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In addition to its well-established role in innate immunity, the complement component C3 is of critical importance in modulating the humoral response. In this study, we examined the effect of C3b linkage to tetanus toxin (TeNT) in the production of antigenic peptides inside human APC. We purified HLA-DR associated peptides isolated either from TeNT or TeNT-C3b pulsed cells. This study revealed that TeNT-C3b derived antigenic peptides are different and more numerous than TeNT derived peptides. This increased production of antigenic peptides correlated with a C3b-induced TeNT modification of proteolysis. These findings argue in favour of a new role for C3b in the modulation of T cell epitope during antigen processing and presentation.

C1q binds to many non-self and altered-self-materials. These include microorganisms, immune complexes, apoptotic and necrotic cells and their breakdown products, and amyloids. C1q binding to amyloid fibrils found as extracellular deposits in tissues, and subsequent complement activation are involved in the pathology of several amyloid diseases, such as Alzheimer's disease. Prion diseases, such as scrapie also involve formation of amyloid by polymerization of the host prion protein (PrP). Complement activation is likely to contribute to neuronal damage in the end stages of prion diseases, but is also thought to participate in the initial infection, dissemination and replication stages. Infectious prion particles are likely to bind C1q and activate the complement system. Bound complement proteins may then influence the uptake and transport of prion particles by dendritic cells (DCs) and their subsequent proliferation at sites such as follicular DCs.

Immunotherapy for cancer aims to generate cytotoxic cells that are capable of eradicating tumour cells. It has been well demonstrated that helper, non-cytotoxic CD4(+) T cells are important for the induction and maintenance of anti-tumour immunity exerted by cytotoxic CD8(+) T cells. In contrast, the existence of direct anti-tumour, effector cytotoxic CD4(+) T cells remains elusive, mainly due to the paucity of reliable experimental data, especially in human B-cell non-Hodgkin lymphomas. This study developed an appropriate, autologous follicular B-cell non-Hodgkin follicular lymphoma model, including the in vitro establishment of a malignant, human leucocyte antigen class I (HLA-I) deficient B-cell line, and the generation of three autologous anti-tumour cytotoxic CD4(+) T-cell clones originating from the peripheral blood of the same patient. These three clones were considered as tumour specific, because they were capable of killing the malignant, HLA-I-deficient B-cell line through a classical HLA-II restricted perforin-mediated pathway, but did not lyse the Epstein-Barr virus-infected autologous normal B lymphocytes. All three CD4(+)clones were T-cell receptor Vbeta17-Dbeta1-Jbeta1.2 and exhibited an identical complementarity-determining region 3, suggesting the immunodominance of a single peptide antigen presented by tumour cells. Such lymphoma models would provide a useful tool for in vivo expansion and the adoptive transfer of selected CD4(+) cytotoxic cells in immunotherapeutic strategies.

Previous studies have shown that transgenic mice overexpressing Cu/Zn superoxide dismutase, a model of Down syndrome, exhibit premature thymic involution. We have performed a flow cytometry analysis of the developing thymus in these homozygous transgenic mice (hSOD1/hSOD1: Tg-SOD). Longitudinal follow-up analysis from day 3 to day 280 showed an early thymic development in Tg-SOD mice compared with controls. This early thymic development was associated with an increased migration of mature T cells to peripheral lymphoid organs. BrdU labeling showed no difference between Tg-SOD and control mice, confirming that the greater number of peripheral T cells in Tg-SOD mice was not due to extensive proliferation of these cells but rather to a greater pool of emigrant T cells in Tg-SOD.

The T cell receptor alpha chain repertoire and the possible influence of the alpha chain on the development and phenotype of naturally arising mouse CD4+CD25+ T cells have not been extensively analysed. We used all available Valpha-specific monoclonal antibodies and a sensitive multiplex genomic DNA PCR assay to study the Valpha repertoire of CD4+CD25+ T cells in normal mice. To address whether CD4+CD25+ T cells express two TCR alpha chains, we have carried out four-colour flow cytometry using combinations of the available anti-Valpha reagents in mice where one allele of the TCRA locus had been inactivated. Results indicate that the Valpha repertoire of CD4+CD25+ T cells is as diverse as their CD25- partners. In addition, CD4+CD25+ T cells develop normally in Tcralpha+/- mice and we show for the first time that despite expressing only one TCRalpha chain, they retain their characteristic CD4(low), CD3(low), TCRbeta(low), CD5(high), CD45RB(low) and cytoplasmic CD152(high) phenotype.

