Nurse shark T-cell receptors employ somatic hypermutation preferentially to alter alpha/delta variable segments associated with alpha constant region.

In addition to canonical TCR and BCR, cartilaginous fish assemble noncanonical TCR that employ various B-cell components. For example, shark T cells associate alpha (TCR-α) or delta (TCR-δ) constant (C) regions with Ig heavy chain (H) variable (V) segments or TCR-associated Ig-like V (TAILV) segments to form chimeric IgV-TCR, and combine TCRδC with both Ig-like and TCR-like V segments to form the doubly rearranging NAR-TCR. Activation-induced (cytidine) deaminase-catalyzed somatic hypermutation (SHM), typically used for B-cell affinity maturation, also is used by TCR-α during selection in the shark thymus presumably to salvage failing receptors. Here, we found that the use of SHM by nurse shark TCR varies depending on the particular V segment or C region used. First, SHM significantly alters alpha/delta V (TCRαδV) segments using TCR αC but not δC. Second, mutation to IgHV segments associated with TCR δC was reduced compared to mutation to TCR αδV associated with TCR αC. Mutation was present but limited in V segments of all other TCR chains including NAR-TCR. Unexpectedly, we found preferential rearrangement of the noncanonical IgHV-TCRδC over canonical TCR αδV-TCRδC receptors. The differential use of SHM may reveal how activation-induced (cytidine) deaminase targets V regions.

An Ectopic CTCF Binding Element Inhibits Tcrd Rearrangement by Limiting Contact between Vδ and Dδ Gene Segments.

Chromatin looping mediated by the CCCTC binding factor (CTCF) regulates V(D)J recombination at Ag receptor loci. CTCF-mediated looping can influence recombination signal sequence (RSS) accessibility by regulating enhancer activation of germline promoters. CTCF-mediated looping has also been shown to limit directional tracking of the RAG recombinase along chromatin, and to regulate long-distance interactions between RSSs, independent of the RAG recombinase. However, in all prior instances in which CTCF-mediated looping was shown to influence V(D)J recombination, it was not possible to fully resolve the relative contributions to the V(D)J recombination phenotype of changes in accessibility, RAG tracking, and RAG-independent long-distance interactions. In this study, to assess mechanisms by which CTCF-mediated looping can impact V(D)J recombination, we introduced an ectopic CTCF binding element (CBE) immediately downstream of Eδ in the murine Tcra-Tcrd locus. The ectopic CBE impaired inversional rearrangement of Trdv5 in the absence of measurable effects on Trdv5 transcription and chromatin accessibility. The ectopic CBE also limited directional RAG tracking from the Tcrd recombination center, demonstrating that a single CBE can impact the distribution of RAG proteins along chromatin. However, such tracking cannot account for Trdv5-to-Trdd2 inversional rearrangement. Rather, the defect in Trdv5 rearrangement could only be attributed to a reconfigured chromatin loop organization that limited RAG-independent contacts between the Trdv5 and Trdd2 RSSs. We conclude that CTCF can regulate V(D)J recombination by segregating RSSs into distinct loop domains and inhibiting RSS synapsis, independent of any effects on transcription, RSS accessibility, and RAG tracking.

Characterization of human αßTCR repertoire and discovery of D-D fusion in TCRß chains.

The characterization of the human T-cell receptor (TCR) repertoire has made remarkable progress, with most of the work focusing on the TCRß chains. Here, we analyzed the diversity and complexity of both the TCRα and TCRß repertoires of three healthy donors. We found that the diversity of the TCRα repertoire is higher than that of the TCRß repertoire, whereas the usages of the V and J genes tended to be preferential with similar TRAV and TRAJ patterns in all three donors. The V-J pairings, like the V and J gene usages, were slightly preferential. We also found that the TRDV1 gene rearranges with the majority of TRAJ genes, suggesting that TRDV1 is a shared TRAV/DV gene (TRAV42/DV1). Moreover, we uncovered the presence of tandem TRBD (TRB D gene) usage in ~2% of the productive human TCRß CDR3 sequences.

Extensive molecular mapping of TCRα/δ- and TCRß-involved chromosomal translocations reveals distinct mechanisms of oncogene activation in T-ALL.

