Assessment of Gene Flow Between Gossypium hirsutum and G. herbaceum: Evidence of Unreduced Gametes in the Diploid Progenitor.

In the framework of a gene flow assessment, we investigated the natural hybridization rate between Gossypium hirsutum (AADD genome) and G. herbaceum (AA genome). The latter species, a diploid progenitor of G. hirsutum, is spontaneously present in South Africa. Reciprocal crosses were performed without emasculation between G. herbaceum and G. hirsutum Neither examination of the morphological characteristics nor flow cytometry analysis of the 335 plants resulting from the G. hirsutum × G. herbaceum cross showed any hybrid features. Of the 148 plants produced from the G. herbaceum × G. hirsutum cross, three showed a hybrid phenotype, and their hybrid status was confirmed by SSR markers. Analysis of DNA content by flow cytometry and morphological traits clearly showed that two of these plants were triploid (AAD). The third plant had a flow cytometry DNA content slightly higher than G. hirsutum In addition, its morphological characteristics (plant architecture, presence and size of petal spots, leaf shape) led us to conclude that this plant was AAAD thus resulting from fertilization with an unreduced AA gamete of the female G. herbaceum parent. Fluorescent In Situ Hybridization (FISH) and meiotic behavior confirmed this hypothesis. To the best of our knowledge, this is the first description of such gametes in G. herbaceum, and it opens new avenues in breeding programs. Furthermore, this plant material could provide a useful tool for studying the expression of genes duplicated in the A and D cotton genome.

Wuschel overexpression promotes somatic embryogenesis and induces organogenesis in cotton (Gossypium hirsutum L.) tissues cultured in vitro.

KEY MESSAGE: This work shows that overexpression of the WUS gene from Arabidopsis enhanced the expression of embryogenic competence and triggered organogenesis from some cells of the regenerated embryo-like structures. Agrobacterium-mediated genetic transformation of cotton was described in the late 1980s, but is still time consuming and largely genotype dependant due to poor regeneration. To help solve this bottleneck, we over-expressed the WUSCHEL (WUS) gene, a homeobox transcription factor cloned in Arabidopsis thaliana, known to stimulate organogenesis and/or somatic embryogenesis in Arabidopsis tissues cultured in vitro. The AtWUS gene alone, and AtWUS gene fused to the GFP marker were compared to the GFP gene alone and to an empty construct used as a control. Somatic embryogenesis was improved in WUS expressed calli, as the percentage of explants giving rise to embryogenic tissues was significantly higher (×3) when WUS gene was over-expressed than in the control. An interesting result was that WUS embryogenic lines evolved in green embryo-like structures giving rise to ectopic organogenesis never observed in any of our previous transformation experiments. Using our standard in vitro culture protocol, the overexpression of AtWUS in tissues of a recalcitrant variety did not result in the production of regenerated plants. This achievement will still require the optimization of other non-genetic factors, such as the balance of exogenous phytohormones. However, our results suggest that targeted expression of the WUS gene is a promising strategy to improve gene transfer in recalcitrant cotton cultivars.

Disrupting Il13 impairs production of IL-4 specified by the linked allele.

Interleukin 13-deficient (IL-13-/-) mice express a defect in priming for IL-4 production that is not corrected by adding IL-13 to the priming culture. This is partly accounted for by the consumption of IL-4 without endogenous replacement during culture of IL-13-/- CD4+ T cells. We examined cells from mice in which disrupted Il13 was linked to wild-type Il4 on one chromosome and wild-type Il13 was linked to a "knocked-in" green fluorescent protein (Gfp) gene in the Il4 locus. Our results show that the deficit in IL-4 production was due, at least in part, to a cis effect, in which disrupted Il13 diminished IL-4 production from the linked Il4 gene.

Regulation of expression of IL-4 alleles: analysis using a chimeric GFP/IL-4 gene.

CD4 cells from mice heterozygous for an IL-4 and a GFP/IL-4 gene frequently express a single allele. Analysis of IL-4 or GFP production by cells from recently primed Th2 cells indicates that essentially all are competent to transcribe either allele but have a low probability of doing so. By contrast, long-term Th2 clones show distinct and heritable ratios in the proportion of cells that express IL-4 or GFP. We conclude that in the course of Th2 priming an early efficient event renders both alleles capable of being inefficiently transcribed; a second, less frequent event occurs that renders one allele more competent, accounting for the differential expression of IL-4 and GFP in different clones.

