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Growing experimental evidence supports a broadening role for the caspases; not only do they participate in the process of apoptosis but also in the control of the cell cycle and cellular proliferation. The biological role of the caspases in the process of T-cell activation and proliferation is still not defined. In the present study, we propose a potential role, by demonstrating an association of T-cell receptor-mediated caspase activity with the development of an apoptosis-resistant memory CD45RO+ T-cell population. As previously shown by us, a time-dependent induction of caspase activity, in the absence of apoptosis, can be observed in CD3-stimulated human peripheral blood lymphocytes. We here show that a population of CD45RO+ cells, with activated caspase-3 and with resistance to tributyltin-induced apoptosis, develops after 3 days of stimulation. A concomitant expression of the anti-apoptotic protein Bcl-xL accompanied the caspase activity and the development of the apoptosis-resistant phenotype. Finally, upon co-culturing with dexamethasone (DEX), the CD3-induced caspase-3 activity was blocked. During this condition, the expression of the activation marker HLA-DR as well as the cellular proliferative response was strongly suppressed. The development of memory cells with a CD45RO+ phenotype was also blocked. Our data support the hypothesis that caspase-3 activity, observed in CD3-stimulated cells, may be an important component in the proliferation process and, furthermore, might play a role for the development of memory T cells, and DEX inhibits this process.

Our data on peripheral blood T cells from Motor neuron disease (MND) patients indicate major immunological disturbances linked to this disease. Both CD4+ and CD8+ T-cell subsets display an increased fraction of cells showing activation markers compared to controls, indicating an unusually high level of activity in both populations. Likewise, an increased number of T-cell expansions were noted in MND patients compared to controls, most dramatically observed in the CD4+ T-cell subset, where 5/144 T-cell V genes analyzed in eight subjects turned out to be expanded in the peripheral blood. In the CD8+ T-cell subset, four out of eight MND patients had peripheral BV gene expansions, 9/144 V genes analyzed. However, the most interesting result was the observation that in three out eight MND patients, expansions concerning the same BV gene were present in both CD4+ and CD8+ subsets (BV8S1 in two and BV12S1 in one patient). Parallel expansions of BV-gene restricted populations in both CD4+ and CD8+ subsets in the same individual, in an major histocompatibility complex (MHC)-unrestricted manner, are normally limited to situations where superantigens are involved. No known superantigen has to date been described with the capacity to simultaneously stimulate both BV8S1 and BV12S1, suggesting that the postulated 'MND-associated' superantigen is a hitherto undefined molecule.

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.

OBJECTIVE: To investigate the T-cell receptor (TCR) repertoire usage in infants born to mothers infected with HIV-1 in order to discern possible perturbations in TCR usage as a consequence of HIV-1 infection. DESIGN: Blood samples from five HIV-1-infected and six non-infected children born to HIV-1-seropositive mothers were collected at two to three timepoints during the first and second year of life and the TCR variable gene usage was determined. METHODS: Triple staining flow cytometry analysis using a panel of monoclonal antibodies (MAb) to TCR V alpha and V beta gene products and antibodies to CD4 and CD8 was performed. RESULTS: Frequent large expansions of CD8+ lymphocyte subpopulations bearing distinct V alpha and V beta gene products was seen in HIV-1-infected children (four out of five) but was rarely detected in uninfected children. CONCLUSION: The study demonstrated the frequent occurrence of persistent and clonal expansions of CD8+ T cells bearing distinct V alpha/V beta gene products in some HIV-1 vertically infected infants similar to those observed during primary infection in adults.

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.

By using mice genomically lacking IFN-γR, IL-12, perforin, and recombination-activating gene-1 (RAG-1), we analyzed the regulation and importance of IFN-γ in the control of infection with Chlamydia pneumoniae. IL-12 participates in resistance of mice to C. pneumoniae, probably by regulating the protective levels of IFN-γ mRNA. In turn, IFN-γ is necessary for the increased IL-12p40 mRNA accumulation that occurs in lungs during infection with C. pneumoniae, suggesting a positive feedback regulation between these two cytokines. In experiments including RAG-1−/−/IFN-γR−/− mice we showed that IFN-γ produced by innate cells controls the bacterial load and is necessary for the increased accumulation of transcripts for enzymes controlling high output NO release (inducible NO synthase), superoxide production (gp-91 NADPH oxidase), and catalyzis of tryptophan (indoleamine 2,3-dioxygenase (IDO)), mechanisms probably related to bacterial killing. Adaptive immune reponses diminish the levels of IFN-γ and IL-12 mRNA and thereby the levels of inducible NO synthase, IDO, and gp91 NADPH oxidase trancripts. By using RAG-1−/−/perforin−/− mice, we excluded the overt participation of NK cell cytotoxicity in the control of C. pneumoniae. However, NK cells and probably other innate immune cells release IFN-γ during the bacterial infection.

Studies were performed to characterize the thymocyte subset responsible for the efficient inhibition of spleen cell interleukin-2 (IL-2) production. By different cell separation techniques, C-mediated cytotoxicity, immunoabsorbance, and cell sorting by flow cytometry, we have identified two phenotypically distinct subpopulations of thymocytes. One subset, belonging to the minor population (3-5%) of the CD4 - CD8 - , i.e., double-negative thymocytes, is defined as the subset from which the suppressive thymocytes are generated. After 28 hr of Con A stimulation, these cells undergo a phenotypical change in vitro and generate a population exerting the inhibitory effect. This latter subset inhibits 95-99% of the IL-2 produced by spleen cells and is characterized by expressing the CD8 antigen, high levels of HSA, low levels of CD3, and being IL-2R positive (HSA + CD4 - CD8 + CD3 low IL-2R + ). Based on the experiments where stimulated CD4 + CD8 + , i.e., double-positive thymocytes, failed to suppress IL-2 production, we conclude that the CD8 + immature single-positive thymocytes are generated directly from the DN subset as an intermediate stage to the DP cells. When CD8 + -stimulated thymocytes were enriched, the suppression was efficient even at thymocyte:spleen cell ratio of 0.01:1. It is suggested that this subpopulation of thymocytes may serve as a regulatory set of cells during critical stages of thymic maturation.

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.