Erica Lantelme

A rapid flow cytometry test based on histone H2AX phosphorylation for the sensitive and specific diagnosis of ataxia telangiectasia

Ataxia telangiectasia (A‐T) is a progressive neurodegenerative disease with onset in early childhood, caused by mutations in the ATM (ataxia‐telangiectasia mutated) gene. Diagnosis relies on laboratory tests showing high levels of serum alphafetoprotein, cell sensitivity to ionizing radiation (IR) and absence or reduced levels of ATM protein. Many tests, however, are not sufficiently sensitive or specific for A‐T, have long turnaround times, or require large blood samples. This prompted us to develop a new flow cytometry method for the diagnosis of A‐T based on the measurement of histone H2AX phosphorylation. We established normal ranges of histone H2AX phosphorylation after 2 Gy IR by testing T‐cell lines, lymphoblastoid cell lines (LCLs) and/or peripheral blood mononuclear cells (PBMCs) or both from 20 genetically proven A‐T and 46 control donors. To further evaluate the specificity and sensitivity of the test, we analyzed cells from 19 patients suspected of having A‐T, and from one Friedreich Ataxia, one Ataxia with Oculomotor Apraxia type 2, and one Nijmegen Breakage Syndrome patients. Phosphorylated histone H2AX mean fluorescence intensity of irradiated A‐T cells was significantly lower than that of healthy donors. The intrastaining, intraassay, and interassay imprecisions were ≤13.22%. Sensitivity and specificity were virtually 100% when the test was performed on PBMCs. Screening of 19 consecutive new patients with suspected A‐T classified 15 patients as non‐A‐T and four as A‐T; diagnosis of the latter four was subsequently confirmed by DNA sequencing to identify ATM mutations. The Friedreich Ataxia patient, the Ataxia with Oculomotor Apraxia type 2 patient and the Nijmegen Breakage Syndrome patient were classified as non‐A‐T. This flow cytometry test is very sensitive, specific and rapid, and requires only 2 ml of blood. It may thus be proposed for the early differential diagnosis of A‐T as an alternative to methods requiring the production of LCLs.

Diverse expansion potential and heterogeneous avidity in tumor-associated antigen-specific T lymphocytes from primary melanoma patients.

While tumor‐associated antigen (TAA)‐specific CD8+ T lymphocytes have been detected in metastatic melanoma patients, immune response in early disease phases has not yet been carefully evaluated. We looked for circulating cytotoxic T lymphocytes (CTL) directed against Melan‐A / MART1, tyrosinase, gp100 and MAGE‐3 antigens in patients with a diagnosis of primary cutaneous melanoma by using fluorescent HLA‐A2 tetramers. In five out of six cases high numbers of CD8+ / tetramer+ cells could be detected by flow cytometry, and in four patients lymphocyte populations specific for two different melanoma antigens (Melan‐A / MART1 and tyrosinase) were contemporarily present. The TAA‐specific cells could represent as much as 1 / 220 T lymphocytes in the circulating CD8+ population. When tetramers were used to monitor the in vitro expansion of TAA‐specific CTL precursors upon antigen‐specific stimulation, a diverse expansion potential was evidenced in CTL from the different donors and, more strikingly, in CTL specific for the different TAA. Melan‐A / MART1‐specific CTL clones derived from two patients exhibited a broad range of avidity. Only the highest avidity clones, representing about 50 % of the cases analyzed, were tumor specific. By correlating tetramer staining with clone avidity, we found that tetramer fluorescence intensity could represent a good indicator of TCR affinity, but not of overall clone avidity.

Cutting Edge: Recombinase-Activating Gene Expression and V(D)J Recombination in CD4+CD3low Mature T Lymphocytes

The recombinase-activating genes, RAG-1 and RAG-2, can be expressed by a subset of B cells within germinal centers, where they mediate secondary V(D)J rearrangements. This receptor revision mechanism could serve either receptor diversification or tolerance-induced functions. Alternatively, it might rescue those cells the receptors of which have been damaged by somatic mutation. Less is known about the occurrence of similar mechanisms in T cells. Here we show that mature T cells with defective TCR surface expression can express RAG genes and are capable of initiating secondary V(D)J rearrangements. The possibility that a cell rescue mechanism based on the generation of a novel Ag receptor might be active in peripheral T cells is envisaged.