Asymptomatic amoebic infection: Entamoeba histolytica or Entamoeba dispar? That is the question.

Two cases of amoebic infection were diagnosed in a heterosexual couple. The cases, a Frenchman with previous trips to various African countries and his sexual partner, a Cameroonese woman immigrant living outside the community, were both asymptomatic; the infection had been diagnosed by chance in the man at the time of his employment in a hospital kitchen. Based on what is known of the epidemiology of amoebic infection, it may be acquired and then transmitted within a couple via the indirect faecal-oral route or, in greater likelihood, by sexual practices. Both amoebic isolates were characterised by isoenzyme electrophoresis as non-pathogenic Entamoeba dispar, zymodemel. Other diagnostic tools, such as ELISA direct stool antigen detection tests and serological assays were employed, confirming the diagnosis of E. dispar infection. Given there are a number of asymptomatic cyst passers of Entamoeba histolytica, besides human carriers of saprophyte E. dispar, we stress the importance of applying, when possible, advanced protocols of diagnosis to distinguish the microscopically identical pathogenic species from the non-pathogenic one.

Size estimate of the alpha beta TCR repertoire of naive mouse splenocytes.

The diversity of the T cell repertoire of mature T splenocytes is generated, in the thymus, by pairing of alpha and beta variable domains of the alpha beta TCR and by the rearrangements of various gene segments encoding these domains. In the periphery, it results from competition between various T cell subpopulations including recent thymic migrants and long-lived T cells. Quantitative data on the actual size of the T cell repertoire are lacking. Using PCR methods and extensive sequencing, we have measured for the first time the size of the TCR-alpha beta repertoire of naive mouse T splenocytes. There are 5-8 x 105 different nucleotide sequences of BV chains in the whole spleen of young adult mice. We have also determined the size of the BV repertoire in a subpopulation of AV2+ T splenocytes, which allows us to provide a minimum estimate of the alpha beta repertoire. We find that the mouse spleen harbors about 2 x 106 clones of about 10 cells each. This figure, although orders of magnitude smaller than the maximum theoretical diversity (estimated up to 1015), is still large enough to maintain a high functional diversity.

Diversity, functionality, and stability of the T cell repertoire derived in vivo from a single human T cell precursor.

In this report, we have analyzed the human T cell repertoire derived in vivo from a single T cell precursor. A unique case of X-linked severe combined immunodeficiency in which a reverse mutation occurred in an early T cell precursor was analyzed to this end. It was determined that at least 1,000 T cell clones with unique T cell receptor-beta sequences were generated from this precursor. This diversity seems to be stable over time and provides protection from infections in vivo. A similar estimation was obtained in an in vitro murine model of T cell generation from a single cell precursor. Overall, our results document the large diversity potential of T cell precursors and provide a rationale for gene therapy of the block of T cell development.

Astrocytoma infiltrating lymphocytes include major T cell clonal expansions confined to the CD8 subset.

Anaplastic astrocytoma and glioblastoma are frequent and malignant brain tumors that are infiltrated by T lymphocytes. Whether these cells result from non-specific inflammation following blood-brain barrier disruption or an antigen-driven specific immune response is unknown. In this study, an in-depth characterization of TCR diversity in tumor and blood RNA biopsies was performed in a series of 16 patients with malignant astrocytoma. Whilst there was no obvious restriction of the AV and BV gene segment usage, complementarity-determining region 3 size analysis and sequencing of amplified TCR transcripts revealed multiple T cell oligoclonal expansions in all astrocytomas analyzed. Unique T cell clones were present in different adjacent areas of a given tumor, but never detected in the blood. Quantification of the number of TCR clonal transcripts per microg of tumor RNA indicated that certain T cell clonal expansions may represent at least 300 cells/10(6) tumor cells. Furthermore, we demonstrated that the in vivo expanded clones were almost exclusively confined to the CD8(+) subset. Overall, these data suggest that spontaneous antigen-driven immune responses may be elicited against human astrocytoma despite the immunosuppressive microenvironment generated by the brain and the tumor itself. However, the ultimate failure of the immune system to control tumor growth could be the consequence of a deficient CD4 T(h) component of the response. This observation could have important consequences for the development of immunotherapies for astrocytoma patients.

