Cytolytic T lymphocytes (CTLs) kill intracellular pathogens by a granule-dependent mechanism. Granulysin, a protein found in granules of CTLs, reduced the viability of a broad spectrum of pathogenic bacteria, fungi, and parasites in vitro. Granulysin directly killed extracellular Mycobacterium tuberculosis, altering the membrane integrity of the bacillus, and, in combination with perforin, decreased the viability of intracellular M. tuberculosis. The ability of CTLs to kill intracellular M. tuberculosis was dependent on the presence of granulysin in cytotoxic granules, defining a mechanism by which T cells directly contribute to immunity against intracellular pathogens.

In order to explore the effect of forest bathing on human immune function, we investigated natural killer (NK) activity; the number of NK cells, and perforin, granzymes and granulysin-expression in peripheral blood lymphocytes (PBL) during a visit to forest fields. Twelve healthy male subjects, age 37-55 years, were selected with informed consent from three large companies in Tokyo, Japan. The subjects experienced a three-day/two-night trip in three different forest fields. On the first day, subjects walked for two hours in the afternoon in a forest field; and on the second day, they walked for two hours in the morning and afternoon, respectively, in two different forest fields. Blood was sampled on the second and third days, and NK activity; proportions of NK, T cells, granulysin, perforin, and granzymes A/B-expressing cells in PBL were measured. Similar measurements were made before the trip on a normal working day as the control. Almost all of the subjects (11/12) showed higher NK activity after the trip (about 50 percent increased) compared with before. There are significant differences both before and after the trip and between days 1 and 2 in NK activity. The forest bathing trip also significantly increased the numbers of NK, perforin, granulysin, and granzymes A/B-expressing cells. Taken together, these findings indicate that a forest bathing trip can increase NK activity, and that this effect at least partially mediated by increasing the number of NK cells and by the induction of intracellular anti-cancer proteins.

We previously reported that a forest bathing trip enhanced human NK activity, number of NK cells, and intracellular anti-cancer proteins in lymphocytes. In the present study, we investigated how long the increased NK activity lasts and compared the effect of a forest bathing trip on NK activity with a trip to places in a city without forests. Twelve healthy male subjects, age 35-56 years, were selected with informed consent. The subjects experienced a three-day/two-night trip to forest fields and to a city, in which activity levels during both trips were matched. On day 1, subjects walked for two hours in the afternoon in a forest field; and on day 2, they walked for two hours in the morning and afternoon, respectively, in two different forest fields; and on day 3, the subjects finished the trip and returned to Tokyo after drawing blood samples and completing the questionnaire. Blood and urine were sampled on the second and third days during the trips, and on days 7 and 30 after the trip, and NK activity, numbers of NK and T cells, and granulysin, perforin, and granzymes A/B-expressing lymphocytes in the blood samples, and the concentration of adrenaline in urine were measured. Similar measurements were made before the trips on a normal working day as the control. Phytoncide concentrations in forest and city air were measured. The forest bathing trip significantly increased NK activity and the numbers of NK, perforin, granulysin, and granzyme A/B-expressing cells and significantly decreased the concentration of adrenaline in urine. The increased NK activity lasted for more than 7 days after the trip. In contrast, a city tourist visit did not increase NK activity, numbers of NK cells, nor the expression of selected intracellular anti-cancer proteins, and did not decrease the concentration of adrenaline in urine. Phytoncides, such as alpha-pinene and beta-pinene were detected in forest air, but almost not in city air. These findings indicate that a forest bathing trip increased NK activity, number of NK cells, and levels of intracellular anti-cancer proteins, and that this effect lasted at least 7 days after the trip. Phytoncides released from trees and decreased stress hormone may partially contribute to the increased NK activity.

Monoclonal antibodies were prepared to anti-HLA-DR cytolytic T lymphocytes (CTLs) and screened for inhibition of CTL-mediated killing. Binding of monoclonal antibodies to four types of molecules, LFA-1, LFA-2, LFA-3, and HLA-DR, inhibited killing, suggesting that these molecules participate in the CTL-target cell interaction. The antigens were characterized by immunoprecipitation, crosslinking, NaDodSO4/polyacrylamide gel electrophoresis, and immunofluorescence flow cytometry. The LFA-1 antigen contains a and ,B polypeptide chains of Mr 177,000 and 95,000 that are noncovalently associated in an alp, structure. It is present on both B and T lymphocytes and marks subpopulations that differ in quantitative expression. Human LFA-1 appears to be the homologue of mouse LFA-1. Human LFA-2 is of Mr 49,000 with a minor component of Mr 36,000. It is expressed on CTL lines but not on a B-cell line and in peripheral blood preferentially on T lymphocytes. Human LFA-3 is of Mr 60,000 and is expressed on both B and T lymphocytes.Antigen-specific T-lymphocyte-mediated killing is a multistep process involving antigen recognition and adhesion of the cytolytic T lymphocyte (CTL) to the target cell, delivery of the lethal hit, and target cell lysis (1)(2)(3). Bystander cells and the CTL itself are unharmed in the killing reaction, and the CTL can detach and engage in further killing encounters. The molecular basis ofCTL-mediated killing is ofconsiderable interest, both in itself and for our understanding of T lymphocyte-mediated immunity in general. In previous studies designed to identify the molecules involved in mouse CTL-mediated killing, rat monoclonal antibodies (MAb) were prepared to mouse CTLs and screened for their ability to inhibit killing (4)(5)(6)(7)(8)(9). MAb to the Lyt-2,3 and lymphocyte function-associated 1 (LFA-1) antigens, but not MAb to many other types ofsurface structures, were found to inhibit killing. The LFA-1 antigen appears generally important in mouse T-lymphocyte immune interactions, since MAb to it block not only allogeneic and xenogeneic CTLmediated killing but also T-helper-cell antigen-specific proliferative responses and T-cell-dependent B-cell responses (4). Anti-LFA-1 MAb exert their effect on killing by binding to the CTL rather than to the target cell and block the formation of CTL-target conjugates and the Mg2+-dependent adhesion step but not the Ca2+-dependent lethal hit stage (4, 7). The mouse LFA-1 antigen contains two noncovalently associated polypeptide chains of Mr 180,000 and 95,000 (10, 11). No human homologue of mouse LFA-1 has been defined, but is the human homologue of Lyt-2,3 (12).We have probed the molecular mechanisms of human T-lymphocyte-mediated killing by preparing MAb to human anti- (14,15) are associated with CTL-mediated killing; however, none of these antigens has thus far been shown to be the T-cell antigen receptor. Direct screening for MAb blocking killing should be a more efficient method ofidentifying the antigen receptor and other molecules involved in C...

