Phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG), a novel ubiquitously expressed transmembrane adaptor protein, binds the protein tyrosine kinase csk and is involved in regulation of T cell activation.

According to a recently proposed hypothesis, initiation of signal transduction via immunoreceptors depends on interactions of the engaged immunoreceptor with glycosphingolipid-enriched membrane microdomains (GEMs). In this study, we describe a novel GEM-associated transmembrane adaptor protein, termed phosphoprotein associated with GEMs (PAG). PAG comprises a short extracellular domain of 16 amino acids and a 397-amino acid cytoplasmic tail containing ten tyrosine residues that are likely phosphorylated by Src family kinases. In lymphoid cell lines and in resting peripheral blood alpha/beta T cells, PAG is expressed as a constitutively tyrosine-phosphorylated protein and binds the major negative regulator of Src kinases, the tyrosine kinase Csk. After activation of peripheral blood alpha/beta T cells, PAG becomes rapidly dephosphorylated and dissociates from Csk. Expression of PAG in COS cells results in recruitment of endogenous Csk, altered Src kinase activity, and impaired phosphorylation of Src-specific substrates. Moreover, overexpression of PAG in Jurkat cells downregulates T cell receptor-mediated activation of the transcription factor nuclear factor of activated T cells. These findings collectively suggest that in the absence of external stimuli, the PAG-Csk complex transmits negative regulatory signals and thus may help to keep resting T cells in a quiescent state.

Commercial LightCycler-based quantitative real-time PCR compared to nested PCR for monitoring of Bcl-2/IgH rearrangement in patients with follicular lymphoma.

Translocation of chromosomes 14 and 18 [t(14;18)] for detection of minimal residual disease in follicular lymphoma patients can be analyzed by nested polymerase chain reaction (PCR) or by quantitative PCR like LightCycler-based assays. We have compared both methods in blood and bone marrow samples of 28 patients enrolled in a clinical study on immunochemotherapy. In 42% of samples, the bcl2-IgH rearrangement was detectable by nested PCR, but not by LightCycler PCR. Nested PCR was able to reveal a significant drop in positive bone marrow or peripheral blood samples after therapy. In contrast, with LightCycler PCR, the detected drop in t(14;18)-positive cells did not reach statistical significance. The majority of patients showed positive results with nested PCR of peripheral blood or bone marrow without any associations to presence or absence of histological bone marrow (BM) infiltration by lymphoma cells. With LightCycler PCR, the numbers of positive cells were higher in samples from patients with BM infiltration of lymphoma cells (1.9 x 10(-2)) compared to samples from patients without involvement (4.08 x 10(-5)). A similar trend was seen in samples derived from the peripheral blood. Positivity for t(14;18) after therapy in two patients correlated with clinical relapse 6 months later. The data shown here demonstrate a lower sensitivity of LightCycler vs. nested PCR for detection of t(14;18). The usefulness of nested PCR for t(14;18) for risk stratification after primary therapy has to be validated in larger trials.

Survivin expression correlates with apoptosis resistance after lymphocyte activation and is found preferentially in memory T cells.

The prevention of apoptosis may be critical for immunological function. Survivin is a recently cloned member of the inhibitor of apoptosis protein family. We analyzed survivin expression before and after lymphocyte activation in isolated cell populations. Prior to activation, survivin was undetectable. After activation with IL-2 and OKT-3, CD3(+) cells expressed survivin. Next, we correlated survivin expression with Fas, FasL and the amount of apoptosis over time in culture. After activation, survivin was readily detected by day 2 and decreased thereafter. Prior to activation (day 0), Fas was present on 60% of the cells and on 100% by days 2-6. Peak FasL mRNA expression was at day 2. During peak survivin expression (days 2-4) the apoptotic fraction was low, but when survivin expression decreased the apoptotic fraction increased rapidly. Finally, we found that CD45RO(+) memory T cells showed a higher expression of survivin than did CD45RA(+) naive T cells after activation. These results suggest that survivin may contribute to T-cell survival early in T-cell responses as well as in memory immune responses.

The apoptotic and proliferative fate of cytokine-induced killer cells after redirection to tumor cells with bispecific Ab.

BACKGROUND: Cytokine-induced killer (CIK) cells are ex vivo expanded T cells with co-expression of CD3 and CD56 and NK activity. They have recently been evaluated in a phase I/II clinical trial against malignant lymphoma. Bispecific Ab (bsAb) redirect CIK cells to tumor targets, thus enhancing their cytotoxicity. While bsAb may improve T-cell mediated anti-tumor activity, little is known about the fate of effector cells upon redirection to tumor targets using a bsAb. METHODS: Using ex vivo-activated CIK cells, Her2/neu expressing breast and ovarian cell lines and a F(ab')2 Her2/neu x CD3 bsAb, we investigated the anti-tumor activity and the proliferative and apoptotic outcome of CIK cells. RESULTS: When redirected to tumor targets with bsAb, there was a significant increase in anti-tumor activity as well as an increase in both CIK cell proliferation and apoptosis. The addition of agonistic Ab against CD28 did not significantly increase proliferation or apoptosis of CIK cells redirected to CD80- and CD86- tumor targets. To attempt to reduce T-cell apoptosis, we incubated CIK cells in the presence of the pan-caspase inhibitor z-VAD-fmk, which led to a partial reduction in T-cell apoptosis without increasing cellular cytotoxicity. DISCUSSION: bsAb are effective in redirecting activated T cells to tumor targets and such redirection leads to both T-cell proliferation and apoptosis that are not altered by co-stimulation through CD28. Effector cell apoptosis can be reduced by using a caspase inhibitor but this does not increase CIK cell cytotoxicity.

Hodgkin disease-derived cell lines expressing ubiquitous mitochondrial creatine kinase show growth inhibition by cyclocreatine treatment independent of apoptosis.

Ubiquitous mitochondrial creatine kinase (uMtCK), a key enzyme in energy metabolism, was identified by differential display PCR to be specifically overexpressed in L1236, the first cell line of definite Hodgkin origin. RT-PCR confirmed overexpression of uMtCK in the L1236 cell line and the absence of cytosolic B-CK, which is co-expressed with MtCK physiologically. Cyclocreatine (cCr), whose phosphorylated form is a very poor substrate for CK, inhibited proliferation of the L1236 cell line nearly entirely. This inhibition by cCr was partially reversed by competition with creatine, which by itself had no effect on proliferation of the L1236 cell line. Although these results support a role of CK activity in the inhibitory action of cCr, it remains open whether the cCr effect is due to its inhibition of CK-linked energy metabolism or if alternative mechanisms have to be considered. Because the anti-proliferative effect of cCr was not due to induction of apoptosis, in contrast to most other anticancer agents, treatment with the creatine analogue cCr may represent an advantageous therapeutic approach for cells resistant to programmed cell death.