Richard G. Miller

Activated, But Not Resting, T Cells Can Be Recognized and Killed by Syngeneic NK Cells

We demonstrate that IL-2-activated NK cells or lymphokine-activated killer cells recognize and kill syngeneic CD4+ and CD8+ T cells that have been activated by APCs. Induction with APC required TCR-specific Ag, and lysis was perforin mediated. Brefeldin A, which disrupts protein transport, inhibited the sensitivity induced by activation. In BALB/c, expression of NKG2D ligands correlated with lysis and could be inhibited by brefeldin A. As well, addition of anti-NKG2D mAb to a killing assay completely abrogated lysis. Transduction of mouse NKG2D into a human NK cell line, YTSeco, conferred upon it the ability to kill activated BALB/c T cells, indicating that NKG2D is necessary for recognition. Our data provide a basis for studying a role for NK cells in T cell regulation.

Oral beta-carotene can increase the number of OKT4+ cells in human blood

Oral doses of beta-carotene (180 mg/day) given for 2 wk to normal human volunteers significantly increased the frequency of OKT4+ (helper/inducer T cells) after 7 days and of OKT3+ (all T cells) after 14 days. The frequency of OKT8+ cells was unaffected, as compared to untreated controls. Plasma levels of beta-carotene were markedly elevated throughout the study. Plasma levels of vitamin A were slightly (but significantly) elevated on days 7 and 14. No toxicity was observed. Oral beta-carotene treatment may be a clinically effective means of enhancing T4+ lymphocytes.

Hoechst 33342 dye uptake as a probe of membrane permeability changes in mammalian cells.

Flow cytometric analysis of the uptake of the DNA-specific and fluorescent probe Hoechst 33342 (HO342) offers a simple and rapid method for measuring membrane transport rates in mammalian cells and identifying cellular subpopulations that differ in their membrane transport rates. In a Chinese hamster ovary wild-type cell line and in three colchicine-resistant lines derived from it, the rate of uptake of HO342 dye decreases as the stepwise resistance to colchicine increases. A colchicine-sensitive revertant cell line shows an increased rate of dye uptake. Drug resistance in these lines has previously been shown to be related to changes in transport rate. In a manner similar to colchicine, the rate of uptake of HO342 dye shows nonsaturation kinetics. The effects of KCN, a metabolic inhibitor, on HO342 dye uptake, both in the presence and in the absence of glucose, is similar to that previously observed for colchicine uptake. When murine spleen cells are stained with HO342 under appropriate conditions, one sees two populations of lymphocytes differing in HO342 fluorescence intensity, a difference not related to DNA content. The two subpopulations show the same relative difference in both colchicine and HO342 uptake. HO342 dye appears, therefore, to enter mammalian cells by the same mechanism as colchicine--i.e., by unmediated diffusion--and can be used as a probe of cytoplasmic membrane permeability. Potential applications of the dye in studies of drug resistance, detection of activated T cells, and recognition of lymphocyte subpopulations are discussed.

Quantitative studies of the activation of cytotoxic lymphocyte precursor cells.

The mixed lymphocyte reaction is widely studied as an in vitro model of T cell activation. For studies in the mouse (reviewed in Cerottini & Brunner 1974, Bach et al. 1976), one cultures lymphoid cells from one strain (responder cells) with inactivated (e.g. by irradiation) cells from an allogeneic or semi allogeneic strain (stimulator cells). The lymphocytes responding to an allogeneic stimulus undergo extensive proliferation and differentiation, leading, after several days in culture, to the production of cytotoxic T lymphocytes (CL) (reviewed in Cerottini & Brunner 1974, Bach et al. 1976). These cells can recognize and destroy cells sharing H-2 alioantigens with the stimulator cells. Only one thing about the production of CL is certain, by definition if nothing else: they arise from cytotoxic lymphocyte precursor cells (CLP) which are themselves also T cells. The basic goal of the studies sunamarized

The correlation of prolonged survival of maternal skin grafts with the presence of naturally transferred maternal T cells

Hypoimmune responses towards noninherited maternal MHC antigens (NIMA) have been observed on both the humoral and cellular level in humans. The mechanism involved is not known but is thought to involve transfer of material from mother to child. Here we use a mouse model to test this hypothesis. Mice exposed to NIMA either in utero and/or via breast milk showed enhanced survival of maternal skin grafts and also had maternal lymphocytes in their lymph nodes. Survival of the maternal skin graft correlated with the number of maternal T cells present and was not correlated with the number of maternal B cells. We propose that the transferred maternal T cells selectively inactivate host T cells capable of recognizing them.

