The interaction between killer cells and target cells can be dissected into three steps. First, the two cells form random conjugates mediated by nonspecific interactions between adhesion molecules (1). Second, specific recognition results in the activation of signal transduction pathways coupled to activating receptors and accessory molecules (2). This leads to the third step consisting of perforin and/or fas-ligand mediated delivery of the lethal hit (3). Lethal hit delivery requires a short time; as a consequence killer cells can recycle and kill multiple targets (4). KeywordsConjugate FormationCell Culture IncubatorEquipment ManualConsequence Killer CellFlow Cytometry StandardThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

A cell line harboring all trans-acting elements necessary for hypermutation was transfected with a plasmid harboring the major cis-acting elements plus a green fluorescent protein gene containing a premature chain-termination codon. Transfected cells do not fluoresce unless the stop codon reverts. When a sizable cell population is purged of revertants by sorting, the frequency of mutants increases linearly with time, and there is no Luria-Delbrück fluctuation effect. Moreover, as mutant frequencies seemed to vary less than cell numbers in replicate cultures, it is suggested that hypermutation might not be coupled closely to cell division.

The protein tyrosine kinase p59(fyn) (Fyn) plays important roles in both lymphocyte Ag receptor signaling and cytokinesis of proB cells, We utilized yeast two-hybrid cloning to identify the product of the tctex-1 gene as a protein that specifically interacts with Fyn, but not with other Src family kinases, Tctex-1 was recently identified as a component of the dynein cytoskeletal motor complex. The capacity of a Tctex-1-glutathione S-transferase fusion protein to effectively bind Fyn from cell lysates confirmed the authenticity of this interaction. Tctex-1 binding required the first 19 amino acids of Fyn and integrity of two lysine residues within this sequence that were previously shown to be important for Fyn interactions with the immunoreceptor tyrosine-based activation motifs (ITAMs) of lymphocyte Ag receptors, Expression of tctex-1 mRNA and protein was observed in all lymphoma lines analyzed, and immunofluorescence confocal microscopy localized the protein to the perinuclear region. Analysis of a T cell hybridoma revealed prominent colocalization of Tctex-1 and Fyn at the cleavage furrow and mitotic spindles in cells undergoing cytokinesis, Our results provide a unique insight into a mechanism by which Tctex-1 might mediate specific recruitment of Fyn to the dynein complex in lymphocytes, which may be a critical event in mediating the previously defined role of Fyn in cytokinesis.

Here, we report data concerning the discovery in adult human peripheral blood of a precursor cell population able to differentiate into CD4+CD3+αβ+ mature T cells. These cells, which represent 0.1–0.5% of total peripheral blood mononuclear cells (PBMC), express substantial levels of CD4, but lack CD3 surface expression. At a molecular level, they express the pre-T cell receptor α (pTα) gene, CD3-γ, CD-δ and CD-ε, and RAG-1 recombination enzyme and have initiated rearrangements in the T cell receptor (TCR)-β locus (D–J). Moreover, low levels of CD3ε protein, but not of TCR-β chain, can be detected in their cytoplasm. Our results suggest that CD4+CD3− cells identified in peripheral blood are different from CD3−CD4+CD8− thymocytes and may contain precursors of an extrathymic T cell differentiation pathway.

Immunoglobulin-like transcript (ILT) 3 is a novel cell surface molecule of the immunoglobulin superfamily, which is selectively expressed by myeloid antigen presenting cells (APCs) such as monocytes, macrophages, and dendritic cells. The cytoplasmic region of ILT3 contains putative immunoreceptor tyrosine-based inhibitory motifs that suggest an inhibitory function of ILT3. Indeed, co-ligation of ILT3 to stimulatory receptors expressed by APCs results in a dramatic blunting of the increased [Ca2+]i and tyrosine phosphorylation triggered by these receptors. Signal extinction involves SH2-containing protein tyrosine phosphatase 1, which is recruited by ILT3 upon cross-linking. ILT3 can also function in antigen capture and presentation. It is efficiently internalized upon cross-linking, and delivers its ligand to an intracellular compartment where it is processed and presented to T cells. Thus, ILT3 is a novel inhibitory receptor that can negatively regulate activation of APCs and can be used by APCs for antigen uptake.