Chromosomal translocations involving the TCR loci represent one of the most recurrent oncogenic hallmarks of T-cell acute lymphoblastic leukemia (T-ALL) and are generally believed to result from illegitimate V(D)J recombination events. However, molecular characterization and evaluation of the extent of recombinase involvement at the TCR-oncogene junction has not been fully evaluated. In the present study, screening for TCRß and TCRα/δ translocations by FISH and ligation-mediated PCR in 280 T-ALLs allowed the identification of 4 previously unreported TCR-translocated oncogene partners: GNAG, LEF1, NKX2-4, and IL2RB. Molecular mapping of genomic junctions from TCR translocations showed that the majority of oncogenic partner breakpoints are not recombinase mediated and that the regulatory elements predominantly used to drive oncogene expression differ markedly in TCRß (which are exclusively enhancer driven) and TCRα/δ (which use an enhancer-independent cryptic internal promoter) translocations. Our data also imply that oncogene activation takes place at a very immature stage of thymic development, when Dδ2-Dδ3/Dδ3-Jδ1 and Dß-Jß rearrangements occur, whereas the bulk leukemic maturation arrest occurs at a much later (cortical) stage. These observations have implications for T-ALL therapy, because the preleukemic early thymic clonogenic population needs to be eradicated and its disappearance monitored.

Long-distance regulation of fetal V(δ) gene segment TRDV4 by the Tcrd enhancer.

Murine Tcra and Tcrd gene segments are organized into a single genetic locus (Tcra/Tcrd locus) that undergoes V(D)J recombination in CD4(-)CD8(-) double-negative (DN) thymocytes to assemble Tcrd genes and in CD4(+)CD8(+) double-positive thymocytes to assemble Tcra genes. Recombination events are regulated by two developmental stage-specific enhancers, E(δ) and E(α). Effects of E(α) on Trca/Tcrd locus chromatin have been well documented, but effects of E(δ) have not. In this regard, E(α) acts over long distances to activate many V(α) and J(α) segments for recombination in double-positive thymocytes. However, in DN thymocytes, it is unclear whether E(δ) functions over long distances to regulate V(δ) gene segments or functions only locally to regulate D(δ) and J(δ) gene segments. In this study, we analyzed germline transcription, histone modifications, and recombination on wild-type and E(δ)-deficient alleles in adult and fetal thymocytes. We found that E(δ) functions as a local enhancer whose influence is limited to no more than ∼10 kb in either direction (including D(δ), J(δ), and TRDV5 gene segments) in adult DN thymocytes. However, we identified a unique long-distance role for E(δ) promoting accessibility and recombination of fetal V(δ) gene segment TRDV4, over a distance of 55 kb, in fetal thymocytes. TRDV4 recombination is specifically repressed in adult thymocytes. We found that this repression is enforced by a developmentally regulated loss of histone acetylation. Constitutively high levels of a suppressive modification, histone H3 lysine 9 dimethylation, may contribute to repression as well.

Dual functions of prostaglandin D2 in murine contact hypersensitivity via DP and CRTH2.

Prostaglandin D2 (PGD2) exerts its effects through two distinct receptors: the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) and the D prostanoid (DP) receptor. Our previous study demonstrated that CRTH2 mediates contact hypersensitivity (CHS) in mice. However, the function of DP receptor remains to be fully established. In this study, we examine the pathophysiological roles of PGD2 using DP-deficient (DP(-/-)) and CRTH2/DP-deficient (CRTH2(-/-)/DP(-/-)) mice to elucidate receptor-mediated PGD2 action in CHS. We observed profound exacerbation of CHS in DP(-/-) mice. CRTH2(-/-)/DP(-/-) mice showed similar exacerbation, but to a lesser extent. These symptoms were accompanied by increased production of interferon-γ and IL-17. The increase in IL-17 producing γδ T cells was marked and presumably contributed to the enhanced CHS. DP deficiency promoted the in vivo migration of dendritic cells to regional lymph nodes. A DP agonist added to DCs in vitro was able to inhibit production of IL-12 and IL-1ß. Interestingly, production of IL-10 in dendritic cells was elevated via the DP pathway, but it was lowered by the CRTH2 pathway. Collectively, PGD2 signals through CRTH2 to mediate CHS inflammation, and conversely, DP signals to exert inhibitory effects on CHS. Thus, we report opposing functions for PGD2 that depend on receptor usage in allergic reactions.

ATM-deficient thymic lymphoma is associated with aberrant tcrd rearrangement and gene amplification.