Fluorescence flow analysis of lymphocyte activation using Hoechst 33342 dye.

The in vitro response of murine lymphocytes to allogeneic and mitogenic stimulation has been studied by using the nontoxic fluorescent DNA probe, Hoechst 33342, and a fluorescence flow cytometer-cell sorter. Under appropriate conditions, two peaks of fluorescent intensity, not related to cellular DNA content, can be seen. As early as 12 hr after culture set up, lymphocyte activation can be identified. It appears that Hoechst-labeled lymphocytes of higher fluorescence intensity represent those cells activated by allogeneic stimulation whereas cells obtained from the lower intensity peak are nonresponding lymphocytes.

Stress Renders T Cell Blasts Sensitive to Killing by Activated Syngeneic NK Cells

Exposure of primary T cell blasts to stress in the forms of heat, hydrogen peroxide, or high-density growth conditions resulted in a state of enhanced susceptibility to killing by syngeneic IL-2-activated NK cells or lymphokine-activated killer cells, but not to killing by CTL. Cytotoxicity was perforin mediated and was not due to decreased target expression of total MHC class I. The levels of stress used had little effect on cell viability. For thermal stress, sensitization increased with temperature, required a minimum exposure time, and disappeared when cells were given a long enough recovery time. Our data support a model that predicts that activated NK cells play a role in the immunosurveillance of nontransformed stressed cells in normal animals.

T cell function and expression are dramatically altered in T cell receptor Vγ1.1Jγ4Cγ4 transgenic mice

Abstract We have characterized transgenic mice carrying a functional T cell receptor (TCR) Cγ4 (Vγ1.1Jγ4Cγ4) gene. Results indicate that active transcription of the Cγ4 transgene can influence expression of the endogenous Cγ4, Cγ1 (Vγ3-, Vγ4-, Vγ2-, or Vγ5Jγ1Cγ1) and Cγ2 (Vγ1.2Jγ2Cγ2) genes, while the ultimate expression of other TCR δ, α, and β chain genes, as well as the adult T cell response, are relatively unaltered. Cells expressing transgenic Cγ4 and endogenous δ TCR transcripts can migrate to the skin as dendritic epithelial cells (DEC) even though Cγ4 cells are rarely, if at all, found in the skin. Transgenic and control mice were compared at 2 weeks, 6–7 weeks, and older. At 2 weeks, the thymus of transgenic mice, particularly the medulla, was much larger than control. Moreover, peripheral lymphoid tissues of younger mice were markedly (as much as 100-fold) more immunoreactive (both Con A response and alloreactivity). These differences, although persistent, became smaller in older mice. The data suggest that transgene expression has a major effect on T cell development and reactivity.

NKG2D splice variants: a reexamination of adaptor molecule associations

NKG2D is a homodimeric C-type lectin-related receptor expressed on natural killer (NK) cells and T cells. In mice, alternative deoxyribonucleic acid (DNA) splicing generates two isoforms of NKG2D that differ in the length of their cytoplasmic domains. Their ability to induce cellular activation is mediated via association with two membrane-bound, signaling adaptor molecules, DAP10 and DAP12. It has been reported that the long form of NKG2D associates exclusively with DAP10, whereas the short variant can interact with either adaptor. The short isoform was reported to be almost undetectable in naïve NK cells. Using two distinct cell types, we demonstrate that like the short isoform, the long variant of NKG2D also associates not only with DAP10 but also with DAP12. Using reporter cells (70Z/3), we demonstrate that DAP12 can compete equally with DAP10 for association with both variants of NKG2D when DAP10 and DAP12 are coexpressed. Cross-linking either isoform of NKG2D induces a calcium flux when associated exclusively with DAP10 or DAP12. Moreover, using quantitative polymerase chain reaction (PCR), we also show that the short isoform of NKG2D is expressed in naïve NK cells. Our data suggest that signaling via mouse NKG2D isoforms is more complex than originally presented.

Measurement of cytoplasmic fluorescence depolarization of single cells in a flow system.

We have built a flow system in which we can analyze and sort individual viable cells on the basis of their cytoplasmic microviscosity. The average cytoplasmic microviscosity (or cytoplasmic structuredness) of a cell can be quantitated by exciting fluorescein molecules in the cytoplasm of the cell with a polarized light source and measuring the extent of depolarization of the fluorescent signal. Changes in the state of a cell (e.g., the reception of a signal inducing the cell to differentiate) often appear to be associated with changes in cytoplasmic microviscosity, these changes being detectable within hours of the inducing signal. An example of one such change is presented.