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Using a novel dual-channel FACS methodology, the organotin compound TBT (2 microM) was shown to induce rapid (maximal by 3 min) and sustained elevations in intracellular calcium levels [Ca(2+)](i) in Jurkat T cells. This was preceded by mitochondrial hyperpolarization (maximal at 1 min), with subsequent loss of membrane potential, (Deltapsi(m)) over the next 15 min and was associated with the release of mitochondrial cytochrome c and the activation of type II caspases. The activation of the caspases was blocked by calcium chelation with EGTA and/or BAPTA. Interestingly, changes in Deltapsi(m) caused by TBT were not affected by chelation of intra- and extracellular calcium or by performing the experiments in a Ca(2+)-free medium. TBT also caused rapid elevation of [Ca(2+)](i) in cells lacking glycolytic ATP production. Despite this, the loss of Deltapsi(m) and the activation of type II caspases were delayed (maximal by 2 h) in these cells. Further, there was a failure to activate type II caspases in cells treated with TBT in a Ca(2+)-free medium, despite rapid release of mitochondrial cytochrome c. Consequently, these cells evaded the induction of apoptosis and were diverted to delayed necrotic deletion. Taken together, these data strongly suggest that the rapid rise in [Ca(2+)](i) caused by TBT in Jurkat T cells is not directly coupled to the induction of mitochondrial permeability transition, which rather results from a direct interaction of TBT with mitochondrial component(s) controlling pore transition. However, the rise in [Ca(2+)](i) is a prerequisite for postmitochondrial events involved in caspase activation prior to the induction of apoptosis.

The principal symptoms of myasthenia gravis (MG), muscle weakness and fatigue due to impaired neuromuscular transmission, are caused by autoantibodies to the muscle nicotinic acetylcholine receptor (AChR). The mechanisms underlying the autoimmune response, however, appear to be initiated by activation of specific HLA class II–restricted CD4+ T lymphocytes. Thus, central to elucidating the causation of MG is determining how T cells are recruited to contribute to misguided immunological assaults on the major autoantigenic target, AChR. By combining a polymerase chain reaction (PCR)–based strategy and Southern blot technique, we have analyzed the frequency of expression of 22 individual T cell receptor (TCR) Vβ gene subfamilies in CD4+ and CD8+ peripheral blood T cell subsets derived from eight MG patients and seven healthy controls. The quantification of relative usage of individual TCR Jβ gene segments was performed by hybridization of PCR-amplified products (specifically Vβ1-Cβ) with a complete panel of 32P-5′-end-labeled Jβ-specific oligonucleotide probes, followed by scanning analysis of autoradiographs. Comparisons of data obtained from Vβ analyses of T cells from MG patients with those from healthy individuals established that MG patients significantly overexpressed Vβ1, Vβ13.2, Vβ17, and Vβ20 gene family members within both CD4+ and CD8+ T cell sub-populations. Moreover, analysis of the relative utilization of individual TCR Jβ gene segments in Vβ1+/CD4+ and Vβ1+/CD8+ T lymphocytes revealed distribution patterns in patients indistinguishable from those recorded in the corresponding cell subsets derived from controls. T lymphocytes from MG patients displayed a biased overexpression of four TCR Vβ gene segments: Vβ1, Vβ13.2, Vβ17, and Vβ20. The relative frequencies of association of individual Vβ1 (Dβ)Jβ combinations revealed that Jβ gene usage in the Vβ1-overrepresented T cell subsets had normal distribution patterns. It can thus be deduced that Jβ gene segment products appear not to have a selective effect on the process leading to overexpression of Vβ1 exons in MG patients. Hence, our observations suggest a possible role for superantigen(s) in the T cell activation in MG patients.

A monoclonal IgM antibody, H17, has been obtained from rats immunized with mouse fibrosarcoma cells from an in vitro established methylcholanthrene (MCA)-induced tumour. H17 shows specific and very selective binding to alpha-N-acetylgalactosamine (GalNAc alpha) when tested for reactivity to a panel of glycolipids. It cross-reacts with GalNAc alpha on the Forssman antigen extracted from dog small intestine, but not from the human blood group A determinant, a finding not commonly observed among antibodies with this specificity. Despite its specificity, H17 does not react with TA3-Ha, a mouse mammary adenocarcinoma, known to express the Tn antigen (GalNAc alpha-O-Ser/Thr). The uniqueness of H17 probably relates to the fact that it has been generated against an MCA-induced tumour rather than against the pure saccharide itself. Minimum energy conformation structures of different GalNAc alpha containing saccharide molecules were computer modelled to allow a plausible interpretation of the accessible site of GalNAc alpha for successful interaction with the H17 paratope as compared to other GalNAc alpha binding antibodies.

