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Abstract: α-Galactosyl-ceramide (1) has been identified as a powerful modulator of immunological processes through its capacity to bind CD1d molecules and specifically activate invariant natural killer (NK)-like T cells (iNKT cells). This paper describes the synthesis of 1, the analogous α-galactosyl-ceramide 3, and its short chain analogue ‘OCH’ (2), by use of the 4,6-di-O-tert-butylsilylene (DTBS) protecting group to produce a powerful α-galactosylating agent. In vivo experiments confirmed these compounds to be potent and selective activators of iNKT cells in a CD1d-dependent manner, each inducing a unique profile of cytokine release. This synthesis strategy will permit the generation of novel derivatives for use in the study of the mechanism of iNKT cell activation. [Copyright &y& Elsevier]

Abstract: The CFSE dye dilution assay is widely used to determine the number of divisions a given CFSE labelled cell has undergone in vitro and in vivo. In this paper, we consider how the data obtained with the use of CFSE (CFSE data) can be used to estimate the parameters determining cell division and death. For a homogeneous cell population (i.e., a population with the parameters for cell division and death being independent of time and the number of divisions cells have undergone), we consider a specific biologically based “Smith–Martin” model of cell turnover and analyze three different techniques for estimation of its parameters: direct fitting, indirect fitting and rescaling method. We find that using only CFSE data, the duration of the division phase (i.e., approximately the S+G2 +M phase of the cell cycle) can be estimated with the use of either technique. In some cases, the average division or cell cycle time can be estimated using the direct fitting of the model solution to the data or by using the Gett–Hodgkin method [Gett A. and Hodgkin, P. 2000. A cellular calculus for signal integration by T cells. Nat. Immunol. 1:239-244]. Estimation of the death rates during commitment to division (i.e., approximately the G1 phase of the cell cycle) and during the division phase may not be feasible with the use of only CFSE data. We propose that measuring an additional parameter, the fraction of cells in division, may allow estimation of all model parameters including the death rates during different stages of the cell cycle. [Copyright &y& Elsevier]

Gamma/delta T lymphocytes cells recognize the antigen in a non-classical way and are considered the third branch of the immune system devoted to defend the integrity of the body. Ageing is characterized by an impairment of the main way of protection (the adaptive branch) but, successfully aged people show compensatory mechanisms of defense such as proneness to inflammation. Moreover, very old subjects show an increased number of NK cells. We have previously demonstrated that γδT lymphocytes are reduced in elderly. In the present paper we have studied some characteristics of these cells to evaluate the possibility that these cells might balance the decreased action of the adaptive branch in successfully aged people. Cytofluorimetric analysis of cells collected from young, old and centenarian subjects has been used to evaluate the ability of these cells to expand in vitro. Here we demonstrate that γδT cells are impaired in the ability to proliferate to different stimuli such as isopentenyl pyrophoshate, that select γδT lymphocytes bearing δ2 chain, other than to phytohemagglutinin and anti-CD3 that are polyclonal activators. Moreover, we demonstrate that γδT cells in aged and centenarians show an enhanced sensitivity to undergo apoptosis induced both by α-Fas and TNF-α. All together these data suggest that γδT lymphocytes are impaired in elderly and suggest that the reduced ability to proliferate and the reduced number of circulating γδT lymphocytes is due to the proneness to apoptosis. Finally on the basis of these data, we conclude that γδT lymphocytes, do not participate in the remodeling of the immune system due to the reduction of classical T cell response and replacement by NK cells in elderly. [Copyright &y& Elsevier]

Interaction of B7 with CD28 and CTLA4 plays an important function in T-cell activation and homeostasis. Disruption of CD28, CTLA4, or both has shown impact on T-cell biology. This paper examined the consequences of overexpressing a tailless mutant form of CTLA4 on T-cell activation and in vivo expansion.Retroviral gene transfer was used to infect bone marrow progenitor cells with either a control vector or a cytoplasmic domain-deleted mutant of CTLA-4 (ΔCTLA4). The cells were subsequently adoptively transferred to RAG-/- mice and allowed to repopulate. The T cells derived from the reconstituted RAG-/- mice were analyzed functionally in vitro and in vivo.The T cells were defective in their ability for IL-2 secretion, survival, and proliferation in response to Ag/APC stimulation in vitro. Addition of exogenous IL-2 or normal T cells was able to rescue the survival defect and allow cell-cycle progression. In adoptive transfer studies, the naïve T cells expressing ΔCTLA4 exhibited compromised capability to expand in RAG-/- mice. Memory ΔCTLA4T cells, however, were capable of proliferating in lymphopenic hosts to a similar extent as control memory T cells, but showed reduced survival.Surface ΔCTLA4 has similar tolerogenic/regulatory activity as CTLA4-Ig. In contrast to CTLA4-Ig, the effect of ΔCTLA-4 is autonomous. The inhibition of in vivo expansion by ΔCTLA4 indicates developmental and/or activation stage dependency of costimulation in T cells. [Copyright &y& Elsevier]

One of the mechanisms ensuring immunological unresponsiveness or tolerance depends on the action of CD8+ lymphocytes. In this paper, we report that, in healthy subjects, a subset of CD8+CD28− T cells suppresses the specific response to TSH receptor (TSHR) of CD4+ clones. Suppression was highly specific, required cell–cell interaction, and was not mediated by cytotoxicity. Co-incubation of CD8+ and CD4+ clones, followed by the removal of the CD8+ cells from the cultures before testing CD4+ responsiveness to TSHR, demonstrated that CD4+ cells were anergic since they showed low response to the antigen and a significant impairment of IL-2 production. In CD8-mediated anergy induction, the T-cell receptor (TCR) on both CD4+ and CD8+ cells seems to play a role. Our results indicate that one of the mechanisms ensuring peripheral tolerance involve CD8+CD28− cells. A disregulation in the control of autoreactive clones by this subset might be important for the onset of autoimmune thyroid diseases. [Copyright &y& Elsevier]