Adhesion measurements between CD8 and 48 point mutants of HLA-A2.1 show that the CD8 alpha-chain binds to the alpha 3 domain of HLA-A2.1. Three clusters of alpha 3 residues contribute to the binding, with an exposed, negatively charged loop (residues 223-229) playing a dominant role. CD8 binding correlates with cytotoxic T-cell recognition and sensitivity to inhibition by anti-CD8 antibodies. Impaired alloreactive T-cell recognition of an HLA-A2.1 mutant with reduced affinity for CD8 is not restored by functional CD8 binding sites on an antigenically irrelevant class I molecule. Therefore, complexes of CD8 and the T-cell receptor bound to the same class I major histocompatibility complex molecule seem to be necessary for T-cell activation.

Permanent cure of acute myeloid leukemia (AML) by chemotherapy alone remains elusive for most patients because of the inability to effectively eradicate leukemic stem cells (LSCs), the self-renewing component of the leukemia. To develop therapies that effectively target LSC, one potential strategy is to identify cell surface markers that can distinguish LSC from normal hematopoietic stem cells (HSCs). In this study, we employ a signal sequence trap strategy to isolate cell surface molecules expressed on human AML-LSC and find that CD96, which is a member of the Ig gene superfamily, is a promising candidate as an LSC-specific antigen. FACS analysis demonstrates that CD96 is expressed on the majority of CD34 ؉ CD38 ؊ AML cells in many cases (74.0 ؎ 25.3% in 19 of 29 cases), whereas only a few (4.9 ؎ 1.6%) cells in the normal HSC-enriched population (Lin ؊ CD34 ؉ CD38 ؊ CD90 ؉ ) expressed CD96 weakly. To examine whether CD96 ؉ AML cells are enriched for LSC activity, we separated AML cells into CD96 ؉ and CD96 ؊ fractions and transplanted them into irradiated newborn Rag2 ؊/؊ ␥c ؊/؊ mice. In four of five samples, only CD96 ؉ cells showed significant levels of engraftment in bone marrow of the recipient mice. These results demonstrate that CD96 is a cell surface marker present on many AML-LSC and may serve as an LSC-specific therapeutic target.hematopoietic stem cell

Contribution of Vgamma9/Vdelta2 T lymphocytes to immune protection against Mycobacterium tuberculosis is still a matter of debate. It was reported earlier that Vgamma9/Vdelta2 T lymphocytes kill macrophages harboring live M. tuberculosis through a granule-dependent mechanism that results in killing of intracellular bacilli. This study found that Vgamma9/Vdelta2 T lymphocytes reduce the viability of both extracellular and intracellular M. tuberculosis. Granulysin and perforin, both detected in Vgamma9/Vdelta2 T lymphocytes, play a major role, which indicates that Vgamma9/Vdelta2 T lymphocytes directly contribute to a protective host response against M. tuberculosis infection.

Granulysin, a protein located in the acidic granules of human NK cells and cytotoxic T cells, has antimicrobial activity against a broad spectrum of microbial pathogens. A predicted model generated from the nuclear magnetic resonance structure of a related protein, NK lysin, suggested that granulysin contains a four α helical bundle motif, with the α helices enriched for positively charged amino acids, including arginine and lysine residues. Denaturation of the polypeptide reduced the α helical content from 49 to 18% resulted in complete inhibition of antimicrobial activity. Chemical modification of the arginine, but not the lysine, residues also blocked the antimicrobial activity and interfered with the ability of granulysin to adhere to Escherichia coli and Mycobacterium tuberculosis. Granulysin increased the permeability of bacterial membranes, as judged by its ability to allow access of cytosolic β-galactosidase to its impermeant substrate. By electron microscopy, granulysin triggered fluid accumulation in the periplasm of M. tuberculosis, consistent with osmotic perturbation. These data suggest that the ability of granulysin to kill microbial pathogens is dependent on direct interaction with the microbial cell wall and/or membrane, leading to increased permeability and lysis.