V(D)J recombination assembles functional T-cell receptor (TCR) genes from V, D and J components in developing thymocytes. Extensive processing of V, D and J extremities before they are ligated creates a high degree of junctional diversity which results in the generation of a large repertoire of different TCR chains. In contrast, the extremities of the intervening DNA segment, which bear the recombination signal sequences, are generally held to be monomorphic, so that signal joints (SJs) consist of the perfect head-to-head juxtaposition of recombination signal extremities. We analyzed the structure of SJs generated during the recombination of TCRAD locus genes in murine and human thymocytes. Junctional diversity resulting from N nucleotide additions or from N nucleotide additions and base loss was found for each type of SJ examined. Different patterns of processing/modification were found, suggesting that different enzymatic activities operate during recombination of TCRA and TCRD genes, although they are located within the same genetic locus. Recombination of the deltaRec-1 element generates a diverse repertoire of SJs exhibiting both combinatorial and junctional diversity in murine and human thymocytes. Therefore, SJ diversity appears to be an intrinsic feature of V(D)J recombination in unmanipulated thymocytes.

We have used human astrocytoma-derived cells to investigate the cellular responses of central nervous system cells to Toxoplasma gondii infection. At 24 h post inoculation, the secretion of CCL-2 (or Monocyte Chemotactic Protein-1) was augmented six-fold over the control. This secretion was down-regulated by D609, a specific inhibitor of phosphatidylcholine-dependent phospholipase C (PC-PLC), but not modulated by gamma interferon (IFN-gamma). Ribonuclease protection assay analyses showed significant down-regulation of CCL-2 mRNA production during infection by Toxoplasma gondii when cells were treated by D609. The mRNA levels of the seven other chemokines studied were not modified by D609. CCL-2 seems to contribute to the cell recruitment during human cerebral reactivation of Toxoplasma gondii. Cellular production of this CC chemokine during toxoplasmosis may be regulated by a PC-PLC-dependent pathway.

Early thymocyte development was compared in normal, recombinase-activating gene 2-inactivated (RAG-2 KO) and pre-T cell receptor alpha-inactivated (pre-Talpha KO) mice, mutants representing either a complete (RAG-2 KO) or partial (pre-Talpha KO) block in progenitor development. Using three colour analysis with antibodies to CD117, CD44 and CD25, cell numbers in each progenitor subset were quantified, demonstrating an accumulation of cells prior to the block. Progenitor number was influenced both by the nature of the genetic block and thymus size, as shown in the enlarged thymus of a transgenic mouse line. By four colour staining for CD3, CD117, CD44 and CD25 and deliberately not gating out CD3(-) cells, a novel aspect of gamma delta T cell development in pre-Talpha KO mice was identified. 5-bromodeoxyuridine labelling and subsequent four colour staining for BrdU, CD117, CD44 and CD25 showed firstly that DN1 cells were cycling, secondly that the developmental block in pre-Talpha KO mice corresponded to a decrease in DN4 cell proliferation, and thirdly provided a novel 'snapshot' of T cell receptor beta-selected cells transiting the DN3 to DN4 compartment. Taken together, these results emphasise the need for a more detailed qualitative and quantitative analysis of the progenitor compartment in the thymus.

In immunocompromised hosts, disruption of toxoplasmic cysts and conversion from bradyzoites to tachyzoites occur in brain. In these areas, infiltrates of mononuclear cells are observed. In the murine toxoplasmosis model, recent data suggest that chemokines may play a role in leukocyte recruitment in the central nervous system (CNS). This study analyzed the monocyte chemotactic protein-1 (MCP-1) secretion and chemokine expression after Toxoplasma gondii infection of human astrocytes, glioblastoma cells (U373) and fibroblasts (MRC5) in vitro. T. gondii infection of these CNS cells, astrocytes and glioblastoma cells significantly increased MCP-1 secretion, particularly for astrocytes. In our cellular models, the pattern of chemokine gene expression is dominated by MCP-1 expression. MCP-1 mRNAs were also quantified by real-time-PCR (LightCycler). The behavior of cells studied after T. gondii infection was different (invasion and growth) and the cell mechanisms of chemokine regulation could be dependent on the type of cells infected, while MCP-1 may contribute to the cell recruitment during human cerebral reactivation of T. gondii.