Transient transfection of primary T helper cells by particle-mediated gene transfer.

The study of the molecular basis of normal CD4+ T cell function, such as the control of commitment to the TH1 or TH2 phenotypes has been difficult due to the resistance of these cells to transfection by conventional methods. We used antibodies specific to T cell surface molecules to immobilize these cells and optimized conditions for transiently transfecting them by means of particle-mediated gene transfer. Using this technique, a construct encompassing - 577 to +1 of the IL-4 promoter allowed transcription of a luciferase reporter gene in recently-differentiated TH2 cells stimulated by anti-CD3, consistent with regulation of endogenous IL-4 gene expression.

Conserved T cell receptor repertoire in primary and memory CD8 T cell responses to an acute viral infection.

Viral infections often induce potent CD8 T cell responses that play a key role in antiviral immunity. After viral clearance, the vast majority of the expanded CD8 T cells undergo apoptosis, leaving behind a stable number of memory cells. The relationship between the CD8 T cells that clear the acute viral infection and the long-lived CD8 memory pool remaining in the individual is not fully understood. To address this issue, we examined the T cell receptor (TCR) repertoire of virus-specific CD8 T cells in the mouse model of infection with lymphocytic choriomeningitis virus (LCMV) using three approaches: (a) in vivo quantitative TCR beta chain V segment and complementarity determining region 3 (CDR3) length repertoire analysis by spectratyping (immunoscope); (b) identification of LCMV-specific CD8 T cells with MHC class I tetramers containing viral peptide and costaining with TCR Vbeta-specific antibodies; and (c) functional TCR fingerprinting based on recognition of variant peptides. We compared the repertoire of CD8 T cells responding to acute primary and secondary LCMV infections, together with that of virus-specific memory T cells in immune mice. Our analysis showed that CD8 T cells from several Vbeta families participated in the anti-LCMV response directed to the dominant cytotoxic T lymphocyte (CTL) epitope (NP118-126). However, the bulk (approximately 70%) of this CTL response was due to three privileged T cell populations systematically expanding during LCMV infection. Approximately 30% of the response consisted of Vbeta10+ CD8 T cells with a beta chain CDR3 length of nine amino acids, and 40% consisted of Vbeta8.1+ (beta CDR3 = eight amino acids) and Vbeta8.2+ cells (beta CDR3 = six amino acids). Finally, we showed that the TCR repertoire of the primary antiviral CD8 T cell response was similar both structurally and functionally to that of the memory pool and the secondary CD8 T cell effectors. These results suggest a stochastic selection of memory cells from the pool of CD8 T cells activated during primary infection.

Selection of T cell clones with restricted TCR-CDR3 lengths during in vitro and in vivo alloresponses.

In a model of heart allograft rejection in adult congeneic rats mismatched for both class I and class II MHC molecules, we analyzed the TCR beta chain repertoire of T cells infiltrating rejected allografts [graft-infiltrating T cells (GITC)]. Although all BV families were used by GITC, oligoclonal expansions reflected by an altered distribution of TCR beta chain CDR3 lengths were detected throughout the rejection process. Interestingly, expansions involving TCR beta chains with common length and BV usage were recurrently found within distinct individuals at late stages of rejection in vivo and after in vitro mixed lymphocyte culture between donor and naive recipient cells. Sequence analysis of the CDR3 regions within recurrent TCR beta chains comprising either BV2 or BV13 gene segments demonstrated a complete sequence identity between BV2-BJ2S3 junctions derived from GITC in all individuals tested and the presence of conserved amino acids at constrained CDR3 positions within GITC BV13+ junctions derived from most individuals. These results suggest the existence of several major alloantigens responsible for expansion of T cell clones bearing a 'public' beta chain rearrangement within rejected allografts. The demonstration that such clones are also expanded during in vitro mixed lymphocyte reactions provides an experimental approach which might allow molecular characterization of the above major alloantigens and their possible in vivo targeting.

