Mechanisms of the natural reactivity of lymphocytes from noninfected individuals to membrane-associated Leishmania infantum antigens.

Membrane-associated Leishmania Ags (MLA) or soluble Leishmania Ags were used in vitro to stimulate cord blood or PBMC from healthy donors noninfected by Leishmania parasites. MLA, but not soluble Leishmania Ags, constantly induce strong proliferation of cord blood mononuclear cells and PBMC from noninfected individuals. Responding cells are CD3+, CD4+, TCRalphabeta+, CD45RO+, and CD45RA+ and secrete IFN-gamma and IL-10, but not IL-4. MLA do not activate NK cells nor NKT cells. Membrane Ags also induce purified macrophages from noninfected individuals to secrete IL-10 and TNF-alpha, but have no effect on IL-1alpha or IL-12 secretion. The effects of MLA are proteinase K-sensitive and resistant to lipid extraction. The lymphoproliferative responses are inhibited by anti-HLA-DR Abs and require Ag processing by APCs, excluding that the biological effect of MLA could be attributed to a superantigen. Finally, TCR repertoire analysis shows that the T cell expansion induced by MLA uses TCR with various variable beta segment rearrangements and CDR3 lengths, features much more characteristic to those observed with a polyclonal activator than with a conventional Ag. These results suggest a particular mechanism developed during the host's natural response to Leishmania parasites that allows direct activation of naive CD4 lymphocytes by parasite membrane-associated Ags.

A profound alteration of blood TCRB repertoire allows prediction of cerebral malaria.

Cerebral malaria (CM) is one of the severe complications of Plasmodium infection. In murine models of CM, Talphabeta cells have been implicated in the neuropathogenesis. To obtain insights into the TCRB repertoire during CM, we used high throughput CDR3 spectratyping and set up new methods and software tools to analyze data. We compared PBL and spleen repertoires of mice infected with Plasmodium berghei ANKA that developed CM (CM(+)) or not (CM(-)) to evidence modifications of the TCRB repertoire associated with neuropathology. Using distinct statistical multivariate methods, the PBL repertoires of CM(+) mice were found to be specifically altered. This alteration is partly due to recurrently expanded T cell clones. Strikingly, alteration of the PBL repertoire can be used to distinguish between CM(+) and CM(-). This study provides the first ex vivo demonstration of modifications of Talphabeta cell compartment during CM. Finally, our original approach for deciphering lymphocyte repertoires can be transposed to various pathological conditions.

Comparative study of brain CD8+ T cells induced by sporozoites and those induced by blood-stage Plasmodium berghei ANKA involved in the development of cerebral malaria.

To obtain insight into the mechanisms that contribute to the pathogenesis of Plasmodium infections, we developed an improved rodent model that mimics human malaria closely by inducing cerebral malaria (CM) through sporozoite infection. We used this model to carry out a detailed study on isolated T cells recruited from the brains of mice during the development of CM. We compared several aspects of the immune response related to the experimental model of Plasmodium berghei ANKA infection induced by sporozoites in C57BL/6 mice and those related to a blood-stage infection. Our data show that in both models, oligoclonal TCRVbeta4(+), TCRVbeta6(+), TCRVbeta8.1(+), and TCRVbeta11(+) major histocompatibility complex class I-restricted CD8 T cells were present in the brains of CM(+) mice. These CD8(+) T cells display an activated phenotype, do not undergo apoptosis, secrete gamma interferon or tumor necrosis factor alpha, and are associated with the development of the neurological syndrome.

Retrovirus-mediated gene transfer in human primary T lymphocytes induces an activation- and transduction/selection-dependent TCR-B variable chain repertoire skewing of gene-modified cells.

In a clinical trial that we recently reported, a suicide gene transfer in human primary T cells required 12 days of ex vivo culture, including activation of peripheral blood mononuclear cells (PBMC) with CD3 monoclonal antibody (CD3 mAb), retrovirus-mediated transduction, and selection of gene-modified cells (GMC) by G418. The aim of the present study was to determine the impact of the initial T cell activation and of the transduction/selection on T cell receptor beta variable chain (TCRBV) repertoire of GMC by using the spectratyping method. The TCRBV repertoires of nontransduced, nonselected control (Co) cells and of GMC generated after an initial stimulation with CD3 mAb, CD3/CD28 beads, or allogeneic PBMC or Epstein-Barr virus-transformed B (B-EBV) cells were compared to the ones of their corresponding PBMC. The TCRBV repertoires were skewed in Co cells generated after CD3 mAb or after allogeneic stimulation, and even more so in their corresponding GMC, demonstrating that both culture-dependent and transduction/selection-dependent events led to TCRBV repertoire alterations. However, TCRBV repertoires were not altered, or to a lesser extent, in Co cells or GMC produced after CD3/CD28 bead activation, demonstrating a protective effect on both culture-dependent and transduction/selection-dependent repertoire alterations. Thus, we suggest to replace the initial CD3 mAb stimulation by CD3/CD28 beads for the production of clinical-grade GMC in the setting of future gene therapy trials.

New methods and software tools for high throughput CDR3 spectratyping. Application to T lymphocyte repertoire modifications during experimental malaria.

Immune repertoires of T or B cells are very often studied by Complementary Determining Region 3 (CDR3) spectratyping. However, data obtained with this method is usually subject to a biased eye analysis. We developed recently the ISEApeaks software package to retrieve and handle peak data from automated sequencers, from which CDR3 spectratype data is obtained. We describe a general strategy for CDR3 spectratype analysis based on two new specific modules and multivariate statistics. The first module addresses the crucial problem of peak smoothing. The second is a toolbox for the analysis of CDR3 spectratypes, which includes perturbation computation, recurrent peak finding, expansion assessment and datamining. To illustrate our approach, we assessed the complex TCRB repertoire modifications induced by Plasmodium berghei ANKA (PbA) infection. This global and exhaustive repertoire analysis approach is of general interest for T- and B-lymphocyte repertoire studies and is currently used in human cohorts in various pathologies and during clinical trials.

A new statistical method for quantitative analyses: application to the precise quantification of T cell receptor repertoires.

In experimental immunology, a situation quite commonly arises in which there are a large number of potential events but the probability of any individual event is small and one wishes to measure the number of events which actually occur. We present a new general statistical method, denoted Continuous Poisson Method (COPOM), for estimating the number of events underlying a quantitative measurement. This situation is well illustrated in the case of quantitative analyses of the immune receptor repertoire in a diverse population of cells. We show that repetition of T cell receptors (TCRs) complementarity determining region 3 (CDR3) length measurements by Immunoscope, on independent samples containing the similar numbers of cells prepared from splenocytes, results in variable profiles. When analyzed by COPOM, this variability provides direct quantification of the lymphocytes expressing any antigen receptor with a given V, J and CDR3 length inside the cell population. Using COPOM, a single dilution was sufficient to cover events over a 100-fold variation in frequency and the sensitivity of the assay was such that a single cell inside a pool of 5 x 10(4) lymphocytes could be quantified. A comparison of the frequency of splenocytes using either Vbeta14-Jbeta or the specific Vbeta8.3-Jbeta1.1 rearrangement, determined either by our or other approaches, revealed the accuracy and convenience of our method. This approach provides the first precise method able to measure the diversity of the antigen receptor repertoire inside a complex cell population by the use of a single straightforward technique.