Ataxia telangiectasia mutated (ATM) deficiency predisposes humans and mice to T lineage lymphomas with recurrent chromosome 14 translocations involving the T cell receptor alpha/delta (Tcra/d) locus. Such translocations have been thought to result from aberrant repair of DNA double-strand breaks (DSBs) during Tcra locus V(D)J recombination, and to require the Tcra enhancer (Ealpha) for Tcra rearrangement or expression of the translocated oncogene. We now show that, in addition to the known chromosome 14 translocation, ATM-deficient mouse thymic lymphomas routinely contain a centromeric fragment of chromosome 14 that spans up to the 5' boundary of the Tcra/d locus, at which position a 500-kb or larger region centromeric to Tcra/d is routinely amplified. In addition, they routinely contain a large deletion of the telomeric end of one copy of chromosome 12. In contrast to prior expectations, the recurrent translocations and amplifications involve V(D)J recombination-initiated breaks in the Tcrd locus, as opposed to the Tcra locus, and arise independently of the Ealpha. Overall, our studies reveal previously unexpected mechanisms that contribute to the oncogenic transformation of ATM-deficient T lineage cells.

Distribution and development of the postnatal murine Vδ1 T-cell receptor repertoire.

Murine γ/δ T cells express canonical Vγ5Vδ1 chains in the epidermis and Vγ6Vδ1 chains at reproductive sites. Both subsets carry an identical Vδ1-Dδ2-Jδ2 chain which completely lacks junctional diversity. These cells are thought to monitor tissue integrity via recognition of stress-induced self antigens. In this study, we showed by reverse transcription-polymerase chain reaction (RT-PCR), complementarity determining region 3 (CDR3) spectratyping and sequencing of the junctional regions of Vδ1 chains from C57BL/6 mice (aged 1 day to 14 months) that the canonical Vδ1-Dδ2-Jδ2 chain is also consistently present at other sites such as the thymus, gut, lung, liver, spleen and peripheral blood. In addition, we found multiple Vδ1 chains with fetal type rearrangements which were also shared among organs and among animals. These Vδ1 chains were typically characterized by a conserved amino acid motif, 'GGIRA'. Furthermore, by analysing the early postnatal period at days 10 and 16, we demonstrated that the diversification of the thymic Vδ1 repertoire is not paralleled by a diversification of extrathymic Vδ1+γ/δ T cells. This indicates that only fetal type rearrangements survive at extrathymic sites. In conclusion, γ/δ T cells expressing the canonical Vδ1-Dδ2-Jδ2 chain are not unique to the skin and reproductive sites. Furthermore, we found other γ/δ T cells expressing fetal type Vδ1 chains which were shared among different organs and animals. Thus, γ/δ T cells expressing conserved Vδ1 chains are likely to have important functions. We suggest a model in which this subset continuously recirculates throughout the organism and rapidly responds to stress-induced self antigens.

Evolutionarily conserved TCR binding sites, identification of T cells in primary lymphoid tissues, and surprising trans-rearrangements in nurse shark.

Cartilaginous fish are the oldest animals that generate RAG-based Ag receptor diversity. We have analyzed the genes and expressed transcripts of the four TCR chains for the first time in a cartilaginous fish, the nurse shark (Ginglymostoma cirratum). Northern blotting found TCR mRNA expression predominantly in lymphoid and mucosal tissues. Southern blotting suggested translocon-type loci encoding all four chains. Based on diversity of V and J segments, the expressed combinatorial diversity for gamma is similar to that of human, alpha and beta may be slightly lower, and delta diversity is the highest of any organism studied to date. Nurse shark TCRdelta have long CDR3 loops compared with the other three chains, creating binding site topologies comparable to those of mammalian TCR in basic paratope structure; additionally, nurse shark TCRdelta CDR3 are more similar to IgH CDR3 in length and heterogeneity than to other TCR chains. Most interestingly, several cDNAs were isolated that contained IgM or IgW V segments rearranged to other gene segments of TCRdelta and alpha. Finally, in situ hybridization experiments demonstrate a conservation of both alpha/beta and gamma/delta T cell localization in the thymus across 450 million years of vertebrate evolution, with gamma/delta TCR expression especially high in the subcapsular region. Collectively, these data make the first cellular identification of TCR-expressing lymphocytes in a cartilaginous fish.

Numerical modelling of the V-J combinations of the T cell receptor TRA/TRD locus.