Local inflammation leads to increased expression of the vascular cell adhesion molecule (VCAM)-1 on vascular endothelium which contributes to the encapture of leukocytes from the circulating blood through the leukocyte ligand alpha4beta1 integrin. Inflammatory vascular endothelium expresses VCAM-1 at high density. We found that the speed of locomotion of activated lymphocytes migrating along surfaces coated with recombinant VCAM-1 at a comparable density to that found on inflammatory endothelium was slow. However, lymphocytes do migrate and extravasate rapidly under inflammatory conditions, indicating that there must be mechanisms that regulate the interaction between alpha4beta1 and VCAM-1 in vivo. Here we show that the lymphocyte alpha(v)beta3 integrin and integrin-associated protein (IAP) is able to regulate this interaction. The occupancy of lymphocyte alpha(v)beta3 integrin by platelet cell adhesion molecule-1 or vitronectin regulated the speed of alpha4beta1 integrin-dependent locomotion of lymphocytes on recombinant VCAM-1. This allowed rapid lymphocyte migration at VCAM-1 densities which are typical of inflammatory vessels. This alpha(v)beta3-mediated enhanced migration of lymphocytes via alpha4beta1 is likely to depend on the interaction of alpha(v)beta3 integrin with the IAP. Furthermore, this motile process correlates with polarization of the actin cytoskeleton in lymphocytes. Our results suggest that cross talk between alpha(v)beta3 integrin and alpha4beta1 integrin is a mechanism in the regulation of lymphocyte locomotion along inflammatory endothelium and subsequent transendothelial migration. This can explain how lymphocytes overcome tight adhesion to the vascular endothelium and start rapid migration along and through the endothelial lining of blood vessels into inflammatory tissue.

Natural killer (NK) cells express killer inhibitory receptors that mediate negative regulation of NK cell cytotoxicity upon binding to MHC class I molecules on target cells. Unrelated inhibitory receptors on B cells have recently been shown to function through recruitment of phosphotyrosine phosphatase 1C (PTP-1C). Here, we show that a human killer inhibitory receptor specific for HLA-C also recruits PTP-1C after phosphorylation induced either by the pharmacological agent phenylarsine oxide or by conjugation with target cells. This recruitment is mediated by the binding of specific cytoplasmic phosphotyrosine-containing sequences to PTP-1C. These results implicate PTP-1C as a cytosolic component of the negative signaling pathway through NK cell inhibitory receptors.

We have investigated the level of TCR occupancy required to elicit different biological responses in human CTL clones specific for an influenza matrix peptide. Specific cytotoxicity could be detected at extremely low peptide concentrations (10(-12) to 10(-15) M). However, IFN-gamma production, responsiveness to IL-2 and Ca++ fluxes were observed only at peptide concentrations > 10(-9) M, while autonomous proliferation required even higher peptide concentrations. In parallel experiments we measured TCR downregulation to estimate the number of TCRs triggered. We observed that at low peptide concentrations, where only cytotoxicity is triggered, TCR downregulation was hardly detectable. Conversely, induction of IFN-gamma production and proliferation required triggering of at least 20-50% of TCRs. Taken together these results indicate that a single CTL can graduate different biological responses as a function of antigen concentration and that killing of the specific target does not necessarily result in full activation.

We have previously demonstrated that in T cell‐antigen‐presenting cell (APC) conjugates many T cell receptors (TCR) are serially triggered by a few peptide‐MHC complexes, resulting in sustained signaling. Here, we investigate the mechanisms that determine the duration and extent of signaling. We show that in the course of the T helper cell‐APC interaction, down‐regulation of triggered TCR leads to extinction of signaling. However, T cells that have been activated by a previous encounter with peptide‐pulsed APC and have extinguished signaling can swiftly repolarize towards APC displaying higher antigen concentrations and dedicate their help to these cells. These results demonstrate that TCR down‐regulation allows T cells to calibrate their response and dedicate their help to APC offering the highest stimulus.