Dominant TCR-α Requirements for a Self Antigen Recognition in Humans

TCR-α and -β chains are composed of somatically rearranged V, D, and J germline-encoded gene segments that confer Ag specificity. Recent crystallographic analyses revealed that TCR-α has more contacts with peptide than TCR-β, suggesting the possibility that peptide recognition predominantly relies on TCR-α. T cells specific for the self Ag Melan-A/MART-1 possess an exceptionally high precursor frequency in human histocompatibility leukocyte Ag-A2 individuals. This provided a unique situation for assessment of the structural relationship between TCR and peptide/MHC ligand at both the pre- and postimmune levels. Molecular and phenotypic analysis of many different Melan-A-specific T cell populations revealed that a structural constraint is imposed on the TCR for engagement with Melan-A peptides presented by HLA-A2, namely the highly preferential use of a particular TCRAV segment, AV2. Examination of CD8 single-positive thymocytes indicated that this preferential use in forming the Melan-A-specific TCR is mainly imposed by intrathymic positive selection. Our data demonstrate a dominant function of TCRAV2 segment in forming the TCR repertoire specific for the human self Ag Melan-A/MART-1 and support the view that Ag recognition is mediated predominantly by TCR-α.

Kinetics of GATA-3 gene expression in early polarizing and committed human T cells.

Different transcription factors have been shown to control the transition of naive T cells into T helper 1 (Th1)/Th2 subsets. The T‐cell‐specific transcription factor GATA‐3 is known to be selectively expressed in murine developing Th2 cells and to exert a positive action on Th2‐specific cytokine production. Investigating GATA‐3 gene regulation in human T cells we have found that naive T cells highly express GATA‐3, and during early T2 or T1 polarization, respectively, they either maintain or quickly down‐regulate expression. In developing T2 cells, as well as in committed Th2 cell lines and clones, we found a positive correlation among GATA‐3, interleukin (IL)‐5 and IL‐4 gene expression kinetics, supporting the positive action of GATA‐3 on Th2‐specific cytokine production. A possible relationship between GATA‐3 gene expression and the down‐regulation of the IL‐12 receptor (β2‐chain; IL‐12Rβ2) gene was evident only in the early phases of T2 polarization (within 24 hr), and not demonstrated at later times. During T‐cell commitment the presence of IL‐4 in the culture was essential to maintain or enhance GATA‐3 transcription, while IL‐12 was not necessary for full repression of GATA‐3. Finally, we showed selective GATA‐3 up‐regulation in human Th2 cell lines and clones and the maintainance of a low basal level of GATA‐3 expression in Th1 cells upon activation.

Increased frequency of RAG-expressing, CD4+CD3low peripheral T lymphocytes in patients with defective responses to DNA damage

Accumulating evidence indicates that peripheral lymphocyte variants with altered antigen receptor expression may be capable of expressing recombination‐activating genes (RAG). We and others recently observed functional RAG gene products in mature T cells with defective TCR expression (MacMahan and Fink, Immunity 1998. 9: 637 – 647; Lantelme et al., J. Immunol., 2000. 164: 3455 – 3459). Here, the association between TCR expression and RAG activity was assessed further in lymphocytes from patients with defective responses to DNA damage. We show that T cells with altered TCR surface expression are present in increased numbers in these patients and that they express RAG genes. The finding of RAG gene expression by TCR variants suggests the possibility that secondary V(D)J rearrangements could be induced in these cells to rescue their defective phenotype and cellular function. Moreover, as V(D)J recombination has been implicated in chromosome translocations involving antigen receptor genes, we discuss a possible relationship between altered TCR expression, RAG activity and the frequent lymphoma‐specific translocations observed in these patients.

Two-tier analysis of histone H2AX phosphorylation allows the identification of Ataxia Telangiectasia heterozygotes

BACKGROUND AND PURPOSE Ataxia Telangiectasia (A-T) heterozygotes constitute 0.36-1% of the general population. They have a higher risk of developing several types of cancer and may be more likely to suffer side-effects following radiotherapy than the general population. Their identification is both labor- and time-consuming and the sensitivity and specificity of the methods employed has not been evaluated. This paper describes a new approach to the identification of A-T heterozygotes based on a two-tier analysis of histone H2AX phosphorylation. MATERIALS AND METHODS We compared the T-cell phenotype after exposure to 2 Gy in nine obligate A-T heterozygotes and 17 normal donors. Examined end points were histone H2AX phosphorylation by flow cytometry 1 h after irradiation (kinase proficiency) and the residual gamma-H2AX foci by confocal microscopy 72 h after irradiation (DSB repair proficiency). RESULTS The sequential use of these two methods results in 100% positive predictive value (PPV), 67% negative predictive value (NPV), 78% sensitivity, and 100% specificity. The overall hit rate, i.e. the ratio between the true positives plus the true negatives and the total number of observations was 85%. CONCLUSIONS A-T heterozygotes can be identified by analysing irradiated T-cell H2AX phosphorylation level and residual gamma-H2AX foci.

