A targeted deletion of a region upstream from the Jkappa cluster impairs kappa chain rearrangement in cis in mice and in the 103/bcl2 cell line.

We have shown previously that a mutation of the KI-KII site immediately 5' to J(kappa)1 on the mouse immunoglobulin light chain kappa locus reduces the rearrangement level in cis, although it does not affect transcription. Here we deleted by homologous recombination in mouse embryonic stem cells a 4-kb DNA fragment, located immediately upstream of the KI-KII element, which contains the promoter of the long germline transcript. Analysis of gene-targeted heterozygous mouse splenic B cells showed a strong decrease in rearrangement for the allele bearing the deletion. When both the KI-KII mutation and the 4-kb deletion were present on the same allele, the overall reduction in rearrangement was stronger than with the 4-kb deletion alone underlying the role of these two elements in the regulation of rearrangement. The same deletion was performed by homologous recombination on one allele of the rearrangement-inducible mouse 103/bcl2-hygro(R) pre-B cell line, and resulted in a similar reduction in the induction of rearrangement of the mutated allele. This result validates this cell line as an in vitro model for studying the incidence of gene-targeted modifications of the kappa locus on the regulation of rearrangement.

Further analysis of the T cell receptor gamma/delta+ peripheral lymphocyte subset. The V delta 1 gene segment is expressed with either C alpha or C delta.

In the present study, we have characterized the reactivity of two mAbs that are directed at the human TCR-gamma/delta. These reagents, designated anti-A13 and anti-TiV delta 2, were found to recognize antigenic determinants encoded by the TCR V delta 1 and V delta 2 gene segments, respectively. Immunofluorescence analyses performed with the antibodies confirmed that, in the TCR-gamma/delta+ cell subpopulation, the expression of V delta 2+ delta chains is largely predominant, as compared with the V delta 1+ counterparts. However, these experiments led to an apparently discrepant finding. Indeed, the total number of cells recognized by the anti-A13 plus the anti-TiV delta 2 antibodies was often greater than that detected with anti-TCR-delta 1, a reagent specific for a constant epitope of the human delta chain. Further investigation showed that the presence of a sizeable peripheral lymphocyte subset coexpressing the BMA031 and the A13 epitopes. Because the former antibody is known to recognize an invariant antigenic determinant of the TCR-alpha/beta dimer, these results suggested that the V delta 1 gene segment may be expressed with either C delta or C alpha. This hypothesis was confirmed using T2, an IL-2-dependent BMA031+ A13+ polyclonal cell line developed from peripheral blood of a healthy adult donor. Indeed, T2 cells were found to have productively rearranged the V delta 1 gene. Together, results of Northern blot analysis and cDNA cloning indicated that V delta 1 was expressed in these cells as part of a 1.6-kb full-length message including J alpha-C alpha segments.

Rearrangement-enhancing element upstream of the mouse immunoglobulin kappa chain J cluster.

Transcriptional regulatory elements have been shown to be necessary but not sufficient for the developmental regulation of immunoglobulin gene rearrangement in mouse precursor B cells. In the chicken lambda light chain locus, additional elements in the V-J intervening sequence are involved in negative and positive regulation of rearrangement. Here, mutation of the mouse homolog of a chicken element, located in the V(K)-J(K) intervening sequence upstream of the J(K) cluster, was shown to significantly decrease rearrangement. This cis-acting recombination-enhancing element affects the rearrangement process without being involved in regulating transcription.

Analysis of T cell receptor variability in tumor-infiltrating lymphocytes from a human regressive melanoma. Evidence for in situ T cell clonal expansion.

Malignant melanomas are often infiltrated by T lymphocytes. It is postulated that the presence of tumor-infiltrating lymphocytes (TIL) reflects ongoing immune responses against transformed cells. Such "responses" appear generally inefficient with the potential exception of infrequent clinical situations characterized by spontaneous tumor regression. We have characterized here the molecular structure of the T cell receptor beta chain expressed by TILs in a case of regressive melanoma. Advantage was taken of the PCR technology to study T lymphocytes directly without cell culture. Experimentally validated V beta subfamily specific primers were used to evaluate the V beta usage in TILs and control samples. Our results reveal that clonal T cell populations, precisely defined by their V-D-J junctional sequences, are amplified at the tumor site. The existence of such local antigen-driven selections support the hypothesis that antitumor responses may indeed take place in regressive melanoma.

