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Previous work by our group showed that Toll-like receptor 2 (TLR2) is essential for activation of innate immunity, playing a major role in the response of macrophages to Candida albicans, triggering cytokine and chemokine expression, and therefore TLR2 -/- mice are more susceptible to systemic primary candidiasis. In this work, we used a murine model of systemic C. albicans infection, in which resistance to reinfection with virulent wild-type cells is induced by prior exposure of mice to a low-virulence agerminative strain of C. albicans (primary sublethal infection), to study the influence of TLR2 gene deletion on (i) the ability to develop an acquired resistance upon vaccination; (ii) the development of the acquired humoral response; and (iii) the production of Th1 cytokines IFN-gamma, IL-12 and TNF-alpha. Our results indicate that, although TLR2 -/- mice have a very impaired production of Th1 cytokines compared with control mice, they are equally capable of mounting a specific humoral response to the fungus and developing a vaccine-induced resistance.

In this work, we studied the role of toll-like receptor-2 (TLR2) in murine defenses against Candida albicans. TLR2-deficient mice experimentally infected intraperitoneally (i.p.) or intravenously (i.v.) in vivo had very significant impaired survival compared with that of control mice. In vitro production of TNF-alpha and macrophage inhibitory protein-2 (MIP-2) by macrophages from TLR2-/- mice in response to yeasts and hyphae of C. albicans were significantly lower (80% and 40%, respectively; P <0.05) than production by macrophages from wild-type mice. This impaired production of TNF-alpha and MIP-2 probably contributed to the 41% decreased recruitment of neutrophils to the peritoneal cavity of i.p. infected TLR2-/- mice. In contrast, in vitro phagocytosis of yeasts and production of reactive oxygen intermediates (ROI) were not affected in macrophages from TLR2-/- animals. Our data indicate that TLR2 plays a major role in the response of macrophages to C. albicans, triggering cytokine and chemokine expression, and it is essential for in vivo protection against infection.

Thioredoxin (TRX) is a ubiquitous dithiol-oxidoreduction enzyme broadly expressed in cells from prokaryote to eukaryote organisms. Human thioredoxin (human TRX) gene, previously cloned in our laboratory, codes for a 12-kDa protein found in the culture supernatant of several hemopoietic human cell lines. This protein is secreted by a nonclassical pathway. The role of the secreted enzyme as a signalling soluble mediator was demonstrated, but nothing is known about a membrane associated form of thioredoxin which could be involved in cell/cell contacts and accessory signal function. Thus, we performed experiments to determine if human TRX is also expressed at the cell surface. We report here positive results based upon indirect immunofluorescence flow cytometric analysis of different human cell lines (HeLa, U 937, Jurkat, 3B6, Daudi and Raji) using a cross reactive sheep anti E. coli TRX polyclonal antibody demonstrating a significant expression of human TRX at the surface of human cells.

The mononuclear cell surface protein IA4 was originally identified in our lab using a mAb selected because of its strong reactivity with three lymphoblastoid variant cell lines which are HLA class I deficient, are LAK susceptible, and form a high number of conjugates with LAK effectors. We previously cloned the cDNA of the IA4 protein, coding for a 267-amino-acid type III integral membrane protein, with four transmembrane domains and three possible N-glycosylation sites. The IA4 protein belongs to the tetra span transmembrane (TST) new family of surface molecules, which also includes CD9, CD37, CD53, CD63, and TAPA-1. IA4 antigen was recently recognized as belonging to a new cluster of differentiation CD82 (International CD Workshop, Boston 1993). The IA4 antigen expression pattern at the surface of immune cells from normal donors was studied. On T lymphocytes, IA4 was barely detectable on resting cells and increased 3.5- to 7-fold following PHA or PHA+PMA stimulation. This IA4 increased expression is correlated with the morphologic change in blast cells and with the expression of activation markers such as CD2 and MHC class II antigens, therefore suggesting that IA4 is an activation marker on T lymphocytes. The expression of IA4 was low on circulating resting monocytes collected by elutriation. However, these monocytes, cultured in medium alone or with GM-CSF, acquired the morphology of macrophage and simultaneously overexpressed MHC Class II, CD14, and IA4 antigens, suggesting that IA4 is a differentiation marker for macrophages, whatever the culture conditions, either adherent (plastic culture dishes) or nonadherent (Teflon culture bags). IA4 stable transfectants of the murine mastocytoma cell line P815 were obtained and used to generate a new mAb. Competitive epitope binding studies have shown that IA4 antigen presents a dominant epitope recognized by most of the mAb prepared either in our lab or elsewhere. This dominant epitope is not shared by any of the other antigens of the TST family. Using this new mAb we were able to biochemically characterize the IA4 antigen as a 28-kDa protein, highly N-glycosylated with different patterns on various cells.

