Administration of daclizumab, a humanized mAb directed against the IL-2R␣ chain, strongly reduces brain inflammation in multiple sclerosis patients. Here we show that daclizumab treatment leads to only a mild functional blockade of CD4 ؉ T cells, the major candidate in multiple sclerosis pathogenesis. Instead, daclizumab therapy was associated with a gradual decline in circulating CD4 ؉ and CD8 ؉ T cells and significant expansion of CD56 bright natural killer (NK) cells in vivo, and this effect correlated highly with the treatment response. In vitro studies showed that NK cells inhibited T cell survival in activated peripheral blood mononuclear cell cultures by a contact-dependent mechanism. Positive correlations between expansion of CD56 bright NK cells and contraction of CD4 ؉ and CD8 ؉ T cell numbers in individual patients in vivo provides supporting evidence for NK cell-mediated negative immunoregulation of activated T cells during daclizumab therapy. Our data support the existence of an immunoregulatory pathway wherein activated CD56 bright NK cells inhibit T cell survival. This immunoregulation has potential importance for the treatment of autoimmune diseases and transplant rejection and toward modification of tumor immunity.CD25 ͉ IL-2 ͉ immunoregulatory natural killer cells M ultiple sclerosis (MS) is an inflammatory͞demyelinating disease of the CNS that is one of the leading causes of neurological disability in young adults (1). It is believed that MS is a T cell-mediated autoimmune disease, and therefore the search for new therapies focuses on agents that affect lymphocyte function. Daclizumab (Zenapax), a humanized mAb that blocks the IL-2 binding site on the IL-2R␣ chain, CD25 (i.e., Tac epitope), is among these novel agents (2). The IL-2R complex is comprised of three subunits: IL-2R␣ (CD25), IL-2R␤ (CD122), and IL-2R␥ (CD132). CD122 and CD132 have intracellular signaling motifs and together form the intermediate-affinity (K dis Ϸ 0.1-1 nM) IL-2R. CD25 binds IL-2 with low (K dis Ϸ 10 nM) affinity, but when it associates with CD122͞CD132 it stabilizes the complex to form the highaffinity (K dis Ϸ 10 pM) receptor (3). CD25 is present at low levels in resting human T cells (with the exception of T regulatory cells) but is significantly up-regulated on activated T cells, enabling them to receive a high-affinity IL-2 signal (4). Therefore, it is believed that the blockade of CD25 will result in selective functional inhibition of activated T cells (5). Although it has been demonstrated that daclizumab (or the original murine anti-Tac mAb) inhibits early IL-2R signal transduction events (6, 7) and blocks T cell activation and expansion in vitro (8), a comprehensive characterization of its in vivo effects is still lacking.We recently concluded a phase II, open-label, baseline-versustreatment crossover trial of daclizumab in 10 MS patients with incomplete therapeutic response to IFN-␤. Daclizumab showed a profound inhibitory effect on brain inflammatory activity (78% reduction) and subsequent stabilization o...

When small, unilamellar lipid vesicles containing a high concentration of the fluorescent dye 6-carboxyfluorescein are incubated with either frog retinas or human lymphocytes, fluroescence distributes widely throughout each cell. Since "self-quenching" largely prevents the dye from fluorescing as long as it remains sequestered in vesicles, it is clear that a considerable amount of dye is released from the vesicles and diluted into the much larger volume of the cell.

Experimental Immunology Branch hypothesis that cytoplasmic proteases are part of the National Cancer Institute core molecular apoptotic death pathway. It does not National Institutes of Health deny the utility of nuclear damage as a readout of apop-Bethesda, Maryland 20892-1360 totic death nor the possibility that cytoplasmic proteases may enter the nucleus and mediate apoptotic damage. The ICE family is defined by sequence homology to It has become clear that selective lymphocyte death ICE. Although this enzyme was characterized as the plays a major role in controlling immune responses in protease that cleaves the inactive precursor of IL-1␤ to both B and T cell compartments. The critical death proyield the active cytokine, its possible connection with cesses occur throughout the life of lymphocytes, from cell death emerged from the cloning of the C. elegans newly emerging precursor cells to fully mature effector death gene ced-3 (Yuan et al., 1993). A database homolcells. Because such cell deaths are generally accompaogy search gave the unanticipated result that human nied by apoptotic features, it is commonly assumed that ICE was the only homologous sequence to Ced-3. The they share a common core of downstream biochemical more recent evidence that ICE family proteases are part steps leading to death, even though the triggering recepof a core apoptotic pathway comes largely from studies tors may differ. However, the molecular characterization of such an apoptotic death pathway has remained elu-showing that inhibitors of these proteases block a wide sive. Much attention has been focused on understand-range of apoptotic death systems. These deaths appear ing the molecular function of Bcl-2, which is capable of at least as diverse as those inhibited by the Bcl-2 family, blocking many diverse apoptotic death systems and but the connections between these two families remain seems to be related to the product of the nematode cloudy. The depiction in Figure 1 of Bcl-2 acting updeath antagonist gene ced-9. Bcl-2 is part of a family stream of the proteases is speculative. To appreciate of related interacting proteins with death-promoting as the biological experiments carried out with inhibitors well as death-inhibiting properties, but their relation to of ICE family proteases, it is necessary to review the the molecular pathway of apoptotic death is unclear. background literature on the biochemisty of these prote-Recent experiments suggest that a novel family of cytoases and the inhibitors themselves. plasmic proteases related to interleukin-1␤ (IL-1␤)-con-The ICE family proteases are summarized in Figure 2. verting enzyme (ICE) plays such a core role, which is While more members will be described in the future, the depicted schematically for thymocytes in Figure 1. The multiplicity of names for individual proteins comes from purpose of this review is to summarize recent findings their identification in multiple laboratories with different on the biochemistry of these proteases and the evidence approaches, suggesting th...

Because mutations in Rab27a have been linked to immune defects in humans, we have examined cytotoxic lymphocyte function in ashen mice, which contain a splicing mutation in Rab27a. Ashen cytotoxic T lymphocytes (CTLs) showed a >90% reduction in lytic activity on Fas-negative target cells compared with control C3H CTLs, and ashen natural killer cell activity was likewise diminished. Although their granule-mediated cytotoxicity pathway is profoundly defective, ashen CTLs displayed a normal FasL–Fas cytotoxicity pathway. The CD4/8 phenotype of ashen T cells and their proliferative responses were similar to controls. Ashen CTLs had normal levels of perforin and granzymes A and B and normal-appearing perforin-positive granules, which polarized upon interaction of the CTLs with anti–CD3-coated beads. However, rapid anti–CD3-induced granule secretion was drastically defective in both CD8+ and CD4+ T cells from ashen mice. This defect in exocytosis was not observed in the constitutive pathway, as T cell receptor–stimulated interferon-γ secretion was normal. Based on these results and our demonstration that Rab27a colocalizes with granzyme B-positive granules and is undetectable in ashen CTLs, we conclude that Rab27a is required for a late step in granule exocytosis, compatible with current models of Rab protein function in vesicle docking and fusion.