T cells express T-cell antigen receptors (TCR) for the recognition of antigen in conjunction with the products of the major histocompatibility complex. They also express two key surface coreceptors, CD4 and CD8, which are involved in the interaction with their ligands. As CD4 is expressed on the early haemopoietic progenitor as well as the early thymic precursor cells, a role for CD4 in haemopoiesis and T-cell development is implicated. Thymocytes undergo a series of differentiation and selection steps to become mature CD4+8- or CD4-8+ (single positive) T cells. Studies of the role of CD4+ T cells in vivo have been based on adoptive transfer of selected or depleted lymphocytes, or in vivo treatment of thymectomized mice with monoclonal antibodies causing depletion of CD4+ T cells. In order to study the role of the CD4 molecule in the development and function of lymphocytes, we have disrupted the CD4 gene in embryonic stem cells by homologous recombination. Germ-line transmission of the mutation produces mutant mouse strains that do not express CD4 on the cell surface. In these mice, the development of CD8+ T cells and myeloid components is unaltered, indicating that expression of CD4 on progenitor cells and CD4+ CD8+ (double positive) thymocytes is not obligatory. Here we report that these mice have markedly decreased helper cell activity for antibody responses, although cytotoxic T-cell activity against viruses is in the normal range. This differential requirement for CD4+ helper T cells is important to our understanding of immune disorders including AIDS, in which CD4+ cells are reduced or absent.

A system for fractionating populations of Jiving cells by velocity sedimentation in the earth's gravitational field i s described. The cells start in a thin band near the top of a shallow gradient of 3% to 30% fetal calf serum in phosphate buffered saline at 4°C. Cell separation takes pIace primarily on the basis of size and is approximately independent of cell shape. A sharply-defined upper limit, called the streaming limit, exists for the cell concentration in the starting band beyond which useful cell separations cannot be achieved. This limit, which varies with the type of cell being sedimented, can be signBcantly increased by proper choice of gradient shape. For sheep erythrocytes (sedimentation velocity of 1.6 -/hour) it is 1.5 X 1 0 ' cells/ml. Measured and calculated sedimentation velocities for sheep erythrocytes are shown to be in agreement. The technique is applied to a suspension of mouse spleen cells and it is shown, using an electronic cell counter and pulse height analyzer, that cells are fractionated according to size across the gradient such that the sedimentation velocity (in mm/hour) approximately equals r2/4 where r is the cell radius in microns. Since cells of differing function also often differ in size, the system appears to have useful biological applications.

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.

As well as being activated or rendered unresponsive, mature T lymphocytes can be deleted, depending on the signals received by the cell. Deletion by programmed cell death (apoptosis) is triggered if a T cell that has received a signal through its T cell receptor complex also receives a signal through the alpha 3 domain of its class I major histocompatibility complex (MHC) molecule. Such a signal can be delivered by a CD8 molecule, which recognizes the alpha 3 domain, or by an antibody to this domain. Precursors of both cytotoxic T lymphocytes (CTL's) and T helper cells are sensitive to this signal but become resistant at some point before completing differentiation into functioning CTL's or T helper cells. Because CTL's carry CD8, they can induce cell death in T cells that recognize them. This pathway may be important in both removal of autoreactive T cells and immunoregulation.

Following exposure to some types of antigen (superantigens), responsive T cells expand and then decline in numbers, a phenomenon that has been called 'peripheral deletion'. This process may play a role in limiting autoimmune reactions and in the maintenance of immune homeostasis. Here we describe experiments on peripheral deletion in mice carrying the lpr/lpr defect, which has been shown to be due to defective production of the CD95/Fas molecule. Young lpr/lpr mice with no apparent immunologic abnormalities display a defect in bacterial superantigen-induced peripheral deletion. Apoptotic death of the expanded T cell population associated with such peripheral deletion. Apoptotic death of the expanded T cell population associated with such peripheral deletion in normal animals is dramatically reduced in the mutant mice. Further, the levels of Fas on responding cells in normal mice increases and decreases together with increases and decreases in cell numbers, suggesting that cells with the highest levels of Fas are preferentially deleted. These observations are consistent with the known ability of CD95 to transduce a signal leading to apoptosis, and they implicate this signal transduction pathway in peripheral deletion. In contrast, bacterial superantigen-induced deletion of thymocytes appears to be fully functional in these mice, and thus Fas/APO-1 does not appear to be required for this process. Further, antibody ligation of the TCR on activated T cells from normal or young lpr/lpr mice can induce apoptosis and therefore under some circumstances this phenomenon is not dependent upon CD95/Fas. Thus, to avoid autoreactivity and ensure immune homeostasis, several different apoptotic mechanisms exist in peripheral T lymphocytes, only some of which involve Fas.(ABSTRACT TRUNCATED AT 250 WORDS)

The immune system does not normally react against self components. Originally, it was postulated that self-reactive cells were somehow deleted or blocked. More recent thinking is that such cells are suppressed by regulatory networks similar to those limiting the immune response against non-self determinants. Both mechanisms may exist. I describe here a type of suppression more closely related to the first postulate. In the in vitro, one-way, mixed lymphocyte reaction (MLR), cytotoxic T-lymphocyte precursor cells (CLP) from the responder population give rise to cytotoxic T-lymphocytes (CL) capable of lysing target cells from the stimulator population. A subpopulation of cells in the spleen of athymic nude mice can, when added to such cultures, inactivate CLP capable of recognizing either the H-2 antigens or TNP modifications of the nude spleen. Regarding the nude spleen cells, activation of self-reactive cells is being prevented.

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Flow cytometric analysis of the uptake of the DNA-specific and fluorescent probe Hoechst 33342 (H0342) offers a simple and rapid method for measuring membrane transprt 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 H0342 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 H0342 dye shows nonsaturation kinetics. The effect of KCN, a metabolic inhibitor, on H0342 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 H0342 under appropriate conditions, one sees two populations of lymphocytes differing in H0342 fluorescence intensity, a difference not related to DNA content. The two subpopulations show the same relative difference in both colchicine and H0342 uptake. H0342 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 ofthe dye in studies ofdrug resistance, detection ofactivated T cells, and recognition of lymphocyte subpopulations are discussed.We describe a sensitive and rapid method for assessing membrane permeability by using a flow cytometer (1) and the bisbenzimidazole dye Hoechst 33342 (H1342) (2). This dye binds both specifically and quantitatively to DNA and, once bound, becomes strongly fluorescent (3). Unlike other DNA-specific fluorescent dyes (4), H0342 is readily taken up by living cells and is nontoxic (5). Although H0342 can be used for flow cytometric (FCM) analysis of DNA content of viable cells (5, 6), H0342 stained murine lymphocyte populations show differences in fluorescence intensity not related to DNA content (7). An attempt to understand these differences led to the present study, in which we conclude that they are due to differences in the rate of H0342 transport across the cytoplasmic membrane. Ling and Thompson (8) computer and the modal fluorescence intensities of the observed peaks were estimated by Gaussian fit. Bidirectional cell sorting was performed using a recently built set of sorting logic circuitry (unpublished).Abbreviations: H0342, Hoechst 33342 dye; FCM, flow cytometric; CHO, Chinese hamster ovary; LI and HI, low-intensity and high-intensity fluorescence populations. 363The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.