Background/aims: During viral chronic hepatitis C (CHC), the intra-hepatic lymphocyte infiltrate is mainly composed of T lymphocytes expressing alphabeta T-cell receptors (TCR). Since little is known about the TCRalphabeta diversity of intra-hepatic T lymphocytes (IHL), we evaluated the IHL repertoire from CHC patients (n=8) as compared to healthy subjects (n=4), total peripheral blood mononuclear cells, and purified peripheral and intra-hepatic CD8(+) cells (n=2)., Methods: The diversity of TCRalphabeta receptors was evaluated by determining the size and the sequence of the TCRbeta chain complementarity determining region 3 (CDR3). The number of total T lymphocytes in liver was estimated by real-time quantitative reverse transcription-polymerase chain reaction of TCRalpha and CD3 epsilon transcripts., Results: Our results show that transcripts encoding all TCR V beta (BV) families and all TCR J beta (BJ) segments were present in healthy and CHC livers. No biased TCR repertoire, in terms of preferential BV or BJ gene use or restricted CDR3 sequence, was observed in infected livers. When corrected for equivalent numbers of T lymphocytes, BJ segments utilization and CDR3 length diversity were similar in IHL and PBMC, indicating that the TCRbeta chain diversity is comparable in both cases. In addition, TCR diversity was similar in both peripheral and intra-hepatic CD8+ T cells., Conclusions: This study shows limited expansions of intra-hepatic T lymphocytes in CHC patients. The increase of T lymphocytes in infected livers correlates with diversification of TCR, arguing for the establishment of a multi-specific immune response.

Complement protein C3 plays a major role in cell regulation and immune response. This last point is mainly due to C3's capacity to act as a bifunctional link between antigen and immunocompetent cells. In a previous work, we have reported the adjuvant effect produced by linking C3 fragments to antigen. In this paper, we characterize the long-term secondary antibody response induced by C3b-antigen complexes. Mice were immunized using either a protein (hen egg lysozyme) or an ovalbumin-derived peptide as antigen. We compared the secondary response elicited by these antigens covalently linked to C3b or emulsified in complete Freund's adjuvant (CFA). Our results provide evidence that C3b linkage induces better long-term adjuvant effect than CFA, resulting in the production of a higher specific IgG titer with a better affinity.

The in vitro regulation of tumour necrosis factor (TNF)-alpha receptors during Toxoplasma gondii infection of human MRC5 fibroblasts and human myelomonocytic THP-1 cells was investigated. Cells were infected with the virulent RH of T. gondii. TNFR membrane receptors were analysed by flow cytometry with biotinylated TNF-alpha. Shedding of the soluble form of TNFR1 and TNFR2 in cell culture supernatants was measured by enzyme-linked immunosorbent assay, and expression of mRNA production of TNFR1 and TNFR2 was analysed by quantitative real-time polymerase chain reaction, 1 h after infection. In the MRC5 cell line, T. gondii infection did not induce any up- or down-regulation of membrane TNFRs, soluble TNFRs or mRNA of TNFRs. However, THP-1 cell infection with living parasites induced a significant soluble TNFR1 release by THP-1 cells after 1 h. We detected an approximately 50% up-regulation (P < 0.01) of soluble TNFR1 in infected THP-1 cells compared to controls. No change in soluble TNFR2 levels was observed in the same conditions. Moreover, infection decreased the level of TNF membrane receptors, but had no effect on TNFR1 and TNFR2 mRNA levels. TNFR modulation by T. gondii infection, in vitro, depends on the cell type. Furthermore, our data suggest that living parasites control the shedding of the soluble form of TNFR1. This mechanism may influence the role of TNF-alpha in toxoplasmosis.