T-Cell antigen Receptor (TR) repertoire is generated through rearrangements of V and J genes encoding alpha and beta chains. The quantification and frequency for every V-J combination during ontogeny and development of the immune system remain to be precisely established. We have addressed this issue by building a model able to account for Valpha-Jalpha gene rearrangements during thymus development of mice. So we developed a numerical model on the whole TRA/TRD locus, based on experimental data, to estimate how Valpha and Jalpha genes become accessible to rearrangements. The progressive opening of the locus to V-J gene recombinations is modeled through windows of accessibility of different sizes and with different speeds of progression. Furthermore, the possibility of successive secondary V-J rearrangements was included in the modelling. The model points out some unbalanced V-J associations resulting from a preferential access to gene rearrangements and from a non-uniform partition of the accessibility of the J genes, depending on their location in the locus. The model shows that 3 to 4 successive rearrangements are sufficient to explain the use of all the V and J genes of the locus. Finally, the model provides information on both the kinetics of rearrangements and frequencies of each V-J associations. The model accounts for the essential features of the observed rearrangements on the TRA/TRD locus and may provide a reference for the repertoire of the V-J combinatorial diversity.

Gamma-delta T-cell lymphomas.

Peripheral T-cell lymphomas (TCLs) are uncommon neoplasms, accounting for about 12% of all lymphoid tumors worldwide. TCLs in which gammadelta T-cell receptors are expressed (gammadelta TCLs) are extremely aggressive and rare (<1% of lymphoid neoplasms). gammadelta TCLs originate from gammadelta T cells, a small subset of peripheral T cells with direct antigen recognition capability acting at the interface between innate and adaptive immunity. Two distinct gammadelta TCL entities are recognized: hepatosplenic T-cell lymphoma (HSTL) and primary cutaneous gammadelta T-cell lymphoma (PCGD-TCL). HSTL is a well-characterized extranodal lymphoma that has a disguised onset, secondary to intrasinusoidal infiltration of the spleen, liver and bone marrow, has a rapidly progressive course that is poorly responsive to chemotherapy, and often ensues in the setting of immune system suppression. PCGD-TCL can present with prominent epidermal involvement or with a panniculitis-like clinical picture that can be complicated by a concurrent hemophagocytic syndrome; the disease shows biological and phenotypic overlap with other extranodal gammadelta TCLs that involve the respiratory or gastrointestinal tract mucosa. The regular application of phenotypic and molecular techniques is crucial for the diagnosis of gammadelta TCLs. In this Review, we discuss the clinical and biological features, the diagnostic challenges and the therapeutic perspectives of HSTL and PCGD-TCL.

Id3 restricts the developmental potential of gamma delta lineage during thymopoiesis.

Most T cell progenitors develop into the alphabeta T cell lineage with the exception of a small fraction contributing to the gammadelta lineage throughout postnatal life. T cell progenitors usually commit to the alphabeta lineage upon the expression of a fully rearranged and functional TCRbeta gene, and most cells that fail to produce a functional TCRbeta-chain will die instead of adopting the alternative gammadelta T cell fate. What prevents these cells from continuing TCRgamma rearrangement and adopting the gammadelta T cell fate is not known. In this study, we show that functional loss of Id3 results in a significant increase of gammadelta T cell production from progenitor cells undergoing TCRbeta rearrangement. The enhanced gammadelta T cell development correlated with increased TCRgamma gene rearrangement involving primarily Vgamma1.1 in Id3 deficient mice. We further show that Id3 deficiency promotes gammadelta T cell production in a manner independent of TCRbeta-chain expression. Our data indicates that Id3 suppresses Vgamma1.1 rearrangement and gammadelta lineage potential among T cell progenitors that have completed TCRbeta gene rearrangement without producing a functional TCRbeta protein.

The pattern of clonal immunoglobulin and T-cell receptor (Ig/TCR) gene rearrangements in Chinese adult acute lymphoblastic leukemia patients.

We have studied forty Chinese adult ALL patients at newly diagnosis, using standard primers and protocols of BIOMED-2 multiplex PCR, to determine the feasibility of Ig and TCR gene rearrangements as diagnostic and patient-specific MRD-RQ-PCR targets for molecular monitoring. Clonal IGH, IGK, IGL, TCRB, TCRG and TCRD rearrangements were found in 86%, 22%, 9%, 19%, 77%, 55% of adult patients with B-lineage ALL, respectively. While in T-ALL, clonal IGH, TCRB, TCRG and TCRD rearrangements were detected in 6%, 83%, 78%, 33% of patients. Several specialties in the pattern of Ig/TCR gene rearrangements in Chinese adult ALL patients in comparison with those reported for children and adult patients in other countries have been noted. These results are useful for further MRD-RQ-PCR detection and quantification for all patients.