Evidence for in situ amplification of cytotoxic T-lymphocytes with antitumor activity in a human regressive melanoma.

We have derived from lymphocytes infiltrating a human regressive melanoma lesion a series of T-cell receptor alpha/beta-dependent, HLA-B14-restricted cytotoxic T-lymphocyte clones reactive against the autologous tumor. Analysis of the T-cell receptor gene expression revealed that all the clones represented a unique cell expressing a V beta 13.1/J beta 1.1 gene segment. T-cell receptor transcripts expressed in the cloned cells were compared to those present in the uncultured tumor tissue. This analysis demonstrated that the specific cytotoxic T-lymphocyte clones characterized in vitro was actually selected and amplified in vivo at the lesion site. These results provide strong evidence that effector T-cells have contributed to tumor regression.

Analysis of T-cell receptor alpha/beta variability in lymphocytes infiltrating a melanoma metastasis.

Multiple experimental and clinical studies have suggested that the immune system may, to some extent, control the development of melanomas. The presence of tumor-infiltrating lymphocytes could reflect an in situ immune reaction directed to the malignant cells. The characterization of T-cell receptor (TCR) expressed by tumor-infiltrating lymphocytes is one way to precisely analyze these local T-cell responses. In this study, we have assessed the TCR alpha/beta variability in tumor-infiltrating lymphocytes from a subcutaneous metastasis of a melanoma patient. Using the anchored-polymerase chain reaction 268 TCR alpha and 266 TCR beta chain transcripts have been cloned and sequenced. Their analysis shows that the T-cell infiltrate is extremely diverse, with no preferential TCR gene segment usage.

Control of apoptosis in Epstein Barr virus-positive nasopharyngeal carcinoma cells: opposite effects of CD95 and CD40 stimulation.

The expression and function of CD95 and CD40 were investigated in malignant cells from EBV-positive undifferentiated nasopharyngeal carcinomas (NPCs). Large amounts of CD95 and CD40 expression were detected in 15 of 16 EBV-positive NPC specimens. In contrast, CD95 was not detected in two biopsies from patients with EBV-negative differentiated NPCs. We tested whether the CD95 apoptotic pathway was functional in NPC cells by treating two EBV-positive NPC tumor lines in vitro with a CD95 agonist. In both cases, NPC cells were extremely susceptible to CD95-mediated apoptosis, despite strong constitutive expression of Bcl-x. Combined CD40 and CD95 stimulation was used to investigate the possible anti-apoptotic activity mediated by CD40. The CD40 receptor was activated by incubating NPC cells with murine L cells producing CD154, the CD40 ligand. This treatment resulted in a strong inhibition of CD95-related cytotoxicity. Such an anti-apoptotic effect of CD40 is well known for B lymphocytes, but has not previously been reported for epithelial cells. These data suggest that NPC tumor-infiltrating lymphocytes, which often produce the CD40 ligand in situ, may increase the survival of malignant cells, thereby enhancing tumor growth in patients.

Alternatively spliced T cell receptor transcripts expressed in human T lymphocytes.

We used the anchored-polymerase chain reaction (A-PCR) procedure to study human TCR transcripts derived from a variety of polyclonal T cell populations. In this series of experiments, 31 'unusual' cDNAs, which do not include exclusively V-J-C, J-C or 5'C genomic sequences, were identified. Ten of these were found to represent distinct types of alternatively spliced TCR alpha transcripts whose structure is derived from unusual splicing of one, two or even three intervening intronic sequences. The splicing events led to either conservation of a novel exon in the mRNA structure (designated aE1 alpha-aE5 alpha) between the V-J and C segments or to deletion of the 3' V region-J segment. In three cases, the alternatively spliced exons (aE1 alpha-aE3 alpha) interrupt the open translational reading frame of the corresponding V-J alpha segment. Nineteen and two cDNA represent sterile C beta or C delta transcripts, respectively. Their structures are derived from the conservation of a non-translatable exon, aE1 beta or aE1 delta, which is precisely spliced at the 5' end of the corresponding C exon sequences. Interestingly, the 3' region of the aE1 beta sequence is homologous to the murine C beta 0 exon. Together, these results led to the characterization of nine novel exons in the TCR alpha, beta and delta loci.