Analysis of Secondary V(D)J Rearrangements in Mature, Peripheral T Cells of Ataxia-Telangiectasia Heterozygotes

Ataxia-telangiectasia (AT) is a rare recessive disease with pleiotropic involvement of the nervous and lymphoid systems. AT heterozygotes have a population frequency of about 1%, and although not manifesting any overt clinical symptoms, they have an increased mortality, mainly because of cancer and ischemic heart disease. We and others have described a mature T lymphocyte population with an altered T cell receptor surface expression (“TCR variant”) that reactivates the recombination activating genes (RAG) and is expanded in the blood of patients with AT. In view of the known role of V(D)J recombination in the onset of tumorigenic translocations, we proposed that the increased RAG activity was responsible for the predisposition of AT homozygotes to develop mature-type T leukemia/lymphoma. In the present report, we used cytofluorimetry to quantify the TCR variant population and the memory/naïve T-cell compartments in the blood of AT heterozygotes compared with AT patients and controls. We assessed the expression of different recombinase genes through RT-PCR/oligotyping and cytofluorometric analysis and searched for rearrangement intermediates by ligase-mediated PCR in T-cell lines from four heterozygous carriers. We found the TCR variant population was increased on average 2 × in AT heterozygotes (vs 10 × in homozygotes) compared with controls, and naïve CD4+ T lymphocytes were reduced on average 0.5 × (vs 0.1 × in homozygotes). We were able to demonstrate recombinase gene expression in all four heterozygous T-cell lines, and rearrangement intermediates, indicative of ongoing V(D)J recombination, in two. These rearrangements were compatible with V-gene replacement, a mechanism of receptor editing described for Ig and TCRα genes, to our knowledge not previously documented for TCRβ. In conclusion, we found that RAG reactivation and secondary V(D)J rearrangements, potential risk factors of mature-type leukemia in AT homozygotes, also take place in AT heterozygous carriers and might place this large population fraction at an increased risk of leukemia/lymphoma.

Clonal predominance, but preservation of a polyclonal reservoir, in the normal alpha beta T-cell repertoire.

We recently demonstrated that the peripheral gamma delta T-cell repertoire becomes oligoclonal with increasing age. Although this junctional homogeneity should not severely affect the ability of gamma delta T cells to respond to foreign antigens, we reasoned that a similar oligoclonal repertoire of alpha beta T cells would lead to a profound impairment of the MHC-restricted response. We used heteroduplex analysis in this research to study the clonal complexity of the peripheral alpha beta T-cell repertoire in human subjects and supply evidence for the presence of alpha beta clonal expansions. Clonal predominance in the alpha beta T-cell repertoire of normal subjects was not simply related to age, since the PBL of young donors also showed clonal expansions and did not always correlate with a numeric increase in the corresponding V beta family. However, the type of alpha beta expansion appears to be strikingly different from the gamma delta expansions. In the case of alpha beta T cells, even in the presence of clonal dominance, evidence for a residual polyclonal background was found in all the donors tested, irrespective of age. The observation that true oligoclonality is exceptionally rare among alpha beta T lymphocytes could mean that maintenance of a highly diversified reservoir of TCR is primary for these cells throughout life.

A novel defect in mitochondrial p53 accumulation following DNA damage confers apoptosis resistance in Ataxia Telangiectasia and Nijmegen Breakage Syndrome T-cells

We have previously shown that whereas T-cells from normal individuals undergo accumulation of p53 and apoptosis when treated with the genotoxic agent Actinomycin D (ActD), those from Ataxia Telangiectasia (AT) and Nijmegen Breakage Syndrome (NBS) patients resist ActD-induced apoptosis [1]. We have now found similar resistance by the p53-null Jurkat T-cell line and by siRNA p53-knockdown normal T-cells. This evidence that ActD initiates a p53-dependent apoptotic responce prompted us to look for defective p53 accumulation by AT and NBS T-cells. Surprisingly the total p53 level was only slightly reduced compared to normal T cells but its intracellular localization was highly defective: p53 was poorly accumulated in the cytosol and nearly undetectable in mitochondria. In accordance with the dependence of ActD-induced apoptosis on a mitochondrial p53 function, in control T-cells specific inhibition of mitochondrial p53 translocation with μ pifithrin reduced apoptosis by 86%, whereas treatment with α pifithrin, which blocks p53-mediated transcription, had no effect. We also showed that nuclear export is not required for mitochondrial p53 translocation. Observation of an altered p53 ubiquitination pattern and Mdm2 accumulation in ActD-treated AT and NBS T-cells provided a mechanistic link to their defective extranuclear p53 localization. Our results disclose an undescribed defect in mitochondrial p53 accumulation in AT and NBS T-cells that makes them resistant to apoptosis following unrepairable DNA damage.