T-cell receptor gamma and delta gene rearrangements and junctional region characteristics in south Indian patients with T-cell acute lymphoblastic leukemia.

Clonal T-cell receptor (TCR) gamma and delta gene rearrangements were studied in 40 T-ALL cases (pediatrics, 29; adults, 11) using PCR with homo-heteroduplex analysis. At least one clonal TCRG or TCRD rearrangement was detected in 34 (85%) cases. TCR gamma (TCRG) rearrangement was detected in 25 (62.5%) cases that included 16 (55%) pediatrics and 9 (81.8%) adults. TCR delta (TCRD) rearrangement was detected in 14/40 (35%) cases, which included 12 (41%) pediatrics and 2 (18%) adults. The frequency of VgammaI-Jgamma1.3/2.3 was significantly more in adults than pediatrics (81.8% vs. 41.3%, P=0.02). In TCRD, Vdelta1-Jdelta1 was rearranged in 10 (25%) cases. The surface membrane CD3 positive cases are significantly associated with absence of TCRD rearrangements (surface membrane CD3+ TCRdelta- 84% vs. surface membrane CD3- TCRdelta- 48%, P value=0.03). Junctional region sequence analyzed with 10 cases each, of TCRG and TCRD, revealed an average junctional region of 7.4 nucleotides (range 2-18 nucleotides) in TCRG and 27 nucleotides (range 14-42 nucleotides) in TCRD-complete rearrangements. In TCRG, trimming at the ends of Vgamma and Jgamma germline nucleotides resulted in deletion, on an average of 9.2 nucleotides. In TCRD, deletion of nucleotides of the Vdelta and Jdelta gene segments on an average was 3.5 nucleotides. The junctional region of TCRD is more diverse than TCRG; nevertheless, the frequency of TCRG was more than that of TCRD and hence we rely more on TCRG clonal markers to quantitate the minimal residual disease in T-ALL.

Gene-specific signal joint modifications during V(D)J recombination of TCRAD locus genes in murine and human thymocytes.

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.

Regulation of TCR delta and alpha repertoires by local and long-distance control of variable gene segment chromatin structure.

Murine Tcrd and Tcra gene segments reside in a single genetic locus and undergo recombination in CD4- CD8- (double negative [DN]) and CD4+ CD8+ (double positive [DP]) thymocytes, respectively. TcraTcrd locus variable gene segments are subject to complex regulation. Only a small subset of approximately 100 variable gene segments contributes substantially to the adult TCRdelta repertoire. Moreover, although most contribute to the TCRalpha repertoire, variable gene segments that are Jalpha proximal are preferentially used during primary Tcra recombination. We investigate the role of local chromatin accessibility in determining the developmental pattern of TcraTcrd locus variable gene segment recombination. We find variable gene segments to be heterogeneous with respect to acetylation of histones H3 and H4. Those that dominate the adult TCRdelta repertoire are hyperacetylated in DN thymocytes, independent of their position in the locus. Moreover, proximal variable gene segments show dramatic increases in histone acetylation and germline transcription in DP thymocytes, a result of super long-distance regulation by the Tcra enhancer. Our results imply that differences in chromatin accessibility contribute to biases in TcraTcrd locus variable gene segment recombination in DN and DP thymocytes and extend the distance over which the Tcra enhancer can regulate chromatin structure to a remarkable 525 kb.

DHPLC based fraction collection of TCR-gamma rearrangements in childhood ALL: direct sequencing of products amplified by a single or a multiplex PCR approach.