Cytolytic T lymphocyte recognition of the immunodominant HLA-A*0201-restricted Melan-A/MART-1 antigenic peptide in melanoma.

The Melan-A/MART-1 gene product is frequently recognized by tumor-specific HLA-A2-restricted CTL. An immunodominant nonapeptide has been localized to the region spanning residues 27-35. However, the decapeptide including residues 26-35 (the nonapeptide extended NH2 terminally by one residue) appeared to be recognized as efficiently as the nonapeptide. In this study, we show that the optimal length immunodominant peptide appears to correspond to the decapeptide 26-35, as assessed by quantitative analyses of both 4 polyclonal and 13 monoclonal populations of specific CTL. Functional assays of peptide binding to HLA-A2 indicate that the decapeptide is significantly a more efficient binder than the nonapeptide. Moreover, analogues of the decapeptide including substitutions at a secondary HLA-A2 peptide anchor further improve decapeptide binding. Finally, we show that the functional (9 CTL clones analyzed) and structural TCR repertoire (7 CTL clones) of a group of specific CTL clones is rather diverse. The findings reported here may have important implications for future peptide-based melanoma vaccination trials as well as for the monitoring of specific CTL responses in vivo.

In vivo longitudinal analysis of a dominant TCR repertoire selected in human response to influenza virus.

Recent studies have demonstrated biased usage of TCR V beta 17 and a high degree of diversity in J beta usage within the influenza virus matrix epitope (M.58-66)-specific CTL response. In contrast, in the course of a study on the cellular response to influenza A virus, we found preferential usage of V beta 17-J beta 2.2 rearrangement in an individual with an unexpectedly high number of CTL precursors (CTLp). We took advantage of such situation to study the longitudinal repertoire of the CD8+ T cell precursors. By limiting dilution analysis combined with the use of a clonotypic primer corresponding to the CDR3 region of this matrix-specific TCR V beta chain, the influenza-specific CTLp were shown to be stable for a period of 6 years. Overall, our results show that virus-specific CTLp can be directly monitored in vivo by molecular fingerprinting without in vitro restimulation. These findings might be extremely important for evaluation of the specific immune response to a given human pathogen.

Expanded CD4+ and CD8+ T cell clones in elderly humans.

The diversity of the human TCR repertoire in aging has been studied by examining the profiles of complementarity-determining region 3 (CDR3) sizes expressed by the BV families. The TCRBV CDR3 profile, which shows size heterogeneity in young adult humans, is significantly restricted in aged humans. Clonal T cell expansions were identified using a PCR-based approach, in one or more BV families from all 14 healthy persons over the age of 65 that we studied. CD4+ T cell expansions were identified in 8 of 11 donors and CD8+ T cell expansions in 7 of 10 donors. These clonal expansions were stable during a 2-year period. Interestingly, more than half of the aged persons had clonal expansions within the BV3, -14, -16, and -23 families. Although there was no homology among the eight CDR3 sequences identified in clonal T cells from 8 aged persons, selective pressure on the expanded T cell clones was suggested by the fact that the BV families used by the T cell clones were not proportional to the number of genes in the different BV families.

Evidence for heterogeneous TCR V beta repertoire expression in mercury-induced immune disorders in rats.

Administration of subtoxic doses of HgCl2 affects differentially the immune system depending on the strain of rats tested. Susceptible Brown-Norway (BN) rats exhibit a CD4+ T cell-dependent polyclonal activation of B cells; in contrast, Lewis (LEW) rats are resistant and develop an immunosuppression mediated by CD8+ T cells recruited by CD4+ T cells. The mechanisms by which mercury induces immune disorders are poorly understood. We were interested in analyzing the diversity and mercury-mediated changes of the TCR Vbeta repertoire in the BN and LEW strains of rats at different times of HgCl2 exposure. Our results obtained after analysis of lymph node T cells by RNase protection assay, flow cytometry or immunoscope assay (i) were not consistent with a superantigen-like stimulus since we observed neither a V beta-selective expansion nor deletion that would have been expected and (ii) showed that in BN rats, as well as in LEW rats, an increase in the number of T cells was associated with the heterogeneous TCR V beta repertoire, thus supporting a polyclonal T cell activation. However, in BN rats the total number of T cells increased very rapidly, whereas in LEW rats only CD8+ T cells accumulated.