Using antigen-specific T cell clones and peptide-pulsed antigen-presenting cells (APCs) we investigated the mechanisms that lead to sustained signaling, known to be required for activation of effector function. Four lines of evidence indicate that the T cell actin cytoskeleton plays a crucial role in T cell activation by antigen-pulsed APCs, but is not required when T cell receptor (TCR) is cross-linked by soluble antibodies. First, addition of antibodies to the major histocompatibility complex molecules recognized by the TCR aborts the ongoing intracellular calcium concentration ([Ca2+]i) increase in performed T-APC conjugates, indicating that the sustained signaling requires the continuous occupancy of TCR. Second, time-lapse image recording shows that T lymphocytes conjugated to peptide-pulsed APCs undergo a sustained [Ca2+]i increase, which is accompanied by the formation of a large and changing area of contact between the two opposing membranes. Third, drugs that disrupt the actin cytoskeleton, Cytochalasin D and and C2 Clostridium botulinum toxin induce a rapid block of [Ca2+]i rise, coincident with a block of the cyclic changes in T cell shape. Finally, the addition of Cytochalasin D or of anti-MHC antibodies to preformed conjugates inhibits interferon gamma production in an 1-antigen dose- and time-dependent fashion. These results identify T cell actin cytoskeleton as a major motor for sustaining signal transduction and possibly for driving TCR cross-linking and offer an explanation for how T cells equipped with low affinity TCR can be triggered by a small number of complexes on APCs.

We investigated the functional role of the cAMP pathway in human cytotoxic T lymphocyte (CTL)-target interaction. Pharmacological increase of intracellular cAMP concentration ([cAMP]i) inhibits killing, especially at low effector-to-target ratios, suggesting an inhibitory effect on CTL recycling. We show that this inhibitory effect is primarily at the level of conjugate formation. Pharmacological increase in [cAMP]i, as well as treatment with cytochalasin D, results in a "rounding up" of the CTL and inhibition of the dramatic changes in shape that occur when a CTL forms a conjugate, even with an irrelevant target. In addition, pharmacological increase in [cAMP]i affects the cytoskeleton of the CTL since it induces a decrease of filamentous actin, as detected by flow cytometry on phalloidin-stained CTL, and a stabilization of microtubules, as detected by increased resistance to the disrupting action of nocodazole. In mature CTL (but not in their immature precursors), T cell receptor triggering by specific targets results in a measurable increase in cAMP levels and strongly synergizes with adenylyl cyclase activators such as prostaglandin E2, cholera toxin and forskolin. We suggest that T cell receptor triggering may induce accumulation of cAMP that interferes with cytoskeleton function and, thus, terminates CTL secretion and adhesion. These effects of cAMP are rapidly reversible and may regulate CTL recycling.

We have analyzed the inducibility of protein kinase C (PKC)-dependent expression of CD 69 molecules in T cell receptor (TCR) transgenic thymocytes developing in the presence or absence of selecting. class I major histocompatibility complex (MHC) molecules. Small CD4+8+ thymocytes developing in the absence of selecting MHC molecules could not be induced to express CD 69 by TCR cross-linking even after spontaneous in vitro up-regulation of their TCR level which resulted in enhanced Ca++ flux. In contrast, a small proportion of CD4+8+TCR(low) and most TCR(high)(CD4+8+ and CD4-8+) thymocytes developing in the presence of selecting MHC ligands could be induced to express CD 69 upon TCR cross-linking. Unlike the anti-TCR antibody, phorbol 12-myristate 13-acetate - a direct activator of PKC - induced the expression of CD 69 on all thymocytes. These results suggest that positive selection of CD4+8+ thymocytes results in coupling of TCR-mediated signals to the CD 69 expression pathway. In vitro analysis of thymocytes before and after positive selection suggests that (1) positive selection does not immediately result in resistance to deletion and (2) that sustained TCR ligation is needed to promote maturation of positively selected CD4+8+ thymocytes resulting in gradual loss of the sensitivity to deletion and acquisition of the ability to proliferate in response to TCR-mediated signals.

We know little about the way mature CD4 (helper) and CD8 (killer) T cells develop from thymic CD4+CD8+ precursors. Here we show that small but not large CD4+CD8+ cells with high levels of the alpha-beta-T cell receptor (TcR(high)) result from positive selection. Neither CD4+CD8+ cells with low TcR levels nor large CD4+CD8+ thymocytes with high TcR levels differentiate in vitro. However, small CD4+CD8+ cells with high TcR levels develop in vitro into mature cells by gradually decreasing the surface levels of one or the other co-receptor and acquiring the potential to respond with proliferation to ligation of the TcR. Small CD4+CD8+ cells with high levels of a major histocompatibility complex (MHC) class I-restricted transgenic TcR develop in vitro exclusively into CD4-CD8+ cells while small CD4+CD8+ TcR(high) cells with heterogeneous TcR from various mice yield both CD4 and CD8 T cells. While these experiments are consistent with an instructive model of CD4/CD8 lineage commitment they do not rule out other mechanisms which require multiple TcR-MHC ligand interactions in the generation of mature alpha-beta-T cells.