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Induction of the TCR signaling pathway terminates the expression of RAG genes, and a link between this pathway and their transcriptional control is evident from the recent demonstration of their re-expression if the TCR is subsequently lost or down-regulated. Since unstimulated T cells display a steady-state level of "tonic" TCR signaling, i.e. in the absence of any antigenic stimulus, it was uncertain whether this control was exerted through ligand-dependent or ligand-independent TCR signaling. Here we demonstrate for the first time that exogenous TCR α and β chains transferred into the human immature RAG(+) T cell line Sup-T1 by lentiviral transduction inhibit RAG expression through tonic signaling, and that this inhibition could itself be reverted by pharmacological tonic pathway inhibitors. We also suggest that mature T cells already expressing an endogenous TCR on their surface maintain some levels of plasticity at the RAG locus when their basal TCR signaling is interfered with. Lastly, we show that the TCR constructs employed in TCR gene therapy do not possess the same basal signaling transduction capability, a feature that may have therapeutic implications.

V(D)J recombination is a mechanism peculiar to the somatic rearrangement of antigen receptor genes. It requires both expression of the RAG-1 and RAG-2 recombinases and accessibility of the substrate to its recombinase and post-cleavage/DNA repair stage. TCR revision is a genetic correction mechanism that changes T cell specificity by re-activating V(D)J recombination in peripheral T cells. This process is now well described in both normal or pathological murine and human settings. Many of its features, such as the question of whether it occurs in truly mature T cells, remain to be elucidated. Its occurrence in human CD8+ T cells is also an open question. We have therefore established an in vitro model of TCR revision in mature human CD8+ T cells to determine whether down-regulation of the TCR/CD3 complex from the cell surface in the presence of IL7 as a factor favouring chromatin remodelling initiates a TCR revision pathway. Only mature CD8+ T cells carrying already-formed antigen receptors were used. CD8+ T cells treated with anti-CD3 and IL7 showed rearrangement intermediates and expressed new Vβ-chains on their surface. Investigation of the molecular pathway thus induced disclosed up-regulation of the RAG-2 transcript, but absence of the ‘canonical’ RAG-1 mRNA. A surprising finding was the demonstration of alternative splice forms of this mRNA, already expressed in untreated CD8+ T cells, encoding for the full-length RAG-1 protein, which was increased three-fold in the treated cells. All the V(D)J requirements were thus fulfilled when mature human CD8+ T cells were stimulated with anti-CD3 and IL7. Induction of TCR revision in vitro in mature T cells is an easily controllable system that could be employed in further studies to elucidate the molecular pathways involved in secondary V(D)J rearrangements in peripheral cells.

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

Modifications in tumour antigen-derived epitopes that stabilize the major histocompatibility complex (MHC)-peptide complex result in enhanced stimulatory capacity and improved immunogenicity of the altered peptide. These epitope analogues are attractive candidates for the development of peptide-based vaccine trials. Any modification, however, in tumour antigens may induce T-cell responses that could either fail to react against the naturally occurring peptides or represent only a subset of the total antigen-specific repertoire. In the present study, we performed a critical analysis of the ability of cytotoxic T-lymphocyte (CTL) clones, derived from two melanoma patients through stimulation with the A27L peptide analogue, to cross-react with the naturally processed Melan-A/MART-1 (Melan-A) peptides in terms of T-cell receptor (TCR) affinity, functional avidity and fine antigen specificity. We found that all the A27L-specific clones analysed possessed a very low avidity for the natural Melan-A peptides, and that their binding affinity for human leukocyte antigen (HLA) tetramers complexed with both the modified and the natural Melan-A peptides did not strictly correlate with their functional avidity. We also observed that these clones were able to cross-recognize both natural Melan-A peptides in one patient, but only one peptide in the second patient. We discuss the capability of the A27L peptide analogue to stimulate all the available Melan-A-specific repertoire.