Selection of T cells reactive against autologous B lymphoblastoid cells during chronic rheumatoid arthritis.

The repertoire and Ag specificity of T cells infiltrating inflamed joints from a chronic rheumatoid arthritis (RA) patient were studied in detail. Repertoire analysis demonstrated a reduced clonality of joint-infiltrating lymphocytes (JIL) as compared with patient's PBL, which was presumably due to an intra-articular expansion of T cell clones with recurrent TCR features. Strikingly, a large fraction of these JIL T cell clones, which were predominantly CD8+, proliferated in vitro when exposed to autologous B lymphoblastoid cells (BLC), unlike randomly chosen PBL clones derived from the same patient. This proliferative response was HLA-restricted, which confirmed a classical TCR-mediated recognition of BLC and was not observed against autologous PHA blasts, suggesting recognition of either EBV or B cell-specific Ags. Finally, a preliminary analysis of synovial lymphocytes derived from another chronic RA patient demonstrated a similar enrichment for T cells reactive against autologous BLC within JILs as compared with patient's PBLs. Taken together, these results, which suggest frequent expansions of autologous BLC-reactive T cells within inflamed joints of chronic RA patients, provide a basis for future studies evaluating the fine specificity and pathogenicity of these lymphocytes.

A fluorescence-based quantitative PCR method for investigation of pseudorabies virus latency.

A quantitative PCR method was developed in order to quantitate the number of copies of Pseudorabies virus (PRV) genome present in tissues from infected pigs. The method is based on the use of an internal standard that differs from the target DNA by a deletion of ten base pairs, and that is co-amplified with the target DNA. The resulting PCR products are labelled with a fluorescent primer and are then separated and detected by means of an automated sequencer. The assay was found to be specific and sensitive, allowing the detection of five copies of viral DNA among 10(6) host cells. The method was used successfully to quantitate the number of PRV DNA copies in trigeminal ganglia samples from infected pigs during the acute and the latent stages of the infection. Between 12 and 3.10(5) copies of viral genome per 10(6) neuronal cells were detected in these tissues which is consistent with data published previously.

Donor-specific blood transfusion-induced tolerance in adult rats with a dominant TCR-Vbeta rearrangement in heart allografts.

Following allotransplantation, determinants encoded within the donor MHC are recognized by recipient T lymphocytes through their Ag receptor. In this study, we investigated the TCR Vbeta chain diversity of T cells infiltrating rejected and tolerated heart allografts in a model of donor-specific blood transfusion-induced tolerance in MHC-mismatched congeneic rats. The PCR-based method that we used allows the diversity of Vbeta chains at the complementarity-determining region 3 level to be analyzed quantitatively. Our results show that the Vbeta repertoire usage in graft-infiltrating T cells was characteristic and different in tolerated compared with rejected grafts, and differed in both cases from the normal distribution of the Vbeta repertoire. An expansion of lymphocytes showing a conserved Vbeta18-Dbetal-Jbeta2.7 gene rearrangement was found, from the first day after grafting onward, in graft-infiltrating cells from all tolerant animals. This clone accounted for as much as 5% of the whole Vbeta repertoire in tolerated hearts, as evidenced by RNase protection assay. In contrast, we demonstrated that, of lymphocytes infiltrating rejected grafts, those with a Vbeta18 chain were diverse, and that even though by day 5 the conserved Vbeta18-Dbeta1-Jbeta2.7 rearrangement was detectable, lymphocytes harboring this rearrangement represented less than 0.6% of the whole TCR-alphabeta+ T cell repertoire. Kinetics analysis revealed that the expansion of lymphocytes bearing this conserved rearrangement was elicited specifically by donor blood transfusion. Indeed, Vbeta18-Dbeta1-Jbeta2.7 transcripts were detected in PBL from transfused animals as early as 7 days after donor-specific blood transfusion. Finally, we provided evidence that this T cell clone belongs to the CD8+ subset. The putative role in inducing and maintaining the allograft tolerance of the CD8+ T cell clone harboring this public Vbeta18-Dbeta1-Jbeta2.7 rearrangement is discussed.