The use of anchored polymerase chain reaction for the study of large numbers of human T-cell receptor transcripts.

Anchored-PCR (A-PCR) is an approach designed to amplify and clone sequences with unknown 5' or 3' extremities. A-PCR is therefore appropriate for studying variable region of T-cell receptors (TCRs) expressed in polyclonal T-cell populations since it does not prejudge which variable gene segments are actually being used. We report here some critical modifications in the initial procedure to make it easy to clone and sequence large series of TCR transcripts. They have been introduced to improve both the yield and specificity of TCR amplified products and include re-amplification, size selections of the material combined with the successive use of nested TCR constant region specific primers. This procedure has been successfully applied to the study of the repertoire of both TCR alpha/beta+ and gamma/delta+ human T-cells. The efficiency of the present A-PCR protocol will help to precisely analyze TCR usage in normal and pathological situations.

[Technological advances in immuno-oncology: from fundamental concepts to patient immunological monitoring]. / L'apport technologique en immuno-oncologie: des concepts fondamentaux au suivi immunologique des patients.

Over the past decade, cancer immunology has known several advances due to both basic research and new technologies recently developed in this field. This review will illustrate the impact of some new immunological technologies and how the latter resulted in the exploration of new territories in cancer immunology and the emergence of new concepts that allowed to revisit the immunosurveillance concept and permitted to improve the patient monitoring.

Rearrangement of the chicken lambda light chain locus: a silencer/antisilencer regulation.

Chicken immunoglobulin lambda light chain rearrangement is regulated by different controlling elements. One negative control element, acting as a strong transcriptional silencer, is located in the V-J intervening sequence which is excised during the rearrangement process. Positive control elements include the V lambda promoter, the enhancer of transcription (3' of C lambda), and one (or two) putative antisilencer element(s) located on each side of the silencer. It is proposed that these antisilencer elements counteract transiently the silencer thus allowing rearrangement of one allele in chicken B cell progenitors. The implications of this regulation for mouse B cell development are discussed.

Negative regulation of Ig gene rearrangement by a 150-bp transcriptional silencer.

We previously showed that the V-J intervening sequence of the chicken lambda immunoglobulin locus contains a strong silencer that acts both on transcription and rearrangement. We show here that the transcriptional silencer activity can be ascribed to a minimal 150-bp fragment. The rearrangement silencing activity was previously shown by the replacement of the V-J intervening sequence with a neutral DNA fragment that dramatically increased the rate of rearrangement of the transgene. Insertion of the minimal silencer in this neutral fragment is shown here to result in a marked decrease in rearrangement of the transgenic construct. Strikingly, deletion of 28 bp from the 150-bp fragment abolished most of the transcriptional silencing activity and had a similar effect on rearrangement. These results conclusively correlate the silencing activity on both rearrangement and transcription.

Studies on the human T cell receptor alpha/beta variable region genes. I. Identification of 7 additional V alpha subfamilies and 14 J alpha gene segments.

The anchored-polymerase chain reaction has been used to study further the diversity of the human T cell receptor alpha chain. The analysis of 308 cDNA transcripts from human peripheral lymphocytes hybridizing with a C alpha probe led to the identification of a series of additional V alpha and J alpha gene segments. The sequences of seven V alpha gene segments which individually define a novel V alpha subfamily (termed V alpha w23 to V alpha w29) are reported. The sequences of some previously described V alpha 1, V alpha 2, V alpha 5, V alpha 7 and V alpha 22 gene segments are also extended. In addition, we report 14 novel J alpha gene segment sequences. Taken together, these data indicate that the contribution of the alpha chain combinatorial diversity to the human T cell receptor alpha/beta variability has not yet been fully appreciated.

Studies on the human T cell receptor alpha/beta variable region genes. II. Identification of four additional V beta subfamilies.