CDT2/L2DTL/RAMP is one of the substrate receptors of the Cullin Ring Ubiquitin Ligase 4 that targets for ubiquitin mediated degradation a number of substrates, such as CDT1, p21 and CHK1, involved in the regulation of cell cycle and survival. Here we show that CDT2 depletion was alone able to induce the apoptotic death in 12/12 human cancer cell lines from different tissues, regardless of the mutation profile and CDT2 expression level. Cell death was associated to rereplication and to loss of CDT1 degradation. Conversely, CDT2 depletion did not affect non-transformed human cells, such as immortalized kidney, lung and breast cell lines, and primary cultures of endothelial cells and osteoblasts. The ectopic over-expression of an activated oncogene, such as the mutation-activated RAS or the amplified MET in non-transformed immortalized breast cell lines and primary human osteoblasts, respectively, made cells transformed in vitro, tumorigenic in vivo, and susceptible to CDT2 loss. The widespread effect of CDT2 depletion in different cancer cells suggests that CDT2 is not in a synthetic lethal interaction to a single specific pathway. CDT2 likely is a non-oncogene to which transformed cells become addicted because of their enhanced cellular stress, such as replicative stress and DNA damage.

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 (beta2-chain; IL-12Rbeta2) 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.

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 contemporaneously 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.

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.

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 γ-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 γ-H2AX foci.

The repair of DNA double-strand breaks is critical for genome integrity and tumor suppression. Here we show that following treatment with the DNA-intercalating agent actinomycin D (ActD), normal quiescent T cells accumulate double-strand breaks and die, whereas T cells from ataxia telangiectasia (AT) and Nijmegen breakage syndrome (NBS) patients are resistant to this death pathway despite a comparable amount of DNA damage. We demonstrate that the ActD-induced death pathway in quiescent T lymphocytes follows DNA damage and H2AX phosphorylation, is ATM- and NBS1-dependent and due to p53-mediated cellular apoptosis. In response to genotoxic 2-Gy gamma-irradiation, on the other hand, quiescent T cells from normal donors survive following complete resolution of the damage thus induced. T cells from AT and NBS patients also survive, but retain foci of phosphorylated H2AX due to a subtle double-strand break (DSB) repair defect. A common consequence of these two genetic defects in the DSB response is the apparent tolerance of cells containing DNA breaks. We suggest that this tolerance makes a major contribution to the oncogenic risk of patients with chromosome instability syndromes.

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/naive 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 2x in AT heterozygotes (vs 10x in homozygotes) compared with controls, and naive CD4(+) T lymphocytes were reduced on average 0.5x (vs 0.1x 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 TCRalpha genes, to our knowledge not previously documented for TCRbeta. 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.

Although there has been extensive analysis on the capacity of MHC-peptide tetramers to bind antigen-specific TCR, there have been comparatively few studies regarding the role of the CD4 and CD8 co-receptors in binding and activation by these multimeric molecules. Here, we start from the observation that different antibodies against human CD8 exert opposite effects on MHC-peptide tetramer binding to the TCR: tetramer staining was enhanced by OKT8 antibody, while it was blocked with SK1 antibody. We used these different anti-CD8 antibodies to modulate CD8 function during tetramer staining of Melan-A/MART1-specific CTL clones. We show that CD8 action could be variably modulated during all the phases of interaction, indicating that CD8 participates in both the initial association of the TCR with MHC-peptide tetramers and the stability of this interaction. While the blocking effect of anti-CD8 antibodies was mostly exerted during the initial binding of the TCR with MHC-peptide tetramers, the enhancing effect was exerted by augmenting the duration of this interaction. Blocking anti-CD8 antibodies were also capable of preventing tetramer-mediated T cell activation. The possibility of variably affecting MHC-peptide tetramer binding and T cell activation using anti-CD8 antibodies confirms the critical role exerted by the CD8 co-receptor in this interaction and supports the notion that TCR engagement by MHC-peptide ligands typically involves CD8.