We recently reported hyperproduction of interleukin-1 (IL1) and hyperexpression of IL1 beta mRNA, after in vitro activation by lipopolysaccharide (LPS) in peripheral macrophages of several neurological mutant mice, i.e. staggerer, lurcher, pcd and reeler, that exhibit patterns of neuronal degeneration in the cerebellum; in the present study, we investigated the expression of several cytokine mRNA in peripheral macrophages of other mutants with neuronal degeneration in the cerebellum or in the spinal cord to determine whether this genetic dysregulation is specific for IL1 beta or whether it reflects a generalized hyperexcitability of these macrophages. Hyperexpression of IL1 beta mRNA was present in the cerebellar mutants nodding and nervous, but not in weaver. A similar phenomenon was found, but to a lesser extent, in the spinal mutants dystonia musculorum, wobbler and motor neuron degeneration. On the contrary, no hyperexpression of IL1 beta mRNA was found in non-genetic models of neuronal degeneration (Wistar rats treated with X irradiation or with 3-acetyl-pyridine). In the heterozygote staggerer +/sg, which exhibits a late onset of cerebellar neuronal loss, hyperexpression was found not only in 12-month old animals but also in 2-month old ones, i.e. when the number of cerebellar neurons is still normal. Synthetic molecules (muramyl dipeptides) like MDP or murabutide (Mu), known as macrophage activators, were also efficient in inducing IL1 hyperexpression in sg/sg macrophages. Hyperexpression of two other cytokine mRNA, i.e. IL1 alpha and tumour necrosis factor alpha mRNA, was also detected in LPS-stimulated macrophages of staggerer and lurcher mutant mice. These data led us to conclude that the macrophages of spinal and cerebellar mutants are in a state of general hyperexcitability. Work is in progress to establish whether the cytokine abnormalities result from a defect intrinsic to the macrophages of the mutant mice or are secondary to the degenerative process ultimately leading to neuronal loss.

We have developed an improved method to analyse stable associations (conjugate formation) between effector and target cells. Hydroethidine (red) stained lymphoblastoid target cells were cocentrifuged with carboxyfluorescein diacetate acetoxymethylester (green) stained human IL-2 activated cytotoxic cells (LAK). In the present studies either enriched or purified CD3 negative large granular lymphocytes (LGL) were used as cytotoxic cells. These fluorescent vital dyes localize intracellularly and therefore do not modify the cell to cell contact which eventually leads to the lytic events. Both dyes can be excited at a common wavelength (488 nm) using a single argon laser. Effectors firmly bound to target(s) (stable conjugates) were detected as two color fluorescent events (red and green). This method has several features: (a) the number of conjugates is recorded with reference to a fixed number of target cells; (b) the composition of conjugates (number of effectors or targets per conjugate) can be studied by analysis of the fluorescence intensities (red or green); (c) conjugate formation can be studied at E:T ratios comparable to those used in the classical 51Cr release cytotoxic assay; (d) it gives reproducible results and permits the study of very weak differences in binding properties. This method was used to study conjugate formation between human IL-2-activated cytotoxic cells (or purified CD3 negative LGL) and various lymphoblastoid target cells. We were able to demonstrate that cell lines susceptible to lysis formed more conjugates and were surrounded by more LAK effectors than their resistant counterparts and that no conjugate contained more than one target.