The human T cell receptor (TcR) beta chain gene segment diversity has been studied using the anchored-polymerase chain reaction. Three hundred and fifty C beta-specific transcripts derived from peripheral lymphocytes were analyzed. Transcripts including V-D beta 1-J beta 2-C2 sequences were found with a high frequency (greater than 10%), suggesting that "illegitimate" joinings may constitute a cis-complementing rearrangement mechanism capable of substantially increasing the TcR beta chain combinatorial diversity. Twelve previously undescribed V beta gene segments have been identified. Five of them delineate four novel V beta subfamilies (V beta w21: two members, V beta w22, 23, 24: one member) which all have a murine homologue. The additional seven gene segments belong to the V beta 5, V beta 6, V beta 12 and V beta 13 subfamilies. In addition, the sequences of two known V beta 7 and V beta 9 gene segments have been extended. Together, the present data support the view that the contribution of the beta chain combinatorial diversity to the TcR alpha/beta variability has not yet been fully appreciated.

In-situ preferential usage of V alpha 8 T-cell receptor gene segments in a patient with bullous pemphigoid.

Bullous pemphigoid (BP) is an autoimmune blistering skin disease associated with the occurrence of autoantibodies directed to a limited series of antigens located at the basement membrane zone of the dermo-epidermal junction. Previous studies have demonstrated the presence of activated T cells in BP lesions although their actual contribution to the pathogenesis of the disease has remained unclear. One approach to better understanding the significance of these T-cell infiltrates is to study the diversity of the recognition receptors (TCRs) expressed at the lesion sites. We report here an extensive analysis, performed in one patient with typical BP, where 187 TCR in frame transcripts from a lesional area, from clinically normal skin or from peripheral blood lymphocytes have been sequenced and compared. The data show preferential usage of the V alpha 8 subfamily gene segments in the lesion. It is therefore suggested that T-cell infiltration in BP may not simply reflect a non-specific inflammatory process but include antigen-specific responses.

Molecular characterization of human T cell receptor alpha chains including a V delta 1-encoded variable segment.

Previously we have shown that a small fraction of human peripheral T cells expresses a surface receptor recognized both by the BMA031 mAb, specific for a TcR alpha/beta framework epitope, and by the A13 mAb, putatively specific for an epitope encoded by the V delta 1 gene segment. An interleukin 2-dependent polyclonal cell line (termed T2) was derived from such A13+BMA031+ circulating lymphocytes. The molecular characterization of the TcR chains expressed by T2 cells demonstrated indeed that the V delta 1 gene (one of the two major V delta genes) was transcribed with the C alpha gene segment. In the T2 polyclonal cell line, distinct V delta 1/C alpha transcripts were all found to include the same J alpha segment suggesting the existence of "hybrid" TcR alpha/delta chains encoded by unique V delta 1/J alpha rearrangements. The present study was designed to characterize further the V delta 1/J alpha rearranged genes expressed in A13+BMA031+ cells. Three additional cell lines were generated from peripheral blood of distinct adult healthy donors. Using the anchored polymerase chain reaction, it was found that 17 different J alpha segments were used in the 20 V delta 1J alpha C alpha transcripts which have been studied. Together, these data indicate that V delta 1 is a "mixed" (i.e. alpha/delta) TcR V segment which can join with most (if not all) J segments in the alpha/delta locus. In addition, it can be definitely concluded that the A13 mAb recognizes a V delta 1-encoded antigenic determinant and not a V delta 1J epitope (i.e. it can be defined and used as an anti-V delta 1 mAb, as opposed to reagents such as for example delta-TCS-1).