We have recently described a monoclonal IgM antibody, H17, to a mouse methylcholanthrene-induced tumor with specificity for a common saccharide, namely α-N-acetyl-galactosamine. Although recognizing a glycosidic determinant most likely to be part of many cell surface glycoproteins, H17 was highly specific for the immunizing tumor. This discriminatory ability was investigated by testing the antibody binding to a panel of normal and transformed mouse and human cells. Cross-linking studies showed that H17 reacts with a glycoprotein with a molecular weight of 40–43 kD, which through blockage during the process of immunization has an impact in vivo on tumor growth. Moreover, the uncommon sensitivity to complement-mediated lysis by this methylcholanthrene-induced tumor was blocked by the H17 monoclonal antibody.

In the present study, we show that the immunotoxicant, tributyltin (TBT), induces a dose-dependent activation of caspases followed by typical apoptotic morphology in resting human peripheral blood lymphocytes. TBT also caused an early loss of mitochondrial membrane potential (Delta(Psi)(m)) and release of cytochrome c, suggesting that apoptosis was triggered by the mitochondrial pathway. When CD4+ T-cells were sorted from peripheral blood and exposed to TBT for 30 min, caspase activation and apoptosis were induced. Interestingly, in the sorted CD8+ T-cell population, caspase activation was not observed until 2 h of TBT exposure, suggesting that these cells were more resistant toward TBT. Moreover, a time-dependent induction of caspase activity was also detected in CD3-stimulated peripheral blood lymphocytes. This caspase activation was not associated with cytochrome c release or loss of mitochondrial Delta(Psi) and did not lead to apoptotic morphology, although it did lead to both PARP and DFF cleavage. We also noticed a concomitant induction of Hsp27, and it awaits to be seen if this chaperone may interfere with the processing of nuclear protein substrates downstream from these primary caspase-3 substrates. Moreover, no increase in caspase activation or induction of apoptosis was observed after TBT treatment in these cells. Instead, the cells were directed toward necrotic deletion. Taken together, these data suggest that TBT-induced deletion of peripheral lymphocytes is likely to be a component in the overall risk for immunotoxic responses in exposed humans.

Objective To investigate T cell receptor (TCR) V alpha/V beta (and in selected cases, J beta) usage in CD4+ and CD8+ peripheral blood lymphocytes of patients with giant cell arteritis (GCA), before and after treatment, as well as to analyze the HLA types of these patients. Methods Flow cytometry, with 10 anti-TCR V-specific monoclonal antibodies (MAb), was used. To analyze J beta usage by cell populations expressing certain V beta, we used the polymerase chain reaction (PCR) technique, with V beta- and C beta-specific primers, Southern blotting of PCR products, and subsequent hybridization with radiolabeled J beta-specific probes. HLA typing was performed using the microlymphocytotoxicity technique. Results Seven of the 9 GCA patients had increased anti-TCR V MAb reactivities (interpreted as T cell expansions), which in many cases, correlated with clinical signs of disease. A strict preference for particular J beta segments was found in 3 of 3 expanded CD4+ T cell populations. Conclusion T lymphocytes expressing specific antigen receptors are implicated in the pathogenesis of GCA.

HIV+ human sera contain antibodies against most HIV proteins, including the envelope glycoprotein Gp120. Some of these antibodies may have an epitope that sterically resembles the CD4 region to which the Gp120 molecule binds. Amongst 58 HIV+ sera tested, we found three with the capacity to block the binding of anti-CD4 monoclonal antibodies to CD4+ cells. The serum with the highest blocking capacity was selected for further analysis. The inhibitor was shown to be an antibody that binds to the Gp120 molecule as well as to the anti-CD4 monoclonal T4.2. These CD4-mimicking antibodies were shown not to interfere with CD4-dependent reactions in vitro. Virus neutralizing experiments in vitro could not show any neutralizing effect with these antibodies alone. The HIV+ individual providing this antibody is still healthy, although HIV+ since 1983.