To verify or to challenge the reports indicating that IL-2 was the only molecule involved in the reconstitution of nu/nu mice alloreactivity in vitro, Balb/c (H-2d) nu/nu spleen cells were primed in culture against C57/B16 (H-2b) in the presence of crude IL-2-containing supernatants or purified IL-2. The generation of cytotoxic effectors was evaluated against a panel of 51Cr-labeled target cells. Although crude IL-2-containing supernatants sustained the generation of cytotoxic effectors, purified "natural" IL-2 (from different origins) and recombinant IL-2 were not able to do so. Con A or PHA were identified as cofactors synergizing with IL-2 to induce effectors from nu/nu spleen cells. These effectors efficiently lysed EL4 (H-2b, tumor line), but not mitogen-induced blast cells from the same strain. They also lysed targets bearing irrelevant allogenic H-2 specificities. Cold competition experiments confirmed the lack of H-2 specificity of such effectors: lysis of EL4 cells (H-2b) was inhibited strongly by YAC-1 cells (H-2a, very sensitive to NK lysis) or P815 cells (H-2d, autologous to the nu/nu effectors). Our results clearly challenge earlier conclusions and indicate that IL-2 alone does not reconstitute nude mice alloreactivity. Crude supernatants containing IL-2 and mitogen induce nonspecific effectors with patterns of reactivity similar to those of activated natural killers. We think that the cytotoxicity observed in these conditions in nude mice results from the mitogenic triggering of some kind of prethymic killer cells which subsequently are expanded by IL-2.

Transfection of DNA into non adherent cells can be achieved by electropermeation. Previously published results, partially successful, were obtained using exponential decaying electric impulsions. However, one limitation of this technique has been the damaging effect of this type of impulsions resulting in poor cell recovery. We report hereby the electropermeation of human lymphoblastoid cell lines using a commercially available electropulsator delivering repeated, short, high voltage, square shaped, electric pulses. The parameters of transfection have been optimized using the "Lucifer Yellow Permeation Assay". With the optimum electric parameters, virtually all the cells were permeated and at least 70% survived the shocking conditions. Both transient expression and permanent integration and expression of DNA was observed.

Several culture parameters were studied in order to establish methods for optimal and reproducible production of interleukin 2 (IL2) by thawed lymphocytes. Standard conditions, considered optimal for production by freshly separated lymphocytes (culture medium RPMI 1640 + 1% normal human serum + 10 micrograms/ml PHA), gave low and poorly reproducible results. An increased concentration of human serum (10 and 20%) in the medium improved production but best results were obtained by adding polyethylene glycol (PEG 6000, 0.1 mg/ml) to the culture medium. Furthermore, with the addition of PEG 6000, results became highly reproducible, thus permitting valid comparison of in vitro IL2 production by lymphocytes from normal donors and patients.

Monoclonal T cell colonies can be grown in agar culture from quiescent T lymphocytes under PHA stimulation, provided that (1) a low number of T lymphocytes (less than or equal to 5 X 10(4)/ml) is seeded, (2) IL-2 is added to the culture, and (3) a high number of accessory B cells (greater than or equal to 5 X 10(5)/ml) is present in contact with the T lymphocytes. Under these culture conditions the colony progenitors can be ascribed to the CD4 subset, whereas CD8 lymphocytes do not generate colonies. This finding is surprising since both CD4 and CD8 lymphocytes may be cloned in liquid culture. We now report the appropriate conditions required to grow cytotoxic CD8 lymphocyte colonies in agar. CD8 colony growth is dependent upon IL-2-IL-2 receptor interaction and is inhibited by anti-IL-2 receptor antibodies. In addition to PHA, accessory B cells and IL-2, an additional signal provided by recombinant IL-1 is necessary for CD8 colony formation. Exogenous IL-1 can be replaced by irradiated CD4 lymphocytes which stimulate the expression of membrane IL-1 activity in the accessory B cells. In addition, colony growth from quiescent but not preactivated CD8 lymphocytes is inhibited by anti-IL-1 antibodies. Altogether, the data show that an IL-1 signal is required for the induction of IL-2 responsive IL-2 receptors on quiescent CD8 colony forming cells.

Mitogen-driven T cell proliferation in liquid culture requires accessory cells that cooperate in interleukin 2 production. We have investigated the accessory cell requirement for human lymphocyte colony formation under PHA stimulation. Semisolid medium limits cell-to-cell contact emphasizing the role of cooperating cells both in growth factor production and in triggering events. Culturing at high T cell density demonstrates that accessory cells can be substituted for colony formation by exogenous IL-2. Culturing at low T cell density in the presence of IL-2 also demonstrates that accessory cells are required for activation of a subset of progenitors into IL-2 responsive colony-forming cells. Consequently, T colony progenitors, contained in the E-rosetting cell fraction of peripheral blood, are heterogeneous in their triggering signals: a minor subset is directly inducible by PHA, and a major subset is inducible by PHA in the presence of accessory cells. We found that monocytes and some leukemic B cells support effective accessory function in both colony growth factor production and colony progenitor sensitization.