Cytotoxic potential despite impaired activation pathways in T lymphocytes infiltrating nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is an epithelial tumor consistently associated with EBV. The histological picture is characterized by a strikingly abundant lymphocytic infiltrate. Furthermore, the epithelial tumor cells present several immunological characteristics which suggest an important role for tumor-infiltrating lymphocytes (TIL) in the biology of this tumor. The present study reports the phenotypic and functional characterization of TIL from NPC obtained after enzymatic digestion of 15 NPC biopsies. Flow cytometric analysis of TIL suspensions indicated that most TIL were mature CD3+ T lymphocytes (mean = 60%) with a variable CD4/CD8 ratio. Most TIL were TCR alpha/beta-positive (mean = 55%) and only a few TCR gamma-delta-positive cells could be identified. A small percentage (mean = 9%) displayed an activated phenotype (CD25+, HLA class II+). Using limiting dilution analysis, we found that the average frequency of proliferative T-lymphocyte precursors (PTL-P) is lower among TIL (1/40) than in autologous (1/7) or normal PBL (1/1.4). Moreover, sorting experiments have shown that this defect is significantly more pronounced in the CD8+ than in the CD4+ TIL subset. Accordingly, the TCR and the CD2-mediated antigen-independent pathways of activation were impaired. Different types of cytotoxic precursor could be detected. These included lectin-dependent cell cytotoxicity (LDCC) and NK-like or lymphokine-activated killer (LAK) activity. Interestingly, some TIL from NPC were able to lyse an NPC tumor (C15) maintained in nude mice. Thus, despite impaired activation pathways, the cytolytic potential of proliferating TIL in NPC is preserved.

Limited T-cell receptor diversity in liver-infiltrating lymphocytes from patients with primary biliary cirrhosis.

Primary biliary cirrhosis is associated with the presence of high-titer anti-mitochondrial autoantibodies as well as T-cell infiltration of the liver, suggesting the involvement of autoimmune mechanisms. We have studied here the sequences of T-cell receptor alpha and beta chains expressed by T-cell clones derived from liver-infiltrating lymphocytes of two patients with primary biliary cirrhosis. Among the eight clones studied from the first patient, four expressed the same member of the V beta 6 subfamily, associated with either V alpha 4 (three clones) or V alpha 21 (one clone) gene segment. Two other clones expressed an identical V beta 12 transcript, and two in-frame alpha chain transcripts, involving V alpha 2 and V alpha 7 gene segments. From the second patient, eight out of the nine clones were found to rearrange V beta 17-J beta 2.1 and V alpha 3 gene segments. The remaining clone expressed distinct T-cell receptor chains, involving V beta 9 and V alpha 11 gene segments. As deduced from the analysis of their junctional regions, the eight T-cell clones expressing V beta 17/V alpha 3 gene segments derived from only three different T cells. Furthermore, conserved amino acid motifs were found to be encoded in both the alpha and the beta-chain junctional regions. Together, these data show a local amplification of unique T lymphocytes in both patients. The use of identical V beta J beta and V alpha gene segments with similar junctional sequences by three different cells, evidenced in one of the two cases, strengthens the view that liver-infiltrating T lymphocytes are selected locally by autoantigens in PBC.

An experimentally validated panel of subfamily-specific oligonucleotide primers (V alpha 1-w29/V beta 1-w24) for the study of human T cell receptor variable V gene segment usage by polymerase chain reaction.

We report here the characterization of a series of T cell receptor (TcR) V alpha or V beta subfamily-specific oligonucleotide primers. Criteria that have guided the design of each oligonucleotide include appropriate thermodynamic parameters as well as differential base-pairing scores with related and unrelated target sequences. The specificity of the oligonucleotides for each V alpha or V beta subfamily was tested by polymerase chain reaction (PCR) on both a series of TcR encoding plasmid DNA and clonal T cell populations. Unexpected cross-reactivities were observed with plasmid cDNA sequences corresponding to unrelated subfamily gene segments. This led to the synthesis of additional series of oligonucleotides to obtain a relevant panel. A series of V alpha 1-w29/V beta 1-w24 TcR subfamily-specific oligonucleotides was eventually selected which generates little, if any, cross-reactivity. The use of C alpha or C beta primers for the amplification of internal positive control templates (i.e. C beta for the V alpha series and C alpha for the V beta series) has been tested in PCR performed with cDNA derived from peripheral blood lymphocytes; it was shown not to alter the amplification of the V subfamily-specific DNA fragments. This panel of oligonucleotides will be helpful in the study of TcRV gene segment usage and, thus, may lead to a better characterization of T cell responses in physiological and pathological situations.