Clonal T-cell receptor gamma (TCR-gamma) rearrangements are frequently used for detection of minimal residual disease (MRD) in childhood acute lymphoblastic leukemia. In approximately 70-80% of cases PCR amplified clonal rearrangements can be sequenced directly. The remaining 20-30% are rearranged on both alleles for the same target and disables direct sequencing. Here we describe a novel HPLC based method for identification and characterisation of TCR-gamma rearrangements either by a single or a multiplex PCR approach. The latter one amplifies several Vgamma segments in two distinct reactions either with a Jgamma1.3/2.3 or a Jgamma1.1/2.1 specific primer. The clonality status was evaluated on a high resolution micropellicular DNASep matrix (WAVE, Transgenomic) at different temperatures. From 331 samples analysed, 151 samples were positive for VgammaI-Jgamma1.3/2.3 including 51 biclonal rearrangements. For characterisation of these biclonal products or for products generated by multiplex-PCR, a second HPLC run was performed utilising a tandem arranged fraction collector. From clearly separated biclonal/biallelic products, several collected fractions were air-dried and afterwards sequenced directly with the appropriate Jgamma primer. We conclude from our results that HPLC is a fast and reliable method for identification of TCR-gamma rearrangements. The fraction collection simplifies the characterisation of single alleles within biclonal or biallelic rearrangements or within multiplex PCR products. The target identification process prior to routine MRD analysis will be shortened due to a simplified screening and sequencing strategy.

New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling.

To gain more insight into initiation and regulation of T cell receptor (TCR) gene rearrangement during human T cell development, we analyzed TCR gene rearrangements by quantitative PCR analysis in nine consecutive T cell developmental stages, including CD34+ lin- cord blood cells as a reference. The same stages were used for gene expression profiling using DNA microarrays. We show that TCR loci rearrange in a highly ordered way (TCRD-TCRG-TCRB-TCRA) and that the initiating Ddelta2-Ddelta3 rearrangement occurs at the most immature CD34+CD38-CD1a- stage. TCRB rearrangement starts at the CD34+CD38+CD1a- stage and complete in-frame TCRB rearrangements were first detected in the immature single positive stage. TCRB rearrangement data together with the PTCRA (pTalpha) expression pattern show that human TCRbeta-selection occurs at the CD34+CD38+CD1a+ stage. By combining the TCR rearrangement data with gene expression data, we identified candidate factors for the initiation/regulation of TCR recombination. Our data demonstrate that a number of key events occur earlier than assumed previously; therefore, human T cell development is much more similar to murine T cell development than reported before.

Heat shock protein induced TCR gammadelta gene rearrangements in patients with oral cancer.

We analyzed the T cell receptor (TCR) gammadelta gene repertoire in peripheral blood and tumor compartment of oral cancer (OC) patients before and after stimulation with heat shock proteins (hsp), which are known ligands for gammadelta T cells. Clonal TCR gamma and delta gene rearrangements in lymphocytes from tumor compartment and peripheral blood were studied using TCR Vgamma and Vdelta gene primers in PCR followed by heteroduplex analysis. Vgamma gene segments derived from VgammaI or VgammaII gene families were most dominantly expressed in peripheral blood lymphocytes (PBL) as compared to tumor infiltrating lymphocytes (TIL) of OC patients. Of the rearranged TCR delta alleles Vdelta1-Jdelta1 and Vdelta2-Jdelta1 gene rearrangements were the most predominant in PBL and TIL of OC patients respectively. Stimulation of gammadelta T cells with hsp 60/70 demonstrated a selective clonal expansion of Vgamma9-Vdelta2 (VgammaII family) subset indicating that, this expanded population of cells could be responsible for eliciting an immune response against oral tumor cells.

IgH DJ rearrangements within T-ALL correlate with cCD79a expression, an immature/TCRgammadelta phenotype and absence of IL7Ralpha/CD127 expression.

cCD79a and IgH VDJ/DJ rearrangements are considered to be relatively specific for B lymphoid precursors. We looked for both in cCD3+, CD7+, CD19- T-ALLs classified by TCR status into alphabeta or gammadelta/immature (IM) lineages, with individualization of HOX11L2+ T-ALLs since they represent an intermediate alphabeta/gammadelta category. cCD79a was expressed at low levels in 47% of T-ALL and was most frequent in IMgamma T-ALLs. IgH rearrangements were common in gammadelta/IM (45%) and HOX11L2+ (35%) T-ALLs compared to HOX11L2-negative cases (3%; P<0.001). CD127 (IL7Ralpha) expression was also more common in the gammadelta/IM lineage but its expression was virtually mutually exclusive of IgH rearrangement. Low-level cCD79a expression alone should therefore not be interpreted as evidence of B lineage affiliation in immature leukemias. gammadelta/IM lineage T-ALLs potentially include two distinct categories: predominantly IgH+, cCD79a+, CD127- cases which retain gammadelta and B lymphoid potential and IgH-, cCD79a-, CD127+ cases with restricted T lineage potential.