no effect on Tc-mediated suppression of the MLR. These results suggest that both Ts and Tc clones can inhibit the MLR but they do so through different mechanisms. Moreover, the maintenance of distinct surface phenotypes on these long term clones suggests that Ts may be a distinct sublineage of CD8+ T cells rather than a variant of CD8+ Tc.     

What are transcellular strands?

Summary The transcellular strands described byThaine (1961, 1962) and interpreted by him as providing the structural basis for a transcellular cytoplasmic streaming are merely lines caused by diffraction of light from walls out of focus. These lines are as clearly visible in dead cells as in living.

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Zusammenfassung Die transcellulären Stränge, dieThaine (1961, 1962) beschreibt und als Wege einer transcellularen cytoplasmatischen Bewegung betrachtet, sind lediglich Linien, die durch Lichtdiffraktion von Zellwänden verursacht werden. Diese Linien können in lebenden sowie toten Zellen beobachtet werden.

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With 15 Figures in the Text     

Fine structure of the proteinaceous substance in sieve tubes

Summary The dense pore-filling substance in the sieve plates of dicotyledons seems to consist of fibrils, rather than systems of membranes.

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Zusammenfassung Die dichte Substanz, welche bei Dicotyledonen die Poren der Siebplatten ausfüllt, scheint eher aus Fibrillen als aus Membransystemen zu bestehen.

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With 6 Figures in the Fext     

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.