Vitiligo is a common skin disease characterized by the presence of well circumscribed, depigmented, milky white macules devoid of identifiable melanocytes. Although the detection of circulating anti-melanocytic antibodies and of infiltrating lymphocytes at the margin of lesions supports the view that vitiligo is an autoimmune disorder, its etiology remains unknown. In particular, it is still a matter of debate whether the primary pathogenic role is exerted by humoral or cellular abnormal immune responses. In this study, the presence of specific cytotoxic T lymphocyte responses against the melanocyte differentiation antigens Melan-A/MART1, tyrosinase, and gp100 in vitiligo patients have been investigated by the use of major histocompatibility complex/peptide tetramers. High frequencies of circulating melanocyte-specific CD8+ T cells were found in all vitiligo patients analyzed. These cells exerted anti-melanocytic cytotoxic activity in vitro and expressed skin-homing capacity. In one patient melanocyte-specific cells were characterized by an exceptionally high avidity for their peptide/major histocompatibility complex ligand. These findings strongly suggest a role for cellular immunity in the pathogenesis of vitiligo and impact on the common mechanisms of self tolerance.

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.

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.

The Src-homology-3 domain (SH3) is an evolutionarily conserved, 50- to 60-amino-acid module carried by intracellular proteins involved in the transduction of signals for cell polarization, motility, enzymatic activation, and transcriptional regulation. The SH3 drives protein-protein interactions through binding to proline-rich ligands. This function relies on the conserved secondary structure, whereas the SH3 primary structure is highly diverse. Taking advantage of the fact that the few conserved amino acids are clustered near the N- and C-terminal ends, we designed degenerate oligonucleotides spanning these two regions and screened by PCR a variety of normal and tumor tissues for the expression of SH3-containing transcripts. Using this strategy, we have identified a novel SH3-containing human gene family of six related transcripts that map to four different chromosomes. The SH3 domain lies at the C-terminal end and shows 56-50% amino acid homology to the C-terminal SH3 of Sem-5/Drk/GRB2. The N-terminal segment of this novel SH3GL (from SH3-containing Grb2-like) gene family does not resemble any known protein. Three of these transcripts are in-frame and show a peculiar tissue distribution: SH3GL2 is preferentially expressed in the brain, SH3GL3 in brain and testis, and SH3GL1 is ubiquitous.

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.

CDT2/L2DTL/RAMP is one of the substrate receptors that binds the CRL4 ubiquitin ligase and confers it specificity for a number of substrates, such as CDT1, p21 and CHK1. Previously, we reported that in ovarian cancer cell lines increased susceptibility to cisplatin is associated to the down-regulation of the CDT2 gene. Here we show that CDT2 depletion was alone able to induce the apoptotic death not only in ovarian cancer cell lines but also in 10/10 other human cancer cell lines from different tissues, irrespective of p53, ATM and ATR status and CDT2 expression level. Conversely, CDT2 depletion did not affect non-transformed human cells, such as immortalized kidney, lung and breast cell lines, and primary cultures of endothelial cells and osteoblasts. After CDT2 suppression, all cancer cells underwent rereplication associated to the inability to destroy CDT1 and to cell death. The ectopic over-expression of an activated oncogene, such as the mutation-activated RAS or the amplified MET in non-transformed immortalized breast cell lines and primary human osteoblasts, respectively, made cells transformed in vitro, tumorigenic in vivo, and susceptible to CDT2 loss. Conversely, the knock-down of the tumor suppressor genes PTEN or RB1 did not transform immortalized breast cells and did not sensitize them to CDT2 loss. These data show that transformation makes cells “addicted” to CDT2. The widespread effect of CDT2 depletion in different cancer cells suggests that CDT2 is not in a synthetic lethal interaction to another specific gene or pathway but becomes dominant in these cells because of their stress phenotype, due to DNA damage, replication and mitotic stress. Citation Format: Martina Olivero, Daniela Dettori, Sabrina Arena, Davide Zecchin, Erica Lantelme, Maria Flavia Di Renzo. The stress phenotype makes ovarian cancer cells addicted to CDT2, a substrate receptor of the CRL4 ubiquitin ligase. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B28.

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.